5. Muniandy Narasiman

APACPH Conference, Bandung, Indonesia 21-23 Oct, 2015
1Muniandy
Narasiman, 1 L. Banusanthar, 1B. Sinnaiah & 2Evie Khoo.
1Universiti
Kuala Lumpur-Royal College of Medicine Perak
2 Veterinary Research Institute, Ipoh, Perak



One of the common re-emerging zoonotic disease
(Demam Kenching Tikus).
Common public health problem - Worldwide.

Annual incidence > 10 per 100,000 per year in the
humid tropics .

Estimated case-fatality rates

Incidence rate in Malaysia= 2 – 7 per 100,000
p p
populations.

More than 90 % of cases reported were sporadic and
occurred throughout the country
country.
= 5% - 30%

Infection caused by pathogenic leptospires - Leptospira
interrogans species.

37 serovars found from wildlife and humans in Malaysia.

Th serovars are divided
The
di id d iinto
t 13 serogroups:

The 13 serogroups : Australis, Autumnalis , Bataviae,
C i l Celledoni,
Canicola,
C ll d i Grippotyphosa,
Gi
h
H bd
Hebdomadis,
di
Icterohaemorrhagiae, Javanica, Pomona, Pyrogenes, Sejroe
and Tarassovi.

Rodents are the main source of spirochetes that are
transmitted to humans .

Humans are known as “dead-end” hosts.
3
Transmission of the organism …………

Transmission of bacteria can be direct or indirect.

Commonly transmitted through urine of carrier animal.

Leptospira enter the body through cuts, wounds, and
mucous membranes (eyes, mouth and nose).

Ingestion of contaminated drinking water and food may
also cause infection.

Rats are the p
principal
p maintenance host of leptospires
p p
in
Malaysia.

Cattle – suspected
p
maintenance host for serovar hardjo
j

Pigs – suspected maintenance host for serovar pomona.
4
Life cycle of Leptospira species
5
Cases of leptospirosis in MOH Hospitals
Year
Cases
Deaths
2004
263
20
2005
378
20
2006
527
22
2007
1418
62
2008
1263
47
2009
1418
62
2011
2268
55
2012
3665
48
2013
4457
71
2014
7806
92
2015
5370
30
Total
28,833
529 CFR= 1.83 %
Rats are reservoirs of leptospirosis and other vector borne diseases.
66
Newspapers Report Continuously 1995…2009, 2010….
Government Acts
Leptospirosis is a notifiable disease in Malaysia under the
Prevention and Control of Communicable Diseases Act 1988
since 2010.
&
DISEASE CONTROL DIVISION
DEPARTMENT OF PUBLIC HEALTH , MINISTRY OF HEALTH MALAYSIA
Produced
GUIDELINES FOR THE DIAGNOSIS,
MANAGEMENT, PREVENTION AND
CONTROL OF LEPTOSPIROSIS IN MALAYSIA
2011
9
GUIDELINES FOR THE DIAGNOSIS,
MANAGEMENT,, PREVENTION AND
CONTROL OF LEPTOSPIROSIS IN
MALAYSIA
Published By:
DISEASE CONTROL DIVISION
DEPARTMENT OF PUBLIC HEALTH , MINISTRY OF HEALTH
MALAYSIA
st
1 Edition - 2011
10
8th October 2013
L t
Leptospirosis
i i C
Cases & D
Deaths
th on th
the Ri
Rise
Kelantan, Selangor & Perak States

Manyy recent deaths ((2013 & 2014)) related to
leptospiral infections in Water Recreational
Spots and

National Service Training
g Camps
p have been
reported in Pahang, Perak and Kedah.
1. To determine if rats in urban areas in Ipoh
harbour pathogenic Leptospira (2014)
2. To determine if pathogenic serovars are still
circulating
i l ti iin National
N ti
lS
Service
i T
Training
i i C
Camps &
Recreational Water Spots (rural) in Perak. (2015)
Methodology
gy
Period
Feb-Oct
2014
Feb- Sept
2015
Location Type
Rats
Water
Soil
95
-
-
58
55
24
Urban
(Houses)
11
-
-
Total
164
55
24
Urban (Ipoh)
(Alleys behind
Wet markets,
Restaurants )
Rural (Perak)
(National Service
Training Camps,
Stagnant Water
Spots)
PLKN- National
Service Training
Camps (7 Camps)
WF- Waterfall
Areas ( 10 Spots)
p )
Urban ( 3 Spots)
METHODOLOGY
Rat species examined:R
Rattus
rattus diardii
di dii (common
(
house
h
rat))
Rattus norvegicus (brown rat)
Suncus murinus ((house shrew))
Bait: cheese or peanut –butter on a slice of bread
or Salted-dried
Salted dried fish.
fish
• Traps: 30 x 22 x 50 cms
19
MethodologyMethodology
- Necropsy of rats

Trapped rats were anaesthesised to moribund stage
with chloroform.

Thoracic cavity cut-opened.

Blood was withdrawn with cardiac puncture using 5 ml
(23G x 11.4 syringe) syringe.

Organs isolated: Spleen, kidneys and bladder.
20
Laboratory Procedures

Kidneys were macerated with pestle and mortar
100 µl of suspension inoculated into EMJH
semisolid medium,
Kept at 28-30 oC. 4-8 weeks, dark
Observed in dark field microscope.
p

Bladder samples
p
with reminiscent urine were
inoculated into Bijou bottles with EMJH medium for
Leptospira growth.

4 drops of blood from cardiac puncture into 4.0 ml
EMJH medium.
di
21
PCR for Leptospira on EMJH inoculates
8 weeks Cultures (Kidney, Bladder, Blood)
Extract DNA using
g innuPREP DNA Mini kit ((Analytik
y JJena))
Bioline Red Mix 2X = 10µl
16S rRNA gene Forward Primer 0.5µl
16S rRNA gene Reverse Primer 0.5µl
RNase free water 13µl
DNA template and 1µl
Final PCR master mix of 25µl
( LipL32 gene Forward and Reverse Primers)
Nexus Gradients PCR thermocycler
Initial denaturation 940C 10 min
Denaturation 940C 1.0 min
Annealing 550C 1.0 min
Extension 720C 1.0 min
Final Extention 720C 10 min
Cycles: 40
Electrophoresis
El
t
h
i off 3µl
3 l off each
h reaction
ti
1.2% agarose gel with 1000bp ladder 30min at 100V.
22
Isolation of Leptospira Species (2015)
No. Positive/ Sample
p Tested
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
U1
U2
U3
Location
PLKN Sentosa , Tapah
PLKN Terkok
PLKN Desa Rimba
PLKN Jiwa Murni
Rats
0/ 2
0/ 3
0/1
0/4
PLKN Kepimpinan Gemilang
PLKN Nilam Ehsan
PLKN Sinaran Suria
Lata Kinjang WF, Tapah
H t Lipur
Hutan
Li
WF Papan
WF,
P
Ayer Hitam WF
Lata Panggung WF
Lata Kebabu WF
Lata Ulu Chepor WF
Lubuk Timah WF
Bukit Jana WF
Gepai WF
Royal Belum Forest
Ipoh
Pasir Puteh
Chemor
Total (148)
0/10
0/3
0/4
0/5
0/4
0/2
0/2
0/12
0/1
0/2
0/3
0/5
0/5
0/1
0/69
Water
5/15
Soil
4/9
0/2
0/2
2/6
3/6
0/5
2/7
0/2
0/2
0/2
0/2
0/2
0/2
0/6
0/2
0/2
0/5
12/55
4/24
ResultsResults
- Positives/ Samples Analysed
Period
FebO t
Oct
2014
FebSept
2015
Location Type
Urban (Ipoh)
(Alleys behind
Wet markets,,
Restaurants )
Rural (Perak)
(National Service
Training Camps,
Stagnant Water
Spots)
Urban
(House)
Positive
Rats
Water
Soil
0/95
-
-
0/58
12/55
4/24
0/11
-
-
0
22 %
17 %
430bp 16SrRNA
279 bp Lip32
Pathogenic strains detecteddetected Highlighted

22 % of water samples and 17 % of soil samples tested
Positive for Leptospira spp.

13/16 isolated were confirmed by PCR as Nonpathogenic
(Preliminary).

4 Pathogenic isolates were confirmed from 2 locations by
PCR.

Leptospira was not isolated from the 106 urban rats and 58
off rurall rats
t captured.
t d

Thus,, Both p
pathogenic
g
and Nonpathogenic
p
g
serovars are
still circulating in National Service Training Camps and
Recreational Water Spots (rural) in Perak.

This poses a Public Health Problem. Cases/Deaths are
increasing despite Efforts by Local Govt Authorities.

More rats, water and soil samples to be
collected from other districts in Perak.

Serotype the isolates, esp Pathogenic spp. by
Microscopic Agglutination Test (MAT).
(MAT)
Preventive and Control Measures

Public Awareness- Alert public or users
regarding
di th
the h
hazards
d off possible
ibl
contaminated areas.

Strict enforcement to restrict Youths from
bathing in contaminated stagment pools.
pools
Youths should not ignore Danger Notices
placed at Waterspot &Waterfall regions

34
What is the intention of Health Authorities here?
1.
Universiti Kuala Lumpur for providing STRG
Grant No.12016. (2014)
2. MARA Research and Inovation Grant (SGPIM)
(SKIM GERAN PENYELIDIKAN & INOVASI MARA)
(2015).
3. UniKL-RCMP and VRI for facilities