WHO/Searo Dengue Bulletin – Vol 28, December 2004

Transcription

D
ENGUE
BULLETIN
Volume 28
December 2004
DENGUE BULLETIN
Volume 28, 2004
WORLD HEALTH ORGANIZATION
South-East Asia Region
Western Pacific Region
Dengue Bulletin
Volume 28
December 2004
World Health Organization
South-East Asia Region
Western Pacific Region
ISBN 92 9022 256 5
© World Health Organization 2004
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the provisions of Protocol 2 of the Universal Copyright Convention. For rights of reproduction
or translation, in part or in toto, of publications issued by the WHO Regional Office for SouthEast Asia, application should be made to the Regional Office for South-East Asia, World Health
House, Indraprastha Estate, New Delhi 110 002, India.
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not imply the expression of any opinion whatsoever on the part of the Secretariat of the World
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Printed in India
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Acknowledgements
Editor, Dengue Bulletin, WHO/SEARO, gratefully thanks the following for peer reviewing
manuscripts submitted for publication.
In-house Review:
Nand L. Kalra: Reviewed the manuscripts in respect of format check, content, conclusions
drawn, including condensation of tabular and illustrative materials for clear, concise and
focused presentation and bibliographic references. He was also involved in the final stages
of printing of the Bulletin.
Peer Reviewers
1. Dana A. Focks
John W. Hock Company
P.O. Box 12852
Gainesville, FL 32604
USA
E-mail: [email protected]
2. Duane J. Gubler
Director
Asia-Pacific Institute of Tropical Medicine and Infectious Diseases
Leahi Hospital
3675 Kilauea Ave
Honolulu, Hawaii 96816
USA
3. Oon Chong Teik
Tropical Medicine & Infectious Diseases
Mt Elizabeth Hospital
Singapore 228510
Dengue Bulletin – Vol 28, 2004
iii
4. Scott Halstead
Uniformed Services University of the Health Sciences
Bethesda, Maryland
USA
5. Siripen Kalyanarooj
WHO Collaborating Centre for Case Management
of Dengue/DHF/DSS
Queen Sirikit National Institute of Child Health (Children’s Hospital)
Bangkok
Thailand
6. Suchitra Nimmannitya
WHO Collaborating Centre for Case Management
of Dengue/DHF/DSS
Queen Sirikit National Institute of Child Health (Children’s Hospital)
Bangkok
Thailand
7. Kevin Palmer
Regional Office for the Western Pacific
P.O. Box No. 2932
12115 Manila
Philippines
8. Michael Nathan
Headquarters
20 Avenue Appia
1211 Geneva 27
Switzerland
The quality and scientific stature of the Dengue Bulletin is largely due to the conscientious
efforts of the experts and also due to the positive response of contributors to comments
and suggestions.
iv
From the Editor’s Desk
O
ver the decades dengue/dengue haemorrhagic fever has emerged as a global public
health problem with countries in Asia and the Pacific sharing more than 70 % of the
disease burden. In some of these countries, DHF is gaining hyper-endemicity causing deaths
among children. During 2004, Indonesia reported a major dengue outbreak encompassing
Central Java, Sumatra and some outer islands. Till the end of July 2004, 69,017 cases of
DF/DHF and 770 deaths were registered by Indonesian health authorities. During this
epidemic DEN-3 was the predominant serotype.
Sri Lanka also reported a major outbreak with 12,400 cases and 71 deaths as of 23
August 2004. A majority of the cases were reported from five cities: Colombo, Kandy,
Gampaha, Kalutara and Kurunegala.
In the South-East Asia Region, Bhutan and Nepal continued to enjoy dengue-free status
till 2003 because of their sub-mountainous location. However, during August 2004, Bhutan
recorded the first-ever outbreak of DF/DHF in Phuntsholing (population 27,000), a border
town with India. During this outbreak a total of 2,544 DF/DHF cases with no deaths were
reported. More than 93 % of those affected were persons above 5 years of age. This sent a
strong signal to the adjoining DF-free north-eastern part of India and Nepal to take appropriate
preventive action.
DengueNet, the WHO Global Surveillance System for management of epidemiological
and virological surveillance data for early detection, planning and response, was launched in
the South-East Asia and Western Pacific countries during 2004. Each country identified
institutions which would participate in the programme.
The current Volume 28 (2004) of the Dengue Bulletin includes contributions from the
South-East Asia Region (13), the Western Pacific Region (7), the American Region (5) and the
European Region (4).
A supplement, featuring experiences from different countries in social mobilization and
communication for dengue prevention and control, is also being issued along with this volume.
We now invite contributions for Volume 29 (2005). The deadline for the receipt of
contributions is 30 June 2005. Contributors are requested to follow the instructions carefully
while preparing the manuscript. Contributions accompanied by computer diskettes using MS
Word for Windows should be sent to the Editor, Dengue Bulletin, WHO/SEARO, Mahatma
Gandhi Road, IP Estate, Ring Road, New Delhi-110 002, India, or by e-mail as a file
attachment to the Editor at [email protected]. Readers desirous of obtaining copies of the
Dengue Bulletin may contact the respective WHO Regional Offices in New Delhi or Manila or
the WHO Country Representative in their country of residence.
Dr Chusak Prasittisuk
Regional Adviser
Vector-borne Disease Control
Regional Office for South-East Asia
New Delhi, India
Contents
1.
Annual Changes of Predominant Dengue Virus Serotypes in Six Regional
Hospitals in Thailand from 1999 to 2002
1
Surapee Anantapreecha, Sumalee Chanama, Atchareeya A-nuegoonpipat,
Sirirat Naemkhunthot, Areerat Sa-ngasang, Pathom Sawanpanyalert
and Ichiro Kurane
2.
A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad:
Demographic and Clinical Features
7
T.U. Brown, K. Babb, M. Nimrod, C.V.F. Carrington, R.A. Salas
and M.A. Monteil
3.
Ecological Study of Rio de Janeiro City DEN -3 Epidemic, 2001-2002
20
Maria Lucia F. Penna
4.
Sero-epidemiological and Virological Investigation of Dengue Infection
in Oaxaca, Mexico, during 2000-2001
28
A. Cisneros-Solano, M.M.B. Moreno-Altamirano, U. Martínez-Soriano,
F. Jimenez-Rojas, A. Díaz-Badillo and M.L. Muñoz
5.
Spatial and Temporal Dynamics of Dengue Haemorrhagic Fever
Epidemics, Nakhon Pathom Province, Thailand, 1997-2001
35
Wutjanun Muttitanon, Pongpan Kongthong, Chusak Kongkanon,
Sutee Yoksan, Narong Nitatpattana, Jean Paul Gonzalez and Philippe Barbazan
6.
Sporadic Prevalence of DF/DHF in the Nilgiri and Cardamom Hills of
Western Ghats in South India: Is it a Seeding from Sylvatic Dengue Cycle
– A Hypothesis
44
Nand Lal Kalra and Chusak Prasittisuk
7.
Autoimmunity in Dengue Virus Infection
51
Chiou-Feng Lin, Huan-Yao Lei, Ching-Chuan Liu, Hsiao-Sheng Liu,
Trai-Ming Yeh, Shun-Hua Chen and Yee-Shin Lin
8.
Inhibition of the NS2B-NS3 Protease – Towards a Causative Therapy for
Dengue Virus Diseases
58
Gerd Katzenmeier
v
Contents
9.
Prognostic Factors of Clinical Outcome in Non-Paediatric Patients with
Dengue Haemorrhagic Fever/Dengue Shock Syndrome
68
Jaime R. Torres, José M. Torres-Viera, Hipólito García, José R. Silva,
Yasmín Baddour, Angel Bajares and Julio Castro M.
10.
Dengue Haemorrhagic Fever with Encephalopathy/Fatality at Petchabun
Hospital: A three-year Prospective Study (1999-2002)
77
Prasonk Witayathawornwong
11.
A New Tool for the Diagnosis and Molecular Surveillance of Dengue
Infections in Clinical Samples
87
C. Domingo, G. Palacios, M. Niedrig, M. Cabrerizo, O. Jabado,
N. Reyes, W.I. Lipkin and A. Tenorio
12.
Clinical and Laboratory Observations Associated with the 2000 Dengue
Outbreak in Dhaka, Bangladesh
96
Monira Pervin, Shahina Tabassum, Md. Mobarak Ali, Kazi Zulfiquer Mamun
and Md. Nazrul Islam
13.
Current Status of Dengue Diagnosis at the Center for Disease Control,
Taiwan
107
Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien,
Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin
and Jyh-Hsiung Huang
14.
Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in
the Presence of Sodium thiocyanate (NaSCN)
118
Masaru Nawa, Tomohiko Takasaki, Mikako Ito, Ichiro Kurane
and Toshitaka Akatsuka
15.
Genetic Influences on Dengue Virus Infections
126
J.F.P. Wagenaar, A.T.A. Mairuhu and E.C.M. van Gorp
16.
Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in
Guangzhou, China, in 2002
135
Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An
vi
Contents
17.
Induction of Cytotoxic T Lymphocytes by Immunization with Dengue
Virus – Derived, Modified Epitope Peptide, Using Dendritic Cells as a
Peptide Delivery System
145
Yoshiki Fujii, Hideyuki Masaki, Takanori Tomura, Kiyohiro Irimajiri
and Ichiro Kurane
18.
Molecular Characterization of Brazilian Dengue Viruses
151
Marize Pereira Miagostovich, Flávia Barreto dos Santos
and Rita Maria Ribeiro Nogueira
19.
Unusual Emergence of Guate98-like Molecular Subtype of DEN-3
during 2003 Dengue Outbreak in Delhi
161
Manoj Kumar, S.T. Pasha, Veena Mittal, D.S. Rawat, Subhash Chandra Arya,
Nirmala Agarwal, Depesh Bhattacharya, Shiv Lal and Arvind Rai
20.
The Animal Models for Dengue Virus Infection
168
Tao Peng, Junlei Zhang and Jing An
21.
Philippine Species of Mesocyclops (Crustacea: Copepoda) as a Biological
Control Agent of Aedes aegypti (Linnaeus)
174
Cecilia Mejica Panogadia-Reyes, Estrella Irlandez Cruz
and Soledad Lopez Bautista
22.
Susceptibility of Aedes aegypti to Insecticides in Viet Nam
179
Vu Duc Huong, Nguyen Thi Bach Ngoc, Do Thi Hien
and Nguyen Thi Bich Lien
23.
Ovipositioning Behaviour of Aedes aegypti in Different
Concentrations of Latex of Calotropis procera: Studies on
Refractory Behaviour and its Sustenance across Gonotrophic Cycles
184
Manju Singhi, Vinod Joshi, R.C. Sharma and Keerti Sharma
24.
Community-based Assessment of Dengue-related Knowledge
among Caregivers
189
Khynn Than Win, Sian Za Nang and Aye Min
25.
Students’ Perceptions about Mosquito Larval Control in a DengueEndemic Philippine City
196
Jeffrey L. Lennon
vii
Contents
Short Notes
1.
Sero-surveillance in Delhi, India – An Early Warning Signal for
Timely Detection of Dengue Outbreaks
207
D. Bhattacharya, Veena Mittal, M. Bhardwaj, Mala Chhabra, R.L. Ichhpujani
and Shiv Lal
2.
Detection of Dengue Virus in Wild Caught Aedes albopictus (Skuse)
around Kozhikode Airport, Malappuram District, Kerala, India
210
B.P. Das, L. Kabilan, S.N. Sharma, S. Lal, K. Regu and V.K. Saxena
3.
Entomological Investigations for DF/DHF in Alwar District, Rajasthan, India
213
Kalpana Baruah, Avdhesh Kumar and V.R. Meena
4.
Essentiality of Source Reduction in both Key and Amplification Breeding
Containers of Aedes aegypti for Control of DF/DHF in Delhi, India
216
B.N. Nagpal, Aruna Srivastava, M.A. Ansari and A.P. Dash
5.
Breeding of Dengue Vector Aedes aegypti (Linnaeus) in Rural Thar Desert,
North-western Rajasthan, India
220
B.K. Tyagi and J. Hiriyan
Book Reviews
1.
A Review of Entomological Sampling Methods and Indicators for
Dengue Vectors
223
2.
DengueNet in India
224
3.
WHO/WPRO/SEARO Meeting on DengueNet Implementation in SouthEast Asia and the Western Pacific, Kuala Lumpur, 11-13 December 2003
226
Instructions to Contributors
viii
232
Annual Changes of Predominant Dengue Virus
Serotypes in Six Regional Hospitals in Thailand
from 1999 to 2002
Surapee Anantapreecha* #, Sumalee Chanama*, Atchareeya A-nuegoonpipat*,
Sirirat Naemkhunthot*, Areerat Sa-ngasang*, Pathom Sawanpanyalert*
and Ichiro Kurane**
*National Institute of Health, Department of Medical Sciences, Ministry of Public Health,
88/7, Tivanond Road, Muang, Nonthaburi, 11000, Thailand
**Department of Virology I, National Institute of Infectious Diseases Toyama, Shinjuku-ku,
Tokyo 162-8640, Japan
Abstract
A virological study was conducted in six hospitals spread across Thailand from 1999 to 2002. All four
dengue serotypes were identified, of which DEN-1 was the most predominant. The predominant dengue
serotypes changed every 1-2 years in three of the six hospitals and the predominant serotypes were
different in different hospitals. DEN-1 was predominant in two hospitals during three years of the study
period (2000-2002). DEN-4 was not isolated, or accounted for only a small percentage of the total
isolates, except in one hospital in 2002.
Keywords: Dengue virus, dengue virus serotypes, Thailand.
Introduction
The dengue virus consists of four serotypes:
DEN-1, DEN -2, DEN-3 and DEN-4. The
clinical
manifestations
range
from
undifferentiated febrile illness to classic
dengue fever (DF), dengue haemorrhagic
fever (DHF) and to dengue shock syndrome
(DSS) [1-3] . Dengue virus infections are now
important public health problems in many
tropical and subtropical areas in the world.
In Thailand, a dengue outbreak first
occurred in Bangkok in 1958. It then
#
occurred in a pattern of 2-year cycle, and
subsequently in irregular cycles as the
disease spread throughout the country. The
largest outbreak was reported in 1987, with
174,284 reported cases, an incidence rate
of 325 cases per 100,000 population. It was
followed by another outbreak in 2001,
which reported an incidence rate of 224
cases per 100,000 population[2,4,5] . In 2002,
114,800 dengue cases were reported with
an incidence rate of 183 cases per 100,000
population. It is important to determine
which of the four serotypes causes the
E-mail: [email protected]; Tel.: 66 29510000 Ext. 99219, 99220; Fax: 66 25915449
1
Dengue Virus Serotypes in Thailand
dengue epidemic each year, and whether
the dominant serotype is the same or
different in each region in the country. In
the present study, we analysed the
predominant dengue serotypes at the six
regional hospitals located in the four regions
of Thailand – north, north-east, central and
south regions – in each year from 1999 to
2002.
Figure 1. Location of six provinces where
the sentinel sites were chosen
Materials and methods
Collection of blood specimens
A total of 5,160 and 3,619 blood specimens
were collected at acute and convalescent
stages, respectively, from suspected dengue
cases who visited Lampang Hospital in
Lampang
(north),
Maharat
Nakhon
Ratchasima Hospital in Nakhon Ratchasima
(north-east), Pathum Thani Hospital in
Pathum Thani, Chareonkrung Pracharak
Hospital in Bangkok, Ratchaburi Hospital in
Ratchaburi (central) and Hadyai Hospital in
Songkhla (south) from 1999 to 2002 (Figure
1). Blood specimens were drawn into tubes
with ethylene diamine tetaacetate (EDTA)
anticoagulant and centrifuged. The number
of specimens ranged from 1 to 3 per patient.
The first samples were collected on the day
of hospitalization and the second samples
were collected on the day of discharge.
Third samples, if any, were collected
between 10-14 days after the onset of
symptoms. Plasma and buffy coat were
separated at each hospital, kept in liquid
nitrogen and then transported to the
Arbovirus Section of the National Institute of
Health, Nonthaburi. Specimens were kept
at -70 °C for virus isolation.
2
1 = Lampang
2 = Nakhon Ratchasima
3 = Pathum Thani
4 = Bangkok
5 = Ratchaburi and
6 = Songkhla
Virus isolation and determination
of dengue virus serotypes
Ten microlitres of acute phase-buffy coat
samples were inoculated onto monolayer
of C6/36 cells in a 24-well plate with
rocking for 90 minutes at room
temperature [6] . The inocula were discarded
and replaced by Leibovitz 15 medium (L15, Gibco BRL) containing 1% heatinactivated fetal bovine serum. After 7 days
of incubation at 28 °C, the cultured media
were collected, the infected cells were
stained by IFA, and dengue virus serotypes
were determined[7,8] . DEN -1 (16007 strain),
DEN -2 (16681 strain), DEN-3 (16562
strain) and DEN-4 (1032 strain) were used
as virus controls in IFA.
Results and discussion
The numbers of samples tested and found
positive for virus isolation are shown in
Table 1.
Table 1. Number of samples used for the analysis
Acute sample
Convalescent
sample
Virus isolation
positive
Year
Hospital
1999
Lampang
Maharat Nakhon Ratchasima
Pathum Thani
Chareonkrug Pracharak
Ratchaburi
Hadyai
128
26
0
0
35
1
108
18
0
0
11
1
74
6
0
0
31
0
2000
Lampang
Pathum Thani
Ratchaburi
Hadyai
35
22
25
15
438
113
33
17
14
15
419
28
15
3
4
10
238
22
2001
Lampang
Pathum Thani
Ratchaburi
Hadyai
262
358
265
224
389
450
238
251
170
216
363
231
150
176
143
99
256
276
2002
Lampang
Pathum Thani
Chareonkrung Pracharak
Ratchaburi
Hadyai
339
930
154
135
490
326
236
600
73
128
346
103
95
292
32
65
170
77
All the four dengue serotypes were
detected in the six hospitals during this
study. When the total numbers of isolates
were analysed, DEN-1 was the predominant
serotype (48%), followed by DEN -2 (27%),
DEN-3 (20%) and DEN -4 (5%).
The
predominant
dengue
virus
serotypes in each hospital were analysed
when more than nine isolates were obtained
in a year. In the Lampang Hospital, the
predominant serotypes were DEN-2 (44%)
in 1999, DEN-1 (86%) in 2000, DEN-1
3
(57%) in 2001 and DEN-2 (79%) in 2002. In
the Maharat Nakhon Ratchasima Hospital,
the predominant serotypes were DEN-1
(47%) in 2001 and DEN-1 (57%) in 2002. In
the
Pathum
Thani
Hospital,
the
predominant serotypes were DEN-1 and
DEN-2 (41% each) in 2001 and DEN-1
(47%) in 2002. In the Chareonkrung
Pracharak Hospital, the predominant
serotypes were DEN-1 (50%) in 2000, DEN 1 (61%) in 2001 and DEN -1 (65%) in 2002.
In the Ratchaburi Hospital, the predominant
serotypes were DEN-1 and DEN-2 (32%
each) in 1999, DEN-3 (52%) in 2000, DEN1 (38%) and in 2001 and DEN -2 (74%) in
2002. In the Hadyai Hospital, the
predominant serotype was DEN-1 (95%,
79% and 44%) in 2000, 2001 and 2002,
respectively.
The predominant serotypes were
compared among six hospitals in each of the
study years (Table 2). In 1999 when data
were analysed for two hospitals (Lampang
and Ratchaburi), DEN-2 was predominant
in one hospital (Lampang), and DEN-1 and
DEN-2 were equally predominant in the
other hospital (Ratchaburi). In 2000 when
data were analysed for four hospitals
(Lampang,
Chareonkrung
Pracharak,
Ratchaburi and Hadyai), DEN-1 and DEN-3
were predominant in three hospitals
(Lampang, Chareonkrung Pracharak and
Hadyai) and one hospital (Ratchaburi),
respectively.
In
2001,
DEN-1
was
predominant in five hospitals (Lampang,
Maharat Nakhon Ratchasima, Chareonkrung
Pracharak, Ratchaburi and Hadyai) and
DEN-1
and
DEN-2
were
equally
predominant in one hospital (Pathum Thani).
In 2002, DEN-1 and DEN -2 were
4
predominant in four hospitals (Maharat
Nakhon
Ratchasima,
Pathum
Thani,
Chareonkrung Pracharak and Hadyai) and
two hospitals (Lampang and Ratchaburi),
respectively. These results suggest that DEN 1 was the most predominant serotype from
1999 to 2002 in Thailand; however, the
predominant serotypes were different in
different hospitals.
The results of the present study are
generally consistent with those previously
reported. The analysis of dengue virus
isolates at Bangkok Children’s Hospital from
1973 to 1999 showed changes in dengue
virus serotypes from year to year[9] . Although
our study leads to a similar conclusion, the
study was conducted in six regional
hospitals throughout the country and not
only in central Bangkok.
Each dengue serotype may possess
unique characteristics in a dengue epidemic
and
disease
severity.
There
were
associations between DEN-1, DEN-2 and
DEN-3 and moderately severe dengue
epidemic years (1984-85, 1989-90, 1997),
and between DEN-3 and severe dengue
epidemic years (1987 and 1998) [9] . In that
sense, it is important to collect more
information on the predominant serotypes
and levels of epidemics. Although the
present study generated information on
isolates only from the patients who visited
the six hospitals, they are located far away
from each other in four regions of
Thailand: north, north-east, central and
south. It is thus assumed that the results of
this study demonstrate, to a certain extent,
the general features of dengue epidemics
in Thailand.
Table 2. Proportion of each of the four dengue serotypes determined by virus isolation
in the six hospitals
Year
1999
2000
2001
2002
Hospital
Total
DEN-1
(%)
DEN-2
(%)
DEN-3
(%)
DEN-4
(%)
Lampang
Maharat Nakhon
Ratchasima
Pathum Thani
Chareonkrug
Pracharak
Ratchaburi
Hadyai
74
6
31
4
(42)
(67)
33
2
(44)
(33)
8
0
(11)
(0)
2
0
(3)
(0)
0
0
0
0
(0)
(0)
0
0
(0)
(0)
0
0
(0)
(0)
0
0
(0)
(0)
31
0
10
0
(32)
(0)
10
0
(32)
(0)
8
0
(26)
(0)
3
0
(10)
(0)
Lampang
Maharat Nakhon
Ratchasima
Pathum Thani
Chareonkrug
Pracharak
Ratchaburi
Hadyai
15
3
13
2
(86)
(67)
1
1
(7)
(33)
0
0
(0)
(0)
1
0
(7)
(0)
4
10
1
5
(25)
(50)
2
4
(50)
(40)
1
1
(25)
(10)
0
0
(0)
(0)
238
22
99
21
(42)
(95)
9
0
(4)
(0)
125
1
(52)
(5)
5
0
(2)
(0)
Lampang
Maharat Nakhon
Ratchasima
Pathum Thani
Chareonkrug
Pracharak
Ratchaburi
Hadyai
150
176
85
83
(57)
(47)
35
68
(23)
(39)
26
16
(19)
(9)
1
9
(1)
(5)
143
99
58
60
(41)
(61)
59
25
(41)
(25)
19
11
(13)
(11)
7
3
(5)
(3)
256
276
98
219
(38)
(79)
43
4
(17)
(2)
96
39
(37)
(14)
19
14
(7)
(5)
Lampang
Maharat Nakhon
Ratchasima
Pathum Thani
Chareonkrug
Pracharak
Ratchaburi
Hadyai
95
292
10
166
(11)
(57)
75
70
(79)
(24)
8
49
(8)
(17)
2
7
(2)
(2)
32
65
15
42
(47)
(65)
8
16
(25)
(25)
7
3
(22)
(5)
2
4
(6)
(5)
170
77
21
34
(12)
(44)
126
13
(74)
(17)
12
13
(7)
(17)
11
17
(7)
(22)
Acknowledgements
We thank Dr Piyaporn Bowonkiratikachorn
and Ms Souvapa Pongsathaporn of
Charoenkrung
Pracharak
Hospital,
Dr Wandhana Sritubtim and Mr Suthichai
Pongmonjit of Pathum Thani Hospital,
Dr Vitaya Jiwariyaves and Ms Vanna
Pengruangrojanachai of Ratchaburi Hospital,
Dr Paiboon Vechpanich and Mr Prayuth
Kaewmalang of Maharat Nakhon Ratchasima
Hospital,
Dr
Wilaiwan
Gulgonkarn,
Dr Aroonrat Suwanarat and Mr Somchai
Niyomthai of Lampang Hospital and
5
Dr Suda Chubuppakarn and Ms Raruay
Jitsakulchaidej of Hadyai Hospital, and other
doctors, nurses and laboratory staff for
assisting us with the collection of samples.
This work was partly supported by grants
from the Department of Medical Sciences,
Ministry of Public Health, Thailand, and the
Japan Health Science Foundation.
References
[1] George R and Lum LCS. Clinical spectrum of
dengue infection. In Gubler D and Kuno G
(Eds.) Dengue and dengue hemorrhagic
fever. Colorado: US Department of Health
and Human Services, 1997: 89-113.
[2] World Health Organization. Monograph on
dengue/dengue haemorrhagic fever. WHO,
Regional Office for South-East Asia, New
Delhi 1993.
[3] World Health Organization. Dengue
haemorrhagic fever. Diagnosis treatment,
prevention and control, 2nd edition.
Geneva: WHO, 1997.
[4] Office of the Permanent Secretary for Public
Health. Annual Epidemiological Surveillance
Report 1993. Ministry of Public Health,
Thailand.
[5] Office of the Permanent Secretary for Public
Health. Annual Epidemiological Surveillance
Report 2001. Ministry of Public Health,
Thailand.
[7] Henchal EA, Gentry MK, McCrown JM and
Brandt WE. Dengue virus – specific and
flavivirus group determinants identified with
monoclonal
antibodies
by
indirect
immunofluorescence. Am J Trop Med Hyg,
1982, 31: 830-836.
[8] Henchal EA, McCown JM, Seguin MC,
Gentry MK and Brandt WE. Rapid
identification of dengue virus isolates by
using monoclonal antibodies in an indirect
immunofluorescence assay. Am J Trop Med
Hyg, 1983, 32: 164-169.
[9] Nisalak A, Endy TP, Nimmannitya S,
Kalayanarooj S, Thisayakorn U, Scott RM,
Burke DS, Hoke CH, Innis BL and Vaughn
DW.
Serotype-specific
dengue
virus
circulation and dengue disease in Bangkok,
Thailand from 1973 to 1999. Am J Trop
Med Hyg, 2003, 68(2): 191-202.
[6] Igarashi A. Isolation of Singh’s Aedes
albopictus cell clone sensitive to dengue and
chikungunya viruses. J Gen Virol, 1978, 40:
531-544.
6
A Retrospective Study of the 1996 DEN-1 Epidemic in
Trinidad: Demographic and Clinical Features
T.U. Brown*, K. Babb*, M. Nimrod*, C.V.F. Carrington*, R.A. Salas**
and M.A. Monteil*#
*Faculty of Medical Science, University of the West Indies, St. Augustine, Trinidad
**Caribbean Epidemiology Centre, Port-of-Spain, Trinidad
Abstract
A retrospective analysis of the 1996 DEN-1 epidemic in Trinidad was undertaken to better understand
the clinical and demographic expression of dengue infection in the island during one of the larger
epidemics in the past 10 years and following the reintroduction of DEN-1 into the island in 1991 after a
gap of 14 years. A total of 393 laboratory-confirmed cases were identified. Of these, notes for 157
patients were available for analysis. The epidemic was island-wide, though most cases occurred in the
most densely populated county of St. George. There was a slight predominance of females (51.6%)
among the cases, and while all age groups were affected, older children and adults comprised the
majority. South Asians among the population predominated. Overall, 27 clinical symptoms were
reported. The most common were: fever (98.7%), generalized pain (96.2%) and anorexia (63.1%). Rash,
arthralgia, retro-orbital pain and haemorrhage (all mentioned in the WHO clinical description for dengue
fever) were reported in <50% of cases. Gastrointestinal symptoms were also very common and occurred
in over two-thirds of cases at presentation. Bleeding manifestations were reported in 30% of patients and
commonly involved the gastrointestinal tract. Features of DHF were noted in only six (4%) patients and
there was one fatality. Deficiencies in documented clinical and laboratory monitoring of patients,
coupled with a lack of population-specific laboratory reference ranges, may contribute to underdiagnosis of DHF in Trinidad.
Keywords: Dengue, DHF, demographic analysis, clinical analysis, Trinidad.
Introduction
The dengue viruses (DEN), of the
Flaviviridae family, are mosquito-transmitted
viruses that can cause dengue fever (DF). DF
is an acute febrile illness characterized by
intense headaches, retro-orbital pain,
myalgia, arthralgia, anorexia and rash.
Additionally, in a minority of cases, a severe
and potentially fatal form of dengue
infection known as dengue haemorrhagic
fever/dengue shock syndrome (DHF/DSS) is
manifested, primarily through increased
vascular permeability and shock. Dengue
exists as four distinct serotypes, DEN-1-4.
Infection with one serotype gives life-long
immunity for that virus but not to the others.
Data suggest that secondary infection with a
#
7
A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
serotype different from the primary infection
enhances
the
risk
of
developing
DHF/DSS[1,2] . The pathological processes
that lead to DHF/DSS remain poorly
elucidated, but epidemiological studies have
highlighted viral and host factors that are
associated with the development of
DHF/DSS[3] .
An estimated 100 million people across
the globe contract dengue annually with
about
250,000
persons
developing
DHF/DSS[4] . In the past 20 years, DF and
DHF/DSS have become significant public
health problems in the Caribbean and in
Latin America. The discontinuation of the
Pan American Health Organization (PAHO)initiated
Aedes
aegypti
eradication
programmes in the 1970s caused a reintroduction of the mosquito vector into
virgin areas and a resurgence of dengue
outbreaks
throughout
the
region.
Furthermore, prior to the 1980s, dengue
outbreaks in the Americas were caused by
single serotypes and were geographically
restricted and largely self-limiting, with no
reports of DHF/DSS[5] . The first cases of
DHF/DSS documented in this region were
identified during the Cuban DEN-2
epidemic in 1981[6] and the pattern of the
disease has changed dramatically since
then[4,5] . Given the strong evidence linking
the disease severity with secondary
heterologous
infection[7-9] ,
increased
hyperendemicity is widely thought to be
one of the most significant factors
contributing to increases in the disease
severity[3] in the region where all the four
serotypes are now present.
The first dengue outbreak in over 20
years in the island of Trinidad was reported
in 1977. That outbreak caused by DEN-1
resulted in a few DF cases with no reported
8
case of DHF/DSS[10] . Since then, increasingly
larger outbreaks have been documented
throughout the 1980s and the 1990s,
caused by DEN -2 and DEN-4. The first
cases of DHF/DSS were reported during the
1993 DEN-1 epidemic[11] , and this was
followed in 1996 by yet another DEN-1
outbreak which was characterized by a
larger number of reported and confirmed
cases of DHF/DSS[12] .
We present here a retrospective analysis
of the 1996 DEN-1 epidemic in Trinidad.
This study was done, firstly, to observe the
clinical and demographic expression of the
dengue infection in the Trinidad population
during one of the largest epidemics of the
past 10 years. In particular, we wished to
assess the ethnic distribution of the disease
since there had been previous anecdotal
reports of a more severe disease in persons
of South Asian ancestry. Secondly, the study
was done to gain an understanding of the
clinical and laboratory infrastructure for
dengue in Trinidad and Tobago as part of
the development of our ongoing denguerelated clinical research in the island.
Methods
A retrospective analysis of the clinical and
demographic features of patients with
laboratory-confirmed dengue from the 1996
DEN-1 epidemic in Trinidad, West Indies,
was conducted. Patients were identified
from the laboratory database of The
Caribbean Epidemiology Centre in Trinidad
(CAREC). The CAREC is a subsidiary of the
Pan American Health Organization (PAHO)
and offers infectious disease testing of sera
from patients with suspected dengue
infections. Since 1975, CAREC has
systematically conducted the isolation and
typing of dengue viruses in support of viral
surveillance programmes[12] .
A total of 1,200 samples from
symptomatic suspected dengue cases in
Trinidad and Tobago were sent to CAREC in
1996 for laboratory confirmation, of which
393 were found positive. We identified
these confirmed cases of dengue infection
and the medical institutions from which the
samples had originated. The demographic,
clinical and laboratory information related
to each sample was collected from the
respective medical records of each patient.
Data were entered into a computerized
Excel database (Microsoft Excel 2000) and
analysed
according
to
demographic
groupings such as ethnicity, county of
residence, age and gender and by
frequencies of clinical symptoms. The
incidence rates per 10,000 persons were
calculated using the 1990 census data[13] .
Proportional data were tested using the Chi
square test and statistical significance was
established at P<0.05. All statistical analyses
of the demographic and clinical data were
done using SigmaStat Statistical Software
for Windows Version 2.03 (Copyright
1992-1997 SPSS Inc.). Ethical approval for
this project was obtained from the Ethics
Committee of the Faculty of Medical
Sciences, University of the West Indies, St.
Augustine.
Results
The names of the 393 laboratory-confirmed
dengue patients were obtained from the
CAREC database for 1996. Of these, 258
(66%) could be traced to the source
institution or the treating doctor. Clinical
records were obtained for a total of 157
patients (40%). Information was incomplete
in 60 (15%) of these cases. The following is
an analysis of the clinical and demographic
data available from the records of the 157
dengue-confirmed cases.
Geographical and temporal
distribution of dengue
virus infections
One hundred and fifty-four patients’
addresses were available. The estimated
county-specific incidence rates (per 10,000
population) based on these addresses
ranged from 0.60 to 1.87, with the extreme
values occurring in the rural counties of
Nariva/Mayaro and St. Andrews/St. David
respectively (Figure 1). The highest
proportion of these 154 confirmed cases
(46%) occurred in the most densely
populated county of St. George, in which
the capital, Port-of-Spain, is situated. There
was no significant difference in the rates of
infection among all the counties (P=0.513;
power of test with α=0.05:0.0.301).
A review of the admission dates to
health care facilities for 136 dengueconfirmed patients showed that there was a
gradual increase in the number of cases
from April to June, followed by a large rise
in July (29 cases) to a maximum in the
month of August (51 cases). In 1996, the
rainfall pattern (Figure 2) consisted of a large
increase in the rainfall (171.7 mm) between
April and May, followed by peak
precipitation in June (333.1 mm). A
comparison of dengue admissions with the
monthly precipitation suggests a direct
relationship between the rainfall and the
number of cases reported, with a twomonth lag between the peak rainfall and the
peak number of admissions.
9
Figure 1. Map of Trinidad showing county-specific incidence rates (per 10,000 persons) for
dengue infections in 1996 (values in lower half of boxes) based on addresses from 154
confirmed cases. Values in the upper half of boxes show the proportion of the 157 cases
found in each county. County divisions are according to the Regional Health Authorities of
Trinidad & Tobago
Figure 2. Monthly admissions of 136 laboratory confirmed dengue cases and precipitation
levels for the year 1996. Rainfall data obtained from the records of the Trinidad & Tobago
Meteorological Office, Piarco, Trinidad
60
45
250
202.6
207.1
203.6
30
150
148.4
20
120.6
16
100
15
9
50
8
30.9
2
Dengue cases
0
Dec
Oct
Nov
Sep
Aug
Jul
2
Jun
Apr
Feb
Mar
28.9
18.9 5
2
1
2
May
51.7
Jan
0
200
174.4
Rainfall 1996 (mm)
287.4
40
10
10
300
51
50
No. of confirmed cases
350
333.1
Rainfall
rate in children (≤15 yrs) was not
significantly different from that in adults
(>15 yrs). An analysis of the estimated
incidence rates by county showed that there
was variation in the age groups with the
highest incidence rates for each county. In
three counties, the highest estimated
incidence rates occurred in persons 40 years
and over; Caroni (50-59 yrs) (2.9%), Victoria
(40-49 yrs) (2.09%) and St. Andrews/St.
David (40-49 yrs) (3.65%). However,
estimated highest incidence rates were
observed in younger patients in the counties
of St George (0-9 yrs) (1.89%) and St.
Patrick (10-19 yrs) (2.46%).
Incidence rates of dengue
infections in relation to
gender, age and ethnicity
Gender and age data were available for all
157 cases. There was a slight female
predominance of 51.6%.
The highest proportion of the cases
occurred in the 30-34-year age group
(15.9% of the cases), followed by 5-9 and
10-14-year olds (14.0% and 14.6%
respectively) (Figure 3). The differences
among the groups were found to be highly
significant (P<0.001; power of test with
α=0.05: 0.998). The estimated incidence
Figure 3. Age distribution of 157 confirmed dengue cases
8.9%
1.9%
4%
3.8%
3.8%
4.5%
6%
2.5%
8%
5.7%
7.0%
10%
8.9%
12%
8.3%
% of confirmed cases
14%
14.6%
14.0%
16%
15.9%
18%
2%
60+
55-59
50-54
45-49
40-44
35-39
30-34
25-29
20-24
15-19
10-14
5-9
0-4
0%
Age group
11
Self-reported ethnicity was available for
136 patients. Of these, 50% were South
Asians, 35% were Africans, 11% were mixed,
and 4% were Chinese and Europeans. As
seen in Figure 4, a comparison of the
incidence rates by ethnicity for the whole
sample and by county showed that the
incidence rates of dengue infection were
the greatest among South Asians for the
group as a whole and in three of the four
counties. A statistically significant difference
was achieved in the county, St. Patrick,
where the number of South Asian patients
was higher than the African patients
(P=0.005; power of test with α=0.05:
0.807), and in the whole group where the
incidence rate for South Asians was
significantly higher than the mixed
population (P=0.016; power of test with
α=0.05: 0.70).
Figure 4. Incidence rates (per 10 000 persons) for 136 confirmed dengue infection patients
by ethnicity for whole population and individual counties.
County Victoria was excluded since ethnicity was reported in <50% of cases in that county
1.42
1.11
0.21
0.56
0
0.5
0
Whole Pop.
St. George
Caroni
St. Patrick
County
South Asians
Summary of clinical and
haemorrhagic manifestations
Overall, 24 clinical symptoms were reported
in the medical records retrieved. The
frequency
of
these
symptoms
at
presentation is shown in Figure 5. Multiple
symptoms at the time of admission were
generally
reported.
In
this
cohort,
12
1.28
1.76
1.58
1.27
0.84
1
1.03
1.5
0.72
2
1.46
Incidence Rate
2.5
2.32
2.44
3
Africans
St. Andrew/ St.
David
Mixed
gastrointestinal (GIT) complaints (nausea,
vomiting, diarrhoea and anorexia) were
commonly reported (66.2%). Symptoms
highlighted in black indicate the symptoms
listed in the WHO clinical description of
dengue fever[14] . Four of these symptoms,
arthralgia, retro-orbital (R/O) pain, rash and
haemorrhage, were recorded in <50% of
cases.
Figure 5. Frequency distribution of clinical symptoms at presentation in 157 dengue
confirmed patients. Symptoms highlighted in black are part of the WHO clinical
description of dengue fever
Fever, 156
Pain, 151
Anorexia, 99
Myalgia, 98
Headaches, 95
Vomiting, 90
Dehydration, 56
Upper resp., 52
Arthralgia, 50
R/O pain, 50
Chills, 45
Rash, 42
Diarrhoea, 38
Haemorrhage, 34
Adenopathy, 25
Conjunctivitis, 25
Dizziness, 20
Petechiae, 17
Malaise, 15
Rhinorrhoea, 14
Itching, 11
Hepatomegaly, 7
Short breath, 6
Photophobia, 4
0
10
20
30
40
50
60
70
80
90
100
Percentage of patients
In 34 patients (21.7%), haemorrhagic
manifestations (HM) other than petechiae
were reported. The reported haemorrhagic
symptoms
and
their
frequency
of
occurrence are illustrated in Figure 6. More
than one form of bleeding was noted in
some patients. In keeping with the high
frequency of gastrointestinal complaints
reported, GIT bleeding including bloody
stool and rectal bleeding occurred in 50% of
the patients.
Six cases (approximately 4% of the 157
cases) could be defined as of DHF based on
the WHO criteria for the diagnosis of
DHF/DSS[14] . Of these, five were female;
five were South Asians and one was mixed;
four were children and one case (29-year
old, South Asian female) died.
13
Figure 6. Type and frequency of haemorrhagic symptoms among 34 dengue confirmed
patients who presented with haemorrhagic manifestations. Symptom highlighted in
black is part of the WHO clinical description for DHF
Gastrointestinal
bleeding, 29.4%
Gingival bleeding,
29.4%
Bloody stool, 17.7%
Urinary tract
bleeding, 14.7%
Nasal bleeding,
11.8%
Not specified, 5.9%
Heavy menstrual
flow, 5.9%
Rectal bleeding, 2.9%
Bloodstained sputum,
2.9%
Discussion
We reviewed the clinical and demographic
features of 157 dengue-confirmed patients
from a DEN-1 epidemic in Trinidad. This
epidemic followed the reintroduction of this
serotype into the island in 1991 after an
absence of 14 years and the first
documentation of DHF in the island in 1993.
Approximately
1,200
samples
from
suspected symptomatic patients were sent
for laboratory confirmation and, of these,
393 (approx. 33%) proved to be positive.
Clinical and demographic data reported
here are from 40% of the confirmed cases
from that outbreak. The outbreak was
island-wide with the estimated incidence
rates being the highest in rural communities,
though most cases occurred in the most
14
densely populated county of St. George.
There was a slight predominance of females
and the infection occurred in all age groups.
The estimated incidence rates were
generally higher in patients of South Asian
ethnicity. Gastrointestinal symptoms were
the most common clinical manifestations at
presentation. Haemorrhagic manifestations,
when present, also commonly involved GIT.
Six patients met the clinical and laboratory
criteria for DHF and, of these, one died.
However, the 157 (40%) available
records closely reflected the distribution of
patients from public and private hospitals,
private doctors and health centres seen for
the 258 names that were traced to their
respective sample sources. In the sample of
258 patients, public hospitals accounted for
71%, private hospitals 16.7%, and health
centres and general practitioners 6.2% each.
In the sample of 157 patients for whom
medical records were obtained, public
hospitals accounted for 67%, private
hospitals 17.2%, health centres 6.4% and
general practitioners 10%. Over 80% of the
patients were seen in hospitals.
The majority of cases (46%) in the
sample lived in the most densely populated
county in Trinidad, St. George’s where
36.5% of the total population of Trinidad
resides and where the Capital, Port-of-Spain
(POS), and its environs are located[13] .
Dengue, as a mosquito-borne infection,
would be expected to spread rapidly in
populous areas, so the presence of a large
proportion of patients in and around POS is
consistent with the experience elsewhere of
dengue
infection
as
an
urban
phenomenon[15] . Interestingly, our estimates
of county-specific dengue incidence (based
on the sample of 157 patients) yielded rates
that did not differ significantly between rural
and urban counties in Trinidad. This
suggests that despite the variation in
population density, DEN-1 infection was
fairly evenly spread across the island. The
island of Trinidad is merely 4,828 km 2 and
the similarity of the incidence rates across
the island may reflect a combination of a
DEN-1 naïve population and easy
accessibility to all parts of the island.
The DEN-1 1996 epidemic occurred in
the rainy season with most admissions taking
place in July and August, 1-2 months after
the peak rainfall for that year in June.
Rainfall is considered to be a risk factor for
the development of dengue outbreaks,
especially where there is improper storage
of water or the presence of receptacles in
the domestic environment such as discarded
plastic containers in which water can collect.
The main mosquito vector for dengue
viruses in the Caribbean and Latin America
is Aedes aegypti, which breeds primarily in
relatively clean, stagnant domestic water
containers. Local research has shown that
important breeding sites for Aedes in
Trinidad and Tobago include outdoor drums,
water tanks, tyres and small discarded
bottles and cans[16] , which get filled easily
with stagnant fresh water during the rainy
season. The lag between the peak rainfall
and the number of confirmed dengue cases
might reflect the period of the mosquitovector population expansion.
All age groups were affected but over
63% of them were adults (>15 years, the
cut off age for paediatric patients in
Trinidad).
Since
DEN-1
had
been
reintroduced in Trinidad in 1991, it is
expected that children aged 5 years or less
would constitute a susceptible population.
Infection in this group would be primary in
which the majority would be asymptomatic.
This may account for children aged 0-4
years comprising only 8% of the sample.
Despite the 1992/93 DEN-1 outbreak in
which over 3,060 cases were reported,
there were still many adults in 1996 who
were susceptible to DEN-1. In total, there
were 3,588 reported cases that year.
However, based on the CAREC experience,
only 33% of the clinically suspected dengue
cases at that time were actually confirmed
by laboratory tests, suggesting thereby that
even during that outbreak, many cases with
clinical features consistent with dengue may
in fact not have been of dengue infection.
Clinical symptoms of dengue are known to
overlap with many other infections such as
measles,
hepatitis
A,
rubella
and
leptospirosis [17-19] .
15
The high proportion of cases in the 3034, 5-9 and 10-14-year age groups suggests
the possibility of spread between children
and parents. In Trinidad and Tobago, 25%
of the heads of households are 30-39 years
of age with an average of 1.99 children per
household[13] . Trinidadian women often start
their families in late teenage or early
twenties, so parents aged 30-34 years will
have children in the age groups 5-9 or 1014 years.
The racial composition of this sample
differs from that reported for the general
population, which is composed of South
Asians (40.3%), Africans (39.6%), mixed
(18.4%) and the rest (1.6%)[13] . Thus, there is
an overrepresentation of South Asians (50%)
in our sample with fewer persons of African
origin (35%) and of mixed race (11%). Since
hospitalized patients account for 84% of the
study population, it may reflect an increased
number of South Asians being hospitalized
for dengue infection as compared with
other racial groups. Teelucksingh[20] has
previously reported a higher incidence of
more severe dengue infection and mortality
in South Asian people (colloquially referred
to as East Indians) in Trinidad.
These data cannot exclude differences
in the environmental factors that might also
increase the exposure of South Asian people
to more mosquito bites. South Asians have
traditionally been the predominant racial
group in agriculture and animal rearing in
Trinidad. In rural communities on the island
there is relatively poor piped water supply,
resulting in more water storage, thus
providing ideal domestic breeding sites for
Aedes aegypti[16] . Therefore, farmers in rural
areas of Trinidad may be at an increased
risk of mosquito bites and consequent
dengue infection. The tendency towards an
16
increased incidence in South Asians is
interesting and merits further analysis with
larger cohorts of dengue-confirmed cases.
The range of symptoms reported in the
157 Trinidadian dengue-confirmed patients
is similar to those described for patients with
acute dengue in other parts of the world[18] .
Recent-onset fever and generalized body
pains occurred in almost all patients.
Headache, anorexia and myalgia, which
often occur in the prodrome, were noted in
60% or more of the cases. Gastrointestinal
and upper respiratory symptoms were also
common complaints at presentation.
Gastrointestinal symptoms have been
described
as
predominant
clinical
manifestations in epidemics in which there
is an adult-susceptible population[21] . The
1996 epidemic was almost wholly due to
DEN-1 and the bulk of the study population
comprised older children and adults.
The symptoms of classic dengue as
described by WHO occurred in fewer than
expected numbers of patients. For example,
retro-orbital pain and arthralgia were
reported in 32% of patients and rash only
noted in 27%. While this may be due to a
true low prevalence of these symptoms in
the study population, the cultural expression
of symptomatology may also contribute to a
falsely low detection of these symptoms. For
example, in Trinidad, all pains in the head
region, including orbital pain, are commonly
referred to as “headache”. Similarly, joint
pains may often be included in “muscle”
pain or be part of a more generalized “body
pains”. The rash, which is usually transient,
may be easily missed and even more so in
dark-skin complexions.
Haemorrhagic manifestations, including
petechiae, were noted in 51 (32.5%)
patients. This is consistent with other reports
of the prevalence of haemorrhage in dengue
fever[18] . In keeping with the global
experience, the bleeding manifestations
occurred at many sites. Despite the bleeding
manifestations in almost a third of the
patients, the WHO criteria for DHF were
fulfilled only by 6 (4%) patients[14] . Of these,
five were female and one was male, five
were South Asian and one was of mixed
ethnicity and four were children and two
were adults. One patient, a 29-year-old
female of South Asian ethnicity died. The
female gender and children were more
prone to the development of DHF/DSS in
dengue-endemic areas [3] . While the 1996
epidemic was predominantly DEN-1, DEN2 was already endemic in the island[12] .
Further, the predominance of persons of
South Asian ancestry in this group was also
consistent with the observation that certain
races were more susceptible to DHF/DSS
than others.
The number of cases of DHF/DSS may
have been underestimated. One of the
essential WHO criteria for establishing the
diagnosis of DHF/DSS is the proof of
increased vascular leakage such as an
increase of at least 20% in average
haematocrit for age and sex; a drop in
haematocrit of 20% or more following
treatment, or clinical evidence of the same
such as pleural effusion, ascites or
hypoproteinaemia[14] . However, since the
plasma leakage can be transient and only
evident by laboratory testing in milder forms
of DHF cases (e.g. Grade 1 and 2 DHF), it is
possible to miss patients with these forms of
DHF if clinical monitoring of the
haematocrit levels or tests such as lateral
decubitus chest X-rays to detect small
pleural effusions are not performed. The
frequency of chest X-rays and serial blood
tests in this cohort of patients varied with
the hospitals that were visited. For example,
chest X-rays were performed in fewer than
10% of the patients admitted to private
hospitals but in over 60% of the patients in
one public hospital. Serial blood tests were
performed in approximately 20% of dengue
cases in private hospitals but in 40-80% of
patients
admitted
to
three
public
institutions[22] .
Furthermore, there are no locally
developed reference ranges for haematocrit
by age and sex in use in many laboratories
in Trinidad where the reference ranges
developed in primarily the Caucasian
populations have been adopted for use.
Moreover, haemoglobinopathies such as
thalassaemia and sickle cell traits are not
uncommon
among
the
Trinidadian
population and these persons tend to have
normal haemtocrit levels below the
reference ranges in use. Consequently, such
patients presenting with 20% or more
increase in haemoconcentration may fall
within the adopted reference range, and if
there is no repeat of haematocrit
concentration in the convalescent period,
evidence of haemoconcentration would
have been completely missed and the
patient wrongly diagnosed as DF instead of
a mild form of DHF[22] .
In summary, the DEN-1 epidemic in
Trinidad in 1996 was characterized by a
large number of clinically suspected but
unconfirmed cases of dengue infection.
Laboratory analysis of a third of all reported
cases revealed that most of these cases were
not of dengue. Furthermore, the variability
in the data available from clinical records,
wherever these could be found, resulted in
less than optimal information retrieval for
further analysis. From the available data set,
the Trinidadian dengue-infected patients
were mainly older children and adults from
17
rural and urban communities. South Asians
were predominant. A wide range of clinical
manifestations was recorded, but the most
common were gastrointestinal. Bleeding
manifestations occurred in over 30% of DF
cases but features of DHF were noted in
only 4%. Improvement in clinical diagnosis
and record-keeping is urgently required to
underpin future clinical research in Trinidad.
References
[1] Halstead SB, Chow J and Marchette NJ.
Immunologic enhancement of dengue virus
replication. Nature New Biology, 1973, 243:
24-26.
[2] Morens DM, Halstead SB and Marchette NJ.
Profiles of antibody-dependent enhancement
of dengue virus type 2 infections. Microbial
Pathogenesis, 1987, October, 3(4): 231-237.
[3] Halstead SB. Epidemiology of dengue and
dengue haemorrhagic fever. In: Gubler DJ,
Kuno G (Eds.), Dengue and dengue
haemorrhagic fever. New York: CAB
International, 1997: 23-44.
[4] Gubler
DJ.
Dengue
and
dengue
haemorrhagic fever: Its history and
resurgence as a global public health problem.
In: Gubler DJ, Kuno G (Ed.), Dengue and
dengue haemorrhagic fever. New York: CAB
International, 1997: 1-23.
[5] Gubler
DJ.
Dengue
and
dengue
haemorrhagic fever. Clinical Microbiology
Review, 1998, 11(3): 480-496.
[6] Pinheiro FP and Corber SJ. Global situation
of dengue and dengue haemorrhagic fever,
and its emergence in the Americas. World
Health Statistics Quarterly, 1997, 50(3-4):
161-169.
[7] Halstead SB. Pathogenesis of dengue:
challenges to molecular biology. Science,
1988, 239 (4839): 476-481.
[8] Kliks SC, Nisalak A, Brandt WE, Wahl L and
Burke
DS.
Antibody-dependent
enhancement of dengue virus growth in
human monocytes as a risk factor for
dengue haemorrhagic fever. American
Journal of Tropical Medicine and Hygiene,
1989, 40(4): 444-451.
18
[9] Thein S, Aung MM, Shwe TN, Aye M, Zaw
A, Aye K, Aye KM and Aaskov J. Risk factors
in dengue shock syndrome. American
1997, 56(5): 566-572.
[10] Le Maitre. Dengue fever epidemic in
Trinidad & Tobago: 1977-1978. Dissertation
for the Diploma in Public Health, University
of the West Indies, Mona, Jamaica, 1978;
Chapter 2: 11-18.
[11] Teelucksingh S, Mangray AS, Barrow S,
Jankey N, Prabhakar P and Lewis M.
Dengue haemorrhagic fever / dengue shock
syndrome: an unwelcome arrival in Trinidad.
West Indian Medical Journal, 1997, 46(2):
38-42.
[12] Campione-Piccardo J, Ruben M, Vaughan H
and Morris-Glasgow V. Dengue viruses in
the Caribbean. Twenty years of dengue virus
isolates from the Caribbean Epidemiology
Centre. West Indian Medical Journal, 2003,
52(3): 191-198.
[13] Central Statistical Office of the Government
of Trinidad & Tobago. http://www.cso.gov.tt.
[14] World Health Organization Regional Office
for South-East Asia. Prevention and control
of dengue and dengue haemorrhagic fever:
comprehensive guidelines. http://w3.whosea.org/
dengue/searono29/pdff.htm.
[15] Kuno G. Factors influencing the transmission
of dengue viruses. In: Gubler DJ, Kuno G
(Ed.), Dengue and dengue haemorrhagic
fever. New York: CAB International, 1997,
61-88.
[16] Focks DA and Chadee DD. Pupal survey: an
epidemiologically significant surveillance
method for Aedes aegypti: an example using
data from Trinidad. American Journal of
Tropical Medicine and Hygiene, 1997,
56(2): 159-167.
[17] Dietz VJ, Nieburg P, Gubler DJ and Gomez I.
Diagnosis of measles by clinical case
definition in dengue endemic areas:
implications for measles surveillance and
control. Bulletin World Health Organization,
1992, 70(6): 745-750.
[18] George R and Lum LCS. Clinical spectrum of
dengue infection. In: Gubler DJ, Kuno G
(Ed.), Dengue and dengue haemorrhagic
fever. New York: CAB International, 1997:122.
[20] Teelucksingh S, Lutchman G, Udit A and
Pooransingh S Childhood dengue shock
syndrome in Trinidad. West Indian Medical
Journal, 1999, 48(3): 115-117.
[21] World Health Organization Regional Office
for South-East Asia. Risk factors of
DEN/DHF epidemics in SEAR countries.
http://w3.whosea.org/Den1/risk.htm.
[22] Nimrod M, Brown TU, Babb K, Carrington
CVF, Salas RA and Monteil MA. An analysis
of laboratory parameters from confirmed
cases of dengue haemorrhagic fever in the
1996 Trinidad epidemic. West Indian
Medical Journal, 2003, 52 (Suppl. 1): 17.
[19] Levett PN, Branch SL and Edwards CN.
Detection of dengue infection in patients
investigated for leptospirosis in Barbados.
American Journal of Tropical Medicine and
Hygiene, 2000, 62(1): 112-114.
19
Ecological Study of Rio de Janeiro City
DEN-3 Epidemic, 2001-2002
Maria Lucia F. Penna#
Escola Nacional de Saude Publica, Fundação Oswaldo Cruz, Rua Leopoldo Bulhões 1480,
DENSP, 21041-210 Rio de Janeiro, RJ, Brazil
Abstract
Dengue virus serotype 3 was introduced in the Rio de Janeiro metropolitan area in January 2001 which
produced a large epidemic during 2001 and 2002. This study looks into the relationship between the
urban socioeconomic organization and the dengue attack rate during the epidemic in Rio. This study
uses secondary data published in the data website of the city administration, including variables related
to sanitation, use of the city area, tax collection, population density, education, income and life
expectancy. The model includes as predictors the proportion of households with a well, the proportion
of the available area in the city used for commerce and services in general, the proportion of city taxes
collected from industries, the mean per capita income and the residential area per inhabitant, all with a
statistical significant level of less than 0.05. The model explains 71% of the attack rate variance. The
variables included in the model indicated that dengue distribution in the city was related to people’s
socioeconomic status and the city organization. Variables that correlate with the movement of people
across towns have emerged as the most significant.
Keywords: Dengue virus, socioeconomic status, urban organization, Rio de Janeiro.
Introduction
After decades of freedom from dengue virus
infection, Brazil experienced an outbreak of
DEN-1 and DEN-4 in areas close to the
borders with Venezuela in 1981-82.
Intensive
vector
control
measures
successfully controlled this outbreak and
checked its spread to other areas in the
country[1] . In 1986, DEN-1 was introduced
in the Rio de Janeiro metropolitan area,
which spread to other areas, causing a
massive epidemic that later covered the
entire country. DEN -2 and DEN-3 were also
#
introduced in the country in 1990 and 2001,
respectively[2] . Once introduced in the Rio
de Janeiro metropolitan area, DEN -3 caused
a large epidemic during 2001 and 2002. In
2003, DEN-1, DEN-2 and DEN-3 were in
circulation in all except the two southern
states of the country, with 341,092 cases
reported (Figure 1) [3] .
Despite the use of a variety of control
strategies, dengue control is a major public
health challenge in the world. Demographic
changes occurring in developing countries
due to widespread rural-urban migration
E-mail: [email protected]; Tel.: 55 21 38829212; Fax: 55 21 38829124
20
Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002
since the 1960s have resulted in
overcrowded cities with multiple deficiencies,
particularly in housing and basic sanitation.
The strategy that resulted in the eradication
of Aedes aegypti in the 70s is no longer
applicable to the reality of the social,
demographic, economic and political
situation in South American countries[4-6].
Figure. Circulation of dengue virus in the Brazilian states, 2003
None
DEN-1 and -3
DEN-1 and -2
DEN-1, -2 and -3
Predicting the risk of dengue correctly
based on sociocultural factors has been the
goal of many authors [7,8] , but the dynamics
of dengue transmission in urban settings are
still poorly understood[9] . The understanding
of the virus transmission dynamics requires a
theoretical
framework
that
includes
individuals, households and behavioural risk
factors as well as the administrative aspects
of city management services, use of city
space and movement of people across the
metropolis. This understanding can help
control measures to be more effective with
responsibilities
divided
between
the
government and communities as per
priorities established on the basis of reliable
data.
The present study looked into the
relationship
between
the
urban
socioeconomic organization and the dengue
attack rate during the DEN-3 epidemic in
Rio de Janeiro city in 2001-2002. It is
mainly an exploratory study, based on
available secondary data, that is likely to
21
throw some light on how urban settings
contribute to dengue transmission.
Methods
Description of study area
Rio de Janeiro city is located at -22°54'23"
south latitude and -43°10'21" west longitude,
at the seashore, with an urban area of 1,255
km 2, including inland and continental
waters. The municipal area was divided into
26 administrative regions (ARs) in 1999
which were used as spatial units for analysis.
The climate is tropical, hot and humid, with
local variations due to altitude differences,
vegetation and proximity to the sea. The
mean annual temperature is 22 °C, with
high daily means in summer (30 to 32 °C).
Rainfall is 1,200 to 1,800 mm per year,
concentrated in summer from December to
March. The city has the lowest rate of
population growth among the Brazilian
capital cities, 6.9% between 1991 and 2000.
Database
This study used secondary data published in
the website of the city administration[10] ,
including the proportion of households
belonging to the sewage collection system
(SEWAGE), the proportion of households
belonging to the water supply system
(WATER), the proportion of households with
a well (WELL), the proportion of households
with waste collection by the city authority
(WASTE), the number of inhabitants per
household (INH/HOUSE), the area of
residential properties (square metres) per
inhabitant (M2/INH), the proportion of
households in slums (SLUMS), the
proportion of the total area built for
22
residence
(HOUSE),
for
industry
(INDUSTRY), for commerce or service
(COMMERCE); the proportion of unused
areas (UNUSED); the proportion of the total
area of parks and squares (PARKS); the
amount of city tax collected per inhabitant
(TAX); the proportion of city tax of
commercial origin (COMTAX), industrial
origin (INDTAX) and service origin
(SERVTAX); the mean per capita income
(INCOME); the life expectancy (LE); the
proportion of literacy among inhabitants
(LITERACY); and the proportion of children
aged 7 to 15 years attending school
(SCHOOL) as independent variables. All
proportions were presented as percentages
and income and taxes as 1,000 reais
(Brazilian currency). The independent
variable was the attack rate for DEN-3
epidemic, calculated by the number of
reported dengue cases during 2001-2002
divided by the population estimate for
January 2001, per 100,000 inhabitants.
Statistical methods
A multiple regression model was adjusted to
the data using stepwise forward approach,
with F to enter = 1 and F to leave = 0.95,
and the author interference to limit the
number of steps in order to prevent a
saturated model. The independent variable
was given a log transformation. The software
used was Statistica, from Statsoft[11] .
Results
Table 1 shows the correlation coefficients
between the independent variable and all
those variables presented in the model,
along with their range. The model included
as predictors the proportion of households
with a well, the proportion of the available
area used for commerce and services, the
proportion of city taxes collected from
industries, the mean per capita income and
the built household area per inhabitant
(Table 2), all with a statistical significant level
less than 0.05. The model explains 71% of
the attack rate variance. Table 3 shows the
partial correlation coefficients for the
variables included in the model, which is a
measure of the association of each variable
when the other variables are controlled for,
making it possible to rank their effect. The
residues fitted well into a normal
distribution and presented no correlation
with the predictors.
Table 1. Descriptive statistics and correlation coefficient with log (attack rate)
Variable
Mean
Minimum
Maximum
Correlation
WASTE
1
0.93
1
0.09
SWAGE
1
0.30
1
0.09
WATER
1
0.88
1
-0,30
WELL
0
0.00
0
0,39
M2/INH
24
5.56
56
0.20
SLAM
15
0.00
41
0.04
3
2.36
4
-0.05
LE
72
65.99
78
-0.01
LITERACY
96
90.74
99
-0.38
SCHOOL
90
67.66
113
-0.36
INCOME
670
212.21
2229
-0.13
HOUSE
69
9.89
89
-0.26
COMMERCE
12
3.40
39
0.27
7
0.11
21
0.01
981
0.99
19729
0.02
PARKS
2
0.00
25
-0.02
COMTAX
0
0.00
3
-0.20
INDTAX
0
0.00
1
0.30
SERVTAX
99
94.30
100
0.03
396
2.78
7157
0.19
INH/HOUSE
INDUSTRY
UNUSED
TAX
23
Table 2. Regression summary
(Standard regression coefficient, regression coefficient, t and P value)
Standard
error of
Beta
Beta
Intercept
Standard
error of B
B
t (20)
P level
6.360585
0.184526
34.46979
0.000000
WHELL
0.33653
0.145309
7.640343
3.299001
2.31596
0.031294
COMMERCE
0.34087
0.136198
0.024121
0.009638
2.50272
0.021111
INDTAX
0.46826
0.126483
2.084555
0.563072
3.70211
0.001410
INCOME
-1.06086
0.273221
-0.001142
0.000294
-3.88279
0.000925
1.12201
0.294558
0.048058
0.012616
3.80912
0.001099
M /INH
2
R= 0.84282066; R2=0.71034667; F(5,20)=9.8096; P<0.00007; Standard error of estimate=0.35169
Table 3. Partial and semi-partial correlation
Beta in
Partial correlation
Semi -partial correlation
WELL
0.33653
0.459859
0.278711
COMMERCE
0.34087
0.488354
0.301187
INDTAX
0.46826
0.637673
0.445527
INCOME
-1.06086
-0.655600
-0.467271
1.12201
0.648419
0.458405
M /INH
2
Discussion
The exploratory aspect of this study justifies
the presentation of a high number of urban
and socioeconomic variables in the model.
These variables present high covariance that
induces the use of the forward stepwise
method. To avoid a saturated model, the
author restrained the number of steps and
assured that only significant variables were
kept in the model by using a relatively high
value of F to leave. The rupture of the
homocedasticy assumption due to the
nature of data (proportions) was successfully
24
dealt with by the logarithmic transformation
of the attack rate[12] , as shown by the
residual analysis.
The proportion of households with a
well is really a proxy variable of the
discontinuity of water supply. The
proportion of households that have access
to the city’s water supply network is 95.07%
for the entire city, indicating a high coverage
of the city’s water supply network. But the
water supply is not equally effective in all
the administrative regions (ARs), with some
areas having chronic problems, mainly
discontinuous supply. The presence of a
well, although small, only in 1.12% of the
households (maximum of 11.85% in
Paqueta AR and 11.06% in Guaratiba AR) is
a proxy variable for the discontinuity
problem, for it is a solution that involves
financial expenses which is only justified in
the face of an important and lasting problem
of supply. These results point to the fact that
water supply is an important issue in dengue
control, but only in the context of lack of or
irregular water supply where the population
is forced to resort to storage, which creates
breeding sites for the vector and not in the
usual context of adequate supply as
suggested by other authors [13] . .
The model included mean per capita
income as a protective factor meaning that
low socioeconomic status of residents of an
AR is a risk factor for dengue transmission. A
study in Brazil found no association
between the socioeconomic status and
dengue risk at the individual level[14] and
suggested that the previous finding of such
an association at the aggregate level[15,16] was
a fallacy. However, it should be noted that
studies that focus on factors at individual
level are insufficient to address the
ecological links in the causal chain.
Ecological studies may be, as pointed out by
Koopman and Languini[17] , the only way to
study risk factors for infectious diseases. The
risk of dengue virus infection is not
dependent
on
the
physiological
characteristics of individuals, but on the
environmental characteristics of the area
where the group of individuals live. This
includes other individuals, the natural
environment and the way it is transformed
by humans. The mean per capita income
has to be interpreted as an environmental
measure concerning the area and not as an
aggregate measure of individuals, because it
impacts the neighbouring households as
well as the nearby public areas, thus
affecting the presence of potential breeding
sites for the vector. This discussion
reinforces the relevance of multi-level
analysis in the evaluation of dengue risk
factors.
The inclusion in the model of the
proportion of the area used for commerce
and services and the proportion of city taxes
paid by industry shows that urban
organization plays an important role in the
distribution of dengue. These two variables
are proxy variables for the movement of
people across ARs. The presence of
commerce and general services was
represented by the proportion of the area
and the presence of industry as the
proportion of city taxes, because those are
the variables correlated to the intensity of
the movement of people. The luxury
commerce and services may collect more
taxes than the popular commerce and
services, but the areas of popular commerce
and services have a much bigger flow of
people. On the other hand, the relative size
of the industrial area is related to the type of
industry, as for instance big industrial
storage areas, and not to the production or
the number of workers. Industries that
collect more taxes have a larger production
and are more likely to have a higher number
of workers. Other authors [18] indicate that
the probability of being reached by a new
dengue virus is correlated to the intensity of
communication among people and the
density of traffic and the road network. It is
important to note that the diffusion of the
epidemic by proximity may be less
important than the diffusion caused by the
circulation of people in central areas in big
cities, which is supported by the present
finding. This area initially imports infected
individuals from the initial foci of the virus
where it is newly introduced, resulting in
higher local transmission, which, in turn,
25
results in exportation of infected individuals
to other areas, reinforcing the epidemic all
over the city. This fact clearly indicates the
priority of mosquito control in areas with
high levels of population mobility, such as
the Rio de Janeiro city central area.
The model also included the area of
residential property per inhabitant as a risk
factor for dengue. This variable is closely
correlated with the socioeconomic status
represented by the mean per capita income
(R=0.864605) that is controlled in the
model, meaning that, for the same
socioeconomic status, a bigger area of
residential property per inhabitant results in
bigger dengue risk. This is possibly due to
the existence of empty spaces in properties,
such as backyards, thus creating greater
opportunities for the existence of the vector
breeding sites. This hypothesis has to be
investigated for its importance in the
selection of priorities for vector control in
private residences as well as in defining the
contents of educational interventions.
The results of the present study allow
the establishment of priorities in vector
control and educational interventions,
highlighting the importance of the flow of
people across cities and its significance in
dengue epidemics.
Conclusion
The current efforts to control dengue
demand a more comprehensive approach,
including health education, community
participation, garbage disposal and proper
urban planning, besides chemical and
biological mosquito control measures. In
order to improve the efficiency of control
efforts, these activities have to concentrate
on areas and populations at higher risk,
which implies early identification of higher
incidence periods and areas and their
characteristics[5] .
In Rio de Janeiro, emphasis has been
placed on bringing about behavioural
changes in the communities to make an
impact on the determinants and risk factors
of dengue through educational interventions.
Health authorities and the press attributed
the main responsibility of the problem to
public behaviour, not clearly distinguishing
between the responsibilities of the
government and that of the private citizen[6] .
References
[1] Osanai CH, Travassos-da-Rosa APA, Amaral
S, Passos ACD and Tauil PL. Surto de
dengue em Boa Vista, Roraima. Revista do
Instituto de Medicina Tropical de Sao Paulo,
1983, 1: 53-54.
[3] Secretaria de Vigilância em Saúde. Dengue
Boletim
semana
16;
2004
online
http://dtr2001.saude.gov.br/svs/epi/dengue/
boletim/pdfs/Boletim%20semana%2016%20
2004.pdf.
[2] Nogueira RM, Miagostovich MP, Schatzmayr
HG, dos Santos FB, de Araujo ES, de Filippis
AM, de Souza RV, Zagne SM, Nicolai C,
Baran M and Teixeira Filho G. Dengue in
the State of Rio de Janeiro, Brazil, 19861998. Mem Inst Oswaldo Cruz, 1999, 94(3):
297-304.
[4] Tauil PL. Urbanização e ecologia do dengue.
Cad Saúde Pública, 2001, 17(1): 99-102.
26
[5] Pan American Health Organization. A
blueprint for action for the next generation:
dengue prevention and control. Washington:
PAHO, 1999, 1-14.
[6] Penna MLP. Dengue control: a challenge for
the public health system in Brazil. Cad
Saúde Pública, 2003, 19(1): 305-309.
[7] Hayes JM, Garcia Rivera, Flores Reyna,
Suarez Rangel, Rodríguez Mata, Coto
Portillo, Baltrons Orellana, Mendoza
Rodríguez, De Garay, Jubis Estrada,
Hernández Argueta, Biggerstaff BJ and Rigau
Perez. Risk factors for infection during a
severe dengue outbreak in El Salvador in
2000. Am J Trop Med Hyg, 2003, 69: 629633.
[8] Ashford DA, Savage HM, Hajjeh RA,
McReady J, Bartholomew DM, Spiegel RA,
Vorndam V, Clark GG, Gubler DG.
Outbreak of dengue fever in Palau, Western
Pacific: risk factors for infection. Am J Trop
Med Hyg, 2003, 69: 135-140.
[9] C‚mara
tecnica
e
cientifica
de
assessoramento ao controle do dengue.
Recomendações à Secretaria de Estado de
Saude. Secretaria de Estado de Saúde do Rio
de Janeiro, Rio de Janeiro, RJ, 2002, 1-11.
[10] Instituto Municipal de Urbanismo Pereira
Passos, Secretaria Municipal de Urbanismo,
Cidade do Rio de Janeiro. Website:
http://www.armazemdedados.rio.rj.gov.br/,
April 2004.
[11] StatSoft Inc. Electronic Statistics Textbook.
Tulsa, OK: StatSoft. http://www.statsoft.com/
textbook/stathome.html, 2004.
[12] Selvin S. Epidemiologic analysis: a caseoriented approach. Oxford University Press,
New York, 2001: 321.
[13] Forattini Oswaldo Paulo and e Brito
Marylene de. Reservatórios domiciliares de
água e controle do Aedes aegypti. Rev
Saúde Pública, 2003, 37: 676-677.
[14] Teixeira M, Barreto ML, Costa M, Ferreira
LD, Vasconcelos PF and Cairncross S.
Dynamics of dengue virus circulation: a
silent epidemic in a complex urban area.
Trop Med Int Health, 2002, 7: 757-762.
[15] Vasconcelos PF, de Menezes, Melo LP,
Pesso ET, Rodríguez SG, da Rosa, Timbo MJ,
Coelho IC and Montenegro F. A large
epidemic of dengue fever with dengue
hemorrhagic cases in Ceará State, Brazil,
1994. Rev Inst Med Trop Sao Paulo, 37:
253-255.
[16] Figueiredo LT, Cavalcante SM and Simoes
MC.
Dengue
serologic
survey
of
schoolchildren in Rio de Janeiro, Brazil, in
1986 and 1987. Bull Pan Am Health Organ,
1990, 24: 217-225.
[17] Longini IM Jr, Koopman JS, Haber M and
Cotsonis GA. Statistical inference for
infectious diseases. Risk-specific household
and community transmission parameters.
Am J Epidemiol, 1988, 128(4): 845-859.
[18] Muttitanon W, Kongthong P, Kongkanon
C, Yoksan S, Gonzalez JP and Barbazan P.
Spatial and temporal dynamics of dengue
haemorrhagic
fever
epidemics
In:
Conference Proceedings of Map Asia
2002,
GIS
Development,
2002,
http://www.gisdevelopment.net/application/
health/planning/healthp0010c.htm.
27
Sero-epidemiological and Virological Investigation of
Dengue Infection in Oaxaca, Mexico, during 2000-2001
A. Cisneros-Solano*, M.M.B. Moreno-Altamirano**#, U. Martínez-Soriano***,
F. Jimenez-Rojas†, A. Díaz-Badillo‡ and M.L. Muñoz‡
*Escuela Nacional de Medicina y Homeopatía
**Escuela Nacional de Ciencias Biológicas-Instituto Poltécnico Nacional
***Universidad Autónoma “Benito Juárez” de Oaxaca
†
Lab. Est. Salud Pública de Oaxaca
‡
Centro de Investigación y Estudios Avanzados-IPN
Abstract
A sero-epidemiological-cum-virological investigation was carried out in Oaxaca, Mexico, during 20002001 to assess the incidence of dengue infection and the circulating viruses.
A total of 200 serum samples reportedly from dengue patients, based on clinical diagnosis, were
collected from Oaxaca´s Central Laboratory of Public Health (in the capital city of the state of
Oaxaca). The samples were initially collected from ten regional health centres located across Oaxaca.
The sample population for the study included both sexes and age groups with clinical signs compatible
with dengue infection. All samples were tested for the presence of dengue virus, mainly by MACELISA and RT-PCR.
Ninety-four out of 100 serum samples suspected of dengue were confirmed to be positive. Thirtytwo were found positive by MAC-ELISA and 58 were positive by RT-PCR. In addition, the RT-PCR
analysis showed that the prevalent serotype in the localities in the study area was DEN-2. However,
one isolate of DEN-1 and another of DEN-4 were also detected. The number of infected females was
higher than that of infected males and the most affected age group was of people aged under 35
years. The study also highlighted that the sensitivity and specificity of diagnostic tools were crucial for
epidemiological studies.
Keywords: Serodiagnosis, MAC- ELISA, RT-PCR, DEN- 2, Oaxaca, Mexico.
Introduction
In recent years, dengue fever (DF) / dengue
haemorrhagic fever (DHF) has emerged as
major health problem in Mexico. In 1960,
the Aedes aegypti mosquito was eradicated
but it reappeared in 1965[1,2]. As pointed out
by Gubler[3] , factors such as demographic
and social changes are responsible for the
re emergence of dengue. Mexico is
considered an endemic country for dengue
and it is reported that major epidemics of
DEN-1 occurred on the eastern coast of
Mexico during 1979-1980. In 1984-1985,
#
E-mail: [email protected], [email protected]; Tel. (5255) 55729-6300 Ext. 62370,
Fax (5255) 55396-3503
28
Dengue Investigation in Oaxaca, Mexico
dengue was diagnosed in 25 of the 32 states
of Mexico. By then, DEN-1, DEN-2 and
DEN-4 were present in the country, and in
1995, DEN-3 was circulating as well. Several
cases of DHF were also confirmed[4] . In
subsequent
years
dengue
achieved
endemicity in the country.
As per the records of the Mexican
Health Office[5] (Secretaría de Salud, SS), a
higher number of dengue cases were
recorded in the states of Nuevo León,
Tamaulipas, Veracruz and Oaxaca during
1998-2001.
Oaxaca is located in the subtropical
region of Mexico at about 1,600 metres
above sea level (Figure 1A). There is high
demographic pressure and migration to
different urban zones is common, resulting
in the establishment of scattered human
settlements with deficient public services. All
these factors contributed to the propagation
of the Aedes aegypti mosquito, resulting in
dengue outbreaks every year in most of
Oaxaca´s communities[6] .
Figure 1. Oaxaca, Mexico
(A) United States of México. Oaxaca is located in the west coast
(B) Oaxaca is divided by the health authorities in six jurisdictions (I-VI)
29
To assess the dengue situation,
epidemiological studies were undertaken to
make an estimate of the incidence of
dengue virus infection and the circulating
serotypes in some selected endemic areas of
Oaxaca during 2000-2001.
Population study
The state of Oaxaca is located on the west
coast of Mexico. The Mexican Health Office
has divided it into six jurisdictions: (I)
Central Valleys, (II) Tehuantepec isthmus,
(III) Tuxtepec, (IV) The Coast, (V) The
Mixteca, and (VI) The Sierra. The presence
of dengue virus has been registered in all six
jurisdictions (Figure 1B). This study was
carried out in ten municipalities distributed
in five jurisdictions in the state of Oaxaca.
The study population included both sexes
and all age groups [7] . Two population groups
were included in this study: one group
consisting of 200 serum specimens from
patients manifesting signs and symptoms of
dengue infection, and another group of 50
serum samples from healthy controls, all
from the same jurisdictions.
Sample collection and diagnosis
of dengue
Human sera were obtained from 200
patients presenting clinical manifestations of
dengue and tested for anti-dengue IgM
antibodies. Serum samples were collected
by venipuncture, using Vacutainer tubes
(Becton-Dickinson). The clinical samples
corresponded with dengue cases reported
during
2000-2001.
Dengue-infected
samples were obtained during the first five
30
days of the onset of fever and were
processed for anti-dengue IgM detection
using IgM capture ELISA (MAC-ELISA) as
described by Vorndam et al.[8] Samples from
healthy donors were obtained at about the
same time.
As a routine practice and with the idea
of recording epidemic data, the suspected
dengue samples already clinically diagnosed
in community health centres were sent to
the Central Laboratories in the city of
Oaxaca (Laboratorio Estatal de Salud
Pública del estado de Oaxaca, Secretaría de
Salud). In this laboratory, the presence of
dengue virus was confirmed by MAC-ELISA
and RT-PCR.
Dengue virus isolates
Aedes albopictus C6/36 cells were grown in
48-well tissue culture plates as described by
Igarashi[9] . Briefly, 2X10 5 cells were plated in
1 ml of minimum essential medium (GibcoBRL, Grand Island, N.Y.) supplemented with
7% fetal bovine serum (Sigma Chemical Co.,
St. Louis, Mo) and 1% glutamine, vitamins
and nonessential amino acids. After 24
hours of culture, 100 µl of every sera diluted
1:10 was added to the corresponding well.
The mixture was then gently shaken and
incubated for 60 minutes at room
temperature. Cells were then washed with
serum-free medium and cultured at 28 °C
with complete medium for at least 10 days.
Cells were harvested for RT-PCR diagnosis.
RNA extraction
Total RNA was extracted either from 100 µl
of serum or from cultured cells by using
Trizol LS (GIBCO BRL, Gaithersburg, MD.)
according
to
the
manufacturers’
recommendations. Ethanol-precipitated RNA
was recovered by centrifugation and air-dried.
The RNA pellet was re -suspended in 50 µl of
Diethyl-pyrocarbonate (Sigma)-treated water
(DEPC water) and used as a template for RTPCR.
RT-PCR
Synthetic oligonucleotide primer pairs were
designed based on published sequence data
for each of the four serotypes of dengue [10,11] .
Four fragments of an expected size of 482
bp (DEN -1), 392 bp (DEN -4), 290 pb (DEN 3) and 119 bp (DEN-2) were obtained by
using the SuperScripTM One Step RT-PCR kit
in conjunction with PlatinumR Taq
polymerase (Invitrogen, Life Technologies).
A mixture of 5 µl of RNA, 25 µM of sense
and anti-sense PCR primers, and DEPC
water to a total volume of 50 µl was
incubated at 85 °C for 5 minutes and then
chilled on ice. The tubes-reaction mixture
containing 2X PCR buffer containing 0.4
mM of each dNTP, 2.4 mM MgSO 4 and
Super ScriptTM RT/platinumR Taq Mix, as
recommended by the manufacturer (In
vitrogen TM Life Technologies), was added
to the RNA and primers -containing tube.
The reverse transcription reaction was
performed at 50 °C for 30 minutes.
Thermocycling began with a hot start at
94 °C for 2 minutes followed by 40 cycles
of annealing at 55 °C for 30 seconds, and
extension at 72 °C for one minute and
denaturing at 94 °C for 15 seconds.
The PCR conditions for serotype
assessment were as follows: 40 cycles of
denaturing at 94 °C for 30 seconds,
annealing at 55 °C for 1 minute, and
extension at 72 °C for 1 minute and, a final
extension at 72 °C for 7 minutes. The
reaction mixtures were electrophoresed and
visualised under UV light after ethidium
bromide staining of the gels.
Results
Diagnosis of the samples by MACELISA
Two hundred serum samples initially
reported as suspected positive for dengue,
based on clinical reports from the hospital
where patients were hospitalised, were
submitted for diagnosis based on antidengue IgM antibodies detection by MACELISA. From these, only 34 samples were
positive for IgM antibodiesψ . As expected,
the 50 negative-control samples resulted
negative for anti-dengue IgM antibodies
(Table 1).
Diagnosis by RT-PCR
Once the serum samples were tested for
anti-dengue IgM antibodies, the results were
confirmed by RT-PCR. In this case, only 25
samples from healthy donors were tested.
By this method 58 samples proved to be
positive for dengue, i.e. 24 more than by
MAC-ELISA. Interestingly, all samples
positive for MAC-ELISA were also positive
by RT-PCR. Those samples showing
positivity for DEN by RT-PCR were further
tested for the four serotypes (DEN-1, -2, -3
and -4). It was found that the main
circulating serotype in Oaxaca during 20002001 was DEN-2. Two other serotypes
(DEN-1 and DEN -4) were also found (only
one case each) (Table 1).
ψ
Out of 200 samples, originally sent, only 100 samples
were found in good condition for evaluation by MACELISA or RT-PCR. Other samples deteriorated under
transportation/storage conditions.
31
Table 1. Positivity for dengue by MAC-ELISA and RT-PCR
From one hundred samples tested, from patients with clinical diagnosis of dengue,
36% proved positive by MAC-ELISA and 61% by RT-PCR. DEN-2 was the prevailing
circulating serotype
Date of
collection
Jurisdiction
Locality
Number
of cases
Nov 2000
Nov 2000
Nov 2000
Jul 2000
Nov 2000
II
III
II
III
II
Salina Cruz
Tuxtepec
Tehuantepec
Temascal
Juchitan
18
9
10
8
3
13+/52+/74+/63+/52+/1-
Total cases
May-Jun
2001
Jun-Nov
2001
Apr 2001
May 2001
Feb 2001
Feb 2001
Feb 2001
IV
II
II
III
V
V
32
Serotype
9+/95+/45+/55+/32+/1-
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
21
6+/15-
12+/9-
DEN-2
DEN-2
Oaxaca
7
2+/5-
7+/0-
DEN-2
DEN-2
Juchitan
Salina Cruz
Tuxtepec
Tonalá
Huajuapan
2
1
7
2
6
0+/20+/12+/50+/20+/6-
2+/01+/02+/52+/06+/0-
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
DEN-2
10+/36-
32+/14-
Table 2. Distribution of dengue cases
by age and sex
Male
Serotype
26+/22-
Total cases
Age
(in years)
RT-PCR
24+/24-
Huatulco
I
MACELISA
Female
1
0
1
2-4
0
2
5-9
7
10
10-14
5
6
15-20
2
3
21-30
0
5
>30
6
6
Total
20 (38%)
33 (62%)
Prevalence of infection by
age and sex
An analysis by age and sex revealed a higher
prevalence (61%) of infection in females
than in males (39%) and that the most
affected group of people was the under-35year-olds (Table 2).
Discussion
The Aedes aegypti mosquito’s adaptability to
changing environmental conditions has
contributed significantly to the increase in
dengue epidemics in the world. Mexico is
considered an endemic country where the
four serotypes of the dengue virus are in
circulation. This study provides some insight
into the dengue epidemic situation in
Oaxaca state, Mexico, in an attempt to
contribute to the prevention and control of
outbreaks of DF/DHF.
From the 200 serum samples collected
from suspected dengue patients initially
considered for the study, only 100 could be
used. The remaining 100 samples were
presumably subjected to non-appropriate
storage conditions. From the 100 suspected
samples tested, nearly 100% proved to be
positive for dengue (36% by MAC-ELISA
and 61% by RT-PCR). However, the data
reported here could be an underestimation
considering the several factors that could
influence the laboratory determination
outcome, such as sample handling and the
diagnosis systems performed at local
hospitals. In some localities of Oaxaca, the
diagnosis
for
dengue
was
being
simultaneously carried out with the
diagnosis for rubella and toxoplasma in a
monoclonal antibodies-based multiplex
assay. In rural communities, however, only
the presence of anti-dengue IgM antibodies
was tested.
In this regard, it is possible that some
patients presenting an early secondary
infection in the absence of strong clinical
manifestations had undetectable levels of
anti-dengue IgM antibodies, since IgG is the
prevalent Ig isotype at this stage of the
infection. For these cases, it would be
necessary to consider some other diagnosis
techniques such as virus isolation or RT-PCR.
Unfortunately, these are difficult to carry out
in rural hospitals due to high costs and lack
of suitably trained personnel.
Additional effort is needed to ensure
appropriate sample collection, handling
and storage in order to send them to the
reference laboratory for adequate diagnosis.
It is worth noting that in several Mexican
states, health authorities are working on
vector control as well as on facilities for
sample collections to be sent to the
Instituto de Referencia Epidemiológica
(InDRE) in Mexico City for a proper
diagnosis. It is still, however, a long way for
good quality medical care to reach most
Mexicans.
This report shows that by MAC-ELISA,
36% of the tested samples were found
positive for dengue, whereas by RT-PCR up
to 64% of the samples proved to be positive.
Although the sensitivity and specificity
reported for MAC-ELISA is reported to be
good enough for a diagnosis system, it is
likely that as a result of inadequate handling
and storage conditions, some samples
reported as negative could in fact be
positive for dengue when tested by RT-PCR.
No false positive results were found. This
raises the question as to how many
laboratory assays must be carried out on a
suspected dengue sample before reporting it
as negative.
The use of RT-PCR makes it possible to
identify the dengue serotype involved; in
this regard this study shows that in Oaxaca,
Mexico, the prevalent serotype of dengue
virus was DEN -2, although isolated cases of
DEN-1 and DEN-4 infections were also
found. Some other local reports had also
mentioned the presence of DEN-3 and
several cases of DHF.
Acknowledgements
We thank Dr F. Javier Sánchez-García for
critically reviewing the manuscript. MMBMA
is an EDI/IPN fellow.
33
References
[1] Novo S. Breve historia y antología de la
fiebre amarilla. Salud Pública de México,
1995, 37: S99-S102.
[7] Martínez-Soriano U. Tipificación Molecular
del virus del dengue en el estado de Oaxaca.
B.Sc. Thesis, 2002, 19-58.
[2] Gómez
H.
Monografía
sobre
la
epidemiología del dengue en México.
Secretaría de Salud, 1992, Vol. 43.
[8] Vorndam V and Kuno G. Laboratory
diagnosis of dengue virus infections. In:
Gubler DJ and Kuno G (Eds.), Dengue and
dengue
hemorrhagic
fever.
CAB
International, New York, NY, 1997, 313-333.
[3] Gubler JD. Epidemic dengue/dengue
haemorrhagic fever as a public health, social
and economic problem in the 21st century.
Trends in Microbiology, 2002, 10: 100-103.
[4] Briseño-García B, Gómez-Dantes H, ArgottRamírez E, Montesano R, Vázquez-Martínez
AL, Ibanez-Bernal S, Madrigal-Ayala G,
Ruiz-Matus C, Flisser A and Tapia-Conyer R.
Potential risk for dengue haemorrhagic
fever: the isolation of serotype dengue-3 in
Mexico. Emerging Infectious Disease, 1996,
2(2): 133-135.
[5] Anonymous. Gaceta informativa de
Secretaría de Salud, SSA-Oaxaca, 1999.
la
[9] Igarashi A. Mosquito cell cultures and the
study of arthropod-borne togaviruses.
Advances in Virus Research, 1985, 30: 21-39.
[10] Lanciotti RS, Calisher CH, Gubler DJ, Chang
GJ and Vorndam V. Rapid detection and
typing of dengue viruses from clinical
samples by using reverse transcriptasepolymerase chain reaction. Journal of
Clinical Microbiology, 1992, 30: 545-551.
[11] Harris E. A low cost approach to PCR. Ed.
Oxford University Press, USA, 1998, 96-105.
[6] Cisneros AS, Martínez US, Cruz GM, Tovar
RG, Moreno MG, Días-Badillo A and Muñoz
ML. Dengue fever in the state of Oaxaca,
Mexico. The American Society of Tropical
Medicine and Hygiene, 50 th Meeting, 2001,
Atlanta, Georgia, USA.
34
Spatial and Temporal Dynamics of
Dengue Haemorrhagic Fever Epidemics,
Nakhon Pathom Province, Thailand, 1997-2001
Wutjanun Muttitanon*, Pongpan Kongthong**, Chusak Kongkanon**,
Sutee Yoksan***, Narong Nitatpattana***, Jean Paul Gonzalez‡
and Philippe Barbazan †#
*Asian Journal of Geoinformatics; Space Technology Application and Research Program,
Asian Institute of Technology. P.O. Box 4, Klong Luang, Pathumthani, Thailand
**Department of Geography, Faculty of Education, Ramkhamhaeng University, Bangkok 10110, Thailand
***Center for Vaccine Development (CVD), Institute of Science and Technology for Research and
Development, Mahidol University, Nakhon Pathom 73170, Thailand
†
Research Center for Emerging Viral Diseases (RCEVD) – IRD – Center for Vaccine Development,
Institute of Science and Technology for Research and Development, Mahidol University,
Nakhon Pathom 73170, Thailand
‡
Institut de Recherche pour le Développement (IRD) Ur034, 213 rue La Fayette, 75480,
Paris cedex 10, France
Abstract
Several environmental factors modulate the distribution of dengue fever (DF), such as climate, density of
vector and human populations in urban areas and distribution of herd immunity. In order to identify
geographical variables involved in the spread of a DHF process, a Geographic Information System (GIS)
has been built to create links between geo-referenced data including medical records and
socioeconomic and environmental data. Applied to a retrospective analytical study of DHF epidemics in
Nakhon Pathom province 1
( 997-2001), the GIS allowed a mapping of spatial variations of DHF
incidence, the recognition of different temporal incidence patterns and the quantification of the dispersal
of outbreaks among defined spatial units. The analysis showed that the diffusion process of these
epidemics was of a contagious type as the distance between epidemic areas (sub-districts) was
significantly lower than the average distance between every sub-district. This result indicates that these
epidemics were likely to be due to the spread of a new or rare virus serotype, from its emergence
location in the province to areas with a sufficient density of vectors and a similar limited immune
protection against this serotype.
Keywords: Dengue haemorrhagic fever, dengue virus, transmission, Geographic Information System, spatial analysis.
#
E-mail: [email protected]; Tel./Fax: (66) 2 441 01 89
35
Spatial and Temporal Dynamics of DHF Epidemics in Thailand
Introduction
Dengue fever (DF) is a viral disease with a
worldwide distribution in all tropical areas.
It is caused by the dengue virus (genus
Flavivirus, family Flaviviridae) which presents
four antigenic forms or serotypes: DEN -1,
DEN-2, DEN -3 and DEN-4. In Thailand,
Dengue haemorrhagic fever (DHF) a severe
form of dengue fever has been endemic
since 1958, with a cumulative total of
1,369,542 cases till date[1] . Epidemics occur
with a periodicity of between two and four
years; these epidemics are of significant
concern for the public health authorities. In
most of the areas where serotype
identifications were performed, two or three
serotypes were found to be co-circulating[2] .
The dengue virus is an arbovirus
(arthropod-borne virus) transmitted by the
mosquito Aedes aegypti (L.). Control of the
spread of the disease focuses on vector
control strategies based mainly on the
elimination of potential breeding sites[3] . A
major attribute of the virus transmission is its
anthropophilic behaviour, as females mainly
bite humans and lay eggs in man-made
containers near houses (for example, water
jars, cans, used tyres). The short flight range
of the vector, less than 1 km, contributes to
the limited spread of the disease by an
infected female. Most of the infections by
dengue viruses are not severe and present
asymptomatically, allowing infected patients
to maintain normal activities.
Two types of viral spread can be
described: (i) the diffusion of human
infections as a function of the spatial
distribution of houses and the limited flight
range of infectious or infected Aedes aegypti
females
(intra-communal,
contagious/
continuous);
and
(ii)
inter-communal
36
dispersion, largely a function of the stochastic
movement of incubating/infectious humans
and the transport via vehicles of virus-positive
females[4].
The understanding of the mechanism of
the inter-community spread of DHF during
epidemic periods is a primary factor likely to
lead to an evaluation of the risk of virus
transmission
and
disease
dispersal[5] .
Moreover, it would provide some guidance
on the distance from the spatial origin of an
epidemic at which preventive control
measures should be applied.
At a monthly time-scale, the main
geographical factors involved in dengue
transmission (urbanization, demography,
cultural and social characteristics) are
stable[6] . A change in the pattern of monthly
DHF transmission, such as the emergence of
epidemics in an endemic area, should then
rather be related to factors evolving with
time: climate, density of vectors, emergence
of a new or rare virus serotype, each type of
factor inducing a specific pattern of diffusion
of the disease[7,8] . The emergence of a new
serotype in a given population is likely to
exhibit particular spatial characteristics. The
outbreak would begin where the serotype
first arrived and then move to places where
a low specific herd immunity (towards this
serotype) and a sufficient density of
mosquito allow a high level of transmission.
The spread of a new serotype is then likely
to follow the main model of contagious
diffusion described for the spread of other
types of moving phenomena[9] . Applied to
the diffusion of an infectious disease, it
means that the probability for an area to be
reached by a contagious disease will be
inversely correlated to the distance to the
formerly contaminated areas, leading to
clusters of epidemic areas.
In order to test the validity of this model
in the frame of dengue dispersal, a study
was conducted to describe the spread of
significantly higher levels of incidence rate
(of epidemic significance) among subdistricts. The study, done in a province of
Thailand, covering the period 1997-2001,
included two DHF epidemics.
Data collection
Data on clinically diagnosed DHF cases
were recorded at the Ministry of Public
Health, the demographic data were
provided by the Administrative Department
of the Ministry of Interior, and the
geographical maps by the Royal Thai Survey
Department. DHF cases were
according to WHO criteria[10] .
defined
Population and study area
Nakhon Pathom province is a part of the
central
plain
region
in
Thailand
encompassing the latitude of 13° 38’45.6” N
to 14° 10’37.2” N and the longitude of
99° 51’10.8” E to 100° 17’6” E. It covers
2,164 sq km, has a population of 774,276
inhabitants and includes 7 districts and 106
sub-districts (Figure 1a). The population
density ranges from 153 to 623
inhabitants/sq km. The average surface area
of sub-districts is 20.4 sq. km. The provincial
health department reported 14,079 DHF
cases during 1983-2001; two DHF
epidemics occurred in 1997-1998 and
2000-2001 (Figure 2).
Figure 1. District scale approach: (i) Administrative limits of districts and sub-districts;
density of population; main roads; (ii) Average incidence observed before the epidemics
(cases/100,000 people), January 1992 – June 1997; (iii) Ratio of the incidence during the
first three months of the DHF epidemic compared to the average incidence from
January 1992 – June 1997
5
0
10
20 km
a) Density of population
(inhabitants / ha)
Main roads
b) Average Incidence
01/1992 - 06/1997
(cases /100,000)
c) Ratio of Incidence
07 – 09 1997 vs
aver. 01/1992 - 06/1997
> 600
22 - 46
1.6 – 2.8
Districts
350 - 450
8 - 10
1.3 – 1.4
Sub-districts
150 - 350
3-7
0.3 – 0.7
37
Figure 2. Monthly DHF incidence in Nakhon Pathom province, Thailand, from January
1992 to August 2001
300
cases
200
100
0
1-92 7-92
1-93 7-93
1-94
7-94 1-95
7-95 1-96 7-96 1-97 7-97
1-98 7-98
1-99
7-99 1-00
7-00 1-01 7-01
month
Method of analysis
The study aimed to describe the spatialtemporal dynamics at a monthly time-scale
of a DHF epidemic among Nakhon
Pathom’s 106 sub-districts considered as the
spatial units. As a first step, epidemics were
defined at the province level as periods of
time (at least two consecutive months) when
the incidence is higher than the average,
plus one standard deviation of the monthly
incidence of each month (i.e. January,
February, etc.). The average was calculated
over the entire 1983-2001 period[11] .
During these epidemic months (EMs),
epidemic sub-districts (ESDs) were those
where the monthly incidence was
significantly higher than in other sub-districts.
The threshold for a significantly higher
incidence was leveled at the average
monthly
incidence
(per
100,000
inhabitants) plus one standard deviation,
observed among every sub-district during
that EM.
38
In a contagious model for an infectious
disease, the spatial entities close to an
infected one were assumed to be more at
risk to become infected than the distant
ones. Applied to the diffusion of an
epidemic phenomenon, it meant that the
distance between the new epidemic subdistricts and the former ones (observed
distance) should be shorter than the average
(expected) distance between all the subdistricts. The distance between sub-districts
was defined as the Euclidian distance
between their centroids. The expected
distance was the average distance between
each ESD and every other sub-district. The
observed distance was the average distance
between each ESD and every other ESD,
during the same month (cluster study), or
from one month to the next (spread study).
H0 (null hypothesis) = the average
observed distance (between ESD) was not
different from the average expected distances.
H1 = average observed
<average expected distance.
distance
The Z test was used to compare the
average distances.
drowned at 5 km; 10 km; 15 km; 20 km
and out of 20 km. This number is compared
to the number of sub-districts centroids
distributed in these surfaces to build a
relative risk index.
The method was applied to the study of
two phenomena: (i) the occurrence of
clusters of ESD during one month; and (ii)
the spread of the epidemic among subdistricts from one month to the next. A
cluster is defined here as an aggregation of
ESD (during one EM) of sufficient size and
concentration to be unlikely to have
occurred by chance, i.e. if the average
distance (between these ESD) is shorter than
the average distance between all subdistricts. The spread of the epidemic is
based on the comparison of observed and
expected distances during two consecutive
EMs, i.e. the average distance between ESD
during one epidemic month (EMm) and ESD
during the next epidemic month (EMm+1),
versus the average distance between ESD
during EMm and every sub-district during
EMm+1.
Relative risk index =
number of ESD in a circle
total number of ESD
number of sub - districts in a circle
total number of sub - districts
Results
At the district scale, the DHF incidence was
higher in the central-west part of the
province. The epidemic broke out in the
northern district with a medium density of
population (Figures 1b and 1c). At the subdistrict scale, the maximum DHF incidence
rate reached 540 cases per 100,000
inhabitants in July 1997.
Nineteen EMs were identified in
Nakhon Pathom province from January
1997 to August 2001 (Table); the number of
EMs in one sub-district ranged from 0
month (in 27 sub-districts) to a maximum of
11 months.
The (discrete) distance, at which an
epidemic can spread in one month, was
estimated by summing the number of ESD
centroid during EMm+1 observed inside
circles centred on each ESD during EMm and
Table. Chronological distribution of epidemic months from January 1997 to August 2001 in
Nakhon Pathom province, Thailand
Year
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
1997
1998
1999
2000
2001
= Epidemic month
39
A total of 49 ESDs were identified
during the first outbreak (1997-1998) and
61 during the second outbreak (20002001); 31 sub-districts were epidemicaffected during the two outbreaks. The
probability of one sub-district being
epidemic-affected during the first outbreak
to be epidemic-affected during the second
outbreak as well is not significantly different
from a random distribution (P<0.05).
the
Cluster study: During EM, 78.67% of
average observed distances were
significantly lower (P<0.05) than the
average expected distance, characterizing
the occurrence of clusters of ESDs according
to the H1 hypothesis.
Spread study: The distance observed
between ESDs during EMm and ESDs during
EMm+1, was significantly smaller than the
expected distance (Figure 3), characterizing
the contagious spread of DHF among ESDs
according to the H1 hypothesis.
Figure 3. Study of the spread of DHF epidemic among sub-districts: observed distances are
calculated between epidemic-affected sub-districts during one month and the epidemicaffected sub-districts during the next month; expected distances are calculated between all
the sub-districts (see text for details)
97-98 epidemic
endemic
00-01 epidemic
20
15
10
5
0
Aug
97
Oct
97
Dec
97
number of epidemic tambon
Feb
98
Apr
98
Jan
01
observed distance
As a consequence, the distribution of
ESDs during EMm+1 in surfaces drowned
round ESD during EMm showed a significant
(P<0.05) aggregation within the first two
circles (5 and 10 km).
40
month
Nov
00
Mar
01
May
01
nb of tambon
distance (km)
25
0
expected distance
Discussion
The method used for the identification of an
epidemic month in the province[11] allowed
a precise framing of epidemics, defined as
periods during which the incidence was
significantly higher than the observed
average over the complete time series of
data (19 years). Meanwhile, this method
could not be used directly at the sub-district
scale because of the lack of long-time data
series on the incidence at this scale.
Moreover, the variance of the DHF
incidence in most of the sub-districts was
very high because of the low values of
incidence often recorded (during the study
period a null monthly incidence was
reported in 66% of the 5,936 months X subdistricts). Similarly, the village scale (from 3
to 24 villages per sub-district) could not be
used as the spatial unit as the addresses of
patients were often consistent only at the
sub-district scale and many students and
pupils did not live in their village.
We assumed in this study that subdistricts
could
be
considered
as
homogeneous small areas and that human
displacements were sufficient to produce a
homogenization of the population, allowing
consideration of the sub-district as a unit
towards DHF transmission. An ‘epidemic’
pattern can then be identified in any subdistrict, whatever its density of population:
the distribution of ESD was not correlated to
the density of population (Pearson’s
correlation = -0.24, P = 0.71). Moreover,
we used the incidence rate per 100,000
inhabitants to reduce the bias related to the
size of the population.
The geographical heterogeneity of the
environment,
e.g.
the
density
of
urbanization or the road network, could also
be at the origin of clusters of ESDs.
Meanwhile, after the two epidemics, ESDs
were found to be uniformly distributed over
the entire province, and the spatial
distribution of all sub-districts having been
epidemic-affected at least during one month
(67% of the sub-districts) was not
significantly different from the spatial
distribution of all sub-districts (average
distances not different, P=0.95). Meanwhile,
the results implied a high degree of spatial
auto-correlation, meaning that neighbouring
sub-districts shared similar characteristics,
such as the level of immunity for the
different serotypes (due to a similar
epidemiological history) or the density of the
vector.
As shown in Figure 3, the observed
distances are smaller than the expected
ones, but exhibit similar monthly variations.
This was mainly because of a border effect,
the propagation in sub-districts located in
neighbouring provinces not being taken into
account. During the months where ESDs
were located on the periphery of the
province, the average distance to other subdistricts was larger than during the months
where ESDs were located near the centre of
the province, as several neighbouring subdistricts located in other provinces
(epidemic or not) were ‘missing’ in the
calculation. The absolute level of expected
and observed distances was then directly
dependent on the location of the ESD in the
province.
The spread of the epidemic between
sub-districts followed Hagerstrand’s model
that has been used to describe many types
of phenomena, such as the spread of new
ideas [9] or the waves of innovation which
lose their ‘energy’ when the distance from
the source increases[12] . In public health
research it has been applied to infectious
influenza[13] . Applied to the DHF epidemic
41
in Nakhon Pathom, it means that during the
epidemic periods the ESDs were the origin
of the emergence of epidemics in
neighbouring sub-districts during the next
month. The probability of this emergence at
m+1 significantly decreased with the
distance from the former ESD. This model is
of a contagious type and may be opposed to
a random or homogeneous model. In the
homogeneous models the occurrence of an
epidemic could be due to a global
phenomenon, such as an increase in
temperature, which should have been
observed in any sub-district, leading to a
random distribution of ESD [14] and an
observed distance not different from the
expected distance.
Inside human communities (villages) it
has been shown that the spread of DHF
viruses from one house to neighbouring
houses due to the displacement of infected
vectors or hosts follows a pattern similar to
what we have described between subdistricts[15] . Meanwhile, among communities
separated by several kilometers, the spread
of viruses cannot be due to the active
dispersal of mosquitoes or to their transport
by car, which is much more rare than the
displacement of infected hosts. More than
80% of infections by dengue virus are
unapparent or not severe, allowing healthy
carriers to travel. The presence of sufficient
densities
of
vectors
in
destination
communities is also necessary to allow the
transmission of the virus after it has been
imported.
The contagious distribution and spread
of the two DHF epidemics among the subdistricts strongly suggests that they were due
to the emergence of a new or rare serotype.
42
DHF is endemic in Thailand and the
different serotypes are largely distributed, as
at least two or three serotypes are generally
found at the same time in the same area[2,16] .
Meanwhile, during epidemic periods the
relative prevalence of the serotypes varies,
as the 2000 epidemic in Bangkok that was
due mainly to the serotypes DEN-1 and
DEN-2 (each reaching 42% of total
isolations), whereas the 1994 epidemic was
due mainly to the rise in DEN -4 (36%). But
as serology and isolation of viruses are rarely
performed, the emergence of a DHF
epidemic cannot be forecast by using these
methods. Indirect methods, such as the
statistical identification of epidemic months,
are then necessary to identify early the
emergence of DHF epidemics.
The epidemiology of DHF in Thailand is
changing[17] .
This
approach
of
the
displacement of epidemics is likely to
contribute to the localization of the origins
of outbreaks and the delineation of areas at
risk during epidemics, as well as to help
public health authorities to focus vector
control activities on selected areas.
Acknowledgements
The study and the preparation of this paper
was supported by the Institut de Recherche
pour le Développement (IRD)-Ur034,
France, by a fellowship to the Center for
Vaccine Development, Institute of Science
and
Technology
for
Research
and
Development, Mahidol University, Thailand,
and by the Department for Technical and
Economic Cooperation, Thailand. We thank
Professor Natth Bramapavarati for his
constant support.
References
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Dengue in the early febrile phase: viremia
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(Eds. P. Elliott, J. Cuzick, D. English and R.
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[9] Hagerstrand T. The propagation of innovation
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1952, 4.
haemorrhagic fever: diagnosis, treatment
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[11] Barbazan P, Yoksan S and Gonzalez JP.
Dengue Hemorrhagic Fever epidemiology in
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epidemics. Microbes and Infection, 2002,
1(4): 699-705.
[12] Gould PR. Spatial diffusion. Washington, US,
Association of American Geographers, 1969.
[13] Cliff AD, Haggett P and Ord JK. Spatial
aspects of influenza epidemic. London, Pion
Limited. 1986.
[14] Jackson EK. Climate change and global
infectious disease threats. The Medical
Journal of Australia, 1995, 163(4): 570-573.
[15] Morrison AC, Getis A, Santiago M, RigauPerez JG and Reiter P. Exploratory spacetime analysis of reported dengue cases
during an outbreak in Florida, Puerto Rico,
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Medicine and Hygiene, 1998, 58(3): 287-298.
[16] Burke DS, Nisalak A, Johnson DE and Scott
RM. A prospective study of dengue
infections in Thailand. American Journal of
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[17] Chareonsook O, Foy HM, Teeraratkul A and
Silarug N. Changing epidemiology of dengue
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Epidemiology and Infection, 1999, 122:
161-166.
43
Sporadic Prevalence of DF/DHF in the Nilgiri and
Cardamom Hills of Western Ghats in South India: Is it a
Seeding from Sylvatic Dengue Cycle – A Hypothesis
Nand Lal Kalra* and Chusak Prasittisuk**#
*A-38, Swasthaya Vihar, Vikas Marg, Delhi – 110 092
**Regional Office for South-East Asia, World Health Organization, New Delhi, India
Abstract
The Western Ghats of south India, encompassing the Nilgiri and Cardamom hills, are the wettest region
of the country. Hills rising upto 3,000 metres receive over 200 cm of rainfall from both the south-west
monsoon (June to September) and north-eastern monsoon (October to January). The eastern slopes of
the Nilgiri Hills (200-500 metres) are bounded by Coimbatore and Erode districts of Tamil Nadu;
whereas the western slopes of the Nilgiri and Cardamom hills are in the state of Kerala. The countryside
has rich forests of teak and sandalwood, interspersed by groves of coconut, rubber, pepper, cardamom
and banana plantations. Apart from the rich flora, monkeys M
( acaca radiata) maintain a strong
association in orchards with humans competing for food.
The emergence of DF/DHF in this hilly region is a recent occurrence. An epidemiological team from
the National Institute of Communicable Diseases (NICD) investigated the first-ever reported outbreak in
Coimbatore in 1998. In all, 20 serological positive (IgM) cases were recorded by the city corporation.
Five cases came from urban towns and 15 cases from rural areas of the two districts of Coimbatore and
Erode. Rural cases were scattered in distantly located villages. Pyramid characterization and clustering of
cases was conspicuously absent. No attempt was made to link urban cases to central/peripheral wards,
nor the history of movement of patients two weeks prior to the onset of fever was investigated. No
increase in fever rate was observed. DF cases did not show any relationship with presence/absence of
Aedes breeding. Aedes aegypti detected in urban centres failed to amplify the infection.
Kerala state also reported 116 cases in 1997, from Kottayam district. Out of these, 14 cases were
confirmed serologically. After a lull of 4 years, 70 probable cases out of 877 were reported from the four
districts famous for rubber plantations. Entomological investigations recorded only Aedes albopictus in
these areas. Considering the high experimental susceptibility of both species of monkeys, viz. Macaca
mulatta and Macaca radiata to yellow fever virus, detection of dengue antigen in field collected Aedes
albopictus in Kozhikode (Kerala) and the evidence of transovarian transmission in Aedes albopictus
reared from soils of tree holes at Jodhpur – Rajasthan (western India) lend support to the hypothesis that
DF in the Western Ghats of South India exists as enzootic monkey – Aedes albopictus – monkey cycle
and causes epizootics among rural human population either during periodic amplification of the enzootic
cycle or as occupational hazards to the people working in orchards.
Keywords: DF/DHF, sporadic cases, Macaca radiata, dengue antigen in Aedes albopictus, transovarial transmission,
sylvatic cycle, Nilgiri and Cardamom hills, south India.
#
44
A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India
Introduction
The Western Ghats of south India
encompass two southern states i.e. Tamil
Nadu and Kerala (Figure). The crests of the
Nilgiri and Cardamom Hills (rising upto
3,000 metres altitude) separate these two
states. The eastern slopes of the Nilgiri Hills
have a gentle slope and include two
important district towns of Tamil Nadu, viz.
Erode and Coimbatore, situated at an
altitude varying between 200 to 500 metres;
each with over one million population. This
part of the region receives rains from both
the south-west and north-east monsoon.
The south-west monsoons become weak,
being on the leeward side of the hills, but
maximum rains come from the north-east
monsoon, the total being <100 cm. The
forests are tropical monsoon, which are
famous for teak and sandalwood, with
patches of arecanut palms[1].
Figure. Physical map of Kerala and
adjoining areas of Tamil Nadu
The western side of the Cardamon hills
encompasses the state of Kerala. Strong
winds of the south-west monsoon lead to
the formation of heavy sand dunes in
coastal areas and the rain water coming
from the steep hills results in formation of
shallow lagoons, all along the coast, at
places connected to the sea. These lagoons
are connected by canals. These backwaters
are the characteristics of Kerala state. The
banks of these backwaters and sand dunes
are dotted with coconut trees[1].
The Region receives heavy rains (>200
cm) during the south-west monsoon. Hence,
the whole region is very wet and supports
luxuriant growth. Large tracts of forests have
been cleared for raising cash crops, viz.
arecanut palms, rubber, banana, pepper
and cardamom plantations[1].
Dengue transmission cycles
Transmission of dengue viruses occurs in two
cycles, viz. enzootic and epidemic cycles.
The enzootic cycle is a primitive sylvatic cycle
maintained by lower primates (monkeys) and
canopy dwelling Aedes mosquitoes, as
reported from South-Asia[2], Africa[3] and Sri
45
Lanka [4]. Current epidemiological evidence
suggests that these viruses do not regularly
move out of the forests to urban centres but
at times are involved in an epidemic cycle in
small rural villages or islands [3]. A number of
Aedes species may act as reservoirs [2].
The epidemic cycle is confined to large
urban centres. The viruses are maintained in
the Aedes aegypti – human – Aedes aegypti
cycles with periodic/cyclic epidemics.
Generally all serotypes circulate and give
rise to hyperendemicity. Virus is maintained
either transovarially by the vectors or by
continuous low-grade transmission in
susceptible hosts added to the population.
DF/DHF in urban cycles is characterized by
‘iceberg’ or ‘pyramid’ phenomenon. At the
base most of the cases are symptom-less,
followed
in
increasing
rarity,
by
undifferentiated fever, DF, DHF or DSS[5].
Occurrence of multiple cases in a single
household or clustering of cases in a locality
is yet another characteristic of this disease[2].
DF/DHF in India
In recent years the first outbreak of DF/DHF
was reported from Kolkata (earlier known as
Calcutta) in 1963[6]. Since then, more than
60 outbreaks have been reported from all
over the country[7]. Aedes aegypti, invariably
has been found to be associated with these
epidemics. All the four serotypes, DEN-1, 2,
3 and 4, are now circulating in the country.
DF/DHF in Nilgiri/Cardamom
hilly areas of south India
Since 1996-97, there have been reports of
sporadic occurrence of DF/DHF cases in
Tamil Nadu and Kerala.
46
DF in hilly regions of Tamil Nadu
During 1998, a team from the National
Institute of Communicable Diseases (NICD),
Delhi, investigated the first-ever reported
outbreak in Coimbatore [8], a town situated
at an altitude varying from 300 to 500
metres on the eastern slopes of the Nilgiri
Hills. The epidemiological characteristics of
the outbreak are summed up below:
•
In all, 20 serologically positive and
compatible to DF/DHF cases were
reported. Eighty percent (16/20) were
children below 10 years and two
patients aged 16 and 5 died of DHF.
•
Fourteen cases were males.
•
Seventeen cases came from Coimbatore
district and three cases from rural areas
of adjoining Erode district.
•
Only five cases came from urban
Coimbatore town and the rest (12
cases) were from rural areas of
Coimbatore district.
•
Rural cases were scattered in distantly
located villages. Clustering of cases and
pyramid phenomenon was conspicuously
absent. No attempt was made to link
urban cases to central or peripheral
wards/zones nor the movement history
of patients two weeks prior to the onset
of fever was investigated.
•
No relationship could be established
between outbreak and increased fever
rate.
•
Eighty-nine percent of blood samples
from healthy contact persons from
urban and rural areas showed dengue
virus IgG antibodies.
•
Entomological investigation recorded
Aedes aegypti in all areas surveyed in
urban and rural areas, but failed to
amplify the infection.
•
DF in Kerala
Epidemiological data
As per investigations undertaken by the
Centre for Research in Medical Entomology
(CRME) [9], the state of Kerala started
reporting DF for the first time in 1997.
Distribution of DF cases are included in
Table 1.
During
the
2003
outbreak
in
Coimbatore town 23 cases of DF were
recorded. Distribution once again
followed the same pattern, i.e. 5 cases
from urban towns and 18 cases from
rural areas (Source: VBDC, New Delhi)
Table 1: Distribution of DF cases in Kerala state during 1997-2001
Year
No. of suspected
cases
No. serologically
positive
Deaths
Districts
1997
116
14
4
Kottayam
1998
67
0
13
Kottayam
1999
1
0
0
Kottayam
2000
0
0
0
Kottayam
2001
877
70
1
4 districts*
* Four districts included Kottayam, Idukki, Ernakulam and Thiruvananthapuram– famous for rubber planatations
Table 2: Results of Aedes Ψ survey of 4 districts of Kerala state during 2001-2003
S.
No.
Name of
district
1.
House index
Container index
%
No.
ex.
No.
+ve
%
Breteau
index
40
26.7
201
63
31.3
42.0
24
10
41.7
54
41
75.9
170.8
Kottayam
70
24
34.2
125
29
23.2
41.4
Kozhikode
3,311
895
26.23
17,912
1,077
6.01
31.57
No.
ex.
No.
+ve
Alappuzha
150
2.
Ernakulam
3.
4.
Remarks
Types of
containers
and sites of
detection of
Aedes
breeding, viz.
domestic/peridomestic;
extradomestic
not mentioned
Ψ
Pool rearing of larval breeding collected from all localities indicated Aedes albopictus as the major species.
Aedes aegypti was encountered in very few places and in scanty numbers (Source: NICD, Delhi, 2004)
47
albopictus, when subjected to IFA test,
showed the presence of dengue antigen,
thereby confirming the transovarial
cycle of the virus [13].
Entomological data
Entomological investigation initiated by a
CRME team in 2 localities of Kottayam
district yielded only Aedes albopictus, and
Aedes aegypti was not detected. Sylvan
environment of rubber plantations was
detected as the unique habitat of the Aedes
albopictus[10,11].
Results of yet another entomological
study carried out by the National Institute of
Communicable Diseases (NICD) field station
located at Kozhikode, in DF affected
districts during 2001-2003, are included in
Table 2.
Antigen detection of dengue virus
During May 2004, a pool of landing
collection of Aedes albopicuts (dessicated),
collected from the fringe of forested villages,
600 metres away from Kozhikode (earlier
known as Calicut) International Airport,
yielded dengue antigen (processed at CRME,
Madurai) [12].
Hypothesis
Occurrence of DF cases in peripheral and
rural areas of Coimbatore and Erode districts
in Tamil Nadu and non-amplification of
infection by Aedes aegypti and sporadic
occurrence of DF cases in Kerala in the
absence of Aedes aegypti points out to
either spillover of enzootic foci of dengue
during periodic amplification of the sylvatic
cycles or occupational hazards in the
presence of vertical transmission as
evidenced by the Kozhikode and Jodhpur
studies.
•
Both the Nilgiri and Cardamom hills are
infested with Macaca radiata, the
bonnet monkeys. Enzootic cycle of
simian malaria caused by Plasmodium
cynomolgi and Plasmodium inui,
transmitted by Anopheles elegans
(Anopheles dirus group) [14,15], has been
detected in the Nilgiri hills. Whereas in
Kerala, a similar simian foci has been
detected at Nilambur district of the
western slope in Macaca radiata, while
in Alappuzha (district in the central
plains, earlier known as Alleppey)
monkeys were found negative for lack
of Anopheles elegans population[16].
•
Both the monkey species, viz. Macaca
mulatta and Macaca radiata have been
found to be highly susceptible to yellow
fever
virus
(flaviviruses)
under
experimental conditions[17]. This lends
support to the susceptibility of these
monkeys to dengue virus as well.
Vertical transmission by
Aedes albopictus
A recent study has been carried out at the
Desert Medicine Research Centre (DMRC),
Jodhpur (Rajasthan), an institution under the
Indian Council of Medical Research, on
possible existence of Aedes albopictus –
monkey – Aedes albopictus cycle. The
highlights of the study included that:
•
In a desert ecosystem, both Aedes
aegypti and Aedes albopictus breed in
tree holes in zoo and monumental
parks, harbouring monkeys, outside the
city limits.
•
Viable eggs retrieved from the soil of
tree holes were reared to adults. Aedes
48
•
•
•
Lack of vectorial competence of Aedes
albopictus in the amplification of urban
dengue epidemic has recently been
demonstrated during the investigation
of the first-ever DF outbreak at
Phuentsholing, Bhutan in 2004[18].
Entomological investigations revealed
that Aedes aegypti occupied domestic
habitats breeding primarily in storage
containers inside houses, while Aedes
albopictus bred in tree holes, 55-gallon
drums and used tyres in the extradomestic habitats. The overlapping
zone was the peridomestic areas where
both species shared breeding in flower
vases/trash.
A
large-scale
source
reduction/larvicidal campaign supported
by deltamethrin fogging in residential
areas largely eliminated Aedes aegypti
and the cases came down to single digits
within a month, while Aedes albopictus
still maintained high larval indices.
Rudnik and Lim [19], while working in
Malaysia, isolated DEN-1, 2 and 3
viruses from monkeys and also
proposed that the rural dengue vector –
Aedes albopictus, may introduce sylvatic
virus into the human population.
Studies in Sri Lanka proved that dengue
virus
causes
epizootics
among
macaques, rather than being enzootic
as observed elsewhere [4].
•
Gubler[20], proposed that at some point
in the past, probably with the clearing
of the forests and development of
human settlements, dengue viruses
moved out of the jungles and into a
rural environment where they were,
and still are transmitted to humans by
peri-domestic mosquitoes such as Aedes
albopictus.
In view of the aforesaid, the land use in
the Nilgiri hills of Tamil Nadu and in the
Cardamom hills in Kerala is under pressure
of deforestation to be replaced with cash
crops. This has brought monkey populations
much closer to human settlements.
Therefore, there is a need for indepth seroepidemiological and entomological studies
with backup of virology support using
molecular tools for genomic sequencing of
viruses obtained from simian and human
sources. Validation of the hypothesis is of
great epidemiological significance as it
would require radical changes in developing
vector control strategies for Aedes
albopictus-transmitted DF.
Acknowledgements
The
author
gratefully
acknowledges
Dr Duane J. Gubler for critically reviewing
the manuscript.
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authorities for control of dengue outbreak
at Phuentsholing, Bhutan (Draft report), 8-9
August 2004, WHO/SEARO, New Delhi.
[19] Rudnik A and Lim TW (Eds.). Dengue fever
studies in Malaysia. Bulletin from the
Institute for Medical Research, Malaysia,
1986, 23: 1-241.
[20] Gubler
DJ.
Dengue
and
dengue
haemorrhagic fever: its history and
resurgence as a global public health
problem. In: Gubler DJ and Kuno G (Eds.)
Dengue and dengue haemorrhagic fever.
Wallingford: CAB International, 1997: 1-23.
Chiou-Feng Lin*, Huan-Yao Lei*, Ching-Chuan Liu**, Hsiao-Sheng Liu*,
Trai-Ming Yeh***, Shun-Hua Chen* and Yee-Shin Lin* #
*Department of Microbiology and Immunology, National Cheng Kung University Medical College,
Tainan, Taiwan
**Department of Paediatrics, National Cheng Kung University Medical College, Tainan, Taiwan
***Department of Medical Technology, National Cheng Kung University Medical College, Tainan, Taiwan
Abstract
Dengue haemorrhagic fever (DHF) is a complicated disease associated with viral and immune
pathogenesis. There is still no effective vaccine to prevent the progression of DHF because of its
undefined pathogenic mechanisms. The generation of autoimmunity in dengue virus (DEN) infection has
been implicated in dengue pathogenesis. Based on our previous studies showing antibodies (Abs) against
DEN nonstructural protein 1 (NS1) cross-reacted with human platelets and endothelial cells, a
mechanism of molecular mimicry may contribute to autoantibody (autoAb) production. Here, the
generation of autoAbs against human endothelial cells in patients infected with different DEN serotypes is
shown. The levels of autoAbs present in different disease stages of DHF and the induction of endothelial
cell apoptosis by patient sera were also determined. The results suggest that autoimmune responses are
implicated in dengue disease pathogenesis and cause concern in vaccine development.
Keywords: Dengue haemorrhagic fever, dengue virus serotype, autoimmunity, autoantibody, endothelial cells.
Introduction
Infection with dengue virus (DEN) causes
dengue fever (DF) – an important
arthropod-borne viral disease in terms of
morbidity and mortality[ 1,2] and may result in
severe dengue haemorrhagic fever and
dengue shock syndrome (DHF/DSS).
Globally, about 2.5 billion people are at risk
of the infection[3]. A recent dengue outbreak
in Indonesia led to a 1.1% case-fatality rate
in 58,301 cases by April 2004[4] . All four
DEN serotypes were present in this outbreak.
#
Presently, the severity of dengue disease is
primarily predicted according to the effect
of antibody-dependent enhancement (ADE)
in different serotype cross-infections[2,5,6]. In
order to effectively control the progression
of the disease, development of an effective
vaccine against DEN infection is needed.
There are several vaccine candidates
undergoing clinical trials[7-10] . Nevertheless,
the role that antibodies (Abs) may play in
increasing
the
severity
of
dengue
[2,7,10]
infections
remains a matter of concern
in vaccine development.
51
In addition to the ADE of DEN infection,
autoantibody (autoAb) production may also
be involved in dengue diseases[11-15] . We
demonstrated that the autoAbs generated in
DEN infection induced endothelial cell
damage[13] and inflammatory activation (in
press). A mechanism of molecular mimicry in
which
Abs
directed
against
DEN
nonstructural protein 1 (NS1) is, at least in
part, responsible for the autoimmunity. The
relationships of the autoAb levels with
dengue serotypes and disease severity are
examined in this study.
Patient sera
DEN-2 and DEN-3 patient sera were
collected during the outbreaks in southern
Taiwan from 1997 to January 1999[16] . DEN4 patient sera were obtained from the
Department of Dengue Hemorrhagic Fever,
Children’s Hospital No. 1, Ho Chi Minh City,
Viet Nam. The disease severity was based on
the WHO definition[3]. Normal control sera
from five healthy individuals were used as
background.
Cell cultures
Human umbilical cord vein endothelial cells
(HUVEC) were cultured in modified M-199
medium as described previously[13] . For
experiments, 1,000 U/ml trypsin and 0.5
mM ethylenediaminetetraacetic acid (EDTA)
were used to detach cells.
Binding activity detection
After detachment, cells were suspended at
5×105 for flow cytometry. The cells were
washed briefly with phosphate-buffered
saline (PBS) and fixed with 1% formaldehyde
in PBS at room temperature for 10 minutes,
then washed again with PBS. Patient sera
52
were 1:25 diluted and incubated with cells at
4 °C for 1 hour. After being washed three
times with PBS, the cells were incubated with
20 µl of fluorescein isothiocyanate (FITC)conjugated anti-human IgG or IgM
(PharMingen, San Diego, CA) at 4 °C for 1
hour. The binding activity of Abs to cells was
analysed using flow cytometry (FACScan; BD
Biosciences, San Jose, CA) with excitation set
at 488 nm.
Cell death detection
For cell viability determination, cells were
stained with eosin Y and counted using light
microscopy. Apoptosis-induced DNA strand
breaks
were
analysed
by
terminal
deoxynucleotidyl
transferase-mediated
dUTP† nick-end-labeling (TUNEL) reaction
using the ApoAlert DNA Fragmentation Assay
Kit (Clontech, Palo Alto, CA). After incubation
with patient sera for 24 hours, endothelial
cells (1×106) were fixed and stained
according to the manufacturer’s instructions,
and then analysed using flow cytometry.
Statistical analysis
The statistical difference was analysed using
unpaired Student’s t-tests in SigmaPlot
version 4.0 for Windows (Cytel Software
Corporation, Cambridge, MA).
Results
Generation of autoAbs in dengue
patients infected with different
serotypes and at different disease
stages
Our previous studies demonstrated the
presence of anti-platelet and antiendothelial cell autoAbs in dengue patient
sera[12,13] . The levels of these autoAbs were
†
deoxyuridine triphosphate
higher in DHF/DSS than in DF patient sera.
The dysfunction of platelets and endothelial
cells caused by the autoAbs was also shown.
The cross-reactivity of patient sera with
endothelial cells was the highest in the acute
stage (3-7 days after fever onset) and
subsequently decreased in the convalescent
(1-3 weeks after acute phase) and later (8-9
months) stages. In our previous study,
patient sera were collected from an
outbreak of DEN-3 infection. In this study,
we further examined the autoAb levels
produced by patients infected with different
DEN serotypes and the relationship
between the autoAb levels and disease
severity. The results showed that the levels
of anti-endothelial cell Abs, as determined
by both the percentages of endothelial cells
reactive with patient sera IgM or IgG and
the mean fluorescence intensity, were
similar in patients infected with DEN -2, 3 or
4 (Table 1). There was no significant
difference between different serotype
infections. The levels of autoAbs were
higher in DHF/DSS than in DF patient sera.
In addition, the levels of IgM isotype of
autoAbs were higher than those of IgG. The
DEN-1 serotype was not tested because we
had no DEN-1-infected patient sera. We
next investigated the endothelial cell crossreactivity of DHF patient sera at different
disease grades. DHF patient sera collected
from Grades I to IV with DEN -4 infection,
according to the WHO definition, were
tested. There was no significant difference
between the four grades of DHF in both
anti-endothelial cell IgM and IgG (Table 1).
Due to our limited sample sizes of patient
sera, especially of Grades I and IV, we were
unable to determine whether there was any
correlation of autoAbs with disease severity.
Table 1. Anti-endothelial cell IgM/IgG levels in the sera of dengue patients infected with
different dengue serotypes and at different disease grades
% of endothelial cells reactive
with patient sera
IgM
IgG
Mean (SD)
Mean (SD)
Mean fluorescence intensity
IgM
Mean (SD)
IgG
Mean (S D)
Normal (n=5)
DEN-2 infection
4.7 (0.5)
2.6 (0.6)
12.8 (1.5)
6.6 (0.8)
DF (n=5)
DEN-3 infection
38.6 (1.4)***
14.8 (5.1)*
62.5 (4.1)***
14.1 (2.1)*
DF (n=6)
DHF/DSS (n=5)
DEN-4 infection
35.1 (3.7)***
54.6 (4.4)***
12.7 (2.1)*
23.7 (1.9)**
65.7 (5.7)***
74.9 (4.6)***
15.7 (1.7)*
24.6 (3.6)**
DF (n=5)
DHF (n=36)
35.9 (1.6)***
50.5 (9.5)***
15.1 (3.5)*
24.9 (8.3)**
66.6 (6.3)***
72.1 (13.9)***
10.0 (2.3)*
11.1 (4.4)*
Grade I (n=1)
Grade II (n=26)
Grade III (n=8)
66.9
51.7 (9.0)***
45.4 (8.6)***
17.8
25.5 (7.7)*
24.7 (10.6)*
83.4
73.6 (10.4)***
64.7 (7.0)***
8.8
11.4 (4.3)*
13.9 (6.4)*
Grade IV (n=1)
42.2
17.9
63.0
11.6
Student’s t-tests: *P<0.05 vs Normal; **P<0.01 vs Normal; ***P<0.001 vs Normal.
53
Induction of endothelial cell
apoptosis by sera of dengue
patients infected with different
serotypes
Anti-endothelial cell autoAbs caused cell
damage which was characterized by
apoptosis [13] . The ability of patient sera with
different DEN serotype infections to induce
endothelial cell apoptosis was tested.
HUVEC were treated with a 1:25 dilution of
dengue patient or healthy-control sera for
24 hours, and cell apoptosis was measured
using TUNEL reaction followed by flow
cytometric analysis. The histogram and the
percentages of apoptotic cells from one set
of duplicate cultures are shown in the Figure
below. The results indicated that cell
apoptosis was induced by all patient sera
and the cells underwent a higher percentage
of apoptosis when induced by DHF patient
sera than by D F patient sera. Healthycontrol sera showed only the background
level. Cell viability detected using eosin Y
staining showed an inverse relationship with
the percentages of apoptosis (Table 2).
There was no significant difference in
endothelial cell apoptosis induced by
patient sera with different serotype
infections.
Figure. Dengue patient sera induced endothelial cell apoptosis
54
Normal
DEN-2 DF
DEN-3 DF
DEN-3 DHF
DEN-4 DF
DEN-4 DHF
Table 2. Endothelial cell apoptosis induced by sera of dengue patients infected with
different dengue serotypes
% of cell viability
Normal (n=5)
% of endothelial cell
apoptosis
94.1 (3.5)
6.9 (2.8)
75.2 (5.4)**
21.9 (5.6)**
81.7 (5.1)**
19.5 (7.1)**
66.6 (10.8)***
29.7 (9.3)***
72.0 (9.5)**
22.0 (5.6)**
63.3 (15.1)***
37.2 (5.5)***
DEN-2 infection
DF (n=5)
DEN-3 infection
DF (n=6)
DHF/DSS (n=5)
DEN-4 infection
DF (n=5)
DHF (n=5)
Student’s t-tests: **P<0.01 vs Normal; ***P<0.001 vs Normal.
Discussion
DHF is a life-threatening disease with poorly
defined pathogenic mechanisms[1,2,5,10] . An
ADE effect in different serotype crossinfection is frequent[2,5,6]. Patients may
develop severe complications of progressive
DHF. Vascular leakage and haemorrhagic
diathesis are the hallmarks in DHF patients. A
number of studies have demonstrated
abnormal immune responses caused by DEN
infection, including cytokine and chemokine
production, complement activation and
immune cell activation[10,11,17-20] . In addition,
autoimmune responses may be involved in
DHF pathogenesis [12-15,21] . Dengue patients
produced Abs which cross-reacted with
human platelets and endothelial cells[12,13] .
Anti-NS1 produced after DEN infection may,
at least in part, account for the crossreactivity of patient sera with endothelial cells.
In this study, we further showed that the
levels of anti-endothelial cell Abs were similar
in patients infected with different DEN
serotypes. The percentages of endothelial
cells reactive with DHF/DSS patient sera
were higher than those with DF patient sera.
However, there was no difference in antiendothelial cell Ab levels at different DHF
disease grades. The sample size of patient
sera needs to be increased to gain an insight
into the role of anti-endothelial cell Abs in
DHF pathogenesis. These autoAbs exerted
similar effects in the induction of endothelial
cell apoptosis of patients infected with
different DEN serotypes.
In dengue pathology, various cytokines
and chemokines including TNF-α, IL-6, IL-8,
and RANTES‡ have been detected in patient
sera with DHF/DSS and in DEN-infected
endothelial cell culture supernatants[17,18,20] .
Our recent studies also demonstrate that
anti-NS1 Abs can stimulate cytokine and
chemokine production (in press). Therefore,
‡
regulated upon activation normal T cell expressed and
secreted
55
both immune activation and apoptosis
occur in endothelial cells after stimulation
by autoAbs.
There are no dengue vaccines available.
Yet, several potential vaccines, including
life-attenuated whole DEN and DNA
vaccines, are undergoing clinical trials[7-10] . It
is hoped that a fusion or a chimera dengue
vaccine will be developed to provide
protection against all serotypes of DEN
infection. In addition, DEN NS1 protein
used as a vaccine candidate in mice showed
resistance to fatal DEN encephalitis [22] .
Passive administration of anti-NS1 Abs also
conferred protection in mice when
challenged with lethal doses of DEN [23] .
However, these previous studies only
monitored the survival rates of mice but did
not examine the potential histopathological
effects. Studies by Falconar[21] showed the
cross-reactivity of anti-NS1 to host antigens
and cells and a haemorrhage-like hallmark
in mice. This, taken together with our
findings, suggests that a potential pathogenic
effect of DEN NS1 vaccine should be taken
into consideration. The possible approaches
include gene modifications of DEN NS1 to
truncate or mutate the epitopes that may
cause the pathogenic effects.
Acknowledgement
We thank Dr N.T. Hung from the
Department of Dengue Haemorrhagic Fever,
Children’s Hospital No. 1, Ho Chi Minh City,
Viet Nam for providing the DEN-4 patient
sera. The authors acknowledge the editorial
assistance of Bill Franke. This work was
supported by grant NSC92-3112-B006-003
from the National Science Council, Taiwan.
References
[1] Gubler
DJ.
Dengue
and
dengue
hemorrhagic fever. Clinical Microbiology
Reviews, 1998, 11: 480-496.
[2] Halstead SB. Dengue. Current Opinion in
haemorrhagic fever: diagnosis, treatment,
prevention and control. 2nd edition.
Geneva: WHO, 1997.
[4] World Health Organization. Dengue fever in
Indonesia-update 4. 11 May 2004.
1988, 239: 476-481.
[6] Halstead SB. Neutralization and antibodydependent enhancement of dengue viruses.
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56
[7] Pang T. Vaccines for the prevention of
neglected diseases – dengue fever. Current
Opinion in Biotechnology, 2003, 14: 332336.
[8] Pugachev KV, Guirakhoo F, Trent DW and
Monath TP. Traditional and novel
approaches
to
flavivirus
vaccines.
International Journal of Parasitology, 2003,
33: 567-582.
[9] Edelman R, Wasserman SS, Bodison SA,
Putnak RJ, Eckels KH, Tang D, KanesaThasan N, Vaughn DW, Innis BL and Sun W.
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tetravalent live-attenuated dengue vaccine.
Hygiene, 2003, 69: 48-60.
[10] Rothman AL. Dengue: defining protective
versus pathologic immunity. Journal of
Clinical Investigation, 2004, 113: 946-951.
[11] Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH
and Liu CC. Immunopathogenesis of dengue
virus infection. Journal of Biomedical
Science, 2001, 8: 377-388.
[12] Lin CF, Lei HY, Liu CC, Liu HS, Yeh TM,
Wang ST, Yang TI, Sheu FC, Kuo CF and Lin
YS. Generation of IgM anti-platelet
autoantibody in dengue patients. Journal of
Medical Virology, 2001, 63: 143-149.
[13] Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS,
Yeh TM, Chen SH and Lin YS. Antibodies
from dengue patient sera cross-react with
endothelial cells and induce damage.
Journal of Medical Virology, 2003, 69: 8290.
[14] Lin YS, Lin CF, Lei HY, Liu HS, Yeh TM,
Chen SH and Liu CC. Antibody-mediated
endothelial cell damage via nitric oxide.
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213-221.
[15] Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM,
Chen SH, Liu CC, Chiu SC and Lin YS.
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antibodies
against
dengue
virus
nonstructural protein 1 via production of
nitric oxide. Journal of Immunology, 2002,
169: 657-664.
[16] Lei HY, Huang JH, Huang KJ and Chang C.
Status of dengue control programme in
Taiwan - 2001. Dengue Bulletin, 2002, 26:
14-23.
[17] Rothman AL and Ennis FA. Immunopathogenesis of dengue hemorrhagic fever.
Virology, 1999, 257: 1 -6.
[19] Lin YW, Wang KJ, Lei HY, Lin YS, Yeh TM,
Liu HS, Liu CC and Chen SH. Virus
replication and cytokine production in
dengue virus-infected human B lymphocytes.
Journal of Virology, 2002, 76: 12242-12249.
[20] Hung NT, Lei HY, Lan NT, Lin YS, Huang KJ,
Lien LB, Lin CF, Yeh TM, Ha DQ, Huong
VTQ, Chen LC, Huang JH, My LT, Liu CC
and Halstead SB. Dengue hemorrhagic fever
in infants: a study on clinical and cytokine
profiles. Journal of Infectious Diseases, 2004,
189: 221-232.
[21] Falconar
AK.
The
dengue
virus
nonstructural-1 protein (NS1) generates
antibodies to common epitopes on human
blood clotting, integrin/adhesin proteins and
binds to human endothelial cells: potential
implications
in
haemorrhagic
fever
pathogenesis. Archives of Virology, 1997,
142: 897-916.
[22] Zhang YM, Hayes EP, McCarty TC, Dubois
DR, Summers PL, Eckels KH, Chanock RM
and Lai CJ. Immunization of mice with
dengue structural proteins and nonstructural
protein NS1 expressed by baculovirus
recombinant induces resistance to dengue
virus encephalitis. Journal of Virology, 1988,
62: 3027-3031.
[23] Henchal EA, Henchal LS and Schlesinger JJ.
Synergistic
interactions
of
anti-NS1
monoclonal antibodies protect passively
immunized mice from lethal challenge with
dengue 2 virus. Journal of General Virology,
1988, 69: 2101-2107.
[18] Avirutnan P, Malasit P, Seliger B, Bhakdi S
and Husmann M. Dengue virus infection of
human endothelial cells leads to chemokine
production, complement activation, and
apoptosis. Journal of Immunology, 1998,
161: 6338-6346.
57
Inhibition of the NS2B-NS3 Protease – Towards a
Causative Therapy for Dengue Virus Diseases
Gerd Katzenmeier#
Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University,
Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand
Abstract
The high impact of diseases caused by dengue viruses on global health is now reflected in an increased
interest in the identification of drug targets and the rationale-based development of antiviral inhibitors
which are suitable for a causative treatment of severe forms of dengue virus infections – dengue
haemorrhagic fever and dengue shock syndrome. A promising target for the design of specific inhibitors is
the dengue virus NS3 serine protease which – in the complex with the small activator protein NS2B –
catalyses processing of the viral polyprotein at a number of sites in the nonstructural region. The NS3
protease is an indispensable component of the viral replication machinery and inhibition of this protein
offers the prospect of eventually preventing dengue viruses from replication and maturation. After nearly
a decade of mainly genetic analysis of flaviviral replication, recent studies have contributed substantial
biochemical information on polyprotein processing including the 3-dimensional structure of the dengue
virus NS3 protease domain, the mechanism of co-factor-dependent activation and sensitive in vitro
assays which are needed for studies on substrate specificity and the development of high-throughput
assays for inhibitor screening. This review discusses recent biochemical findings which are relevant to the
design of potential inhibitors directed against the dengue virus NS3 protease.
Keywords: Dengue virus, NS2B/NS3, polyprotein, protease, inhibitor, treatment.
Viral polyprotein processing
Dengue viruses, members of the Flaviviridae
family, possess single-stranded, positive
sense RNA genomes and generate mature
viral proteins by co- and post-translational
proteolytic processing of a polyprotein
precursor catalysed by host cell and virusencoded proteases [for review see refs. 1, 2
and references herein]. The genomic RNA
of dengue virus serotype 2 contains 10,723
#
nucleotides
and
encodes
a
single
polyprotein precursor of 3,391 amino acid
residues[3] . Individual viral proteins are
arranged in the order C-prM-E-NS1-NS2ANS2B-NS3-NS4A-NS4B-NS5.
Proteolytic
cleavages in the N-terminal region of the
viral polyprotein are mediated by a host
signal peptidase and yields three structural
proteins C, prM and E, which constitute the
virion[4] . Before the virion exits the cell, prM
is cleaved by a cellular furin-type
E-mail: [email protected]; Tel.: (662)-800-3624-1237; Fax: (662)-441-9906
58
Inhibition of NS2B/NS3 Protease
prohormone convertase in the post-Golgi
acidic compartment to yield the M protein[5] .
Cleavages
at
the
NS1/NS2A
and
NS4A/NS4B junctions are catalysed by a
signalase bound to the membranes of the
ER [6,7] . Proteolytic cleavages in the
nonstructural region of the polyprotein are
mediated by a heterodimeric complex of
NS3 with the activator protein NS2B which
catalyses in cis (intramolecular) cleavages at
the NS2A/NS2B and NS2B/NS3 sites and in
trans (intermolecular) cleavages at the
NS3/NS4A and NS4B/NS5 polyprotein
junctions[8-10] .
Additional
cleavages
mediated by the NS3 protease within the C,
NS2A, NS4A and within a conserved Cterminal portion of NS3 itself have been
described in the literature [11-13] .
Figure 1. The 3-dimensional structure of
the dengue virus NS3 protease
(shown here is a superimposition of a
ribbon-presentation of the Cα trace on a
space-filling surface model. Residues of
the catalytic triad (His51, Asp75, Ser135)
are shown as sticks. The figure was
generated by Deepview Swiss-Pdb Viewer)
Ser135
His51
Asp75
Cleavage sites recognized by the
NS2B/NS3 protease consist of ‘dibasic’
residues Lys-Arg, Arg-Arg and Arg-Lys at the
(nonprime) P1 and P2 positions of the
cleavage site sequence followed by short
chain residues such as Gly, Ala and Ser at
the (prime) P1’ position. The “noncanonical” NS2B/NS3 site contains a Gln
residue at the P2 position (Figure 1).
The NS3 protease domain
The existence of a trypsin-like serine
protease domain in the N-terminal region of
the flaviviral NS3 proteins was originally
predicted by sequence comparisons between
cellular and virus-encoded proteases[14]. The
NS2B-NS3 endopeptidases of the Flavivirus
genus which at present comprises at least 68
known members, are now commonly
designated as flavivirin (EC 3.4.21.91) [15,16] .
The dengue virus 69 kDa NS3 protein is a
multifunctional protein with a serine
protease domain located within the Nterminal 167 amino acid residues[17] and
activities of a nucleoside triphosphatase
(NTPase) and RNA helicase in the Cterminal moiety[18] . A catalytic triad
consisting of residues His51, Asp75 and
Ser135 was identified by site-directed
mutagenesis experiments and replacement
of the catalytic serine by alanine resulted in
an enzymatically inactive NS3 protein[19] .
The NS3 protease is an essential component
for maturation of the virus and viable virus
was never recovered from infectious cDNA
clones carrying mutations in the NS3
sequence
which
abolished
protease
activity[20] . Interaction of the helicase
portion of NS3 with the viral RNAdependent RNA polymerase NS5 may
promote the association of the viral
replicase complex to the membranes of the
ER [21] .
59
Figure 2. Schematic of dengue virus polyprotein processing
(shown here are sites on the flavivirus polyprotein cleaved by host-encoded proteases and
the virus-encoded two-component protease NS2B-NS3)
C
prM
E
NS4BNS5
NS1 NS2A
NS2BNS3 NS4A
NS2B/NS3 protease
Signal peptidases
Furin protease
Unidentified protease
The 3-dimensional structure of the Nterminal 185 residues of the dengue virus
NS3 protease domain (NS3pro) was
resolved at a resolution of 2.1 Å[22] . The
overall folding of the protein resembles the
6-stranded β-barrel conformation typical for
chymotrypsin-like
serine
proteases.
Interestingly, the structure of the dengue
virus NS3 protease is closer to that of the
hepatitis C virus NS3/NS4A co-complex
than to the unliganded HCV NS3 protease,
an observation which is suggestive of major
structural differences in the co-factordependent activation mechanism of the two
proteases[23] . The substrate binding site of
NS3pro is relatively shallow and contourless
and specific enzyme-substrate interactions
were not predicted to extend beyond the
P2 and P2’ positions of the substrate
peptide in the absence of the NS2B cofactor[22] (Figure 2).
The NS2B co-factor
The presence of a small activating protein or
co-factor is a prerequisite for optimal activity
of the flaviviral NS3 proteases with their
60
natural polyprotein substrates. Although the
dengue virus NS3 protease exhibits NS2Bindependent activity with model substrates
for serine proteases, enzymatic cleavage of
dibasic peptides is markedly enhanced with
the NS2B-NS3 co-complex and the
presence of the NS2B activation sequence is
indispensable
for
the
cleavage
of
polyprotein substrates in vitro[24] . The initial
characterization
of
the
co-factor
requirement for the dengue virus NS3
protease had revealed that the minimal
region necessary for protease activation was
located in a 40-residue hydrophilic segment
of NS2B[25] . The hydrophobic flanking
regions of the 14 kDa NS2B protein are
likely to be involved in targeting the
protease complex to the membranes of the
ER where genome replication occurs. Fusion
of the NS2B core sequence to the NS3
protease domain yielded a catalytically
active NS2B(H)-NS3p protein, which, upon
expression in E. coli and subsequent
refolding,
displayed
autoproteolytic
processing at the NS2B/NS3 site conducive
to the formation of a non-covalent adduct[24] .
Incorporation of a flexible nonamer linker,
Gly4 -Ser-Gly4, between the NS2B core
segment and the protease domain resulted
in a cleavage-resistent protease with
optimized
enzymatic
activity
against
hexapeptide substrates representing native
polyprotein
cleavage
junctions[26] .
A
recombinant construct representing the fulllength NS2B co-factor linked to the NS3
protease domain was enzymatically active
with peptide substrates derived from the
polyprotein; however, this protein was
completely resistant to proteolytic selfcleavage[27,28] .
An essential requirement for the correct
association of the co-factor with the
protease is the presence of hydrophobic
residues which act as ‘anchor’ for the
protease – co-factor interaction. Recently,
based on mutagenesis experiments and
sequence comparisons of known flaviviral
co-factors, the “Φx3Φ” - motif was proposed
as the common structural element involved
in co-factor binding to the protease[29] . The
“Φx3Φ” - motif is comprised of two bulky
hydrophobic residues separated by three
unspecified
residues
and
it
was
hypothesized that additional residues
located at the N-terminus of the activation
sequence would contribute to the stringent
specificity of the protease for the
polyprotein substrate. A mutagenesis study
with the dengue virus NS2B co-factor had
revealed that substitutions of the “Φ” residues (Leu75 and Ile79 in NS2B of
dengue virus type 2) by alanine resulted in
preponderant effects on the catalytic activity
of the NS3 protease rather than on substrate
binding [P. Niyomrattanakit, unpublished
data]. A single residue in the N-terminal
region of NS2B, Trp62, was critical for
protease activation and replacement of this
residue yielded a NS3 protease which was
catalytically inactive in autoproteolysis and
reaction with the synthetic substrate peptide
GRR-AMC.
For the HCV NS3-NS4A protease
complex, large structural rearrangements
leading to a catalytic triad, which is
conformationally optimized for proton
shuttle during catalysis, were observed as a
result of co-factor binding[30] . No 3dimensional structure is available for the
dengue virus NS2B-NS3 co-complex and
the precise mechanism of co-factordependent activation is not fully elucidated
as yet. In particular, it is an open question as
to whether binding of the substrate
contributes to the formation of an ‘induced
fit’ conformation as observed with the HCV
NS3 protease[31,32] .
Substrate specificity
So far, only very limited efforts have been
undertaken to analyse the precise substrate
requirements and determinants of cleavage
efficiency for the dengue virus NS3 protease.
This is surprising in the light of the fact that
development of inhibitors against serine
proteases usually starts with optimal peptide
substrates derived from the nonprime side
wherein the scissile amide bond is replaced
by an electrophile which reacts with the
catalytic serine residue.
The NS3 protease reacts with small
model substrates for serine proteases such as
N-α-benzoyl-L-arginine-p-nitroanilide (BAPA)
and activity of the unliganded NS3 protease
towards this substrate is higher than that of
the NS2B-NS3 co-complex[24] , a finding
which suggests that substrate recognition in
the complex requires additional interactions
extending beyond the P1 side for optimal
activity. A number of fluorogenic tripeptides
containing dibasic residues at the P1 and P2
61
positions are cleaved by NS2B(H)-NS3pro
protease and the best substrate identified in
these experiments was GRR-AMC, which
had a Km value of 180 µM, a kcat value of
0.031 s-1 and a catalytic efficiency expressed
as kcat / Km of 172 s-1 M-1 [24] .
Chromogenic p-nitroanilide peptides
representing hexameric sequences of the
native polyprotein cleavage junctions were
tested in a photometric assay and the most
efficiently cleaved substrate derived from
the NS4B/NS5 site (Ac-TTSTRR-pNA) had a
Km of 346 µM, kcat of 0.095 s-1 and a kcat / Km
of 275 s-1 M-1 [26] .
Recently, we have shown by a HPLCbased assay with fluorometric detection that
the NS2B-NS3pro protease incorporating a
full-length NS2B co-factor could cleave Nterminally dansylated 12mer peptides
mimicking native polyprotein junctions in
the absence of microsomal membranes[28] .
However, this protein was completely
inactive in autocleavage and the efficiency
of this recombinant protease with the
peptide substrate was markedly reduced
when compared to constructs incorporating
the 40-residue NS2B core sequence, likely
due to structural distortions induced by the
flanking regions of NS2B and inefficient
activation of the protease.
Currently, a detailed study on substrate
specificity of the dengue virus NS3 protease
is in progress, which uses combinatorial
libraries of internally quenched fluorogenic
peptides labelled with the aminobenzoyl /
p-nitrotyrosine reporter pair. For a substrate
peptide based on the NS3/NS4A cleavage
site,
Abz-AAGRK?SLTLY(NO2)R-NH2 (?
denotes the cleavage site), a Km value of 141
µM, a kcat of 0.18 s-1 and a kcat / Km of 1262
s-1 M-1 was found, which is approximately
62
10-fold better than the best commercially
available substrate tested so far, GRR-AMC
[J. Wikberg, unpublished data]. In the near
future, these investigations will likely lead to
the identification of NS3 substrates with
optimized sequence length and improved
binding affinities, which can be applied as
sensitive probes for enzyme activity in highthroughput inhibitor screenings.
Perspectives for inhibitor
development
Principally,
every
step
of
viral
morphogenesis, from cell-entry, uncoating,
replication and assembly of new virus
particles, is a potential target for antiviral
inhibitors. However, the molecular events in
the infectious cycle of flaviviruses such as
dengue virus are characterized only to a
very limited extent, making the design of
specific inhibitors an adventurous task. In
contrast, proteases and their inhibitors have
been intensively studied because of their
potential for the development of selective
antiviral compounds. Although tremendous
progress in the field is indicated by the
design and clinical use of antiviral drugs
against HIV (AIDS) and hepatitis C virus
proteases, the potential of dengue and
related flaviviral proteases for inhibitor
discovery is largely unexploited. In response
to the global problem of the dengue virus
epidemics, considerable efforts are now
being devoted to the development of drugs
which will eventually be suitable for a
chemotherapeutic intervention in acute
dengue diseases, not only by academic
institutions but also by pharmaceutical
companies
(for
example
see
http://www.nitd.novartis.com).
In a first step towards a rational inhibitor
design for the dengue virus NS3 serine
protease, inhibition by synthetic peptides
mimicking uncleavable transition state
isosteres of the P6-P2’ residues of the native
polyprotein sites, was demonstrated[26] . The
peptides with an α-keto amide in place of
the scissile amide bond acted as competitive
inhibitors of the NS3 protease with Ki values
in
the
micromolar
range.
Replacement of the P1’-P2’ residues by a
carboxyl-terminal
aldehyde
in
the
NS3/NS4A-derived peptide (Ac-FAAGRRCHO) yielded a competitive inhibitor with a
Ki of 16 µM. For the dengue virus protease,
the hexapeptides displayed Ki - values which
were only 2- to 6-fold lower than the Km value for the corresponding substrate, a
feature which discriminates dengue virus
NS3 from the HCV protease, where product
inhibitors had binding affinities which were
one order of magnitude lower than those for
the substrates[33,34] .
Product inhibition of the HCV NS3
protease by cleavage-site derived peptides
led to the discovery of very potent inhibitors
of this enzyme with IC50 – values in the
nanomolar range by cyclic optimization of
the inhibitor structures[33,34] . Recently, we
have shown that peptides representing nonprime-side residues of the dengue virus NS3
protease act as competitive inhibitors of the
enzyme, whereas prime-side peptides
appeared to have negligible effects on
enzyme inhibition at concentrations >1.0
mM. (S. Chanprapaph, unpublished data). Ki
- values for hexapeptides derived from all 4
dengue virus cleavage sites were in the low
micromolar range and the best inhibitor was
based on the NS2A/NS2B site (Ac-RTSKKRCONH2) and gave a Ki of 12 µM. In analogy
to the HCV NS3 protease, these findings
suggest the existence of a high-affinity
binding site in the non-prime region of the
enzyme and offer the prospect of
developing effective inhibitors against the
dengue virus protease by combinatorial
optimization based on the structure(s) of
native polyprotein cleavage site peptides.
However,
in
general,
peptide-based
inhibitors exhibit poor pharmacokinetic
properties and usually the conversion of
these structures into less “peptide-like”
compounds (“peptidomimetics”) is required
to generate drug-like entities.
Inhibitor discovery for the dengue virus
NS3 protease is currently limited by the lack
of a 3-dimensional structure for the NS2BNS3 co-complex; however, it can be
expected that crystallographic studies and
NMR-experiments will provide more insight
into the structure and catalytic mechanism of
the enzyme in the near future. In addition,
powerful computer modelling approaches
exist which may help to obtain information
required for a rational drug design even in
the absence of crystallographic structures.
Proteochemometric modelling (PCM) is
currently explored as a tool to analyse the
structural and physicochemical properties
which are necessary for the interaction of
potential inhibitors with the dengue virus
NS3 protease target structure [35] . Preliminary
data obtained by this approach suggest that
the proteochemometric models are valid
and useful for the accelerated design of
novel inhibitors. This approach does not
only circumvent the traditional erratic dug
discovery process with its high attrition rates,
but also allows to incorporate potential
resistance of the target and the development
of ‘drug resistance – resistant’ compounds as
an initial consideration in the design process.
The existence of large conformational
63
ensembles is particularly challenging in the
case of rapidly mutating viral enzymes,
where a design against a moveable target
would require large sets of corresponding
inhibitors. In the future, these problems are
likely to be addressed by ‘shotgun
approaches’ to the structure of enzymeinhibitor complexes and the identification of
hot spots of ‘interaction flexibility’ by the
use of fast, high-resolution methods such as
NMR. Detailed accounts on this strategy are
given in Reference 36.
Conclusions
Substantial progress has been made over the
past few years in the biochemical
characterization of the dengue virus NS2BNS3 two-component protease. The data,
which are available now, make the dengue
virus NS3 protease a valid molecular target
for the development of antiviral compounds.
A large repertoire of powerful methods for
inhibitor development and evaluation exists
which includes state-of-the-art technology in
organic synthesis and computer-aided
molecular design. Although there is no
suitable animal model available for dengue
virus diseases, initial screening of potential
antiviral compounds would be facilitated by
well-established insect and mammalian cell
culture systems which are useful to monitor
the effects of anti-NS3 inhibitors on the
propagation of the virus.
Moreover, alternative drug targets
which are present on the dengue virus NS3
protein can be exploited for inhibitor
development. These include the binding site
of the NS2B co-factor to the NS3 protease,
the NS3 NTPase / helicase portion and the
interaction surface of NS3 with the NS5
replicase. The presence of multiple
64
biomedical targets in the dengue virus
polyprotein would even make a therapy
feasible, which uses combinations of
different inhibitors and therefore could
minimize the risk of rapid resistance
development.
It can also be expected that progress for
inhibitors against the dengue virus protease
will be of large benefit for drug design
against related human pathogens of the
flavivirus complex such as yellow fever virus,
Japanese encephalitis virus and West Nile
virus.
However, in order to bring an effective
anti-dengue drug from the ‘bench to the
bedside’, several questions and limitations
need to be addressed. These include the
evaluation of prognostic markers for disease
severity, the pathobiochemistry of dengue
haemorrhagic fever and shock syndrome,
the
problem
of
selectivity
against
pharmacologically relevant human proteases
such as furin and the risk of adverse effects.
Potential complications may also arise from
the presence of four related dengue
serotypes in the case that their NS3
proteases show marked differences in their
inhibition profiles. Intensive efforts and
sustained multidisciplinary research is
required in the future to cope with the
challenging task of a causative treatment for
dengue virus diseases.
Acknowledgements
We thank Dr J. Wikberg, Department of
Pharmaceutical
Biosciences,
Uppsala
University, Sweden, for intensive discussions
and access to data prior to publication. This
work was supported by grants from the
Thailand Research Fund (TRF).
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67
Prognostic Factors of Clinical Outcome in
Non-Paediatric Patients with
Dengue Haemorrhagic Fever/Dengue Shock Syndrome
Jaime R. Torres*#, José M. Torres-Viera**, Hipólito García**, José R. Silva**,
Yasmín Baddour**, Angel Bajares** and Julio Castro M.*
*Tropical Medicine Institute, Infectious Diseases Section, Universidad Central de Venezuela
**Clínica Santa Sofía, Caracas, Venezuela
Abstract
A total of 112 adults with dengue haemorrhagic fever (DHF) admitted at Clínica Santa Sofía, Caracas,
Venezuela, were studied during June 1998–June 2001. Capillary leakage (CL) occurred in 28.8% cases,
21.6% experienced bleeding, 9.2% developed pleural effusion (PE) and 9.2% developed acute
acalculous cholecystitis (AAC). High correlation was noticed between the length of illness prior to
admission (IPA) and length of hospitalization (LH) and levels of Hct, Hb and leukocytes. Significant
differences were seen in the length of IPA, LH and level of platelets for patients with or without bleeding
(P<0.05) or CL (P<0.005), and in LH for patients with or without PE (P<0.005), or CL (P<0.05).
Patients with AAC reached higher leukocyte counts (P<0.05). ANOVA showed an association between
IPA and LH, between either of them and levels of Hb, Hct or leukocytes, and between platelets levels
and PE, CL or bleeding. The length of IPA and degree of alteration of Hb, Hct, leukocytes and platelets
predicted a more severe course in adults with DHF.
Keywords: Dengue, dengue haemorrhagic fever, adults, prognostic factors.
Introduction
Over the last decade, dengue fever has
dramatically spread in virtually all Latin
American and Caribbean countries which
are infested with Aedes aegypti. During this
period, the number of cases reported every
year in these countries jumped from
250,000 to more than 750,000[1] .
Furthermore, recent serological surveys
suggest the occurrence of millions of such
infections[2] . After its emergence in Cuba in
1981[3] , epidemics or sporadic cases of
dengue haemorrhagic fever (DHF) have
#
been reported in at least 25 countries in the
Americas [4] . Venezuela has recorded large
numbers of DHF cases every year, and, in
1995, the country reported the largest
outbreak in the region with almost 30,000
dengue cases and 5,000 DHF cases.
Although DEN-1, DEN -2 and DEN -4 had
been isolated during this epidemic, DEN-2
was the predominant serotype [5] .
In contrast with observations made in
Asian countries, where DHF is almost
completely restricted to young children, in
the Americas, older age groups are widely
involved[6- 8] .
E-mail: [email protected]; Fax: + (58-212) 9876590
68
Prognostic Factors in Adult Patients with Haemorrhagic Dengue
The host’s immune response appears to
be a major factor influencing the type and
severity of disease, as sequential infection
with different dengue virus serotypes in the
presence of non-neutralizing antibodies has
been
strongly
incriminated
in
the
occurrence of DHF/DSS[8-10] , and cases of
DHF/DSS are seldom documented in
patients
with
primary
infection[11-13] .
Individual factors, such as age, sex, genetic
background and underlying diseases, may
also play a role[14] .
Since most of the currently available
clinical and epidemiological data on
DHF/DSS derives from observations on
infected children, information is sparse
regarding prognostic factors of poor
evolution among adult patients. In an
attempt to identify potential prognostic
factors in this specific setting, a retrospective
study was carried out among non-paediatric
inpatients with DHF/DSS followed at a
single South American private medical
institution.
A total of 112 non-paediatric patients
(male/female ratio: 64/48; age range: 15-92
years; median 36 years) admitted to Clínica
Santa Sofía, Caracas, Venezuela, during a
36-month period from June 1998 to June
2001, who were attended to by the same
team of physicians, were included in the
study. The endemic dengue transmission
season in the country typically extends from
May to October, matching the yearly cycle
of rains.
All cases
of DHF/DSS,
definitions:
evidence of
fulfilled the diagnostic criteria
according to WHO and PAHO
acute febrile illness with
bleeding, thrombocytopenia
<105 per µL, and evidence of plasma
extravasation, such as haemoconcentration
(20% increase over base Hct, or 20%
decrease after rehydration), polyserositis, or
hypoproteinemia, with or without signs of
circulatory failure, including narrowing of
pulse pressure (≤ 20 mm Hg), hypotension,
or shock[15] . The level of severity of the
condition was established according to the
following scale:
(i)
Grade I, fever + nonspecific
constitutional symptoms + positive
tourniquet test + evidence of
haemoconcentration and thrombocytopenia;
(ii) Grade II, all of the above +
spontaneous
bleeding,
usually
restricted to the skin ± other sites;
(iii) Grade III, all of the above +
circulatory failure manifested by
rapid and weak pulse, narrowing of
pulse pressure (20 mm Hg or less),
or hypotension with the presence
of cold, clammy skin, and
restlessness or agitation, and
(iv) Grade IV, all of the above +
profound shock with undetectable
blood pressure and pulse[15] .
Clinical evidence of gross capillary
leakage (CL) was defined as the occurrence
of polyserositis, expressed by any of the
following: symptomatic pleural effusions,
ascytis, pericardial effusions, gallbladder wall
edema and/or acute acalculous cholecystitis.
Acute acalculous cholecystitis (AAC) was
diagnosed according to the following
criteria: fever; persistent abdominal pain;
nausea and vomiting. On physical
examination, occurrence of tenderness or
muscle rigidity in the right upper abdominal
quadrant, epigastrium, or both, and
69
Murphy's sign. Additional relevant findings
were a palpable mass in the region of the
gall-bladder, jaundice, and mild elevations
in the serum levels of bilirubin, alkaline
phosphatase, and/or transaminases. On
ultrasound, demonstration of an enlarged
gall-bladder with thickened wall (≥ 6 mm)
and pericholecystic fluid appearing as a halo,
or the presence of a diffuse, homogeneous,
non-shadowing, medium level echogenicity
within the gall-bladder lumen, were all
considered ‘positive’ findings[16] .
Either
specific
IgM
or
IgG
seroconversion over a period of 15 days was
documented by means of a rapid
commercial
qualitative
immunochromatographic test (PanBio Dengue ®,
Windsor, Australia).
Statistical analysis was performed using
a StatSoft, Inc. (1995), STATISTICA for
Windows, Computer programme manual,
Tulsa, OK. Either Student’s t-test for the
comparison of means from independent
samples of unknown variances, Pearson’s
correlation
analysis,
univariate
and
multivariate logistic regression analysis was
performed with CAA, vascular leakage or
bleeding as main outcomes, by means of a
forward stepwise independent variable entry
in the final model. All calculations were
two-tailed, and 0.05 significant criteria were
used. ANOVA analysis of variables was
performed as required.
Results
Out of 112 patients, 71 (63.4%) developed
DHF grade I, 23 (20.5%) had DHF grade II,
and 18 (16.1%) had either DHF grade III or
grade IV. In 37 patients (33%), clinical signs
of vascular leakage were evident, 25
(22.3%) experienced moderate to severe
70
bleeding, and 14 (12.5%) developed pleural
effusion on plain chest X-ray films. AAC
ensued in 14 (12.5%) cases. No deaths
occurred (Table 1).
Table 1. Degree of severity of DHF and
clinical complications in 112 non-paediatric
Venezuelan patients
Severity of
disease and
clinical
complication
Number
of cases
Percentage
(%)
DHF grade I
71
63.4.9
DHF grade II
23
20.5
DHF grade III
or IV
18
16.1
Bleeding
25
22.3
Pleural effusion
14
12.5
Acalculous
cholecystitis
14
12.5
Pearson’s correlation analysis results
according to clinical complications are
depicted in Table 2. A significant correlation
was found between the levels of Hb and
Hct (r=0.762; P<0.0001), and between the
level of platelets and Hb (r=-0.280;
P<0.01). Correlation was also observed
between the number of days of illness prior
to admission (IPA) and the length of
hospitalization (r=-0.233; P<0.05), as well
as between the length of hospitalization
(LH) and the degree of alteration in platelets
level (r=-0.198; P<0.05). A higher
correlation was seen between the length of
hospitalization and the number of days of
IPA (r=-0.426; P<0.005) for patients with
dengue grade II or higher, or those with
bleeding (r=-0.427, P<0.005).
Significant differences were seen in the
length of IPA (mean 4.32 days vs. 3.60 days;
P<0.05), and the minimum level of platelets
(mean 43,000 per µL vs. 65,816 per µL;
P<0.05) for patients with or without clinical
bleeding, as well as those with or without
dengue type 2 (mean 4.43 vs. 3.56 days,
and 48,870 vs. 70,800 per µL; P<0.05,
respectively). Significant differences were
also found in the length of hospitalization
(mean 5.77 days vs 3.61 days; P<0.005) for
patients with or without pleural effusion, as
well as for those with or without clinical
vascular leakage (mean 4.85 days vs. 3.53
days; P<0.005). The length of IPA was
longer (mean 4.35 days vs. 3.26 days;
P<0.05), the length of hospitalization was
longer (mean 4.85 days vs. 3.61 days;
P<0.001), the serum levels of alkaline
phosphatase were higher (mean 183 µ/L vs.
91 µ/L; P<0.05), and the minimum level of
leukocytes was higher (mean 12,890 per µL
vs. 5,026 per µL; P<0.001) in patients
developing vascular leakage. Patients with
AAC exhibited a significantly longer hospital
stay (4.71 days vs. 3.71 days; P<0.05), and
a higher mean level of peripheral blood
leukocytes (15,986 x mm3 vs. 5,071 x mm3;
P<0.001).
Table 2. Significant differences in disease outcome in 112 non-paediatric Venezuelan patients
with dengue haemorrhagic fever according to type of clinical complication
Clinical complication
Bleeding
Variable
Mean value
Significance
Yes
No
Illness prior to admission
4.66 days
3.70 days
P<0.05
Yes
No
Minimum platelet level
43,619 per µL
61,128 per µL
P<0.05
Pleural
effusion
Yes
No
Length of hospitalization
5.0 days
3.6 days
P<0.005
Signs of
capillary
leakage
Yes
No
Length of hospitalization
4.24 days
3.53 days
P<0.05
Yes
No
Illness prior to admission
4.71 days
3.56 days
P<0.001
Yes
No
Minimum platelet level
45,178 per µL
68,784 per µL
P<0.01
Yes
No
Peripheral blood leukocytes
11,300 per µL
3,418 per µL
P<0.05
AAC
Both one-way and multivariate logistic
regression analysis revealed that the only
variables significantly associated with
bleeding were illness severity on admission
according to WHO scale (OR=5.3;
P<0.001), and length of IPA (OR=1.7;
71
P<0.05). Vascular leakage was also
associated with illness severity on admission
in the multivariate regression analysis
(OR=14.3; P<0.005), as well as with the
length
of
hospitalization
(OR=1.79;
P=0.001), illness severity on admission
(OR=26.5; P=0.001), leukocytes level
(OR=1.33; P=0.001), and a prolonged
aPTTA† (OR=18.9; P=0.001), in the
univariate regression analysis. Of note was
the finding that whereas patients with
bleeding did not remain hospitalized longer,
those with vascular leakage did (OR=1.79;
P=0.001). Statistically significant results for
the one-way and multivariate logistic
regression analysis are summarized in Tables
3, 4 and 5.
OR = odds ratio
Blank cells indicate values that should not be included
since they are not significant
Table 3. Logistic regression analysis of
clinical variables associated significantly
with capillary leakage in 112 non-paediatric
Venezuelan patients with dengue
haemorrhagic fever
clinical variables significantly associated with
acute achalculous cholecystitis (AAC) in 112
non-paediatric Venezuelan patients with
dengue haemorrhagic fever
Variable
Univariate
Multivariate
OR
OR
P
Length of
hospitalization
1.79 0.001
>0.05
Blood leucocytes
level
1.33 0.001
>0.05
Abnormal PTT
18.9 0.001
>0.05
Bleeding
3.51 0.015
>0.05
OR = odds ratio
Activated partial thromboplastin time
72
Univariate
Variable
OR
P
Multivariate
OR
P
Degree of severity
5.85
0.001 5.3
0.01
Length of illness
prior to admission
1.37
0.032 1.7
0.13
Platelets (nadir)
0.99
0.015
>0.5
Platelets <50.000
3.83
0.005
>0.05
Pleural effusion
6.3
0.02
>0.05
Univariate
Variable
Multivariate
OR
P
Degree of severity
50.5
0.001
Blood leucocytes
level
1.58
0.001 1.34 0.04
Abnormal PTT
1.81
0.001 11.3 0.01
Pleural effusion
9.75
0.001
P
Degree of severity 26.5 0.001 14.3 0.002
†
clinical variables associated significantly with
bleeding in 112 Venezuelan patients with
dengue haemorrhagic fever
OR
P
>0.05
>0.05
OR = odds ratio
Discussion
The age distribution for DHF cases in the
Americas differs from that observed in
South-East Asia[1,3,6,7,8,17,18] , where young
children continue to be the age group
almost exclusively affected. In contrast, an
age range of 31 to 45 years has been
reported for Brazilian patients with
DHF/DSS[6] , while in Puerto Rico, the mean
age of the patients reported in 1990-91 was
38 years [7] . Furthermore, during the
outbreaks in Cuba in 1981 and in
Venezuela in 1989, about one third of the
deaths were among patients older than 14
years [8,19] , and in the 1997 Cuban outbreak,
all registered deaths were seen among
adults[8] .
The main pathogenic feature of dengue
is an increase in vascular permeability
leading to loss of plasma from blood vessels,
which causes haemoconcentration, low
blood pressure and shock. This may also be
accompanied by haemostatic abnormalities
such as thrombocytopenia, vascular changes
and coagulopathy[15] .
The clinical spectrum of dengue virus
infection may range from an asymptomatic
infection to a severe and rapidly fatal
disease[15,20-24] . The most severe end of the
spectrum of dengue virus infection in
children is represented by dengue shock
syndrome (DSS) [15] . Adults seem less likely
than children to suffer from DSS. Indeed, in
a retrospective study of 108 adult
Malaysians with DHF, the overall morbidity
was significant (29.4%) but the case-fatality
rate remained low (2.0%)[25] .
Haemorrhagic manifestations in DHF
usually consist of petechiae, ecchymoses,
easy
bruising
and
bleeding
from
venipuncture sites. Epistaxis, gum bleeding
and gastrointestinal haemorrhage are less
common[23-24] . If improperly treated, shock
leads
to
metabolic
acidosis,
severe
generalized bleeding and, eventually,
death[15] . Unlike children, many adult
patients show severe bleeding of the
gastrointestinal (GI), or of other sites,
preceding the shock, which may be severe
enough to cause death[20-21,25] .
Relevant laboratory findings in DHF
cases include thrombocytopenia, haemoconcentration and hypoproteinemia[15] . A
drop in platelet count to below 100,000 per
µL and an increase of 20% or more in the
haematocrit, both resulting from increased
vascular permeability, are consistent findings.
Other signs of plasma leakage include pleural
effusion, ascites and hypoproteinemia.
Leukopenia and leukocytosis are common,
and aminotransferases are usually elevated.
Thrombin,
prothrombin
and
partial
thromboplastin times are often prolonged.
Fibrinogen levels decrease and fibrin
degradation products may increase. In
patients with DSS, the severity of laboratory
abnormalities described for DHF tends to be
worse.
Dilutional
hyponatremia
and
hypoproteinemia correlate with disease
severity[1] .
In the current series, patients with
longer length of hospitalization exhibited
significantly lower levels of platelets, as well
as a higher tendency to severe capillary
vascular leakage (CVL) ‡. Overall, a shorter
duration of IPA associated surprisingly with
a longer length of hospitalization, probably
reflecting the fact that more severely ill
patients tended to seek medical attention
earlier. Nevertheless, patients with dengue
type II, as well as those with evidence of
CVL, exhibited significantly more protracted
IPA. The occurrence of the latter two
conditions most likely reflect the delay in
‡
CVL may induce many other clinical manifestations
and complications besides clinical bleeding, such as
pleural effusion, ascitis, joint swelling, etc.
73
initiating a proper and adequate fluidreplacement treatment, which is the key to
treating DHF in order to compensate for the
loss of plasma from blood vessels due to
increased vascular permeability[1,15] .
Of note is the finding that a
considerable percentage of the cases
(12.5%) developed AAC. Recent data
suggest that in children with DHF, a gallbladder wall thickening =5 mm on
ultrasonography correlates with a higher risk
of hypovolemic shock[26] . However, despite
a few scattered reports of AAC complicating
adult DHF patients[27- 30] , little information
exists in medical literature on the
pathological and clinical implications of this
newly recognized condition. It is worth
mentioning that while our nine patients with
AAC exhibited a significantly increased level
of peripheral blood leukocytes during
hospitalization, their clinical outcome in
terms of IPA, length of hospitalization or
occurrence
of
other
life-threatening
complications, did not differ from that of
patients without AAC. Details of the clinical
aspects and imaging techniques findings for
this set of patients will be discussed
elsewhere.
Viral, serological and genetic factors
may influence virulence. Molecular studies
have identified genetic variation among all 4
dengue virus serotypes[31-34] . Of note here is
the finding that DEN-2 strains associated
with grade II DHF or DSS grow to higher
titers in peripheral blood leukocytes than do
DEN-2 strains isolated from mildly ill
patients[35,36] . Although characterization of
the viral serotypes involved in these patients
was not performed, DEN -1, 2 and 4 all
circulated in Venezuela during the period
when the cases occurred, DEN-2 being the
most prevalent one [5] .
In conclusion, the number of days of
IPA, the length of hospitalization and the
degree of alteration in the level of Hb, Hct,
leukocytes and platelets were all predictors
of a more severe and complicated course in
adult patients with DHF/DSS. The onset of
leukocytosis must suggest the occurrence of
inflammatory complications such as AAC.
DHF/DSS in the Americas continues to
occur in a significant number of adults, but
it is not clear whether this relates with the
genetic background of the populations, the
epidemiological events or, else, with other
unknown factors.
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[27] Van Troys H, Gras C, Coton T, Deparis X,
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76
Dengue Haemorrhagic Fever with
Encephalopathy/Fatality at Petchabun Hospital:
A three-year Prospective Study (1999-2002)
Prasonk Witayathawornwong#
Department of Paediatrics, Petchabun Hospital, Petchabun Province, Thailand
Abstract
During the three-year period from 1 May 1999 to 30 April 2002, there were 1,465 cases of dengue
haemorrhagic fever (DHF) admitted to the Department of Paediatrics, Petchabun Hospital. The male to
female ratio was 722:743 (1:1.03). Their ages ranged from 80 days to 15 years with a median of 9 years.
Thirty-two patients (2.2%) were under one year of age with a median of 8 months, and all except two
had primary dengue infection.
There were 34 DHF patients with encephalopathy (2.3% of all DHF cases). The male to female ratio
was 17:17 (1:1). The median age was 8 years and 11 months (range 10 months to 13 years). Thirty
patients (88.2%) were older than 5 years. Thirty and four patients respectively developed
encephalopathy in shock and convalescent stages. All 17 fatal cases (1.16% of the total DHF cases,
male:female = 8:9) had both prolonged shock and massive gastrointestinal haemorrhage since
admission. About 64.7%, 76.4% and 58.8% of the seventeen non-fatal cases (male:female = 9:8) had
gastrointestinal haemorrhage, shock state and massive fluid overload since admission respectively. The
risk factors for encephalopathy included prolonged shock, severe gastrointestinal haemorrhage, severe
hepatic dysfunction and prior fluid overload.
Keywords: Dengue haemorrhagic fever, encephalopathy, fatality, gastrointestinal haemorrhage, shock, fluid overload,
hepatic dysfunction.
Introduction
The major pathophysiological hallmarks in
dengue haemorrhagic fever (DHF) are
leakage and abnormal haemostasis that
leads to hypovolemic shock and/or
haemorrhage. Generally, vital organs are not
primarily involved in DHF but unusual
manifestations, mainly the involvement of
the central nervous system (CNS) and severe
hepatic dysfunction, are increasingly being
detected[1-6] . The incidence of CNS
involvement in dengue infection was about
0.88-5.4%[7-11] first reported in 1976[12] .
Although dengue encephalitis existed as
evident from the direct dengue viral
invasion[13-17] , the more common conditions
were encephalopathy secondary to fluid
extravasation,
cerebral
oedema,
hypoperfusion, haemorrhage, hyponatremia,
liver failure and renal failure [2,18-19] . The
treatment of DHF with CNS involvement is
supportive and symptomatic. Early detection
and proper fluid management of DHF
should be done to prevent any risk factors.
#
77
DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002)
Results
There were 1,465 cases of DHF admitted to
the Department of Paediatrics, Petchabun
Hospital, Petchabun province, Thailand,
from 1 May 1999 to 30 April 2002. The
diagnosis methods used followed the WHO
criteria[20] and about 82% of cases were
serologically confirmed using either enzymelinked
immunosorbent
assay
or
haemagglutination inhibition tests. The
treatment consisted of general measures
(closely observed vital signs, general
appearance
and
serially
recorded
haematocrit in 24-48 hours after fever, no
medication except antacid for moderate to
severe abdominal pain) and fluid therapy
(minimal amount to normalize vital signs
and haematocrit: 5% dextrose saline for
infants, 5% dextrose Ringer acetate for older
patients, Dextran-40 in normal saline for
impending or fluid overload and fresh whole
blood or packed red cells for significant
bleeding, platelet concentrate and plasma
not used).
Of the 1,465 cases of DHF admitted to the
Department of Paediatrics, Petchabun
Hospital, from 1 May 1999 to 30 April 2002.
The male to female ratio was 722:743
(1:1.03). The patients’ age ranged from 80
days to 15 years with the median of 9 years.
The highest incidence was in the 5-10-year
age group (57%) and the second highest was
in the 10-15-year age group (26.2%). Thirtytwo patients (2.2%) were under one year of
age with the median of 8 months, and all of
them except two had primary dengue
infection. Eighty-seven per cent of all cases
were found in the rainy season during MayOctober. The patients were from all 11
districts in the Petchabun province, 84%
from the central district alone. There were
DHF patients from all villages (178) of all the
subdistricts (17) of the central district.
Patients with encephalopathy (drowsy,
stuporous, comatose and convulsion) were
closely observed for blood sugar or
dextrostix
every
six
hours.
Serum
transaminase was done once a day until
recovery. Vitamin K 1 and 10% calcium
gluconate were administered for three days.
Lumbar puncture was performed cautiously
if there was no other risk.
The statistical analysis included the
percentage, mean, standard deviation and
range for demographic data and Student’s t
and chi-square tests for comparing noncategorized and categorized variables,
respectively.
78
There were 34 DHF patients with
encephalopathy (2.3%). The male to female
ratio was 17:17 (1:1). The median age was
8 years 11 months (range 10 months to 13
years). Thirty patients (88.2%) were older
than 5 years. Thirty and four patients
developed encephalopathy in the shock and
convalescent stages respectively. There were
7, 18 and 9 patients with encephalopathy
stage II (drowsy), stage III (stuporous) and
stage IV (comatose) respectively. Twentyeight patients (82.3%) were referred from
district hospitals and 17 patients (50%) had
a massive fluid overload. Lumbar puncture
was performed in 2 non-fatal cases with
normal findings. Their serology indicated
secondary dengue response.
About 17 fatal cases (1.6% of total DHF
cases, male:female = 8:9 , median age 8
years and 4 months, range 10 months to 12
years) had the median duration of 13 hours
for deaths (range 1-26 hours, mean±SD =
13.4±8.4 hours). Sixteen of the 17 cases
(94.1%) were dead within 24 hours.
Profound shock and massive gastrointestinal
haemorrhage since admission were detected
in all patients. Hepatic failure, comatose
stage and massive fluid overload were
detected since admission in 12 (70.6%),
9 (52.9%) and 7 (41.1%) cases respectively.
There
was
no
history
of
taking
acetaminophen more than 60 mg per kg/day.
Aspirin and non-steroidal anti-inflammatory
drug (NSAID), Ibuprofen, were taken by each
patient for many days during the febrile stage.
Convulsion was detected in 4 cases (Tables 1
and 2).
Table 1. Clinical manifestations of 17 fatal cases
Patients
1NW
Age
Weight
L/S DHF Haemorrhage Encephalopathy Fluid
Sex
TDF/DI
(years)
(kg)
(cm) grade manifestations signs/onset
overload
1.3
F
81
3/3
3/-
4
GIH
Comatose/s
+
Associated
diseases
Diarrhea
convulsion
Referral*
+
2SS
11.7
F
42
6/7
2/-
4
GIH
Comatose/s
–
-
–
3TR
13
M
37
4/3
2/-
4
GIH
Stuporous/s
+
ASA
+
4KB
6
F
132
3/3
10/4
4
GIH
Comatose/s
–
Thalasemia
convulsion
+
5SM
7
M
20
5/6
3/-
4
GIH
Stuporous/s
–
Pneumonia
G6PDdef
+
6KC
7
F
191
4/4
2/-
4
GIH
Comatose/s
+
7UA
2.11
F
21
7/7
3/-
4
GIH
Comatose/s
+
8WT
10
M
50
5/5
2/-
4
GIH
Stuporous/s
+
6-36.3 M
9KF
+
NSAID
convulsion
+
+
40
5/6
3/-
4
GIH
Stuporous/s
–
–
0.10
F
71
4/4
4/-
4
GIH
Stuporous/s
–
+
11JP
10
F
31
4/5
3/-
4
GIH
Stuporous/s
+
12DP
9.0
M
17
5/6
3/-
4
GIH
Comatose/s
+
+
13BP
9.5
F
231
4/4
5/-
4
GIH
Comatose/s
–
+
14NO
8.4
F
24
6/7
3/-
4
GIH
Comatose/s
–
–
1
10RM
ARDS, DIC
+
15TW
8.10
M
20
5/6
3/-
4
GIH
Drowsy/s
–
16WK
10
M
35
4/4
3/-
4
GIH
Comatose/s
–
+
17PL
12
M
45
5/6
2/-
4
GIH
Stuporous/s
–
–
Hypoglycemia
convulsion
+
TDF = total duration of fever; DI = duration of illness
GIH = gastrointestinal haemorrhage; S = shock stage; kg = kilograms
1,2
= first, second degree protein energy malnutrition
* or prior medications especially excessive fluid replacement
ARDS = A dult respiratory distress syndrome
DIC = Disseminated intravascular coagulation
NSAID = Non-steroidal anti inflammatory drug
L/S = liver/spleen
79
Table 2. Laboratory investigations of 17 fatal cases
Patients
1NW
Platelets/
Hct (max-min)
AST/ALT
(IU/L)
PT/PTT*
(sec)
Sodium
(mEq/L)
DHF
serology
Duration
in hospital
23,00 (37-)
481/161
20.2/96.6
129
SDI
6h
2SS
27,000 (50-34)
10,081/2,410
20.9/97.9
135
SDI
24 h
3TR
98,000 (39-21)
1,880/636
29.0/>180
130
SDI
26 h
4KB
35,000 (20-12)
1,346/420
137.0/58.0
130
SDI
7.5 h
5SM
15,000 (28-8)
50/20
26.2/42.0
138
SDI
2h
6KC
33,000 (47-27)
9,729/4,821
24.0/170
134.9
SDI
21 h
7UA
24,000 (48-37)
10,043/3,734
19.7/171
129.6
SDI
13 h
8WT
17,000 (49-34)
3,537/1,599
20.1/>180
134
SDI
8.5 h
9KF
18,000 (57-47)
600/1,524
13.8/77.7
136
SDI
17.5 h
10RM
22,000 (29-27)
2,582/671
23.1/119.6
128
SDI
6h
11JP
46,000 (42-6)
3,298/1,274
43.9/472
136.7
SDI
16 h
12DP
12,000 (48-31)
1,452/797
17.1/84.8
137
SDI
24 h
13BP
25,000 (42-34)
10,215/5,015
43.1/172
135
SDI
21 h
14NO
10,675 (35-26)
1,486/631
35.6/>180
138
SDI
1 h 10 m
15TW
2,000 (48-28)
3,940/1,592
35.3/>180
134.0
SDI
12 h
16WK
2,000 (56-32)
234/99
12.6/41.1
136
SDI
20 h
17PL
89,000 (41- )
17,950/10,446
45.2/160.8
135
SDI
2 h15 m
* normal (10-14)/(23-35) sec
h = hours, m = minutes
SDI = secondary dengue infection
There were 17 non-fatal cases
(male:female = 9:8, median age = 9 years,
range = 3-12 years). Massive gastrointestinal
haemorrhage, shock and massive fluid
overload were detected since admission in
11 (64.7%), 13 (grade 3-9, grade 4-4,
76.4%) and 10 (58.8%) cases respectively.
There were 5 cases with acute renal failure
and one with liver failure since admission.
Fifteen of the 17 cases were referred from
80
district
hospitals.
Haemodialysis
and
plasmapheresis were done in 3 renal failure
cases and 2 cases with only supportive
treatment.
All
17
cases
recovered
completely without neurological sequelae.
The average (mean ± SD) hospitalization
duration and recovery period from
encephalopathy were 8.3 ± 5.31 days and
5.1 ± 3.7 days respectively (Tables 3, 4).
Table 3. Clinical manifestations of 17 non-fatal encephalopathy cases
TDI/DI
L/S
(cm)
DHF
grade
Hemato
manifes
Enceph
signs/onset
25
121
4/4
5/3
3/3/-
4
4
GIH
Petichiae
Stuporous/s
Stuporous/s
Fluid
overload
+
–
F
F
29
151
6/6
7/7
3/3/-
3
3
GIH
GIH
Stuporous/s
Stuporous/s
+
+
5.9
9.9
6.6
9
M
F
M
F
17
43
181
41.5
7/7
5/5
3/3
5/5
1/JP
3/2/-
2
3
3
3
GIH
GIH
Petichiae
GIH
Stuporous/s
Stuporous/s
Stuporous/s
Stuporous/s
–
+
+
–
9SN
10PP
11Sma
12WL
11
11
9.11
6.11
F
M
M
F
301
34
28
19
4/7
5/5
5/5
5/5
4/2/5/8/6
2
4
3
3
GIH
GIH
GIH
GIH
Stuporous/s
Stuporous/s
Drowsiness/s
Stuporous/s
–
+
+
+
13ThP
14SL
7.9
5.9
M
M
22
251
5/5
5/6
4/5/-
2
3
GIH
Petichiae
Stuporous/s
Stuporous/s
11.11
10.4
3
M
M
F
232
49
13.5
5/5
6/6
5/7
1/2/2/-
4
3
2
Petichiae
Petichiae
Petichiae
Drowsiness/s
Drowsiness/s
Drowsiness/s
Age
(y.m)
Sex
1YB
2PS
9
4.4
M
F
3ST
4SU
10.3
6
5CP
6WD
7WB
8SSr
Patients
15KN
16MK
17WTh
Weight
(kg)
Associated
diseases
Referral
ARF
Pneumonia
+
–
+
+
–
+
Pneumonia,
Tracheitis
ARF
Hypoglycemia,
ARF
ARF
Thalasemia,
ARDS,
pneumonia
ARF
–
–
+
Rhinitis
+
–
+
+
+
+
+
+
+
+
+
+
+
1,2
= first, second degree protein energy malnutrition
ARF = acute renal failure; ARDS = Adult respiratory distress syndrome
GIH = gastrointestinal haemorrhage; JP = just palpable
Table 4. Laboratory investigations of 17 non-fatal encephalopathy cases
Patients
1YB
2PS
3ST
4SU
5CP
6WD
7WB
8SSr
9SN
10PP
11Sma
12WL
13ThP
14SL
15KN
16MK
17WTh
Platelets/
Hct (max-min)
62,000(51-30)
17,000(50-26)
16,000(50-38)
44,000(46-34)
90,000(52-30)
36,000(41-30)
4,000(58-34)
20,000(48-32)
68,000(49-27)
21,000(42-27)
34,000(48-33)
20,000(42-27)
56,000(42-30)
45,000(56-33)
40,000(49-32)
3,000(51-35)
60,000(32-30)
AST/ALT
(IU/L)
9,420/2,239
2,023/458
1,098/452
255/148
168/110
13,895/5,200
2,128/918
14,580/5,852
6,987/3,789
3,810/1,935
2,250/448
3,436/1,841
1,315/549
4,746/1,470
8,150/4,450
2,996/1,482
275/599
PT/PTT *
(sec)
23.4/>120
14.4/59.9
14.7/121.6
16.8/48.5
18.4/64.5
24.6/77.6
15.1/67.8
16.4/56.0
32.5/57.2
15.3/52.5
14.9/78.9
15.0/89.3
12.4/61.5
15.9/1,133
16/72.2
13.8/64.0
15.4/66.7
Sodium
(mEq/L)
128
135
130
135
140. 3
127
128
134
138
129
128
143
134
131.6
137
132.8
128
DHF
serology
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
SDI
Duration **
13/7
9/5
4/2
17/10
13/10
11/7
6/3
10/7
7/3
4/2
6/3
21/15
2/2
2/2
7/4
5/3
4/3
* normal: (10-14 )/(23-35 ) sec
** duration in hospital/consciousness change (days)
81
A comparison between the clinical
manifestations and laboratory investigations
of the fatal and non-fatal cases, usual
manifestations and encephalopathy cases
are shown in Tables 5, 6 and 7 respectively.
Table 5. Comparison between clinical manifestations and laboratory investigations
of fatal and non-fatal cases
Clinical manifestations or laboratory
Fatal cases
investigations*
(n=17)
Shock (grade IV)
17
Internal bleeding
17
Onset of encephalopathy in shock stage
17
Coma since admission
9
Platelets (/cubic millimeters)**
29,33.8 (26,662.1)
AST (IU/L)
4,641.4 (5082.7)
ALT (IU/L)
2,108.8 (2,655.1)
PT in sec
25.5 (11.1)
PTT in sec
146.1 (98.8)
Serum sodium (mEq/L)
133.9 (3.3)
Non-fatal cases
P-value
4
11
13
0
37,411.7 (24,153.3)
4,560.7 (4,545.8)
1,878.8 (1,838.6)
17.3 (5.0)
75.9 (25.5)
132.8 (4.8)
<0.001
0.007
0.035
<0.001
0.362
0.961
0.771
0.011
0.011
0.471
* mean (standard deviation, SD)
** minimum value
PT=Prothrombin time
PTT=Partial thromboplastin time
Table 6. Comparison between clinical manifestations of usual manifestations
and encephalopathy cases
Clinical data
Age <1 yr
1 – 4 yr
5 – 10 yr
10 – 15 yr
Female
Shock
Referal or prior medications
Internal bleeding
Malnutrition[21]
Death
Duration in hospital (days):mean
(standard deviation)
Usual manifestations
(n=1431)
31 (2.1%)
210 (14.6%)
812 (56.7%)
378 (26.4%)
726 (50.7)
332 (23.2)
(grade 3=330, 4=2)
266 (18.6%)
130 (9.1%)
716 (50.0%)
(1°=511, 2°=192, 3°=13)
–
3.4 (1.38)
Encephalopathy
(n=34)
1 (2.9%)
4 (11.7%)
23 (67.6%)
6 (17.6%)
17 (50.0%)
30 (88.2%)
(grade 3=9, 4=21)
28 (82.3%)
28 (82.3%)
12 (35.3%)
(1°=10, 2°=2)
17 (50%)
83 (5.31)*
P-value
0.775
0.661
0.219
0.252
>0.993
<0.001
<0.001
<0.001
0.091
<0.001
0.002
* only non fatal cases
82
Table 7. Comparison between laboratory investigations of usual and encephalopathy cases
Usual manifestations
(n=1431)
Encephalopathy
(n=34)
P-value
AST (IU/L)
257.3 (250.6)
4601.0 (4748.3)
<0.001
ALT (IU/L)
119.5 (138.1)
1993.8 (2251.8)
<0.001
70697.9 (2975.7)
33372.8 (2538315)
<0.001
PT (10-14 sec)
11.2 (1.1)
21.4 (9.4)
<0.001
PTT (23-35 sec)
51.9 (13.4)
111.0 (79.5)
<0.001
Laboratory data*
Platelets (minimum)
* mean (standard deviation, SD)
Discussion
DHF patients from all the 11 districts in the
Petchabun province were included in this
study. The minors were referred from 10
other districts. The adults were from the 178
villages in all 17 subdistricts of the central
district. This epidemic was similar to the
previous dengue epidemic in 1997[22] . This
data implied that the dengue virus had
spread nationwide. The percentage of DHF
patients with CNS involvement in this study
was 2.3%, the same as in the previous
study[22] as well as in other studies[7-11] .
Female patients were reported to be more
severely affected and accounted for more
fatalities than male patients but without any
significant difference[18,23-25] . In this study,
the number of both sexes was equal and
both had usual manifestations and
encephalopathy cases. Young patients,
especially those less than 1 year of age, had
the tendency to be more severely and fatally
affected[18,23-25] . Age itself was not a risk
factor of disease severity in this study.
Although there was one fatal case aged 10
months (10 RM), 85% of those who died
were older than 5 years. Most of the
DHF/DSS patients were well-nourished but
patients with CNS involvement were more
undernourished
without
significant
difference[24,26] . In this study, patients with
usual manifestations and encephalopathy
were underweight[21] by about 50% and
35.3%
respectively.
Patients
with
encephalopathy were mainly in stage
III [7,8,18,27] (88.2%) initially developed in
shock stage (52.9%), more than in febrile or
convalescent stage[7,9,28] . Seizure in DHF
with encephalopathy had been reported in
about 18.8-100%[7- 9,13,18,24,27,29 ,30] . In this
study seizure was detected in 4 out of 34
patients (11.7%). Two patients (1NW, 7UA),
under 5 years, developed seizure in afebrile
stage, indicating that the seizure might have
had a specific primary cause[10] . However,
some children had possible confounding
factors such as hyponatremia (3 patients,
1NW, 4KB, 7UA), hypoglycemia (15TW),
and liver failure (3 patients, 1NW, 7UA,
15TW). Other factors could be a history of
previous febrile seizure, co-infection[10] and
drug ingestion.
The causes or factors contributing to
CNS manifestations included the following:
direct CNS infection – a rare entity, CNS
83
bleeding[6,18] and severe hepatic dysfunction.
Lumbar puncture was done in only two
patients (5CP, 15KN) in the convalescent
stage with normal findings. Direct CNS
infection could not be evaluated in this
study. There was massive gastrointestinal
haemorrhage in 28 out of 34 cases (82.3%).
Acute liver failure could be the direct or
indirect cause of encephalopathy[31,32] and
an important cause of death in DHF[33] .
Twelve of the 17 fatal cases (70.5%) had this
severe
condition.
Severe
hepatic
dysfunction could be due to profound shock,
massive gastrointestinal haemorrhage or
immune complex mechanism[34] . The liver
pathology in profound shock stage might be
centrilobular hepatocellular necrosis [35] or
extensive necrosis of hepatocytes usually in
a massive or submassive distribution[33] .
Massive gastrointestinal haemorrhage might
cause hepatic dysfunction and vice versa.
Immune complexes, detected in 80% of
DHF patients[36] , might be deposited in
hepatocytes and then destroyed as in
hepatitis B virus infection[34] . Excessive use
of hepatotoxic drugs such as acetaminophen,
an antiemetic drug, might interfere with
liver function. There was no history of
excessive use of such drugs in this study.
Two fatal cases with liver failure and massive
gastrointestinal haemorrhage had taken
aspirin (3TR) and non-steroidal antiinflammatory drug, Ibuprofen (7UA), for
many days during the febrile stage. Both
drugs
aggravated
the
gastrointestinal
haemorrhage and aspirin could have
induced Reye syndrome[37] .
Thalassemia, of which two cases with a
large liver and spleen were included in this
study (4KB, 12WL), was a risk factor for
hepatic failure [18] . Acute renal failure (ARF)
was a rare complication of DHF but could
84
occur in severe cases with prolonged shock,
DIC and also hepatorenal syndrome[37] .
There were five patients with ARF in this
study. All of them recovered completely,
three (6WD, 8SSr, 14SL) with haemodialysis
and plasmapheresis and two (1YB, 9SN)
with only supportive and symptomatic
therapy. Hyponatremia was a factor in CNS
involvement.
Serum
sodium
of
encephalopathy patients in this study was
slightly low. It was due to excessive
hypotonic solution replacement. The risk
factors of encephalopathy in this study were
profound shock, massive gastrointestinal
haemorrhage, excessive fluid overload and
severe hepatic dysfunction. Among the fatal
and non-fatal encephalopathy cases,
laboratory
investigations
except
for
coagulogram, were not significantly different.
But fatal cases had more severe shock
conditions, gastrointestinal haemorrhage
and onset and depth of encephalopathy
(Table 5).
Conclusion
In conclusion, encephalopathy in DHF was
a severe complication with high mortality,
although neurological sequelae in recovered
patients were rare. Prevention should be
attempted by early diagnosis and proper
management of fluid therapy.
Acknowledgements
The author thanks officials of the Regional
Medical Science Centre, Phitsanuloke,
Thailand, for DHF serology testing and all
nursing staff and workers of the Paediatrics
Department of Petchabun Hospital for
taking good care of the patients.
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A New Tool for the Diagnosis and Molecular
Surveillance of Dengue Infections in Clinical Samples
C. Domingo* #, G. Palacios**, M. Niedrig***, M. Cabrerizo*, O. Jabado**,
N. Reyes*, W.I. Lipkin** and A. Tenorio*
*Laboratorio de Arbovirus y Enfermedades Víricas Importadas, Centro Nacional de Microbiología,
Instituto de Salud Carlos III, Carretera de Pozuelo Km 2 (28220 Majadahonda), Madrid, Spain
**Jerome L and Dawn Greene Infectious Disease Laboratory, Columbia University, New York, USA
***Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany
Abstract
Dengue fever and dengue haemorrhagic fever are amongst the most important challenges in tropical
diseases due to their expanding geographical distribution, increasing outbreak frequency,
hyperendemicity and evolution of virulence.
Here, the use of a RT-nested PCR for both the diagnosis and genetic characterization of dengue
infections in clinical samples is described.
Keywords: Dengue, dengue haemorrhagic fever, diagnosis, molecular epidemiology, surveillance, glycoprotein E
gene, NS1 gene.
Introduction
Dengue fever (DF), dengue haemorrhagic
fever (DHF) and dengue shock syndrome
(DSS) are considered to be the most
important arthropod-borne viral diseases due
to the high rates of morbidity and mortality
caused by them. Over 2.5 billion people are
at risk of the infection and more than 100
countries are home to endemic dengue
transmission with an increasing incidence of
DHF cases[1-3], making dengue an archetypal
“emerging” disease. International travel,
urbanization, overpopulation, crowding,
poverty and a weak public health
infrastructure in most endemic areas are the
likely factors contributing to the surge in new
cases[4] . A major concern is the potential
spread of dengue fever into the United States
of America and Europe due to climate
warming and the spread of its vector.
Travellers to areas where dengue is
endemic are a potential source of the spread.
Most infections manifest as a mild febrile
illness during travel that coincides with the
peak of viral shedding and risk for
transmission.
Imported
dengue
virus
infections have been reported in several nonendemic countries; and dengue virus
infection is one of the most frequent causes
#
E-mail: [email protected]; Phone: (+34) 918223954, Fax: (+34) 915097966
87
A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples
of febrile illness in tourists and people
working in dengue-endemic areas [5,6,7,8] . As
early symptoms of DF mimic those of other
diseases such as malaria or leptospirosis,
rapid laboratory diagnosis is important for
proper patient care. Dengue fever can be
diagnosed by virus isolation, genome and
antigen detection, or serological studies.
Samples obtained for the detection of
dengue virus by cell-culture require proper
handling of the sample for viral viability.
Serology, even for anti-dengue IgM
antibodies, is feasible only after 5 days
following the onset of the symptoms. Thus,
molecular techniques fulfil an important role
in the diagnosis of dengue infection during its
early stages.
The characterization of circulating
dengue virus serotypes is important in
surveillance, since the introduction of a new
variant to areas affected by pre-existing
serotypes constitutes a risk factor for
DHF/DSS[9] . By defining intra-serotypic
genetic variation, the global distribution and
spread of virus strains can be mapped and
followed up[10-13] , and the genetic differences
associated with disease severity can be
identified[10,14-16] .
Moreover,
recent
epidemiological analyses suggest that the
more virulent genotypes are now displacing
those of lower epidemiological impact[17] ,
resulting in dengue outbreaks [18]. In this
context, a methodology for real-time,
worldwide surveillance is needed to track
dengue strains and help anticipate changes in
the epidemiology of the infection.
Here we report the amplification and
analysis of a genomic interval spanning the
E/NS1 junction of the dengue genome for
the detection and typing of all four dengue
virus serotypes in clinical specimens. This
sensitive, specific and rapid alternative assay
requires only a single acute phase serum
sample.
88
Virus isolates and clinical samples
Viral RNAs were provided by the National
Collection of Pathogenic Viruses (Porton
Down, Salisbury, UK): serotype 1 dengue
virus (DEN-1; strain Hawaii), serotype 2
dengue virus (DEN-2; strain New Guinea C),
serotype 3 dengue virus (DEN-3; strain H87),
and serotype 4 dengue virus (DEN-4; strain
H241); the RNAs from prototype strains of
Japanese encephalitis (JEV), yellow fever
(YFV), tick-borne encephalitis (TBEV),
Murray Valley encephalitis (MVEV) and St.
Louis encephalitis (SLEV) viruses were used
to check the specificity of the dengue virus
assay. Serial dilutions of this genome material
were prepared to obtain the standards to
assess the sensitivity of the assay.
Viremic human sera samples were
obtained from patients with a clinical
diagnosis of dengue infection (Sera
1794F02; 13VI02; 366VI03; 438VI03).
These were travellers who presented at the
Spanish Tropical Medicine Units with
dengue-compatible symptomatology on
their return from the Dominican Republic,
India, Indonesia and Nicaragua, respectively,
and suffered from classical DF as defined by
the WHO criteria[19] .
Selection and synthesis of
oligonucleotide primers
A RT-nested PCR protocol was developed
for the detection of the four serotypes of
dengue virus in clinical samples. Dengue
virus primers for amplification and/or
sequencing (Table) were designed based on
dengue virus sequences in the public
sequence databases, using a computerassisted analysis (MACAW version 32
software, 1995, NCBI, Maryland) to
determine consensus sequences. The Table
shows the sequences and the respective
primer positions in the prototype strains of
the four dengue serotypes. To address the
natural variability of dengue viruses, mixtures
of degenerated primers were used to enable
hybridization with all known serotypes.
RT-nested PCR
Using purified dengue virus RNA as a
template, relevant aspects of the RT-PCR and
nested PCR assay (Mg2+ concentration,
primers, RT temperature, number of cycles,
annealing temperatures) were initially
optimized to achieve the greatest sensitivity.
A PTC-200 Peltier thermal cycler (MJ
Research) was used throughout. 5 µl of viral
RNA solution were added to 45 µl of a
medium compatible with both the reverse
transcription and PCR amplification steps
(QIAGEN OneStep RT-PCR kit). The RTPCR mix contained 1×OneStep RT-PCR
buffer, 400 mM of each dNTP, 20 pmol of
each sense or antisense degenerated primer
(S1871DEN1,
1871DEN2,
1871DEN3,
1871DEN4, AS2622DEN1, AS2622DEN2,
AS2622DEN3, AS2622DEN4) and an
optimized combination of Omniscript and
Sensiscript reverse transcriptases and HotStar
Taq DNA polymerase. The RT-PCR reactions
were carried out using an initial reverse
transcription step at 41 °C for 45 minutes
followed by a denaturation and Hot Star Taq
polymerase activation step (94 °C, 15
minutes) and 40 cycles of denaturation
(94 °C, 30 seconds), primer annealing
(55 °C, 1 minute), and primer extension
(72 °C, 30 seconds). A final incubation was
carried out at 72 °C for 5 minutes. A second
amplification reaction (nested PCR) was
seeded with 1 µl of the initial amplification
product. The reaction mixture contained 1×
buffer B (60 mM Tris-HCl pH 8.5, 2 mM
MgCl2, 15 mM (NH4) 2SO 4, 40 pmol of each
sense and antisense primer (S2176DEN1,
S2176DEN2, S2176DEN3, S2176DEN4,
AS2504DEN) and 2.5 U of DNA Taq
Polymerase (Perkin-Elmer). The samples were
subjected to a denaturation step (94 °C, 2
minutes) followed by 40 cycles of
denaturation (94 °C, 30 seconds), primer
annealing (57 °C, 4 minutes), and primer
extension (72 °C, 30 seconds) and a further
extension step at 72 °C for 5 minutes.
Dengue virus sequence database
A dengue sequence database was
constructed by extracting sequences from
the NCBI GenBank. Each sequence was
identified by name, place, date and
serotype. Previously described genotypes
were taken from the references listed:
dengue strains genotypes were noted as
described by Rico-Hesse for DEN-1, 3 and
4[17] and by Twiddy et al. for dengue virus
type 2[20] . A manual search was employed
for all the sequences in GenBank
encompassing the targets of selected primers.
Next, we used BUSSUB, a new tool
developed at the Bioinformatics Unit of the
Institute of Health Carlos III[21] . This software
simplifies and boosts the process of retrieving
sequences contained between two given
flanking regions, improving the final results of
a search. Genetic characterization was
performed on a total data set of 113 DEN-1,
191 DEN-2, 102 DEN-3 and 153 DEN -4
sequences.
Sequence analysis of amplified
products
Original sequence data were first analysed by
the CHROMAS software (version 1.3,
McCarthy 1996; School of Biomolecular and
Biomedical Science, Faculty of Science and
Technology, Griffith University, Brisbane,
Queensland, Australia); forward and reverse
sequence data of each sample were aligned
using the programme EDITSEQ (DNASTAR
89
Inc. Software, Madison, Wisconsin, USA).
The consensus sequence was compared and
aligned to other samples or DNA database
sequences using the programme CLUSTAL X,
version 1.83[22]. Programmes from the MEGA
package[23]
were
used
to
produce
phylogenetic trees using NJ as the method to
reconstruct the phylogeny and Kimura-2p as
nucleotide substitution calculation method.
The statistical significance of a particular tree
topology was evaluated by bootstrap resampling of the sequences 1,000 times.
Published
sequences
used
in
the
comparisons were obtained from the
GenBank databases. Pair-wise comparisons
of the dengue virus database were done by
global alignment using the Needleman
Wunsch[24]
algorithm
using
the
implementation from EMBOSS, the European
Molecular Biology Open Software Suite[25] . Z-
Scores were calculated to test the significance
of each pair-wise alignment by Monte Carlo
simulation on the shuffled sequences.
Statistical analysis was conducted with the
SPSS statistical package (SPSS Software,
Chicago, IL).
Results
Design of the primers
The E/NS1 region of the genome was
chosen for the development of a RT-nested
PCR. The primers selected specifically
amplify the four dengue viruses with no
cross reactivity to other members of the
flavivirus family. A mix of degenerate
primers representing each serotype was
used to ensure coverage for the highly
variable dengue serotypes (Table).
Table. Primers used in RT-nested PCR assays and sequencing
Primer*
SequenceØ
Genome position ¶
PCR
S1871DEN1
S1871DEN2
S1871DEN3
S1871DEN4
5’-TGGCTGAGACCCARCATGGNAC-3’
5’-TAGCAGAAACACARCATGGNAC-3’
5’-TCTCCGAAACGCARCATGGNAC-3’
5’-TGGCAGAAACACARCAYGGNAC-3’
1869 to 1890
1871 to 1889
1863 to 1884
1873 to 1894
RT-PCR
AS2622DEN1
AS2622DEN2
AS2622DEN3
AS2622DEN4
5’-CAATTCATTTGATATTTGYTTCCAC-3’
5’-CAATTCTGGTGTTATTTGYTTCCAC-3’
5’-CAGTTCATTRGCTATTTGYTTCCAC-3’
5’-TAGCTCGTTGGTTATTTGYTTCCAC-3’
2620 to 2644
2622 to 2646
2614 to 2638
2624 to 2648
RT-PCR
S2176DEN1
S2176DEN2
S2176DEN3
S2176DEN4
5’-ATCCTGGGAGACACYGCNTGGG-3’
5’-ATTTTRGGTGACACAGCNTGGG-3’
5’-ATCTTGGGAGACACAGCNTGGG-3’
5’-ATTCTAGGTGAAACAGCNTGGG-3’
2174 to 2195
2176 to 2197
2168 to 2189
2178 to 2199
Nested
AS2504DEN
5’-TGRAAYTTRTAYTGYTCTGTCC-3’
2506 to 2527 DEN-1
2504 to 2525 DEN-2
2496 to 2517 DEN-3
2506 to 2527 DEN-4
Nested
*Primers name s beginning with “S” indicate a genome (plus)-sense orientation; names beginning with “AS” indicate a
complementary sense orientation.
¶
The genome positions are given according to each dengue virus serotype prototype strain (DEN-1; strain Mochizuki,
DEN-2; strain Jamaica N-109, DEN-3; strain H87, DEN-4; strain 814669)
Ø
Degenerate positions: N:A/C/g/T, R:A/g, Y:T/C
90
Dengue virus RT-nested
PCR specificity
The specificity of the RT-nested PCR was
determined by analysing serial dilutions of
RNA from related flavivirus (JEV, MVEV,
SLEV, TBEV, WNV, YFV) and no
amplification was obtained (data not shown).
The amplification was successful with
both commercial RNA and serum samples
for all dengue virus serotypes as shown
(Figure 1), yielding a distinct DNA product
of the expected size (328-pb) in agarose gels.
Figure 1. Amplification products obtained
through PCR analysis of sera from subjects
with acute dengue virus infection
396
344
298
366VI03
1794F02
13VI02
438VI03
MWM
[Arrow indicates 328 bp E/NS1 products. 1%
agarose gel. MWM: Molecular weight markers;
438VI03 (DEN-1); 13VI02 (DEN-2); 1794F02
(DEN-3); 366VI03 (DEN-4)]
One hundred and sixty-four serum
samples from cases of febrile illness
associated with travel were tested with the
assay. Thirty-seven cases were diagnosed as
of classical dengue fever by the WHO
criteria[19] . Sixteen of these cases were found
positive by using our E/NS1 assay.
Convalescent sera were available for 13 of
these cases; all were later confirmed to be
seroconvert. All serum samples found
positive by RT-nested PCR were collected in
the first week after the onset of the
symptoms.
Sequence analysis results
The phylogenetic trees obtained by the
analysis of the representative strains of the
four serotypes and unknown sample
sequences allowed rapid differentiation of
the corresponding serotype (Figure 2).
Pair-wise sequence analysis using
Needleman Wunsch global alignment was
carried out on the 220bp sequence where a
higher amount of sequences were available
for comparison. As expected, comparisons
between serotypes showed a low sequence
similarity and could be easily grouped. An
all-against-all sequence comparison was
done within each serotype to evaluate the
possibility of using sequence similarity to
classify genotypes. Significant sequence
similarity was observed when comparing
sequences within the same genotype. This
was evaluated by an analysis of variance
between groups (ANOVA), comparing the
scores of sequence comparisons within
genotypes
to
comparisons
between
genotypes. Each group was significant to the
P<0.001 level. Genotypes with only one
member sequence were excluded from the
analysis.
91
Figure 2. Phylogenetic tree constructed with the E/NS1 fragment which identifies
the four dengue serotypes
[Phylogenetic analysis was performed using the Kimura-two parameter model as a model of nucleotide
substitution and using the neighbor joining method to reconstruct the phylogenetic tree (MEGA version
2.1 software package)]
Den1-Peru9615-00-PERU
Den1-FGA89-FRENCHGUYANA1989
Den1-Peru9581-00-PERU
Den1-BR90-BRASIL1990
Den1-Br01MR-BRASIL2001
Den1-233-BRASIL1997
Den1-111-BRASIL1997
Den1-409-BRASIL1997
Den1-Peru5198-97
Den1-SingaporeS275-90-SINGAPORE1990
Den1-CAMBODIA-CAMBODIA1998
Den1-GZ80-CHINA1980
Den1-DJIBOUTI1998
Den1-MOCHIZUKI-JAPAN1953
Den1-16007-THAYLAND1964
Den1-A88-INDONESIA1988
Den1-clonePDK27
Den1-WestPac-NAURU1974
Den3-80-2-CHINA1980
Den3-H87-PHILIPPINES1956
Den3-11069
Den2-IQT2913-PERU1996
Den2-539-96-PERU1996
Den2-IQT1797-PERU1995
Den2-131-MEXICO1992
Den2-VEN2-VENEZUELA1987
Den2-44-CHINA1989
Den2-NewGuineaC-NEWGUINEA1944
Den2-43-CHINA1987
Den2-04-CHINA1985
Den2-Mara4-VENEZUELA1990
Den2-N.1409-JAMAICA1983
Den2-CookIslands-AUSTRALIA1997
Den2-16681-THAYLAND1964
Den2-ThNH54-93-THAYLAND1993
Den2-ThNHp36-93-THAYLAND1993
Den2-CO371-THAYLAND1995
Den2-K0008-THAYLAND1994
Den2-K0010-THAYLAND1994
Den4-TRI94-TRINIDAD1994
Den4-HON91-HONDURAS1991
Den4-MON94A-MONTSERRAT1994
Den4-TRI99-TRINIDAD1999
Den4-BAH98A-BAHAMAS1998
Den4-MON94B-MONTSERRAT1994
Den4-SUR94A-SURINAM1994
Den4-BDS93A-BARBADOS1993
Den4-MEX91-MEXICO1991
Den4-JAM83-JAMAICA1983
Den4-TRI82A-TRINIDAD1982
Den4-814669-DOMINICA1981
Den4-SUR82D-SURINAM1982
Den4-JAMAICA1981
Den4-TRI84A-TRINIDAD1984
YellowFever
WestNile
JVE
DENGUE SEROTYPE 1
DENGUE SEROTYPE 3
DENGUE SEROTYPE 2
DENGUE SEROTYPE 4
0.1
Sequences that had no known genotype
were classified with respect to the group to
which they were most similar. To verify the
utility of this method, a phylogenetic tree
was built in parallel with the unknown and
characterized sequences. Bootstrap values
in the 220bp region were too low to
92
generate a full taxonomy tree, but did fully
differentiate the genotypes (data not shown).
Even with this simple method, all unknowns
were classified correctly into their genotypic
group (Figure 3 illustrates one example
result for each serotype, compared to
known sequences).
Figure 3. Pair-wise analysis of four dengue strains detected by PCR amplification of 328 bp
E/NS1 products from patient sera. Samples are (a) 438VI03, DEN-1 AMERICAN-AFRICAN
genotype, (b) 13VI02, DEN-2 INDIAN genotype, (c) 1794F02, DEN-3 COSMOPOLITAN
genotype, and (d) 366VI03, INDONESIAN DEN-4 genotype
a) Dengue 1 - 438VI03 Nicaragua 2003
(a)
d) Dengue 2 - 13VI02 India 2002
(b)
1100
1100
1000
NW Score
NW Score
1000
900
800
900
800
700
700
AMAF
ASIAN
MAL
SP
COS
THAI
Genotype
ASIAN
AMERICAN
AMERICAN
ASIAN
GENOTYPE 1
AFRICAN
Genotype
b) Dengue 3 - 1794F02 Dominican Republic 2002
(c)
c) Dengue 4 - 366VI03 Indonesia 2003
(d)
1200
1200
1100
1100
1000
1000
NW Score
NW Score
ASIAN
GENOTYPE 2
900
800
900
800
700
700
SE Asia/SP
THAILAND
INDIAN
SUBCONTINENT
AMERICAS
INDONESIA
SE ASIA
MALAYSIA
Genotype
Genotype
Discussion
The efficient worldwide control of dengue
virus requires the definition of sources of
epidemic
viruses
and
the
precise
identification of virus genotypes. A key
objective of DF and DHF surveillance
programmes is early detection of outbreaks
to permit the implementation of control
measures.
DHF
outbreaks
can
be
anticipated by monitoring the emergence of
new genotypes in a region. The need for
surveillance is warranted, since air travellers
can quickly move viruses from an endemic
area to a receptive area. Dengue virus
surveillance should be implemented in
endemic and non-endemic areas to aid
governments and healthcare workers in
planning for potential outbreak situations.
The advent of a simple and accurate
method for diagnosis and surveillance could
improve the establishment of these
programmes in developing countries
affected by the disease, and in non-endemic
areas where dengue is a travel-acquired
infection.
The RT-nested PCR described here
allows rapid direct diagnosis of acute
dengue infection in laboratories without
BSL-3 (bio-safety level 3) facilities.
Pair-wise
comparison
to
classify
sequences has been used for enteroviruses
and potyvirus [26-28] . Multiple alignment and
93
rigorous phylogenetic methods are preferable
to establish exact lineages of sequence strains
and discover recombination events. Pair-wise
comparisons can substitute if only a high level
of taxonomic classification is desired. Our
method allows classification of dengue
genotypes using the sequence of the 220bp
region amplified by the PCR assay. The
advantage of pair-wise comparison for
classification is its speed, simplicity and
availability. The database and classification
scheme provides a repository for sequences,
complementing efforts in tracking dengue
genotype distribution. A website could be
deployed wherein clinical laboratories post
their sequences, location and circumstances
of isolation. This would allow rapid
centralized analysis detailing the genotype,
date and location of the most similar
sequence isolate in the database. New
genotypes could be rapidly identified by
failure to relate them to a described group.
Acknowledgements
This investigation received financial support
from the Instituto de Salud Carlos III (ISCIII)
through research project grants (MPY
1194/02 and C03/04). G. Palacios and WI
Lipkin were supported by the Ellison Medical
Foundation and the National Institutes of
Health (AI 51292 and U54 AI57158-Lipkin).
C. Domingo was contracted by an agreement
between the Public Health Division of the
Spanish Ministry of Health (DGSP-MSC) and
the Instituto de Salud Carlos III (ISCIII) for the
development of the Haemorrhagic Viral
Fevers Surveillance and Control Programme.
The authors thank Dr J. Gascón, Dr
R. López-Vélez and Dr S. Puente and the
many scientists who contributed dengueinfected patient samples for this work. The
authors are grateful to Dr J.E. Mejia for
assisting in manuscript preparation.
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95
Clinical and Laboratory Observations Associated with
the 2000 Dengue Outbreak in Dhaka, Bangladesh
Monira Pervin* #, Shahina Tabassum**, Md. Mobarak Ali***,
Kazi Zulfiquer Mamun* and Md. Nazrul Islam**
*Department of Virology, Dhaka Medical College, Dhaka, Bangladesh
**Department of Virology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh
***Shahid Suhrawardi Hospital, Dhaka Bangladesh
Abstract
A large outbreak of dengue fever (DF)/dengue haemorrhagic fever (DHF) occurred in Dhaka city,
Bangladesh, in 2000. The present study was conducted on 105 clinically-suspected cases of DF to
confirm the diagnosis, determine the major clinical manifestations and correlate the haemorrhagic
manifestations with different dengue serotypes circulating during the outbreak. A total of 97 cases were
positive for anti-dengue IgM and were considered as recent dengue infection; 52.6% patients had
secondary and 47.4% had primary dengue infection. According to WHO case-definition, 79 cases were
classified as DF, 17 as DHF and 1 as DSS. Among the 18 DHF/DSS cases, 14 had secondary and 4 had
primary type of antibody response. The mean age of the dengue patients was 29.2±12.9 years and most
of them (37.1%) belonged to the 20-29-year age group. All the clinically-suspected patients had fever
ranging from 100-104 ºF, but the secondary dengue fever patients had higher (101.6±1.4 ºF) mean
body temperature. Common complaints included myalgia (84.5%), headache (82.5%), arthralgia (68.0%),
lethargy (80.4%) and retro-orbital pain (49.5%). Rash, especially maculopapular type, was significantly
higher in primary infection (P<0.01), while hepatomegaly was higher in secondary infections (P<0.01).
Haemorrhagic manifestations were observed both in primary and secondary dengue patients and were
mostly associated with serotypes DEN-3. In 22.7% of cases, the platelet count was <1x105/mm3 and was
associated more with secondary infection. Haematocrit more than 45% was found in 16.5% patients and
a significantly higher association was detected among the secondary dengue fever patients (P=0.02).
Although all 4 dengue serotypes were prevalent during the outbreak, DEN-3 was the predominant
serotype (70.5%) and was associated with more severe clinical manifestations.
Keywords: Primary dengue fever, secondary dengue fever, dengue haemorrhagic fever, serotypes, Bangladesh.
#
96
Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka
Introduction
Dengue was first reported in Bangladesh in
1964 and the outbreak came to be known
as ‘Dacca Fever’ [1] . For a long period after
that, dengue cases remained undetected. A
few cases were reported in 1999 before a
large outbreak occurred in 2000, during
which 5,551 cases and 93 dengue-related
deaths were reported[2,3] . Cases reported
during the outbreaks were mostly diagnosed
clinically, except for a few serologically
diagnosed cases[4] .
The study was conducted on clinically
suspected cases of dengue fever attending
the inpatient and outpatient departments of
Medicine and Paediatrics, Bangabandhu
Sheikh Mujib Medical University (BSMMU)
Hospital, and patients admitted at the Dhaka
Medical College Hospital during June –
December 2000. Some patients seeking
diagnostic facilities at the Department of
Virology, BSMMU, were also included in the
study.
Several studies have observed that
sequential or secondary dengue infections
are more likely to produce a severe form of
the disease. DHF and DSS occur mostly in
persons with pre-existing dengue antibodies
acquired actively or passively[5-7] .
Detection of anti-dengue IgM indicates
the diagnosis of recent dengue infection in
both primary and secondary cases. Antidengue IgM develops earlier than IgG in
primary infection and is usually detectable
by day 5 of illness and wanes after 1-2
months[8] . The ratio of IgM and IgG
antibodies determined by ELISA is useful for
distinguishing primary and secondary
infections[8,9] . In primary infection, the
IgM/IgG ratio generally exceeds 1.8 OD
units in acute or convalescent sera[8] .
Detection of an early and excess of IgG
characterizes secondary infection. In the
present study, dengue patients were
classified into primary and secondary cases
on the basis of this concept and it was
carried out to confirm the clinical diagnosis,
correlate the clinical manifestations with
laboratory findings and establish the
association of haemorrhagic manifestations
with different dengue virus serotypes
responsible for the outbreak of 2000.
According to specific inclusion criteria,
105 clinically suspected patients with fever
(presenting within 5 days of onset with body
temperature above 100 °F at the time of
blood sample collection) and fulfilling the
case-definition criteria of dengue fever (DF)
and dengue haemorrhagic fever (DHF) of
WHO[10] were enrolled in the study. The
exclusion criteria defined cases of febrile
illness of more than 5 days and/or with
definite sources of infection, chronic
illnesses like tuberculosis, bronchial asthma,
congenital heart disease, renal failure,
history of bleeding tendency since birth and
patients who refused to give two blood
samples.
Clinical data were collected through
interviewing the patients or their attendants
and meticulous physical examination of the
patients conducted by a doctor. The
tourniquet test was performed in all patients
by conventional method[11] . Hepatomegaly
and ascites were ascertained by physical
examination
and
on
reports
of
ultrasonography and X-rays. 5 ml of venous
blood was collected aseptically from all
patients during both the early and
convalescent stages of fever irrespective of
age and sex. This was processed and stored
appropriately for virus isolation, antibody
assay, platelet count and haematocrit
97
estimation. Detection of IgM and IgG antidengue antibody, isolation of dengue viruses
by mosquito inoculation technique and
serotyping of the isolated viruses were done
as described previously[12,13] . Patients were
classified into DF and DHF or DSS
according to WHO, 1997[10] .
Statistical analysis
The numerical data obtained from the study
were analysed and the significance of the
difference was estimated by using statistical
methods. The data were expressed in
frequency, percentage, mean and standard
deviation as applicable. The comparison
between groups was done by Student’s ‘t’
test and ‘Chi square’ and ‘Z’ test as
applicable. All data were analysed by using
the computer-based SPSS programme.
Probability less than 0.05 was considered as
significant.
Results
Of the 105 clinically suspected dengue
patients, 39 (37.1%) were positive for either
anti-dengue IgM or IgM and IgG antibodies
and 38 (36.2%) patients developed only
anti-dengue IgG in acute stage by ELISA.
When these patients were tested in the
convalescent stage, 21 (20%) were positive
for only IgM and 76 (72.4%) were positive
for both IgM and IgG antibodies. Eight
patients did not develop anti-dengue IgM
and IgG in either of the specimens (Table 1)
and were also negative on virus isolation.
Thus, a total of 97 (92.4%) cases were
diagnosed serologically as current dengue
infection, while 8 (9.8%) cases were
considered as non-dengue febrile illness and
were excluded from further analysis. Of the
97 dengue patients, 46 (47.4%) had primary
and 51 (52.6%) had secondary infection
depending on anti-dengue IgM and IgG
antibody level of acute and convalescent
stage sera (Table 2). Among them, 17
patients developed DHF and one developed
DSS. Of the 18 DHF/DSS patients, 14 had
secondary type of infection and 4 had
primary infection.
Table 1. IgM and IgG dengue antibody positive cases by ELISA
Time of serum
collection
Only IgM
Only IgG
Both IgM and IgM and IgG
antibody
antibody
IgG antibody
antibody
positive cases positive cases positive cases negative cases
n (%)
n (%)
n (%)
n (%)
Total
n (%)
Acute stage serum
(within 5 days of
fever)
21 (20.0)*
38 (36.2)
18 (17.1)*
28 (26.7)
105 (100)
Convalescence
stage serum
(within 14-21
days of fever)
21 (20.0)**
00 (0.0)
76 (72.4)**
8 (7.6)***
105 (100)
* Total number of dengue infection cases detected in acute stage serum (21+18) = 39 (37.1%).
**Total number of dengue infected cases detected in convalescence serum (21+76) = 97 (92.4%).
*** Non-dengue febrile illness cases = 8 (7.6%).
Figures in parenthesis indicate percentage
98
Table 2. Age distribution of dengue patients by primary and secondary dengue infection
Primary infection
n (%)
Secondary infection
n (%)
Total
n
<10
3 (42.9)
4 (57.1)
7
10-19
4 (44.4)
5 (55.6)
9
20-29
30-39
20 (55.6)
11 (45.8)
16 (44.4)
13 (54.2)
36
24
40-49
7 (53.8)
6 (46.2)
13
>50
1 (12.5)
7 (87.5)
8
Total
46 (47.4)
51 (52.6)
97 (100.0)
*Mean±SD
27.5±11.0
30.7±14.4
29.2±12.9
Age in years
P value 0.214 (unpaired student’s ‘t’ test)
* Mean of age
The involvement of all age groups,
especially an adult predominance, was
observed. The mean age of the dengue
patients was 29.2±12.9 years and most
belonged to the 20-29-year age group. The
mean age of primary dengue fever patients
was 27.5±11.7 years and that of secondary
patients was 30.7±14.4 years.
Dengue viruses were isolated from 44
of the 97 dengue patients. The rate of
dengue virus isolation was significantly
higher (68.2% vs 31.2%) among primary
than secondary infection patients (P=0.018).
The isolation rate decreased gradually with
the increasing duration of fever (see Figure).
Virus isolation rate (%)
Figure. Isolation rate of dengue virus between primary and secondary dengue patients by
day of fever
100
4/4
5/6
9/10
8/11
80
60
3/5
1/3
40
5/18
4/14
4/15
20
1/1
0
1st
2nd
3rd
4th
5th
Days of fever
Primary
Secondary
(P=0.018)
P value reached from Z test
99
The distribution of clinical manifestations
in dengue cases is given in Table 3. The
mean body temperature of the dengue
patients was 101.5±1.4 ºF but there was no
significant difference in the mean body
temperature between primary and secondary
DF patients. Other common symptoms
included myalgia (84.5%), headache (82.5%),
arthralgia (68%), lethargy (80.4%) and retroorbital pain (49.5%). Patients with primary
dengue infection presented more commonly
with headache, arthralgia and retro-orbital
pain, whereas lethargy was commonly
associated with secondary dengue infection.
The most common presenting sign was rash,
especially maculopapular type, and its
association was significantly higher with
primary DF cases (P=0.016). Ascitis was
observed in 5 (9.8%) cases; all had
secondary DF. Hepatomegaly was present in
13 (13.4%) patients and its association was
significantly higher (P=0.002) in secondary
DF patients. Abdominal pain (6.2%) and
lymphadenopathy (4.1%) was less frequent
among our study patients though abdominal
pain was more common (7.8%) in secondary
DF patients.
Table 3. Distribution of clinical manifestations in dengue patients
Types of dengue
Clinical characteristics
Primary infection
n=46
Secondary
infection
n=51
Total
n=97
Mean temperature (°F)
101.4±1.4
101.6±1.4
101.5±1.4
Headache
41 (89.1)
39 (76.5)
80 (82.5)
Arthralgia
34 (73.9)
32 (62.7)
66 (68.0)
Retro-orbital pain
25 (54.3)
23 (45.1)
48 (49.5)
Myalgia
39 (84.8)
43(84.3)
82 (84.5)
Lethargy
36 (78.3)
42 (82.4)
78 (80.4)
Rash
19 (41.3)
13 (25.5)
32 (32.9)
Maculopapular
17 (36.9)
8 (15.7)
25 (25.8)
2 (4.3)
5 (9.8)
7 (7.2)
16 (34.8)
19 (37.3)
35 (36.1)
Abdominal pain
2 (4.3)
4 (7.8)
6 (6.2)
Ascitis
0 (0.0)
5 (9.8)
5 (5.1)
Enlarged lymph node
2 (4.3)
2 (3.9)
4 (4.1)
Hepatomegally
1 (2.2)
12 (23.5)
13 (13.4)
Petechial
Anorexia, nausea and
vomiting
P-value
0.016S
0.002S
P-value reached from chi-square analysis
100
Haemorrhagic manifestations in primary
and secondary dengue infections are
indicated in Table 4. The most common
sign of bleeding manifestation, i.e. a positive
tourniquet test, was observed in 18 (18.6%)
patients. Petechiae 15 (15.5%), purpura 12
(12.4%), gum bleeding 12 (12.4%) and
haematemasis/malaena 11 (11.3%) were the
other bleeding manifestations. Besides these,
conjunctival bleeding, haematuria and per
rectal bleeding also occurred in a small
number of patients.
Table 4. Haemorrhagic manifestations among primary and secondary dengue cases
Types of dengue
Primary infection
n=46
Secondary
infection
n=51
Total
n=97
Positive tourniquet test
7 (15.2)
11 (21.6)
18 (18.6)
Petechiae
8 (17.4)
7 (13.7)
15 (15.5)
Purpura
5 (10.9)
7 (13.7)
12(12.4)
Epistaxis
1 (2.2)
2 (3.9)
3 (3.1)
Haematemesis/melaena
8 (17.4)
3 (5.9)
11 (11.3)
Gum bleeding
5 (10.9)
7 (13.7)
12 (12.4)
Per vaginal bleeding
2 (4.3)
1 (2.0)
3 (3.1)
Conjunctival bleeding
1 (2.2)
1 (1.0)
2 (2.0)
Haematuria
1 (2.2)
0 (0.0)
1 (1.0)
Per rectal bleeding
0 (0.0)
2 (3.9)
2 (2.0)
The haematological features in primary
and secondary dengue infections are given
in Table 5. Platelet counts of <1x10 5/mm3
was detected in 22 (22.7%) patients and
was more frequently 16 (31.4%) associated
with secondary DF (Table 5). Haematocrit
value of >45% was observed in 16 (16.5%)
patients
and
a
significantly
higher
association of >45% haematocrit level was
detected among secondary DF patients
(P=0.02).
Haemorrhagic manifestations were
mostly associated with DEN-3 and DEN-4
infections; only one patient with DEN-1
infection had per rectal bleeding (Table 6).
101
Table 5. Haematological features in primary and secondary dengue infection
Types of dengue infection
Primary infection
n=46
Secondary
infection
n=51
Total
n=97
P-value
<1×105
6(13.1)
16(31.4)
22(22.7)
0.059
>1×105
40(86.9)
35(68.6)
75(77.3)
>45%
3(6.5)
13(25.5)
16(16.5)
<45%
43(93.5)
38(74.5)
81(83.5)
Haematology
Platelets/cu mm
Haematocrit
0.020S
P-value reached from chi-square analysis
Table 6. Haemorrhagic manifestations in different serotypes of dengue virus infections
Serotypes of dengue viruses
DEN-1
(n=6)
DEN-2
(n=3)
DEN-3
(n=31)
DEN-4
(n=4)
Positive tourniquet test
0 (0.0)
0 (0.0)
6 (19.4)
3 (75.0)
Petechiae
0 (0.0)
0 (0.0)
1 (3.2)
1 (25.0)
Purpura
0 (0.0)
0 (0.0)
3 (9.7)
1 (25.0)
Epistaxis
0 (0.0)
0 (0.0)
1 (3.2)
0 (0.0)
Haematemesis/melaena
0 (0.0)
0 (0.0)
4 (12.9)
1 (25.0)
Gum bleeding
0 (0.0)
1 (33.3)
4 (12.9)
1 (25.0)
Haematuria
0 (0.0)
0 (0.0)
1 (3.2)
1 (25.0)
Per rectal bleeding
1 (16.7)
0 (0.0)
0 (0.0)
1 (25.0)
Discussion
The secondary dengue infection is usually
associated with more severe manifestations
than the primary infection. In our study, out
of the 97 dengue cases, 46 (47.4%) had
primary and 51 (52.6%) had secondary
102
dengue infection. The presence of
secondary cases indicated that DF was
present in the area and perhaps a low-grade
transmission was continuing during the
previous years. Thus, it may be speculated
that a considerable proportion of cases,
which were diagnosed as “viral flu” in the
past, may have been due to dengue virus
infection. On serological test in acute stage,
only 39 (37.1%) cases were found to be
positive for dengue antibody. However,
when the serum from the patients was
tested again in the convalescence stage, 97
(92.4%) cases were found to be positive,
indicating that they had recent dengue virus
infection (Table 1). Thus, the detection of a
significant number of cases could have been
missed if the tests were not done again at
the convalescence stage. A second test
should therefore be considered at the
convalescence stage to delineate the actual
numbers of infection due to dengue.
Our study indicates the involvement of
all age groups of the population with adult
predominance. This finding is not consistent
with the epidemiological data from other
endemic countries in Asia where young
children were affected[14,15] . However, adults
may also be the major victims of DHF as
reported in different epidemics where
dengue was endemic[16,17,18] . No significant
age or sex difference among patients was
observed between primary and secondary
infections in the present study. Similar
observations were also reported from an
outbreak in Fiji[11] .
Anorexia, nausea, vomiting, abdominal
pain and ascitis were associated more with
secondary than primary dengue infections
(Table 3). In the present study, a statistically
significant (P=0.01) association of rash
(32.9%) among primary dengue infection
cases was demonstrated. The predominant
type of rash in primary infection was
macular
or
maculopapular
whereas
petechial rash was found frequently in
secondary infection. Other studies also
reported similar association of rash in
DF[11,19] . Petechiae were frequently found in
DHF in Cambodian[20] and Thai children[21] ,
while it was less frequent in patients in our
study. In the present study, statistically
significant (P=0.002) hepatomegaly (13.4%)
in secondary dengue infection was noted.
Hepatomegaly was also a common clinical
finding in several other studies[22-24] . Acute
abdominal pain, which is considered as an
early sign of shock in DF/DHF[10] , was
present in only 6 (6.2%) patients in the
present study. This is similar to a study from
Lucknow, India, where abdominal pain was
found in only 5% of cases in an epidemic of
DHF[19] . In some studies, acute abdominal
pain was strongly correlated with DHF and
DSS[10,24,25] . In the present study, ascitis, a
sign of plasma leakage, was present in only
5 (9.8%) secondary infection cases and was
in agreement with the findings of other
studies[25,26] .
The positive tourniquet test, which
reflects capillary fragility and is used as a
guiding test for detecting dengue illness[27] ,
was found in only 18.6% cases in the
present study. There was no statistically
significant difference in test positivity
between primary and secondary DF patients
although it was more frequently positive in
secondary DF. In most studies, comparing
DHF with classic DF, either a higher
incidence of tourniquet test positivity in
DHF[23,26,28] or no difference between the
two groups [24,29] , was reported. These
variable findings are probably because the
pathogenesis of tourniquet positivity and
other bleeding manifestations in dengue
cases are different. Moreover, the defects
which lead to increase in capillary fragility
may also predispose to the development of
shock, thus indicating a better correlation of
the test to shock rather than to haemorrhage.
103
In our study, platelet counts of
<1×105/mm3 were observed among 22
patients and were associated more with
secondary than primary infections (Table 4).
Lack of correlation between a moderately
low
platelet
count
and
bleeding
manifestation in dengue patients has been
noted by other investigators [8]. The bleeding
in dengue is probably due to other factors
with or without the additive effect of
thrombocytopenia. Haemoconcentration, the
evidence of plasma leakage and shock, was
observed in 16 patients with secondary
infection and the association was statistically
significant (P=0.02). Increased haematocrit
was found in 95.5% patients with shock in
contrast to 31.7% cases without shock[21] .
In the present study, the virus isolation
rate was significantly higher in patients with
primary (68.2%) than secondary (31.2%)
infections (P=0.018). Similar results have
been reported in a study from Fiji[11] . In
Bantal, Indonesia, the isolation rate was
100% among patients with primary dengue
infection and 57% with secondary dengue
infection[30] . However, a study from
Thailand reported a high rate of virus
isolation in both primary (98%) as well as
secondary (93%) infections[31] . The reason
for a higher isolation rate in secondary
infection was probably because blood
samples were collected at an early febrile
phase (within 72 hours). In our study, the
isolation rate was also high in both primary
(100%) and secondary (68%) infections in
early febrile period, i.e. on day 1 of fever.
Thereafter, the rate of isolation was always
higher in primary than in secondary dengue
infection in the subsequent days of fever
(Figure).
In conclusion, the clinical diagnosis is
not very reliable in dengue infection as
104
there is a wide variation in the presence of
various symptoms, which may misguide the
physician regarding the severity of the
disease. Therefore, the use of clinical casedefinition results in inaccuracies. Thus, in
order to identify the cases accurately, one
should take the help of a simple and cheap
diagnostic test that is able to diagnose
accurately in the laboratory. In this context
ELISA is clearly very promising for the
confirmation of clinical diagnosis and to
differentiate
between
primary
and
secondary infections. This will guide the
clinician to take prompt and meticulous
clinical management and early hospitalization
of severe cases. Indeed, it is hoped that
accurate laboratory diagnosis will not only
reduce the morbidity and mortality but will
also reduce the economic burden of the
patient and the government.
Acknowledgements
We express our sincere thanks to
Dr Vorndam Vance, CDC Dengue Lab, San
Juan, Puerto Rico, USA, and Dr Ichiro
Kurane, Arbovirus Research Laboratory,
NIID, Japan, for their kind support in
providing cell line and anti-dengue
monoclonal antibodies. We are also grateful
to the patients who participated in the study.
We would like to thank Mr Tauhid Uddin
Ahmed, Ex-PSO, IEDCR, Bangladesh, and
Noor-e -Jannat for providing mosquito
colony and rearing techniques. We would
also like to thank Dr Bijon Kumar Sil, ExSenior
Scientific
Officer,
Bangladesh
Livestock Research Institute, Savar, Dhaka,
Bangladesh. We also express our gratitude
to the Department of Medicine and
Paediatrics, BSMMU, and Dhaka Medical
College, Bangladesh, for their support in the
study.
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Philippines: clinical and virological findings
in 517 hospitalized patients. American
1988, 39: 110-116.
[29] Kalayanarooj S, Vaughn DW, Nimmannitya
S, Green S, Suntayakorn S, Kunentrasai N,
Viramitrachai
W,
Ratanachu-eke
S,
Kiatpolpoj S, Innis BL, Rothman AL, Nisalak
A and Ennis FA. Early clinical and laboratory
indicators of acute dengue illness. The
Journal of Infectious Diseases, 1997, 176:
313-321.
[30] Gubler DJ, Suharyono S, Lubis I, Eram S and
Gunarso S. Epidemic dengue 3 in Central
Java, associated with low viremia in man.
Hygiene, 1981, 30(5): 1094-1099.
BL, Nimmannitya S, Suntayakorn S,
Rothman AL, Ennis FA and Nisalak A.
Dengue in early febrile phase: viremia and
antibody responses. The Journal of
[25] Guzman MG, Kouri G, Morior L and
Fernandez A. A study of fatal haemorrhagic
dengue cases in Cuba 1981. Bulletin of Pan
American Health Organization, 1984, 18:
213-220.
106
Current Status of Dengue Diagnosis at the
Center for Disease Control, Taiwan
Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien,
Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin
and Jyh-Hsiung Huang#
Center for Research and Diagnostics, Center for Disease Control, Department of Health,
161, Kun-Yang Street, Taipei, Taiwan
Abstract
A national-level diagnostic laboratory has been set up in Taiwan for routine diagnosis of reported cases of
dengue fever (DF)/dengue haemorrhagic fever (DHF), Japanese encephalitis (JE) and yellow fever (YF).
The facilities include serological diagnosis, virus isolation by cell culture, molecular diagnosis and
molecular tools for epidemiological investigations. To detect and differentiate dengue, JE and YF virus
infections, a differential diagnostic system has been developed. For acute-phase sera, virus isolation by
cell culture and real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) has been
established. For all of the serum samples reported, serological diagnosis of specific antibodies based on
envelope and membrane (E/M)-specific capture IgM and IgG enzyme-linked immunosorbent assay
(ELISA) are performed. In this report, a case study from Taiwan has been presented with the analysis of
959 serum samples (including some paired sera) collected between day 1-30 of illness from 799
confirmed dengue cases reported in 2002. The results demonstrated that 94.5% of acute-phase serum
samples of confirmed dengue cases could be identified as positive or probable with the combined use of
real-time one-step RT-PCR and E/M-specific capture IgM and IgG ELISA. Furthermore, a nonstructural
protein NS1 serotype-specific indirect IgG ELISA has been developed and used to analyse dengue NS1specific IgG antibodies. Both E/M-specific capture IgM and IgG ELISA and the NS1 serotype-specific
indirect IgG ELISA have been used to detect and differentiate primary and secondary dengue virus
infections. In addition, the NS1 serotype-specific indirect IgG ELISA has the potential of replacing the
plaque-reduction neutralization test (PRNT) and is being used for a large-scale seroepidemiological study.
Keywords: Dengue virus, virus isolation, real-time one-step RT-PCR, E/M-specific capture IgM and IgG ELISA, NS1
serotype-specific indirect IgG ELISA.
#
E-mail: [email protected]; Tel.: 886-2-26531374, Fax: 886-2-27883992
107
Dengue Diagnosis in CDC, Taiwan
Introduction
The dengue viruses cause a broad spectrum
of illness ranging from inapparent infection,
mild undifferentiated fever and classic
dengue fever to a more severe form, dengue
(DHF/DSS), resulting in high morbidity and
mortality. The diagnosis of dengue virus
infection based on clinical syndromes is not
reliable, and a confirmation of the infection
should rely on laboratory diagnosis with the
detection of the specific virus, viral antigen,
genomic sequence and/or antibodies.
A rapid, simple, sensitive and specific
assay system to detect the virus in the acutephase serum is essential to improve the
clinical treatment, etiological investigation
and disease control of dengue virus
infection. Among the various assays for virus
detection, virus isolation by cell culture and
dengue virus antigen detection by ELISA[1,2]
suffer from some disadvantages – while the
former needs a longer time, the latter has
low sensitivity. However, recent advances in
molecular diagnosis have demonstrated that
various RT-PCR protocols can be reliably
used to detect the viral genomic sequence
with high sensitivity and specificity. More
recently, several investigators have reported
real-time RT-PCR assays for the detection of
dengue virus in acute-phase serum
samples[3-7]. The real-time RT-PCR assay has
many advantages over the conventional RTPCR methods, which include rapidity,
quantitative
measurement,
lower
contamination rate, higher sensitivity, higher
specificity and easy standardization.
For convalescent sera, detection of
specific IgM and IgG antibodies based on
108
haemagglutination inhibition (HI) test and
E/M-specific capture IgM and IgG ELISA[8]
are the two most commonly used serological
techniques for the routine diagnosis of
flavivirus infection. The serodiagnosis of
flavivirus is rather complicated due to the
high cross-reactivity of IgG antibodies to
homologous and heterologous viruses. We
have attempted to set up an ELISA system
that can be easily and reliably used to detect
and differentiate various flavivirus infections.
To accomplish this goal, three different
forms of ELISA were developed including:
(i) E/M-specific capture IgM and IgG ELISA;
(ii) E/M-specific antigen-coated indirect IgM
and IgG ELISA; and (iii) NS1 serotypespecific indirect IgG ELISA[9-11] .
Case study of 2002 outbreak of
DEN-2 in southern Taiwan
We present here a case study of the analysis
of biological material obtained during a
DEN-2 outbreak in 2002 in southern
Taiwan, by utilizing the facilities available in
the national diagnostic laboratory.
A major DEN-2 epidemic occurred in
southern Taiwan, affecting Kaohsiung city,
Kaohsiung county and Pingtung county
between October 2001 and December 2002,
with more than 5,000 confirmed cases.
Among these, 227 cases were classified as of
DHF with 21 deaths. This outbreak was a
repeat of the 1987-1988 DEN-1 epidemic in
many aspects[11,12] . In this report, we present
the results of a total of 959 acute- and
convalescent-phase sera collected from 799
confirmed dengue patients reported to the
Kun-Yang office of the Center for Disease
Control (CDC), Taiwan, 2002.
Human serum samples
The serum samples used in this study were
collected from the confirmed cases of
dengue patients reported to the Arbovirus
Laboratory in the Kun-Yang office, CDC,
Department of Health, 2002. A total of 959
acuteand
convalescent-phase
sera
collected from 799 confirmed dengue
patients were analysed. Most of these serum
samples were from the major DEN-2
outbreak, together with a few serum
samples from imported cases contracted
during travel to the neighbouring South-East
Asian countries.
Case definitions
A confirmed case of dengue virus infection
was defined as febrile illness associated
with: (i) the isolation of dengue virus;
(ii) positive test of real-time one-step RTPCR; (iii) positive seroconversion or ≥ fourfold increase in dengue-specific IgM or IgG
antibody from appropriately timed paired
serum; or (iv) high-titer dengue-specific IgM
and IgG antibody in a single serum
specimen where cross-reaction to Japanese
encephalitis (JE) had been excluded. Sera
collected during day 1-7 after the onset of
symptoms are referred to as acute-phase
sera. Early and late convalescent sera refer
to the specimens collected during day 8-13
and day 14-30, respectively.
Virus isolation by cell culture and
virus antigen preparation
The isolation of dengue virus by cell culture
and virus antigen preparation from culture
supernatants of DEN-1, DEN-2, DEN -3,
DEN-4 or JE-virus infected Vero cells were
performed as previously described[7,9] . The
culture supernatants were used as the
source of E/M and NS1 antigens for ELISA.
The control antigen was prepared by the
same procedure from Vero cells culture
without viral infection.
One-Step SYBR Green I Real-Time
RT-PCR
One-step SYBR Green I real-time RT-PCR
for dengue virus was performed in the
Mx4000 TM
quantitative
PCR
system
(Stratagene) as recently described[7] . Briefly,
a set of flavivirus- (in the NS5 gene region),
dengue- and serotype-specific primer pairs
(in the core gene region) was selected and
used for analysis. To assure the specificity of
amplicons produced from SYBR Green I
real-time RT-PCR in daily routine screening,
both flavivirus- and dengue -specific primer
pairs were used for each of the serum
samples tested. Serum samples found
positive for initial screening were then
tested for serotype by each of the four
serotype-specific primer pairs.
ELISA
E/M-specific capture IgM and
IgG ELISA
A modified E/M-specific capture IgM and
IgG ELISA was performed to measure the
dengue-specific IgM and IgG antibodies as
recently
described[13] .
Briefly,
each
microtiter 8 wells strip was coated with
5 µg/ml, 100 µl/well of affinity purified goat
anti-human IgM (µ-specific) or IgG (γspecific) antibodies, followed by incubation
with 1:100 diluted serum, incubation with
cocktail contained 1:3 diluted pooled virus
antigens from culture supernatants of DEN-1,
DEN-2, DEN-3 or DEN -4 infected Vero cells
109
and 1 µg/ml mAb D56.3, incubation with
1:1,000 diluted alkaline phosphataseconjugated goat anti-mouse IgG (γ-specific).
The enzyme activity was developed with the
substrate
p-nitrophenyl-phosphate
and
optical density (OD) was taken 30 minutes
later. For routine screening, culture
supernatant from JE virus-infected Vero cells
was used as negative control antigen due to
the limited cross-reactivity between dengueand JE-specific IgM antibodies measured by
E/M-specific capture IgM ELISA. This was in
contrast to the high cross-reactivity of
dengue- and JE-specific IgG antibodies
among dengue patients with secondary
infection.
E/M-specific antigen-coated indirect
IgM and IgG ELISA
E/M-specific antigen-coated indirect IgM
and IgG ELISA were performed as previously
described[10] . Two-fold serial dilutions of
appropriately timed paired sera diluted from
1:100 to 1:12,800 were analysed to
determine whether ≥ four fold increase of
dengue-specific IgM or IgG antibody could
be found. This assay has the advantage of
better sensitivity in the detection of IgM and
IgG antibody increase than E/M-specific
capture IgM and IgG ELISA.
NS1 serotype-specific indirect
IgG ELISA
NS1 serotype-specific indirect IgG ELISA
was performed as previously described[9,13] .
Briefly, each microtiter 8 wells strip was
coated with 5 µg/ml, 100 µl/well of mAbs
D2/8-1, followed by incubation with 1:3
diluted NS1-containing culture supernatants
of DEN-1, DEN -2, DEN-3, DEN-4 or JE
viruses-infected Vero cells, incubation of
serum samples at a 1:50 dilution, incubation
110
with goat anti-human IgG conjugated to
alkaline phosphatase. The enzyme activity
was developed and OD was taken 30
minutes later.
Data analysis
For E/M-specific capture IgM and IgG ELISA,
primary dengue virus infection was defined
if the IgM:IgG OD ratio was ≥1.2, or
secondary if the OD ratio was <1.2. For
those sera with positive NS1-specific IgG
antibody response, NS1 serotyping was
calculated by the ratio of the highest OD
value and the second highest OD value read
from the four dengue serotypes. Positive
serotype-specificity is defined if the OD
ratio is ≥1.2 and negative serotypespecificity is defined if the OD ratio is <1.2.
Based on NS1 serotype-specific indirect IgG
ELISA, primary dengue virus infection was
defined if: (i) negative NS1-specific IgG
antibody response was found for sera
collected between day 1 and 14 of illness,
or (ii) positive serotype-specificity for sera
collected ≥9 days of illness. Secondary
dengue virus infection was defined if:
(i) positive NS1-specific IgG antibody
response was found for sera collected
between day 1 and 8 of illness, or
(ii) positive NS1-specific IgG antibody
response and negative serotype-specificity
was found any time after the onset of
infection.
Results
Dengue surveillance system and
laboratory diagnosis in Taiwan
Taiwan has an integrated programme for
dengue surveillance and control. The
dengue prevention and control centre is a
mission-oriented structure jointly sponsored
by the Department of Health and the
Environment
Protection
Administration
responsible for the planning and execution
of dengue control. To assure the
effectiveness of dengue surveillance, three
report systems are currently associated with
the
dengue
surveillance
programme
including: (i) hospital-based passive report
system; (ii) syndrome report system (under
the classification of viral haemorrhagic
fever); and (iii) active surveillance system.
The Arbovirus Laboratory in Kun-Yang office,
Taipei City, CDC, Department of Health, is
responsible for the diagnosis of various
flavivirus. In addition, a second dengue
diagnostic laboratory was set up in the
Fourth Branch, Kaohsiung City, CDC, in July
2002 to provide prompt service to
Kaohsiung city, Kaohsiung county and
Pingtung county due to the large samples
generated by the DEN-2 outbreak. For
routine diagnosis, serum samples from the
reported cases were sent to the laboratory
on a daily basis and tested according to the
flow chart shown in Figure 1. The periods of
time required to complete these tests were
7 days, 6 hours and 4 hours for virus
isolation, real-time one-step RT-PCR and
E/M-specific capture IgM and IgG ELISA,
respectively. The results were reported as
positive, negative or probable cases. The
probable case was referred to ELISA result
with only IgM or IgG antibody positive. For
negative
and
probable
cases,
the
convalescent serum samples collected after
day 14 of the illness were demanded and
tested for the presence of or increase in IgM
and/or IgG antibodies.
Figure 1. Flow chart of laboratory diagnosis of dengue virus infection
1-7 days
Serum
1-30 days
Virus isolation
(+) Result
Probable or
(-) Result
Real-Time
RT-PCR
E/M-specific
Capture
IgM/IgG ELISA
(+) Result
Current
dengue
infection
1:100 dilution
>=14 days serum
Probable or (-) Result
E/M-specific
Capture
IgM/IgG ELISA
Final
Result
1:100 dilution
111
provided a good example of the dynamic
change of dengue virus and specific IgM and
IgG antibodies in the acute- and
convalescence-phase sera from patients with
primary or secondary dengue virus infection
covering all four serotypes. As shown in
Figure 1, all of the three assays were
performed for acute-phase sera, whereas
only E/M-specific capture IgM and IgG
ELISA was tested for convalescence-phase
sera.
Representative results of routine
diagnosis measured by virus
isolation, real-time one-step
RT-PCR, and E/M-specific capture
IgM and IgG ELISA
Table 1 shows the representative results of
serum samples analysed by virus isolation,
real-time one-step RT-PCR and E/M-specific
capture IgM and IgG ELISA. The results
Table 1. Representative results of routine diagnosis of serum samples from reported dengue cases
Dengue
infection
Dengue
serotype
Primary
infection
Secondary
infection
Onset
Virus
days isolation
Real-time one-step RT-PCR
Ct value
E/M-specific capture IgM and
IgG ELISA
OD 405 nm
Flavi- Dengue- Serotype- Dengue- JE-IgM Denguespecific specific specific
IgM
IgG
JE-IgG
DEN-1
3
16
+
28
25
25
0.526
2.791
0.237
0.951
0.200
1.687
0.330
1.453
DEN-2
2
15
+
26
33
29
0.209
3.484
0.192
0.245
0.182
1.165
0.174
0.708
DEN-3
3
16
+
25
31
32
0.478
3.074
0.104
0.329
0.148
2.535
0.228
1.704
DEN-4
3
15
–
26
35
35
0.094
2.225
0.082
0.148
0.057
1.301
0.063
0.096
DEN-1
1
16
+
27
28
24
0.166
1.949
0.151
0.469
0.226
3.606
0.176
1.699
DEN-2
2
18
+
17
18
20
0.267
0.496
0.217
0.157
0.222
3.482
0.164
1.069
DEN-3
1
7
+
18
23
24
0.175
3.631
0.142
0.691
0.180
3.494
0.216
3.474
DEN-4
7
12
+
20
28
29
1.298
1.468
0.105
0.108
2.883
3.424
0.273
0.637
+ = positive
– = negative
Due to the long time needed to isolate
virus using the cell culture method, it has
limited value in rapid diagnosis. The isolated
virus, however, is the key material for the
later studies of molecular epidemiology and
pathogenesis. The real-time one-step SYBR
Green I RT-PCR we developed is a simple,
112
reliable and universal RT-PCR protocol that
can be used to systemically detect and
differentiate various flavivirus. To assure the
specificity of amplicons produced from
SYBR Green I real-time RT-PCR in routine
screening, both flavivirus- and denguespecific primer pairs were run (Table 1).
Those serum samples positive for initial
screening were then tested for serotype by
each of the four serotype-specific primer
pairs. The analysis of acute-phase serum
samples demonstrated that the one-step
SYBR Green RT-PCR was more sensitive to
the virus isolation method and could detect
two-times more the acute-phase sera with
positive dengue-specific IgM and/or IgG
antibodies[7] .
Therefore, a positive dengue-specific IgM
and IgG antibody response can be easily
used to detect and differentiate primary and
secondary dengue virus infections.
The E/M-specific capture IgM and IgG
ELISA has several advantages in the
detection of dengue-specific IgM and IgG
antibodies including: (i) high sensitivity;
(ii) high specificity (only for IgM antibody);
(iii) analysis of isotype-specific antibody
responses; (iv) easy automation to test large
amount
of
serum
samples;
and
(v) differentiation of primary and secondary
dengue infections. The results shown in
Table 1 demonstrated the low crossreactivity between dengue - and JE-specific
IgM antibody and inverse pattern of IgM:IgG
OD ratio of primary and secondary infection.
Occasionally, there were acute-phase sera
which tested positive with E/M-specific
capture IgM or IgG antibody response, but
did not show an apparent increase in
antibody titers in convalescent sera. Due to
the higher sensitivity of E/M-specific indirect
IgM and IgG ELISA (especially for IgG
antibody), it can be reliably used to
determine whether ≥ four-fold increase of
dengue-specific IgM or IgG antibody were
presented. Figure 2 shows an example
where significant dengue-specific IgG
antibody increase in a convalescent serum.
E/M-specific antigen-coated
indirect IgM and IgG ELISA
for the detection of dengue
virus infection
Figure 2. E/M-specific antigen-coated indirect IgM and IgG ELISA to detect the increase of
dengue-specific IgM and/or IgG antibodies in the pair sera. The serum samples showed a
= four-fold increase in dengue-specific IgG antibody titer in the convalescent sera
3.0
2.5
OD 405nm
2.0
S9100837A-day 5 IgM
S9100837B-day 15 IgM
S9100837A-day 5 IgG
S9100837B-day 15 IgG
1.5
1.0
0.5
0.0
50x
100x
200x
400x
800x
1600x
3200x
6400x
Patient serum dilution (S9100837)
113
respectively. The positive rate for E/Mspecific capture IgM and/or IgG ELISA was
31.6%, 32.6%, 30%, 39%, 52.6%, 87.5%
and 80% for day 1-7 of illness, respectively.
Thus, the combined results of real-time RTPCR and E/M-specific capture IgM and IgG
ELISA (IgM and/or IgG positive) could detect
an average 94.5% (89.7% to 97.5%) of
acute-phase serum samples of confirmed
dengue cases. The results also showed that
the real-time RT-PCR was more sensitive
than virus isolation although very few sera,
which were virus-isolation positive, were
missed by real-time RT-PCR.
Statistical analysis of results of
serum samples from confirmed
dengue cases reported in 2002
Table 2 shows the comprehensive analysis
of the results of serum samples from
confirmed dengue cases reported to KunYang office in 2002. The results of virus
isolation, real-time RT-PCR and E/M-specific
capture IgM and IgG ELISA were analysed
separately or in combination from the sera
samples collected on 1-30 day of illness.
The positive rate for real-time RT-PCR was
74.7%, 69.5%, 72.3%, 76.6%, 57.7%,
36.3% and 22.2% for day 1-7 of illness,
Table 2. Statistical analysis of results of serum samples from confirmed dengue cases
reported in 2002
Assays
Days after onset
1
2
Total serum no.
tested
95
V.I.+ (Virus
isolation)
53
% of V.I.+
3
4
5
6
7
8
9
95
130
52
64
10
11
128
97
80
45
58
27
24
58
32
9
2
–
–
–
–
–
55.8 54.7 49.2 45.3 33.0 11.3
9
12
13
16
7
14-30 Total
148
959
–
–
270
4.4
–
–
–
–
–
–
–
–
RT-PCR+ and
V.I.+
52
50
60
57
31
8
2
–
–
–
–
–
–
–
260
RT-PCR+ or
V.I.+
72
68
98
99
57
30
10
–
–
–
–
–
–
–
434
RT-PCR+ (Realtime)
71
66
94
98
56
29
10
–
–
–
–
–
–
–
424
74.7 69.5 72.3 76.6 57.7 36.3 22.2
–
–
–
–
–
–
–
–
–
–
–
–
246
% of RT-PCR+
-
RT-PCR (Real time)
24
29
36
30
41
51
35
–
–
–
ELISA + (E/Mspecific capture
IgM+IgG+)
20
25
21
27
29
41
27
45
19
23
8
12
7
140
444
Probable (ELISA
IgM+ or IgG+)
10
6
18
23
22
29
9
9
8
1
1
3
0
8
147
31.6 32.6 30.0 39.1 52.6 87.5 80.0 93.1
100
100
100 93.8 100
100
0
0
% of IgM+
and/or IgG+
RT-PCR -, V.I.-,
ELISA IgM-IgG-
114
3
3
5
3
9
1
3
4
0
1
0
0
–
32
Assays
+
Days after onset
1
2
3
4
5
6
7
8
9
10
11
12
13
14-30 Total
RT-PCR and
IgM+IgG+
3
2
4
5
6
7
2
–
–
–
–
–
–
–
29
RT-PCR+ and
IgM+IgG-
6
2
4
7
9
13
1
–
–
–
–
–
–
–
42
RT-PCR+ and
IgM-IgG+
1
3
4
12
5
1
1
–
–
–
–
–
–
–
27
RT-PCR+ and
IgM-IgG-
61
59
82
74
36
8
6
–
–
–
–
–
–
–
326
RT-PCR - and
IgM+IgG+
17
23
17
22
23
34
25
45
19
23
8
12
7
140
415
RT-PCR - and
IgM+IgG-
3
0
8
3
4
14
6
8
7
0
0
3
0
6
62
RT-PCR - and
IgM-IgG+
0
1
2
1
4
1
1
1
1
1
1
0
0
2
16
RT-PCR - and
IgM-IgG-
4
5
9
4
10
2
3
4
0
0
0
1
0
0
42
RT-PCR+ or
ELISA +
88
89
111
120
79
63
35
45
19
23
8
12
7
140
839
92.6 93.7 85.4 93.8 81.4 78.8 77.8 77.6 70.4 95.8 88.9 75.0 100
94.6
–
7
148
917
100 93.8 100
100
–
% (RT-PCR +or
ELISA +) / Total
serum no.
tested
RT-PCR+ or
ELISA IgM+
and/or IgG+
91
90
121
124
87
78
54
27
24
% (RT-PCR +or 95.8 94.7 93.1 96.9 89.7 97.5 93.3 93.1
ELISA IgM+
and/or IgG+) /
Total serum no.
tested
100
100
NS1 serotype-specific indirect IgG
ELISA in the differentiation of JE,
primary and secondary dengue
virus infections and for the DEN
serotyping of primary infection
More recently, we have developed a NS1
serotype-specific indirect IgG ELISA in the
detection and differentiation of primary and
secondary infections. Comparisons of E/M-
42
9
15
specific capture IgM and IgG ELISA and NS1
serotype-specific indirect IgG ELISA showed
good correlation with 95.90% agreement[13] .
Most importantly,
retrospective seroepidemiological studies on serum samples
collected from Liuchiu Hsiang, Pingtung
county and Tainan city in southern Taiwan,
demonstrated that NS1 serotype-specific
indirect IgG ELISA could replace plaquereduction neutralization test (PRNT) for
seroepidemiological study to differentiate JE,
115
primary and secondary dengue virus
infections and for the DEN serotyping of
primary infection[11] .
Discussion
Recent advances in molecular and
serological assays have revolutionized the
laboratory
diagnosis
of
flavivirus
infection[7,13,14]. Rapid diagnosis of dengue
virus infection in the acute-phase sera,
which is important for disease control
measures and potential treatment, will
require very sensitive and specific assays.
With the maturation of real-time RT-PCR
technique, its routine application to clinical
and laboratory diagnosis has now become a
reality. For serodiagnosis, E/M-specific
capture IgM and IgG ELISA has become the
new standard assay for the detection and
differentiation of flavivirus infection.
The large DEN -2 epidemic in southern
Taiwan, was uncontrolled despite vigorous
attempts to contain it by the central and
local health governments during October
2001 – December 2002. Although
insecticide -resistance was blamed as an
important factor for this disaster, other
elements
including,
political,
social,
environmental, community and human
factors were also responsible for this setback.
This epidemic was a strong warning to us
and suggested that more effective measures
should be sought and applied. There is an
urgent need to improve the surveillance
system and laboratory diagnosis which
would help to identify confirmed cases in
the acute-phase sera and respond promptly
and effectively to control the transmission
chain.
Along with the progress of the DEN-2
outbreak in 2002, we have developed and
116
evaluated the real-time RT-PCR method for
rapid detection of dengue virus in the acutephase sera. In this report, we have
presented a detailed analysis of a total of
959 acute- and convalescent-phase sera
collected from 799 confirmed dengue
patients reported to the Kun-Yang office of
CDC, Taiwan, in 2002. The results
demonstrated that 94.5% of acute-phase
serum samples of confirmed dengue cases
could be identified as positive or probable
with the combined use of real-time one-step
RT-PCR and E/M-specific capture IgM and
IgG ELISA. The results are very encouraging
and suggest that these two assays are wellsuited for routine tests for the early diagnosis
of dengue virus infection.
Conclusion
The real-time RT-PCR assay has many
advantages over conventional RT-PCR
methods, which include rapidity, quantitative
measurement, lower contamination rate,
higher sensitivity, higher specificity and easy
standardization.
Therefore,
real-time
quantitative assay might eventually replace
virus isolation and conventional RT-PCR as
the new gold standard for the rapid diagnosis
of virus infection in the acute-phase serum
samples.
Acknowledgements
We wish to thank Hsiu-Ling Pan, Yun-Yih
Chang and Chih-Heng Chen for their expert
technical assistance. This work was in part
supported by grants DOH91-DC -2007 and
DOH91-DC -2016 from the Center for
Disease Control, Department of Health,
Taiwan.
References
[1] Alcon S, Talarmin A, Debruyne M, Falconar A,
Deubel V and Flamand M. Enzyme-linked
immunosorbent assay to dengue virus type 1
nonstructural protein NS1 reveals circulation
of the antigen in the blood during the acute
phase of disease in patients experiencing
primary or secondary infections. J Clin
Microbiol, 2002, 40: 376-381.
[2] World
Health
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Dengue
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[3] Callahan JD, Wu SJ, Dion-Schultz A,
Mangold BE, Peruski LF, Watts DM, Porter
KR, Murphy GR, Suharyono W, King CC,
Hayes CG and Temenak JJ. Development
and evaluation of serotype- and groupspecific fluorogenic reverse transcriptase
PCR (TaqMan) assays for dengue virus. J Clin
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[4] Drosten C, Gottig S, Schilling S, Asper M,
Panning M, Schmitz H and Gunther S.
Rapid detection and quantitation of RNA of
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[5] Houng HH, Hritz D and Kanesa-thasan N.
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[6] Laue T, Emmerich P and Schmitz H.
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LJ, Sue CL, Lin TH and Huang JH.
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[8] Innis BL, Nisalak A, Nimmannitya S,
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Suntayakorn S, Puttisri P and Hoke CH. An
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[9] Shu PY, Chen LK, Chang SF, Yueh YY, Chow
L, Chien LJ, Chin C, Lin TH and Huang JH.
Dengue NS1-specific antibody responses:
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Japanese encephalitis virus: potential
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L, Chien LJ, Chin C, Yang HH, Lin TH and
Huang JH. Potential application of
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[12] Ko YC, Chen MJ and Yeh SM. The
predisposing and protective factors against
dengue virus transmission by mosquito
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Comparison of capture immunoglobulin M
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Lab Immunol, 2004, 11: 642-650.
117
Detection of Dengue Virus Serotype-specific IgM
by IgM Capture ELISA in the Presence of
Sodium thiocyanate (NaSCN)
Masaru Nawa* #, Tomohiko Takasaki**, Mikako Ito**, Ichiro Kurane**
and Toshitaka Akatsuka*
*Department of Microbiology, Saitama Medical School, 38, Moroyama, Saitama 350-0495, Japan
**Division of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases,
1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Abstract
Dengue virus serotype-specific IgM was detected by IgM-capture enzyme-linked immunosorbent assay
(IgM-ELISA) in the presence of a chaotropic agent, sodium thiocyanate (NaSCN). NaSCN did not affect the
reactions between anti-human IgM and patients’ IgM, and between dengue viral antigens and detecting
antibody, peroxidase-conjugated flavivirus-specific monoclonal antibody D1-4G2 IgG. Among 18 dengueconfirmed cases, highest IgM responses were detected to infecting serotypes in 14 cases in the presence of
0.5 M of NaSCN. The results indicate that: (i) the protein-denaturing agent, NaSCN, affects antigenantibody reaction in IgM-ELISA, and enables the differentiation of serotype-specific IgM from cross-reactive
IgM; and (ii) IgM responses against the infecting serotypes are higher than those against the other three
serotypes in most primary dengue virus infection. In conclusion, the addition of NaSCN to IgM-capture
ELISA is useful for highlighting serotype-specific IgM responses in primary dengue virus infections.
Keywords: Dengue, IgM-capture ELISA, serotype-specific IgM response, sodium thiocyanate, NaSCN.
Introduction
Dengue is currently one of the most
important arboviral disease in humans.
Dengue viruses, belonging to the family
Flaviviridae,
are
comprised of
four
antigenically cross-reactive serotypes and are
responsible for epidemics in tropical and
subtropical countries. Since the dengue
outbreaks in Osaka, Kobe, Hiroshima and
Nagasaki from 1942 to 1945, dengue has not
occurred in an epidemic form in Japan[1].
However, imported dengue cases have been
reported[2]. Approximately five million
Japanese people annually visit countries in
#
tropical and subtropical areas and nearly two
million people visit Japan from these areas.
Therefore, dengue fever (DF) and dengue
haemorrhagic fever (DHF) has become an
infectious disease of significance and worthy
of more attention from the medical
community in Japan.
We have earlier reported the laboratory
diagnosis of dengue by reverse transcription
polymerase chain reaction (RT-PCR) and
IgM-ELISA[3-5]. We demonstrated that IgMELISA was a reliable diagnostic method and
that IgM responses were generally serotype
cross-reactive but often highest against
E-mail: [email protected]; Tel.: +81-49-276-1438; Fax: +81-49-295-9107
118
Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN
infecting virus serotype in most Japanese
cases[4]. The serotype specificity of IgM
responses in dengue patients has been
controversial[6-8]. Burke had reported that
serotype-specific
IgM
responses
corresponding to the isolated virus type were
detected in primary dengue virus infection[6].
Gubler had reported that in dengue infection,
frequent monotypic IgM responses were not
correlated with the virus serotype isolated
from patients[7]. In 1984, Inouye et al. [9]
demonstrated a new technique for the
differentiation of antibody avidity after virus
infection, i.e. rubella, rota and Japanese
encephalitis viruses. They estimated the
antibody avidity to viral antigen using a low
concentration of a protein-denaturing agent,
guanidine hydrochloride, in the diluent of
antibody in the ELISA. They concluded that
the “stringent immunosorption” technique
was useful for investigating the antigenic
relationship among closely-related viruses.
In the present study, we examined
serotype-specific IgM responses under
stringent conditions in the presence of a
chaotropic agent, sodium thiocyanate
(NaSCN), in the reaction mixture of dengue
viral antigens and patients’ sera. The
development of a simple method to
distinguish serotype-specific reaction from
cross-reaction will be useful not only for
laboratory
diagnosis
but
also
for
seroepidemiological studies.
Twenty-eight serum specimens from 18
confirmed Japanese dengue cases were used
in the study. Serum samples from 22
Japanese subjects with other illnesses, who
had never been to areas where dengue was
epidemic-prone or endemic, were used as
the control. These sera were obtained for
diagnostic purposes in clinics and hospitals in
Japan from 2000 to 2002 and sent to the
Department of Virology 1, National Institute
of Infectious Diseases, Tokyo, Japan.
Prototype
dengue
viruses
were
propagated in the Aedes albopictus
mosquito cell clone C6/36, and the infected
cell culture supernatants were inactivated by
incubation with beta-propiolactone at a final
concentration of 0.2% for 30 minutes at
37 °C as previously reported[10]. Tetravalent
and four monovalent dengue viral antigens
were prepared by the method as previously
reported[4,5].
The IgM capture ELISA was carried out
according to the method described
previously[3-5]. Anti-human IgM (µ chain
specific) goat serum and peroxidaseconjugated anti-human IgM were purchased
from Sigma-Aldrich, Inc, USA. The IgG
fraction of the flavivirus-specific monoclonal
antibody, D1-4G2, was prepared by the
Protein G affinity chromatography kit
(ImmunoPure G IgG Purification kit, PIERCE,
USA), and then conjugated with horseradish
peroxidase by a commercial kit (EZ-Link Plus
Activated Peroxidase kit, PIERCE, USA).
In order to detect the serotype-specific
reaction between dengue viral antigen and
dengue virus-specific IgM antibody, patient
serum was treated with the Protein G affinity
chromatography kit described above. The
IgG fraction in the serum specimen was
removed by adding Protein G beads
according to the manufacturer’s instruction.
Unless otherwise stated, data were presented
as the mean of independent two-to-three
assays.
One of the authors has previously reported
that the addition of NaSCN to the reaction
mixture of ELISA highlights serotype-specific
119
reaction between crude dengue viral antigen
and anti-dengue hyperimmunized mouse
sera[11]. The antigen-antibody reaction was
affected not only by the concentration of
NaSCN but also by the procedures of the
NaSCN treatment. A concentration higher
than 0.7 M inhibited the reaction nonspecifically, while a concentration lower than
0.3 M had no effect on the discrimination of
serotype-specific reaction from the cross-
reactive one. According to these results, we
decided to use NaSCN in IgM-capture ELISA
at the following conditions: (i) NaSCN was
included at a final concentration of 0.5 M in
10% normal calf serum-PBS; (ii) peroxidaseconjugated flavivirus- specific monoclonal
antibody D1-4G2 (D1-4G2) was diluted in
0.5 M NaSCN; and (iii) viral antigens
captured by patients’ IgM were detected by
the detection antibody in 0.5 M NaSCN.
Figure 1. The effects of NaSCN treatment on antigen-antibody reaction
(a)
1.5
Control
NaSCN
ELISA O.D.
1
0.5
0
50 60 70 80 90100
200
300
400
Serum dilution
(b)
2
Control
NaSCN
ELISA O.D.
1.5
1
0.5
0
1
2
3
4
5
6 7 8
Antigen dilution
(a) Human serum was serially diluted from 1:50 to 1:400 with 10% CS-PBS containing 0.5 M NaSCN, and IgM was
captured on anti-human IgM goat serum-coated wells. IgM was detected with peroxidase-conjugated anti-human
IgM goat serum. (b) Serially diluted tetravalent dengue viral antigen was captured on the solid phase sensitized
with D1-4G2 IgG, and then detected with peroxidase-conjugated D1-4G2 IgG in the presence of 0.5 M NaSCN.
120
Figure 1 shows the reactions between
anti-human IgM and patients’ IgM (a), and
dengue viral antigen and the detection
antibody (b), in the presence of 0.5 M
NaSCN in the ELISA. These two reactions
were not affected by 0.5 M NaSCN,
suggesting that IgM capture and detection of
dengue viral antigens were not affected in
IgM-capture ELISA.
Figure 2 shows anti-dengue IgM titration
curves in the presence or absence of 0.5 M
NaSCN in the ELISA. The IgG fraction in the
serum specimen was removed by adsorption
with the Protein G beads (UltraLink
Immobilized Protein G Plus, capacity: ~25
mg IgG/ml of the gel, PIERCE). The levels of
cross-reaction between dengue viral antigens
and patients’ IgM antibody (a) were
decreased after the treatment with 0.5 M
NaSCN in the ELISA (b): The level of reaction
with homologous dengue-1 antigen was less
affected. The results suggested that the
addition of 0.5 M NaSCN to the reaction
mixture of ELISA may highlight a serotypespecific reaction between crude dengue viral
antigen and anti-dengue IgM antibody.
Figure 2. Titration curves of dengue virus specific IgM antibody in the presence or
absence of 0.5 M NaSCN in the ELISA
(a)
2
v.s.D1 Ag
v.s.D2 Ag
v.s.D3 Ag
v.s.D4 Ag
1.5
1
0.5
0
100
1000
Serum dilution
(b)
2
1.5
1
0.5
0
100
1000
Serum dilution
A serum specimen obtained from the dengue virus type 1-infected patient was used in the study. The IgG fraction
was removed according to the method described in the Materials and Methods section. Protein G-treated serum
specimen was serially diluted from 1:20 to 1:2560 in the absence (a) and in presence of 0.5 M NaSCN (b) in 10%
CS-PBS, and then reacted on the plates coated with each of 4 dengue viral antigens. Dengue virus-specific IgM
antibody was detected with 1:500 diluted peroxidase-conjugated anti-human IgM goat serum.
121
We examined the IgM levels by ELISA
with the antigen of 4 dengue virus serotypes.
The table below shows the results of 28
serum samples from 18 Japanese dengue
patients. The infecting dengue virus
serotypes were determined by RT-PCR. The
data were presented as the index value
according to the method described
previously[4] . We defined index values of
2.28 or greater and 26.60 or greater as
positive, respectively, in the absence and
presence of 0.5 M NaSCN in the ELISA. In
all the tested cases, the serotype-specific
IgM levels were the highest against the
infecting dengue virus serotype than against
three other serotypes in the presence of
NaSCN.
The
serotype-specific
IgM
responses were more highlighted in the
presence of NaSCN than in its absence.
These data suggest that IgM responses to the
infected dengue virus serotype determined
by RT-PCR in primary dengue infection
were the highest. These results agreed with
the report by Burke [6] .
Table. Serodiagnosis of dengue by IgM-ELISA with and without 0.5M NaSCN
Index values*
Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60)
1
2
3
4
5
6
7
122
D3
D2
D3
D3
D2
D3
D1
D1
D2
D3
D4
D1
D2
4
1.20
1.47
2.29
1.42
5.33
9
8.58
7.73
16.40
7
4.89
52.18
7.96
4.49 128.00 1,246.00 670.00 239.00
14
5.27
37.93
8.87
5.42
68.00
6
5.34
3.34
9.60
2.35
50.25
15.00 283.00
17
6.79
7.07
12.38
4.00
78.00
46.25 413.25 108.50
8
5.61
2.84
9.20
2.63
67.25
12.75 305.50
77.00
10
4.77
3.16
9.10
3.45
54.00
14.00 274.20
97.20
6
1.13
4.77
1.71
0.81
2.78
9.36
8.00
0.00
13
2.65
12.31
4.49
2.15
10.27
46.27
28.73
14.45
6
3.07
1.75
6.55
0.89
11.83
4.00 140.33
2.50
8
4.06
2.40
9.23
1.34
21.00
6.83 198.33
11.83
5
2.76
1.46
6.33
1.39
7.86
0.29
20.71
0.00
7
13.51
6.65
11.49
3.67 127.14
72.86
11.43
25.71
8
16.91
9.53
13.28
4.43 143.37
100.00
20.37
42.00
14
24.51
11.49
9.86
3.63 214.50
68.25
34.00
51.13
10.46 110.30
3.67
D3
45.67
D4
5.67
47.67 471.67 273.00
395.00 243.70 179.00
39.50
Index values*
Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60)
8
D1
D1
D2
D3
D4
D1
7
1.73
3.89
1.66
1.45
5.54
11
6.95
10.19
2.27
1.76
D2
D3
D4
6.38
6.00
1.62
26.42
20.25
19.00
10.17
9
D2
5
1.93
3.02
1.28
0.72
1.80
4.70
3.90
0.00
10
D2
ND
0.75
2.98
0.92
0.28
2.13
4.63
3.75
0.00
11
D1
5
1.25
1.28
1.32
0.69
4.00
1.57
10.71
0.00
12
D1
ND
20.35
15.44
15.02
8.00 161.28
36.71
39.90
97.71
13
D1
19
18.37
10.69
10.02
12.75 244.43
14
D1
8
7.55
6.78
6.41
3.37
50.25
11.67
27.92
19.92
15
D4
19
1.12
1.44
1.19
2.41
2.56
3.44
7.22
35.44
16
D1
5
1.36
1.13
1.27
0.27
6.27
1.45
2.45
0.00
17
D1
6
4.74
2.01
5.20
0.98
58.67
3.71
17.58
0.00
18
D1
15
14.18
8.39
8.52
3.58 137.38
33.27
31.00
49.88
22.86 138.14 135.00
D1 to D4, dengue virus type 1 to 4, respectively. ND, not determined.
* index values were calculated by the formula A492 with the viral antigen/A492 with uninfected control antigen.
The chaotropic agent, NaSCN, is known
to denature protein structure and inhibit the
formation of immune complexes as was
exploited
for
immunoaffinity
chromatography[12,13]. There are multiple
factors which affect NaSCN’s ability to
highlight
serotype-specific
reaction
in
comparison with cross-reaction in the ELISA.
One of the factors is a characteristic of
dengue viral antigens, which are prepared
from the infected mosquito cell culture. It
was reported that a rapidly sedimenting
haemagglutinin (RHA) was cross-reactive and
labile, but the soluble complement-fixing
antigen (SCF) was serotype-specific and
relatively stable against the NaSCN treatment
in the ELISA[11,14]. The predominant
polypeptides in RHA and SCF fractions
represent the envelope glycoprotein and NS1,
respectively[11]. Trent et al.[15,16] observed
three antigenic determinants on the envelope
glycoprotein: (i) flavivirus group- reactive;
(ii) complex-specific; and (iii) serotypespecific. Henchal et al.[17] characterized four
antigenic determinants on the envelope
glycoprotein: (i) flavivirus group-reactive;
(ii) dengue complex-specific; (iii) dengue
subcomplex-specific; and (iv) dengue
serotype-specific.
Brandt et al. [14] reported that the DEN-2
SCF antigen extracted from infected mouse
brains was resistant to the treatment with
protein denaturing agents. Falconar and
Young[18] reported serotype-specific epitopes
on NS1. These results suggest that
123
heterologous antigenic determinants on
dengue viral antigens contribute to the crossreactivity among the four dengue serotypes,
and that the presence of a chaotropic agent
in the reaction mixture induces changes of
viral antigens and may decrease the crossreactive antigenicity.
reactive IgM. This procedure may be useful
for determining infecting dengue virus
serotypes, especially in primary dengue virus
infection.
In conclusion, we demonstrated that the
addition of NaSCN to IgM-capture ELISA
highlighted the detection of serotype-specific
IgM in comparison with serotype cross-
This work was supported by a grant for
research on emerging and re-emerging
infectious diseases from the Ministry of
Health, Labour and Welfare, Japan.
Acknowledgement
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J.F.P. Wagenaar #, A.T.A. Mairuhu and E.C.M. van Gorp
Department of Internal Medicine, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam,
The Netherlands
Abstract
Dengue virus infections are an important cause of morbidity and mortality in the tropics, with 100
million people infected annually and an estimated 2.5 billion people at risk. Human infections can be
asymptomatic or can manifest as the self-limited febrile dengue fever, or the more severe and lifethreatening dengue haemorrhagic fever (DHF). There are several possible reasons why some infected
individuals might develop a more severe form of the disease than others. Antibody enhancement and
viral virulence have been implicated in the pathogenesis of DHF but host genetic factors may also be
relevant and predispose some individuals to DHF. This review discusses the possible involvement of a
variety of genetic polymorphisms on the course of dengue virus infections. It has been shown that several
common genetic polymorphisms can influence the susceptibility to dengue haemorrhagic fever. Gene
polymorphisms concerning human leucocyte antigens, antibody receptors, inflammatory mediators and
other factors with immunoregulatory effects are described. The study of genetic polymorphisms might
provide important insights into the pathogenesis of a more severe disease and could have an impact on
the design of future vaccines.
Keywords: Dengue haemorrhagic fever, genetic polymorphisms.
Introduction
Dengue has become one of the most
important arthropod-borne diseases in
tropical and subtropical regions of the world.
Approximately 100 million cases of dengue
infections occur annually, and an estimated
2.5 to 5 billion people are at risk of dengue
virus infection[1] . The four serotypes of
dengue virus (DEN-1, 2, 3 and 4) are
transmitted to humans through the bite of
an infective female Aedes mosquito and
may result in dengue fever (DF), an acute
viral infection characterized by fever, rash,
#
headache and muscle and joint pain.
Occasionally, dengue virus infections
progress to dengue haemorrhagic fever
(DHF) and dengue shock syndrome (DSS).
These are potentially life-threatening
illnesses characterized by haemorrhagic
manifestations and the loss of plasma from
the vascular compartment, which may give
rise to shock in severe cases.
Central in the pathogenesis of DHF and
DSS is the loss of endothelial integrity that is
believed to be the result of an abnormal
immune response and a disturbance in
E-mail: [email protected]; Tel.: + 31-(0)20-5124591; Fax: + 31-(0)20-5124783
126
immune regulation. Elevated levels of
several cytokines and chemical mediators,
which cause capillary leakage and may lead
to shock, have been found in those suffering
from DHF and DSS. Replication of dengue
viruses occurs primarily in mononuclear
phagocytes, which are a major source of
tumor necrosis factor (TNF)-α and other
vasoactive inflammatory mediators. Several
studies have demonstrated that TNF-α and
other cytokines that are produced
downstream of TNF-α in the inflammatory
cascade, e.g. IL-1β, IL-6 and IL-8, are
related with disease severity[2-4] . Other
inflammatory mediators, like IL-2 and
interferon (IFN)-γ, are released from T
lymphocytes that are activated during
dengue virus infections. The levels of these
cytokines are significantly higher in DHF
and DSS patients than in DF patients[5,6] .
There are several possible reasons why
some infected individuals might produce a
greater inflammatory response than others.
The most favoured hypothesis concerns the
antibody-dependent enhancement theory.
Several epidemiological studies demonstrate
that prior infection with a different viral
serotype constitutes the largest risk factor for
DHF[7-11] . In vitro studies demonstrated that
the presence of anti-dengue antibodies at
sub-neutralizing concentrations augment
dengue virus infection of Fcγ receptorpositive cells, such as monocytes[12,13] . Based
on these epidemiological and laboratory
observations, it has been hypothesized that
dengue cross-reactive antibodies may
increase the number of dengue virusinfected monocytes during secondary
infections, and lysis of these dengue virusinfected monocytes may lead to DHF and
DSS. Another possibility why some infected
individuals might produce a greater
inflammatory response is related to viral
virulence. Several studies have found that
infection with DEN-2 caused more severe
disease than other serotypes, suggesting that
the virus phenotype influences the
outcome[7,8,11] . In addition, genetic variations
within a specific serotype may also account
for differences in disease severity, although
reports remain scanty[14].
Hyperendemic transmission of multiple
DEN serotypes in a Haitian population and
the apparent absence of DHF and DSS, in
addition to the observation that black people
were hospitalized less frequently with DHF
and DSS than the whites during epidemics in
Cuba, led to the hypothesis that human
genetic factors, e.g. gene mutations and gene
polymorphisms, may contribute to variable
susceptibility[15-17] . Genetic polymorphisms
are stable gene variants that usually have
minor effects on the regulation or function of
proteins. These subtle chances might very
well have important consequences for
susceptibility to the disease[18] . Several studies
have
confirmed
that
some
genetic
polymorphisms may protect or predispose an
individual to DHF and DSS. Understanding
the molecular basis for these differences in
susceptibility should provide useful insight in
the pathogenesis of DHF and DSS and aid in
the development of effective therapies and
vaccines. This review attempts to describe
the current knowledge of the role of genetic
influences on dengue virus infections.
Human leucocyte antigen
genes
The function of the human leukocyte
antigens (HLAs), encoded by the major
histocompatibility complex (MHC) and
whose genes are on chromosome 6, are to
127
display antigen-proteins to antigen receptors
of host T-lymphocytes in order to activate
cellular host immune responses. HLA genes
show great variability and it could well be
that specific polymorphisms seen in human
HLA gene regions influence peptide epitope
binding[18] . A number of studies have looked
at the variation in HLA genes and found
some of them to be associated with the
severity of dengue virus infections (Table).
Table. Effect of HLA and non-HLA alleles on the severity of dengue virus infections
Alleles
HLA alleles
Non-HLA alleles
Class
Effect
Population
Reference
Class I
A1
A2
A*0203
A*0207
A24
A29
A33
B blank
B13
B14
B44
B46
B51
B52
B62
B76
B77
Susceptibility
Susceptibility
Protective
Susceptibility
Susceptibility
Protective
Protective
Susceptibility
Protective
Protective
Protective
Susceptibility
Susceptibility
Protective
Protective
Protective
Protective
Cubans
Thai
Thai
Thai
Vietnamese
Cubans
Vietnamese
Cubans/Thai
Thai
Cubans
Thai
Thai
Thai
Thai
Thai
Thai
Thai
20
19, 22
22
22
21
20
21
19, 20
19
20
22
22
22
22
22
22
22
Class II
DRB1*04
Resistance
Mexicans
29
Fc gamma-receptor
Vitamin D receptor
Resistance
Resistance
Vietnamese
Vietnamese
30
30
HLA class I
HLA class I alleles consist of HLA-A, -B, and C; its products have a wide distribution and
are present on the surface of all nucleated
cells and on platelets. Antigens associated
with HLA class I products will interact with
CD8 cells during an immune response.
128
Polymorphisms in this class I region gene
were found to be associated with DHF
disease susceptibility. Chiewslip and Paradoa
were the first to report an association
between HLA class I and the severity of
dengue virus infection[19,20] . In two ethnically
and geographically distinct populations
evidence was presented suggesting that HLA-
A1, HLA-A2 and HLA-B blank increased in
frequency in DHF patients. A negative
relationship was found for HLA-B13, HLAB14 and HLA-A29. However, these studies
had a small sample size and additional
studies with a larger number of patients were
needed. Subsequently, a large case control
study in 560 study subjects was performed,
which mainly confirmed the observations
made by the two previous studies that HLA
class I was important[21] . The data
demonstrated that polymorphisms in the
HLA class I region, particularly of the HLA-A
gene, were significantly associated with
susceptibility to DHF. Of the 15 alleles
studied, two particular alleles were relevant:
patients with HLA-A33 were less likely to
develop DHF (odds ratio 0.56; 95%
confidence interval 0.34-0.39), whereas
those with HLA-A24 were at an increased
risk to develop DHF (odds ratio 1.54; 95%
confidence interval 1.05-2.25). The HLA-B
alleles were not associated with DHF disease
susceptibility.
Another case control study, in a Thai
population, also demonstrated that the HLAA2 locus serotype was associated with
disease susceptibility[22]. HLA-A*0203 was
associated with the less severe DF, whereas
HLA-A*0207
was
associated
with
susceptibility to the more severe DHF.
Interestingly, these associations were only
found in immunologically primed persons,
but not in immunologically naïve patients
with primary infection. Dengue virus-specific
associations were also observed within the
HLA-B5 group of related alleles, whereby
molecularly-determined HLA-B51 alleles
were associated with the development of
DHF after secondary infections. HLA-B51restricted CTL responses to a variety of
viruses have been described, including
Hantaan virus which also causes a
haemorrhagic fever[23] . HLA-B52 showed a
strong association with less severe DF. The
reduced frequency of the HLA-B15 group of
serotypes, including HLA-B62, B76 and B77,
in patients with secondary infections, suggests
that they may be protective against
developing
clinical
disease
in
immunologically primed individuals. By
contrast, HLA-B46 that also belongs to the
HLA-B15 group of serotypes, was increased
in DHF patients with secondary infections.
Since HLA-B46 is in strong equilibrium with
HLA-A*0207, it is believed that the effect of
B46 was likely to be an adjunct to that of
A*0207. Finally, HLA-B44 appeared also to
be protective against the development of
severe disease in patients with secondary
dengue virus infections.
HLA class II
Class II HLA products consist of HLA-D, -DR,
-DP, and -DQ; they have a more limited
distribution
on
B-cells,
macrophages,
dendritic cells, Langerhans cells and activated
T cells. HLA class II alleles have shown to
play a role in mycobacterial diseases, and
their association with hepatitis clearance is
also established[24-26] . HLA-DRB1, which is
one of the most polymorphic loci of the HLA
complex in the Mexican population[27,28] , was
studied in Mexican patients suffering from a
dengue virus infection[29] . Although the
sample size was relatively small, the
investigators found that the frequency of
HLA-DRB1*04 was lower in DHF patients.
Persons homozygous for DRB1*04 were less
likely to develop DHF than persons who
were DRB1*04 negative (odds ratio 0.28;
95%
confidence
interval
0.12-0.66),
suggesting a protective effect. The envelope
protein (E) of the virus is responsible for viral
entry into target cells. The immunological
determinants of protein E are probably
129
processed and presented by HLA class II
antigens. The HLA-DRB1*04 molecule may
present these viral antigens to CD4+
lymphocytes leading to an effective immune
response and therefore protection from DHF.
These findings are in contrast to the findings
of Loke et al., who studied polymorphisms in
the HLA-DRB1 gene but did not find an
association[21] .
gene
has
been
associated
with
meningococcal disease and recurrent
respiratory tract infections[33,34] . It chances the
IgG binding affinity of the receptor with
reduced opsonisation of IgG2 antibodies
causally associated with the arginine variant.
Loke et al. found that homozygotes for the
arginine variant at position 131 of the FcγRIIA
gene may be less susceptible to DHF[30] .
HLA class III
Vitamin D receptor (VDR)
Genes in the class III region encode a
number of proteins, including complement
proteins (C4A, C4B, C2 and Bf), TNF-α and
TNF-β[18] . Loke and colleagues studied
promotor polymorphisms in the TNF-α gene
but did not find an association[21] . No other
studies are reported to have studied HLA
class III polymorphisms.
This gene mediates the immuno-regulatory
effects of 1.25-dihydroxyvitamin D3, which
include activating monocytes, stimulating
cellular immune responses and suppressing
immunoglobulin production and lymphocyte
proliferation[35] . Recently the tt genotype of a
single nucleotide polymorphism at position
352 of the VDR gene has been associated
with
tuberculoid
leprosy,
enhanced
clearance of HBV infection and resistance to
pulmonary tuberculosis [36,37] . Expression of
VDR may affect susceptibility to DHF since
activated B and T lymphocytes express VDR
and 1,25D3 affects monocytes, the main sites
of dengue virus infection and replication[12] .
The t allele at position 352 of the vitamin D
receptor (VDR) gene was associated with
resistance to severe dengue, although the
exact mechanism needs to be explored.
Non-HLA host genetic factors
The number of studies on polymorphisms
within non-HLA genes remains low. Loke
and colleagues investigated the association
between susceptibility to DHF and
polymorphic non-HLA alleles: vitamin D
receptor (VDR), Fcγ receptor II (FcγRII),
Interleukin-4 (IL-4), Interleukin-1 repeat
alleles (IL-1RA), and mannose-binding lectin
(MBL) [30] . Two of the five genes assessed
showed evidence of association with altered
risk of severe dengue.
Fcγ receptor
The Fcγ receptor is a widely distributed
receptor for all subclasses of IgG and is able
to
mediate
antibody
dependent
enhancement in vitro by binding to virus-IgG
complexes[31,32] . An arginine to histidine
substitution at position 131 of the FcγRIIA
130
Interleukin-4 (IL-4)
IL-4, primarily produced by Th2 subset of
CD4+ T-cells, regulates B-cell growth, IgG
class switching and suppresses Th1-type
responses as well[38,39] . Since this gene affects
both antibody responses and inflammatory
responses during disease, IL-4 promotor
polymorphisms were studied in order to find
a relationship in susceptibility to DHF.
However, no associations were found in this
context[30] .
Interleukin-1 repeat allele (IL-1RA)
IL-1RA was thought to be a good candidate
gene as well because IL-1RA is involved in
the regulation of IL-1-mediated inflammatory
responses by competitive binding to ILreceptors [40] . But no significant difference
could be found in the DHF group in addition
to the controls[30] .
Mannose-binding lectin (MBL)
Several mutations in the MBL gene, which
encodes for a protein involved in the
activation of the classical complement
pathway[41,42] , have been associated with a
marked reduction in serum MBL levels and
MBL-mediated complement activation[43,44] .
Polymorphisms in this gene were not proved
to have any effect on the susceptibility to
DHF. However, this variant allele was
relatively low in the observed population,
which limits the statistical power of the
analysis [30] .
Discussion and future
perspective
The number of candidate susceptibility and
protective genes is expanding rapidly, but
what is the use of studying these genes in
relation to DHF? Studying host genetic factors
will clearly contribute to our understanding of
the pathophysiology of dengue virus
infections but also of viral infections in
general. The finding of a protective
association with particular HLA or non-HLAtypes may encourage the design of future
vaccines, whereas polymorphisms associated
with the susceptibility to develop a more
severe disease may help to identify certain
risk groups in a population. It is therefore of
great importance to stimulate the study of the
interaction
of
single
and
multiple
polymorphisms in severe dengue virus
infections.
The few studies performed thus far have
demonstrated that host genetic factors can be
important in susceptibility to DHF. It is most
likely that classical HLA class I and class II
gene products play a crucial role in
determining the severity of dengue virus
infections. Two polymorphic non-HLA alleles,
the FcγRII receptor and VDR, could also play
an important role in susceptibility to DHF.
Some polymorphic HLA alleles were
observed in several studies, e.g. HLA-A2 in a
Thai and Vietnamese population, but
differences in susceptibility to DHF were
observed[19,21,22] . An explanation for the
observed difference may be that a genetic
polymorphism is more frequent in a
population whereas another is relatively
infrequent. Overall, such disease associations
warrant further analysis, but also emphasize
the need to expand the scope of investigation
to other candidate genes within and outside
of the HLA region.
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Identification and Phylogenetic Analysis of DEN-1 Virus
Isolated in Guangzhou, China, in 2002
Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An#
Department of Microbiology, College of Medicine, Third Military Medical University,
Chongqing, 400038, People’s Republic of China
Abstract
Virus was isolated and identified from the serum samples of patients with suspected classical dengue
fever (DF) in Guangzhou province, China, in 2002. The serum was incubated with the Aedes albopictus
cell line, C6/36, for isolation and 5 of 20 serum samples caused cytopathologic effects on C6/36 cells. A
539-nucleotide (nt) fragment in the NS1 region of the isolated virus genome was amplified using
universal primers of 4 serotypes of DEN viruses. By sequencing the primer-extension production and
blasting in the GenBank, the isolated strain was the closest to rDEN-1 dalte30 (AY145123), which was an
attenuation strain of DEN-1 virus. A 593nt fragment from the Envelope-Nonstructural protein 1 (E/NS1)
of these isolates was also sequenced to compare it with published sequences of other DEN-1 viruses. The
240nt from the E/NS1 region of this DEN-1 virus genome was the closest to DEN-1/T14 strain (M32931),
which was genotype IV. The phylogenetic analysis of the NS1 (480nt) and E/NS1 (240nt) regions of the
gene nucleotide sequences was performed using neighbour-joining methods: 11 strains of DEN-1 virus
were divided into three genotypes: I, IV and V, as defined by Rico-Hesse, or Asia, South Pacific and
Americas/Africa, as defined by Goncalvez. Based on the sequence information and the definition, the
isolated virus (named DEN-1/GZ 2002) strain belonged to DEN-1 and IV or South Pacific genotypes. The
suckling mice died on days 10 to 11 after intracerebral inoculation with the isolated virus. TCID50 of the
virus was 4.37 log pfu/0.2ml. Small plaques with an unclear edge were seen on Vero cells on days 8 to 9
after infection. Combined with clinical data that thousands of patients only showed DF manifestations,
the results suggested that DEN-1/GZ 2002 might be a low-virulence strain.
Keywords: Dengue virus, DEN-1/GZ2002, virulence, phylogenetic analysis.
Introduction
Dengue (DEN) viruses belong to the genus
Flavivirus. There are four closely related, but
antigenically distinct, virus serotypes (DEN-1,
DEN-2, DEN -3 and DEN-4). The viral
genome is a positive-sense single-stranded
#
RNA, approximately 11,000 nucleotides
long, encoding 10 distinct proteins. The
gene order is 5’-C-prM(M)-E-NS1-NS2ANS2B-NS3-NS4A-NS4B-NS5-3’, expressed
as a single polyprotein that is cleaved by
both viral and cellular proteases to form the
viral polypeptides. The three 5’ proteins are
E-mail: [email protected]; Tel.: +86-23-68752774; Fax: +86-23-65463259
135
Identification and Phylogenetic Analysis of Dengue-1 Virus
three structural proteins: capsid (C),
membrane (M) and envelope (E), and the
remaining seven are nonstructural (NS)
proteins[1]. The DEN viruses cause classical
dengue
fever
(DF)
and
dengue
(DHF/DSS). DF is a non-specific viral febrile
illness while DHF/DSS is a severe and fatal
haemorrhagic disease. Up to 100 million
cases of DF occur annually in the world,
with some 2 billion people at risk of the
infection in tropical and subtropical regions
of Africa, Asia and the Americas [2,3].
The pathogenesis of the DEN virus
infection is not clear. Although many factors
are proposed to be involved in the DEN
virus epidemiology and occurrence of
DHF/DSS, the evolution of the DEN viral
virulence and the spread of DEN viruses in
different geographical areas are two
important features in the epidemiology and
pathogenesis of the disease. Studies have
suggested that specific viral structures might
contribute to the replication capability in
the target cells and to their transmission[4] .
Co-circulation of novel DEN genotype,
another genotype of the same serotype or
different serotypes of DEN viruses in the
same area, might lead to the displacement
of the native genotype by a new genotype
and thus to the occurrence of DHF/DSS[5] .
Several studies have shown that the
phylogenetic trees obtained from small gene
fragment sequences are congruent with the
trees obtained from an entire gene
sequence, although there might be minor
rearrangements in the terminal branches[6-13] .
Using a partial sequence from EnvelopeNonstructural protein 1 (E/NS1) junction
region, Rico-Hesse defined five genotypes
for DEN-1 viruses isolated worldwide:
136
I) America, Africa and South-East Asia;
II) Sri Lanka; III) Japan; IV) South-East Asia,
the South Pacific, Australia and Mexico; and
V) Taiwan and Thailand[14]. Recently,
Goncalvez set up the phylogeny of 36 DEN 1 viruses according full E gene sequences.
Statistical analyses of the validity of
branching
patterns
(bootstrap)
also
suggested five classifications: 1) from Asia;
2) from Thailand; 3) from sylvatic/Malaysia;
4) from South Pacific; and 5) from the
Americas/Africa, in which three genotypes
indicated as 1), 4) and 5) were consistent to
I), IV) and V) mentioned by Rico-Hesse[15] .
Aviles-G and collaborators sequenced
Capsid-pre Membrane (C/prM) and E/NS1
regions of 24 recent isolates of DEN-1 from
South America, suggesting that the recent
epidemics in Argentina and Paraguay were
due to the re-emergence of a previously
circulated strain[ 16] . This indicated that small
gene fragment sequences are available for
analysis of viral genetic characterization.
Current methods for obtaining DEN
virus sequences no longer require a viable
virus isolate. In fact, the entire genome
sequences can be obtained by enzymatic
amplification of viral RNA template in the
patient’s blood sample. Unfortunately, there
are numerous sequences that contain errors,
which have led to serious mistakes in their
interpretation. Therefore, it is wise to obtain
virus
isolates
for
further
genetic
characterization[17]. In this study, a viral
strain that circulated in Guangzhou, China,
in 2002 was isolated first, then identified by
amplifying and sequencing 539 nucleotide
(nt) in NS1 region of DEN virus genome
using universal primers. Also, E/NS1 regions
of the isolated virus were sequenced to
determine their origin.
Collection of blood samples
Twenty patients with suspected DEN virus
infection admitted to the Eighth People’s
Hospital of Guangzhou were enrolled – 12
males and 8 females aged between 23-57
years. The blood samples were collected on
day 1 to 11 after the onset of fever. The sera
were separated by centrifugation and stored
at –70 °C until use.
Viral isolation
Aedes albopictus mosquito cell line, C6/36,
was cultured in RPMI 1640( pH6.8~ 7.0)
containing 10% fetal bovine serum (FBS)
and 2 mM glutamine with 5% CO 2 at 28 °C.
30µ l of the serum sample was diluted with
1 ml RPMI 1640 containing 2% FBS and
was incubated with overnight C6/36 cells for
1 hour at 28 °C. After removing the serum,
the cells were cultured in RPMI 1640
( pH6.8 ~ 7.0 ) containing 2% FBS at
28 °C. The cells were observed daily for
cytopathic effect (CPE) [18] . If CPE was not
seen initially, the cells were passaged several
times to confirm whether there was virus in
the serum samples or not.
RNA extraction
Viral RNA was extracted from the culture
supernatants of infected C6/36 with
ISOGEN (Nippon Gene, Toyama, Japan)
according to the manufacturer’s instructions.
RT-PCR amplification
Nucleotides from positions 2503~3041
coding a fragment of the NS1 gene were
amplified using RT-PCR. The universal
primer sequences are: 5'GTG CAC ACA
TGG ACA GAA CA 3'(forward) and 5'CTT
TCT ATC CAA TAA CCC AT 3'(reverse).
Their combination generated a 539nt
fragment. Briefly, 5 µl of the extracted RNA
was mixed with 50 pmol (2µl) of reverse
primer at 70 °C for 5 minutes and cooled
down on ice. Then 13 µl of RT mix
containing 4µl of 5X RT XL buffer, 0.5 µl of
10mM dNTP, 6.0 µl of sterile UHQ water,
0.5µl RNase inhibitor and 20 U of AMV
reverse transcriptase XL (TaKaRa, Japan)
were added. The mixture was incubated at
42 °C for 1 hour for RT reaction, then
heated to 95 °C to inactivate AMV and
cooled down on ice. PCR was subsequently
carried out by adding 24 µl of PCR mix
containing 2.5 µl of 10X Taq Polymerase
buffer (PROMEGA, USA), 2.5 µl of 25mM
MgCl2, 12.5 pmol each of sense and reverse
primers, 0.5 µl of 10mM dNTP, 2U of Taq
DNA Polymerase (PROMEGA, USA) and
17.5 µl of sterile UHQ water to the 1st
strand synthesis tube containing 1 µl of
cDNA. PCR with denaturation at 94 °C for
30 seconds; annealing at 53 °C for 30
seconds; and extension at 72 °C for 1
minute 30 seconds for 30 cycles. The RTPCR conditions for the amplification of
nucleotides from positions 2107~2701
coding for the E/NS1 fragment were similar
to those used for NS1 fragment, but with an
annealing temperature of 58 °C instead of
53 °C.
DNA purification and sequencing
PCR-amplified
DNA
products
were
electrophoresed in 1% agarose gels and
stained with 1µg/ml ethidium bromide. The
bands of predicted size (539nt for the NS1
fragment and 593nt for E/NS1 fragment)
were excised from the gel and purified using
137
a Golden Beads Product Purification Kit
(Songon,
CN)
according
to
the
manufacturer’s instructions. Purified PCR
products were cloned on to pMD18-T
Vector (TaKaRa, Japan) and sent to Co.
Bioasia, Shanghai, CN, in 15% Glycerol to
perform sequencing using an ABI Prism 377
Genetic Analyzer. Amplifying primers were
M13-48.
Computer analysis
The
multiple
sequence
alignment
programme Clustalx, version 1.8[19] , was
used to obtain an optimal nucleotide
sequence alignment file. Phylogenetic
analysis of nucleotide sequences from the
NS1 region and the E/NS1 junction of our
isolates were carried out with the neighborjoining method (NJ)[20] , calculating bootstrap
confidence intervals of 1,000 replicates.
Character state tree-building algorithms
(PHYLIP package) were also tested. A strict
consensus bootstrap tree was obtained by
using
the
following
programmes:
(i) SEQBOOT to generate 100 replicas,
(ii) DNADIST and NEIGHBOR to acquire
the tree of each reiterated data, and
(iii) CONSENSE to build a strict consensus
bootstrap tree. Phylogenetic trees were
drawn
using
TreeView[21] .
Published
sequences used in the analysis are listed in
Table 1.
Table 1. Dengue viral sequences from GenBank used in the phylogenetic analysis
Virus
Strains
Origin
Year isolated
Accession number
DEN-1
FGA/89
French Guiana
1989
AF226687
DEN-1
BR/90
Brazil
1990
AF226685
DEN-1
S275/90
Singapore
1990
M87512
DEN-1
Abidjan
Ivory Coast
1999
AF298807
DEN-1
WestPac/74
Nauru
1974
U88535
DEN-1
A88
Indonesia
1988
AB074761
DEN-1
GD05/99
Guangdong, CN
1999
AY376738
DEN-1
GD23/95
Guangdong, CN
1995
AY373427
DEN-1
GZ/80
Guangzhou, CN
1980
AF350498
DEN-1
Mochizuki
Japan
1943
AB074706
DEN-1
Djibouti
Djibouti
1998
AF298808
DEN-2
TR1751*
Trinidad
1954
M32969
* The full-length gene segment sequence of this viral strain is unavailable in GenBank and the NS1 region in the
phylogenetic analysis was sequenced by us
138
The resulting unrooted trees were outgrouped to the sequence of DEN-2 (TR1751),
which was kindly provided by Dr Oya A
(National Institute of Infectious Diseases,
Japan) and isolated from a DF patient[22,23] .
The RT-PCR amplification of the NS1 region
of this virus strain using universal primers and
sequencing of amplified products was done
as described above.
Figure 1. Cultured C6/36 cells:
(a) C6/36 cells without DEN-1 infection;
(b) Cell-cell fusion and syncytia on C6/36
cells was seen on day 6 after infection
with DEN-1/GZ2002
Biological character detection
The virus isolated from the serum samples
was propagated in C6/36 cells (MOI = 1)
and the titer was determined by the plaque
assay using Vero cells monolayer culture
under methylcellulose overlay medium.
TCID50 of the virus was also measured by
the plaque assay and calculated using ReedMuench method[24] . Eighteen suckling mice
were intracerebrally (ic) inoculated with
104 pfu/mouse of the virus for observing
clinical signs and their survival time.
Figure 1 (a)
Results
General observation
Figure 1 (b)
After two passages, 5 of the 20 serum
specimens caused CPE on C6/36 cells.
Infected C6/36 cells became syncytia and
cell-cell fusion. Some cells showed necrosis
and got detached from the plate at a late
stage of the infection (Figure 1). The virus
was propagated in C6/36 cells and the
highest titer was 5×105 pfu/ml at day 6 postinfection (pi). Using the Reed-Muench
method, TCID50 of the virus was 4.37 log
pfu/0.2ml. Plaque formation on Vero cells
was detected on day 8 or 9 after infection
and they were small and unclear (Figure 2).
After ic inoculation with isolated virus, all of
the suckling mice showed kyphoscoliosis
and paralysis of hind legs and died on days
10 to 11 after infection.
Figure 2. Plaque formation on Vero cells
was detected on day 8 after infection
A. Blank control; B. Vero cells were
infected with DEN-2/Tr1751; C and D. Vero
cells were infected with DEN-1/GZ2002
139
Comparison of nucleotide
sequences
As shown in Figure 3, a 539nt fragment
from the NS1 region (positions 2503 to
3041) was amplified using the universal
primer by RT-PCR and the amplified
products were sequenced to determine the
relationships among the 5 isolates and their
origin. Comparisons of the NS1 region of
the 5 isolates virus strains showed little
divergence, so we chose one as the
representative strain to compare with the
sequences published in GenBank. The
nucleotide sequence of this strain was the
closest to rDEN-1 dalte30 (AY145123),
which was an attenuation strain of DEN-1
virus. According to the full-length gene
sequences of rDEN-1 dalte30 (AY145123),
we designed the primers to amplify the
nucleotides of a fragment from the E/NS1
region (positions 2107 to 2701). The 240nt
from the E/NS1 region (position 2309 to
2548) of this DEN-1 virus genome is the
closest to DEN-1/T14 strain from Australia
isolated in 1981 (M32931), which was
genotype IV, their divergence was 2%.
Based on sequence information and
definition by Rico-Hesse, our isolates
belonged to DEN-1 and genotype IV,
named DEN -1/GZ2002.
Figure 3. Agarose gel analysis of the cDNA products from RT-PCR of RNA samples isolated
from the supernatant of the infected C6/36 cells
(a) After amplification with the universal primer, a band of 539nt for the NS1 fragment
was seen. Lanes 2-6: PCR amplified DNA products from different samples respectively;
lane 7: blank control; lanes 1 and 8: 1Kb DNA Ladder marker (GeneRulerTM )
(b) After amplification with a primer for 593nt fragment of E/NS1 region, an expected
band about 593nt was seen (lane 1), Lane 2: 1Kb DNA Ladder marker (GeneRulerTM )
Figure 3 (a)
Figure 3 (b)
140
Phylogenetic analysis
We compared 480nt from the NS1 region
(position 2516 to 2995) and 240nt from the
E/NS1 region (position 2309 to 2548) of this
DEN-1 virus genome with sequences of
other published DEN -1 viruses. The
phylogenetic analysis of the NS1 (480nt)
and E/NS1 (240nt) regions of the gene
nucleotide sequences was performed using
NJ methods respectively. Two NJ trees were
set up and they showed a similar result,
except for a little difference in terminal
branches (Figure 4). From the NJ trees in our
study, 11 strains of the DEN-1 virus were
divided into three genotypes coinciding with
I, IV and V as defined by Rico-Hesse or Asia,
South Pacific and Americas/Africa, as
defined by Goncalvez. Our isolated DEN 1/GZ2002 strain belonged to genotype IV or
South Pacific genotype. In addition, it was
found that an arrangement of the S275/90
strain showed a major difference between
the two trees, which supports the previous
hypothesis that the S275/90 strain is a
recombined strain[25] .
Figure 4. Phylogenetic relationship of DEN-1/GZ2002 to previously characterized DEN-1
viruses. Two phylogenetic trees were set up using 240nt from the E/NS1 region, position
2309 to 2548 (a) and using 480nt from the NS1 region, position 2516 to 2995 (b) of these
DEN-1 virus genomes. The resulting unrooted trees were out-grouped to the sequence of
DEN-2 (TR1751)
Discussion
It is evident that the DEN virus
epidemiology is determined by many factors,
including those in the host, the virus, the
vector and the environment. DEN virus
evolution is also determined by many
complex interactions. Some genetic changes
that occur during the natural transmission
cycles of the DEN virus might affect its
141
virulence and cause the disease. Therefore,
understanding DEN virus variation is
especially
important
to
clarify
the
pathogenesis of DEN virus infection.
Although current methods of obtaining DEN
virus sequences no longer require viable
virus isolates, virus isolates are necessary for
further studies of the biological and genetic
characteristics[17]. In this study, we isolated
DEN-1 virus that circulated in Guangzhou in
2002 and analysed its possible origin and
partial biological features.
in DEN virus evolution and the NS1
sequences were a highly conserved region.
In our study, after sequencing E/NS1 (240nt)
and the NS1 (480nt) gene region, we
analysed the phylogenetic relationship of 11
DEN-1 virus representatives of three
genotypes coincidence with I, IV, and V as
referred to by Rico-Hesse (1990), or Asia,
South Pacific and Americas/Africa, as
defined by Goncalvez. This indicated that
the isolated virus belonged to genotype
IV/South Pacific.
In an In vitro experiment, the virus
caused typical CPE on C6/36 cells such as
syncytia and cell-cell fusion, which was
continuously shown when the virus
passaged
several
times.
Their
titer
maintained a level about 5 × 105 pfu/ml
during passages. On Vero cells, a small and
unclear plaque, a biological marker of
attenuation, was seen. The suckling mice
survived for 11 days after ic inoculation.
Interestingly, the sequences of 539nt from
the NS1 were very close to rDEN-1 dalte30
(AY145123), which was an attenuation
strain of DEN-1 virus. Combined with
clinical data where thousands of patients
only showed DF clinical signs, our results
indicated that the isolated virus might be a
low-virulence strain.
Tolou completed the genome sequence
analysis to demonstrate the likelihood of
recombination between different strains of
DEN-1 virus. The region of 1295~2592nt
was considered to be a hot spot for the
recombination[25] . We sequenced one
region outside (2516~2995nt) and one
region inside (2309~2548nt) the possible
recombination site. No recombination event
occurred in our strains, at least in the
regions analysed. However, no virus isolates
meeting
the
stringent
criteria
for
recombination have yet been described and
it remains to be determined whether and at
what frequency DEN viruses undergo
recombination in nature [17].
As is known, a comparison of full-length
gene segment sequences is impractical,
especially when looking at a large number
of samples. Recently, several studies have
shown that the phylogenetic trees obtained
from small gene fragment sequences are
highly congruent with those obtained from
an entire gene sequence, except for a minor
rearrangement in the terminal branches,
suggesting thereby that small gene fragment
sequences were effective for the study of
viral genetic characteristics[6-13] . The E/NS1
junction sequences were a useful indicator
142
The evolution of DEN viruses has had a
major impact on their virulence for humans
and on the epidemiology of the DEN virus
around the world. It is necessary that a more
complete and systematic survey of DEN-1
samples be undertaken before a link
between specific genotypes and the
virulence of these viruses can be established.
Acknowledgment
This work was partially supported by grants
nos. 30170848 and 30300303 from the
National Science Foundation of China
(NSFC).
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Induction of Cytotoxic T Lymphocytes by Immunization
with Dengue Virus – Derived, Modified Epitope Peptide,
Using Dendritic Cells as a Peptide Delivery System
Yoshiki Fujii*, Hideyuki Masaki** #, Takanori Tomura**, Kiyohiro Irimajiri*
and Ichiro Kurane***
*Department of Pharmacotherapy, Kinki University School of Pharmaceutical Sciences,
Higashi-Osaka, Osaka, Japan
** First Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama,
Osaka 589-8511, Japan
***Department of Virology 1, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan
Abstract
A single 9-amino acid peptide of the defined murine cytotoxic T lymphocyte (CTL) epitope (named
peptide-1), which corresponds to the amino acid residues 298-306 (GYISTRVEM) of NS3 of dengue virus
serotypes DEN-2 and 4, was examined for induction of specific CTLs. Immunization of BALB/c mice
subcutaneously with the peptide-1 emulsified with complete Freund adjuvant (CFA) did not induce
specific CTLs. The peptide-2 (GYISTRVEL), in which the residue (M) at 9th position of the peptide-1 was
substituted for L, was prepared. The peptide-2 possessed the complete H-2Kd-binding motif. Intravenous
immunization with 5x10 5 dendritic cells (DCs) pulsed with the peptide-2-induced specific CTLs.
Furthermore, subcutaneous immunization with the peptide-2 emulsified with CFA-induced CTLs which
lysed peptide-1-pulsed target cells as well as peptide-2-pulsed ones. These results indicate that
immunization with dengue virus-derived CTL epitope peptide induces specific CTLs, and that DC can be
used as a vehicle for the modified epitope peptide.
Keywords: Dengue virus, cytotoxic T lymphocyte, dendritic cell, epitope, peptide, binding motif.
Introduction
Major histocompatibility complex (MHC)
class I – restricted, CD8+ cytotoxic T
lymphocytes (CTLs) are known to play an
essential role in the recovery from viral
infection by lysing virus-infected cells[1] .
There are two major strategies to induce
CTL-mediated protective immunity. One is to
#
have CTL epitope expressed in host cells by
infection with live viruses, or by
administration of an expression plasmid
vector (i.e. DNA vaccine) in which the
epitope gene is incorporated. The other is
immunization with a defined CTL epitope
peptide. The former strategy is more
physiological; however, the preparation of
immunogen is often difficult and there is a
E-mail: [email protected]; Tel.: +81-72-366-0221, Ext.3152; Fax: +81-72-366-0206
145
Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus
potential risk that the immunogen may be
pathogenic. Immunization with a CTL
epitope peptide is relatively easy; however,
the epitope varies depending on T cell
receptor repertoires and MHC class I
haplotypes. Furthermore, the peptide
administrated into the body may be
degraded or washed away soon. Thus, the
peptide is usually less immunogenic for CTL
induction, and an appropriate delivery
system is necessary for induction of CTLs [2,3].
Dendritic cells (DCs), which are potent
antigen-presenting cells, are postulated to be
one of the peptide delivery systems for
inducing CTLs [2,4] . It has been reported that
intravenous immunization with DCs pulsed
with virus-derived peptides, or tumourderived peptides, elicits specific CTLs [5-7] .
Dengue viruses cause dengue fever and
dengue haemorrahgic fever/dengue shock
syndrome (DHF/DSS). Vaccine development
against dengue virus infection has not been
accomplished yet. It is important to analyse
CTL responses elicited by a single epitope in
order to understand the role of CTLs in
dengue virus infection. In the present study,
we employed a single 9-amino acid (a.a.)
peptide, in which C-terminal residue was
replaced to provide the complete H-2K dbinding motif. This peptide was a derivative
of the defined H-2K d-restricted 9-a.a. CTL
epitope peptide that corresponds to the
residues 298-306 of NS3 of dengue virus
types 2 and 4[8] . We examined whether
intravenous
immunization
with
bone
marrow-derived DCs pulsed with this peptide
elicited specific CTL response.
Mice
Female BALB/cAJcl mice were purchased
from Clea, Japan, and were maintained in
146
the Animal Facility of Kinki University School
of Medicine under conventional conditions.
Mice were used at the age of 6 to 12 weeks.
Cells
Murine mastcytoma line, P815 (H-2d), was
used as target cells in CTL assays. The cells
were maintained in RPMI 1640 medium
(Sigma, St. Louis, MO) with 5x10-5M 2mercaptoethanol (2-ME), 100U penicillin,
100µg/ml streptomycin, 10mM HEPES, and
10% heat-inactivated fetal calf serum
(Complete medium) at 37 °C in 5% CO2.
Peptides
The
peptide-1
(GYISTRVEM),
which
corresponds to the amino acid residues 298306 of NS3 of dengue virus types 2 and 4,
and the peptide-2 (GYISTRVEL) were
synthesized with 9-fluorenylmethoxycarbonyl
chemistry by Sigma Genosis, Japan. The
purity was determined to be 95.0% for the
peptide-1 and 96.2% for the peptide-2 by
reverse phase HPLC.
Induction of dendritic cells
BALB/c mouse bone marrow cells (9x105)
were cultured in 1ml of AIM-V medium
(Invitrogen, Carlsbad, CA) supplemented with
20ng/ml mouse GM-CSF (R&D Systems,
Minneapolis, MN) in 24-well plate at 37 °C
in 5% CO2. On days 4 and 6, 50 to 75%
volume of the culture medium was changed
with the fresh one supplemented with the
same amount of GM-CSF. On day 7, cells
were harvested, subjected to flow cytometry
analysis and used as DCs for immunization.
Flow cytometry analysis
Bone marrow-derived cells (4x105) were
incubated with 1µg of FITC-conjugated antimouse I-Ad/I-Ed antibody (BD PharMingen,
San Diego, CA), PE-conjugated anti-mouse
CD86 antibody (BD PharMingen), and
biotinylated
anti-mouse
CD11c
(BD
PharMingen) in 100µl of phosphate buffered
saline (PBS) containing 0.02% NaN 3
(PBS/NaN3) at 4 °C for 20 minutes. Same
amounts of FITC-conjugated rat IgG2a,κ (BD
PharMingen), PE-conjugated rat IgG2a,κ (BD
PharMingen) and biotinylated hamster IgG
group 1, λ (BD PharMingen) were used as
isotype controls. The cells were washed three
times with PBS/NaN 3 at 4 °C, and then
incubated with 0.1µg of streptoavidinconjugated Cy-ChromeTM (BD PharMingen)
in 100µl of PBS/NaN 3 at 4 °C for 20 minutes.
The cells were washed three times, fixed with
1ml of PBS containing 1% paraformaldehyde,
and analysed by a FACS Calibur (Becton
Dickinson, San Jose, CA) and CELL QuestTM
version 3.3 software.
Immunization and CTL induction
Bone marrow cells (10x106) stimulated with
GM-CSF for 7 days were incubated in the
presence of 10µM peptide-2 in 1ml of AIM-V
at 37 °C for 2 hours. The cells were washed
two times with RPMI-1640. Peptide-2-pulsed
cells (2x106) were injected intravenously into
BALB/c mice. Four weeks later, the spleens
were collected, minced into single cell
suspension, erythrocyte-lysed, and treated
with anti-CD4 antibody (BD PharMingen) at
the rate of 1µg/1x107 cells and 10% baby
rabbit complement (Cederlane, Hornby, Ont,
Canada) to deplete CD4-positive cells. Five
million cells were then co-cultured with the
same number of peptide-2-pulsed, 33Gy Xray-irradiated syngeneic spleen cells in 2ml of
EHAA medium (Sigma) supplemented with
100µg/ml nucleic acid precursors, 2mM Lglutamine, 5x10-5M 2-ME, 100U penicillin,
100µg/ml streptomycin, 10mM HEPES, and
10% fetal calf serum in 24-well plate at
37 °C in 5% CO2. On day 4, half volume of
the medium was replaced with fresh one,
and 10 U recombinant mouse IL-2 was
added. On day 7, the cells were harvested
and used as CTL (cytotoxic T lymphocyte)
effector cells. Mice were also immunized by
subcutaneous injection with 1 n mole of the
peptide emulsified with complete Freund
adjuvant (CFA) into two-foot pads. In this
immunization protocol, draining lymph node
(popliteal lymph nodes) cells were used as
the effector cells after stimulation in vitro with
the peptide as described above. When mice
were immunized with the peptide-1, the cells
were stimulated in vitro with peptide-1pulsed spleen cells.
Cytotoxicity assays
P815 cells (1x106) were pulsed with the
peptide at a concentration of 10µM in
complete medium at 37 °C for 3 hours. The
cells were labelled with 100 µCi of
Na251CrO 4 (NEN Life Science Products,
Boston, MA) for one hour, then washed three
times and suspended in complete medium.
Peptide-pulsed, 51Cr-labelled cells were
seeded in 96-well V-bottom plate at 1.5x103
cells in 100µl of complete medium per well.
Effector cells were added to the plate to
make various effector/target ratios (E/T ratios)
in a total volume of 0.2ml per well, and the
plate was incubated at 37 °C in 5% CO2 for
four hours. The supernatant fluids were
harvested with a Supernatant Collecting
System (Skatoron, Lier, Norway), and 51Cr
content was measured by a gamma counter
(Aloka model ARC-300). Maximum 51Cr
release was determined by adding 0.1%
Triton X, and spontaneous 51Cr release was
determined with the wells that contained
target cells and medium only. Assays were
performed in triplicate, and the mean value
was used to calculate percent-specific lysis
with the following formula: % specific lysis =
100 x [(release with effector cells –
spontaneous release) / (maximum release –
147
spontaneous release)]. Spontaneous release
did not exceed 28.9% of the maximum
release.
It is known that mature murine DCs strongly
express major histocompatibility complex
(MHC) class II antigen, co-stimulatory
molecules CD80 and CD86, and CD11c, the
α chain of p150/95 β2-integrin[9,10] . We
examined the expression of MHC class II
antigen I-Ad/I-Ed, CD86, and CD11c on
BALB/c mouse bone marrow cells, which
were cultured with GM-CSF for seven days,
by flow cytometry three colour analysis, and
evaluated the purity of DC. As shown in
Figure 1a, 25.07% of the bone marrow cells
strongly expressed both I-Ad/I-Ed and CD86.
The percentage of CD11c-positive cells in
these double positive cells was 95.81%
(Figures 1b and 1c). In contrast, freshly
isolated bone marrow cells did not express
these surface molecules (data not shown).
These results suggest that bone marrow cells
were differentiated into DCs during the
culture with GM-CSF for seven days and that
DCs accounted for one-fourth of the entire
population.
Figure 1. Expression of the dendritic cell markers on bone marrow cells after culture
in the presence of GM-CSF for 7 days
(a)
(b)
(c)
R1
CD86
CD11c
I-Ad/I-E d
CD11c
I-Ad/I-E d
CD86
[BALB/c mouse bone marrow cells were cultured for 7 days in AIM-V medium with 20 ng/ml of
murine GM-CSF. The cells were stained with anti – I-Ad/I-Ed – FITC, anti-CD86 – PE, anti-CD11c –
biotin, and streptoavidin – Cy-Chrome TM. (a); The cells in the region (R1) strongly expressed I-Ad/I-Ed
and CD86, and accounted for 25.07% of the entire cultured bone marrow cells. (b) & (c): The cells
in the region (R1) were examined for the expression of CD11c. 95.81% expressed CD11c.]
We first attempted to induce specific
CTLs by two foot pad immunizations with
the peptide-1 (GYISTRVEM) emulsified
with CFA. Specific CTL activity was not
detected in draining lymph node cells.
(Table, Experiment no. 1). We speculated
that the inability of the peptide-1 to
148
induce specific CTLs might partly be due
to the low binding affinity to H-2K d MHC
class I molecule, because the peptide-1
possesses only one anchor residue (Y) for
binding to H-2K d molecule. We, therefore,
prepared the peptide-2 (GYISTRVEL) in
which the last residue M of the peptide-1
was substituted for L in order to provide
the complete H-2K d-binding motif [11].
It was reported that immunization
with virus epitope peptide–pulsed DCs
efficiently induced virus -specific CD8+
CTLs and protective immunity[5,6] . We
attempted to induce specific CTLs by
immunization with peptide-2-pulsed DCs.
We intravenously injected 2x106 bone
marrow cells, which were stimulated with
GM-CSF for seven days and pulsed with
the peptide-2, into BALB/c mice. Onefourth of the cultured bone marrow cells
were DCs and it was reported that
intravenous immunization with 1x105 to
5x10 5 purified DCs pulsed with peptides
induced antiviral immunity[5] . Spleen cells
from the mice immunized with peptide-2pulsed DCs lysed peptide-2-pulsed P815
cells in a dose dependent fashion after
stimulation in vitro with X-ray-irradiated,
peptide-2-pulsed syngeneic spleen cells in
the presence of recombinant IL-2 for
seven days (Table, Experiment no. 2). This
result demonstrates that intravenous
immunization with peptide-2-pulsed DCs
induced peptide-2-specific CTLs.
Table. Induction of specific CTLs by immunization with peptide-2-pulsed dendritic cells
and the peptide-2 emulsified with complete Freund adjuvant
Experiment
no.
% Specific lysis*
Immunization
E/T ratio
Non-pulsed
Peptide-1pulsed
1.
Peptide-1/CFA
20
100
5.4
22.9
2.
Peptide-2/CFA
10
20
40
6.5
8.4
9.9
Not done
Not done
Not done
36.8
55.2
65.4
Peptide-2-pulsed DC
10
20
40
20.9
18.7
24.0
Not done
Not done
Not done
24.5
35.8
44.2
Peptide-2/CFA
5
10
20
12.7
20.8
28.7
3.
7.9
21.9
Peptide-2pulsed
Not done
Not done
23.0
30.4
46.3
23.9
34.2
48.3
* 51Cr–labeled P815 mastcytoma (H-2d) pulsed with the peptide (10µM, 3hours) were used as target cells.
Specific CTL activity was observed in
the draining lymph node cells after
immunization
with
peptide-2/CFA.
Interestingly, CTLs induced by peptide2/CFA demonstrated lower levels of nonspecific cytotoxic activity to P815 cells than
those induced by peptide -2-pulsed DCs.
Peptide-2/CFA-induced CTLs lysed peptide1-pulsed target cells as well as peptide-2Dengue Bulletin – Vol 28, 2004
pulsed ones. (Table, Experiment nos. 2 and
3). These results suggest that the peptide-2
which has the complete binding motif to H2kd molecule can induce specific CTLs
when used with CFA, and that peptide-2specific CTLs also lyse original peptide-1pulsed target cells. Induction of higher levels
of non-specific cytotoxicity by immunization
with DCs may be due to the high antigen
149
presentation ability of DCs to prime various
repertoires of T cells. The other possibility is
the difference in the source of lymphocytes.
We
observed
that
spleen-derived
lymphocytes tended to show higher levels of
non-specific lysis than lymph node -derived
cells (data not shown). It seems that the
draining lymph node cells from mice
immunized with the peptide and CFA are a
better source of CTLs than the spleen cells
from those immunized with peptide-pulsed
DCs because of low non-specific cytotoxic
activiity. The data, however, only suggest
how efficiently measurable specific CTLs
can be induced in vitro. It is plausible that
intravenous immunization with peptidepulsed DCs induces higher levels of specific
CTLs in vivo and protective immunity
against viral infections. Moreover, CFA is not
accepted
for
human
use.
Thus,
immunization strategy using peptide-pulsed
DCs is still worth investigating for induction
of
CTL-mediated
anti-dengue
virus
immunity.
References
[1] Gotch F, Gallimore A and McMichael A.
Cytotoxic T cells – protection from disease
progression – protection from infection.
Immunol Lett, 1996, 51: 125-128.
[2] Toes RE, van der Voort EI, Schoenberger SP,
Drijfhout JW, van Bloois L, Storm G, Kast
WM, Offringa R and Melief CJ.
Enhancement of tumor outgrowth through
CTL tolerization after peptide vaccination is
avoided by peptide presentation on
dendritic cells. J Immunol, 1998, 160: 44494456.
[3] BenMohamed L, Belkaid Y, Loing E, Brahimi
K, Gras-Mass H and Druihe P. Systemic
immune responses induced by mucosal
administration of lipopeptides without
adjuvant. Eur J Immunol, 2002, 32: 22742281.
[4] Steinman RM. The dendritic cell system and
its role in immunogenicity. Annu Rev
Immunol, 1991, 9: 271-296.
[5] Ludewig B, Ehl S, Karrer U, Odermatt B,
Hengartner H and Zinkernagel RM.
Dendritic cells efficiently induce protective
antiviral immunity. J Virol, 1998, 72: 38123818.
[6] Takahashi H, Nakagawa Y, Yokomuro K and
Berzofsky JA. Induction of CD8+ cytotoxic T
lymphocytes
by
immunization
with
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syngeneic irradiated HIV-1 envelope derived
peptide-pulsed dendritic cells. Int Immunol,
1993, 5: 849-857.
[7] Porgador A, Snyder D and Gilboa E.
Induction of antitumor immunity using bone
marrow-generated dendritic cells. J Immunol,
1996, 156: 2918-2926.
[8] Spaulding AC, Kurane I, Ennis FA and
Rothman AL. Analysis of murine CD8+ T-cell
clones specific for the dengue virus NS3
protein: flavivirus cross-reactivity and
influence of infecting serotype. J Virol, 1999,
73: 398-403.
[9] Maraskovsky E, Brasel K, Teepe M, Roux ER,
Lyman SD, Shortman K and McKenna HJ.
Dramatic increase in the numbers of
functionally mature dendritic cells in Flt3
ligand-treated mice: multiple dendritic cell
subpopulations identified. J Exp Med, 1996,
184: 1953-1962.
[10] Son YI, Egawa S, Tatsumi T, Redlinger RE Jr,
Kalinski P and Kanto T. A novel bulk-culture
method for generating mature dendritic cells
from mouse bone marrow cells. J Immunol
Methods, 2002, 262: 145-157.
[11] Romero P, Corradin G, Luescher IF and
Maryanski.
H-2Kd-restricted
antigenic
peptides share a simple binding motif. J Exp
Med, 1991, 174: 603-612.
Marize Pereira Miagostovich#, Flávia Barreto dos Santos
and Rita Maria Ribeiro Nogueira
Laboratory of Flavivirus, Department of Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation,
Fiocruz Avenida Brasil 4365 Manguinhos, Rio de Janeiro 21040-360, RJ, Brazil
Abstract
Many countries in Central and South America as well as Brazil have been characterized by a rise in
dengue endemicity. Since 1986, dengue infection has gained endemicity in these countries and more
than 3 million dengue cases have been reported along with the emergence also of the severe forms of
the disease. Once intratypic variations among dengue virus (DEN) serotypes have been associated with
the disease severity, the molecular characterization of DEN becomes an indispensable tool for the
laboratories performing virological surveillance programmes. In countries endemic for DEN, as in Brazil,
the monitoring of DEN activity should be an ongoing programme to detect the eventual introduction of
new serotypes/genotypes to curb the impact of the circulating strains. Here, the molecular
epidemiological studies performed on Brazilian DEN strains are presented in order to contribute to a
better understanding of the dengue epidemiology in the country.
Keywords: Dengue viruses, molecular epidemiology, genotypes, Brazil.
Introduction
The dengue (DEN) virus belongs to the
Flaviviridae family, genus Flavivirus, and it has
four distinct antigenic serotypes (1 to 4) that
cause a spectrum of diseases ranging from
asymptomatic, mild, undifferentiated fever
and classic dengue fever (DF) to more severe
forms known as dengue haemorrhagic fever
(DHF) and dengue shock syndrome (DSS) [1,2].
The DEN virus is a spherical particle of
approximately 500 Å in diameter, lipid
enveloped that includes one segment of a
single-stranded positive sense RNA with
~11,000 nucleotides in length. The genomic
RNA contains a single long open reading
#
frame (ORF) of over 10,000 nucleotides that
encodes a polyprotein precursor of about
3,400 amino acid residues which is co- and
post-translationally processed by the host cell
and virus-specific protease to yield structural
and nonstructural proteins. The coding
protein region starts with the sequence for
the core (C), precursor of membrane (prM/M)
and the envelope (E) structural proteins,
followed by a series of seven nonstructural
(NS) proteins ordered as follows: NS1, NS2A,
NS2B, NS3, NS4A, NS4B and NS5. The ORF
is flanked by two untranslated regions (5’ and
3‘ UTR) and has a type I cap at its 5’ end
(m7GpppAmp) and appears to lack a 3’ terminal poly A tract[3,4].
E-mail: [email protected]; Fax: 55-21-2598-4373
151
Electron micrographs show that the
virion is characterized by an electron dense
core that consists of an isometric
nucleocapsid, made up of a single C (100
amino acids) protein, surrounded by a
double lipid layer, whereas both E (495
amino acids) and M (75 amino acids)
proteins are associated[5] . E-glycoprotein is
the major surface protein and as showed by
crystallography, the flat elongated dimmer
extends parallel to the viral membrane [6] . The
E protein is associated with a number of
biological activities, being the most important
antigen with regard to virus biology and
immunity[7] .
The intratypic variation of DEN was
demonstrated by the fingerprinting method
that determined genetic variants within each
serotype and employed the term topotype to
define variants representing samples from the
same geographical region[8] . From 1990
onwards, the molecular analysis by the partial
sequencing of the DEN genome gathered the
topotypes in genomic groups (genotypes) and
became an important tool to determine their
genetic variation and to identify risk factors
associated with the transmission of particular
strains[9] .
The E gene has been the most
commonly surveyed gene in dengue
molecular epidemiology[10-15] , although genes
that encode nonstructural proteins and noncoding regions have also been used in the
phylogeny studies[16-22].
Once intratypic variations among
different serotypes were associated with the
disease severity, technologies for the
molecular characterization of DEN became
an indispensable tool for laboratories
performing
virological
surveillance
programmes. This paper presents the most
relevant
results
of
the
molecular
152
characterization of DEN strains isolated in
Brazil during the last 18 years since dengue
became
endemic
and
surveillance
programmes were implemented in the
country.
DEN-1
The first molecular analysis for DEN Brazilian
strains characterization was performed by an
analysis on genome fragments from DEN-1
by using restriction endonuclease (RE)
enzymes. In this study, restriction fragment
heterogeneity by Hae III digestion of cDNA
products was used to map the distribution of
DEN-1 topotype found in the American
region. The strains isolated in the state of Rio
de Janeiro from human serum specimens
from 1986 to 1994[23,24] were grouped in the
American (Caribbean) topotype, recognized
as the only one circulating in the
Americas [8,25] . The percentage of the similarity
observed among DEN-1 Brazilian strains
ranged from 60% to 94%, showing the
evolution of those samples since its
introduction in the state in 1986.
By the sequencing of 240-nucleotides
(nts) spanning the E/NS1 junction (111 nts
from the 3’ end of E gene and 129 nts from
the 5’ end of NS1 gene), the DEN-1 Brazilian
strains were classified as belonging to
genotype I which comprises of strains from
the Americas, Africa and South-East Asia[9] .
The DEN-1 Brazilian strains were also
analysed by using one-step amplification with
four primers that target regions spanning
polymorphic
endonuclease
restriction
specific sites (RSS-PCR) and all of them were
grouped into subtype C, which corresponds
to the largest genotypic group of DEN-1
described as genotype 1[9,26] . The RSS-PCR
has become an alternative tool routinely used,
which has allowed the characterization of
DEN strains for molecular epidemiological
studies performed in endemic countries,
providing rapid identification of viruses
currently circulating[27-30] .
Another study performed with the DEN1 Brazilian strains compared the complete
sequences of three strains isolated in 1990,
1997 and 2001. The genome analysis of
those strains revealed a remarkable
conservation of the structural proteins and 27
amino acids substitutions in the nonstructural
genes, and 12 of them in the NS4B-NS5 and
nine specific to strains BR/97 and BR/01.
Those
findings
also
suggested
that
recombinant events might have occurred,
since some amino acids substitutions were
previously identified in DEN-1 strains
sequenced so far[31]. The evidence that the
genetic diversity of DEN might be generated
by recombination among those viruses has
been described[32-34].
DEN-2
The DEN-2 fingerprinting analysis of the
American strains showed that this serotype
exists in the American continent as two
topotypes representing strains from the
Caribbean region (Puerto Rico) and from the
Americas, India and South Pacific[8]. By the
sequencing of the E/NS1 junction those
topotypes could be related to genotype I
(Native American) and to genotype III (SouthEast Asian/American), respectively. This latter
genotype was introduced into the Americas
in 1981, and was responsible for the first
DHF/DSS epidemic that occurred in the
continent and spread throughout the region
over the next two decades[35,36] . The direction
of the transmission from South-East Asia to
the Americas was demonstrated as well, since
DEN-2 from Brazil, Colombia, Mexico and
Venezuela have a common progenitor with
those from South-East Asia[36] .
In
Brazil,
the
first
molecular
characterization of DEN-2, introduced in the
country in 1990[37,38] , was performed by RE
analysis and showed a similarity of 80% with
the 1981 Jamaica isolate, suggesting the
spread of those viruses from the Caribbean
region to South America[25] .
The geographical origin of DEN-2
Brazilian strains was also established by the
direct sequencing of cDNA fragments
amplified by the polymerase chain reaction
of a fragment encoding amino acids 29 to 94
in the E gene. Considering a divergence of
6% between the nucleotide sequences as a
cut-off for genotype classification, it was
demonstrated that the Brazilian strains
belonged to the South-East Asian/American
genotype. The comparison of the three DEN2 strains isolated in Rio de Janeiro, two of
them obtained from classic dengue cases and
one from a fatal case, did not identify the
markers for virulence in the region studied[10] .
The analysis of DEN-2 samples isolated
in the states of Rio de Janeiro, Ceará, Bahia,
and Alagoas between 1990 and 1995 was
performed by the partial sequencing of nts
1685 and 2504 encompassing the E gene
and demonstrated the spread of this serotype
from Rio de Janeiro to other states[39] .
All the characteristics observed in the
Brazilian DEN-2 genotype were confirmed by
the full-length analysis of the nucleotide and
amino acids sequence (GenBank access #
AF489932) [40] . The Asian-specific nonconserved amino acid differences, previously
described by Leitmeyr et al.[41] as well as
additional differences specific to the Brazilian
strain were found in E, NS3, and NS5
153
genes[40]. Changes in the E protein could
affect
the
immunogenicity
or
cell
entry/tropism, whereas changes in NS3
(helicase/protease) and NS5 (RNA-dependent
RNA polymerase) could affect replication
efficiency. In addition, differences in the
predicted secondary structure of the 5’ and
3’ untranslated regions were found between
the South-East Asian/American and native
American genotypes; in these regions, the
Brazilian isolate was identical to the SouthEast Asian/American strains in sequence and
consequently in the predicted secondary
structures[40]. These similarities with the
South-East Asian/American genotype were
also reported recently for the Martinique
703/98 strain after a complete analysis of the
genome[42] .
In Brazil, this DEN-2 genotype was
responsible for some clinical features, mainly
related to the severity of the disease. In
regions where DEN-2 accounted for primary
infections, as in the states of Bahia and
Espirito Santo[43] , the most common clinical
feature consisted of classic fever, with
frequent exanthema, pruritus and a few
severe cases. However, in other states where
DEN-2 circulated after extensive epidemics
caused by DEN-1, as in Rio de Janeiro, Ceará,
Pernambuco and Rio Grande do Norte, an
increase in the number of severe cases was
observed. The first DHF/DSS case was
reported in Rio de Janeiro after the
introduction of DEN-2 in 1990, and it was
accompanied by an increasing number of
hospital admissions resulting from DEN-2
secondary infections[44,45] .
By using RSS-PCR we were able to
analyse geographically and temporally
distinct Brazilian DEN-2 strains encompassing
ten years (1990 to 2000). The analysis of the
RSS-PCR products showed that all Brazilian
154
strains presented the same pattern,
presenting consistent and reproducible
amplicons of 582bp and 100bp and,
occasionally, extra amplicons of 676 bp or
150 bp[29] . The DEN-2 Brazilian RSS-PCR
pattern was consistent; however, it did not
match any of the RSS-PCR patterns
previously described by Harris et al.[46] Once
the method was developed with DEN-2
isolates obtained from 1964-1986, the
ongoing evolution of those viruses over the
last 15 years could explain the genetic
diversity
observed.
Despite
those
observations, the sequence of the E/NS1
gene junction (GenBank Access # AF529064
to AF529078) showed that the Brazilian
DENV-2 strains still belonged to the SouthEast Asian/American genotype [29].
DEN-3
Different from the DEN-3 genotype IV
(topotype
Caribbean)
responsible
for
epidemics in the ‘60s and ‘70s, the DEN-3
re-introduced in the American continent after
an absence of 17 years belonged to genotype
III (topotype Sri Lanka), represented by
strains from Sri Lanka and India, which are
associated with DHF/DSS cases in those
countries[13,47] .
By the time of DEN-3 isolation in
Brazil[48] , RSS-PCR was extremely valuable
which
once
allowed
the
rapid
characterization of the first strain as subtype
C, confirming the introduction and direction
of transmission of those viruses from Central
to South America[28,46] .
After the RSS-PCR analysis, th e nucleic
acid sequencing from positions 278 to 2550
of DEN genome was performed (Access
number AY038605) and the parsimony
analysis generated a phylogram assigning a
Brazilian DEN-3 strain to genotype III,
reconfirming those data[13,28] . The similarity
rate of a Brazilian DEN-3 strain to others
represented by the same subtype III ranged
from 96% to 98% and 98% to 99% for
nucleic acid and deduced amino acid
sequences, respectively. The comparison of
the Brazilian DEN-3 with strains isolated in
Guatemala showed a total of 14 nucleic acid
substitutions, with one of them resulting in an
amino acid change from histidine to
arginine [28] .
As a result of several DEN-3 epidemics in
Latin American countries, a large number of
DEN-3 genome sequences have been
recently deposited in the GenBank[49-51] . A
phylogenetic study compromising DEN-3
strains isolated in Sri Lanka, East Africa and
Latin America confirmed the establishment of
the new DEN-3 genotype[51] . According to
the author, there are two separate lineages
formed within genotype III: Group A
consisting of isolates from 1981 to 1989 in Sri
Lanka and Group B which was expanded in
three distinct clades including isolates from
1989 to 1998 in Sri Lanka, strains isolated in
East Africa from 1985 to 1993 and isolates
from 1994 in Latin America. The
phylogenetic analysis suggested that genotype
III was introduced from the Indian
subcontinent into East Africa in the 1980s
and from Africa into Latin America in 1994,
showing a single genotype introduction in the
continent and its subsequent diversification[52] .
In the same year, Peyrefitte et al.[53] showed a
high similarity between the DEN-3
Martinique and the Brazilian strains.
Furthermore,
the
complete
genome
characterization of the Brazilian DEN-3
sequence (AY679147) strain confirmed an
insertion of 11 nts in the 5´ non-coding
region of the genome as previously described
for the Martinique strain[53] .
The severity of the disease and the
occurrence of deaths resulting from primary
infections during the DEN-3 epidemic in the
state of Rio de Janeiro in 2002 could be
explained partially by the virulence of this
particular genotype [28,54] . Fatal cases, resulting
from primary dengue infections, were
previously described[55] before the DEN-3
genotype III introduction in Brazil. However,
the highest number of DHF/DSS cases that
occurred in the state were due to secondary
infection by the South-East Asian/American
DEN-2
genotype [45] .
Those
findings
corroborated the previous observations that
some DEN strains can be more virulent than
others, representing an important risk factor
for DHF/DSS and that antibody-dependent
enhancement (ADE) itself does not explain all
cases of severe disease [51,56-58] . Recently, it
was suggested that the more virulent
genotypes were now replacing those that had
a lower epidemiological impact throughout
the world[59] .
Conclusion
In the last few decades, dengue has spread as
a pandemic in the American continent,
starting in the Caribbean islands and
expanding to North, Central and South
America[60] . In this context, the dengue
epidemiological profile in Brazil has changed
from a non-endemic to a hyperendemic one.
Since the 1980s when the first DEN strains
were isolated, more than 3 million dengue
cases and nearly 2,090 DHF/DSS cases have
been
reported
in
the
country
(www.funasa.gov.br) [61,62] .
The endemicity of dengue in 25 out of
the 27 federative units, the remarkable
virulence of the DEN-2 and DEN-3
genotypes and the risk of the introduction of
155
DEN-4 in the country highlight the alarming
dengue epidemiological picture in Brazil. In
this scenario, use of rapid methods for DEN
identification and molecular characterization
are indispensable tools in the virological
surveillance laboratories, mainly due to an
obvious need to characterize dengue
genotypes before a major outbreak
occurs [26,46,63] . The partial sequencing of DEN
strains genome has also been used routinely
for DEN molecular epidemiological studies,
and it recently characterized the co-infecting
genotypes of DEN-1 and DEN-2 in a patient
presenting classic dengue fever in São
Paulo[64] .
The knowledge of the virus genotype
circulating in a particular region has also
implications for the potential introduction of
vaccines, allowing the evaluation of the
genomic relations between the viruses used
in vaccine development and the circulating
strains. The ability of pre-existing dengue
antibodies to neutralize better certain DEN
variants than others has been demonstrated.
Some strains may produce a more severe
disease, not because of the virulenceinherited properties but because antibodies
from a primary infection may enhance
infection
with
one
genotype
while
neutralizing infection with a distinct one[52,65] .
Given the limited options available for
dengue
control,
active
surveillance
programmes with continuous monitoring of
dengue infection in communities is still one
of the strategies available to detect the
introduction of new serotypes/genotypes, and,
consequently, to prevent the occurrence of
epidemics, thus minimizing the impact of the
circulating strains.
Acknowledgements
To Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Fundação
de Amparo a Pesquisa do Estado do Rio de
Janeiro (FAPERJ), Fundação Oswaldo Cruz
(FIOCRUZ), PAPES III – FIOCRUZ. Thanks
are also due to the staff of the Laboratory of
Flavivirus for their technical assistance.
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160
Unusual Emergence of Guate98-like Molecular Subtype
of DEN-3 during 2003 Dengue Outbreak in Delhi
Manoj Kumar*#, S.T. Pasha*, Veena Mittal*, D.S. Rawat*, Subhash Chandra Arya**,
Nirmala Agarwal**, Depesh Bhattacharya*, Shiv Lal* and Arvind Rai*
*National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi – 110 054, India
**Sant Parmanand Hospital, 18 Shamnath Marg, Delhi – 110 054, India
Abstract
With a view to identifying the molecular subtype of the circulating dengue virus responsible for a major
outbreak of dengue fever (DF) / dengue haemorrhagic fever (DHF) in and around Delhi during the postmonsoon period in 2003, 32 serum samples were collected from clinically suspected cases. These were
subjected to reverse transcription/polymerase chain reaction (RT/PCR) for amplification of 511 bp CPreM gene region of the dengue virus. Seven specimens, yielding a satisfactory quantum of viral RNA,
were subsequently processed for automated nucleotide sequencing. Five of the seven analysed isolates
showed close DNA sequence homology with Guate96-98 strains of DEN-3 virus, whereas two turned out
to be genotype IV of DEN-2. Earlier, DEN-2 (genotype IV) had been identified as the etiological agent
during a major DF/DHF outbreak in Delhi in 1996 and also in 2000. Though DEN-2 continues to prevail,
DEN-3, having a close sequence homology with Guate96-98 strains, seems to have entered India for the
first time in late 2003, resulting in a major DF/DHF outbreak. How the Guate96-98 strain of DEN-3
entered India remains to be linked epidemiologically.
Keywords: Dengue outbreak, molecular typing, CpreM gene, DEN-3, Delhi.
Introduction
Dengue fever/dengue haemorrhagic fever
(DF/DHF) is caused by one or more of the
four antigenically-related dengue virus
serotypes DEN-1 to DEN-4. It is widespread
in tropical and subtropical countries in the
world and is a serious cause of morbidity
and mortality, threatening about one third
of the total human population[1-3] . Many
outbreaks and epidemics of DF/DHF have
been reported in different parts of India
during the past four decades[4-8] . In Delhi
#
alone, a number of outbreaks of dengue
virus infection were recorded in 1967, 1970,
1982, 1988 and 1990[9-13] . Again in 1996, a
major DHF outbreak, resulting in 10,252
cases with 432 deaths, occurred in and
around Delhi. DEN -2 genotype IV was the
predominant etiological agent[14,15] .
Delhi and its adjoining areas were again
struck by a major outbreak of DF/DHF
between September and December 2003.
The present study was undertaken to unveil
the predominant molecular subtype of the
dengue virus involved in this outbreak.
E-mail: [email protected]; Tel./Fax: 91-11-23912960
161
Emergence of DEN-3 in Delhi in 2003
Virus RNA isolation
Clinical specimens
Thirty-two serum samples were subjected to
dengue viral RNA isolation. 140µl sera
sample was processed for RNA isolation using
QIAamp viral RNA Kit (QIAGEN, Germany)
using standard kit protocol. Finally, viral RNA
was eluted in 30µl nuclease-free water.
A total of 32 serum samples from clinically
suspected acute cases of DF/DHF were
collected from different hospitals in Delhi
between September and December 2003.
Most of the serum samples were collected
within the first five days of the clinical onset.
Samples were transported to the laboratory
within six hours of collection and stored at –
70 °C until processed. Thirty-two sera are
considered adequate to establish the
predominant molecular subtype of dengue
virus.
Reverse transcription/polymerase
chain reaction (RT/PCR)
RT-PCR was carried out using previously
reported D1 and D2 primers that were M13
tailed for the convenience of nucleotide
sequencing. This primer set is capable of
amplifying all the four types of dengue viruses
(DEN-1 to DEN-4).
Primer D1 (with M13F tail)
(5’-TGTAAAACGACGGCCAGTTCAATATGCTGAAACGCGCGAGAAACCG-3’)
(M13 forward primer sequence underlined)
Primer D2 (with M13R tail)
(5’-CAGGAAACAGCTATGACCTTGCACCAACAGTCAATGTCTTCAGGTTC-3’)
(M13 reverse primer sequence underlined)
Complementary DNA (cDNA) synthesis
and gene amplification of 511bp CpreM
gene region of the dengue virus was
performed using one step GeneAmp RNA
Gold RT PCR Kit (Applied Biosystems, USA)
with D1 and D2 primers for detection and
typing of all the four types of dengue virus [16] .
Briefly, 50µl reaction mix containing final
concentration of 1X of 5X RT buffer, 1.5mM
MgCl2, 200µM dNTPs, 5mM dithiotheratol
and 10pmol of D1 and D2 primers, 10U
RNAase inhibitor, 15U of MuLV MultiScribe
reverse transcriptase, 2.5U of Amplitaq Gold
DNA polymerase and 5 µl of extracted viral
RNA. RT was performed at 42 °C for 20
minutes on GeneAmp 9700 PCR System.
Then pre-hold at 95 °C for 10 minutes
162
followed by 40 cycles of 95 °C for 30
seconds, 55 °C for 30 seconds, 72 °C for 1
minute and final extension for 72 °C for 10
minutes and hold at 4 °C. Appropriate
positive and negative controls were used in
RT/PCR.
PCR
products
were
electrophoresed on 1.5% agarose gel along
with 100bp DNA ladder marker (MBI
Fermentas, USA) and were visualized on gel
documentation system (Biometra, Germany).
Gene sequencing and phylogenetic
analysis
PCR amplicons were purified using
Centricon-100 columns (Millipore, USA)
and subjected to automated dideoxy chain
termination nucleotide cycle-sequencing
using commercial ABI PRISMTM Big Dye
Terminator Cycle Sequencing Kit with
Amplitaq DNA Polymerase FS, following the
manufacturer’s protocol and run on ABI
PRISM TM 310 Genetic Analyser (Applied
Biosystems, USA). Nucleotide sequences
were edited and aligned using Sequence
Navigator Software. Subsequently, blast
search (http://www.ncbi.nlm.nih.gov/blast)
and phylogenetic analysis using DNA Star
software, were done to reveal the dengue
virus molecular subtype.
Result and discussion
During the study, a majority of the subject
patients had clinical symptoms of DF and
only sporadic cases presented symptoms of
DHF/DSS {DF: 24 (75%), DHF: 7 (21.9%)
and DSS: 1 (3.1%)}. The samples belonged
to all age groups ranging from 5 to 50 years.
The male-female ratio was 18:14. The mean
platelet count was 77,120, which ranged
from 18,000 to 250,000. Most of the
samples collected within five days from the
onset of the fever were selected for RT-PCR
testing.
Out of the 32 serum samples subjected
to RT-PCR, only seven yielded amplification
of 511bp C-PreM gene region of dengue
virus. Lane 1-14 in the Figure included
clinical samples, 100bp DNA ladder marker
in lane M and negative control in lane Neg.
Automated nucleotide sequencing of these
seven RT/PCR products revealed two groups
of sequences, the first group had five almost
identical sequences, while the second group
had two similar sequences. All samples were
subjected to blast search, which revealed
that five isolates had a very close sequence
homology (=98%) with Guate98 AB038478
strain of dengue type 3 (DEN -3), while two
turned out to be DEN-2 and showed =99%
homology with Delhi96 AF047394 strain
Delhi 2000 strains (Table). These Cpre M
gene sequences were submitted in the Gene
Bank wide accession AY 706094-AY706099.
Figure. PCR products visualization on 1.5% agarose gel
163
Table. Clinical and molecular analysis data of seven samples, yielding RT-PCR positivity for
511bp CpreM gene of dengue virus
RT-PCR
511bp
Assigned
genotype
on blast
search
89,000
+ve
DEN-3
20/M
79,000
+ve
DEN-2
DHF
15/M
39,000
+ve
DEN-3
08.10.03
DF
14/F
NA
+ve
DEN-3
16DEL03
01.10.03
DF
34/F
18,000
+ve
DEN-2
6
19DEL03
04.10.03
DHF
40/M
38,000
+ve
DEN-3
7
26DEL03
13.10.03
DHF
16/M
36,000
+ve
DEN-3
S.
No.
Sample
ID*
Serum
collection
Clinical
state
Age/Sex
1
04DEL03
22.09.03
DF
30/F
2
06DEL03
24.09.03
DF
3
10DEL03
07.10.03
4
11DEL03
5
Platelet
count
Out of 32 samples subjected to RT-PCR for 511bp Cpre M gene of dengue virus, only 7 turned positive, while 25 did
not show any amplification.
All five DEN-3 strains had =98% nucleotide sequence homology with Guate98 strains.
Both DEN-2 strains had =99% nucleotide sequence homology with already circulating Delhi96 and Delhi2000
strains.
NA: Information not available
Several studies have shown that dengue
virus infection has been endemic in
different parts of India, as documented for
over four decades[17,18] , and almost all the
four known serotypes of dengue virus (DEN 1 to DEN-4) have been reported. The
metropolitan city of Delhi witnessed several
outbreaks of DF/DHF in 1967, 1970, 1982,
1988,1996 and 2000. DEN -1 and DEN-3
viruses were associated with the 1970
epidemic, DEN-1 and DEN -2 with the 1988
epidemic, while genotype IV of DEN-2 was
responsible for the major DHF outbreak in
1996[15] . Our previous findings revealed
genotype IV of DEN-2 as the predominant
type circulating from 1996 onwards, based
on RT-PCR and C-PreM gene sequencing,
164
although cases
detected[19-21] .
of
DEN-1
were
also
During the present study, the majority
of the patients had clinical symptoms of DF
and only sporadic cases presented with
symptoms of DHF or DSS. The samples
referred to our laboratory for molecular
characterization were accompanied by IgM
serology results. When cross-checked, we
found that five RT/PCR-positive samples
were IgM-negative, while two RT/PCRpositive samples were dengue IgM-positive.
Previous studies had also shown that most,
but not all, RT/PCR-positive samples had
negative IgM serology. The reason for this is
attributed to the fact that the virus had not
been recovered from most of the DF/DHF
patients beyond the 5th day of the onset of
the symptoms, while detectable levels of
dengue-specific IgM antibodies appear after
the 4th or the 5th day. Our findings reestablished that the ideal time for the
collection of samples for molecular test was
between days 1-5.
The nucleotide sequence alignment and
blast search of the seven RT/PCR-positive
samples in the present study, when
compared with those of earlier ones,
revealed that only ~29% (2/7) belonged to
the already prevalent genotype IV of DEN2; whereas the majority ~71% (5/7) showed
close genomic homology (=98%) with
GUATE98 AB038478 strain of dengue type
3 (DEN-3) which caused a widespread
dengue outbreak in Guatemala in the late
Nineties[22] . A similar strain is also reported
to have been reintroduced in Marlinique
(French West Indies)[23] and in Rio de
Janerio, Brazil (unpublished data vide Gene
Bank Accession No. AY679147). The first
reported evidence of DEN-3 in Delhi was in
1970, based on serotyping, but no genomic
data of the 1970 strain of DEN -3 is available.
It is difficult to ascertain, after a long gap of
33 years, whether the old 1970 strain of
DEN-3 had re-emerged during the current
outbreak; or Guate98 DEN -3 strain
(prevalent in South American countries) had
been introduced for the first time in India.
The changing epidemiology of different
subtypes of dengue virus and their coexistence and/or replacement of one type
by the other is well documented[24] . During
the present outbreak, we found DEN-3 as
the predominant type, but it did not seem
to completely replace the previously
circulating DEN -2. Prior to 1977, coexistence of DEN-2 and DEN -3 in the
Americas had also been reported[25] . The
first epidemic of DEN -3 in Jamaica and
Puerto Rico was witnessed in 1963, which
was followed by another epidemic of DEN 3 in Colombia and Puerto Rico in the mid1970s, and in the Pacific islands in early
1980s [26] . In 1994, a new strain of DEN-3
was introduced in the Americas, causing a
major epidemic of DF/DHF in Nicaragua
and an outbreak of DF in Panama[27] . But
this DEN-3 was genetically different from
the DEN-3 strains which circulated in the
Americas earlier. Interestingly, in 1994, this
DEN-3 genotype was reported to have a
close identity with those strains which
caused DHF epidemic in some of the SouthEast Asian countries around the same
period[28] . This DEN -3 strain subsequently
spread from Asia to Central America and
Mexico in 1995 and caused major
epidemics. A classic example of the
replacement of one type by the other is
evident from the fact that, in 1971, DEN-2
was introduced into the Pacific areas
followed by a new strain of DEN-1 in 1975
and DEN-4 by 1979, and in early 1980s by
yet another new strain of DEN -3[29] . These
reports support our current findings that,
although DEN -2 (genotype IV) has been
predominant in northern India over the past
few years, Guate96-98-like DEN-3 strain
dominated during the major outbreak of DF
in Delhi in 2003.
Acknowledgements
We thank Ms Priyanka, Ms Kamini Singh
and Ms Seema George for their technical
assistance. The secretarial assistance of
Mr A.K. Manchanda, Ms Kiran Bhatt and
Ms Sarita Kumar is acknowledged.
165
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167
Tao Peng, Junlei Zhang and Jing An #
Department of Microbiology, College of Medicine, Third Military Medical University, Chongqing, 400038,
People’s Republic of China
Abstract
Currently, the mechanisms involved in the pathogenesis of DHF/DSS remain poorly understood and
there is no effective vaccine available to prevent infection with DEN virus. The lack of a reliable small
animal model that mimics dengue disease is a major obstacle. In this paper, the development of small
animal models such as mice for dengue virus infections is reviewed.
Keywords: Dengue virus, animal model, mice.
Introduction
Dengue (DEN) viruses are mosquito-borne
RNA viruses, which belong to the genus
Flavivirus (family Flaviviridae), and are
grouped into four antigenically distinct types
(DEN-1, DEN-2, DEN-3, and DEN-4). Every
year, they infect millions of people and can
cause a mild-to-debilitating febrile illness
(classical dengue fever, DF) or lifethreatening
syndrome
(dengue
haemorrhagic fever/dengue shock syndrome,
DHF/DSS). In recent years, the geographical
range of dengue in tropical and subtropical
regions of the world has extended and
DHF/DSS is occurring in new areas and with
increased incidence[1] . Cardinal signs of
DHF/DSS include haemorrhage, abrupt
onset of vascular leakage and shock,
accompanied by severe thrombocytopenia
and massive complement activation.
#
However, the mechanisms involved in the
pathogenesis of DHF/DSS remain poorly
understood and there is no effective vaccine
available to prevent infection with any of
the four serotypes of DEN virus. A major
technical barrier is the absence of a suitable
animal model that mimics DEN disease,
including DHF/DSS. So far, there are only
three known hosts for DEN virus infections:
mosquitoes, humans and lower primates[2] .
Although these lower primates infected with
wild type DEN viruses develop viremia, they
generally manifest only very mild or no
clinical signs of disease[3] . Since the
appearance of the severe combined
immunodeficiency (SCID) mice in 1983[4] ,
efforts have been made to develop new
small animal models that may be useful for
the development of a future DEN virus
vaccine and for studying the pathogenesis of
DEN virus infections.
E-mail: [email protected]; Tel.: +86-23-68752774; Fax: +86-23-65463259
168
Animal models based on
SCID mice
The SCID mice, which do not produce
functional T and B cells and lack detectable
immunoglobulin (Ig), can support DEN susceptible human cell lines xenografts, and
this system has been employed to study
DEN virus infection in vivo. SCID mice
reconstituted with human peripheral blood
lymphocytes (hu-PBL-SCID) have been used
for studies on the pathogenesis of infection
with the human immunodeficiency virus
(HIV)[5,6] and for research on treatment of
HIV infection[7] .
The hu-PBL-SCID mice were firstly
evaluated as an animal model for DEN viral
infection in 1995[8] . SCID mice were
injected intraperitoneally (ip) with hu-PBL
for
reconstitution
and
successful
engraftment was demonstrated by the
presence of a high serum level of human
IgG. Hu-PBL-SCID mice were ip-infected
with DEN-1 virus. Unfortunately, only 5 of
19 hu-PBL-SCID mice showed sensitivity to
DEN-1 virus.
It was suggested that the main reason
for the low DEN infection rate was a scanty
number of appropriate human target cells in
the reconstituted mice. Thus, investigators
searched for more DEN-susceptible human
cell lines to improve the infection rate of the
SCID mouse model. One promising
candidate
was
K562
cell,
an
erythroleukemia cell line. SCID mice were
engrafted ip with K562 cells (K562-SCID
mice)[9] . After intratumor injection into the
peritoneal tumor masses of DEN-2 virus,
K562-SCID mice showed neurological signs
of paralysis and died at approximately 2
weeks post-infection (pi). In addition to
being detected in the tumour masses, high
virus titers were detected in the peripheral
blood and the brain tissues, indicating that
DEN virus had replicated in the infected
K562-SCID mice. Other serotypes of DEN
viruses were also used to infect the K562SCID mice, and the mortality rates of the
infected mice varied with different challenge
strains, suggesting that this animal system
might potentially be utilized to define the
virulence of various human DEN isolates
and to characterize the molecular
determinants for such viral virulence. K562SCID mice were also challenged with DEN 2 virus and received antibody administration
at the same time or one day earlier, and the
results revealed that these mice exhibited a
reduction in mortality and a delay of
paralysis onset after DEN virus infection.
These results indicated that an in vitro
neutralizing antibody also defended K562SCID mice against DEN-2 virus infection.
Target cells and organs for DEN virus
replication in humans remain unclear.
Unusual clinical manifestations, mostly
cerebral and hepatic symptoms, have
become more common in patients with
DEN virus infection in recent years [10,11] . The
involvement of liver cells in the
pathogenesis of DEN virus infection has
been indicated by abnormal liver function,
pathological findings and detection of viral
antigen in hepatocytes and Kupffer’s cells at
biopsies[12] . It was reported that DEN virus
could replicate in a human heptocarcinoma
cell line, HepG2, and infectious particles
were released into the culture medium[13,14] .
Therefore, HepG2 cells were transplanted
into SCID mice to develop an animal model
for studying the pathogenesis of DEN virus
infection[15] . The replication of HepG2 cells
in host mice was confirmed by an increase
of serum human albumin and propagation
of HepG2 cells in the liver. At 7-8 weeks
169
after transplantation, HepG2-grafted SCID
mice were ip-infected with DEN-2 virus. A
high titer of the virus was detected in the
liver and serum but not in the brain in the
early stage of the infection. When the mice
showed paralysis, the highest titer of virus
was detected in the serum and brain. DEN 2 antigens were also found in HepG2 cells
of the liver in the early stage and some
neurons of the brain in the late stage. Upon
clinical examination, thrombocytopenia,
prolonged partial thromboplastin time,
increased haematocrit, blood urea nitrogen
and tumour necrosis factor a (TNF a) were
seen in the paralyzed mice. Moreover, mild
haemorrhages in the liver and tarry stool in
the small intestine were observed in some
mice.
All of above animal models based on
SCID mice with transplanted DEN susceptible human cells mimic some of the
aspects of human disease, which may be
helpful for studying DEN virus infection,
especially in the areas of viral pathogenesis,
virus-host
interaction
and
vaccine
development against DEN infections.
However, it is generally agreed that
DHF/DSS is an immune-mediated disease,
and since SCID mice are unable to produce
the innate immune response, this may
impose some limitations on the use of these
animal models to extrapolate the situation in
human DHF/DSS.
BALB/c mouse model
Inbred four-week-old BALB/c mice were
found sensitive (haplotype H-2d) to the
challenge with dengue virus type 2 (strain
P23085) [16] . Mice were ip-infected with a
dose of 5 LD 50 of the mouse-adapted DEN 2 virus, and the first clinical manifestations
170
such as arching of the back, ruffling of the
fur and slowing of activity appeared at end
of day 4 pi. The presence of DEN-2 virus in
the blood was confirmed on day 2 pi by
reverse
transcriptase-polymerase
chain
reaction (RT-PCR). The development of the
experimental DEN -2 virus infection in
mouse model was accompanied by the virus
reproduction in all animals. Within 5 and 6
days pi, all mice showed severe sickness
with anorexia and weight loss ending in
limb paralysis and 100% mortality rate was
noted at 7 days pi. The most impressive
changes were seen with TNF-a, which
abruptly and steeply increased 24 h before
death. Serum levels of interleukin (IL)-1ß, IL6, IL-10, IL-1 receptor antagonist and
soluble TNF receptor I continuously
increased during the time of infection.
Treating animals with anti-TNF-a serum
reduced the mortality rate down to 40%.
This model supports the view that the
activation of innate immune response is at
least partially responsible for mortality in
DEN-2 virus infection, and in line with this
concept, anti-TNF treatment significantly
reduces the mortality rates. Therefore,
inbred 4-week-old BALB/c mice are useful
models to research the immune activation
of host in DEN-2 virus infection.
Gene knockout mouse
(AG129) model
There is evidence that alpha and beta
interferons (IFN-a/ß) and gamma IFN (IFN-?)
might be involved in human DEN virus
infection[17,18] . In addition, exogenously
administered IFN appears to protect mice
from
DEN
virus
challenge[19] .
This
information suggested that mice defective in
their IFN response might provide a suitable
model
for
DEN
virus
infection.
Intraperitoneally
administered
mouseadapted DEN -2 virus was uniformly lethal in
AG129 mice[20] , which lack IFN-a/ß and
IFN-? receptor genes, regardless of age. The
mice showed neurological abnormalities,
including hind-leg paralysis and blindness at
7 days pi, and died at 12 days after infection.
The immunized mice were protected from
virus challenge, and the survival time
increased following passive transfer of antiDEN polyclonal antibody. To determine
which aspect of the IFN response was
critical in protecting these mice from DEN
virus infection, animals individually deficient
in either IFN-a/ß (A129) or IFN-? (GKO)
functions as well as BALB/c controls were
subjected to a similar DEN virus challenge.
None of these mice exhibited any overt
symptoms of illness, indicating that for DEN
virus infection, IFN-a, -ß, and -?
abnormalities
in
combination
were
necessary for the mouse-adapted virus to be
lethal when the ip challenge route was used.
These results demonstrated that AG129
mice were a promising small animal model
for DEN virus vaccine trials.
A/J mouse model
DEN virus infection causes DF and
DHF/DSS. No animal model is available that
mimics this clinical manifestation. The
immunocompetent mouse (A/J strain) was
reported as a mouse model for DEN virus
infection that resembles the thrombocytopenia manifestation[21] . Intravenous
injection of DEN-2 virus into A/J mice
induced paraplegia at 2-3 weeks, while the
mock-infected controls were normal.
Viremia detected by RT-PCR was found
transiently at two days but at no other time
after infection. Although A/J mice developed
paraplegia after virus infection, they
recovered after one month. However, there
was transient thrombocytopenia at 10-13
days pi. When the mice were re-infected
with the same DEN-2 virus two months later,
thrombocytopenia was manifested again at
10 days after infection. Anti-platelet
antibody was also generated after injection.
And there was strain variation in DEN-2
virus infection; the A/J strain was more
sensitive than BALB/c or B6 mice. These
results show that this DEN-2 virus-infected
mouse system accompanied by thrombocytopenia and anti-platelet antibody may be
a suitable model to study the pathogenicity,
especially immune activation in DEN virus
infection. On the other hand, A/J mice had
to be inoculated with a large quantity of
DEN-2 virus; a dose of less than 1×10 8 pfu
per mouse was not effective in causing
paraplegia. Furthermore, viremia was low
and transient in A/J mice compared with
that in SCID or IFN-deficient AG129 mice.
DEN-2 virus could not be isolated from the
blood of infected mice; it could only be
detected by sensitive RT-PCR in A/J mice.
Conclusion
Although the above-mentioned new small
animal models, which mimic some of the
aspects of human DEN virus disease, may
facilitate not only the study of DEN
pathogenesis but also the evaluation of antiDEN virus as well as vaccine development,
there are still drawbacks in each model,
especially in mimicing DHF/DSS. Presently,
the molecular mechanisms underlying the
pathogenesis of DHF/DSS remain unknown,
such as the receptors of DEN virus which
are still not clear. Even though the use of
transgenic animals has been proposed in the
quest for an animal model, it is apparent
171
that one needs to know more about the
mechanisms involved in the pathogenesis of
DHF/DSS at the molecular level before one
can construct a transgenic animal to serve as
a model for use in research on the
pathogenesis, vaccine development and
therapy for DHF/DSS.
Acknowledgment
This work was partially supported by grants
nos. 30170848 and 30300303 from the
National Science Foundation of China
(NSFC).
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173
Philippine Species of Mesocyclops (Crustacea:
Copepoda) as a Biological Control Agent of
Aedes aegypti (Linnaeus)
Cecilia Mejica Panogadia-Reyes*#, Estrella Irlandez Cruz**
and Soledad Lopez Bautista***
*Department of Biology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines
**Research Institute for Tropical Medicine, Alabang, Muntinlupa, MM, Philippines
***Department of Medical Technology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines
Abstract
The predatory capacity of two local populations of Mesocyclops aspericornis (Daday) and Mesocyclops
ogunnus species were evaluated, for the first time in the Philippines, as a biological control agent for
Aedes aegypti (L) mosquitoes. Under laboratory conditions, Mesocyclops attacked the mosquito first
instar larvae by the tail, side and head. The mean of first instar larvae consumed by M. aspericornis and
M. ogunnus were 23.96 and 15.00, respectively. An analysis of the variance showed that there was a
highly significant difference between the mean number of first instar mosquito larvae consumed by
M. aspericornis and by M. ogunnus, which indicated that the former is a more efficient predator of
dengue mosquito larvae.
The results of the small-scale field trials showed that the mean number of surviving larvae in
experimental drums was 63.10 and in control drums was 202.95. The Student t-test of means indicated
that there was a significant difference between the mean number of surviving larvae in the drums with
and without M. aspericornis. The findings indicated that M. aspericornis females were good biological
control agents, for they destroyed/consumed about two-thirds of the wild dengue mosquito larvae
population.
Keywords: Mesocyclops aspericornis, Mesocyclops ogunnus, biological control agent, Aedes aegypti, Aedes albopictus,
Philippines.
Introduction
Copepods feed on paramecium (ciliates
protozoans) while naupli feed on
Chilomonas spp. The adult planktonic
copepods utilize diatoms as the principal
food, while predacious adult copepods
feed on protozoa, rotifers and several
#
aquatic animals of their own size including
small fishes and mosquito larvae. Some
adults are able to tear pieces out of the
body of their victims with their strong
mandibles. In the Philippines, around 14
species of Cyclops have been recorded, of
which Mesocyclops aspericornis and
M. ogunnus are more common[1,2] .
174
Mesocyclops as a Biological Control Agent of Aedes aegypti
In Brazil, a study[3] reported that under
laboratory conditions, four different strains
of M. aspericornis showed the potential as
biological control agents of Aedes aegypti
larvae. In Viet Nam, under laboratory
conditions, M. aspericornis consumed a
mean 23.75 L1 and killed a mean 13.43
L1, or a total of 37.18 L1 within 24 hours.
M. ogunnus, on the other hand, consumed
a mean 8.48 L1 and killed a mean 7.54 L1
or a total of 16.02 L1 within 24 hours [4] . In
Australia, six species of Mesocyclops were
evaluated as biological control agents of
Aedes aegypti[5] . Of these, M. aspericornis
was found to be the most effective
predator. This study attempted to evaluate,
for the first time, the potential of the local
population of two species of Mesocyclops
as biological control agents of Aedes
aegypti (L) under laboratory and field
conditions in the Philippines.
Methodology
Mesocyclops culture
Mesocyclops aspericornis and M. ogunnus
were raised in laboratory following the
techniques adopted by Marten and
Thomson[6] . To ensure the establishment of
a single species culture, a female with egg
sacs was captured and placed in a petri
dish for examination under a dissecting
microscope before it was transferred to a
wide-mouthed beaker containing 100 ml
of mixed culture of Paramecium caudatum
and Chilomonas sp. (food of Mesocyclops).
Populations of Chilomonas sp. and
P. caudatum in the culture bottle were
maintained using sterile wheat seeds.
Sample specimens from copepods culture
were sent to Maria Holynska, Museum
and Institute of Zoology, Warsaw, Poland,
for identification.
Laboratory trials
In a 600 ml beaker, 500 ml filtered tap
water with pH 7 was poured. Then 50
Aedes aegypti L1 obtained from laboratory
culture and a female M. aspericornis were
added at the same time. The same
procedure was done for M. ogunnus. The
control group did not receive Mesocyclops.
The experiment was replicated six times
and was observed every day for five
consecutive days, with daily replacements
of new L1. The daily number of L1
destroyed or consumed by M. aspericornis
and M. ogunnus and those that died in the
control
group
were
determined.
Copepods’
feeding
behaviour
was
observed.
Field trials
Permission to conduct the study in Estero
de Tanque located at P Nieto Street,
Barangay 674, Zone 73, Paco, Manila, was
secured from the community health
officials
and
Barangay
chairperson.
Members of the households were
informed regarding the procedures to be
undertaken and the possible benefit they
could derive from it. Consent from the
caretakers of the sample households was
also secured.
A preliminary study was conducted for
15 days. Twenty houses were chosen as
study sites. Two drums per household or a
total of 40 drums were emptied and filled
almost to the brim with tap water, pH 7.2.
A litre of water as sample from each drum
container was collected in sterile bottles
and brought to the laboratory to exclude
the presence of fungi, bacteria and
indigenous copepods. Mosquito eggs that
hatched into larvae in drums were
monitored and collected daily for
175
recording and identification. Water pH,
water
temperature
and
ambient
temperature were taken daily. Drums
were checked daily for the presence of
mosquito eggs and larvae with the aid of a
magnifying glass. Drums with mosquito
larvae were marked and female copepods
were introduced in experimental drums
using
the
ratio
one
Mesocyclops
aspericornis per 50 Aedes aegypti first
instar larvae (L1). No copepods were
introduced in the 20 control drums.
Observation was made daily for 15
consecutive days. Surviving larvae were
collected from all drums and brought to
the laboratory for recording and species
identification.
Laboratory trials
The results of the predatory capacity of
M. aspericornis and M. ogunnus as
evaluated under laboratory conditions and
analysis of variance are presented in
Tables 1 and 2. The mean L1 consumed
by M. aspericornis was 23.96 while that of
M. ogunnus was 15.00. Control means
were 0.63 L1 and 0.60 L1, respectively.
The findings showed that there was a
highly significant difference between the
mean number of Ae. aegypti L1 consumed
by M. aspericornis and that of M. ogunnus
and the control group.
Table 1. Predatory capacity of female Mesocyclops aspericornis vs Aedes aegypti first instar
larvae in 500 ml filtered water under laboratory conditions
Treatment
Experimental group
Control group
Mean
F value
23.96
338.83
Tabular f
7.12
Statistical
significance
Highly significant
0.63
Table 2. Predatory capacity of female Mesocyclops ogunnus vs Aedes aegypti first instar
larvae in 500 ml filtered water under laboratory conditions
Treatment
Experimental group
Control group
Mean
F value
15.00
319.18
7.12
Statistical
significance
Highly significant
0.60
To determine if there was a difference
between the mean number of L1
consumed by M. aspericornis and
M. ogunnus, the analysis of variance was
carried out (Table 3). The findings showed
that there was a highly significant
176
Tabular f
difference between the mean number of
L1 consumed by M. aspericornis and by
M. ogunnus. The study showed that
M. aspericornis was a more efficient
predator of Aedes aegypti larvae.
Table 3. Comparison of the predatory capacity of M. aspericornis and M. ogunnus
under laboratory conditions
Treatment
Mean
M. aspericornis
23.96
M. ogunnus
15.00
F value
Statistical
significance
Tabular f
36.11
7.12
Highly significant
Table 4. Predatory capacity of Mesocyclops aspericornis vs Aedes larvae in small-scale
field trials using drum containers
Treatments
# Replicates
Mean + SD
Experimental group
20
63.10 + 59.43
Control group
20
202.95 + 140.43
Field trials
The results of the field trials are presented in
Table 4.
The mean L1 in experimental drums was
63.10 and 202.95 L1 in control drums. To
determine the difference between the mean
number of surviving larvae in the drums with
M. aspericornis and in the drums without M.
aspericornis, a Student t-test was used. The
findings indicated that there was a significant
difference between the mean number of
surviving larvae in the drums with and
without
Mesocyclops.
This
significant
difference suggests that M. aspericornis is a
good biological control agent, for it
consumed about two-thirds of the wild,
dengue mosquito larvae population.
Conclusion
The results of the study showed that
Mesocyclops aspericornis was an efficient
predator of Aedes aegypti larvae both in
Statistical
significance
P value
0.0002
Significant
laboratory and field conditions. These
copepods could be effectively used for the
control of Aedes breeding in non-removable
containers, viz. drums and used tyres.
Acknowledgements
The World Health Organization and the
Yaman Lahi Foundation, Inc. – Emilio
Aguinaldo College provided financial
support for this project. We acknowledge
the kind assistance of Dr Kevin Palmer of
the WHO Western Pacific Region; Dr Jose
Paulo E. Campos of Yaman Lahi Foundation,
Inc. – Emilio Aguinaldo College (YLFI-EAC),
Philippines; Dr Remigio M. Olveda of the
Department of Health – Research Institute
for
Tropical
Medicine
(DOH-RITM),
Philippines; Dr Maria Holynska of Museum
and Institute of Zoology – Polish Academy
of Science (MIZ-PAS), Poland; Dr Vu Sinh
Nam of the National Institute of Hygiene
and Epidemiology (NIHE), Viet Nam; and
Dr Brian H. Kay of the Queensland Institute
of Medical Research (QIMR), Australia.
177
References
[1] Mamaril AC and Fernando CH. Freshwater
zooplankton of the Philippines (Rotifera,
Cladocera, and Copepoda). Natural and
Applied Science Bulletin, 1978, 30(4): 109221.
[2] Tuyor JB and Baay MO. Contribution to the
knowledge of freshwater copepods of the
Philippines. Asia Life Sciences, 2001, 10(1):
35-43.
[3] Kay BH, Cabral CP, Sleigh AC, Brown MD,
Ribeiro ZM and Vasconcelos AW.
Laboratory
evaluation
of
Brazilian
Mesocyclops (Copepoda: Cyclopidae) for
mosquito control. J Med Entomol, 1992,
29(4): 599-602.
178
[4] Nam VS, Tien TV, Huan TQ, Yen NT, Kay B,
Marchand R, Marten G, Holynska M and
Reid J. Mesocyclops of Vietnam Part I laboratory evaluation as biological agent for
control of Aedes aegypti. Dengue Bulletin,
1999, 23: 89-93.
[5] Brown MD, Kay BH and Hendrikz JK.
Evaluation of Australian Mesocyclops
(Cyclopoida: Cyclopidae) for mosquito
control. J Med Entomol, 1991, 28(5): 618623.
[6] Marten G and Thompson G. Copepod
production and application for control
procedure for New Orleans Mosquito Board,
City of New Orleans, USA, 1997.
Susceptibility of Aedes aegypti to Insecticides
in Viet Nam
Vu Duc Huong#, Nguyen Thi Bach Ngoc, Do Thi Hien and Nguyen Thi Bich Lien
Department of Entomology, National Institute of Malariology, Parasitology and Entomology,
B.C. 10 200 Tu Liem, Hanoi, Viet Nam
Abstract
During 2000-2002, studies on the susceptibility of Aedes aegypti to insecticides were conducted at 22
places in 11 provinces and cities in four different regions of Viet Nam. Aedes aegypti was found
susceptible to malathion, but resistant to DDT in almost all the study sites. It continues to be susceptible
to the pyrethroid group of insecticides (permethrin, lambda-cyhalothrin, deltamethrin and alphacypermethrin) in many places in the North and Centre regions, but is resistant to these insecticides in
many places in the South and Central Highlands in Viet Nam. However, the species was found highly
and widely resistant to etofenprox.
Keywords: Aedes aegypti, pyrethroids, insecticides, Viet Nam.
Introduction
Insecticidal measures, especially in the
outbreak-risk areas, are the most important
for the control of Aedes aegypti, the main
vector of DHF. Many insecticides of the
group organochlorine (DDT), organophosphorous (fenthion, malathion and
temephos) and pyrethroid (permethrin,
deltamethrin, lambda-cyhalothrin, etc.) have
been used for the malaria control programme
and for Aedes aegypti. Aedes aegypti has
been resistant to DDT since the early 1960s
and cross-resistant to many insecticides of the
pyrethroid and temephos groups in many
countries, but is not yet resistant to
malathion[1-3] . When this species is resistant
to the insecticides of the pyrethroid group,
the organophosphorous ones could take their
place[4] .
#
Both malaria and DHF were endemic in
many mountainous, forested and coastal
plain areas of Viet Nam where house spray
and bednet treatment were applied for
years. DDT was widely used before 1990
and later lambda-cyhalothrin, permethrin
and deltamethrin[5] were introduced. In
1999, Aedes aegypti was found resistant to
DDT and some insecticides of the
pyrethroid group in many places in Nam Bo
(the South), Central Highlands, but not yet
to malathion[6] . This study provides more
data on the susceptibility of Aedes aegypti to
insecticides in different regions of Viet Nam.
Time and study regions
During 2000-2002, studies were conducted
at 22 places (located in 11 provinces and
179
cities) – six places in the North, six in the
Centre, six in the South and four in the
Central Highlands.
Methods
The susceptibility to the insecticides was
evaluated on the following criteria:
•
Mortality 98-100%:
insecticide.
•
Mortality
80-97%:
Possibility
resistance to insecticide.
•
Mortality <80%: Resistant to insecticide.
[7]
The WHO standard bioassay tests (1998)
were followed and the papers treated with
DDT 4%, the control paper with OC
(organochlorine control), malathion 5% and
the control paper with OP (organophosphate
control) and 5 insecticides of the pyrethroid
group
(permethrin
0.75%,
lambdacyhalothrin 0.05%, deltamethrin 0.05%,
alpha-cypermethrin 30mg/m2 and etofenprox
0.5%) with the same control paper PY
(pyrethroid control).
The tests were done at a temperature of
25 °C ± 2 °C and humidity of 75-85%.
The unfed, F1 mosquitoes, one or two days
old – at least 150 mosquitoes for each
insecticide, 100 for the test and 50 for the
control, were used. Twenty-five mosquitoes
were put in each test tube and the per cent
mortality count was done 24 hours after the
exposure. The mosquitoes in the resting
tubes were then fed with glucose 10% in
soaked cotton.
Susceptible
to
of
Results
North
Of the six study places, Aedes aegypti was
found resistant to DDT at five places and
possibly resistant to DDT at one place; it
was susceptible to malathion at five places
and possibly resistant to this insecticide at
one place. It was susceptible to all the tested
insecticides of the pyrethroid group such as
permethrin, lambda-cyhalothrin, deltamethrin
and alpha-cypermethrin in at five places and
the possibility of resistance to these four
insecticides existed at one place. Aedes
aegypti was resistant to etofenprox at four
places and possibly resistant to etofenprox at
two places (Table 1).
Table 1. Results of suceptibility tests on Aedes aegypti to insecticides
in the North and Centre, Viet Nam
% Mortality
S. No.
Places
Lambda AlphaPermethrin
Deltamethrin
Etofenprox
cyhalothrin
cypermethrin
0.75%
0.05%
0.05%
2
0.05%
30mg/m
DDT Malathion
4%
5%
1.
Thi Cau (Co)
Bac Ninh (T)
Bac Ninh (P)
100
100
100
100
68
57
100
2.
Phu Lang (Co)
Que Vo (D)
Bac Ninh (P)
100
100
100
100
92
94
100
3.
Cat Ba (S)
Cat Hai (D)
Hai Phong (C)
100
100
100
100
42
14
100
180
% Mortality
Deltamethrin
Etofenprox
cyhalothrin
cypermethrin
0.75%
0.05%
0.05%
2
0.05%
30mg/m
S. No.
Places
4.
Niem Nghia (Co)
Le Chan (D)
Hai Phong (C)
91
92
94
95
92
21
100
5.
Ly Thai To (Co)
Hoan Kiem (D)
Ha Noi (C)
99
96 (200)
100
100
18
21
97.33
(150)
6.
Thinh Liet (Co)
Thanh Tri (D)
Ha Noi (C)
100
99
98
98
37
60
98
7.
Thach Phu (Co)
HaTinh (T)
Ha Tinh (P)
100
100
100
100
100
63
100
8.
Duc Tho (S)
Duc Tho (D)
Ha Tinh (P)
100
100
100
100
93
37.39
(115)
100
9.
Song Cau (S)
Song Cau (D)
Phu Yen (P)
100
100
100
98
51
4
100
10.
No. 6 (Co)
Tuy Hoa (T)
Phu Yen (P)
100
100
100
91
76
2
100
11.
Dong Luong (Co)
Dong Ha (T)
Quang Tri (P)
94
96
97
95
16
2
100
12.
Trieu Do (Co)
Trieu Phong (D)
Quang Tri (P)
100
100
100
100
86
11
100
DDT Malathion
4%
5%
No. 1 - 6 in the North
No. 7 - 12 in the Centre
Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City
Figures in parenthesis indicate the number of mosquitoes tested
Central
Aedes aegypti was found resistant to DDT
but was susceptible to malathion in all six
places. It was susceptible to four insecticides
of the pyrethroid group such as permethrin,
lambda-cyhalothrin,
deltamethrin
and
alpha-cypermethrin in five places and
possibly resistant to them in one place, and
resistant to etofenprox in three places, and
possibly resistant and susceptible to
etofenprox at two and one places,
respectively (Table 1).
South
Aedes aegypti was resistant to DDT at all six
places; but susceptible to malathion at four
places and possibility of resistance to
malathion at two places; it was resistant to
181
permethrin and lambda-cyhalothrin at four
places and the possibility of resistance to
permethrin and lambda-cyhalothrin at twor
places. It also showed resistance to
deltamethrin at one place and possibility of
resistance to deltamethrin at five places;
resistance to alpha-cypermethrin at two
places and possibility of resistance to alphacypermethrin at four places as well as
resistance to etofenprox at all six places
(Table 2).
Table 2. Results of susceptibility tests on Aedes aegypti to insecticides
in the South and Central Highlands, Viet Nam
% Mortality
No.
Places
Deltamethrin
Etofenprox
cyhalothrin
cypermethrin
0.75%
0.05%
0.05%
2
0.05%
30mg/m
DDT Malathion
4%
5%
1.
No. 6 (Co)
Ben Tre (T)
Ben Tre (D)
62
67
90
82
4
2
81
2.
BinhThuan (Co)
Binh Dai (D)
Ben Tre (P)
94
90
97
92
53
20
100
3.
Binh Khanh (Co)
Can Gio (D)
Ho Chi Minh (C)
18
24
84
75
1
0
99
4.
Binh Trung Tay (Co)
No. 2 (D)
Ho Chi Minh (C)
96
93
97
96
20
11
95
5.
An Loc (S)
Binh Long (D)
Binh Phuoc (P)
79
56
84
96
1
0.8
(125)
90
6.
Tan Xuan (Co)
Dong Xoai (T)
Binh Phuoc (P)
8
28
19.33
(150)
25
7
2
99
7.
Plei Can (S)
Ngoc Hoi (D)
Kon Tum (P)
36
32
51
28
1
1
100
8.
Quyet Thang (Co)
Kon Tum (T)
Kon Tum (P)
57
61
66
47
1
1
100
9.
Buon Trap (Co)
Krong Ana (D)
Dak Lak (P)
5
11
41
40
0
6
97
24
36
63
34
23
0
98
10. Thang Loi (Co)
Buon Me Thuot (C)
Dak Lak (P)
No. 1 - 6 in the South
No. 7 - 10 in the Central highlands
Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City
Figures in parenthesis indicate the number of mosquitoes tested
182
Central Highlands
Aedes aegypti was resistant to DDT,
permethrin, lambda-cyhalothrin, deltamethrin,
alpha-cypermethrin and etofenprox at all
four places in this region and susceptible to
malathion at three places and showed
possibility of resistance to malathion at one
place (Table 2).
Discussion
Aedes aegypti was susceptible to malathion
and resistant to DDT at almost all study
places in Viet Nam. It was still susceptible to
the four insecticides of the pyrethroid group
(permethrin, lambda-cyhalothrin, deltamethrin
and alpha-cypermethrin) in many places in
North and Centre Viet Nam, but resistant to
these insecticides in many places in the
South and Central Highlands. These results
were not completely comparable with the
observations made by Reiter and Gubler
(1997) [3] , and Vu Duc Huong and Nguyen
Thi Bach Ngoc (1999) [6] . Aedes aegypti was
possibly resistant to malathion in some
places. This discrepancy in different regions
was possibly due to the longer and more
extended use of the insecticides of the
pyrethroid group in malaria and dengue
haemorrhagic fever control programmes and
in agriculture in the Southern and Central
Highlands. It is therefore suggested that the
susceptibility tests should be conducted on
all insecticides before use. Moreover, the
cross-resistance of Aedes aegypti to
insecticides belonging to the pyrethroid
group should also be checked. Aedes
aegypti was highly and widely resistant to
etofenprox and further studies should be
conducted in this context.
References
[1] Yap HH, Cheng NL, Foo AES and Lee CY.
Dengue vector control: present status and
future prospects. Kaoshiung J Med Sci,
1994,10: 102-108.
[5] Phan VT. Malaria epidemiology and malaria
control in Vietnam. Medical Publishing
House, Hanoi, 1996: 218-241 (in
Vietnamese).
haemorrhagic fever: diagnosis, treatment,
prevention and control. 2nd edn. Geneva:
WHO, 1997: 48-59.
[6] Huong VD and Bach Ngoc NT.
Susceptibility of Aedes aegypti in south
Vietnam. Dengue Bulletin, 1999, 23: 85-88.
[3] Reiter P and Gubler DG. Surveillance and
control of urban dengue vectors. Dengue
and dengue haemorrhagic fever. Colorado:
CAB International, 1997: 425-454.
[4] Kantachuvesiri A. Dengue haemorrhagic
fever in Thai society. Southeast Asian J Trop
Med Public Heath, 2002, 33(1): 56-67.
[7] World Health Organization. Report of the
WHO
informal
consultation.
Test
procedures for insecticide resistance
monitoring in malaria vector, bio-efficacy
and persistence of insecticides on treated
surfaces. Geneva: WHO, 1998. Document
WHO/CDC/CPC/MAL/98.12: 1-43.
183
Ovipositioning Behaviour of Aedes aegypti in
Different Concentrations of Latex of Calotropis procera:
Studies on Refractory Behaviour and its Sustenance
across Gonotrophic Cycles
Manju Singhi, Vinod Joshi#, R.C. Sharma and Keerti Sharma
Desert Medicine Research Centre (Indian Council of Medical Research), New Pali Road,
Jodhpur 342 005, India
Abstract
Dengue fever associated with dengue haemorrhagic fever is gaining endemicity in India. Due to lack
of any chemotherapy against this arboviral infection, the control of the disease depends largely on
preventive measures against Aedes mosquito vectors. A wild shrub, Calotropis procera, commonly
growing in the desert areas of Rajasthan has shown a remarkable effect as a larvicide against Aedes
aegypti. However, different water concentrations of this biocide have also brought forward very
important observations on the ovipositioning behaviour of Aedes aegypti. At 0.7% concentration of
latex, the ovipositiong was avoided by the gravid female mosquitoes and this behaviour continued till
three gonotrophic cycles. However, at lower concentrations (0.2% and 0.1%) of the larvicidal latex,
the refractory behaviour of ovipositioning could not be retained up to the third gonotrophic cycle. The
concentration of latex such as 0.7% and 0.2% were observed as ovicidal also and this effect continued
across all the gonotrophic cycles. The behavioural observations reported in the present study may
serve as significant information on choosing bio-larvicides for vector control against dengue.
Keywords: Dengue, Aedes aegypti, Calotropis procera, ovipositioning, refractory behaviour, gonotrophic cycle.
Introduction
Dengue fever and dengue haemorrhagic
fever (DF/DHF) is gaining endemicity in
many states in India[1] . In the absence of
chemotherapy and vaccines, vector control,
largely based on larval control, is the only
option available. In Rajasthan, a number of
epidemics of dengue associated with DHF
#
have been reported[2-4] . It has also been
established that dengue virus undergoes
transovarial transmission across generations
of Aedes aegypti under natural as well as
experimental
conditions[5,6] .
Latex
of
Calotropis procera, a milky weed plant
growing all across the desert areas in the
state has shown promising larvicidal
properties in a series of laboratory
184
Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera
experiments carried out against Aedes
aegypti (unpublished data, Desert Medicine
Research Centre). In an attempt to assess
the value of this agent as an oviposition
attractant for use in ovitraps, it was
discovered that the agent showed
oviposition refractoriness instead. The
present communication incorporates the
findings of these studies.
The experiments were conducted at 2530 °C (room) temperature and at relative
humidity of about 60-70%. The eggs of each
generation were counted under a dissecting
microscope in the control as well as
experimental sets. All the eggs were
immersed in plain water to observe whether
exposure to larvicide had any ovicidal effect
on them.
Six experimental sets were designed for the
study. Cages of 30 cm3 size, with wooden
frame and iron mesh with muslin cloth on
one side, were used as units of present
experiments. In each cage, 16 gravid
females of Aedes aegypti were released. The
concentration of latex in water, which
showed the highest (0.7%), moderate (0.2%)
and no mortality (0.1%) effect in the
larvicidal
efficacy
experiments,
were
prepared and put in beakers in the cages A,
B, C, D and E while cage F was kept without
latex solution and contained only water. A
beaker containing plain water was also
placed in each of the six cages to serve as
control. While in cage D all the
experimental concentrations were placed
along with the control, in cage E all the
concentrations were placed without choice
of a control. In the sixth cage (cage F) two
beakers with plain water were placed
without
any
experimental
lethal
concentration. The eggs laid by female
Aedes aegypti were counted after 48 hours
in each of the experimental cages. Second
and third blood meals were provided to
facilitate G2 and G3 (gonotrophic cycles) to
mosquitoes.
Table 1 shows relative observations on
different concentrations of latex of
Calotropis procera on the ovipositioning
behaviour of Aedes aegypti. In experimental
cage A, while in the control set, 65 eggs
were laid, in the corresponding lethal
concentration of 0.7% of the same cage, no
eggs were laid. In this cage all the
mosquitoes showed persistent refractiveness
of ovipositioning across all gonotrophic
cycles from G1-G3. In cage B where 0.2%
concentration was offered along with
control, refractive behaviour up to two
cycles only was observed while in the third
cycle (G3), 127 eggs were laid. Similar
observations were made in cage C. It was
interesting to note that in cage D where all
the experimental concentrations were
offered to mosquitoes along with choice of
control, even in G1 cycle no refractiveness
was shown. In experiment E where choice
of control (plain water) was not offered,
maximum preference for ovipositioning was
shown in lowest concentration (0.1%). The
different preference among different
concentrations continued across all the
gonotrophic cycle (Table 1).
185
Table 1. Ovipositing preference of Aedes aegypti in different larvicidal concentrations of latex of
Calotropis procera
Latex concentration (%) in experimental cages
Cage
A
B
C
D
Eggs laid
within 48
hrs
C
0.7
C
0.2
C
0.1
C
G1
65
0
156
0
180
0
109 18
G2
93
0
156
1
149
0
100
G3
275
0
E
0.1 0.2 0.7 0.1
F
0.2 0.7
C
C
18
6
101
51
11 102 110
0
0
0
33
10
230 127 275 52 270 98
31
0
215 150 20 233 197
7
96
83
C: Control
G1-G3: Gonotropic cycles
Table 2. Effect of latex on the percentage viability of eggs of Aedes aegypti
Experimental cages
Cage
A
B
C
D
E
Latex (%)
C
0.7
C
0.2
C
0.1
C
Eggs
immersed
65
0
156
0
180
0
109
18
18
6
Eggs
hatched
55
0
78
0
126
0
78
0
0
0
50
0.0
70
0.0
73
% eggs
hatched
84.6 0.0
F
0.1 0.2 0.7 0.1 0.2 0.7
101 51
6
2
C
C
11
102
110
0
96
103
0.0 0.0 0.0 5.9 4.0 0.0 93.1 93.6
C: Control
Table 2 shows the results of the
experiment carried out to study the viability
of eggs that were exposed to different
larvicidal concentrations and to control
groups. The eggs laid in experiments A, B, C
and D showed no hatching, while in
experimental cage E where no control had
been kept, the eggs laid up to a
concentration 0.2% showed some viability
and in cage F where only controls were kept
the laid eggs showed 93% viability.
186
Dengue fever associated with DHF has
become a problem of public health
importance. Available evidence shows that
the virus undergoes vertical transmission
across generations of mosquitoes[6] . Larval
control, therefore, is the most effective
approach to restrict the vector and virus
sustenance in nature. The wildly grown
plant, Calotropis procera, has shown very
encouraging results in its different lethal
concentrations. However, if this plant
species is to be used as a material of choice,
its other aspects such as the ovipositioning
behaviour of gravid females towards the
larvicide can also become known.
The significance of the reported
observations is that the refractiveness
developed by the species is sustained across
two gonotrophic cycles when one larvicidal
concentration
was
offered
with
a
corresponding control (Table 1). However,
when all the concentrations viz. 0.1 %,
0.2 % and 0.7% were of fered with one
control, such avoidance was not shown.
Similarly, in the experimental set E where all
concentrations of latex were made available
without any control, the maximum egg
laying was preferred in 0.1 % of latex. The
data suggest fine chemo-sensation in Aedes
aegypti where the ovipositioning female
could distinguish to choose the least
larvicidal concentration for its egg laying.
The observation showed that if in the
various types of domestic containers where
water is stored a non-lethal concentration of
latex (0.1%) is used, the refractiveness of
ovipositioning will not be there and the
domestic mosquito fauna in such premises
will lay the eggs but they will lose their
viability to hatch into larvae (Table 2).
The role of Calotropis procera as a
larvicide has been reported by other workers
from India[7] . However, contrary results have
been reported by some workers[8] where
insect growth regulators actually enhanced
ovipositioning.
The observations reported by us not only
present the results of the ovipositioning
behaviour induced by a bio-larvicide, but
also add the information on relative
preference in ovipositioning and its
sustenance across gonotrophic cycles when
different
larvicidal concentrations were
offered.
Acknowledgements
The authors gratefully acknowledge the
guidance and inspiration received from
Mr N.L. Kalra, Member, Scientific Advisory
Committee of the Desert Medicine Research
Centre, Jodhpur, India.
References
[1] Dengue alert in South-East Asia Region.
New Delhi: World Health Organization,
Regional Office for South-East Asia, 2004
(http://w3.whosea.org/index.htm, accessed
25 August 2004).
[2] Ghosh SN and Sheikh BH. Investigations on
the outbreak of dengue fever in Ajmer city,
Rajasthan. Part II: Results of serological tests.
Indian Journal of Medical Research, 1974,
62: 523-533.
[3] Padbidri VS, Dandawate CN, Goverdhan
MK, Bhat UK, Rodrigues FM, D'Lima LV,
Kaul HN, Guru PY, Sharma R and Gupta NP.
An investigation of the etiology of the 1971
outbreak of febrile illness in Jaipur city, India.
Indian Journal of Medical Research, 1973,
61(12): 1737-1743.
[4] Chouhan GS, Rodrigues FM, Shaikh BH,
Ilkal MA, Khangaro SS, Mathur KN, Joshi KR
and Vaidhye NK. Clinical and virological
study of dengue fever outbreak in Jalore city,
Rajasthan, 1985, Indian Journal of Medical
Research, 1990, 91: 414-418.
[5] Joshi V, Singhi M and Chaudhary RC.
Transovarial transmission of dengue 3 virus
by Aedes aegypti. Transaction of Royal
Society of Tropical Medicine and Hygiene,
1996, 90: 643-644.
187
[6] Joshi V, Mourya D and Sharma RC.
Persistence of vertical transmission of
dengue-3 virus through vertical transmission
passage in successive generations of Aedes
aegypti mosquitoes. American Journal of
67(2): 158-161.
188
[7] Girdhar G, Deval K, Mittal PK and
Vasudevan P. Mosquito control by
Calotropis latex. Pesticides, 1984, 26-29.
[8] Moore CG. Insecticide avoidance by
ovipositioning Aedes aegypti. Mosquito
News, 1977, 37: 291-293.
Community-based Assessment of Dengue-related
Knowledge among Caregivers
Khynn Than Win* #, Sian Za Nang** and Aye Min***
*Health Systems Research Division, Department of Medical Research (Lower Myanmar), Myanmar
**Health Education Bureau, Department of Health Planning, Myanmar
***Vector Borne Disease Control Programme, Department of Health, Myanmar
Abstract
The study was conducted in Thaketa township in Myanmar involving 405 respondents aged 18 years and
above. It was aimed: (i) to explore the extent of dengue-related knowledge among caregivers; (ii) to
identify the exposure of community members to the existing IEC materials; and (iii) to find out the factors
related to high knowledge scores. The findings were triangulated by results from personal interviews,
focus group discussions and observational checklist. The difference of mean scores among males and
females was not statistically significant. Knowledge scores of the caregivers were not statistically different
whether there was a primary DHF case at home or not. Almost 60% of the interviewees had received
information on DHF by watching television and they observed that television was the most effective
medium. Females with more than six years of schooling, persons who had access to pamphlets/posters,
television, newspapers and journals got higher scores than the unexposed group. Less than 15% were not
exposed to any of the IEC materials. Aedes aegypti larvae were found in 67% of water storage tanks and
15.9% of flower vases when using observational checklist. Focus group discussions were held for drafting
IEC materials. Community members were more interested in the mode of DHF transmission to children
rather than in the elimination of the Aedes mosquitoes. A low practice score was observed in those with
high knowledge level, which means that high knowledge does not necessarily lead to high practice. Less
than half of the respondents had seen posters and pamphlets. IEC materials need to be improved so that
they present the message most effectively and they should be extensively distributed in the community.
Keywords: Dengue, caregivers, community, IEC material, knowledge score, Myanmar.
Introduction
Dengue haemorrhagic fever (DHF) is
endemo-epidemic in 12 out of 14 states and
divisions in Myanmar and is transmitted by
Aedes aegypti. Most of the reported cases
are under 15 years of age[1] . The Myanmar
National Health Plan (NHP) (1996-2001)
#
termed DHF as one of the diseases under
national surveillance. DHF is also listed as
the 17th priority disease in Myanmar. One of
the strategies devised in NHP for the
prevention and control of DHF is
production of guidelines for basic health staff
(BHS) as part of the information, education
and communication (IEC) programme.
189
Assessment of Dengue-related KAP among Caregivers
IEC initiatives are based on the concepts
of prevention and primary health care. They
create awareness, increase knowledge,
change attitudes and motivate people to
adopt new ideas [2] .
materials; and (iii) to find out the factors
responsible for high knowledge scores.
Communication participation appears
to be one of the most promising innovative
means to prevent and control DHF. Simple
elimination of vector-breeding water
collections or “source reduction” is the
possible answer to the problem. Community
activities are identified mainly as reduction
of non-essential water containers, protection
of water containers from larvae breeding,
larviciding and release of larvivorous fish.
Community participation needs to be
sustained by dissemination of health
messages through various channels[3] .
This community-based cross-sectional study
was based on multistage sampling to identify
405 caregivers in Thaketa township, Yangon.
This area was one of the dengue endemic
regions in the Yangon division and the casefatality rate (CFR) was 1.65% in 2002. Both
quantitative and qualitative data collection
methods, including observation checklist,
were used in the study. The study sample
included household members aged 18 years
and above. The households with children
were
selected
randomly.
In
these
households, we chose one subject from
each household, regardless of sex. The
respondent was a relative of the child
(mother/father/grandfather/grandmother/
brother/sister/uncle/aunt). Mothers were the
key persons to be interviewed after taking
their consent. The questionnaire was pilottested for clarity and validity; all questions
were reviewed by epidemiologists, public
health experts, health educators and by
investigators experienced in conducting
community-based surveys in DHF. In order
to ensure the accuracy and completeness of
data, our surveyors were trained before and
after pre-testing.
Existing IEC materials in Myanmar
included health talks routinely carried out in
schools and in the community. Health
messages were distributed through radio,
television, newspapers and journals before
and during the epidemic season. Pamphlets
were developed locally in states and
divisions. However, it is necessary to find
out the most appropriate IEC materials and
means that would be relevant for various
communities.
This study attempted to improve the
existing IEC materials on DHF control based
on the knowledge and practice of child-care
providers in the Thaketa township of the
Yangon division in Myanmar. DHF control
will be more effective in the future by
strengthening community participation on
case information and source reduction.
This research aimed to: (i) explore the
extent of dengue-related knowledge among
caregivers; (ii) identify the exposure of
community members to the existing IEC
190
Methods and materials
Ten
sessions
of
Focus
Group
Discussions (FGDs) were performed among
basic health staff (BHS), general practitioners,
Maternal and Child Welfare Association
(MCWA)
members,
other
volunteers
including Ward Law and Order Restoration
Council members, voluntary fire brigade
members, etc., for recommendation of
existing IEC activities (including television,
radio, newspapers, local journals and
pamphlets). In every FGD, the moderator
explained thoroughly the purpose of
conducting the discussions. The Township
Health Centre, Ward Law and Order
Restoration Council offices and homes were
chosen for holding FGDs.
Caregivers were those who took care of
children at home, or supervised at the
health centre or clinic, or advised parents on
home care of a child with fever.
Data checking, cleaning and validation
were performed using Epi-info 6.0, and data
analysis was conducted using SPSS 10.0.
P<0.05 was used as the definition of
statistical significance. The study period
lasted one year starting in June 2002.
Results
About respondents
The sex ratio of the respondents was 7:1 for
females and males. The mean age was
35.9±10.3. The majority of the respondents
were aged 18-35 years, literate and
dependants. About 8.6% of them had
received ≥ 12 years’ school education.
Nearly 40% lived in their own wooden
houses. Most of the households (77.8%) had
1 to 2 children under 15 years of age. Only
34 (8.4%) of the households that
participated in the interview had a child
with history of DHF.
The difference of mean score among
males and females was not statistically
significant (P=0.271).
Dengue-related knowledge
responses
The responses of caregivers are included in
Table 1.
Table 1. Dengue-related knowledge
responses of caregivers
Responses
Frequency Percent
DHF is common in
children 3-8 years of age
254
44.4
DHF is transmitted by
the mosquito
331
81.7
Mosquito species of
DHF vector is Aedes
204
61.6
Biting time of mosquitoes
is at daytime
266
80.4
135
166
33.3
41.0
The Aedes mosquito
breeds inside the house
• flower vases
• ant traps
253
144
96
62.5
35.6
23.7
The Aedes mosquito
breeds outside the house
• water containers
• old tyres, broken pots,
and coconut shells
• blocked gutters
292
72.1
178
75
61.0
25.7
9
2.2
DHF transmission is
highest in the rainy
season
348
85.9
DHF may be fatal
382
94.3
There is a vaccine for
prevention of DHF
243
60.0
A previously infected
child may get DHF again.
293
72.3
If the child is febrile,
DHF should be
observed
262
64.7
Aedes breed in
• clear water
• polluted water
The total knowledge scores were
categorized as low (0-19) and high (20-39).
The percentage of the high score group was
more than that of the low score group
191
(68.6% vs 31.4%). Signs and symptoms of
DHF were fever (57.3%), vomiting (51.6%),
purpura (36.3%), drowsiness (28.1%), cold
extremities (17%), etc.
Knowledge score of respondents
with and without history
of DHF case in their homes
Knowledge scores with and without past
history of primary DHF cases at home are
given in the Figure.
Figure. Past history of primary DHF cases
at home and knowledge scores
Low
High
300
258
Knowledge scores
250
200
150
113
100
50
14
20
0
Present
Absent
Past history of primary DHF cases at home
The figure illustrates that the knowledge
scores of the caregivers were not statistically
different according to the presence of
primary DHF case at home (P=0.137).
Prevention of mosquito bites
To prevent and protect children from
mosquito bites, the following measures were
taken: use of mosquito nets (47.9%), use of
repellants (47.2%), wearing of long sleeves
(12.6%), others (8.6%), and none (5.2%).
192
Existing exposure to IEC material in
the community
Table 2 contains the responses of the
communities to IEC materials.
Table 2. Exposure to IEC materials in the
community
Community members exposed
to IEC materials
(n)
Percent
197
137
246
133
48.6
33.8
60.7
32.8
• Percent exposed to any type 352
of IEC
• Percent not exposed to any
53
IEC
• Percent exposed to all types 87
of IEC
87.0
•
•
•
•
had seen pamphlets
listened to radio
watched on television
read in newspapers/journals
Are the facts easily recognized?
• Yes
317
• No
23
13.0
21.5
78.3
5.7
The percentage of people watching
television was the highest as compared to
other types of exposure. Nearly half of the
respondents had seen the pamphlets about
DHF (48.6%). Exposure to radio talks and
information in newspapers and journals was
very low. The extent of respondents
exposed to any type of IEC was 87%. The
facts in those IEC materials were concise
and easily recognized (78.3%) (Table 2).
Almost 60% of the interviewees felt that
television was the most effective medium for
dissemination of knowledge on DHF in the
community.
The logistic regression model of
knowledge score by the respondents’
characteristics and exposure to health
education media is given in Table 3.
Assessment of Dengue-related KAP Among Caregivers
Table 3. Logistic regression model of knowledge scores, by respondents’ characteristics and
exposure to health education materials
Variables
(n)
Percent
Odds ratio
(95% confidence interval)
Respondent's characteristics
Sex
• Male
• Female
Years of formal schooling
• 0 to 5 (r)
• 6 to 20
Mean years of schooling
54
351
13.3
86.7
1
0.379 (0.198-0.727)**
133
272
7.6±3.6
32.8
67.2
1
0.588 (0.364-0.950)*
Exposure to health education media
Pamphlets/posters
• Not seen
• Seen
197
208
48.6
51.4
1
0.478 (0.291-0.784)**
Television
• Not watched (r)
• Watched
159
246
39.3
60.7
1
0.353 (0.218-0.571)***
Newspapers/journals
• Not read (r)
• Read
272
133
67.2
32.8
1
0.443 (0.245-0.803)**
* Significant at P<0.05; ** P<0.01; *** P<0.001; (r) = Reference category
An analysis of the findings suggested
that females with over six years of schooling
were significantly related to total knowledge
scores. Survey respondents with 6-20 years
of schooling were more likely to obtain high
scores than those with 0-5 years of
schooling. Respondents who were exposed
to health education media such as
pamphlets/posters, television, newspapers
and journals obtained higher scores than the
unexposed group. The respondents who
watched television were more likely to score
higher than those who did not (Table 3).
Cross-check by observation
checklist
The researchers used an observation
checklist to support the findings. Nearly
55% of the households had two to three
water containers. Although 46.5% of the
water containers had lids, only 19% were
covered tightly. Larvae were found in 67%
of the water storage tanks and 15.9% of the
flower vases at the time of interviews. Gutter
blockage was observed in 3.2% of cases.
Old tyres, coconut shells and tins were
found in the compounds and larvae existed
in half of these solid wastes.
193
Approaches considered for
improvement of IEC activities
Many recommendations were extracted
from FGDs for the improvement of existing
IEC materials. It was suggested that messages
should be short and clear. Pamphlets should
be widely distributed among the community,
especially in schools. The same messages
could be published in newspapers and
journals once a week before and during the
rainy season. Health magazines and
magazines on astrology were the most
preferred media. They also pointed out the
most suitable times for telecast and broadcast
of health messages. Regular clearing of
gutters should be mentioned in the health
message as well as in the book, “Facts for
Life”. The fact that “DHF may attack again a
previously-infected child” was important to
remind mothers of the danger. Cartoons and
art competitions and exhibitions on DHF
would be the most effective media for
schoolchildren and mothers.
Discussion
The mean scores of male and female
respondents were not significantly different
in the community. Community members
knew a lot (more than 80%) about
transmission of DHF by mosquito bite, the
biting habit of Aedes and the resultant
fatality. Nevertheless, only 61.6% correctly
identified the main vector of DHF. Nearly
20% answered that Aedes usually bit at
nighttime. Some people were still confused
that polluted water was also a breeding
place. Most of the interviewees responded
that the common breeding sites of Aedes
were water containers and flower vases. Not
more than 30% identified ant traps, broken
pots, tins, old tyres and coconut shells as the
194
vector breeding places. The subjects knew
little of blocked gutters as possible breeding
sites. Nearly 80% thought that DHF could
be prevented by immunization. A majority
of the interviewees used measures to
prevent children from being bitten by
mosquitoes. A very small percentage did not
use any measure. Some points that require
emphasis for community awareness include:
•
Aedes is the main vector for DHF.
•
Aedes only breeds in clear water, not in
polluted water.
•
The biting time of Aedes is daytime.
•
Blocked gutters should be mentioned as
possible breeding sites for Aedes in
existing IEC materials.
•
There is no vaccine available for the
prevention of DHF.
The chance to get high scores was
better in females with high education.
Caregivers with low educational levels
should be targeted for health education. The
level of knowledge scores was not related to
the history of a previously infected child at
home. Knowledge scores may be changed
through exposure to various media. Also,
sufficient numbers of IEC materials should
be distributed in the community. Only a
small percentage of the interviewees were
not exposed to any type of existing IEC. Ways
and means should be found to improve
people’s exposure by further discussions with
community members and health workers
using participatory approaches.
Pamphlets/posters, television, newspapers
and journals are still popular media for the
public. Television is the most effective
medium and various types of programmes,
such as songs, comedies, short movies with
famous actors, actresses, etc., apart from
discussions and health talks can be telecast.
However, radio is still a valuable tool in the
health education process because of its easy
availability ad popular use in semi-urban
and rural areas. The effectiveness of various
existing IEC materials should be reviewed for
further improvement.
Many water containers were not
covered tightly to prevent larval breeding.
Social mobilization for sustainability of
larvae
control
activities
should
be
implemented. Coordination and cooperation
with volunteers and local NGOs should also
be strengthened.
Acknowledgement
We express our gratitude to the Township
Medical Officer and basic health staff in
Thaketa township for their support. We are
grateful to volunteers, NGO members and
the study area residents for their active
participation in the study.
References
[1] Tha NO. A study on the larval control
operations for DHF prevention in
Sanchaung township, Yangon, Myanmar,
2000-2001 (Unpublished report).
[3] Yoon SY. Community participation in the
control and prevention of DF/DHF: Is it
possible? Dengue Newsletter, 1987, (13): 714.
[2] World Health Organization. Information,
education and communication. Lessons
from the past: perspectives for the future.
Department of Reproductive Health and
Research, World Health Organization,
Geneva, 2001.
195
Students’ Perceptions about Mosquito Larval Control
in a Dengue-Endemic Philippine City
Jeffrey L. Lennon #
Foundation University, School of Education, Dumaguete City, Negros Oriental, The Philippines
Abstract
A study was carried out among university students to find out their perceptions about mosquito larval
control in a dengue-endemic Philippine city. This formative research was conducted to obtain
information for formulating future school-related dengue control strategies. The study was carried out inclass through a semi-structured, open-ended question format. The study yielded information on students’
perceptions about the most important measures for mosquito larval control and perceived reasons why
people did not implement them. The study also explored the opinions of the students by gender. The
study yielded students’ knowledge on the types of mosquito larval habitats. Little was expressed about
specific indoor mosquito larval control nor about the frequency of conducting source-reduction activities.
Perceived barriers to constructing mosquito larval control centred on themes such as apathy, laziness and
lack of time. Further studies are necessary to follow-up on these themes in depth.
Keywords: Dengue, formative research, mosquito larval control, students, open-ended questionnaire, Philippines.
Introduction
The control of Aedes aegypti mosquito
larvae is essential for the control of dengue
fever (DF) and dengue haemorrhagic fever
(DHF) [1] . The need to know the perceptions
of key informants is necessary in order to
better address the dengue-related control
issues in a specific area or community[2-5].
Schools are potential mosquito breeding
sites[6-8] . Also, primary, secondary and
tertiary school-age students are principal
targets of the Aedes mosquitoes[9-11].
Dengue has become a steadily increasing
health problem in the Philippines[9] .
#
Consequently, it has become endemic in
Dumaguete city, Philippines[12].
Aedes control is largely based on source
reduction. Therefore, knowledge of the
types of mosquito breeding sites is a
prerequisite
for
health
personnel,
schoolteachers and children and the
community at large for the control of
dengue. Various types of containers have
been identified as potential mosquito
breeding sites. These include plastic and
metal containers, animal-feeding dishes,
tyres, flower vases, coconut shells and water
storage drums[3,4,13,14] .
196
Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City
The knowledge about the types of
breeding containers alone is not enough to
achieve mosquito control. Attitudes and
beliefs impact a person’s knowledge about
mosquito control. For example, the belief
that dengue is not a fatal or serious problem
impairs a person from carrying out adequate
mosquito control practices. Some people
believe that mosquitoes within the home
and outside are different. So it is believed
that mosquitoes inside the house do not
carry disease[15] .
Gender-related
responsibilities
for
control of certain mosquito breeding
containers might also exist. There may be
different responsibilities for one gender
inside the residence compared to the
outside surroundings. Or, there may be a
distribution of clean-up activities depending
upon ownership of specific items such as
tyres[15] .
One study in Mexico indicates that
informants devalued the use of screens in
dengue prevention and believed that the
screens were not effective in keeping out
mosquitoes[2] .
Since dengue is already endemic in this
study city in the Philippines, it was
presupposed that there would already be a
high level of awareness about dengue in
general. So, this study sought to explore the
more specific topic related to dengue
control,
through
source
reduction.
Therefore, this objective of the study was to
explore the opinions of Philippine university
education students about the mosquito
larvae control in a dengue endemic city.
Subjects
The subjects consisted of 43 university
major students at Foundation University,
College of Education, Dumaguete city,
Philippines. There were 36 female and 7
male subjects. All subjects were Filipinos.
The participants were students in a collegebased “personal and community hygiene”
course. This study took place prior to any
course coverage or discussions about
dengue fever or any of the mosquito-borne
diseases. The participation was voluntary
and no student in the class declined it. The
results and data were confidentially held. All
results were tabulated and reconciled so
that the responses would be anonymous.
Procedure
Two open-ended semi-structured questions
were administered to the subjects on
January 16, 2003. The students’ instruction
was the facilitation of the questionnaire.
The two questions were as follows:
(i)
What is the best way to control
mosquito larvae?
(ii) Why don’t some people use the
measure you suggest for question
No. 1?
The questions were given verbally. The
students were also told that they were
allowed to give more than one response per
question. The students gave written
responses to the questions. The responses
were classified by gender. There were no
predetermined categories. Categories of
responses were created from post-survey
results. As categories emerged, subcategories were created according to their
relationship to the broader response
categories. The time allotted for the whole
process of issuing instructions to the
students and completion of response-writing
was approximately 15 minutes.
197
Results
On the responses to Question 1, the
principal
categories
that
emerged
concerning mosquito larvae control activities
were as follows: outside activities, inside
activities, activities not specified as inside or
outside, activities related to specific types of
containers, use of insecticide, unrelated
activities, and not familiar with mosquito
larvae (Table 1).
Table 1. Responses to Question 1
Category
I.
Outside activities
A.
General outside activities
A.1.
A.2.
Clean surroundings or yard
Clean surroundings daily
B.
Specific water-containing items outside
B.1.
B.2.
B.3.
B.4.
B.5.
Turn over coconuts
Dispose of water in coconut shells
Put tyres in a safe place
Dispose of tyres
Dispose of stagnant water in drums
C.
Other environmental activities
C.1.
C.2.
C.3.
C.4.
Clean canals
Burn grass
Put trash cans in a safe place (to avoid rain-water entry)
Throw garbage properly
D.
Activities involving other participants
D.1.
Clean-up drive participation
II.
Inside activities
A.
Clean house (No specific activities mentioned)
III.
Activities not specified as inside or outside
A.
Non-specific cleaning
A.1.
A.2.
A.3.
A.4.
A.5.
A.6.
A.7.
Clean things with stagnant water
Clean or eliminate stagnant water
Empty all stagnant water
Avoid or prevent stagnant water
Dispose of anything where mosquito eggs are laid
Kill mosquito larvae in stagnant water (not specified)
Clean things where mosquito larvae are laid
(non-specific item)
198
Male
Female
Total
5
1
22
1
27
2
1
0
0
1
0
0
1
2
0
1
1
1
2
1
1
1
0
0
0
5
1
1
5
6
1
1
5
0
1
1
0
4
4
1
1
0
0
0
0
0
0
5
2
4
1
2
2
1
6
2
4
1
2
2
Category
Male
Female
Total
B.
Activities related to non-specific types of containers
B.1.
B.2.
B.3.
B.4.
B.5.
Prevent water from entering open containers
Cover any water container
Clean water containers
Dry water containers
Empty containers
0
0
0
0
0
1
8
1
1
1
1
8
1
1
1
C.
Clothes-hanging arrangement
0
1
1
IV.
Activities related to specific types of containers
A.
B.
C.
D.
E.
F.
G.
H.
I.
J.
K.
L.
M.
Avoid open cans
Cover water containers such as gallon containers
Cover water barrels and tanks
Put open cans in a safe place
Dispose of cans
Cover jars
Cover basins
Clean bottles
Replace flower vase water (no time mentioned)
Replace flower vase water daily
Replace flower vase water weekly
Cover garbage
Put garbage in a can (No mention of placing over)
0
0
0
0
1
0
0
0
1
1
0
0
0
1
1
2
3
0
1
1
1
0
1
1
2
1
1
1
2
3
1
1
1
1
1
2
1
2
1
V.
Other control-related activities
A.
Spraying insecticide
0
4
4
VI.
Unrelated activity
A.
Remove an injured person
0
1
1
VII.
Not familiar with mosquito larvae
0
1
1
14
94
108
n
7
36
43
No. of students with multiple responses
5
29
34
Total
199
Table 2. Responses to Question 2
Category
I.
A.
B.
C.
D.
E.
E.1.
E.2.
E.3.
E.4.
E.5.
Knowledge -related categories
Lack of education
Lack of knowledge
Lack of consciousness of surroundings or environment
Lack of consciousness of health
Specific responses related to knowledge
Do not know that the suggested method of larval control
is effective
Do not know the cause and effect of the mosquito to
dengue
They think that they know everything about mosquito
control already
Not aware of the consequences
Do not know that the mosquito is dangerous
II.
A.
B.
Attitude-related categories
Don’t care – apathy
Don’t care because they have enough money to pay
hospital bill
C. Lazy
D. Busy
E. Since people are busy, they only use mosquito sprays to
kill adult mosquitoes instead of mosquito larvae
F.1. No time
F.2. Insufficient time or little time
G. No money especially to pay for insecticide spray
H. Tired
III. Practice-related categories
A. Lack of cooperation with others
B. They just use insecticide spray instead of source
restriction environment control
IV. Invalid or unrelated responses
A. Misunderstood the question (Answered in the affirmative)
B. Either answered Question 1 incorrectly or unfamiliar
with the term “mosquito larvae”
V.
No response
Total
n
Students with multiple responses
200
Male
Female
Total
1
0
0
0
–
1
1
5
3
3
–
1
2
5
3
3
–
2
0
1
1
1
0
1
0
0
2
2
2
2
1
0
9
1
10
1
3
2
1
7
6
0
10
8
1
0
0
0
1
6
1
1
1
6
1
1
2
0
0
2
1
2
1
1
0
8
2
9
2
1
0
1
13
63
76
7
4
36
20
43
24
For the responses to Question 2, the
principal
categories
that
emerged
concerning reasons why people do not
undertake mosquito larvae control were as
follows:
knowledge-related
categories,
attitude-related categories, practice-related
categories, invalid or unrelated responses
and no response (Table 2).
Discussion
In response to Question 1, both female and
male students rated “clean surroundings”
(Table 1, Category I A.2) with the largest
number of responses. This reflects the need
for emphasis on specific mosquito control
efforts while developing training and health
education strategies[5,14] .
The higher rating of outside activities
compared to inside activities reflects the
perceived importance of outside clean-up,
and conversely, the relative lack of
importance of inside clean-up and control
of inside mosquito breeding sites. This may
follow the perception as recorded in
literature that “house mosquitoes” do not
transmit dengue [2,5] . Perhaps students
believe that the Aedes mosquitoes do not
live inside houses or other living quarters.
No male subject gave any response
related to indoor mosquito control measures
(Table 1, Category II). Besides the
perception that the indoors lack importance
in mosquito control, the males may have
deemed the indoors as the domain of
females. The role of women as homemakers
needs to be considered in vector control
programmes. This was reviewed in a
number of international settings[15] .
Female subjects responded to all but
one response, “activities not specified as
inside or outside” (Table 1, Category III).
Perhaps, the male respondents were not
carrying out these activities. The male
response emphasis on general response
items Table 1 A, “General outside activities”
may suggest their lack of specificity in the
details necessary to conduct proper denguerelated mosquito control. The largest
response in Table 1, Category III, was on the
section “cover any water container”
(Category III B.2). This practice is consistent
with recommendations throughout the
literature [4,6,7,13,14] . However, these responses
in Table 1, Category III B.2, are less than
one-third the number of the responses given
in Table 1, Category I A.1.
The
training
of
students
in
environmental
surveys
and
in
the
environmental action plan known as “4
o’clock habit”, has previously been
successful[6] . It is suggested that this be
projected on a larger scale to include other
members of the community.
The responses in “activities related to
specific types of containers” Table 1,
Category IV, were scattered across the item
sub-categories by the respondents. Many of
these response items could be used as
inside containers. Not only is it necessary to
cover, empty, and clean containers, but also
to indicate the frequency of the activity in
order to conduct adequate mosquito control.
Only the responses in Table 1, Category IV J,
and Category IV K, both dealing with
flower-water replacement, and Category I
A.2 “clean surroundings daily” mentioned
the frequency and timing of activities. This
amounted to four responses out of a total of
108 responses in Table 1. Perhaps,
201
programmes need to stress health education
on the mosquito life-cycle and include
emphasis on the time interval to conduct
successive mosquito control clean-ups. The
lack of responses on the frequency of
environmental clean-up is in agreement
with previous literature on the incomplete
understanding about the mosquito life-cycle,
and thus, the need to include this topic in
future health education programmes[2,5] .
Only four female respondents and no
male respondent in Table 1, Category V A,
indicated that insecticide spraying was the
most important mosquito control measure.
The overwhelming majority of the
respondents discussed general or specific
environmental clean-up activities. Source
reduction of mosquito breeding sites
without the use of adult insecticides has
been stressed in dengue-related health
education programmes in Honduras [3] ,
Mexico[4] , and the Philippines[6,13,16] . Since
larvicides are not currently being used or
promoted in this Philippine study site, the
respondents most likely were referring to the
use of adult insecticides. In contrast, dengue
control programmes in Puerto Rico utilized
both source reduction of mosquito breeding
sites and insecticide use[14] . However, for
developing countries, regular adult aerosol
insecticides were deemed to be too
expensive for regular home use, and thus
not recommended for routine control[13] .
Only one respondent as seen in Table 1,
Category VI, gave a response to an activity
unrelated to mosquito control. Also, there
was only one respondent as seen in Table 1,
Category VII, who responded to the
category, “Not familiar with mosquito
larvae”. These responses validated that the
respondents in general had awareness about
202
mosquito larvae. However, only a minority
gave specific details either in types of
control measures or in the timing of control
activities.
For Question 2, the greatest number of
responses among the knowledge-related
category was the general category, “Lack of
knowledge”, Table 2, Category I B. There
was no concentration of specific knowledgerelated responses. There were nearly twice
as many attitude -related responses as
knowledge-related
responses.
Attitude
responses were greater than knowledge
responses for both male and female subjects
(Table 2). Since dengue is endemic in
Dumaguete City[12] , and various health
education programmes have continued for
years, it is reasonable to agree that there is a
high general awareness about dengue.
However, knowledge alone is generally not
sufficient
to
change
attitudes
and
behaviour[17] . Health education to increase
only knowledge without addressing health
behaviours has also been ineffective in the
dengue control experience[5] .
The greatest numbers of responses were
in the attitude-related categories of “Don’t
care – apathy”, Table 2, Category II A, and
“Lazy”, Table 2, Category II C. The
responses of apathy or laziness could have
been a result of a weak belief in the
effectiveness of the proposed measures from
Table 1 to control mosquitoes and dengue.
The health belief model may help to explain
these responses. Two key components of
the health belief model are “perceived
benefits” and “perceived barriers.” A lower
perception of benefits coupled with
elevated barriers may result in a lower
possibility for change[18] . Likewise, people
having low self-efficacy (a construct of the
Social Cognitive Theory and also the Health
Belief Model), or, in other words,
confidence in doing something[18,19] could
have resulted in their lack of interest to carry
out the suggested mosquito control tas ks.
The large number of responses found in
Table 2, Categories II D, II E, II F1 and II F2,
related to the factors of insufficient time to
perform clean-up tasks is also suggested in
the literature [15] . While people may know
about various individual mosquito breeding
sites source reduction tasks, they may lack
the self-confidence necessary to perform a
regular,
comprehensive
environmental
clean-up task. Thus, skill development on
how to conduct the steps of an
environmental action plan for dengue
control should be emphasized in order to
increase self-efficacy and mosquito control
behaviours. This promotion of skill
development may, in turn, increase
personal efficiency to perform source
reduction tasks and thus, decrease the
perception of time as a limitation to perform
mosquito control activities.
There were only two responses to Table
2, Category III A, “Lack of cooperation with
others”. This low number may have been
due to a high value placed on the important
relational imperative or supportive Filipino
norm of “bayanihan” or cooperation[20] . Yet,
there were still responses to perceived lack
of cooperation as a cause for lower rate of
mosquito larval control activities. Addressing
this norm through various communications
and other health promotion means may
help in increasing mosquito larvae control
activity.
Increasing the self-efficacy of individuals
in the community may help in increasing
the collective efficacy for the desired
behavioural change activities. High levels of
perceived collective efficacy may increase
the likelihood of a group carrying out
desired behavioural change activities[21].
Therefore, promoting strategies to increase
collective efficacy may enhance the
likelihood that community mosquito control
activities will be carried out and sustained.
Asking the questions orally, rather than
in a written form, had some limitations. This
may have contributed to the nine
respondents who misunderstood Question 2.
They answered Question 2 by explaining
their reason of choice for the best mosquito
larval control, rather than explain why
people were not using the best mosquito
larval control method. See these responses
in Table 2, Category IV A.
This form of questioning was also
limited in the lack of in-depth follow-up.
The procedure in this study did not allow for
follow-up of such responses as the reason
for “apathy” as a response to Question 2.
Unlike other studies that used interviews[25,14]
or focus groups [4,5] , the facilitator of the
questions did not interact with the
respondents , nor probe for follow-up
responses.
The method and procedure also had its
strengths. In spite of utilizing open-ended
questions, the procedure was very efficient
in time and in organization. Unlike a
previous study where interviews lasted for
an hour per respondent, and at home[5] ,
respondents in this study were able to
complete the questions in a matter of
minutes, and in one sitting.
All questions were completed at the
same time, reducing potential biases that
203
could result from interaction with others in
the outside environment. Also, having the
students complete the questions individually,
without interaction with fellow students and
reduced biased or blended responses.
•
Efforts are needed to create awareness
regarding frequency of mosquito
activities.
•
There
should
be
an
in-depth
exploration of the reasons behind the
perceived causes of non-participation in
mosquito larval control. Especially, the
reasons behind “apathy” and “laziness”
as
perceived
causes
for
nonparticipation in mosquito larval control
should be further explored.
•
Activities such as an environmental
control action plan and creation of a
mosquito control “checklist” and
implementation for houses and schools
as a means to increase self-efficacy
should be promoted.
•
The determinants of collective efficacy
in mosquito larval control and dengue
control should be explored.
The procedure also demonstrated the
strength of producing a large variety of
response categories, multiple responses and
overall total responses in a very short period
of time. This was evident for both sexes and
in response to both questions.
Summary and
recommendations
•
The study is suggestive that there was an
understanding by most students of the
term
“larvae”,
and
its
general
relationship to mosquito and dengue
control. This was exemplified by the
low number of incorrect responses to
content unrelated to mosquito larval
control as indicated in Table 1,
Categories VI and VII, and Table 2,
Categories III B, IV B and V.
•
There were no male responses for
indoor control measures to Question 1.
Also, there were almost no specific
mosquito control measures mentioned
by the male respondents. Further
studies should explore the possible
gender relationship to mosquito control
practices.
•
The majority of male and female
respondents did not mention indoor
mosquito larval control. Therefore,
future mosquito control programmes
should stress the importance of indoor
mosquito larval control measures.
204
Teachers play an important role in
facilitating of health promotion in dengue
endemic areas. Students and teachers
should be properly oriented to carry out
personal, school and community mosquito
and dengue control measures. Antecedent
to this is an understanding of students’
perceptions about mosquito-related dengue
control.
The
in-class
semi-structured
question method is one tool to carry out this
type of formative research.
Acknowledgement
The author is associated with the
International Technical Assistance Group
(ITAG), Seattle, WA, USA. ITAG’s support is
gratefully acknowledged. Also, thanks to
Chona F. Lennon and Fernando N.
Florendo for their assistance.
References
[1] Gubler DJ. Aedes aegypti and Aedes aegyptiborne disease control in 1990s: top down or
bottom up. American Journal of Tropical
[2] Kendall C, Hudelson P, Leontsini E, Winch P,
Lloyd L and Cruz F. Urbanization, dengue
and the health transition: anthropological
contributions to international health.
Medical Anthropology Quarterly, 1991, 5:
257-268.
[3] Leontsini E, Gil E, Kendall C and Clark GG.
Effect of a community-based Aedes aegypti
control programme on mosquito larval
production sites in El Progreso, Honduras.
Transactions of the Royal Society of Tropical
[4] Lloyd LS, Winch P, Ortega-Canto J and
Kendall C. The design of a communitybased health education intervention for the
control of Aedes aegypti. American Journal
of Tropical Medicine and Hygiene, 1994,
50: 401-411.
[5] Winch P, Lloyd L, Godas MD and Kendall C.
Beliefs about the prevention of dengue and
other febrile illnesses in Merida, Mexico.
1991, 94: 377-387.
[6] Lennon JL. Knowledge of dengue
haemorrhagic fever by Filipino University
students. Dengue Bulletin, 1996, 20: 82-86.
[7] Swaddiwudhipong W, Chaovakiratipong C,
Nguntra P, Koonchote S, Khumklan P and
Lerdlukanavonge P. Effect of health
education on community participation in
control of dengue hemorrhagic fever in an
urban area of Thailand. Southeast Asian
Journal of Tropical Medicine and Public
Health, 1992, 20: 200-206.
[8] Strickman D and Kittayapong P. Dengue
and its vectors in Thailand: introduction to
the study and seasonal distribution of Aedes
larvae. American Journal of Tropical
[9] Custodio GGV. (Ed.). National objectives for
health, Philippines, 1999-2004: Kalusugan
Para sa Masa. HSRA Monograph Series
(Series No. 1), 1999 (On-line Serial).
Retrieved on 1 November 2000, from
http/www.doh.gov.ph/noh/default.shtm.
[10] Gubler
DJ.
Dengue
and
dengue
haemorahagic fever. Clinical Microbiology
Reviews, 1998, 11: 480-496.
of dengue and dengue haemorrhagic fever
Health Statistics Quarterly, 1995, 50: 161169.
[12] Dengue cases by Barangay, Dumaguete City,
Philippines, City Health Office, 2003.
[13] Advocacy and Health Promotion Service.
Best way to prevent dengue haemorrhagic
fever. Cebu City, Philippines, Department of
Health.
[14] Winch PJ, Leontsini E, Rigau-Perez JG, Clark
GG and Gubler DJ. Community-based
dengue prevention programmes in Puerto
Rico: impact on knowledge, behavior and
residential mosquito infestation. American
2002, 67: 363-370.
[15] Winch PJ, Lloyd LS, Hoemeke L and
Leontsini E. Vector control at the household
level: An analysis of its impact on women.
Acta Tropica, 1994, 56: 327-339.
[16] Lennon JL and Gonzales MJCP. The
utilization of college of education students in
a dengue health education campaign.
Philippine Journal of Education, 1995, 73:
393, 421, 425-426.
[17] Green LW and Kreuter MW. Health
promotion planning: an educational and
environmental approach, Second Edition.
Mountain View, CA, USA: Mayfield
Publishing, 1991: 155.
205
[18] Strecher VJ and Rosenstock IM. The health
belief model. In: K Glanz, FM Frances and
BK Rimer (Eds). Health behavior and health
education: Theory, research and practice,
Second Edition, San Francisco, CA, USA:
Jossey-Bass Pub, 1997: 41-59.
[19] Bandura A. Self-efficacy: the exercise of
control. New York: WM Freeman Pub. 1997.
206
[20] Andres
JD
and
Ilada-Andres
PB.
Understanding the Filipino. Quezon City,
Philippines: New Day Pub, 1987: 55-56.
[21] Bandura, A. Exercise of human agency
through
collective
efficacy.
Current
Directions in Psychological Research, 2000,
9: 75-78.
Short Note 1
Sero-surveillance in Delhi, India – An Early Warning
Signal for Timely Detection of Dengue Outbreaks
D. Bhattacharya*, Veena Mittal* #, M. Bhardwaj*, Mala Chhabra*,
R.L. Ichhpujani** and Shiv Lal*
*National Institute of Communicable Diseases, 22-Sham Nath Marg, Delhi – 110 054, India
**Directorate General of Health Services, Nirman Bhavan, New Delhi – 110 011, India
In India the first major outbreak of dengue
fever (DF) accompanied with dengue
haemorrhagic fever (DHF) was reported in
Kolkata (Calcutta) in 1963[1] . More than 60
outbreaks have been reported since 1956 to
date[2] . Of these two major outbreaks of
DF/DHF occurred in 1996 and 2003 in
Delhi and its adjoining states. Surveillance is
the most cost-effective approach for
prevention and control of dengue. A strong
surveillance system will help in detecting
early warning signals of an outbreak,
instituting timely and appropriate control
measures,
assessing
the
impact
of
intervention measures and early containment
of the outbreak. Considering the above facts,
the arbovirus laboratory at the National
Institute of Communicable Diseases, Delhi,
has started sero-surveillance and monitoring
of dengue fever in Delhi since 1996 as an
ongoing activity. It was intended to develop
an early warning signal for timely detection of
an impending outbreak and institution of
preventive and control measures in high-risk
areas.
Sera samples of clinically suspected
cases of DF and/or DHF are received from
various hospitals of Delhi round the year.
#
These samples were tested for dengue by
haemagglutination inhibition (HI) test[3] or
IgM Capture ELISA Test[4] . A titre of
≥ 1:1280 in HI test in acute phase serum is
considered a presumptive diagnosis of a
current dengue infection[5] . Samples positive
for IgM antibodies against dengue virus
indicate recent infection with dengue virus.
The results of the sentinel serosurveillance from 1996 to 2003 are
summarized in the Table. The analysis of
data over the period of eight years shows that
dengue strikes Delhi every year. The
positivity ranges from approximately 13%33%, except in 1996 and 2003 when dengue
fever occurred in epidemic proportions along
with DHF. The positivity in these two years
was 53.4% and 57.8% respectively.
The month-wise distribution of samples
tested from 1996-2003 shows that the
positivity for dengue starts appearing in the
month of August and reaches a peak in
October and continues till mid-November
and then a decline starts and the last cases
are reported upto 2nd week of December.
The data for 2003 also shows a similar trend
(Figure).
207
208
11
931
Total
3
AUG
DEC
0
JUL
104
0
JUN
NOV
3
MAY
643
0
APR
OCT
3
MAR
159
4
FEB
SEP
1
Tested
0
0
0
0
0
0
0
0
493
(53.4%)
3
74
343
73
+ve
1996
JAN
Year/
Month
764
15
81
369
169
20
15
8
33
39
4
6
5
Tested
3
0
0
0
1
0
0
0
255
(33.3%)
1
26
159
65
+ve
1997
868
47
293
201
70
48
31
27
67
28
24
11
21
Tested
5
0
1
0
0
0
0
0
1
206
(23.7%)
21
140
38
+ve
1998
482
40
130
145
71
27
11
12
11
7
12
11
5
Tested
0
0
0
1
0
0
0
0
93
(19.2%)
5
35
35
17
+ve
1999
696
77
254
188
56
32
18
9
26
8
9
8
11
Tested
1
0
0
0
0
0
0
0
213
(30.6%)
13
112
74
13
+ve
2000
852
35
225
304
127
43
22
22
13
10
11
21
19
Tested
6
1
0
0
0
0
0
0
283
(33.2%)
9
94
129
44
+ve
2001
Table: Dengue serology during the years 1996 – 2003 in Delhi
328
34
60
55
31
17
23
12
6
34
19
19
18
Tested
1
0
0
0
0
0
0
0
0
42
(12.8%)
11
18
12
+ve
2002
4,072
35
877
2,351
701
43
11
11
10
8
13
5
7
Tested
0
5
0
0
0
0
0
2,356
(57.8%)
14
450
1,438
433
16
+ve
2003
Sero-surveillance in Delhi – An Early Warning Signal for Timely Detection of Dengue Outbreaks
Sero-surveillance in Delhi – An Early Warning Signal for Timely Detection of Dengue Outbreaks
Figure. Month-wise samples tested/positive for dengue antibodies in Delhi
during the year 2003
2,500
2,000
1,500
1,000
500
Tested
The studies for the estimation of the
House Index (HI) of mosquitoes also shows
that the house index for Aedes aegypti, the
vector of dengue fever, starts building-up
during the rainy season, i.e. from July and
reaches a peak in August-September[6].
A regular monitoring of suspected
dengue cases by detection of IgM antibodies
December
November
October
September
August
July
June
May
April
March
February
January
0
Positive
to dengue virus can act as an early warning
signal for an impending outbreak. The
above observations show that serological
surveillance throughout the year, especially
during the outbreak-prone period, can play
an important role in the detection of early
cases.
References
[1] Ramakrishnan SP, Gelfand HM, Bose PN,
Sehgal PN and Mukherjee RN. The
epidemic of acute haemorrhagic fever,
Calcutta. Ind J Med Res, 1964, 52: 633-650.
[4] Lam SK, Devi S and Pang T. Detection of
specific IgM dengue infection. Southeast
Asian J Trop Med Public Health, 1987, 18:
532-538.
[2] Epidemic preparedness – dengue fever,
DHF and DSS. CD Alert, Monthly
Newsletter,
National
Institute
of
Communicable Diseases, Delhi, June 2001,
Volume 5 (16).
[5] Gubler DJ. Serological diagnosis of
dengue/dengue haemorrhagic fever. Dengue
Bulletin, 1996, 20: 20-23.
[3] Clark DH and Casals J. Techniques for
haemagglutination and haemagglutination
inhibition with arthropod borne viruses. Am
J Trop Med, 1958, 7: 561-573
[6] Katyal R, Singh K and Kumar K. Seasonal
variation in Aedes aegypti population in
Delhi, India, Dengue Bulletin, 1996, 20: 7881.
209
Short Note 2
Detection of Dengue Virus in Wild Caught Aedes
albopictus (Skuse) around Kozhikode Airport,
Malappuram District, Kerala, India
B.P. Das*, L. Kabilan**, S.N. Sharma*, S. Lal*, K. Regu*** and V.K. Saxena*
*National Institute of Communicable Diseases, 22 Sham Nath Marg, Delhi – 110 054, India
**Centre for Research in Medical Entomology, 4 Sarojini Street, Chinna Chokkikulam,
Madurai – 625 002, India
***National Institute of Communicable Diseases, Kozhikode Branch, Kerala, India
Introduction
In India, outbreaks of dengue fever
(DF)/dengue haemorrhagic fever (DHF) have
been reported in various parts of the
country during the past four decades[1] .
Aedes aegypti is the only vector that has so
far
been
implicated
in
dengue
transmission[1,2] ,
even
though
Aedes
albopictus is known to be present in some
of the peri-urban and rural areas [2] . Recently,
a survey was carried out in the Kozhikode
(earlier known as Calicut) airport area of
Malappuram district, Kerala. During 2002
and 2003 (up to July), 75 and 150 clinical
dengue fever cases, respectively, were
reported from the district[3] . Earlier, reports
of Aedes survey in Kerala had shown the
presence of Aedes albopictus in rubber
plantation areas [4] and in plastic cups [5] .
This communication presents the results
of the detection of dengue virus from the
wild and dry preserved, adult females of
Aedes albopictus and their breeding indices
210
in and around the airport area. The survey
was carried out in May 2004.
The Kozhikode airport is situated at 11° .15'
N latitude and 75° .49' E longitude, in a hilly
area of Malappuram district, Kerala. It
became functional as an international
airport in 1988. A larval survey was carried
out in various types of water-holding
containers to detect the breeding of Aedes
(Stegomyia) mosquitoes, both inside the
airport premises and its periphery up to
about 600 metres. The larvae were
identified as per the method described
earlier[6,7] . Adults of Aedes (Stegomyia)
mosquitoes were collected while landing on
human baits by aspirator tube in a forested
residential area about 600 metres away
from the airport.
The wild Aedes albopictus females
caught from outside the Kozhikode airport,
the adults reared from larval collections
Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport
from inside the Kozhikode airport and the
city, and the adults of Aedes aegypti reared
from
larval
collection
from
the
Thiruvananthapuram international airport
were separated sex-wise, pooled by species
(about 15 adults per pool) and transported
to the Centre for Research in Medical
Entomology (CRME), Madurai, Tamil Nadu,
in a dry state, for detection of dengue virus.
The methodology followed was similar to
that used for the detection of JE virus and
based on the protocol developed and
standardized by CRME[8] . However, the
antibody (D3-5C9-1) was diluted at 1:5000
as being followed by CRME.
Aedes survey
The survey for larval infestation in 52
houses/premises around the airport area
revealed 16 premises as positive for Aedes
albopictus breeding (house index 30.7%). A
total of 272 wet containers searched for
Aedes breeding revealed 41 containers as
positive for Aedes albopictus and two for
Aedes vittatus (container index 15.1% and
0.7% respectively). The most preferred
containers for Aedes albopictus breeding
were discarded tyres, coconut shells and
plastic containers. The average landing rate
of Aedes albopictus on humans was 20
females/human bait/hour.
Dengue virus detection in
Aedes albopictus
Of the three pools of Aedes albopictus
tested for dengue virus infection following
antigen-capture enzyme immunoassay (EIA),
one pool was found positive for dengue
virus (OD-0.32), thereby indicating dengue
viral activity in this mosquito species. The
mosquitoes in the positive mosquito pool
were collected as landing collection around
the Kozhikode airport on 28 May 2004, and
transported as dry specimens to the CRME
laboratory and processed on 8 June 2004
(Table). Earlier dengue virus was also
isolated from Aedes albopictus collected in
a village in Vellore district of Tamil Nadu[9] .
Table. Aedes mosquito pools tested for dengue virus infection by ELISA
Mosquito
species
Aedes
albopictus
Locality
Collected on
(Processed
on)
No. of pools tested/No. of adults/No. of
pools positive
Wild caught adults
(landing)
Reared adults
(immature)
Kozhikode airport
27/05/04
(08/06/04)
–
1/15/0
Residential area
around the airport
28/05/04
(08/06/04)
1/10/1*
–
City area
(2 kms from
airport)
28/05/04
(08/06/04)
–
1/20/0
* Positive pool
211
Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport
The present study confirms that antigencapture enzyme immunoassay is a useful
surveillance tool for monitoring dengue virus
infection in mosquitoes.
Centre for Research in Medical Entomology,
Madurai, Tamil Nadu, is gratefully
acknowledged. The authors are thankful to
Mrs V. Thenmozhi and Mr S. Venkatesan,
CRME, for providing laboratory assistance.
Acknowledgements
The provision of laboratory facilities and the
permission for the study by the Director,
References
[1] Yadava RL and Narasimham MVVL.
Dengue/dengue hemorrhagic fever and its
control in India. Dengue Newsletter, 1992,
17: 3-8.
[2] Das BP, Sharma SK and Datta KK.
Prevalence of Aedes aegypti at the
International port and airport, Kolkata (West
Bengal), India. Dengue Bulletin, 2000, 24:
124-26.
[3] Anonymous. Annual Report of the Centre
for Research in Medical Entomology, Indian
Council of Medical Research, 2002-2003:
82.
[4] Sumodan PK. Potential of rubber plantation
as breeding source for Aedes albopictus in
Kerala, India. Dengue Bulletin, 2003, 27:
197-98.
[5] Hiriyan J, Tewari SC and Tyagi BK. Aedes
albopictus (Skuse) breeding in plastic cups
around tea-vendor spots in Ernakulam city,
Kerala state, India. Dengue Bulletin, 2003,
27: 195-96.
212
[6] Das BP. A simple modified method for
mounting mosquito larvae. J Commun Dis,
1986, 18: 63-64.
[7] Das BP and Kaul SM. Pictorial key to the
common
Indian
species
of
Aedes
(Stegomyia) mosquitoes. J Commun Dis,
1998, 30: 123-127.
[8] Tewari SC, Thenmozhi V, Rajendran R,
Appavoo NC and Gajanana A. Detection of
Japanese encephalitis virus antigen in
desiccated mosquitoes: an improved
surveillance system. Trans Roy Soc Trop
Med Hyg, 1999, 93: 525-26.
[9] Tewari SC, Thenmozhi V, Katholi CR,
Manavalan R, Munirathinam A and
Gajanana A. Dengue vector prevalence and
virus infection in a rural area in south India.
Trop Med Int Health, 2004, 9(4): 499-507.
Short Note 3
Entomological Investigations for DF/DHF in
Alwar District, Rajasthan, India
Kalpana Baruah #, Avdhesh Kumar and V.R. Meena
National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi-110 054
Introduction
During 2001, small outbreaks of DF/DHF
were reported in many districts in
Rajasthan, including the capital city of
Jaipur and the industrial city of Alwar.
There was a total of 1,820 laboratoryconfirmed cases (based on serology using
kits for IgG and IgM antibodies) with 30
deaths (CFR: 1.65%)[1] . The present
communication
deals
with
the
entomological investigations carried out by
the National Institute of Communicable
Diseases, Delhi, during the outbreak
period in a few urban and rural areas of
Alwar district that were affected.
Study area
Alwar district is situated in the northeastern part of Rajasthan between 27°4′
and 28°4′ north latitude and 76°7′ and
77°13′ longitude. The central part of the
district is occupied by the Aravali hills. The
population of the district is 29,90,862
(2001 census), of which 85% is
predominantly rural. The monsoon season
#
is usually of a short duration (July-August),
the average rainfall being 61.16 cm. The
highest temperature during June goes up
to 47 °C whereas the lowest may go down
to freezing point.
Alwar town has a number of industrial
units. Migration of labour thus poses an
increased threat for malaria and other
vector-borne diseases, including DF/DHF.
The city has irregular piped water supply
resulting in water storage practices for
household purposes. In rural areas no such
piped water supply system exists; therefore,
water from wells, bore-wells and natural
streams is used for household purposes
with minimal storage practices.
Larval survey
An Aedes survey, as per WHO guidelines[2] ,
was carried out in four localities out of 10
in urban areas and in five localities under
four primary health centres (PHCs) in rural
areas, all reporting fever cases. The results
of the Aedes survey are given in Table 1.
213
Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India
Table 1. Aedes aegypti larval indices in the urban and rural areas, Alwar, Rajasthan
Name of
Total
the locality
houses
(or villages) searched
Houses
found
positive
House
Index
Containers
searched
Containers
positive
Container
Index
Breteau
Index
Urban areas
Sonwa
30
10
33.3
62
17
27.4
56.7
Karaulikund
30
9
30.0
57
13
22.8
43.3
Arya Nagar
20
4
20.0
47
4
8.5
20.0
Kalakuan
25
9
36.0
111
15
13.5
60.0
Indok
25
0
–
47
0
–
–
Madhogarh
20
0
–
32
0
–
–
Malakhera
(Kalachara)
25
0
–
34
0
–
–
Bhartahari
Tiraha
25*
5
20.0
20
5
25.0
20.0
Kushalgarh
10*
1
10.0
15
1
6.7
10.0
Rural areas
* Shops and nearby houses just outside the villages
Larval surveys
In urban areas, the House, Container and
Breteau indices ranged from 20.0% to
36.0%, 8.5% to 27.4% and 20.0 to 60.0,
respectively. Mixed breeding of Aedes
aegypti and Anopheles stephensi was also
detected in cement tanks in some areas.
In comparison, the House Index was nil
in rural residential areas as no mosquito
breeding could be detected; however,
shops in the marketplaces near the villages
and their adjacent houses were found to be
positive. The House/Premise Index in these
localities ranged from 10.0% to 20.0%,
214
Container Index 6.7% to 25.0% and Breteau
Index from 10.0 to 20.0 only. The shops
used earthen pots for storing drinking water
wherein co-breeding of Aedes aegypti and
Aedes albopictus was detected. The
dwelling houses along the shops were also
found positive, as Aedes breeding was
detected in cement containers used for
providing drinking water to cattle.
The area-wise infestation by containers
is given in Table 2. In the urban areas, out
of the total of 49 positive containers,
67.35% were domestic water-storing
containers (like cement tanks, clay pots and
overhead tanks), followed by 30.61%
evaporation coolers and the remaining
2.04% trash.
Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India
Table 2. Area-wise infestation of Aedes aegypti in the urban areas, Alwar district, Rajasthan
Type of container
Area
Evaporation
coolers
S
%
+ve
Cement
tanks
%
+ve
S
Clay pots
S
%
+ve
Overhead
tanks
S
Others
%
+ve
S
%
+ve
0
–
–
Sonwa
27
18.5
16
56.3
12
16.7
Karaulikund
19
21.1
21
38.1
17
5.9
–
–
–
–
Arya Nagar
24
8.3
9
11.1
14
7.1
–
–
–
–
Kalakuan
37
10.8
27
25.9
41
4.9
4
25.0
2
50.0
107
14.0
73
34.2
84
8.3
11
9.1
2
50.0
Total
7
S=Searched
Adult surveys
Landing collections were also undertaken in
the same urban and rural areas where larval
surveys were carried out. In urban areas the
adult density of Aedes aegypti ranged from
2.0 to 7.0 per man-hour. The houses with
poor ventilation and light yielded higher
numbers.
In rural areas both Aedes aegypti and
Aedes albopictus were detected in shopping
areas only. The cases reported from these
rural areas could be attributed to population
movement from rural to urban areas (city)
during daytime for earning their livelihood
or shopping purposes. A similar observation
was made earlier by Kalra et al.[3] in Ajmer
(Rajasthan), where people from the
periphery picked up the infection during
their day visit to the city area where Aedes
aegypti breeding indices were very high.
Acknowledgments
The authors are grateful to the Director,
National
Institute
of
Communicable
Diseases,
Delhi,
for
providing
the
opportunity and necessary facilities to
undertake
this
investigation.
The
cooperation extended by the staff of the
Chief Medical Officer’s office, Alwar district,
and the technical support of the staff of the
NICD,
Alwar
branch,
is
gratefully
acknowledged.
References
[1] Anonymous. Report of State Health
Authority, Jaipur, 2001 (unpublished).
[2] World Health Organization. Prevention and
control of dengue and dengue haemorrhagic
fever – comprehensive guidelines, 1999,
WHO Regional Publication, SEARO 29.
[3] Kalra NL, Ghosh TK, Pattanayak S and
Wattal
BL.
Epidemiological
and
entomological study of an outbreak of
dengue fever at Ajmer, Rajasthan (1969). J
Com Dis, 1976, 8(4): 261-279.
215
Short Note 4
Essentiality of Source Reduction in both Key and
Amplification Breeding Containers of Aedes aegypti
for Control of DF/DHF in Delhi, India
B.N. Nagpal, Aruna Srivastava #, M.A. Ansari and A.P. Dash
Malaria Research Centre, 20 Madhuban, Delhi - 110 092
Delhi, the Capital of India, reported the
first-ever outbreak of dengue fever in
1967[1] . Since then the city has experienced
cyclic epidemics every 2-3 years. During
1996, a large epidemic swept the city when
10,252 cases were hospitalized and 423
deaths were recorded[2] . The disease has
now become endemic and the yearly
incidence has varied between 160 and 300
cases of DF/DHF, with a couple of deaths[3] .
Aedes aegypti has been invariably found to
be associated with these outbreaks in Delhi.
Overhead tanks (OHTs) and ground level
tanks (GLTs) as key containers, and
evaporation coolers have repeatedly been
reported as seasonal amplification sites[4,5] .
The key containers maintain mosquito
breeding throughout the year, whereas
coolers amplify the A edes population from
May to November, and thereafter they go
dry[6] .
During 2003, DF/DHF resurged in some
localities in Delhi and a total of 2,604
DF/DHF cases and 33 deaths were
registered up to 9 November 2003 by the
#
city Municipal Corporation of Delhi (MCD).
The MCD organized a campaign aimed at
source reduction, which was backed by
vehicle-mounted thermal fogging for the
control of the outbreak. However, the
reduction in the DF incidence was not
found to be commensurate with the inputs
made into the campaign. At the request of
the MCD, a team of experts from the
Malaria Research Centre undertook a
comprehensive survey in five affected
localities, viz. Dayanand Colony (Lajpat
Nagar Phase IV), Lajpat Nagar (Phases I and
II), Kotla Mubarakpur, Dayalpur Extension
and Harsh Vihar – Tulsi Niketan, to identify
both the key containers (OHTs and GLTs)
and
the
amplification
containers
(evaporation coolers, earthen pots, flower
vases, household ornamental fountains, etc.)
during November 2003 as per WHO
techniques. The results of the study are
included in this communication.
The results of the larval survey of Aedes
aegypti in five dengue-affected localities in
Delhi as mentioned above are given in the
E-mail: [email protected], [email protected]
216
Source Reduction for DF/DHF Control in India
Table. The results revealed that among the
key containers, out of 243 OHTs and 20
GLTs, 64 OHTs (26.34%) and 8 GLTs (40%)
were found positive for breeding of Aedes
aegypti. On the other hand, out of the 533
evaporation coolers, which are identified as
the major amplification-breeding site,
checked, none was found positive for
breeding of Aedes aegypti. Ornamental
fountains inside the drawing rooms and
mud-pots containing water for birds were
supporting the heavy breeding (>1,000
larvae in each site) of Aedes aegypti. The
maximum positivity of OHTs was found in
Kotla Mubarakpur (39.39%), followed by
Phases I and II of Lajpat Nagar (30%) and
Dayanand Colony (20.69%). Two OHTs
located on the roofs of the market area were
found supporting the breeding of Aedes
albopictus. In a children’s home for boys
belonging to the Social Welfare Department
of the Delhi Government, five out of 12
OHTs were found positive for Aedes aegypti
while two were found dry. It would thus
appear that during the MCD campaign,
source reduction efforts had concentrated
on evaporation coolers, and key containers
and other trash materials breeding the
species remained intact in domestic habitats.
IEC Activities of MCD
The IEC activities of the MCD generally
covered the following aspects:
(1) Creating awareness through media and
spreading vocal messages through the
use of loudspeakers in DF-affected
localities, distribution of pamphlets,
etc.;
(2) MCD workers who visit houses for
physical verification of breeding sites
also interact with householders and
impress upon them to remove small
water collections indoors/outdoors,
specially water evaporation coolers.
During the survey it was found that
fogging generally lacked a ‘pre-fogging
public information campaign’ requiring the
public to keep the house doors/windows
open to permit the entry of fog. Health
workers,
while
interacting
with
householders, invariably talked about the
removal of breeding from coolers and tanks
but did not point out other sites of breeding.
The crosschecking teams of the MCD also
laid emphasis on inspection of evaporation
coolers as focal points and did not verify
OHTs/GLTs and trash materials, as they
found it a time-consuming affair. In urban
areas, for security reasons or houses being
locked, access by health workers into the
houses was also a constraint.
In view of the aforesaid, it can be
concluded that: (i) source reduction should
cover both the key containers as well as the
amplification breeding sites, viz. evaporation
coolers and other trash articles breeding the
vector species; (ii) the fogging operation
should be preceded by a pre-fogging
information campaign in order to seek full
cooperation of communities to derive
optimal benefits from fogging; (iii) the IEC
campaign based on KAP studies should be
prepared with particular emphasis on
community participation; (iv) special efforts
should be focused on behavioural change in
accordance with the guidelines laid down
by WHO[7] ; and (v) health staff of the MCD
should be trained in entomological
surveys/techniques
related
to
Aedes
breeding, prevention and its control.
217
218
0
2
5
34
* Ornamental fountain and trash
–: NIL
Total
Harsh Vihar –
Tulsi Niketan
64
26
66
Kotla
Mubarakpur
243
24
80
Lajpat Nagar
(Phases I and II)
Dayalpur
Extension
12
+ve
58
Examined
26.34
5.88
0.0
39.39
30.00
20.69
%
Overhead tanks (OHTs)
Dayanand
Colony
(Lajpat Nagar
Phase IV)
Locality
–
–
–
20
7
13
Examined
8
–
–
3
–
5
+ve
40.00
–
–
42.86
–
38.46
%
Ground level tanks
(GLTs)
Key containers
533
45
69
134
138
147
Examined
0
0
0
0
0
0
+ve
0
0
0
0
0
0
%
Evaporation coolers
–
–
5
2
2
1
Examined
5
–
–
2
2
1
+ve
100
–
–
100
100
100
%
Other domestic containers
Amplification containers
Table. Breeding of Aedes aegypti in key and amplifier containers in dengue-affected localities of Delhi during November 2003
Acknowledgement
The authors are thankful to Dr K.N. Tiwari,
Municipal Health Officer-cum-Director,
Health Services, Municipal Corporation of
Delhi, for providing assistance in the
conduct of the study, and to Mr N.L. Kalra,
former Deputy Director, National VectorBorne Diseases Control Programme, for his
valuable suggestions and guidance. We
acknowledge the efforts of the field staff for
assisting in carrying out the survey. Thanks
are also due to Mr Sanjeev Gupta for his
help in various ways.
References
[1] Balaya S, Paul SD, D'lima LV and Pavri KM.
Investigations of an outbreak of dengue in
Delhi in 1967. Indian Journal of Medical
Research, 1969, 57: 767-774.
[5] Ansari MA and Razdan RK. Seasonal
prevalence of Aedes aegypti in five
localities of Delhi, India. Dengue Bulletin,
1998, 22: 28-32.
[2] Sharma S, Sharma SK, Mohan A, Wadhwa
J and Dar L. Clinical profile of dengue
haemorrhagic fever in adults during the
1996 outbreak in Delhi, India. Dengue
Bulletin, 1998, 22: 20-27.
[6] Katyal R, Gill KS and Kumar K. Seasonal
variation in Aedes aegypti population in
Delhi, India. Dengue Bulletin, 1996, 20:
78-81.
[3] Katyal R, Kumar K, Gill KS and Sharma RS.
Impact of intervention measures on
DF/DHF cases and Aedes aegypti indices
in Delhi, India: an update 2001. Dengue
Bulletin, 2003, 27: 163-167.
[7] Parks W and Lloyd L: Planning social
mobilization and communication for
dengue fever prevention and control. A
step-by-step guide. WHO, Geneva, 2004.
[4] Krishnamurthy BS, Kalra NL, Joshi GC and
Singh NN. Reconaissance survey of Aedes
mosquito in Delhi, Bull Ind Soc Mal Com
Dis, 1965, 2: 56-67.
219
Short Note 5
Breeding of Dengue Vector Aedes aegypti (Linnaeus) in
Rural Thar Desert, North-western Rajasthan, India
B.K. Tyagi# and J. Hiriyan
Centre for Research in Medical Entomology, Indian Council of Medical Research, 4 Sarojini Street,
Chinna Chokkikulam, Madurai 625 002, India
Aedes aegypti, the vector of dengue fever, is
widely present in India[1,2] , including the
Thar desert in north-western Rajasthan.
Jalore town in the Thar desert experienced
the first-ever epidemic of dengue fever in
1985. Entomological studies carried out in
Jalore during 1990 and subsequently in
1996 observed extensive breeding of Aedes
aegypti[3,4] . Recently, dengue fever again
struck the Thar, this time in Jodhpur district,
warranting a serious review of the vector
ecology in the region, particularly in view of
the prodigious breeding of Aedes aegypti in
discarded household and community-based
underground water reservoirs called tankas,
of which thousands are formed in different
forms in the Thar[3] . These tankas, which
originally attracted only the malaria vector
Anopheles stephensi as long as the water
remained potable, started breeding Aedes
aegypti only after being discarded by local
populations in the wake of the recent
availability of conduit-based water supply
under Indira Gandhi Nahar Pariyojana
(IGNP canal project). It is, therefore,
#
considered worthwhile to highlight the
association of Aedes aegypti and tankas in
sustaining the vector population under
extreme xeric conditions.
A total of 33 villages in the three
districts of Jodhpur, Jaisalmer and Sri
Ganganagar (now incorporated partly in the
newly created Hanumangarh district) were
surveyed for the presence of tankas (Table
1). Compared to those of Jodhpur and
Jaisalmer, most villages in Sri Ganganagar
are highly irrigated and adequately supplied
with the conduit-water system. Four major
types of water storing facilities were
identified which supported the breeding of
Aedes aegypti. About 13.6% of the tankas,
which constituted 77.1% of the four water
bodies, were found to be breeding Aedes
aegypti. It is noteworthy that the typical
century-old traditional earthen-type tanka
was almost invariably present in most
villages of Jodhpur (88.8%) and Jaisalmer
(85.7%) but less so in Sri Ganganagar (17%)
where canal-based water storage sources
220
Aedes aegypti Breeding in ‘Tankas’ in the Thar Desert
had greatly reduced the existence of the
tankas. As many as 11 (24.4%) out of a total
of
45
community
tankas
existing
peripherally in Kanasar village were
abandoned for want of proper water storage
facility and rendered uncared for by the
villagers. All such tankas, wherein the water
turned turbid in course of time including
vegetation growth, Aedes aegypti was
invariably found to breed, replacing in the
process its earlier and original occupant, the
malaria vector Anopheles stephensi. The first
instar larvae abounded the most (52.3%),
while the fourth instar larvae (7.8%) was
present the least, followed by pupae (2.5%).
This situation clearly indicated a sustained
breeding of Aedes aegypti in the tankas.
Cement tanks, invariably constructed in
close vicinity of bore-wells, for cattle
drinking bred Aedes aegypti as soon as the
water there turned turbid due to prolonged
use by cattle, sometimes with Anopheles
subpictus. None of the beris, another type of
earthen reservoir in the desert environment,
supported the breeding of Aedes aegypti.
Table 1. Distribution of different kinds of tankas and beris in various villages in three districts
currently under varying degrees of irrigation and/or conduit water supply from canals
No. of villages with different types of ‘tankas’
District
No. of
villages
Typical intradomestic
earthen tankas
–
Sri
Ganganagar
+
Metallic
mobile
tankas
++ –
+
Central
community
tankas
++ –
Peripheral
village tankas
assembly
+
++
–
+
++
Cement
tanks
Beris
–
+ ++ –
+ ++
17
9
5
3
4
13
0
0
17
0
17
0
0 17 0
0 2 12
3
Jodhpur
9
1
5
3
0
8
1
0
9
0
0
8
1
8 1
0 0
7
2
Jaisalmer
7
1
6
0
0
7
0
0
7
0
0
6
1
6 1
0 0
6
1
33
11
16
6
4
28
1
0
33
0
17
14
0 2 25
6
Total
2 31 2
– = Absence of breeding habitat
+ = Presence of breeding habitat, with potable water breeding Anopheles stephensi
++ = Presence of abandoned breeding habitat positive for Aedes aegypti breeding
Conclusion
The dengue vector, Aedes aegypti, has so far
been collected from the Thar desert only
from townships and/or desert fringe areas in
the vicinity of urban environment, breeding
mostly in household pitchers and cement
tanks [2,4,5] . The breeding of Aedes aegypti in
the tankas in the rural areas of the Thar
desert is considered to be a rather recent
phenomenon, possibly due to the easy
accessibility of newly provided conduitwater supply to villages, which has led to
the abandoning of the traditional water
reservoirs.
221
Aedes aegypti Breeding in ‘Tankas’ in the Thar Desert
References
[1] Kalra NL, Wattal BL and Raghvan NGS.
Distribution pattern of Aedes aegypti in India
and some ecological considerations. Bull Ind
Soc Mal Comm Dis, 1968, 5: 307-334.
[4] Joshi V, Mathur ML, Dixit AK and Singhi M.
Entomological studies in a dengue endemic
area Jalore, Rajasthan. Indian J Med Res,
1996, 104: 161-165.
[2] Kalra NL, Kaul SM and Rastogi RM.
Prevalence of Aedes aegypti and Aedes
albopictus vectors of DF/DHF in north,
north-east and central India. Dengue
Bulletin, 1997, 21: 84-92.
[5] Tyagi BK. A note on the breeding of vector
mosquitoes in cement tanks and pit latrines.
J Appl Zool Res, 1994, 5: 149-151.
[3] Chouhan GS, Rodrigues FM, Sheikh BH,
Ilkal MA, Khangaro SS, Mathur KN, Joshi KR
and Vaidhye NK. Clinical and virological
study of dengue fever outbreak in Jalore city,
Rajasthan, 1985. Indian J Med Res, 1990,
91: 414-418.
222
Book Review 1
A Review of Entomological Sampling Methods and
Indicators for Dengue Vectors
Dana A. Focks
Infectious Disease Analysis, Gainsville, Florida, USA
TDR / IDE / Den / 03.1
This review was developed in response to a
recommendation of the WHO Informal
Consultation
on
Strengthening
Implementation of the Global Strategy for
Dengue Fever/ Dengue Haemorrhagic Fever
Prevention and Control, held in October of
1999, urging “the refinement of existing
entomological
indicators
and/or
the
development of new indicators that better
reflect
transmission
potential.”
The
Consultation “recommended that such
indicators should provide clear, meaningful
information for communities as well as for
programme managers and policy-makers.”
Whereas the traditional Stegomyia indices
(the House, Container, and Breteau indices,
and various related derivations) are of some
operational value for measuring the
entomological impact of larval control
interventions against the mosquito vectors of
dengue virus, they are not proxies for adult
vector abundance. Neither are they useful
for assessing transmission risk because they
do not take into consideration the
epidemiologically
important
variables,
including adult vector and human
abundance,
temperature,
and
seroconversion rates in the human population.
The document reviews and critiques
current methods, focusing especially on
sampling methods that provide information
on (1) the risk of transmission as a function
of vector abundance, and (2) the relative or
absolute importance of the various types of
containers in the environment. This second
aspect is essential when considering a
suppression strategy designed to minimize
costs or to improve sustainability by
targeting only a subset of the breeding
containers for control or elimination –
specifically those container types that are
responsible for the majority of adult
production. In reviewing current and
generally-used sampling methods, each is
discussed with respect to transmission risk
assessment and evaluated in terms of being
useful for either “research or special studies”
or as a practical operational tool providing
useful information for planning and
management of vector control programmes.
223
Book Review 2
DengueNet in India
Weekly Epidemiological Record, 2004, 79(21): 201-203
Epidemic dengue fever (DF) and dengue
haemorrhagic fever (DHF) have emerged as
a global public health problem in recent
decades. In fact, the problem has become
hyperendemic in many urban, periurban
and rural areas, with frequent epidemics.
The South-East Asia Region is one of the
regions at highest risk of DF/DHF,
accounting for 52% of the global risk.
Dengue outbreaks now occur in India, as in
other high-burden countries in the Region,
such as Indonesia, Myanmar and Thailand.
Following the pilot use of DengueNet in
the
Americas,
a
joint
WHO
HQ/SEARO/WPRO meeting on DengueNet
implementation in South-East Asia and the
Western Pacific was held in Kuala Lumpur
on 11-13 December 2003 2 . The objective
of the meeting was to expand the pilot
project to these two regions, building upon
the lessons learned from the pilot project in
the
Americas.
Based
on
the
recommendations of this meeting, two
country workshops were organized in India
in March 2004, supported by the
WHO/CSR and USAID project to strengthen
surveillance in India. The first took place in
New Delhi on 11-12 March 2004 with the
northern states and the second in Bangalore
on 16-17 March 2004, with the southern
states. WHO collaborating centres attended
both meetings. The proceedings and
recommendations from the New Delhi
meeting were discussed at the Bangalore
meeting to ensure that the consensus
recommendations addressed national issues,
needs and priorities. Experts from health
service departments of all states and the
Delhi City Corporation, the National
Institute for Communicable Diseases (NICD),
the National Institute of Virology (NIV) in
Pune,
the
WHO
Department
of
Communicable Disease Surveillance and
Response, WHO Representative for India
and SEARO participated.
1
2
Strengthening
epidemiological
and
laboratory surveillance of dengue and
dengue haemorrhagic fever including, the
implementation of DengueNet 1 , is one of
the priorities of the global and regional
strategies for dengue prevention and control.
DengueNet
is
WHO’s
global
data
management system created on the Internet
to collect and analyse standardized
epidemiological and laboratory surveillance
data with the objective to improve capacity
for effective national and international
planning for the prevention and control of
epidemic dengue and DHF.
1 See No. 36, 2002, pp. 300-304.
224
See No. 6, 2004, pp. 57-62.
DengueNet in India
The main objective of the workshops
was to strengthen disease surveillance and
response to vector-borne diseases using
DengueNet as an entry point. Work focused
specifically on assessing current surveillance
practices (including the use of case
definitions,
reporting
formats
and
mechanisms for flow of information),
laboratory facilities and tests for DHF, on
identifying and strengthening regional
collaborative laboratories and on establishing
a framework for participation in DengueNet.
Experiences both from India and from
the region on surveillance and control were
discussed. The need for an integrated
approach to surveillance of vector-borne
diseases and application of lessons and
experiences from malaria and other vectorborne diseases was identified. The
consensus was to implement DengueNet in
accordance with the Integrated Disease
Surveillance Programme (IDSP) that is
starting in India. This would require capacity
building for disease surveillance and
response at national, state and district levels,
through training of health workers and
programme managers and strengthening of
laboratory services.
Given the vastness of the country, the
heterogeneity of the disease burden and the
organizational
network,
the
group
recommended piloting the participation of
selected states in global DengueNet through
focal points at state and national level.
States in which implementation would be
piloted
included
Delhi,
Karnataka,
Maharashtra, Tamil Nadu and Uttaranchal.
Maharashtra and Tamil Nadu have a well
developed disease surveillance system and a
network of public health laboratories at both
district and state level, with strong linkages
to
national-level
laboratories.
Delhi,
following the recent dengue outbreak, has
significantly improved its surveillance system,
including strengthening of laboratory
services. Uttaranchal, although lacking a
good network of laboratories, was included
because of its close proximity to Delhi.
It was also decided to designate two
WHO laboratory collaborating centres
(WHO CC) for northern and southern states
to ensure practical use of these facilities.
Accordingly, NIV-Pune will continue as
WHO CC for the south, and it is
recommended that WHO designate NICD
to serve as WHO CC for the northern states
(following a request from NICD). The two
centres would be responsible for training,
quality control, use of standard procedures
and networking at national and international
level.
Based on the recommendations of the
workshops, a follow-up meeting has been
planned to develop an activity plan for 2004
focusing on laboratory strengthening,
training, disease and vector surveillance,
networking,
information
sharing
and
reporting to DengueNet. NICD will take the
lead role in this follow-up activity, which is
scheduled for the end of May 2004,
working closely with all major stakeholders 3.
3
Major stakeholders include the Indian Council for
Medical Research, IDSP Cell, Delhi City Corporation,
NICD – WHO CC for Training and Epidemiology,
WHO CC for Rabies Epidemiology, WHO Regional
Reference Laboratory for Polio Surveillance, National
Reference Laboratory for SARS and Avian Influenza,
state nodal officers of the pilot states and states that
were not represented in the earlier meetings, national level institutes such as the Central Bureau of Health
Intelligence, Vector Control and Research Centre –
WHO CC for Filariasis and Vector Control, National
Vector Borne Disease Control Programme and NIV –
WHO CC for Arboviral Disease Diagnosis and Research
and National Influenza Surveillance Centre.
225
Book Review 3
WHO/WPRO/SEARO Meeting on DengueNet
Implementation in South-East Asia and the
Western Pacific, Kuala Lumpur, 11-13 December 2003
Weekly Epidemiological Record, 2004, 79(6): 57-62
Dengue/DHF – Global public
health problem
Epidemic dengue fever and dengue
haemorrhagic fever (DHF) have emerged as a
global public health problem in recent
decades,
with
the
development
of
hyperendemicity in urban and peri-urban
centres of many tropical and subtropical
countries. Asia-Pacific countries have more
than 70 % of the disease burden; in several
of them, DHF has become a leading cause of
hospitalization and death among children.
Latin America and the Caribbean appear to
be following the same DHF epidemic trend,
with the disease affecting all ages and casefatality rates as high as 10-15 % in areas with
limited health service infrastructure. The
African and Eastern Mediterranean regions
are much less affected. Air travel is also
facilitating the rapid global movement of
dengue viruses and increasing the risk of
DHF epidemics through the introduction of
new serotypes. Globally, 2.5 billion people
live in areas where dengue viruses can be
transmitted: an estimated 50 million dengue
infections occur each year, with 500,000
cases of DHF and at least 22,000 deaths,
mainly among children. Although dengue is a
notifiable disease in many endemic countries,
only a small proportion of cases are reported
to WHO.
226
Rationale for DengueNet1
DengueNet, WHO’s global surveillance
system for dengue fever and DHF, has been
created as a web-based central data
management system to collect and analyse
standardized epidemiological and virological
data in a timely manner and to present
epidemiological trends as soon as new data
are entered. Strengthening epidemiological
and virological surveillance of dengue and
DHF,
including
implementation
of
DengueNet, for early detection, planning and
response is one of the four main priorities of
WHO’s global prevention and control
strategy, adopted in resolution WHA55.17 in
May 2002. DengueNet, when fully
implemented, will facilitate WHO’s global
outbreak and response activities and support
the GOARN2.
Epidemiological and laboratory-based
surveillance is required to monitor and guide
dengue/DHF
prevention
and
control
programmes whether these are based on
vector control or possible future vaccination
with a safe, effective and affordable vaccine.
Recent
and
encouraging
research
developments have made it likely that a
dengue vaccine will become available. As a
consequence, the public health community
1
See No. 36, 2002, pp. 300-304.
2
Global Outbreak Alert and Response Network.
DengueNet Implementation in South-East Asia and the Western Pacific
needs to define the burden of dengue for
society, so that adequate cost-benefit
analyses can be presented to government
leaders before they decide to use the vaccine.
Standardized global dengue surveillance data,
one of the principal results expected from the
establishment of DengueNet, have become
critical.
Phased implementation of
DengueNet
First meeting in San Juan,
Puerto Rico, July 2002
The first DengueNet meeting was held jointly
with
WHO/PAHO
and
the
WHO
collaborating centre for dengue/DHF at the
Centers for Disease Control and Prevention
(CDC Dengue branch; San Juan, Puerto Rico).
Its objective was to describe and demonstrate
DengueNet to prospective users and to
launch a pilot, building on the existing
reporting systems and network of dengue
laboratories in the Americas. Epidemiologists
and virologists from eight countries in the
Americas, three countries in Asia and five
WHO
collaborating
centres
provided
recommendations for the administrative and
technical procedures involved in making
DengueNet operational.
Second meeting in Kuala Lumpur,
Malaysia, December 20033
representing 20 island countries in the
Americas, and after changes to the
supporting computer hardware, software,
and routines, a second meeting was
convened jointly with WHO/WPRO/SEARO
and the WHO collaborating centre for
dengue/DHF at the University of Malaya in
Kuala Lumpur, 11-13 December 2003. The
objective was to expand the pilot to SouthEast Asia and the Western Pacific, building on
the lessons learned from the pilot conducted
in the Americas. About 70 participants
included national epidemiologists, laboratory
specialists, and clinicians from 19 Asia-Pacific
countries, three countries in the Americas, six
WHO collaborating centres, and WHO HQ,
regional and country staff4.
The
plenary
presentations
and
discussions focused on: (1) the challenges
and
need
for
standardized
global
epidemiological and laboratory surveillance
of dengue and DHF; (2) the activities of the
Pediatric Dengue Vaccine Initiative (PDVI);
(3) the national surveillance and reporting
systems in the participating countries in
South-East Asia and the Western Pacific;
(4) the activities of the participating WHO
collaborating centres; (5) the WHO global
4
South-East Asian and Western Pacific regions:
national programmes from Bangladesh,
Cambodia, China, Fiji, French Polynesia, India,
Indonesia, Lao People’s Democratic Republic,
Maldives, Malaysia, Myanmar, Nepal, Philippines,
Sri Lanka, Singapore, Thailand, Viet Nam; the biregional Mekong Basin Disease Surveillance
Network; WHO collaborating centres and
research institutes in Australia, India, Japan,
Malaysia, Thailand.
§
Americas: DengueNet pilot country Brazil; WHO
collaborating centres in Cuba and USA; interim
director of the Dengue Pediatric Vaccine Initiative
(PDVI).
§
WHO: HQ; regional offices (PAHO, SEARO,
WPRO); country offices (India, Malaysia).
After pilot use of DengueNet by four
Member
States
and
one
network
3
This meeting was organized by the WHO Department
of Communicable Disease Surveillance and Response,
Global Alert and Response, jointly with the WHO
Regional Offices for South-East Asia and the Western
Pacific, and the WHO Collaborating Centre for
dengue/DHF at the University of Malaya in Kuala
Lumpur, Malaysia, with technical and financial support
from the US Centers for Disease Prevention and Control.
Participants included:
§
227
strategy
and
regional
programmes;
(6) WHO’s global outbreak and response
activities and GOARN; (7) the DengueNet
pilot and lessons learned; (8) presentation of
DengueNet and a “hands on” session with
the “new” prototype web site in Global Atlas.
•
to provide early warning of potential
outbreaks of dengue disease or of the
introduction of dengue viruses into
epidemiologically silent areas, for the
purpose of implementing timely control
measures and notifying decision-makers
in institutions whose occupations or
livelihood may be affected;
•
to
strengthen
and
standardize
epidemiological surveillance of DF and
DHF;
•
to promote the use of standardized
clinical case definitions and reporting
criteria for dengue illnesses, permitting
comparisons between countries and
over time;
•
to
strengthen
the
network
of
collaborating centres and national
laboratories for serotype determination
and strain characterization;
•
to promote improvement in the quality
of laboratory data reported at national
and international levels;
•
to provide a standardized database for
epidemiological research and analysis;
•
to provide data useful for estimating the
burden of disease (including the social
and economic burden) on a national,
regional, or global scale;
•
to support the improvement in national
and international alert and response
capacity for dengue/DHF outbreaks;
and
•
to promote the free and timely exchange
of epidemiological information between
affected countries, their neighbours, and
other stakeholders in order to facilitate
and promote dengue control activities
within the region.
Two working groups were convened. The
first reviewed and defined the epidemiological
data and reporting requirements for
DengueNet, modifications needed to the
present format, identification of countries
for expanding the DengueNet pilot to AsiaPacific regions, and roles and responsibilities
of national and international partners. A
subgroup also reviewed and defined the
objectives of DengueNet. The second
working group reviewed the existing
laboratory capacity in South-East Asian and
Western Pacific countries in relation to
DengueNet, identifying the current needs
(and gaps) for laboratory standards, quality
control, and dengue serological diagnosis
and virus isolation, as well as for reporting
and information exchange. The group made
recommendations
that
focused
on
strengthening regional dengue laboratory
diagnosis capacity, so that laboratories
participating in DengueNet are able to
report data of the highest quality possible
within their working environment.
Meeting outcomes
Objectives of DengueNet
The participants agreed that the overall
objective of DengueNet is to improve
capacity for effective national and
international planning for the prevention
and control of dengue and that the specific
objectives for implementing this global
surveillance system are:
228
Recommendations of the
laboratory working group
•
To strengthen the regional dengue
laboratory
diagnosis
capacity,
the
participants of this group made the
following recommendations for national
laboratories, WHO collaborating centres,
WHO, and government health authorities.
Quality control
•
•
•
Quality
control/proficiency
testing
should be undertaken by the national
laboratory/WHO collaborating centre
for other laboratories in the country
concerned.
A
reference
centre
should
be
established at the WHO Collaborating
Centre for Tropical Viral Diseases,
Nagasaki,
Japan,
to
undertake
coordination of quality assurance/
control for other WHO collaborating
centers
and
designated
national
laboratories in the two Regions.
The Nagasaki reference centre should
coordinate, organize, and distribute a
WHO panel of reference sera for
validation of tests/kits/rapid tests by
WHO, national laboratories, and WHO
collaborating centres.
Reference services
•
Countries that do not have facilities for
virus isolation should send appropriate
samples to a WHO collaborating centre
of their choice after consultation with
that centre.
•
WHO should recommend capacitybuilding for virus isolation to the
ministries of health of countries that
lack facilities.
In collaboration with WHO country and
regional offices, WHO collaborating
centres should provide reference
reagents to national laboratories –
standard
inactivated
antigens,
monoclonal antibodies, standard sera,
cell lines for virus isolation, and
prototype dengue virus strains.
Laboratory training
•
WHO should organize regional training
courses on laboratory diagnosis of
dengue and other flaviviruses.
•
WHO should develop a laboratory
manual for dengue diagnosis.
•
WHO HQ should establish a global
technical advisory group, including
representatives
from
collaborating
centres, to meet annually to advance
laboratory training, capacity building,
reagents, quality issues, and DengueNet.
Reporting and information exchange
With regard to collection of laboratory data
and information transfer, the group
identified a strong need for government
health authorities to develop a reporting
system
to
collect,
centralize,
and
disseminate these data, identify key
laboratories to participate in this system, and
designate a focal point for DengueNet.
The group recommended that WHO
support national health ministries to assess
current laboratory status in Asia-Pacific
countries and to plan mechanisms to
strengthen laboratories for DengueNet. A
draft DengueNet laboratory assessment tool
is available for review and use.
The group expressed appreciation of
the efforts made to develop DengueNet and
229
recommended that WHO work with
partners to develop strategies for raising
crucial resources.
-
Recommendations of the
epidemiology working group
Rate calculations
The group reviewed currently available data
and reporting practices in the Asia-Pacific
countries in relation to DengueNet. The
discussion was organized around the
principal epidemiological variables of time,
place and personal characteristics, plus
information about the virus. The group
made
recommendations
on
the
modifications to be made to the present
format of the DengueNet prototype in
Global
Atlas,
on
strengthening
epidemiological surveillance, and on a
framework for implementation of the
DengueNet in Asia-Pacific regions with
emphasis on the quality of available data
and the active participation of national
programmes.
Data collection
-
Both incidence and mortality rates
should be expressed per 100 000
mid-year population; countries
should provide updates to
DengueNet in the event of
significant change.
-
The system should not show
incidence and mortality rates for
countries that report data only from
sentinel sites.
Virus serotype data – all available
-
Data should be entered (when
provided by the laboratory) as the
cumulative number of isolations of
each serotype in the country from 1
January.
General recommendations
Epidemiological data
For countries
-
Countries should promote implementation
of the WHOrecommended surveillance
standards for dengue. (Participants were
provided with copies of these standards.)
-
230
Countries should provide annual
epidemiological data by sex and
age groups.
To accommodate currently available
case classification reporting practices,
countries should provide three
categories – DF cases, DHF/DSS
(dengue shock syndrome) cases,
total dengue cases (DF/DHF/DSS).
These data should be provided
monthly, at state/department level
by large countries and at island
level by island nations.
Countries should provide data,
monthly when available, on
“dengue deaths” (probable or
confirmed).
For DengueNet
A country information page should be
provided on the DengueNet web site for all
country-specific information, definitions,
and methods used (e.g. sentinel site
information, reporting by time of onset or
notification, case classification other than
according to the WHO definition, etc.).
WHO-recommended
surveillance
standards should be made available on the
DengueNet web site.
Roles and responsibilities of
the partners in this network
Countries will collect, validate, and provide
epidemiological and laboratory data. They
will designate the participating centres and
focal points, and WHO country offices will
facilitate the process. WHO collaborating
centres will provide laboratory support,
proficiency panels, and training to national
laboratories. WHO regional offices will
implement the country support activities,
and WHO HQ will maintain and moderate
the DengueNet web site. WHO regional
offices and HQ will seek financial support
for dengue surveillance activities.
Country participation
A major outcome of the meeting was that
representatives of all participating countries
showed interest in collaborating with
DengueNet and agreed to present the
meeting’s recommendations to their health
ministries. Country participation will require
a letter of request from WHO to the
ministry of health; ministry authorization for
participation and designation of a national
DengueNet focal point; and, for some
countries, an external budget.
The DengueNet pilot will be expanded
to countries in American, South-East Asian,
and Western Pacific regions in 2004 after
modifications to the system have been
made in Global Atlas. The lessons learned
from the pilot will be used to develop a
consensus framework for DengueNet
implementation for standardized global
surveillance of dengue and DHF.
231
Instructions for Contributors
The Dengue Bulletin welcomes all original research papers, short notes, review articles, letters
to the Editor and book reviews which have a direct or indirect bearing on dengue
fever/dengue haemorrhagic fever prevention and control, including case management. Papers
should not contain any political statement or reference.
Manuscripts should be typewritten in English in double space on one side of white A4
size paper, with a margin of at least one inch on either side of the text and should not exceed
15 pages. The title should be as short as possible. The name of the author(s) should appear
after the title, followed by the name of institution and complete address. E-mail address of the
corresponding author should also be included.
References to published works should be listed on a separate page at the end of the
paper. References to periodicals should include the following elements: name and initials of
author(s); title of paper or book in its original language; complete name of the journal,
publishing house, or institution concerned; volume and issue number, relevant pages and date
of publication, and place of publication (city and country). References should appear in the
text in the same numerical order (Arabic numbers in parenthesis) as at the end of the article.
For example:
(1) Nimmannitaya S. Clinical spectrum and management of dengue haemorrhagic
fever. The Proceedings of the International Conference on Dengue Haemorrhagic
Fever, Kuala Lumpur, September 1-3, 1983:16-26.
(2) Gubler DJ. Dengue and dengue haemorrhagic fever: Its history and resurgence as
a global public health problem. In: Gubler DJ, Kuno G (ed.), Dengue and dengue
haemorrhagic fever. CAB International, New York, NY, 1997, 1-22.
(3) Nguyen Trong Lan, Nguyen Thanh Hung, Do Quang Ha, Bui Thi Mai Phuong, Le
Bich Lien, Luong Anh Tuan, Vu Thi Que Huong, Lu Thi Minh Hieu, Tieu Ngoc
Tran, Le Thi Cam and Nguyen Anh Tuan. Treatment of dengue haemorrhagic
fever at Children's Hospital N.1, Ho Chi Minh City, 1991-1996. Dengue Bulletin,
1997, 22: 150-161.
Figures and tables (Arabic numerals), with appropriate captions and titles, should be
included on separate pages, numbered consecutively, and included at the end of the text with
instructions as to where they belong. Abbreviations should be avoided or explained. Graphs or
figures should be clearly drawn and properly labeled, preferably using MS Excel, and all data
clearly identified.
232
Instructions for Contributors
Articles should include a self-explanatory abstract at the beginning of the paper of not
more than 300 words explaining the need/gap in knowledge and stating very briefly the area
and period of study. The outcome of the research should be complete, concise and focused
conveying the conclusions in totality. Appropriate keywords and a running title should also be
provided.
Articles submitted for publication should be accompanied by a statement that they
have not already been published, and, if accepted for publication in the Bulletin, will not be
submitted for publication elsewhere without the agreement of' WHO, and that the right of
republication in any form is reserved by the WHO Regional Offices for South-East Asia
(SEARO) and the Western Pacific (WPRO).
One hard copy, original and clear figures/tables and a computer diskette indicating
the name of the software, of the manuscript should be submitted to:
The Editor
Dengue Bulletin
WHO Regional Office for South-East Asia
Mahatma Gandhi Road
New Delhi 110002
India
Telephone: 91-11-23370804
Fax: 91-11-23379507, 23370972
Manuscripts received for publication are subjected to in-house review by a
professional expert and are peer-reviewed by experts in the respective disciplines. Papers are
accepted on the understanding that they are subject to editorial revision, including, where
necessary, condensation of the text and omission of tabular and illustrative material.
Original copies of articles will not be returned. The principal author will receive 10
reprints of his article published in the Bulletin. A PDF file can be supplied on request.
233

WHO/Searo Dengue Bulletin – Vol 28, December 2004

Transcription

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