1 - Instituto de Medicina Tropical de São Paulo

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Established: 1959.
The year 2015 is the 57th anniversary
of continuous publication
ISSN0036-4665
ISSN 1678-9946 on line
EDITOR‑IN‑CHIEF
Prof. Dr. Thales F. de Brito Associate Editors:Prof. Dr. Pedro Paulo Chieffi Prof. Dr. Thelma S. Okay
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UNIVERSIDADE DE SÃO PAULO - BRAZIL
FACULDADE DE MEDICINA
Instituto de Medicina Tropical de São Paulo
Director: Prof. Dr. Paulo C. Cotrim
I
The purpose of the “Revista do Instituto de Medicina Tropical de São Paulo” (Journal of the
São Paulo Institute of Tropical Medicine) is to publish the results of researches which contribute significantly to knowledge of all transmissible diseases.
REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO
(JOURNAL OF THE S. PAULO INSTITUTE OF TROPICAL MEDICINE).
São Paulo, SP-Brasil, 1959 v. ilust. 28 cm
1959-2014, 1-56
1973-2002 (supl. 1-12)
2003 (supl. 13 - on-line only)
2005-2012 (supl. 14-18)
2015, 57 (1)
ISSN 0036-4665
II
Impact Factor: 0.907
5-year Impact Factor: 1.213
ISSN0036-4665
Rev. Inst. Med. Trop. Sao Paulo
Vol. 57
No. 1
P. 1-92
January-February, 2015
CONTENTS
REVIEW
RABIES
1
63 Bat-borne rabies in Latin America
Origin and prevalence of human T-lymphotropic virus type 1 (HTLV1) and type 2 (HTLV-2) among indigenous populations in Americas
A. PAIVA & J. CASSEB
L.E. ESCOBAR, A.T. PETERSON, M. FAVI, V. YUNG & G. MEDINA-VOGEL
PARASITOLOGY
15 Leprosy nephropathy: a review of clinical and histopathological
features
73 Assessment of the presence of Toxocara eggs in soils of an arid
area in Central-Western Argentina
MICROBIOLOGY
BRIEF COMMUNICATION
G.B SILVA JUNIOR, E.F. DAHER, R.J. PIRES NETO, E.D.B. PEREIRA, G.C.
MENESES, S.M.H.A. ARAÚJO & E.J.G. BARROS
M.V. BOJANICH, J.M. ALONSO, N.A. CARABALLO, M.I. SCHÖLLER, M.A.
LÓPEZ, L.M. GARCÍA & J.A. BASUALDO
21 Survival, induction and resuscitation of Vibrio cholerae from the
viable but nonculturable state in the Southern Caribbean Sea
77 Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis
27 Enteropathogens detected in a daycare center, Southeastern Brazil:
bacteria, virus, and parasite research
81 Identification of Pseudomonas spp. as an amoeba-resistant microorganism in isolates of Acanthamoeba
M. FERNÁNDEZ-DELGADO, M.A. GARCÍA-AMADO, M. CONTRERAS, R.N.
INCANI, H. CHIRINOS, H. ROJAS & P. SUÁREZ
E.D.R. CASTRO, M.C.B.Y. GERMINI, J.D.P. MASCARENHAS, Y.B. GABBAY,
I.C.G. LIMA, P.S. LOBO, V.D. FRAGA, L.M. CONCEIÇÃO, R.L.D. MACHADO
& A.R.B. ROSSIT
LEISHMANIASIS
33 Historical series of patients with visceral leishmaniasis treated
with meglumine antimoniate in a hospital for Tropical Diseases,
Maceió-AL, Brazil
L.J.D. SILVEIRA, T.J.M. ROCHA, S.A. RIBEIRO & C.M.S. PEDROSA
EPIDEMIOLOGY
39 Study of the prevalence of Capillaria hepatica in humans and rodents in an urban area of the city of Porto Velho, Rondônia, Brazil
E.J.G. ROCHA, S.A. BASANO, M.M. SOUZA, E.R. HONDA, M.B. CASTRO, E.M.
COLODEL, J.C.D. SILVA, L.P. BARROS, E.S. RODRIGUES & L.M.A. CAMARGO
47 Seropositivity for ascariosis and toxocariosis and cytokine expression among the indigenous people in the Venezuelan Delta region
Z. ARAUJO, S. BRANDES, E. PINELLI, M.A. BOCHICHIO, A. PALACIOS, A.
WIDE, B. RIVAS-SANTIAGO & J.C. JIMÉNEZ
MYCOLOGY
57 Toll-like receptors (TLR) 2 and 4 expression of keratinocytes from
patients with localized and disseminated dermatophytosis
M.A. CORRAL, F.M. PAULA, M. GOTTARDI, D.M.C.L. MEISEL, P.P. CHIEFFI &
R.C.B. GRYSCHEK
V.J. MASCHIO, G. CORÇÃO & M.B. ROTT
85 Elevated trans-mammary transmission of Toxocara canis larvae in
BALB/c mice
P.L. TELMO, L.F.C. AVILA, C.A. SANTOS, P.S. AGUIAR, L.H.R. MARTINS, M.E.A.
BERNE & C.J. SCAINI
CORRESPONDENCE
88 Neurocysticercosis and afebrile seizure
V. WIWANITKIT
LETTER TO THE EDITOR
89 Loose and compact agglomerates of 50 nm microvesicles derived
from Golgi and endoplasmic reticulum membranes in pre- and inapoptotic mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope
A. SESSO, E.H. YAMASHIRO-KANASHIRO, N.M. ORII, N.N. TANIWAKI, J.
KAWAKAMI & S.M. CARNEIRO
92 Influenza virus surveillance by the Institute Adolfo Lutz, influenza
season 2014: antiviral resistance
K.C.O. SANTOS, D.B.B. SILVA, M.A. BENEGA, R.S. PAULINO, E.R.E. SILVA Jr.,
D.S.PEREIRA, A.D.H. MUSSI, V.C. SILVA, L.V. GUBAREVA & T.M. PAIVA
C.B. OLIVEIRA, C. VASCONCELLOS, N.Y. SAKAI-VALENTE, M.N. SOTTO, F.G.
LUIZ, W. BELDA JÚNIOR, M.G.T. SOUSA, G. BENARD & P.R. CRIADO
ADDRESS
INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO
Av. Dr. Enéas de Carvalho Aguiar, 470
05403-000 São Paulo, SP - Brazil
Phone/Fax: 55.11.3062.2174; 55.11.3061-7005
e-mail: [email protected]
SUBSCRIPTIONS
FOREIGN COUNTRIES
One year (six issues)......... U$200.00
Single issue....................... U$50.00
III
Impact Factor: 0.907
5-year Impact Factor: 1.213
ISSN0036-4665
Vol. 57
No. 1
P. 1-92
Janeiro-Fevereiro, 2015
CONTEÚDO
REVISÃO
RAIVA
1
63 Rabia transmitida por murciélagos en Latino América
Origem e prevalência do vírus linfotrópico de células T humanas
em populações indígenas das Américas
A. PAIVA & J. CASSEB
L.E. ESCOBAR, A.T. PETERSON, M. FAVI, V. YUNG & G. MEDINA-VOGEL
PARASITOLOGIA
15 Nefropatia da hanseníase: revisão dos aspectos clínicos e histopatológicos
73 Evaluación de la presencia de huevos de Toxocara en suelos de una
zona árida en la región centro-oeste Argentina
MICROBIOLOGIA
COMUNICAÇÃO BREVE
G.B SILVA JUNIOR, E.F. DAHER, R.J. PIRES NETO, E.D.B. PEREIRA, G.C.
MENESES, S.M.H.A. ARAÚJO & E.J.G. BARROS
M.V. BOJANICH, J.M. ALONSO, N.A. CARABALLO, M.I. SCHÖLLER, M.A.
LÓPEZ, L.M. GARCÍA & J.A. BASUALDO
21 Supervivencia, inducción y resucitación de Vibrio cholerae del
estado viable no cultivable en el sur del Mar Caribe
77 Frações de membrana de Strongyloides venezuelensis para o imunodiagnóstico da estrongiloidíase humana
27 Enteropatógenos detectados em crianças de creche no Sudeste do
Brasil: pesquisa de bactérias, vírus e parasitos
81 Identificação de Pseudomonas spp. como microrganismo resistente
a ameba em isolados de Acanthamoeba
M. FERNÁNDEZ-DELGADO, M.A. GARCÍA-AMADO, M. CONTRERAS, R.N.
INCANI, H. CHIRINOS, H. ROJAS & P. SUÁREZ
E.D.R. CASTRO, M.C.B.Y. GERMINI, J.D.P. MASCARENHAS, Y.B. GABBAY,
I.C.G. LIMA, P.S. LOBO, V.D. FRAGA, L.M. CONCEIÇÃO, R.L.D. MACHADO
& A.R.B. ROSSIT
LEISHMANIOSE
33 Série histórica dos pacientes com leishmaniose visceral tratados com
antimoniato de meglumina em um hospital de Doenças Tropicais,
Maceió-AL, Brasil
L.J.D. SILVEIRA, T.J.M. ROCHA, S.A. RIBEIRO & C.M.S. PEDROSA
EPIDEMIOLOGIA
39 Estudo da prevalência da Capillaria hepatica em humanos e roedores em área urbana da cidade de Porto Velho, Rondônia, Brasil
E.J.G. ROCHA, S.A. BASANO, M.M. SOUZA, E.R. HONDA, M.B. CASTRO, E.M.
COLODEL, J.C.D. SILVA, L.P. BARROS, E.S. RODRIGUES & L.M.A. CAMARGO
47 Seropositividad para ascariosis y toxocariosis y expresión de citocinas entre la población indígena de la región del delta Venezolano
Z. ARAUJO, S. BRANDES, E. PINELLI, M.A. BOCHICHIO, A. PALACIOS, A.
WIDE, B. RIVAS-SANTIAGO & J.C. JIMÉNEZ
MICOLOGIA
57 Expressão de receptores do tipo Toll 2 e 4 nos queratinócitos de
pacientes com dermatofitose localizada e disseminada
M.A. CORRAL, F.M. PAULA, M. GOTTARDI, D.M.C.L. MEISEL, P.P. CHIEFFI &
R.C.B. GRYSCHEK
V.J. MASCHIO, G. CORÇÃO & M.B. ROTT
85 Elevada transmissão transmamária de larvas de Toxocara canis em
camundongos BALB/c
P.L. TELMO, L.F.C. AVILA, C.A. SANTOS, P.S. AGUIAR, L.H.R. MARTINS, M.E.A.
BERNE & C.J. SCAINI
CORRESPONDÊNCIA
88 Neurocysticercosis and afebrile seizure
V. WIWANITKIT
CARTA AO EDITOR
89 Loose and compact agglomerates of 50 nm microvesicles derived
from Golgi and endoplasmic reticulum membranes in pre- and inapoptotic mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope
A. SESSO, E.H. YAMASHIRO-KANASHIRO, N.M. ORII, N.N. TANIWAKI, J.
KAWAKAMI & S.M. CARNEIRO
92 Influenza virus surveillance by Institute Adolfo Lutz, influenza
season 2014: antiviral resistance
K.C.O. SANTOS, D.B.B. SILVA, M.A. BENEGA, R.S. PAULINO, E.R. SILVA Jr,
D.S.PEREIRA, A.D.H. MUSSI, V.C. SILVA, L.V. GUBAREVA & T.M. PAIVA
C.B. OLIVEIRA, C. VASCONCELLOS, N.Y. SAKAI-VALENTE, M.N. SOTTO,
F.G.LUIZ, W. BELDA JÚNIOR, M.G.T.S. SOUSA, G. BENARD & P.R. CRIADO
IV
ENDEREÇO
INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO
Av. Dr. Enéas de Carvalho Aguiar, 470
05403-000 São Paulo, SP - Brasil
Fone/Fax: 55.11.3062.2174; 55.11.3061-7005
e-mail: [email protected]
57(1):1-13, January-February, 2015
http://dx.doi.org/10.1590/S0036-46652015000100001
REVIEW
ORIGIN AND PREVALENCE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1)
AND TYPE 2 (HTLV-2) AMONG INDIGENOUS POPULATIONS IN THE AMERICAS
Arthur PAIVA(1,2) & Jorge CASSEB(2)
SUMMARY
Human T-lymphotropic virus type 1 (HTLV-1) is found in indigenous peoples of the Pacific Islands and the Americas, whereas type
2 (HTLV-2) is widely distributed among the indigenous peoples of the Americas, where it appears to be more prevalent than HTLV-1,
and in some tribes of Central Africa. HTLV-2 is considered ancestral in the Americas and is transmitted to the general population and
injection drug users from the indigenous population. In the Americas, HTLV-1 has more than one origin, being brought by immigrants
in the Paleolithic period through the Bering Strait, through slave trade during the colonial period, and through Japanese immigration
from the early 20th century, whereas HTLV-2 was only brought by immigrants through the Bering Strait. The endemicity of HTLV‑2
among the indigenous people of Brazil makes the Brazilian Amazon the largest endemic area in the world for its occurrence. A
review of HTLV-1 in all Brazilian tribes supports the African origin of HTLV-1 in Brazil. The risk of hyperendemicity in these
epidemiologically closed populations and transmission to other populations reinforces the importance of public health interventions
for HTLV control, including the recognition of the infection among reportable diseases and events.
KEYWORDS: HTLV-1; HTLV-2; Indians; Origin; Americas.
INTRODUCTION
The human T-lymphotropic virus type 1 (HTLV-1) and type 2
(HTLV-2), although closely related, have different geographical
distributions8,21,23,35,59,63,72,93,110,119, pathogenesis10,95,97,98, and clinical
manifestations8,10,21,23,35,59,63,97,110. Although types 3 (HTLV-3) and 4
(HTLV-4) have been found in populations of central Africa, they have
not yet been associated with disease in humans19,133.
HTLV-1, the causative agent of HTLV-associated myelopathy/tropical
spastic paraparesis (HAM/TSP), uveitis, infective dermatitis, and other
inflammatory disorders21,35,59,110, is endemic in many parts of the world,
including southwestern Japan, some of the Caribbean islands, South
America, and foci in western and central Africa and Australo-Melanesia59.
In turn, HTLV-2, despite there being no clear indications associating
it with well-defined clinical manifestations, has been associated with
sporadic cases of neurological disorders similar to HAM/TSP8,63, and has
been observed as prevalent in native populations, such as the indigenous
peoples of the Americas, certain tribes of pygmies in Africa23,63,72,93,119, and
injection drug users (IDUs) in urban areas of the United States, Europe,
and Latin America27,42,45,82,89,114,124,138.
these areas (78% of whom live in urban areas), for an estimated total
population of 896,917 counted as indigenous by the 2010 census29.
Both HTLV-1 and HTLV-2 are prevalent among Brazilian indigenous
populations, with HTLV-2 being the most predominant among these
individuals14,51,52,69,72,91,93,100,102,119,126.
The search strategy adopted in this review was intentionally broad so
as to ensure the identification of all relevant studies published between
1980 and 2014 relating to infection by HTLV in indigenous populations
in Latin America. Searches were made via the PubMed, Lilacs and
Google Scholar electronic databases using the following terms: “HTLV”,
“Indians”, “natives”, “Americas”. Lastly, multiple relevant articles
were used to carry out the ‘Snowball’ method to supplement the review.
However, the results of most of the studies published on this thematic
should be taken with caution since the majority of them include rather
small populations and not all the studies were carried out using stringent
criteria of positivity, Western blot or PCR. In addition, HTLV prevalence
tends to increase with age and is higher in women, but frequently
information pertaining to age and sex of the studied populations has not
been specified.
ORIGIN OF HTLV-1 AND HTLV-2 IN THE AMERICAS
In Brazil, approximately 517,000 Indians live in officially recognized
indigenous lands, with an estimated additional 380,000 living outside
HTLV-1 is endemic in South America, present in all 13 countries
(1) Universidade Federal de Alagoas. Hospital Universitário. Maceió, Alagoas, Brazil. E-mail: [email protected]
(2) Institute of Tropical Medicine of São Paulo, University of São Paulo, São Paulo, SP, Brazil. E-mail: [email protected]
Correspondence to: Arthur Paiva. E-mail: [email protected]
PAIVA, A. & CASSEB, J. - Origin and prevalence of human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) among indigenous populations in the Americas. Rev. Inst. Med.
Trop. Sao Paulo, 57(1): 1-13, 2015.
and prevailing in all ethnic populations22,26,59. In Brazil, for example,
HTLV-1 is found in immigrants from other endemic foci, such as Africa
and Japan53,79, and in those who are descended from Amerindians who
have inhabited South America for thousands of years46,9,72,100,119.
There are two hypotheses about the origin of HTLV-1 in the Americas.
The first considers the prehistoric migration of infected populations
about 11,000 to 13,000 years ago across the Bering Strait into North
America. These people migrated through Central America and spread
into South America, where they settled mainly in the Andes and along
the Amazon. The other hypothesis suggests that HTLV-1 originated in
Africa and was brought to the Americas (including the Caribbean islands,
United States, and South America) through the slave trade between the
16th and 19th centuries.
It is estimated that the separation of African and non-African human
populations occurred around 75,000 to 287,000 years ago28,112, with gene
flow occurring from pygmies to neighboring populations28. HTLV-1 and
HTLV-2 infections among pygmies are the oldest, and although frequent
transmission of simian T-lymphotropic virus type 1 (STLV-1) from apes
to humans in Africa could suggest that these infections were the result
of interspecies transmissions over the years87,90,115,130, the absence of nonhuman primates in Melanesia and Australia suggests that HTLV-1 existed
among the Australoid people who first populated Australo-Melanesia
around 60,000 years ago56,135.
The transcontinental migration of HTLV-1 is supported by studies
showing that two strains of human leukocyte antigen (HLA)-A alleles
are associated with HTLV-1 in endemic regions around the world. Both
strains (HLA-A*26 and HLA-A*36) originally evolved in Africa and
dispersed through Asian populations and indigenous North and South
Americans120. This transcontinental dispersal of HTLV-1 partially
overlapped the migration pattern of southeastern Asian mongoloids120,123.
Interestingly, these alleles are associated with adult T-cell lymphoma
(ATL), and phylogenetic analysis revealed that the ancestral genes of
these alleles came from primate major histocompatibility complex genes,
including those of gorillas, chimpanzees, and monkeys113. Taking this
into consideration, it is possible that the HLA-A*26 and HLA-A*36
evolved > 50 million years ago and that carriers have become natural
hosts of HTLV-1 due to a low immune responsiveness to the virus120.
Phylogenetic analysis of bone marrow samples from the femur of a
mummified specimen revealed that the isolated clones were similar to
those of the Indians of South America and belonged to a transcontinental
subgroup closely related to HTLV-1 carriers, the Ainu of northern Japan,
and some mongoloid Asian subgroups. These observations suggest
that the Andean mummy’s HTLV-1 could have originated from Asian
paleo-mongoloids86.
The debate over the origin of HTLV-1 has primarily focused on
empirical-level molecular phylogenetic analysis, particularly on how
to better explain the phylogenetic origin of the cosmopolitan subtype
of HTLV-1, to which the indigenous strains found in South America
belong and which is the predominant subtype among the other isolates
from various endemic and non-endemic areas of the Americas22. The
subtypes, which do not appear to differ in pathogenicity, likely reflect
the geographical origins and migrations of ancient populations77,80,118.
Analysis of genetic sequencing has divided HTLV-1 into subtypes 1a
2
(cosmopolitan), 1b (Central African), 1c (Melanesian), and 1d, 1e, 1f,
and 1g (found in Central Africa). The cosmopolitan subtype is divided
into five subgroups, depending on geographical location: transcontinental
(A), to which most strains of HTLV-1 isolated from South America
belong; Japanese (B); West African (C); North African (D)55,94; and
Afro-Peruvian (E)128.
The transcontinental subgroup (A) has been characterized both in
North America (United States and Canada) and South America (Argentina,
Brazil, Chile, Colombia, French Guiana and Peru)23,53,59,77,118,122,127. The
Japanese subgroup (B) has been found in the north, northeast, and
southeast regions of Brazil; Canada; Colombia; and Peru and can be
explained by the thousands of years of migration from Asia to the
Americas and by recent Japanese immigration53,59,118,127,129. The West
African subgroup (C) was identified in the Caribbean and French
Guiana but not in Brazil, despite its introduction being associated with
slave trafficking from West Africa and the high rate of infection among
black South Americans23,53,59,122. The Afro-Peruvian subgroup (E) was
characterized in two black individuals presenting a type of mitochondrial
DNA identical to that found in some populations of West Africa55,128.
The migration of African people to the Americas through slave trade
took place from mainly western and central Africa, and 40% of the
approximately 10 million Africans arrived at Brazilian ports, making it
intriguing that subgroup C is not found in Brazil53. Aiming to clarify the
origin of HTLV-1 in Brazil, GALVÃO-CASTRO et al.53 analyzed 243
sequences of the long terminal repeat region of isolates from descendants
of various ethnic groups and geographical regions of the country, all of
which were classified as the cosmopolitan subtype. Of these, 98% were
in the transcontinental subgroup (A) and 3.3% in the Japanese subgroup
(B), which is discordant with historical data indicating that the majority
of Africans who came through Salvador were the original carriers of the
West African subgroup (C).
The migration of African populations comprised several cycles:
Guinea Cycle during the second half of the 16th century; Angola and
Congo Cycle in the 17th century; Mina Coast Cycle during the first threequarters of the 17th century; and Bay of Benin Cycle in the 18th and 19th
centuries. It is possible that the occurrence of multiple introductions
of some sequences of HTLV-1 in the post-Columbian era are clustered
in Latin American groups with sequences of southern African ancestry
that were segregated from the same ancestor of another group with a
central African string. Thus, this relationship of ancestry suggests that
this group was first introduced in South Africa due to the migration of
the Bantu people of Central Africa to South Africa about 3,000 years
ago and then to Brazil during the slave trade period between the 16th and
17th centuries53. Analysis of the distribution of haplotypes linked to the
group of β-globin genes demonstrated that 29.4% of the Bantu haplotype
could explain why the majority of HTLV-1 isolates are grouped with
those from southern Africa2. It is known that Africans of Bantu ethnicity
were brought to Bahia between 1678 and 1810 and that approximately
2,400 African Bantu (with 100 coming from Angola and 2,300 from
Madagascar) were brought between 1817 and 184337. During the
colonization of South Africa by the British in the 17th and 18th centuries,
many Africans migrated to neighboring regions currently known as
Angola, Madagascar, and Mozambique, where they were captured and
transported to Salvador37. In addition, there is evidence that the ports
of departure of African slave ships often were not related to the ethnic
Trop. Sao Paulo, 57(1): 1-13, 2015.
and geographic origins of African transportees37. Thus, taken together,
these results corroborate the hypothesis of multiple introductions of
post-Columbian subgroup A in Brazil.
Most of non-indigenous persons infected by HTLV-1 in the Americas
have probably been infected by virus strains originating from the African
slave trade during the post-Columbian period2,37,44,53,57,59,75,122,128,130 and,
it is today accepted that HTLV-1 in the Americas has more than one
source: through migration from Asia through the Bering Strait86,94,104,134
during the Paleolithic era; the trafficking of slaves during the colonial
period2,53,122,128,130; and more recently, the Japanese immigration in the
early 20th century (Fig. 1)79,127,128.
HTLV-1 and HTLV-2 are similar, with approximately 60% of their
structures based on the same sequence of nucleic acids and 70% on the
same sequence of amino acids20. Both viruses are very old and have
evolved independently through transmission of STLV from nonhuman
primates to the first humans62. HTLV-2 is divided into four subtypes:
2a, 2b, 2c, and 2d. Molecular studies confirm the presence of HTLV2c in Brazil, the only country to identify this subtype43. The Brazilian
variant of HTLV-2 has a tax region similar to that of subtype 2b and the
env-like subtype 2a genomic long terminal repeat region33,43,72. Only the
2c variant was identified in Brazilian tribes, and this subtype of HTLV-2
is prevalent in IDU and non-IDU populations in Brazil3,43,69,70,72,119,125,126.
HTLV-2 is considered an ancestral virus in the Americas because it is
endemic among isolated indigenous groups, having been inherited by the
general population and transmitted to IDUs from indigenous populations
(Fig. 1)3,43,70,72,125,126.
The endemicity of HTLV-2 among various indigenous peoples of
the Americas and the lack of evidence of infection with STLV-2 in
New World monkeys have led to the conclusion that HTLV-2 has been
present on the American continents since ancient times11,66,81,101,105. The
hypothesis of independent development of HTLV-1 and HTLV-2 from a
common ancestor after human migration to the Americas has a very low
possibility of support due to the fact that HTLV-2 has been identified in
isolates from pygmies in northwestern Zaire and Cameroon58,61, where
HTLV-1 has not been identified, as well as the above mentioned absence
of STLV-2 in New World monkeys75 and the slow evolutionary potential
of HTLV-2116.
HTLV IN INDIGENOUS GROUPS OF OTHER COUNTRIES
OF THE AMERICAS
Among native populations, HTLV-1 is found in the Pacific countries,
including Australia and Melanesia, as well as in North, Central, and
South America10, whereas HTLV-2 is endemic and widely distributed
among the indigenous peoples of North, Central, and South America,
where it appears to be more prevalent than HTLV-1, and in some tribes
of Central Africa.
The population of American Indians and Alaska Natives is estimated
at 5.2 million or 2% of the general US population, of which approximately
49% are not mixed with other races5. There are 566 tribes and 325 Indian
reservations recognized by the US federal government5. In Canada, about
1.4 million Indians make up 4.3% of the total population, with the most
populous group being the First Nations (61%) followed by Métis (32.3%)
and Inuit (4.2%)1. In North America, there are few large, well-sampled
studies of HTLV infection among Native Americans, and some have
additional feature limitations, as shown by earlier serologic screening
and confirmatory assays, or lack precise HTLV typing and molecular
characterization of the virus23. A database of 2,047,740 blood donors
was examined in North America for the period of 2000 to 2009 for both
viruses, suggesting a prevalence of HTLV in the general population of
0.1% to 0.2% and showed a higher prevalence of HTLV-2 (14.7/100,000)
than HTLV-1 (5.1/100,000) in the western and southwestern United
States31, which could be attributed to endemic foci among American
Indians30.
A previous study in blood donors had already demonstrated a rate of
HTLV infection of 0.72/1,000 in New Mexico (most cases of which were
attributed to HTLV-2) and, in turn, a higher prevalence among American
Fig. 1 - Origin of HTLV-1 and HTLV-2 in the Americas. Based on the references 53, 116, 120 and 128.
3
Trop. Sao Paulo, 57(1): 1-13, 2015.
Indian blood donors (1.0%-1.6%) than among non-Hispanic white donors
(0.009%-0.06%)65. In the United States, HTLV is endemic among the
Navajo and Pueblo people of New Mexico and Seminole people of
Florida, with HTLV-2 presenting a higher prevalence85,88 (Table 1 and
Fig. 2). In Alaska, the prevalence of HTLV among native peoples is
0.5% (two out of 380)34. This was confirmed as HTLV-1 in a subsequent
study involving five other native individuals seropositive for HTLV from
various geographical areas of Alaska, one being a blood donor with ATL,
one with HAM/TSP, one with a neurological disease characterized by gait
disturbance and urinary incontinence, and one with Hodgkin disease; all
subjects with HTLV-1 had no risk factors for infection78.
In Canada, studies in indigenous populations also have been limited.
PICARD et al. (1995)108 observed that phylogenetic analysis of HTLV-1
strains recovered from indigenous people living on the coast of British
Columbia in western Canada suggested multiple origins for the virus.
Subsequently, ANDONOV et al. (2012)6 performed a phylogenetic
analysis on various strains of HTLV-1 from the Nunavut population of
Canada, of which 85% are Native Americans of British Columbia’s Pacific
coast, and Canadian non-indigenous people. The study demonstrated
that strains in Nunavut (the cosmopolitan subtype) are related to those
from East Asia and consistent with the presence of HTLV-1 in ancient
indigenous peoples of the Canadian Arctic, noting the diversity of other
strains of HTLV-1 analyzed from other Indians and reinforcing previous
evidence of multiple incursions of this virus in indigenous populations
of coastal British Columbia.
PETERS et al. (2000)107 found a prevalence of 2.8% for HTLV-1 and
1.6% for HTLV-2 among 494 serum samples from the Nuu-Chah-Nulth
tribe of Vancouver Island in British Columbia, which is known for a high
incidence of rheumatic disease. Although they found no association
between arthropathy and HTLV-1, diseases such as ATL and HAM/TSP
have been reported in this region36,54,103,107,109.
In Latin America, there are more than 400 indigenous groups,
ranging from 45 million to 48 million individuals97. Most live in Bolivia,
Guatemala, Peru, Ecuador, and Mexico9, comprising the majority of the
population in Bolivia (62%) and Guatemala (60%)68, and less than 3%
live in Paraguay, Venezuela, and Brazil29,68,96 (0.4% of the population)29.
In South America, only Uruguay has no remaining indigenous population.
In Mexico, the only Latin American country in North America, a 0.23%
prevalence rate of HTLV-2 was found among 440 native Maya Indians
and Mayans living in six communities on the Yucatan Peninsula60.
geographical distribution among the various indigenous groups (Fig. 3).
FUJIYOSHI et al. (1999)51 conducted a seroepidemiological study on
indigenous peoples of the Andes of Colombia, Peru, Bolivia, Argentina,
and Chile; Chiloé Island (Chilean coast); Easter Island (Chilean province
of Polynesia); and the plains along the Atlantic coast from Colombia
to the Orinoco, Amazon, and Patagonia, demonstrating an ethnic and
geographically independent distribution between HTLV-1 and HTLV2, with foci of HTLV-1 prevalent mainly in the Andean highlands and
that of HTLV-2 in the coastal plains. FUJIYOSHI et al. (1995)50 also
observed that HLA haplotypes in indigenous Andean groups with HTLV-1
and indigenous groups with HTLV-2 of the lower Orinoco (Venezuelan
Amazon) were mutually exclusive. HLA haplotypes associated with
HTLV-1 are commonly found in the known HTLV-1 endemic Indian and
Japanese populations, whereas the haplotypes associated with HTLV-2
are specifically found among indigenous Orinoco and North American
groups, suggesting that ethnic HLA haplotypes are separate from those
native to South America and may be involved in the susceptibility to
infection by HTLV-1 or HTLV-2. In southern Colombia, for example,
HTLV-1 was first detected among natives belonging to the Paez people
of the Andes136. Subsequently, HTLV-2 has been identified among the
Wayuu, Guahibo, and Tunebo groups in the Guajira Peninsula in extreme
northeastern Colombia (Caribbean Sea), with prevalence rates between
4.1% and 31.5%39,50,121,136. Other foci of HTLV-1 are found in various
isolated indigenous populations (Wayuu, Waunana/Noanama, Inga,
Kamsa, Embera), with prevalence rates from 1.0% to 8.5%7,39,40,137. Both
viruses have been detected within some of these groups7,39, although in
most, HTLV-1 and HTLV-2 appear to be mutually exclusive40,41,49,136,137,
including a 31.5% HTLV-2 rate among Guahibo natives49 (Table 1).
These observations suggest that the natives of South America could
be divided into two major ethnic groups by an HTLV-1 and HTLV-2
carrier state that evolved among mongoloid populations and transmitted
independently as two different strains among the indigenous peoples of
the Andes highlands and coastal Atlantic plains41,50,51,137.
In Venezuela, a high prevalence was found for HTLV-2, reaching
61% among Guahibo and Yaruro83,84,106, whereas in central and southern
Bolivia, only HTLV-1 was detected among the Quechua and Aymara
indigenous peoples, with a prevalence of 6.8% and 5.3%, respectively51.
In the Peruvian Andes, Quechua and Aymara, the most populous
indigenous groups in the region, also present solely HTLV-1, with
prevalence rates from 1.6% to 2.82%51,73,92,117, whereas in the Amazon,
the Peruvian Shipibo-Konibo notably carry both viruses, with high
prevalence rates ranging from 1.43% to 5.9%4,15,92 and 2.1% to 3.8%4,15
for HTLV-1 and HTLV-2, respectively. A few cases of only HTLV-2
have also been reported in two other indigenous groups of the Peruvian
Amazon92 (Table 1).
In the seven countries of Central America (Belize, Costa Rica,
El Salvador, Guatemala, Honduras, Nicaragua, and Panama), some
of which with strong commercial and cultural ties to the Caribbean
islands, GESSAIN & CASSAR59 reviewed the high endemicity for
HTLV-1 infection and associated diseases and demonstrated a very low
HTLV-1 seroprevalence, with significant differences observed among
the populations tested. In indigenous populations, HTLV-2 was endemic
only among Guaymi people, with prevalence rates of 7.9%-8.5% in
Panama46,81,105,111,132 and 8.0% in Costa Rica131(Table 1 and Fig. 2).
In Chile, where HTLV-1 seems to be endemic among groups of
isolated indigenous peoples living in the Andes or in the southernmost
region of the country59, prevalence ranges from 0.5% to 0.8% among the
Mapuche and Rapa Nui24,51,67 and up to 4.1% among the Atacama were
found51. Foci of HTLV-2 were reported among the Alacalf (34.8%)51,
Yaghan (9.1%)51, and Huilliches/Mapuche (1.0%) peoples24.
In South America, HTLV-2 predominates among indigenous
groups (Table 1), with subtype 2b clearly prevailing in Amerindian
populations121, except in Brazil, where it is characterized by subtype
2c23,43,69,72,119,126 (Fig. 1). HTLV-1 and HTLV-2 also differ in their
In Argentina, ethnic and geographic restriction are also seen for
both viruses13, with HTLV-1 prevalent among the Qulla and Puná of
northwestern Argentina38,44,51 and HTLV-2 variably, but generally more
prevalent among the Chorote, Wichi, Chulupi, and Toba peoples of
4
Trop. Sao Paulo, 57(1): 1-13, 2015.
Table 1
Prevalence of positivity for HTLV in indigenous groups of other countries of the Americas
Country
United
States
Canada
Mexico
Panama
Costa Rica
Author *
Levine et al. 199385
Lowis et al. 199988
Davidson et al. 199034
Peters et al. 2000 107
Gongora-Bianchi et al. 199760
Lairmore et al. 199081
Reeves et al. 1988111
Pardi et al. 1993105
Feigenbaum et al. 199446
Vitek et al. 1995132
Visoná et al. 1997131
Inostroza et al. 199167
Cartier et al. 199324
Chile
Fujiyoshi et al. 199951
Bolivia
Zamora et al. 1990136
Dueñas-Barajas et al. 199239
Fujiyama et al. 199349
Duenas-Barajas et al.199340
Colombia
Zaninovic et al. 1994137
Arango et al. 19997
Egea41
Ferrer et al. 199447
Bouzas et al. 199416
Biglione et al. 199912
Medeot et al. 199992
Dipierre et al.38
Argentina
Eirin et al. 201044
Population
Seminole
Seminole
Alaska Natives
Nuu-Chah-Nulth
Maya
Guaymi
Guaymi
Guaymi
Guaymi
Guaymi
Guaymi
Mapuche
Huilliches/Mapuche
Atacama
Alacalf
Yahgan
Rapa Nui
Aymara
Quechua
Paez
Wayuu
Guahibo
Waunana/Noanama
Tunebo
Inga
Kamsa
Wayuu
Embera
Inga
Wayuu
Toba and Wichi
Toba
Wichi
Toba
Toba
Indians in Puna Jujeña
Mapuche
Chorote
Toba
Wichi
Chorote/Wichi
Chorote/Chulupi
Kolla
Puna
N
106
46
380
494
440
8
317
317
109
3686
405
199
217
23
22
132
151
96
32
523
92
143
40
62
59
123
1014
155
157
175
222
205
105
72
86
94
171
21
204
14
10
112
88
HTLV-1 (%)
1
2
14
2.17
0.5
2.8
3
1
9
0.7
0.5
4.1
1
8
6
2
1
0.8
5.3
6.2
6.3
1.6
3
2.1
1
5
1.6
8.5
10
2
1.0
1.2
1
1
0.45
2
2
2.78
2.32
11
2
HTLV-2 (%)
14
13.2
11
23.9
8
1
1
25
25
9
352
1.6
0.23
12.5
7.9
7.9
8.3
9.5
8.0
2
1.0
8
2
34.8
9.1
3
29
4.8
31.5
2
5.0
5
7
1
11
24
22
62
23
2
4.1
0.7
0.7
7.0
13.7
9.91
3.0
21.9
2.78
2
61
5
26
4
8
2.1
35.6
23.8
12.7
28.5
80
9.8
2.3
5
Trop. Sao Paulo, 57(1): 1-13, 2015.
Table 1
Prevalence of positivity for HTLV in indigenous groups of other countries of the Americas (cont,)
Country
Author *
Population
Chulupi
94
32
34
Ayoreo
51
2
3.9
Lengua
49
5
10.2
1
4.8
24
16.4
Paraguay
Cabral et al. 199818
51
Medeot et al. 199992
Peru
French
Guiana
Sanapaná
30
Angaité
21
HTLV-1 (%)
2
Chaco
146
Quechua
40
1
2.5
Shipibo-Konibo
70
1
1.43
Harakmbet
22
1
4.54
Huambisa
42
1
2.38
6
2.1
47
3.8
Aymara
62
1
1.6
Sanchez-Palacios et al. 2003117
Quechua women
198
5
2.5
Alva et al. 20104
Shipibo-Konibo
290
12
4.1
Quechua
389
11
2.82
Blas et al. 201315
Shipibo-Konibo women
1253
74
5.9
Perez et al. 1993
73
Pume (Yaruro)
210
12
5.7
Leon-Ponte et al. 199683
Guahibo
166
41
24.7
Leon-Ponte et al. 199884
Yaruro/Guahibo
41
25
61
Arawack
54
1
1.8
Talarmin et al. 1999122
Palikur
78
2
2.6
Wayampi
138
2
1.4
106
* Numbering as given in the references.
Fig. 2 - HTLV-1 and HTLV-2 among indigenous populations of North America and Central America. Based on the references from Table 1.
6
HTLV-2 (%)
6.7
Ita et al. 2013
Venezuela
N
Trop. Sao Paulo, 57(1): 1-13, 2015.
the Chaco region, covering parts of Bolivia, Argentina, Brazil, and
Paraguay12,13,16,47,48,51,92 (Table 1).
HTLV-2 is found almost exclusively among the indigenous population
of Paraguay, who mostly live in the Gran Chaco region in the northwest,
with a prevalence ranging from 3.9% to 34%18,48,51 (Table 1). In French
Guiana (not included in Latin America), TALARMIN et al. (1993)122 did
not detect HTLV-2 among the 847 indigenous peoples of the Arawak,
Palikur, Wayampi, Galibi, Emerillon, and Wayana, but HTLV-1 was
detected in 1.81% (5 out of 270) of the Arawak, Palikur, and Wayampi,
with prevalence ranging from 1.4% to 2.6% (Table 1), with the virus
probably acquired through contact with people of African descent during
slave trade, according to phylogenetic analyses128.
HTLV IN INDIGENOUS POPULATIONS OF BRAZIL
The first description of HTLV infection among indigenous people
in Brazil dates back to 1990 when, while assessing the prevalence of the
virus in some human populations at risk, NAKAUCHI et al.99 reported a
39% positivity for HTLV-1 in the serum of 82 Mekranoiti subjects and
20% in 55 Tiriyo subjects from Pará. Confirmatory tests of the final
results of this study were positive in 3.63% (two out of 55), 12.19%
(10 out of 82), and 13.88% (10 out of 72) for HTLV-1 among Tiriyo,
Mekranoiti, and Xicrin subjects, respectively99,100.
Subsequent studies showed that HTLV-2 is predominant among
Brazilian indigenous groups14,51,52,69,91,93,102,119,126, with an area of high
endemicity in the Amazon region. HTLV-1, on the other hand, is present
in isolated clusters69,119 (Table 2 and Fig. 3).
ISHAK et al. (1995) 69 investigated sera collected from 1,382
individuals belonging to 26 indigenous communities within endemic
regions of HTLV-2. These communities are distributed through the
states of Maranhão (Urubu-Kaapór), Amapá (Galibi, Palikur, Wayampi),
Amazonas (Yamamadi), Roraima (Yanomami), Rondônia (Cinta Larga,
Surui, Karitiana), and Pará (Wayana-Apalai, Tiriyo, Assurini Kuatinemo,
Assurini Trocará, Zoé, Arara Laranjal, Arara Kurambê, Arara, Iriri,
Araweté, Parakanã, Munduruku, and six different Kayapo tribes).
The authors found HTLV-2 to be present in 17 of the 26 communities,
providing evidence that the Amazon region of Brazil is the most endemic
area in the world for HTLV-269,72. This study also found the presence of
antibodies to HTLV-1 limited to five individuals: one Galibi (Amapá),
three Yanomami (Roraima), and one Kayapo from the village of Aukre
(Pará) (Table 2). The highest prevalence for HTLV-2 was observed among
the Kayapo (32.2%), followed by Tiriyo (15.4%), Mundukuru (8.1%),
and Arara Laranjal (11.4%) peoples69. Several other studies have also
shown a very high prevalence of HTLV-2 among the Kayapo, at rates
ranging from 28% to 57.9%14,51,91,102,126. Moreover, MALONEY et al.
(1992)91 reported the presence of HTLV-2 in 12.2% (21 out of 172) of
Krahos people in Tocantins.
Tiriyo and Wayampi indigenous peoples live at the border between
Brazil, Suriname and French Guiana, respectively (approximately 1,700
Tiriyo, 750 of whom live in Brazil and are spread over 15 villages, and
1,200 Wayampis, 450 of whom live in Brazil in the state of Amapá)119.
The presence of both HTLV-1100,119 and HTLV-269,119,126 was confirmed
among Wayampi and Tiriyo from Brazil in whom HTLV-2 was
predominant69,119,126, in contrast to what is observed among the indigenous
population of the Amazon region of French Guiana, where only HTLV-1
is present and probably brought from Africa during the post-Columbian
slave trading period74,122.
The south of Brazil, on the other hand, is directly related both
geographically and ethnically to northern Argentina and southern
Paraguay, areas known to be endemic for HTLV-212,16,18,47,48,51,92. A study
of the Guarani Indians in southern Brazil reported a prevalence of 5.76%
for HTLV-2 among 52 individuals examined, suggesting that the Guarani
is another endemic indigenous group for this retrovirus and the need for
molecular and phylogenetic studies with larger numbers of samples93.
The prevalence of infection reported in Brazil for the various ethnic
groups ranges from 0.48% to 13.9% for HTLV-1 (Xicrin, Mekranoiti,
Tiriyo, Yanomami, Galibi, Wayampi, and Kayapo)69,100,119 and 0.44%
to 57.9% for HTLV-2 (Kayapo, Tiriyo, Xicrin, Kraho, Arara Laranjal,
Mundukuru, Guarani, Yamamadi, Karitiana, Yanomami, Parakanã, Galibi,
Wayana-Apalai, Cinta Larga, and Wayampi)14,51,52,69,91,93,102,119,126 (Table 2).
In addition to approximately two dozen cases initially reported in
1992 by NAKAUCHI et al.100 among the Xicrin, Mekranoiti, and Tiriyo
peoples of Brazil, only seven cases were confirmed as HTLV-1 infection
among Brazilian indigenous69,119 (Table 2). The possibility of posttransfusion infection cannot be ruled out as a means of introduction of
the virus to these ethnic groups with a low prevalence for retrovirus and
in whom malaria is frequent76. These data support the African origin
of HTLV-1 in Brazil introduced in the post-Columbian period through
slave trade, similar to what would have occurred in French Guiana and
the Caribbean basin2,37,53,57,74,122.
Fig. 3 - HTLV-1 and HTLV-2 among indigenous populations of South America. Based on
the references from Table 2.
The importance of maintaining the endemicity of HTLV in these
epidemiologically closed populations by transmission through sexual
7
Trop. Sao Paulo, 57(1): 1-13, 2015.
Table 2
Prevalence of positivity for HTLV in indigenous populations of Brazil
State
Amazonas
Amapá
Tribe/Nation
Yamamadi
Galibi
Wayampi
Kayapo
Kayapo **
Tiriyo
Pará
Xicrin
Mekranoiti
Parakanã
Roraima
Rondônia
Tocantins
Paraná
Arara Laranjal
Munduruku
Wayana-Apalaí
Yanomami
Karitiana
Cinta-Larga
Kraho
Guarani
Author *
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Shindo et al. 2002119
Maloney et al. 199291
Black et al. 199414
Ishak et al. 199569
Vallinoto et al. 2002126
Novoa et al. 1997102
Nakauchi et al. 1992100
Ishak et al. 199569
Vallinoto et al. 2002126
Shindo et al. 2002119
Gabbai et al. 199352
Gabbai et al. 199352
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Ishak et al. 199569
Maloney et al. 199291
Menna-Barreto et al. 200593
N
36
148
71
321
264
703
207
19
27
141
55
26
150
683
72
206
82
89
52
44
161
50
102
50
50
172
52
HTLV-1 (%)
1
0.67
2
0.62
HTLV-2 (%)
2
5.6
3
2.0
1
1.4
88
1
0.48
2
3.6
10
13.9
10
12.2
3
2.94
67
11
6
59
33.3
28.0
32.2
57.9
22.2
41.8
4
3
3
15.4
2.0
0.44
31
15.0
2
1
5
13
1
4
2
1
21
3
2.25
1.92
11.4
8.1
2.0
3.9
4.0
2.0
12.2
5.8
* Numbering as given in the references. ** Children born to HTLV-2-positive mothers.
relations and vertical pathways is supported by evidence from the
Kayapo Indians, who are experiencing a gradual increase in antibody
prevalence with age and constant and continuous transmission between
couples14,72. Molecular biology has confirmed a high prevalence (42%)
of HTLV-2 among children born to HTLV-2-positive mothers 71,102.
Breastfeeding (and cross-feeding) is a primary source of nutrition
for indigenous children, which increases the spread of the virus72. If
transmission is not stopped, HTLV-2 will become hyperendemic14,69,
and transmission to non-indigenous populations is increasingly possible
despite the relative geographical isolation of the Kayapo. For example,
some villages of Kayapo (Kubenkokre, Kokraimoro, Aukre, Kararaô,
Gorotire, and Kikretum) are located near small towns maintained by
commercial industries, agriculture, and mining, and it is common practice
of indigenous Brazilian men to visit these towns to have sexual relations
with non-indigenous women72.
Ignorance about the virus greatly increases the risk of transmission;
thus, preventive measures to reduce the spread and transmission
of retroviruses in indigenous populations initially depend on the
identification of cases and educational programs 17,23,32,139,140. The
8
indigenous community should be adequately informed about the
modes of HTLV transmission and the risks associated with prolonged
breastfeeding and cross-feeding. Pregnant women should be routinely
screened for HTLV infection and, if positive, should be given access
to other alternatives to breast milk, such as formulas17,23,32,64. Woman
who are likely to breast feed other children should be also screened for
HTLV.
Regarding the prevention of sexual transmission of HTLV, programs
should emphasize the importance of condom use, systematic screening
for infection, and individual counseling17,23,32,139,140. Screening should also
include the parents of the infected individual and siblings if the mother
tests positive; children born to mothers with the virus should receive
appropriate follow-up17,32,64.
In Brazil, HTLV is not considered a public health problem and,
thus, has been widely neglected25,32,139. Like disease, injury, and public
health events, cases of HTLV should be reportable to better approximate
its incidence among indigenous peoples and to track its spread to the
general population.
Trop. Sao Paulo, 57(1): 1-13, 2015.
RESUMO
Origem e prevalência do vírus linfotrópico de células T humanas
em populações indígenas das Américas
O vírus linfotrópico de células T humanas do tipo 1 (HTLV-1) é
encontrado em populações indígenas de países do Pacífico e Américas
enquanto o tipo 2 (HTLV-2) é amplamente distribuído entre as populações
indígenas das Américas, nas quais aparenta ser mais prevalente que o
HTLV-1, e em algumas tribos da África Central, sendo considerado
ancestral nas Américas e transmitido à população geral e de usuários
de drogas injetáveis a partir da população indígena. No continente
americano o HTLV-1 teria mais de uma origem, sendo trazido na era
paleolítica pelos imigrantes através do estreito de Bering, através do
tráfico de escravos no período colonial e com a imigração japonesa a partir
do início do século XX, enquanto para o HTLV-2 teria sido trazido pelos
imigrantes através do estreito de Bering. A endemicidade do HTLV-2
entre os indígenas do Brasil tornam a região amazônica brasileira a maior
área endêmica do mundo para sua ocorrência e a revisão da infecção
pelo HTLV-1 em todas as tribos brasileiras apoiam a origem africana
do HTLV-1 no Brasil. O risco de hiperendemicidade nestas populações
epidemiologicamente fechadas e de transmissão a outras populações
reforçam a importância de medidas no âmbito da saúde pública para
seu controle, incluindo o reconhecimento da infecção entre os agravos
e eventos de notificação compulsória.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Augusto Cesar Penalva de
Oliveira and Jerusa Smid for helpful discussions in the last years. They
also thank Prof. Melchior Carlos do Nascimento (Universidade Federal
de Alagoas), who prepared the maps used in this manuscript. FAPESP:
2012/23397-0; 2008/56427-4.
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http://dx.doi.org/10.1590/S0036-46652015000100002
REVIEW
LEPROSY NEPHROPATHY: A REVIEW OF CLINICAL AND HISTOPATHOLOGICAL FEATURES
Geraldo Bezerra da SILVA JUNIOR(1), Elizabeth De Francesco DAHER(2), Roberto da Justa PIRES NETO(3), Eanes Delgado Barros PEREIRA(2),
Gdayllon Cavalcante MENESES(4), Sônia Maria Holanda Almeida ARAÚJO(1) & Elvino José Guardão BARROS(5)
SUMMARY
Leprosy is a chronic disease caused by Mycobacterium leprae, highly incapacitating, and with systemic involvement in some
cases. Renal involvement has been reported in all forms of the disease, and it is more frequent in multibacillary forms. The clinical
presentation is variable and is determined by the host immunologic system reaction to the bacilli. During the course of the disease
there are the so called reactional states, in which the immune system reacts against the bacilli, exacerbating the clinical manifestations.
Different renal lesions have been described in leprosy, including acute and chronic glomerulonephritis, interstitial nephritis, secondary
amyloidosis and pyelonephritis. The exact mechanism that leads to glomerulonephritis in leprosy is not completely understood.
Leprosy treatment includes rifampicin, dapsone and clofazimine. Prednisone and non-steroidal anti-inflammatory drugs may be used
to control acute immunological episodes.
KEYWORDS: Leprosy; Hansen disease; Kidney dysfunction; Chronic kidney disease; Glomerulonephritis.
INTRODUCTION
Leprosy is a chronic disease caused by Mycobacterium leprae, an
acid-fast bacilli, intracellular parasite, with predilection to Schwann cell
and skin. The disease is highly incapacitating, and systemic involvement
is reported in some cases45. Renal involvement has been reported in all
forms of the disease, and it is more frequent in multibacillary forms51.
The present paper presents a review of the clinical and histopathological
aspects of leprosy nephropathy.
EPIDEMIOLOGY: The number of leprosy patients is estimated to
be between 10 and 15 million, distributed across more than 100 countries.
In 2007, a total of 254,525 new cases were reported all over the world45.
Brazil is considered as having a high endemicity index and is the country
with the second highest number of cases, with 37,610 new cases registered
in 200958. Leprosy prevalence in Brazil was reduced by 85%, going from
17 to 3.8 cases/10,000 population in the period between 1985 and 200135.
LEPROSY PATHOPHYSIOLOGY: Infected persons with M.
leprae are thought not to develop clinical disease. M. leprae is slow
growing and the incubation period is long at 2-12 years. The M. leprae
has a high infective power, but low pathogenic power3. Person-to-person
spread via nasal droplets is believed to be the main route of leprosy
transmission. Most people with leprosy are non-infectious. Patients
with lepromatous leprosy excrete M. leprae from their nasal mucosa
and skin and are infectious before starting treatment with multidrug
therapy. Contacts of these patients are, therefore, at increased risk of
developing the disease. There may be a genetic predisposition to disease
manifestation. Infection with M. leprae leads to chronic granulomatous
inflammation in skin and peripheral nerves 46. Single-nucleotide
polymorphism (SNP) association studies showed a low lymphotoxin-α
(LTA)-producing allele as a major genetic risk factor for early onset
leprosy. Other SNPs to be associated with disease and/or the development
of reactions in several genes, such as vitamin D receptor (VDR), TNF-α,
IL-10, IFN-γ, HLA genes, and TLR1 have also been suggested6. The type
of leprosy that patients develop is determined by their cell-mediated
immune response to infection. Patients with tuberculoid disease have a
good cell-mediated immune response and few lesions with no detectable
mycobacteria. Patients with lepromatous leprosy are anergic towards M.
leprae and have multiple lesions with mycobacteria present46. Schwann
cells (SCs) are a major target for infection by M. leprae leading to
nerve injury, demyelination, and consequent disability. Binding of M.
leprae to SCs induces demyelination and loss of axonal conductance.
Macrophages are one of the most abundant host cells to come in contact
with mycobacteria. Phagocytosis of M. leprae by monocyte-derived
macrophages can be mediated by complement receptors CR1 (CD35),
CR3 (CD11b/CD18), and CR4 (CD11c/CD18) and is regulated by protein
kinase6. The inflammation present in nerves is driven by mycobacterial
antigens that activate a destructive inflammatory immune response
mediated by CD4+ cells and macrophages, and with involvement of
multiple pro-inflammatory cytokines, such as tumor necrosis factor α46.
In the tuberculoid lesions there is a predominance of CD4+ auxiliary
Financial Support: This research was supported by the Brazilian Research Council (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil, Protocol 475040/2011-2).
(1) School of Medicine, Master in Collective Health, Health Sciences Center, University of Fortaleza. Fortaleza, Ceará, Brazil.
(2) Post-Graduation Program in Medical Sciences, Department of Internal Medicine, Federal University of Ceará. Fortaleza, Ceará, Brazil.
(3) Department of Community Health, School of Medicine, Federal University of Ceará. Fortaleza, Ceará, Brazil.
(4) School of Pharmacy, Federal University of Ceará. Fortaleza, Ceará, Brazil.
(5) Department of Internal Medicine, School of Medicine, Federal University of Rio Grande do Sul. Porto Alegre, Rio Grande do Sul, Brazil.
Correspondence to: Elizabeth De Francesco Daher. R. Vicente Linhares 1198, 60135-270 Fortaleza, Ceará, Brasil. Phone/Fax: +55 85 32249725, +55 85 32613777. E-mails: ef.daher@uol.
com.br, [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]
SILVA JUNIOR, G.B.; DAHER, E.F.; PIRES NETO, R.J.; PEREIRA, E.D.B.; MENESES, G.C.; ARAÚJO, S.M.H.A. & BARROS, E.J.G. - Leprosy nephropathy: a review of clinical and
histopathological features. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 15-20, 2015.
T cells and Th1 cytokines such as IL-2 and IFN-gamma, while in
lepromatous (Virchowian) lesions suppressant T cells, CD8+, and Th2
cytokines such as IL-4, IL-5 and IL-103 predominate. In the tuberculoid
type, the exacerbation of cellular immunity and the production of
pro-inflammatory cytokines (IL-1 and TNF-alpha) prevents the bacilli
proliferation, but can cause injury to the host due to the lack of regulator
factors. In the Virchowian type, the production of PGL-1 (phenolic
glycolipid antigen-1) and LAM (lipoarabinomannan) antigens by the
bacilli, inside macrophages, favors the escapade of the bacilli from the
intramacrophage oxidation, because these antigens have a suppressant
effect over macrophage activity, and then favors bacilli proliferation3.
CLINICAL MANIFESTATIONS: Leprosy is characterized by
tegumentary lesions and nervous system injury. The clinical presentation
is variable and is determined by the host immunologic system reaction
to the bacilli. During the course of the disease there are the so called
reactional states, in which the immune system reacts against the
bacilli, exacerbating the clinical manifestations. There are two types
of reactional states: reversal reaction (type I), which is more common
in the paucibacillary forms, and erythema nodosum (type II), more
common in multibacillary forms45. The disease is divided into four forms,
according to the criteria established by the World Health Organization:
indeterminate, tuberculoid, dimorphous and virchowian. The diagnosis
and classification are based on clinical findings and complementary tests,
such as baciloscopy, which allow the classification in multibacillary and
paucibacillary.
EFFECTS OF IMMUNOSUPPRESSION, HIV AND
TRANSPLANT IN LEPROSY: At the beginning of the HIV epidemic
there was a fear that HIV infection could increase the risk of leprosy
development or that the co-infection (HIV-leprosy) would cause a more
severe disease46. This hypothesis was not confirmed, since some studies
have shown that patients receiving highly active antiretroviral therapy are
more likely to develop borderline tuberculoid leprosy than other types of
leprosy46. HIV infection has not been reported to increase susceptibility to
leprosy, impact on immune response to M. leprae, or to have a significant
effect on the pathogenesis of neural or skin lesions to date. The initiation
of antiretroviral treatment has been reported to be associated with
activation of subclinical M. leprae infection and exacerbation of existing
leprosy lesions (type I reaction) likely as part of immune reconstitution
inflammatory syndrome6. Leprosy has also been reported to occur after
organ transplantations, but this is not frequent and immunosuppressant
therapy did not seem to affect the course of leprosy manifestations4,55.
The course of leprosy seems not to be affected by immunosuppression55.
RENAL INVOLVEMENT: Renal involvement in leprosy was
first described in the beginning of the XX century, through necropsy
studies, in which glomerulonephritis and tubulointerstitial lesions were
described28,36. Different renal lesions have been described in leprosy,
including acute and chronic glomerulonephritis, interstitial nephritis,
secondary amyloidosis and pyelonephritis19,41,48. There are several reports
of renal involvement in leprosy, as summarized in Table 1.
The exact mechanism that leads to glomerulonephritis in leprosy
is not completely understood. The M. leprae may be directly involved
in renal injury and it has already been detected in glomeruli of infected
patients. The glomerular lesion is probably caused by immunologic
mechanism, with complement decrease and immune complexes
16
deposition in glomerular basement membrane, subendothelial and
subepithelial space, detected by electronic microscopy19,41,48. Some studies
have also detected mesangial proliferation and the presence of IgA in the
mesangial area53. The pathophysiology of renal involvement in leprosy
is illustrated in Fig. 1.
A consistent relation between the lepromatous form, erythema
nodosum and kidney disease has been described in some studies18. Although
leprosy nephropathy is more frequent in the multibacillary form, it can also
occur in other forms and in the absence of the reactional state19.
A large retrospective study with 923 leprosy patients followed in a
tertiary hospital in Brazil found acute kidney injury in 3.8% of cases, and
65% of them had the multibacillary form. Risk factors for kidney injury
were reactional state, multibacillary classification and advanced age10.
RENAL LESION MECHANISM: Erythema nodosum leprosum
is a reactional state characterized by immune complexes formation in
circulation and subsequent deposition in vessels and tissues. Sometimes
they are determined by Hansen’s bacilli antigens which are released into
circulation after the beginning of antibiotic therapy9. The antigens are
recognized by host antibodies, and then immune complexes are formed.
After this, immune complexes can deposit in the glomerulus or can
occur by the formation of immune complexes in situ. However, not all
glomerulonephritis in leprosy are associated with erythema nodosum,
which raises the hypothesis of multifactorial influence in the development
of leprosy nephropathy. In the virchowian form there is a cellular
immunity decrease and a hyperactivation of humoral immunity, which
makes the patient susceptible to the formation of immune complexes30.
The antigen that can induce the formation of immune complexes
can originate from Hansen’s bacilli or even from therapeutic agents.
Anti-dapsone antibodies have been detected in the circulation of leprosy
patients. Auto-antibodies have also been described in leprosy, mainly
cryoglobulins with IgG and IgM13.
Some patients with lower limb ulcers and secondary infections by
Streptococcus presented a higher frequency of glomerulonephritis7.
URINARY FINDINGS: Hematuria has been described in leprosy,
mainly in the virchowian form and during erythema nodosum state, even
in the absence of evident glomerulonephritis18. Microscopic hematuria
is found in 12-16% of cases, which is higher than what is found in the
general population (0.5-2%)7,17,57. This complication can disappear after
a few months of specific treatment9.
Proteinuria has been described in several studies and its prevalence
varies from 2.1 to 68%, and it is also more frequent in the multibacillary
forms7,27,29,39,50. Proteinuria varies from 0.4 to 8.9g/day. Nephrotic
syndrome is not frequent in leprosy. RAMANUJAM et al.44 reported
five cases in the virchowian form, four were in reactional state and only
two had amyloid deposits detected.
Other urinary abnormalities, such as cylindruria and leukocyturia,
are more frequently found in the virchowian form with reactional state.
In the milder forms these abnormalities are uncommon44.
GLOMERULONEPHRITIS: Glomerulonephritis represents the
Table 1
Studies and case reports on kidney involvement in leprosy
Number of
cases
Age (years)
Gender
Iveson (1975)23
1
19
M
Date (1977)12
19
Singhal (1977)53
3
Gupta (1981)20
21
Phadnis (1982)42
50
Chugh (1983)7
60
Reference
Proteinuria
Hematuria
AKI
35
74
Al-Mohaya (1988)
1
17
M
Madiwale (1994)34
2
30-45
M
Ahsan (1995)1
1
79
M
Lau (1995)31
1
71
M
AKI
Nakayama (2001)38
199
47-74
M (79.3%)
Oliveira (2008)40
59
43 ± 15
M (51%)
1
25
F
1
58
M
923
41 ± 19
M (53.3%)
Sharma (2010)49
Silva Junior (2010)
52
Daher (2011)10
Poliarthritis
AKI
Proteinuria
Hematuria
AKI
Jayalakshmi (1987)25
2
Clinical picture
Proteinuria
Proteinuria
Hematuria
Hematuria
AKI
AKI
Drug hepatitis
Kidney biopsy
Diffuse proliferative lesion
Amyloidosis
Acute tubular necrosis
Crescentic nephropathy
Amyloidosis
Membranoproliferative nephropathy
Membranous nephropathy
Amyloidosis
Mesangial proliferative lesion (8.3%)
Diffuse proliferative lesion (8.3%)
Amyloidosis (5%)
Interstitial nephritis
Amyloidosis
Membranoproliferative nephropathy
Interstitial nephritis
Amyloidosis (31%)
Diffuse proliferative lesion (5%);
Focal proliferative (4%);
Membranoproliferative (2%);
Membranous (1%);
Mesangial proliferative lesion (0.5%)
Glomerular sclerosis (11%)
Tubulo-interstitial nephritis (9%)
Granulomata (1%)
Concentration defect (84%)
Acidification defect (30%)
Function loss (50%)
AKI
Proteinuria
CKD
Proteinuria (4.8%)
Hematuria (6.8%)
Function loss (3.8%)
No
AA Amyloidosis
No
M: Male; AKI: Acute kidney injury; CKD: Chronic kidney disease.
most frequent type of kidney disease in leprosy. In renal biopsy studies
glomerulonephritis was found in more than 30% of patients30, which is
higher than what is found in necropsy studies (7%)13. In the multibacillary
form, the prevalence of glomerulonephritis is higher8. Erythema nodosum
has a strong correlation with the occurrence of glomerulonephritis,
although there are some reports of its occurrence in reactional state
type I7,9. Almost all kinds of glomerulonephritis have been described in
leprosy7,13, and there is no specific histopathological pattern in leprosy
nephropathy. There is a discrete predominance of membranoproliferative
glomerulonephritis, which are in general associated with infectious
disease-associated nephropathies20,24,42,49.
HISTOPATHOLOGICAL FINDINGS: The diversity of
histopathological lesions found in leprosy suggests a heterogeneous
17
Fig. 1 - Pathophysiology of renal involvement in leprosy. AKI = acute kidney injury; CKD
= chronic kidney disease.
d i s e a s e bu t n o t n e c e s s a r i l y w i t h d i ff e r e n t e t i o l o g i e s 1 3 .
Immunohistochemical studies with renal tissue have identified the
presence of granular deposits of IgG and C3, and less frequently IgA,
IgM and fibrin in the mesangium and in the glomerular capillaries,
which is characterized by immune complex deposits or in situ formation.
Electronic microscopy confirms the presence of dense granular deposits
in mesangial-subendothelial and subepithelial regions14,26. Complement
consumption in some cases reinforces the hypothesis of an immune
complex-mediated disease30.
A study by GROVER et al.19, with 72 leprosy patients undergoing
renal biopsy found the following histopathological patterns: membranous
nephropathy (31.5%) and mesangioproliferative glomerulonephritis
(11.1%). VALLÉS et al.56 reported one case of IgA nephropathy in a patient
with the virchowian form, with reduction in glomerular filtration rate.
Several renal biopsy studies have been performed in leprosy. JOHNY
et al.26 performed renal biopsies in 35 patients with leprosy and identified
histological abnormalities in 45% of them, and the most frequent was
proliferative glomerulonephritis. GUPTA et al.20 performed renal
biopsies in 21 patients with virchowian leprosy, and found proliferative
glomerulonephritis in 13 of them. GROVER et al.19, in a study with
54 renal biopsies found 12 cases (22.2%) of diffuse proliferative
glomerulonephritis (11 virchowian and one tuberculoid). They also
found two cases of rapidly progressive glomerulonephritis, with acute
kidney injury. Membranous nephropathy was found in 17 cases (31.5%).
PHADNIS et al.42 performed 50 renal biopsies and identified membranous
nephropathy in two cases and membranoproliferative glomerulonephritis
in six cases, of whom 45 had the lepromatous form and had reactional
state. Interstitial nephritis was observed in 10 patients and amyloidosis
in one case. Chronic kidney disease caused by secondary amyloidosis
has also been described in leprosy52 (Fig. 2).
TUBULOINTERSTITIAL LESION: Interstitial nephritis is one
of the most common histological findings in leprosy12,20,37. This has been
described in patients with lepromatous leprosy, and is present in more
than 20% of cases19. It seems to be related to disease duration and the
long-term treatment with nephrotoxic drugs26.
The identification of specific lesions in leprosy is described as
the presence of granulomas in renal interstitium, with evidence of
18
Fig. 2 - Kidney biopsy from a patient with leprosy and chronic kidney disease showing amyloid
deposits (A), H&E, x200; glomeruli without mesangial proliferation, with amyloid deposit
in mesangium, H&E, x400; amyloid deposit, H&E x200; tubules without abnormalities,
H&E x200. Reprinted from Silva Junior et al. Rev Soc Bras Med Trop. 2010;43:474-6.52
mononuclear cells with vacuolized cytoplasm, without the presence
of Hansen’s bacilli43,47. Epithelioid granuloma and the Hansen’s bacilli
have already been detected in renal parenchyma38. The low incidence of
granulomas in renal tissue is due to the fact that renal tissue presents a
resistance to M. leprae or the fact that the bacteria has a low affinity to
renal tissue42.
The occurrence of tubular dysfunction is frequent, varying from 25 to
85% of cases, in both multibacillary and paucibacillary forms7,40. Urinary
acidification defect has been described in 20 to 32% of patients, and
urinary concentration defect in 85% of cases16,40. Renal tubular acidosis
has also been described16,21,40.
CHRONIC KIDNEY DISEASE: Chronic kidney disease (CKD)
has been reported as one of the causes of death in leprosy, mainly in the
first studies of leprosy nephropathy5,28,36,43,. CKD is mainly caused by
amyloidosis and is also more frequent in the virchowian form26,33,52. It has
also been reported in patients with the tuberculoid form33. A correlation
between the duration of the disease and the development of amyloidosis
has not been observed26. A positive correlation was detected between
the occurrence of erythema nodosum and secondary amyloidosis in
leprosy15,32,33. Serum levels of amyloid protein A increases in erythema
nodosum episodes and remains high for several months. LOMONTE et
al.32 described the evolution of eight patients with leprosy who developed
CKD and required renal replacement therapy.
DRUGS TOXICITY: Despite not being common, renal abnormalities
due to leprosy specific treatment have been described. There are reports
on acute tubular necrosis, interstitial nephritis and papillary necrosis
causing acute kidney injury in leprosy7,15.
Acute kidney injury can occur due to interstitial nephritis secondary
to rifampicin use, which is more common with higher doses (9001200mg) than the usual (450-600mg)22. Dapsone can induce hemolysis
and intravascular coagulation, which can lead to acute tubular necrosis54.
TREATMENT: Leprosy treatment encompasses specific therapy
to overturn M. leprae, avoid immunological complications and
prevent physical deformities, simultaneously promoting physical and
psychosocial rehabilitation. Additionally, health authority notification
is mandatory35. WHO-standardized leprosy therapy includes rifampicin,
dapsone and clofazimine. Prednisone (1 to 2 mg/kg/day) and nonsteroidal anti-inflammatory drugs (NSAI) may be used to control acute
immunological episodes. Erythema nodosum leprosum may sometimes
have a protracted course (months, or years) and is usually treated with
NSAI, steroids, thalidomide, clofazimine and pentoxiphyline. It must be
kept in mind that all are potentially nephrotoxic. Hemodialysis or kidney
transplant are alternatives in treating leprosy ESRD. Post-transplant
immunosuppression apparently does not modify leprosy response to
drugs. However, acute transitory deterioration of its course has been
reported4.
CONCLUSION
Renal involvement is an important complication in leprosy, which
should be investigated in every patient. Multibacillary status seems to
be the main risk factor for kidney dysfunction in this disease. Different
kinds of glomerulopathy have been described in association with leprosy.
Specific treatment seems to impact on renal function improvement.
RESUMO
Nefropatia da hanseníase: revisão dos aspectos clínicos e
histopatológicos
A hanseníase é doença crônica causada pelo Mycobacterium leprae,
altamente incapacitante e com envolvimento sistêmico em alguns casos.
O envolvimento renal tem sido relatado em todas as formas da doença,
sendo mais frequente nas formas multibacilares. A apresentação clínica é
variável e determinada pela reação do sistema imunológico do hospedeiro
ao bacilo. Durante o curso da doença podem ocorrer os chamados estados
reacionais, nos quais o sistema imune reage contra o bacilo, exacerbando
as manifestações clínicas. Diferentes lesões renais tem sido descritas na
hanseníase, incluindo glomerulonefrites, nefrite intersticial, amiloidose
secundária e pielonefrite. O mecanismo exato que leva à glomerulonefrite
na hanseníase ainda não está completamente esclarecido. O tratamento da
hanseníase inclui o uso de rifampicina, dapsona e clofazimina. Prednisona
e antiinflamatórios não-hormonais podem ser usados no controle dos
episódios imunológicos agudos.
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Received: 19 March 2014
Accepted: 2 June 2014
http://dx.doi.org/10.1590/S0036-46652015000100003
SURVIVAL, INDUCTION AND RESUSCITATION OF Vibrio cholerae FROM THE VIABLE
BUT NON-CULTURABLE STATE IN THE SOUTHERN CARIBBEAN SEA
Milagro FERNÁNDEZ-DELGADO(1,2), María Alexandra GARCÍA-AMADO(2), Monica CONTRERAS(2),
Renzo Nino INCANI(3), Humberto CHIRINOS(4), Héctor ROJAS(5) & Paula SUÁREZ(1)
SUMMARY
The causative agent of cholera, Vibrio cholerae, can enter into a viable but non-culturable (VBNC) state in response to unfavorable
conditions. The aim of this study was to evaluate the in situ survival of V. cholerae in an aquatic environment of the Southern Caribbean
Sea, and its induction and resuscitation from the VBNC state. V. cholerae non-O1, non-O139 was inoculated into diffusion chambers
placed at the Cuare Wildlife Refuge, Venezuela, and monitored for plate, total and viable cells counts. At 119 days of exposure to
the environment, the colony count was < 10 CFU/mL and a portion of the bacterial population entered the VBNC state. Additionally,
the viability decreased two orders of magnitude and morphological changes occurred from rod to coccoid cells. Among the aquatic
environmental variables, the salinity had negative correlation with the colony counts in the dry season. Resuscitation studies showed
significant recovery of cell cultivability with spent media addition (p < 0.05). These results suggest that V. cholerae can persist in the
VBNC state in this Caribbean environment and revert to a cultivable form under favorable conditions. The VBNC state might represent
a critical step in cholera transmission in susceptible areas.
KEYWORDS: Vibrio cholera; In situ survival; VBNC state; Resuscitation; Aquatic environments; Cholera.
INTRODUCTION
Vibrio cholerae is one of the most important waterborne pathogens
and the causative agent of cholera8, a disease of great public health
concern in developing countries with low socio-economic status7. This
microorganism is a natural inhabitant of aquatic environments, which
could act as a source and reservoir for human infections8. It has been
found to survive for extended periods in estuarine and brackish waters
and to undergo conversion to a dormancy or viable but non-culturable
state (VBNC)8,35. This state is a survival strategy adopted by many
bacteria when environmental conditions are unsuitable for sustaining
normal growth. In this physiological condition bacteria exhibit detectable
metabolic function, but are not culturable by conventional laboratory
culture methods27. It has been shown that VBNC cells are reduced in
size and become coccoid6,36, and sustain certain functions like metabolic
activity, specific gene expression18, antibiotic resistance27, virulence28
and their pathogenic potential for a prolonged time36. Environmental
conditions are involved in the induction of VBNC state, notably low
nutrient concentrations8, suboptimal and downshift temperatures3,28,
elevated salinity, extreme pH or solar radiation11.
Since the concept of the VBNC state was introduced 30 years ago
a significant body of research has been done, serving V. cholerae as the
prototype8. Several in vitro induction studies have been carried out in
autoclaved water8, salt water, buffered saline10, alkaline seawater3,18,
freshwater microcosms25 and conditioned medium22. Resuscitation of V.
cholerae from the VBNC state has been demonstrated in the intestines
of human volunteers10 and recently in vitro by temperature upshift25
and co-culture with eukaryotic cells32. However, little information is
available on the natural behavior of this microorganism, the dynamics
of the VBNC state in the environment and the mechanisms whereby
non-culturable cells become culturable to initiate seasonal epidemics of
cholera, especially from the Caribbean Sea.
Many recent cholera outbreaks have occurred in this part of the world
as large outbreaks or as sporadic cases19,30. In Venezuela, there have been
several epidemics of this disease caused by V. cholerae O1 biotype El
Tor16. The pathogen has not been recovered from the environment during
interepidemic periods, but instead non-O1, non-O139 strains have been
isolated from seawater and planktonic organisms on the Northwestern
coast of this country17. These serogroups were associated with occasional
outbreaks of cholera-like diseases close to the area23. Because of the
public health importance of the VBNC state and the existing cholera
risk in the Caribbean Sea19, the objective of this study was to evaluate
the in situ survival of V. cholerae by using a diffusion chamber (DC)
approach to allow the exposure of the microorganism to the natural
conditions of this environment. Moreover, the study aimed to examine
in vitro resuscitation procedures to test the recovery of the VBNC cells,
(1) Departamento de Biología de Organismos, Universidad Simón Bolívar, Caracas, Venezuela.
(2) Centro de Biofísica y Bioquímica, Laboratorio de Fisiología Gastrointestinal, Instituto Venezolano de Investigaciones Científicas, Altos de Pipe, Edo. Miranda, Venezuela.
(3) Departamento de Parasitología, Universidad de Carabobo, Valencia, Edo. Carabobo, Venezuela.
(4) Asociación de Lancheros de Chichiriviche, Edo. Falcón, Venezuela.
(5) Instituto de Inmunología, Universidad Central de Venezuela, Caracas, Venezuela.
Correspondence to: Dr. Paula Suárez, Departamento de Biología de Organismos, Universidad Simón Bolívar, 1080 Caracas, Venezuela. Phone: +58.212.9063070. Fax: +58.212.9063047.
E-mail: [email protected]
FERNÁNDEZ-DELGADO, M.; GARCÍA-AMADO, M.A.; CONTRERAS, M.; INCANI, R.N.; CHIRINOS, H.; ROJAS, H. & SUÁREZ, P. - Survival, induction and resuscitation of Vibrio
cholerae from the viable but non-culturable state in the Southern Caribbean Sea. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 21-6, 2015.
due to the possibility that the bacterium may resuscitate and start dividing
upon access to the host.
MATERIALS AND METHODS
Bacterial strain and culture conditions: Vibrio cholerae non-O1,
non-O139 strain D3-TCBS was obtained from seawater samples collected
in December 2004 at Cueva de la Virgen, Cuare Wildlife Refuge
(10°54´23´Ń, 68°18´10´´W), a protected environment also designated
as a touristic and shellfish-growing marine area at the Northwestern
coast of Falcon State, Venezuela. The strain was cultured according to
FERNÁNDEZ-DELGADO et al.17, stocked in nutrient broth (HIMEDIA)
supplemented with 15% glycerol at -80 °C and deposited at the Centro
Venezolano de Colecciones de Microorganismos (CVCM) (No. 1742).
In situ survival study: Vibrio cholerae D3-TCBS was grown in
BHI (HIMEDIA) at 37 °C in mid-logarithmic growth phase. The cells
were harvested by centrifugation at 327 g for 15 min at 4 °C and washed
twice with artificial seawater (ASW)5, previously autoclaved at 121 °C
for 15 min and filtered through a 0.22-µm pore-size filter (Millipore). A
bacterial suspension in nutrient-free ASW (final concentration 107 cells/
mL) was aseptically injected into sterile three mL DC, a modification
of those of KAEBERLEIN et al.20, and fitted with 0.03-µm pore-size
polycarbonate membranes (GE Water & Process Technologies), as
described20. A number of three DCs filled with ASW without bacterial
inoculum were considered as negative controls of the study. Time zero
samples were taken for further culturability and microscopic analysis.
At the study site, a total of thirty-three chambers were placed vertically
in open containers, immersed at approximately one m below the surface
in Caño Las Carmelitas (10°55´63´Ń, 68°17´50´´W) near the sampling
site where the study’s V. cholerae strain was isolated, and exposed to
the natural environment for 119 days. During this period, sets of three
DCs were sampled at various intervals, kept in containers with the
natural seawater under refrigeration and returned to the laboratory to
be processed.
The seawater in situ values of pH (pHep1, Hanna Instruments),
salinity (RHS-10ATC refractometer, Westover Scientific), temperature
and dissolved oxygen (OXDP-02 oxygen meter, VWR International,
Inc.) were monitored throughout the study.
Culturability, cell counting and viability assays: Culturability
was determined in triplicates by spread plate count. One milliliter of
the material inside the chambers was removed and serial dilutions of
suspensions were plated onto BHI agar (HIMEDIA). All the colonies
on plates containing fewer than 300 colonies were counted to estimate
the colony-forming unit (CFU) per milliliter, after 48 h of incubation at
37 °C. V. cholerae cells were considered to be in a non-culturable state
when counts reached < 10 CFU/mL1,26.
The number of total cells per milliliter was determined by direct
microscopic count method using the blue fluorescent dye 4’,6-diamidino2-phenylindole (DAPI, Sigma). Aliquots of bacterial suspensions from
the chambers were fixed with formaldehyde (3% v/v), diluted in filtersterilized ASW, stained with DAPI (5.0 µg/mL, final concentration) for
three min and filtered onto 0.22-µm pore-size black polycarbonate filters
(Millipore) in the dark. After three rinses in filter-sterilized distilled water,
the membrane filter was placed on a slide and a cover slip was placed
22
directly on top of the filter. Additionally, the viability or membrane
integrity of bacterial cells was assessed by the LIVE/DEAD BacLight kit
(Molecular Probes Inc.). This kit utilizes a mixture of the reagents Syto-9
(a green fluorescent nucleic acid stain) and propidium iodide (PI, a red
fluorescent nucleic acid stain). Syto-9 generally labels all bacteria in a
population (both cells with intact membranes and damaged membranes).
In contrast, PI penetrates only bacteria with damaged membranes and
causes a reduction in the Syto-9 stain fluorescence when both dyes
are present. Ideally, healthy living bacteria with an intact cytoplasmic
membrane stain with a green fluorescence, and dead or injured cells with
a compromised membrane stain fluorescent red34. These reagents were
prepared according to the manufacturer instructions and mixed in equal
proportions. A minimum of 15 random fields were visualized for total
and viable cell counts under a Nikon TE 2000 fluorescent microscope
(Nikon Instrument Inc.). For LIVE/DEAD BacLight stain, a xenon lamp
of 100-W was used to deliver light to two filter sets, one set of filters
with 485/530 nm of excitation and emission, respectively, and another
with 550/615 nm of excitation and emission filters. For DAPI dye a set
of 330/450 nm of excitation and emission filters was used. Samples were
observed using an oil-immersion objective (100X/0.5-1.3 NA Nikon).
All the experiments were carried out in triplicate.
Resuscitation studies: To attempt the recovery of culturability in V.
cholerae non-O1, non-O139 cells a series of two in vitro approaches were
performed when the titer of colony counts in the DC samples declined <
10 CFU/mL. Total cell counts were performed as described previously. To
evaluate the effect of nutrients, initial resuscitation assays were performed
in 96-well microplates (Corning Incorporated) containing either 50 µL
BHI or 50 µL HP broth13 modified without the addition of antibiotics.
The use of HP selective medium originally designed for the isolation of
Helicobacter pylori from freshwater samples13 allowed the isolation of
V. cholerae from this aquatic environment17. Bacterial cells from three
DCs were serially diluted 10-fold into filter-sterilized ASW (10-1-10-7)
and 50 µL samples were taken from undiluted and from each dilution
sample and added to twelve replicate wells. A number of two plates
were considered for each medium and DC replicate. Wells containing
the two media without bacterial inocula and wells with media inoculated
with an active growing culture of the same strain (with a known number
of total cells and plate counts) were reserved as negative and positive
controls, respectively. Plates were incubated at 37 °C, with shaking (150
rpm) for seven days. Evidence of growth was registered by measuring
optical density (600 nm) of cultures using a microplates reader (Tecan),
considering day 0 of the study as the starting point.
Secondly, the effect of spent media (SM, growth media consisting
of filter-sterilized culture supernatant) on the recovery of V. cholerae
cells was investigated. In this study, SM was obtained from V. cholerae
D3-TCBS cultures harvested at mid-logarithmic and stationary phases
and subjected to centrifugation (327 g, 15 min). Cell-free supernatants
were filtered twice with disposable syringe filters of 0.22-μm pore
size (Millipore) and stored at -80 °C before use. The wells from plates
containing nutrient media and DC samples without evident growth
at seven days of incubation were amended with SM, considering one
plate for each stage and DC replicate. A number of these wells were
left without addition of SM, as controls for spontaneous resuscitation.
Other controls of contamination consisted in each stage of SM alone
and amended with BHI and HP media. The plates were incubated at
37 °C with agitation for another seven days. Growth was monitored
by measuring the optical density as described previously, including the
time zero for this assay.
Statistics: The linear dependence between two variables (Pearson
correlation analysis) in the in situ survival study and the effect of nutrients
and SM addition on the resuscitation of VBNC cells (Student test of
unpaired data) were analyzed by OrigenPRO 7.5 SR6 (Origen Lab
Corporation). p values < 0.05 were considered significant.
The environmental parameters: temperature, pH, salinity and dissolved
oxygen of seawater registered during the present survival study ranged
from 27.2-31.8 °C, pH 6.5-7, 2-32‰ and 4-7.9 mg/L, respectively. The
most important variation of these seasonal conditions was the salinity of
seawater, which was found with two distinct patterns during the rainy (from
0 to 21 days) and dry periods (from 21 to 119 days) considered in this study.
When salinity was between two and 18‰, the culturability of V. cholerae
was higher than 1 x 104 CFU/mL, whereas salinities higher than 18‰
produced cultivability of up to three orders of magnitude fewer (Fig. 2).
RESULTS
V. cholerae showed declining recoverability on exposure to the
aquatic environment and to nutrient depletion conditions. A large
population of this microorganism progressively became non-culturable
over a period of 119 days when the titer of culturable or colony counts
decreased four orders of magnitude (from 2 x 105 to 1 x 101 CFU/mL),
and the number of live cells with membrane integrity was 5.2 x 105
cells/mL. Regardless of whether the cells could be grown on agar, they
could be seen under the microscope by direct total count within 106-107
cells/mL. A great difference between colony counts and total cell counts
was observed since time zero of the study. Increases in the period of V.
cholerae exposure to the natural environment (up to 119 days) resulted
in a progressive enhancement of non-culturable cells (Fig. 1). From these
data, three subpopulations of cells could be inferred at the end of the
survival studies: culturable (0.00013%), VBNC (6.80%), and nonviable
(93.20%). Morphological changes and decreased size of bacterial
cells were observed since the first days of incubation in the natural
environment, comprising a large population of coccoid forms. These
results indicate physiological changes during the prolonged exposure
of V. cholerae cells to the aquatic environment which could promote
the bacterial survival but decrease the recovery of stressed cells on BHI
agar. Preliminary in situ survival studies and microcosm experiments of
V. cholerae non-O1, non-O139 in ASW at 17 °C were performed with
similar viability and cultivability results (data not shown).
Fig. 2 - Culturability of Vibrio cholerae cells associated with seawater salinity during
sampling time. The broken line distinguishes the two seasons: rainy (from 0 to 21 days) and
dry (from 21 to 119 days). Data are mean ± SE values of triplicate samples. Colony counts
() and salinity ().
There was no significant correlation between the colony counts and
salinity for the first 21 days (r = 0.704; p = 0.295), while a significant
inverse correlation was found for the second period of the study after day
21 until day 119 (r = -0.980; p = 0.019). The reduced levels of salinity
registered in the first month of this study coincided with the local rainy
season which increases the drainage of adjacent freshwater bodies and
may modify the physicochemical conditions of this coastal area.
Results from resuscitation experiments initially showed regrowth of V.
cholerae non-culturable cells in the undiluted samples by the addition of
BHI and HP media. However, after serial dilutions no growth was evident
with both media. The addition of SM at logarithmic and stationary phases
to those wells containing 10-2 diluted samples, previously supplemented
with either HP or BHI, showed the recovery of non-culturable cells and
was found to be significant (p < 0.05) (Fig. 3).
DISCUSSION
Fig. 1 - In situ survival of Vibrio cholerae on exposure to natural conditions of the Cuare
Wildlife Refuge. Data are mean ± SE values of triplicate samples. Total cell counts (),
viability cell counts (), and culturable counts ().
Cholera is a disease of great public health concern in developing
countries and has recently re-emerged in a Caribbean coastal area
with severe outbreaks or sporadic cases. The reasons of the unusual
dynamic of cholerae outbreaks and the status regarding the V. cholerae
population in this region are not completely clear19,30. Although the
ecology of V. cholerae in marine and estuarine ecosystems as well as
its viability in laboratory microcosms has been well studied8, only one
23
might be important in understanding the local environmental drivers of
cholera outbreaks in the Caribbean region.
Fig. 3 - Resuscitation of Vibrio cholerae cells from the VBNC state by adding spent media
(SM) at logarithmic (SM1) and stationary (SM2) phases in samples diluted 10-2. SM day 0 ( )
and SM day 7 ( ). Data are mean ± SE of a minimum of 23 and a maximum of 36 replicate
values. The symbol * shows significant effect (p < 0.05) for paired data of SM addition on
the resuscitation of VBNC cells.
piece of research has reported the in situ survival of this microorganism
in aquatic environments29. However, several studies have employed
diffusion chamber approaches to assess the VBNC state in other Vibrio
and bacterial species27. The present work reveals, for the first time, the
capacity of V. cholerae to enter the VBNC state when exposed to the
real conditions of a coastal area of the Southern Caribbean Sea where
this bacterium was isolated, in order to better understand its behavior
in this environment as a potential natural reservoir. V. cholerae non-O1,
non-O139 survived for extended periods of time (approximately four
months), demonstrating a decrease in culturability and viability, as has
been reported for this species10. The bacterial cells reduced their size and
changed their morphology from rods to coccoid. This is in agreement
with the description of the V. cholerae coccoid morphology in the VBNC
state as an adaptation of the cells to environmental constraints6,22,25.
The entrance of V. cholerae to this state was probably mainly induced
by the exposure to the constant nutrient depletion conditions inside
the chambers during the study. Starvation has been recognized as an
important stimulus to enter the VBNC state and represents a common
strategy for survival among bacteria in nutrient-poor environments8.
Moreover, the study found a starvation response of V. cholerae in
combination with high salinity values of seawater during the dry season
(after day 21 to 119) that caused a decrease on the cell culturability
with a significant inverse correlation, although in the first 21 days the
colony counts were not affected by the salinity decline during the rainy
season. Prior to the authors’ research, many surveys were conducted on
the Northwestern coast of Venezuela in search of culturable forms of V.
cholerae, and only non-O1, non-O139 strains were recovered during an
intense rainy season with low salinity waters17. Similar reduced levels
of salinity registered in the bacterial cultivability period of this research,
occurred during the local rainy season; such low salinity has been widely
reported as optimal (between 5‰ and 25‰) for this microorganism in
aquatic environments24. Uncovering the influence of rainfall and salinity
fluctuations on V. cholerae recoverability from these marine environments
24
The role of the VBNC state in cholera epidemiology is vital, not just
because the bacterium can persist in harsh environmental conditions, but
also because of its potential to revert back to a fully potent pathogenic
form and contribute to the spread of the disease25, as has been shown with
other Vibrio spp. in mouse models where non-culturable cells remained
virulent and were capable of causing fatal infections following in vivo
resuscitation28. Therefore, it is important to define the mechanisms by
which non-culturable cells return to culturability2. Resuscitation studies
reported here show recovery of cell cultivability when SM at logarithmic
and stationary phases were used along with 10-2 diluted cells, whereas
nutrient addition did not show true resuscitation suggesting the presence
of culturable cells in the undiluted samples. It has often been questioned
whether resuscitation of apparently non-culturable cells represents ‘true’
resuscitation of all cells of the initial inoculum that had become VBNC,
regrowth of only a few non-culturable cells remaining viable (able to revert
to active growth), or merely growth of a very few culturable cells5,25,27. In
the present study, the resuscitation of non-culturable cells was attempted
after dilution with several resuscitation procedures, along with different
enrichment media and SM addition. A previous step of either HP or BHI
addition did not increase the recovery of V. cholerae cells in the diluted
samples. However, significant growth at 10-2 dilution was observed after
the addition of SM at logarithmic and stationary phases. The culture
supernatants could contain components that apparently aided in the
recovery of a number of non-culturable V. cholerae cells. These findings
are likely related to observations of other authors who report an increase
in the recovery of bacteria after adding cell-free supernatants from active
cultures, whose results indicate that intercellular communication or growthpromoting factors are likely to improve their culturability4,21,31,33.
The observations of the present study’s authors suggest that a fraction
of the cell population is able to recover culturability. A physiological
heterogeneity could exist within this V. cholerae population, as has been
reported with V. cholerae O1 cells resuscitated by temperature upshift24
and with other Vibrio species12. Recently, EPSTEIN14 described this
heterogeneity as a percentage of cells that are different from the rest of the
population due to the lack of growth restrictions typical of the majority,
and proposed a signaling scout model to explain the cell heterogeneity14.
In this model, a few viable cells or scouts have a signaling function in
the dormant population and may start a new population by waking up
the dormant cells15. Considering this principle, the resuscitation of V.
cholerae presumably occurred in those diluted samples that had scouts
or possible growth-inducing factors. These in vitro resuscitation studies
could mimic what happens with non-culturable V. cholerae strains during
times of stress or interepidemic periods if eventually the presence of
favorable environmental conditions or the availability of nutrients and
the appearance of signaling cells, enhance their recovery from the VBNC
state in these aquatic environments. More work is required to study the
resuscitation of VBNC cells and the compounds possibly present in these
culture supernatants.
In conclusion, these results emphasize the need to study nonculturable V. cholerae in areas of the Caribbean Sea susceptible to
cholera epidemics, considering that this bacterium can persist in the
environment in a VBNC state and revert to a transmissible form in the
presence of suitable conditions. Because the VBNC state might represent
a critical step in cholera transmission around the world and particularly
in the Caribbean and Latin American region, this research encourages
investigators, governments and communities involved in public health
to implement and not neglect the programs for prevention, systematic
environmental monitoring and surveillance of culturable and VBNC V.
cholerae through the global networks.
RESUMEN
Supervivencia, inducción y resucitación de Vibrio cholerae del
estado viable no cultivable en el sur del Mar Caribe
El agente causal del cólera, Vibrio cholerae, puede entrar a un estado
viable no cultivable (VNC) en respuesta a condiciones desfavorables.
El objetivo de este estudio fue evaluar la supervivencia in situ de V.
cholerae en un ambiente acuático al sur del Mar Caribe y su inducción
y resucitación del estado VBNC. V. cholerae no-O1, no-O139 fue
inoculado en cámaras de difusión ubicadas en el Refugio de Fauna Cuare,
Venezuela, y monitoreado para contaje de colonias, células totales y
viables. En 119 días de exposición al ambiente, el contaje de colonias
fue < 10 UFC/mL y una fracción de la población bacteriana entró al
estado VBNC. Adicionalmente, la viabilidad disminuyó dos órdenes
de magnitud y ocurrieron cambios morfológicos de células bacilares
a cocoides. Entre las variables del ambiente acuático, la salinidad
presentó correlación negativa con el contaje de colonias. Los estudios
de resucitación mostraron recuperación significativa de la cultivabilidad
celular con adición de sobrenadantes de cultivos en crecimiento activo
(p < 0.05). Estos resultados sugieren que V. cholerae puede persistir en
estado VBNC en este ambiente de Caribe y revertir a una forma cultivable
bajo condiciones favorables. El estado VBNC podría representar un paso
crítico en la transmisión del cólera en áreas susceptibles.
ACKNOWLEDGEMENTS
This work was partially funded by grants from Decanato de
Investigación y Desarrollo (DID) to P.S. and Decanato de Postgrado
to M.F. of Universidad Simón Bolívar, and grants from the Instituto
Venezolano de Investigaciones Científicas to M.A.G. and M.C. The
authors gratefully acknowledge S. Epstein for his valuable advice
on scientific aspects of the project and supplies facilities, and also to
reviewers of this manuscript for their useful suggestions.
AUTHOR CONTRIBUTIONS
M. Fernández-Delgado: Sampling, experimental procedures, results
analysis and manuscript preparation. M. A. García-Amado: Results
analysis. M. Contreras: Results analysis. R. N. Incani: Sampling,
laboratory support and manuscript preparation. H. Chirinos: Sampling. H.
Rojas: Microscopic and statistical analysis, and manuscript preparation.
P. Suárez: Sampling, results analysis and manuscript preparation.
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Accepted: 28 May 2014
http://dx.doi.org/10.1590/S0036-46652015000100004
ENTEROPATHOGENS DETECTED IN A DAYCARE CENTER, SOUTHEASTERN BRAZIL: BACTERIA,
VIRUS, AND PARASITE RESEARCH
Edna Donizetti Rossi CASTRO(1)*, Marcela Cristina Braga Yassaka GERMINI(1)*, Joana D’Arc Pereira MASCARENHAS(2),
Yvone Benchimol GABBAY(2), Ian Carlos Gomes de LIMA(2), Patrícia dos Santos LOBO(2), Valéria Daltibari FRAGA(1),
Luciana Moran CONCEIÇÃO(1), Ricardo Luiz Dantas MACHADO(1,3) & Andréa Regina Baptista ROSSIT(1,4)
SUMMARY
Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children
from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government
daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of
246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventythree strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of
mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive
of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for
the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm
was observed. A significant association was found between food consumption outside home and daycare center and the presence of
intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have
contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of
geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the
modulation of the frequency of different enteropathogens.
KEYWORDS: Childhood diarrhea; Gastroenteritis; Daycare center; Enteropathogens.
INTRODUCTION
In 2009, the World Health Organization (WHO) estimated that
approximately nine million under 5-year-old children die each year.
Diarrhea is reported to be the second most important disease in the
etiology of infant death33. In developing countries, gastrointestinal
disorders are directly associated with higher infant morbidity
and mortality in this age group, resulting in 2.5 million deaths
per year28.
Infectious diarrhea is generally caused by bacterial, viral and
parasitic pathogens whose actions may result in malabsorption of water,
electrolytes and nutrients30. Intestinal infections are closely correlated
to young age, immune status, nutritional deficiencies, inadequate food
hygiene practices, early weaning, level of schooling of guardians/
caregivers, gatherings at home and in institutions such as daycare centers
and schools, lack of basic sanitation, access to treated water and the high
temperatures during the year23.
Recently, profound changes in some urban centers’ workforce have
been reported in Brazil, and as a result, a large number of children are
being cared for outside the familial environment in daycare centers29. Due
to the greater urbanization and the effective participation of women in
the economically active working force, these institutions have become
the main place frequented by children outside their home environment
and as such, a potential environment for contamination36.
Although infectious enteric diseases are more evident in children
with severe infections that culminate in hospitalization, a large number
of infections are endemic in the community, presenting as asymptomatic
or with mild clinical symptoms. This is particularly true for parasites,
as the progression is often slower30. In Brazil few official data about
the in depth prevalence of infectious intestinal diseases in children are
available, especially those including the infectious agents implicated
in the etiology of these diseases in different regions of the country3.
Nevertheless, such knowledge is essential for the development of effective
prevention strategies.
The objective of the current study was to determine the prevalence of
these infections and the etiology of diarrhea in the infant population of a
daycare center situated in an urban area of disadvantaged socioeconomic
conditions.
*The authors contributed equally to this work.
(1) Microorganisms Research Centre, Sao Jose do Rio Preto Faculty of Medicine, Sao Paulo, Brazil.
(2) Virology Section, Evandro Chagas Institute, Ananindeua, Pará, Brazil.
(3) Parasitology Section, Evandro Chagas Institute, Ananindeua, Pará, Brazil.
(4) Department of Microbiology and Parasitology, Biomedical Institute of the Fluminense Federal University, N iterói, Rio de Janeiro, Brazil.
Correspondence to: Prof. Dr. Ricardo Luiz Dantas Machado. Seção de Parasitiologia, Instituto Evandro Chagas, BR153, Km 7, s/n, Levilândia, 67030-000 Ananindeua, Pará, Brasil.
Phone: +55.91.32142089. E-mail: [email protected]
CASTRO, E.D.R.; GERMINI, M.C.B.Y.; MASCARENHAS, J.D.P.; GABBAY, Y.B.; LIMA, I.C.G.; LOBO, P.S.; FRAGA, V.D.; CONCEIÇÃO, L.M.; MACHADO, R.L.D. & ROSSIT, A.R.B.
- Enteropathogens detected in a daycare center, Southeastern Brazil: bacteria, virus, and parasite research. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 27-32, 2015.
MATERIAL AND METHODS
Study region and participants: In the period from October 2010 to
February 2011, stool samples of 100 children were tested in a government
daycare center in São José do Rio Preto (latitude 20º89’72’’ S and
longitude 49°37’44’’ W at an altitude of 489 m above sea level) located
in the Northwest São Paulo State region, 450 km from the city of São
Paulo. The population of this city is 419,632 with an area of 431 km2.
Participants were allocated in two groups: Diarrheal Group - defined by
the occurrence of three or more liquid or semi- liquid evacuations in the
24 hours before fecal collection and Non-diarrheal Group - comprised of
children from the same daycare center paired by age and gender. Fecal
samples were collected in clean flasks and then sent immediately to the
Microorganisms Research Centre of FAMERP for parasitological and
microbiological analysis. A standard questionnaire was applied to assess
the children’s socioeconomic conditions including information on age,
gender, family structure, hygiene and water consumption source and
treatment in their homes. Structural characteristics of the daycare center
were also obtained.
The project was approved by the Research Ethics Committee of
FAMERP (Protocol CEP 6332/209) and written informed consent was
provided by parents or guardians after a detailed explanation of the
objectives of the work.
Laboratorial analysis: Stool samples were evaluated using Cary-Blair
transport medium for bacterial analysis. A second clean flask was used
to collect stools to investigate parasites and viruses.
Mon 431/433 partial region of the RNA- dependent RNA polymerase
-RdRp) that detects the NoVs genogroups I and II, respectively35. The
human astrovirus (HAstV) study was performed using the PCR and
primers Mon 269 and Mon 270 (ORF2 region)35. PCR products were
subjected to 1% agarose gel electrophoresis using SYBR Safe DNA Gel
Stain (Invitrogen, Eugene, Oregon, USA). Photodocumentation was
performed using the Gel Doc 1000 imaging system (BioRad, Hercules,
CA). Samples with an amplicon of 213 and 449 base pairs (bp) were
considered positive for norovirus and astrovirus respectively.
The RNA obtained in the fecal specimens was tested for rotavirus
species A and C using polyacrylamide gel electrophoresis as described by
PEREIRA et al.38. RT- PCR was employed to detect rotavirus species C
as described by GOUVEA et al.21 and KAZUYA et al.25 for the VP6 and
VP7 genes, respectively. Products were stained using SyBR® safe DNA
Gel Stain (Invitrogen TM, Eugene, Oregon, USA) in the loading buffer,
run in 1.5% agarose gel and documented in a Doc Gel 1000 imaging
system (BioRad, Hercules, CA). Samples that presented fragments of
356 bp and 1027 pb were considered positive, for the VP6 and VP7
genes, respectively.
Statistical analysis: Statistical analysis was performed using the
Epi-Info statistics program (version 6.0). The v2 test or the Fisher exact
test was applied to obtain independence between proportions. The
relationship between the clinical characteristics of the participants and the
presence of enteropathogens was assessed using the Wilcoxon Rank Sum
test. The level of significance adopted for statistical inference was 5%.
RESULTS
Enterobacteria: All samples were analyzed on the day of collection.
Briefly, colonies were isolated using McConkey agar, ShigellaSalmonella agar, brilliant green (after enrichment in tetrathionate
broth) and Columbia Agar to isolate and identify the following bacteria:
enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli
(EHEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC),
Shigella spp., Salmonella spp., Yersinia spp. and Campylobacter jejuni.
Isolates identified by biochemical tests (EPM-Milli and Oxidase stripes)
were serotyped by standard techniques in addition to commercially
available antisera (Probac, Brazil).
Parasites: For each patient, a fecal sample was collected in a universal
sterile container with no preservative solution and maintained at 4 °C
until laboratory analysis on the same day. Flasks were labeled with the
name of the patient and data on collection. The Hoffman-Pons-Janer24,
centrifugal flotation in zinc sulfate solution13 and Baermann-Moraes
methods were used to prepare samples. Two slides were examined for
each stool sample to detect parasites using optical microscopy (Nikon,
Japan) with magnifications of 100 × and 400 ×, by two researchers.
Enteric viruses: The detection of rotavirus, norovirus and astrovirus
was performed by molecular methods using the same fecal suspension
used in the investigation of parasites, which was diluted in water and kept
at a low temperature until the test. In brief, viral RNA was extracted as
described by BOOM et al. with modifications4,6. Reverse transcription
(RT) was performed to obtain complementary DNA using a random
primer [hexamer pd(N)6-50 A260 units; Amersham Biosciences,
Freiburg, Germany]. For norovirus detection the polymerase chain
reaction (PCR) was used with the pool of primers Mon 432/434 and
28
Of the 100 fecal samples tested, 50 were in the Diarrheal Group and
50 in the Non-diarrheal Group. There were no differences between the
two groups regarding gender. As summarized in Table 1, most individuals
lived in treated water and sanitary sewer system available houses. No
significant association was observed between these variables and the
presence of enteropathogens (data not shown) or diarrhea, except for
food consumption outside home or the daycare center and the presence
of intestinal parasites (Chi-square; p-value = 0.0053).
A total of 246 bacteria were isolated in 99% of all fecal samples; 129
were in the Diarrheal Group and 117 in the Non-diarrheal Group. Of this
total, 44 children had at least one species of bacterium, 37 children had
two, while 17 children had three. Four distinct bacteria were isolated in the
fecal material of one of the children. Seventy-three strains of Escherichia
coli were isolated (73.7% of the E. coli O157 strain), 19 Enterobacter
(19.1%), one Alcaligenes (1.0%) and one Proteus (1.0%). There were
14 cases of mixed colonization with Enterobacter and E. coli (14.1%).
Noroviruses (2%) and Astrovirus (1%) were detected in children
with clinical signs suggestive of diarrhea (Table 2). These viruses
were detected exclusively among children residing in urban areas
(p-value < 0.0001). All fecal samples were negative for the presence of
the rotavirus species A and C.
Parasites were detected in 49 of the fecal samples evaluated, as shown
in Table 2. The prevalence of at least one parasite in the entire study
population was 42.0%. There were seven cases of parasitism involving
two organisms: five were associations between G. lamblia and E. coli
Table 1
Social indicators, hygiene, water source and food consumption habits of 100
children of a government daycare center in the city of São José do Rio Preto,
São Paulo grouped as Diarrheal (n = 50) or Non-diarrheal (n = 50)
Dia rrheal
(n)
%
Non-diarrheal
(n)
%
Table 2
Detection of bacterias, virus and parasites in fecal specimens from a
daycare center located in São José do Rio Preto, São Paulo. October 2010 to
February 2011
Diarrheal
(n=50)
p-value
25
50.0
27
54.0
Male
25
50.0
23
46.0
%
(n)
%
-
0.0
1
2.0
1.0000
EPEC
12
24.0
8
16.0
0.4533
EIEC
3
6.0
1
2.0
0.6098
E. coli O157
1
2.0
2
4.0
1.0000
Pseudomonas
2
4.0
4
8.0
0.6737
Norovirus
2
4.0
0
0.0
0.4751
Astrovirus
1
2.0
0
0.0
1.0000
Rotavirus A
0
0.0
0
0.0
0.7025
Rotavirus C
0
0.0
0
0.0
0.7025
Giardia lamblia
20
40.0
24
48.0
0.5456
Entamoeba coli
1
2.0
6
12.0
0.1169
Endolimax nana
3
6.0
1
2.0
0.6098
Hookworm
1
2.0
0
0.0
1.0000
Bacteria
Acinetobacter
Treated drinking water
Yes
2
4.0
8
16.0
Source of water
Public reservoir
44
88.0
44
88.0
Viruses
Pets at home
Yes
22
44.0
25
50.0
Sewer system at ho me
Yes
50
100.0
48
96.0
Ingestion of raw food
Yes
35
70.0
30
Parasites
60.0
Meals outside the residence or daycare center
Yes
p-value†
(n)
Gender
Female
Non-diarrheal
(n=50)
23
46.0
09
18.0
0.0053*
*Chi-square test.
(5.0%), one of G. lamblia and E. nana (1.0%) and the other between G.
lamblia and hookworm (1.0%).
Table 3 shows no statistically significant differences after stratified
analysis of the samples divided into Diarrheal and Non-diarrheal Groups
versus clinical signs suggestive of diarrhea or versus the presence of
enteropathogens (data not shown).
EPEC: enteropathogenic Escherichia coli; EIEC: enteroinvasive E. coli; †: Fisher
exact test.
Table 3
Clinical data obtained of children in a government daycare center of São José
do Rio Preto, São Paulo State, grouped according to clinical signs suggestive of
diarrhea in Diarrheal and Non-diarrhea l Groups
Diarrheal (n=50)
DISCUSSION
This study was carried out during a period without any registers of
diarrhea outbreak. Despite the presence of EPEC, EIEC, E. coli O157 and
Pseudomonas spp., no association was observed between bacteria and
diarrhea clinical symptoms. Similar results were observed in a case-control
study conducted in a HIV-1 positive infant40 population as well as in a HIV1 seropositive adult population20, both from the same region. However, in
another investigation conducted in the 1990s, diarrhea was associated with
these enterobacteria in a population treated in the Pediatrics Outpatient
Clinic of a Hospital in São José do Rio Preto, Sao Paulo2. This implies
that strains of bacteria with different virulence circulate in the population
of this region and, therefore, they may pose a risk of diarrhea. Intestinal
infections caused by EIEC are rare but are more common in children over
two, as observed in the current study, and also in adults20.
Noroviruses were detected in 2% of the Diarrheal group. In a
retrospective study, conducted in a daycare center in Rio de Janeiro
(n)
%
Non-diarrheal (n=50)
(n)
%
Fever
Yes
14
28.0
13
26.0
No
34
68.0
37
74.0
Blood in the feces
Yes
0
0.0
1
2.0
No
49
98.0
49
98.0
Abdominal pain
Yes
11
22.0
4
8.0
No
38
76.0
46
92.0
Vomits
Yes
8
16.0
5
10.0
No
42
84.0
45
90.0
29
involving children under five, with acute gastroenteritis, the occurrence of
noroviruses was higher ranging from 23 to 67%. Furthermore, norovirus
(GII) was responsible for three out of four of the studied outbreaks18. In
Brazil, the HAstV caused 3 to 11% of the cases of acute gastroenteritis
in children under 56,16. The prevalence of HAstV obtained in this study
(1%) is lower than that reported by other authors (2-5%)42 and quite
different to the prevalences described in other developing countries
(2-26%) or even in developed countries (2-11%)9-11. Rotavirus species
A and C were not detected in the population of the daycare center here
evaluated. This may be explained by the high vaccination coverage in
São José do Rio Preto (99%), which provides protection against the most
common rotavirus, species A37. Indeed, studies in several countries have
shown that rotavirus species C is not as uncommon as thought, however
its prevalence is low (0.4-35%)15,17,26,34.
Despite the absence of any association with diarrhea, these viruses
were detected in the studied population. It should be noted that the
transmission of norovirus is very efficient, with rapid dispersal during
outbreaks, primarily due to the high infectivity of this agent when an
inoculum of only 10 to 100 virions is enough to cause an infection.
Furthermore, it is of concern that infected people can transmit the virus
after recovering from the symptoms as they continue to eliminate viral
particles for up to three weeks39,41. Contamination of drinking water and
water used for recreational activities can serve as a primary source of
outbreak as noroviruses are resistant to treatment with chlorine and may
remain infectious for long periods in this environment37.
In daycare centers of different Brazilian cities, there is a wide
variation in the prevalence of intestinal parasitic diseases, ranging from
15.2%19 to 53.4%29. This is in accordance with the 49.0% verified in
this study, where approximately 14 children have parasitism involving
infection by two parasites. The different enteric parasite incidences
observed in this study may be due to the wide diversity of geographical,
social, economic characteristics and climate in Brazil, which are reported
as critical factors in the modulation of the frequency of different enteric
parasites40. Although most children received treated water, a higher
prevalence of water-borne parasites was found. These results suggest
that the use of treated water is not a protective factor against water-borne
parasites or that water treatment does not follow the ideal procedures to
eliminate parasites.
The prevalence of giardiasis in Brazil7,22 and in the state of São
Paulo8,29 varies, on average, between 4% and 30%, with variation also
reported in daycare centers. The highest frequency for this protozoan
was associated with the 1- to 2-year age range followed by the over
3-year-old population, which is probably related to the high rate of
fecal-oral transmission of the pathogen1. Due to the common detection of
G. lamblia cysts on fingers and under nails, it is possible that caregivers
in daycare centers are the main form of transmission of this parasite
among children.
This study identified low prevalence of Entamoeba coli (2.0%) and
Endolimax nana (2.0%) demonstrating that these amoebae may not be
endemic in the region. However, it should be pointed out that the detection
of commensal amoebas may indicate that the children ingested water or
food contaminated with fecal waste and that they are therefore at risk for
contamination by Entamoeba histolytica, which has a high prevalence
in tropical regions32.
30
In this study, just one case of hookworms associated with G. lamblia
was identified in a non-diarrheal one-year-old child. This is actually one
of the most common helminths transmitted worldwide; higher rates have
been reported in daycare centers in Northeastern Brazil23,31. Furthermore,
the majority of children included in this study have piped water and
a sewer system in their homes. There is a historical trend toward the
reduction of these parasites in the state of São Paulo attributable to
improvements in these services14,27.
The exposure to intestinal pathogens of children studied at this
daycare center does not seem to contribute to the occurrence of diarrhea
or other intestinal symptoms. However, these results raise the question as
to the real reasons that children in this daycare center manifested diarrhea.
In fact, previous work on the etiological agents associated with diarrhea
indicate that the relative importance of the various enteropathogens varies
greatly depending on the season of the year, area of residence (urban or
rural), socioeconomic class, geographical location and in particular, the
age of the host42. Associated with this situation, diarrhea may be related to
other non-infectious diseases or even by other enteropathogen infections,
not investigated in this study. On the other hand, given the fact that many
infants were asymptomatic carriers, it should be pointed out that this can
be due to immunological tolerance mechanisms or intraspecific variations
of bacterial communities that comprise the virulence of the parasite5.
Thus, the present study provides a warning to authorities responsible
for community healthcare concerning asymptomatic children, which can
potentially pose a risk for outbreaks of gastroenteritis.
Finally, further investigations should be planned in the city of São
José do Rio Preto and others within the diverse regions of the country, in
order to increase knowledge and provide appropriate responses to these
clinical infections with the elaboration of effective measures to prevent
and control enteropathogens as a public health policy in Brazil.
RESUMO
Enteropatógenos detectados em crianças de creche no Sudeste do
Brasil: pesquisa de bactérias, vírus e parasitos
Introdução: O objetivo deste estudo foi determinar a prevalência
e o perfil etiológico de enteropatógenos em crianças de uma creche.
Métodos: No período de outubro de 2010 a fevereiro de 2011
foram coletadas e analisadas amostras de fezes de 100 crianças
matriculadas em creche do governo no município de São José do
Rio Preto, Estado de São Paulo. Resultados: Um total de 246 bactérias
foram isoladas em 99% das amostras de fezes; 129 eram diarreicas e
117 não-diarreicas. Foram isoladas setenta e três cepas de Escherichia
coli, 19 de Enterobacter, uma de Alcaligenes e uma de Proteus. Foram
detectados 14 casos de colonização mista com Enterobacter e de E. coli.
Norovírus e Astrovirus foram detectados em crianças com sinais clínicos
sugestivos de diarréia. Estes vírus foram detectados exclusivamente
entre as crianças residentes em áreas urbanas. Todas as amostras fecais
foram negativas para a presença das espécies de rotavírus A e C. Foi
observada a presença de Giardia lamblia, Entamoeba coli, Endolimax
nana e ancilostomídeos. Foi encontrada associação significativa entre
o consumo de alimentos fora do centro da casa e creche e a presença
de parasitos intestinais. Conclusões: Para as crianças desta creche, a
infecção intestinal por patógenos não parece ter contribuido para a
ocorrência de diarreia ou outros sintomas intestinais. As diferenças
observadas podem ser atribuídas à grande diversidade de características
geográficas, sociais e econômicas e o clima do Brasil, as quais tem
sido relatadas como fatores críticos para a modulação da frequência
de diferentes enteropatógenos
ACKNOWLEDGEMENTS
To all individuals enrolled in this study. The authors thank the
Research Center of Microorganisms/FAMERP, state of São Paulo and
Instituto Evandro Chagas - Section of Virology, MS/SVS, Ananindeua,
state of Pará, for the partnership in carrying out bacteriological,
parasitological, and viral analyzes.
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Received: 9 January 2014
http://dx.doi.org/10.1590/S0036-46652015000100005
HISTORICAL SERIES OF PATIENTS WITH VISCERAL LEISHMANIASIS TREATED WITH MEGLUMINE
ANTIMONIATE IN A HOSPITAL FOR TROPICAL DISEASES, MACEIÓ-AL, BRAZIL
Lindon Johoson Diniz SILVEIRA(1), Thiago José Matos ROCHA(2), Sandra Aparecida RIBEIRO(3) & Célia Maria Silva PEDROSA(4)
SUMMARY
Introduction: Visceral leishmaniasis is an endemic protozoan found in Brazil. It is characterized by fever, pallor, hepatosplenomegaly,
lymphadenopathy, and progressive weakness in the patient. It may lead to death if untreated. The drug of choice for treatment is
meglumine antimoniate (Glucantime®). The aim of this study was to evaluate patients with visceral leishmaniasis according to criteria
used for diagnosis, possible reactions to Glucantime® and blood pressure measured before and after treatment. Methods: 89 patients
admitted to the Teaching Hospital Dr. Hélvio Auto (HEHA) in Maceió-AL, in the period from May 2006 to December 2009 were
evaluated. Data were collected on age, sex, origin, method of diagnosis, adverse effects of drugs, duration of hospitalization, duration
of treatment and dosage up to the onset of adverse effects. Results: There was a predominance of child male patients, aged between
one and five years old, from the interior of the State of Alagoas. Parasitological diagnosis was made by bone marrow aspirate; three
(3.37%) patients died, 12 (13.48%) had adverse reactions and treatment was changed to amphotericin B, and 74 (83.14%) were cured.
Changes that led to replacing Glucantime® were persistent fever, jaundice, rash, bleeding and cyanosis. Conclusion: During the
study, 89 patients hospitalized for VL were analyzed: 74 were healed, 12 were replaced by amphotericin B treatment and three died.
Most of them were under five years old, male and came from the interior. The dosage and duration of treatment with Glucantime®
were consistent with that advocated by the Ministry of Health. Persistence of fever, jaundice, rash, cyanosis and bleeding were the
reactions that led the physician to modify treatment. No change was observed in blood pressure before and after treatment. This study
demonstrated the work of a hospital, a reference in the treatment of leishmaniasis, which has many patients demanding its services
in this area. It demonstrates that this disease is still important today, and needs to be addressed properly to prevent injury and death
due to the disease.
KEYWORDS: Glucantime; Visceral leishmaniasis; Hepatosplenomegaly.
INTRODUCTION
Visceral leishmaniasis (VL) is a major international public health
problem, affecting approximately 65 countries with an estimated annual
incidence of 500,000 new cases, 90% of which occur in India, Nepal,
Sudan, Bangladesh and Brazil. The fatalities are high, and an estimated
59,000 people die from the disease each year2. Currently, it is among the
six endemic diseases prioritized in the world1.
The increased occurrence of Leishmania and HIV co-infection may
be related in part to the geographical distribution of both pathologies3. In
areas in which VL is endemic, people who are immunocompromised due
to infection by HIV are more likely to develop clinical LV as compared
to those without HIV co-infection. In fact, L. infantum co-infection is
now the third most common infection in HIV-infected individuals in
endemic areas of VL4. Rates of HIV infection are 5% in Brazil, from
2-5% in India and vary between 25 and 40% in Ethiopia (WHO, 2010)28.
In Alagoas, 1076 cases of VL were reported in the last ten years
(1999-2008), according to the Ministry of Health. The years with the
highest number of cases were 1999 (171 cases), 2000 (285 cases) and
2001 (234 cases). The disease predominates in rural areas, affecting
mainly children who live in these areas. Of the 102 municipalities in the
State, VL has been reported in 89% of them. Over the past five years
the municipalities that most reported cases were Arapiraca, Palmeira
dos Índios, Traipú, Cacimbinhas, Igaci, Santana do Ipanema and São
José da Tapera.
In Alagoas, the Teaching Hospital School Dr. Hélvio Auto (HEHA),
maintained by the public service, is considered a reference by the National
Health Service (NHS) for hospitalization of patients with infectious and
parasitic diseases in the state of Alagoas.
In Brazil, pentavalent antimonials are the drug of choice for treatment
of VL due to their proven therapeutic effectiveness6. Meglumine of
(1) Specialist in Health Sciences from the University of Health Sciences of Alagoas-UNCISAL. University Center Cesmac. E-mail: [email protected]
(2) PhD in Therapeutic Innovation, Federal University of Pernambuco-UFPE. E-mail: [email protected]
(3) PhD in Medicine (Pulmonology) Federal University of São Paulo. Associate Professor, Department of Preventive Medicine, Federal University of São Paulo. E-mail: [email protected]
(4) PhD in Tropical Medicine from the Federal University of Pernambuco. III Associate Professor at the Federal University of Alagoas. E-mail: [email protected]
Correspondence to: Thiago J.M. Rocha, Universidade Federal de Pernambuco, Depto. Antibióticos, 50670-901 Recife, Pernambuco, Brasil. E-mail: [email protected]
SILVEIRA, L.J.D.; ROCHA, T.J.M.; RIBEIRO, S.A. & PEDROSA, C.M.S. - Historical series of patients with visceral leishmaniasis treated with meglumine antimoniate in a hospital for
Tropical Diseases, Maceió-AL, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 33-8, 2015.
antimoniate (Glucantime®) is especially effective in the treatment of
cutaneous, mucocutaneous and visceral leishmaniasis. The drug causes
rapid regression of clinical manifestations and hematological disease and
causes sterilization of the parasite7. However, recent research has shown
an increase in the number of reports of adverse reactions to Glucantime®.
Knowing that VL is a frequent cause of hospitalization in HEHA and
that most patients are treated with Glucantime®, the authors developed an
interest in seeing how these patients would present, taking into account
the general characteristics, the criteria used for the diagnosis, possible
effects of the Glucantime® and blood pressure measured before and after
treatment. The research aimed to study the cases of VL admitted to the
HEHA from 2006 to 2009 in Maceió-AL.
Study area: The State of Alagoas is located in Northeastern Brazil,
and consists of 102 municipalities having an area of 27,767 km2 and a
population of 3,156,108 inhabitants. Alagoas is the most populous state
in the Northeast of Brazil with a population density of 101.3 inhabitants
per km2. The urban population accounts for 68.01% of the total group
of children and adolescents and accounts for 28.26% of the population
with 797,931 inhabitants.
Study type: Observational, cross-sectional and descriptive.
Study site: The site where the research was conducted HEHA,
belonging to the State University of Health Sciences of Alagoas
(UNCISAL), located in the city from Maceió, AL.
Data collection: Data collection began in May 2006 and ended in
December 2009. All patients admitted to HEHA diagnosed with VL were
selected, and they were treated with Glucantime®. During this period,
89 patients were included. All patients, after being diagnosed with VL,
were invited to sign a consent form (ICF).
Research subjects: In the period from May 2006 to December 2009
patients admitted with a diagnosis of VL were invited to participate in the
study and were prescribed treatment with Glucantime®. For children, an
authorized guardian permitted their participation in the research.
Inclusion criteria: The study included all clinical-epidemiological,
serological or parasitological patients confirmed to have VL, who were
admitted and prescribed treatment with Glucantime®. Clinical and
epidemiological diagnosis was considered for patients who had fever and
Hepatosplenomegaly and were native to areas considered endemic areas.
Exclusion criteria: The study excluded patients who were prescribed
treatment with other medicines than Glucantime®. There were no refusals.
Patient sex: Gender was raised in the study with the proposition
to investigate the distribution of VL cases according to the respective
groups: male and female.
Age of the patient: In the study, patients were divided into groups
as follows: patients < 1 year, patients one to five years, patients six to
10 years, patients 11 to 20 years, patients 21 to 30 years, patients 31-40
years, patients 41-50 years, 51-60 years, 61-70 years and 71-80 years.
34
Weight: The study used a variable weight, whereby the daily dose of
Glucantime® is calculated according to the weight of the patient.
Patient’s origin: As HEHA caters to all patients referred with
suspected VL, in another 44 municipalities in Alagoas beyond the
capital, the record of this variable will allow a better characterization of
the study population as there are endemic municipalities. The data were
computed with the name of the municipality and then were cataloged in
the physiographic regions of the State (Coastal, Zona da Mata, Wasteland
and Hinterland).
Type diagnosis: Patients with VL can be diagnosed using two
criteria: clinical criteria and laboratory clinical epidemiological criteria.
Therefore this variable was recorded so that they could relate them to
other variables.
Period of stay: Treatment for VL using Glucantime® as a first-choice
drug can last 30 days on average and depending on the patient’s condition,
they may be hospitalized during this period. The date of admission and
date of discharge was noted.
Treatment period: Knowing that the drug research topic is
Glucantime® and the duration of the treatment is the same, on average
30 days, the date of initiation of treatment and end date of the treatment
was computed. The annotation of this variable was important for the
Glucantime® treatment because various adverse reactions may lead to
treatment discontinuation.
Dosage: The recommended dose in the study and used for the
treatment of visceral leishmaniasis with Glucantime® was 20 mg Sb +5
kg/day with intravenous application (IA) or intramuscular (IM) for at
least 20 and up to 40 days, using the maximum two to three vials/day.
The Glucantime® ampoules had 5 mL containing up to 1500 mg (300
mg/mL) equivalent to 405 mg (81 mg/mL) pentavalent antimony (Sb
+5
). In adult patients, Amphotericin B was used at a dose of 1 mg/kg on
alternate days (maximum total dose of 3 g). In children, it was used at a
dose of 15 to 25 mg/kg, also administered on alternate days.
Amendment(s) that motivated (plow) treatment interruption:
All clinical and adverse reactions that led the doctor to replace treatment
with Glucantime® with another drug were noted.
Cure criteria: The cases were followed up to six months after
treatment completion and the cure criteria used was a good clinical
response to treatment with disappearance of fever and reduction of
hepatosplenomegaly.
Data analysis: After collection, the data were stored in a spreadsheet
(Microsoft Excel 2003®. Redmond, WA, USA) as the database. The
results were tabulated and frequencies of the variables in each group were
calculated and arranged in tabular and graphic formats. The tabulated data
were processed by the application Microcomputer Statistical Package
for Social Sciences (SPSS ©) (version 15.0 for Windows, SPSS Inc). The
descriptive statistics for numeric variables included calculations of the
mean and standard deviation (SD).
Ethical considerations: This research project was developed
according to the international guidelines that deal with human research,
notably resolution 196/96 of the National Health Council (CNS).
Table 1
Distribution of VL patients by sex and age in HEHA, Maceió, 2006-2009
RESULTS
During the research following a specified protocol, patients admitted
to the HEHA in Maceió-AL, in VL diagnosis were treated with the drug
Glucantime® from May 2006 to December 2009.
After confirmation of the diagnosis of VL, 89 patients were included
who started treatment with Glucantime®. Three patients (3.37%) died, 12
patients (13.48%) had adverse reactions and treatment was changed to
amphotericin B, 74 patients (83.14%) who continued to be treated with
Glucantime ® were cured of the disease.
The duration of treatment for all 89 patients ranged from one to 40
days with a mean of 24.42 (SD = ± 10.04) and the cumulative dose in mg
ranged from 340 mg to 44640 mg with an average of 13808 (SD = ±11950).
Of the total 89 patients studied, 52 (58.4%) were male and 37
(41.5%) female. During the observation period the patients were from
33 municipalities in Alagoas. The municipality with the highest number
of cases was São José da Tapera with 12 (13.48%), followed by Girau
Ponciano with seven (7.87%), Palmeira dos Índios and Traipú with six
(6.74%) and other municipalities between five and one patients. The
highest incidence was found in the municipalities of the state.
The patients’ ages ranged from nine months to 73 years, with a mean
of 13.58 (SD = ± 17.22) and a median of five. In this research the most
affected age group was that of patients 1-5 years and decreased with
increasing age: < 1 year (1), 1-5 years (45), 10-20 years (12), 20-30
(-10), 30-40 years (7), 40-50 years (6), 50-60 years (1), 60-70 (4), 70-80
years (2), > 80 (1).
During the research period there were three deaths (3.37%) in those
patients who used Glucantime®, only one was aged under five years and
the other two over 60 years.
Of the patients who died (three from 89), all with positive
parasitological tests, two patients had treatment duration for a period
shorter than 10 days and the third had 25 days of treatment. The
cumulative dose of Glucantime® administered in mg/kg until death in
the first patient was 400 mg, in the second patient 8544 mg, and the
third 28555 mg.
Patients who were admitted to the HEHA, from May 2006 to
December 2009 with VL showed the following distribution according
to age and sex (Table 1).
For the diagnosis of VL all patients underwent bone marrow
aspiration which identified 68 patients (76.40%) as positive, 18 (20.20%)
were considered positive by clinical epidemiological criteria and only
three (3.40%) were positive by serological method (IIF) (Table 2).
Of the total 89 patients, 12 (13.48%) had clinical changes that led
to replacement of Glucantime®. The main changes were persistent fever
in five patients (41.68%), jaundice in three patients (25%), rash in two
patients (16.66%), bleeding in one patient (8.33%) and also cyanosis in
one patient (8.33%).
Age (years)
<1
1-5
6 - 10
11 - 20
21 - 30
31 - 40
41 – 50
51 - 60
61 - 70
71 - 80
Total
Male
1
21
6
8
5
5
0
4
1
1
52
Female
0
24
6
2
2
1
1
0
1
0
37
Total
1
45
12
10
7
6
1
4
2
1
89
%
1.12
50.56
13.48
11.23
7.86
6.74
1.12
4.49
2.24
1.12
100
Table 2
Method for VL diagnosis by age, HEHA, Maceió, 2006-2009
DIAGNOSIS
ClinicalParasitological
epidemiological
< 1 year
0
0
1 - 5 years
38
6
6 - 10 years
7
5
11 - 20 years
6
3
21 - 30 years
6
1
31 - 40 years
4
2
41 - 50 years
0
1
51 - 60 years
4
0
61 - 70 years
2
0
71 - 80 years
1
0
TOTAL
68
18
Age group
I.F.I
TOTAL
1
1
0
1
0
0
0
0
0
0
3
1
45
12
10
7
6
1
4
2
1
89
It can be seen that 11 patients with clinical abnormalities were aged
below five years and only one above 70. The length of treatment before
the onset of the reactions ranged from one to 11 days with a mean of
5.08 (SD = ± 2.84) and the cumulative dose in mg/kg of Glucantime®
ranged from 340 to 7644 mg with a mean of 1775.
Of the 12 patients who had some type of clinical change after they
received Glucantime®, 11 (91.66%) of them reacted up to 10 days of
hospitalization and only one (8.34%) showed a reaction between the tenth
and twentieth day of treatment. Patients who were cured of the disease (74
from 89) during the search using treatment as Glucantime® had a mean
hospital stay of 19.6 days (SD = ± 11.4), the duration of treatment ranged
from 21 to 40 days with an average of 29.18 days. When correlated with
the time of admission, age was a negative correlation between length of
stay and age (Fig. 1).
The measurement of blood pressure before treatment occurred shortly
after the diagnosis of VL and verification after treatment, which occurred
on average 30 days after starting treatment with the drug Glucantime®. All
patients were normotensive and did not show any significant change in
35
since São José da Tapera has one of the lowest Human Development
Indices (HDI) in the state at 0.588, and may also be due to a lack of
sanitation and the custom of the inhabitants of rural communities to
raise domestic dogs6.
Most patients in this study were from the state (81.8%). A similar
percentage (77.0%) was also found in another study of children under
15 years old coming from Alagoas, where they studied clinical and
epidemiological aspects of these children20. A survey in the state of
Pernambuco found that 82.5% of patients’ origins were concentrated
within the state and 14.8% from the metropolitan region of Recife21.
In Tunisia, a study researching VL cases between 1996 and 2006
found that 65.3% lived in rural areas4. Although there is a work related
urbanization VL17, one must realize that it is still a disease coming
from inside. It is worth noting that there are factors associated with this
internalization that make for a breeding reservoir for the disease: dogs, a
lack of sanitation, and the low education and precarious socioeconomic
conditions of the affected population that causes ignorance about the
form of transmission16.
Fig. 1 - Correlation length of stay with the age of patients hospitalized for LV, HEHA,
Maceio, 2006-2009.
systolic values and diastolic BP. No patient was taking antihypertensive
drugs.
DISCUSSION
From May 2006 to December 2009 a total of 130 patients with a
diagnosis of VL in the HEHA were seen and treated, and 89 (68.5%)
were prescribed treatment with the drug of choice Glucantime®. 41
patients were treated with amphotericin B, which is the drug of second
choice for treatment of VL; this occurred because some patients presented
contraindications and other unsatisfactory responses to Glucantime®. This
substitution occurred mostly because the patients had a liver problem
and/or renal impairment.
The ages of the 89 patients in this study ranged from nine months to
73 years with a mean of 13.58 and a median of five. In a similar study
of 114 patients in New Delhi, India, ages ranged from six months to 68
years and the average was 31 years13.
In research conducted on the expansion of VL in the state of Mato
Grosso-BR, between January 1998 and December 2005, predominance
was found in males (58%) and in ages 0-9 years (51.5 %)15. The same
happened with the present study, verifying the predominance of males
at 58.42% and of age with 0-9 years at 65.16%. Even in developed
countries like France the prevalence of cases occur in children under
five years of age (77%)12.
As HEHA, where the research was conducted, is a referral hospital for
the entire state of Alagoas for VL treatment, 33 patients were studied from
(32.35%) municipalities in Alagoas for a total of 102. The municipality
of Alagoas had a higher incidence of cases with 12 (13.48%); São José
da Tapera was located in the hinterland of the state. This may be justified
36
All 89 patients were submitted to parasitological diagnosis by bone
marrow puncture. Of this total 68 (76.40%) were positive and 21 (23.60%)
negative. Among those who were negative in the bone marrow puncture,
three were confirmed by immunological method IFIs and the others were
treated based on clinical epidemiological criteria. A study in Madrid,
Spain, confirms this finding, predominantly diagnosed by bone marrow
aspirate with 77% of patients in research. Based on these findings the
diagnosis of VL by finding the parasite in bone marrow aspirate remains
the diagnostic test performed in the area26.
The diagnosis made by the finding of developmental forms of
Leishmania in bone marrow smears is a quick and easy one, while the
IFI technique, despite the presence of some false-negatives, emerged as
the most specific27.
Some studies confirmed the efficacy of treatment with Glucantime®,
although there is still little known about its mechanism of action3.
During the last two decades, the emergence of resistance to
pentavalent antimonials had a huge impact on the epidemiology of
leishmaniasis10. Authors consider pentavalent antimony safe for the
treatment of leishmaniasis, although the reports of adverse effects are
increasing24. Adverse effects such as elevated serum liver and pancreas
enzymes were found in a study comparing patients who used Glucantime®
with patients who received pentamidine25. They are described in further
research as electrocardiographic changes, myalgia, headache, rash,
nephritis, gastrointestinal and respiratory disorders7,14,22.
The cardiotoxicity associated with high doses and duration of treatment,
is the most serious side effect with the use of Glucantime®23. Other authors
also suggest that it is necessary to evaluate electrocardiographic tests
during treatment with pentavalent antimonials regardless of the presence
of factors that increase the risk of cardiac disease24.
During the study 89 patients were evaluated using the drug
Glucantime® for treatment of VL. Treatment was interrupted in 12
(13.48%) patients because of clinical alterations that compromised the
continuity of treatment with Glucantime®. In a study with 55 patients,
seven (12.72%) of these patients had poor response to antimony,
characterized by continuing of signs and symptoms and/or worsening
of the clinical picture18.
Of the 12 patients who had clinical changes with the use of
Glucantime®, two (16.66%) had a rash; the same occurred in Research
Study 18 in which one patient out of 11 was found with the same reaction
and in Study 3, 21 subjects presented with a rash19. Failure of treatment
with Glucantime® was also found in another study, involving 16 (10.1%)
of 158 patients in a retrospective analysis in the Protozoology Unit of
the Istituto Superiore di Sanità (Italian National Institute of Health)8.
The other reactions that led to treatment failure were persistent fever,
jaundice, cyanosis and bleeding. The reactions presented occurred in the
vast majority, 11 of 12 patients during the first 10 days of treatment and
the age group affected by these reactions was 0-5 years, with five patients
less than one year, six patients in the range one - five years and only one
patient over 70 years old. As a result of these reactions, treatment was
replaced by amphotericin B, the second drug of choice for treatment of
VL recommended by the Ministry of Health.
Among the 89 patients, 74 (83.14%) were treated with Glucantime® for
the period and dose according to Ministry of Health and were considered
cured according to clinical and laboratory criteria. Clinical criteria were:
disappearance of fever and reduction of hepatosplenomegaly. At the end
of treatment the spleen usually decreased by 40% or more compared
to baseline. With respect to laboratory criteria the improvement of
hematological parameters (hemoglobin, leukocytes and platelets) and a
return to normal reference values of liver enzymes (AST and ALT) were
taken into account. Patient follow-up was done at 3, 6 and 12 months
after treatment and if, at the last evaluation he remained stable, the patient
was considered cured.
During the treatment, three (3.37%) patients died. Of the three patients
that died after the use of Glucantime®, two of these occurred within ten days
after the start of treatment. These deaths were not attributed to medication
because these two patients were one and 63 years old, respectively,
and upon showing clinical signs were admitted to the hospital; drug
administration occurred only at two and seven days, respectively. The third
death occurred in a patient 65 years old who had a stroke (cerebrovascular
accident) during hospitalization and died after 25 days of treatment with
Glucantime®. It can be observed that the deaths are not related to dosage.
old, treated with Glucantime® died on day 18 of treatment from coronary
artery disease, likely acute9.
When the blood pressure was observed before and after treatment
during research it was seen that there was no significant increase or
decrease in normotensive remaining and no patient had hypertension prior
to the start of the treatment, nor was there any use of antihypertensive
drugs. It had already been observed in another study that while nine of
23 patients studied had hypertension, patients were mostly adults and
the sample was small, while in the present study the sample was bigger
and patients were mostly younger than 10 years old11.
Given the prevalence of cases of VL in Alagoas it should guide the
medical profession operating there to the appreciation of the signs and
symptoms of the disease so that specific therapy can begin as soon as
possible, thereby avoiding a worsening of the patient’s prognosis. Early
treatment reduces the risk of death, especially in children, and increases
the cure rate.
The reporting of adverse reactions must be continuous and efficient
and well publicized so that everyone can know all those cases that are not
reported in the current literature. The city of São José da Tapera, inside
Alagoas, should plan reduction targets of cases of VL since both this
study and in others in this municipality, Alagoas remains among those
with a higher incidence of new cases.
CONCLUSIONS
During the research, 89 patients with visceral leishmaniasis were
included in the study, treated with Glucantime®, and 74 patients were
considered cured, 12 had reactions that led to the replacement of
Glucantime® with amphotericin B for treatment, and three died. The
mean hospital stay was 22 days. Among the patients studied most were
less than five years of age, male, and from the interior of the state. The
dose of Glucantime® and the period of treatment during the study were
consistent with the recommendations of the Ministry of Health. The
reactions that led the doctor to replace treatment by amphotericin B were:
persistent fever, jaundice, rash, cyanosis and bleeding. All patients were
normotensive and there was no change in blood pressure before and after
treatment with Glucantime®.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
A similar finding was observed in a study in Pernambuco, which
recorded 44 deaths, in which the average hospital stay was 10 days
(SD = ± 9), and the main immediate causes of death were associated
infections, bleeding and liver failure21. In the account given by other
authors, a 45-year old patient was treated with Glucantime® for 30 days,
and death did not occur until ten days after the end of this thirty day
treatment and the cause of death was considered sudden death18.
In a retrospective analysis of four confirmed cases of VL who were
admitted to the Hospital Municipal de Santo André, it was seen that two
patients died in the first week of treatment, indicating that these deaths
were caused by the phenomenon of hypersensitivity or more likely the
development of septicemia in patients already immunocompromised5. In
another study, one hypertensive and diabetic patient who was 58 years
RESUMO
Série histórica dos pacientes com leishmaniose visceral tratados
com antimoniato de meglumina em hospital de Doenças Tropicais,
Maceió-AL, Brasil
A Leishmaniose visceral é doença infecciosa causada por protozoários
das espécies chagasi e donovani sendo transmitida pela picada de insetos
fêmea dos gêneros Lutzomyia e Phlebotomos. Constitui doença febril,
determinando amplo aspecto de manifestações clínicas e prognóstico
variável, que pode levar à morte se não for tratada. É doença endêmica
encontrada no Brasil e nos últimos anos verificou-se intenso processo
de urbanização da endemia e aumento da letalidade por leishmaniose
37
visceral. O estudo teve como objetivo avaliar pacientes com leishmaniose
visceral de acordo com os critérios utilizados para o diagnóstico, possíveis
reações ao Glucantime® e pressão arterial, medidos antes e após o
tratamento. Métodos: Foram avaliados 89 pacientes internados no Hospital
Universitário Dr. Hélvio Auto (HEHA), em Maceió-AL, no período de
maio de 2006 a dezembro de 2009. Foram coletados dados sobre idade,
sexo, origem, método de diagnóstico, efeitos adversos da droga, duração da
hospitalização, duração do tratamento e dose até o aparecimento de efeitos
adversos. Resultados: Houve predomínio de crianças do sexo masculino,
com idade entre um e cinco anos, a partir do interior do Estado de Alagoas.
O diagnóstico parasitológico foi feito pelo aspirado de medula óssea, três
(3,37%) pacientes morreram, 12 (13,48 %) apresentaram reações adversas
e o tratamento foi alterado para anfotericina B, e 74 (83,14 %) foram
curados. As alterações que levaram à substituição de Glucantime® foi
febre persistente. A dosagem e duração do tratamento com Glucantime®
foi seguido como preconizado pelo Ministério da Saúde. A persistência
de febre, icterícia, prurido, cianose e sangramento foram as reações
que levaram o médico a modificar o tratamento. Nenhuma mudança foi
observada na pressão arterial antes e após o tratamento. O estudo realizado
demonstrou o perfil de um Hospital, que recebe grande demanda de casos
de leishmaniose visceral. Isso demonstra que essa doença continua sendo
importante na atualidade, precisando ser abordada de maneira adequada,
evitando assim agravos e mortes pela doença.
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38
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Received: 19 July 2013
http://dx.doi.org/10.1590/S0036-46652015000100006
STUDY OF THE PREVALENCE OF Capillaria hepatica IN HUMANS AND RODENTS IN AN URBAN
AREA OF THE CITY OF PORTO VELHO, RONDÔNIA, BRAZIL
Elierson José Gomes da ROCHA(1), Sérgio de Almeida BASANO(1), Márcia Maria de SOUZA(2), Eduardo Resende HONDA(3),
Márcio Botelho de CASTRO(4), Edson Moleta COLODEL(5), Jéssica Carolinne Damasceno e SILVA(1), Lauro Prado BARROS(1),
Elisa Sousa RODRIGUES(1) & Luís Marcelo Aranha CAMARGO(6)
SUMMARY
Introduction: Hepatic capillariosis, caused by Capillaria hepatica (Calodium hepaticum) (Bancroft, 1893), Travassos, 1915
(Nematoda, Trichinelloidea, Capillariidae), is a common zoonosis in rodents but is rare in humans. Seventy-two cases in humans
have been reported worldwide since the first case was described by MACARTHUR in 192417,27. This study aimed to determine the
prevalence of Capillaria hepatica in humans and rodents in an urban area of Porto Velho, the capital of Rondônia, in Brazil. Methods:
After conducting a census of the area, 490 residents were randomly selected, and, after signing a term of consent, provided blood
samples that were screened for anti-Capillaria hepatica antibodies. Simultaneously, rats were captured to assess the prevalence of
this parasite in rodents by histopathological examination in liver sections. Results: A prevalence of 1.8% was found among residents
who had specific antibodies at a dilution of 1:150, indicating exposure to parasite eggs; 0.8% of the subjects also had positive titers
at a dilution of 1:400, indicating true infection. The prevalence in rats was 2%. Conclusions: The prevalence of infection with this
parasite among humans and rats was low. While the prevalence encountered among humans was within the limits reported in the
literature, the prevalence among rodents was much lower.
KEYWORDS: Capillariasis; Capillaria hepatica; Rondônia; Amazônia.
INTRODUCTION
Capillaria hepatica has a wide geographic distribution and is able to
colonize a diverse array of environments and mammals, including wild
and domestic rodents.
Certain authors reported finding Capillaria sp. eggs in canine
coprolites from 6,500 BC in Patagonia18, and certain studies reported
evidence of human infections in France in the Neolithic and Paleolithic
periods3 and in the region that is now Belgium in the 16th century and
the Middle Ages13,35. This parasite has been detected from the icy regions
of Canada, where studies have shown that eggs can withstand the six
winter months16, to Africa, Asia and South America, passing through
the US and Europe17.
This nematode has a unique esophageal structure consisting of
specialized cells known as stichocytes (characteristic of the superfamily
Trichinelloidea). The adult forms of this parasite are very slender, small
and morphologically similar to parasites of the genus Trichuris that live
in the hepatic parenchyma. The male is 30 to 50 mm long42 and the eggs
are bi-operculate with a tray-like shape36. The completion of the life cycle
of the worm does not depend on an intermediary host as embryonated
eggs are ingested directly from the soil or animal carcasses. L1 larvae
hatch in the cecum, penetrate the mucosa, travel to the portal system
and establish themselves in the hepatic parenchyma, where they develop
from L2 to L4 larvae and finally into the adult form. Fertilized eggs are
released in groups around the female, and the female perishes after an
average period of 30 days. The viable, but still immature eggs remain for
up to 120 days. For embryogenesis to occur, the egg must be outside the
host, which can occur: a) when the host dies and its carcass disintegrates
and/or b) when the viscera of the host are ingested by predators; in the
latter case, the non-embryonated eggs are eliminated in the feces and thus
returned to the environment and embryonate12,25. There is also evidence
that arthropods (flies and beetles) can spread eggs from the soil29,30,32.
(1) Faculdade São Lucas. R. Alexandre Guimarães, 1927, Areal, 78916-450 Porto Velho, Rondônia, Brazil. E-mails: [email protected], [email protected], jessica.damascenow@hotmail.
com, [email protected], [email protected]
(2) Laboratório de Patologia Experimental, Centro de Pesquisas Gonçalo Moniz, CPqGM/FIOCRUZ. R. Waldemar Falcão 121, 40296-710 Salvador, Bahia, Brazil. E-mail: msouza@pqvisitante.
bahia.fiocruz.br
(3) Laboratório Central de Saúde Pública de Rondônia, LACEN/RO. R. Anita Garibaldi 4130, 78903-770 Porto Velho, Rondônia, Brazil. E-mail: [email protected]
(4) Laboratório de Patologia Veterinária, Via L4 Norte s/nº, Hospital Universitário, UnB Universidade de Brasília, Campus Universitário Darcy Ribeiro, 70910-970 Brasília, DF, Brazil. E-mail:
[email protected]
(5) Departamento de Clínica Médica Veterinária, CLIMEV. Faculdade de Agronomia e Medicina Veterinária, FAMEV. Universidade Federal de Mato Grosso/UFMT, Av. Fernando Correia da
Costa 2367, 78068-900 Cuiabá, Mato Grosso, Brazil. E-mail: [email protected]
(6) University of Sao Paulo. Rua Francisco Prestes 2827, Monte Negro, 78068-900 Rondonia, Brazil. E-mail: [email protected]
Correspondence to: Elierson José Gomes da Rocha, Faculdade São Lucas/Pediatria, R. Getúlio Vargas 2614, Apto. 203, 76804-060 Porto Velho, Rondônia, Brasil. Tel:55.69.99555965.
ROCHA, E.J.G.; BASANO, S.A.; SOUZA, M.M.; HONDA, E.R.; CASTRO, M.B.; COLODEL, E.M.; SILVA, J.C.D.; BARROS, L.P.; RODRIGUES, E.S. & CAMARGO, L.M.A. - Study
of the prevalence of Capillaria hepatica in humans and rodents in an urban area of the city of Porto Velho, Rondônia, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 39-46, 2015.
Seventy-two cases of hepatic capillariosis have been reported
in humans in the literature17 since the first case was described by
MACARTHUR in 192427. Infections have been documented on all
continents, and five were reported in Brazil33,34,39. In addition, C.
hepatica eggs have reportedly been found in the feces of Suruí Indians9,
ethnicities of the Guaporé-Mamoré valley38 and a riverine population in
Rondônia, Brazil4. As a rule, the infection produces the classical triad
of fever, hepatomegaly and eosinophilia and can progress to death if
left untreated34,39.
Some sero-epidemiological surveys of hepatic capillariasis have
been performed: JUNCKER-VOSS et al.25 surveyed employees of the
Vienna zoo and encountered a total prevalence of 1.8%, and GALVÃO21
documented a prevalence of approximately 1.6% in Salvador, Bahia.
CAMARGO et al., documented a C. hepatica seroprevalence of 0.8%
in the local riverine population of Rio Preto at the intersection of the
Machado and Madeira Rivers in the state of Rondônia in the Amazonian
Region of Brazil (300 km away from Porto Velho, the capital city of
Rondônia)4.
The logical follow-up to the study of CAMARGO et al. (2010)4
would be to assess the seroprevalence of this parasitic disease in the
urban area of Porto Velho, a city that suffers from many urban problems,
such as unhygienic living conditions that force many of its inhabitants to
coexist with a large population of domestic rats (an important reservoir
for the disease in urban areas)19. This study aimed to verify the findings
of CAMARGO et al.4 and propose specific intervention measures.
Because adult worms and their eggs remain confined to the liver, they
are not normally eliminated with feces. Eggs are sometimes found in the
stool of “spurious carriers” (i.e., those that swallowed immature eggs in
the viscera of other animals) that are not necessarily sick. This makes the
diagnosis of capillariasis somewhat more complex. However, available
serological techniques (Enzyme-Linked Immunosorbent Assay - ELISA
and indirect immunofluorescence - IFI) with different antigens1,24 can
identify “spurious carriers” of C. hepatica or diseased individuals while
ruling out cross-reactions with other similar helminths22,24.
Because this disease is seldom suspected, it is thought that less
severe cases are rarely detected2,24. Furthermore, clinical manifestations
of mild cases are likely to produce signs and symptoms common to other
pathologies. Therefore, the detection of these cases will heavily depend
on the degree to which C. hepatica infection is suspected.
No consensus exists on the treatment of this disease. CHEETHAM
& MARKUS6 used mebendazole and albendazole in an experimental rat
model and showed that these drugs prevented oviposition in the hepatic
parenchyma. EL GEBALY et al. and EL NASSERY et al.10,11 successfully
used ivermectin and mebendazole in an experimental model to stop
larvae from developing into adult form and thus prevent oviposition.
SAWAMURA et al.39 successfully treated three infected patients with
mebendazole and albendazole.
GENERAL AIM
The objective of this work was to study the prevalence of C. hepatica
infection in humans and rodents in the urban area of Porto Velho, the
capital of the Northern Brazilian state of Rondônia.
40
METHODS
Ethical parameters: All procedures described and performed in
this study were approved by the Ethics Committee on Research with
Human Beings of the Biomedical Sciences Institute of the University
of São Paulo through process 1051/ICB and by the Ethics Committee
on Animal Use (Comissão de Ética no uso de Animais - CEUA) of the
same institute, according to the protocol registered under number 149
on page 136 of book 02.
Study site: The study took place in the urban area of the Tucumanzal
district of the municipality of Porto Velho, Rondônia, Brazil. To improve
the distribution of samples and participants in this study, the area was
divided into five subareas: AO, A1, A2, A3 and A4 (Table 1). The region
chosen for the study has an epidemiological profile that is compatible
with the transmission of C. hepatica19-21: it is an old district of slums
populated by low/medium income residents and has irregular garbage
collection and inadequate sanitation.
Table 1
Sample stratified random sub-areas, the total area of study. Tucumanzal district,
city of Porto Velho, Rondônia, 2011
Subarea
AO
A1
A2
A3
A4
Total
No. Inhabitants
323
546
442
301
737
2,349
Sample
66
117
90
66
151
490
% (sample)
13.5
23.9
18.4
13.5
30.7
100
A UBS (Unidade Básica de Saúde - Basic Health Unit), a college and
a variety of commercial establishments are found in the area.
Census: A census of the population residing in the study area
(Tucumanzal District, city of Porto Velho (8°46’44.9”S 63°53’43.8”W
- Google Maps accessed on 03/04/2014), was conducted during the
months of February and March 2011. A total of 2,349 inhabitants
were catalogued, and the following information was gathered from
each inhabitant: name, monthly family income, address, age, gender,
information about the sanitation conditions of households, ingestion of
game meat and time residing in the area.
A sample size calculation was performed using the appropriate
statistical analysis in the Open Epi software (Open Source Epidemiologic
for Public Health) version 2.3.1 (www.openepi.com accessed on
10/01/2011). From a total of 2,349 registered inhabitants, given an
estimated prevalence of 2% and a confidence limit of 5%, a sample size
(N) of 383 individuals was obtained. This sample size was inflated by
30% to compensate for potential drop-out or refusal to participate, thus
raising the total to 498 inhabitants. The sample was randomized using
the Random Number Generator program (www.random.org accessed on
10/01/2011). Blood samples were collected from 490 of 498 randomly
selected individuals (Table 1).
The population was willing to provide blood samples, most likely
because health care in the region is lacking. To avoid concentrating the
ground and in bushes or trees up to 2 m high 20 to 40 m apart, depending
on the irregularity of the terrain.
sampling within a single region of the district, the sample was randomly
drawn from five subareas in proportion to the population of each subarea.
This process resulted in a stratified random sample.
Sedation of the rodents: After rodents were captured, the animals
were sedated using ketamine (10 mg/kg) and xylazine (1 mg/kg) using
lethal doses of the same sedatives16. Liver samples were then collected
and immediately stored in numbered tubes containing 10% buffered
formaldehyde.
Human blood collection: The blood of the 490 volunteers was
collected according to the following methodology. First, information
on the study and the parasite was provided to the residents; in addition,
participants received an informational leaflet. Next, pre-registered
residents - who had been randomly selected and agreed to take part in the
study - signed an Informed Term of Consent. Residents then underwent
a general clinical examination, and blood was collected from the cubital
fossa after the collection site was disinfected with 70% alcohol and a
tourniquet was placed on the proximal arm region using a latex strap. The
collected blood was allowed to stand to coagulate and then centrifuged
at 5,000 RPM for 10 minutes, and the serum was aliquoted into PVC
tubes and stored at -20 °C until the time of analysis.
Slides were prepared by fixing the sample material to preserve
tissue morphology, dehydrating the tissue in increasing concentrations
of ethyl alcohol, cleaning the tissues in xylol, impregnating them with
melted paraffin and embedding them. Afterwards, the tissues were cut
with a microtome, stained and mounted. Slides were prepared using
histological sections from liver samples of 50 rats (Rattus rattus and
Rattus norvegicus).
Serological technique: The technique previously proposed by
ASSIS et al.1 was used: serum samples were diluted in 50% phosphate
buffered saline (PBS) solution up to a concentration of 1:150 and tested
using histological sections of rat livers containing C. hepatica worms
and eggs embedded in paraffin supplied by the LAPEX-FIOCRUZ
Brazilian government laboratory. Seropositive samples at a dilution
of 1:150 were classified as either weakly positive (PI), moderately
positive (PII) or strongly positive (PIII) according to the intensity
of fluorescence assessed by two different examiners. Serum samples
considered either moderately or strongly positive at a dilution of 1:150
were tested again at a dilution of 1:400, i.e., at the highest dilution
used as a cut-off point for the elimination of false positives (spurious
infections).
Disposal of material: Sharp/cutting material was discarded in rigid
containers while gloves and cotton were disposed of in 10-micrometerthick opaque white plastic garbage bags according to the the Brazilian
Health Surveillance Agency for disposal.
RESULTS
Socio-economic conditions of the population: From an economic
point of view, the large majority of the residents are workers with little
or no specialization who perform manual labor in commerce, the public
service, private companies or informally. Table 2 gives an overview of the
population characteristics of the studied area. As can be seen, the sample
is composed of people of low socioeconomic status, with an average
monthly income of around US$ 400 (up to twice the minimum wage).
As a result, residents belong to classes D and E of the classification of
the Brazilian Institute of Geography and Statistics i.e., they have low
incomes (Table 2).
System for capturing rodents: Rodents were captured using 20
“Tomahawk” live traps (20 x 20 x 40 cm) with banana, bread and peanuts
as bait. All traps were mounted and remained active from 6:00 pm on
Wednesday to 6:00 pm on Friday. Traps were checked and bait was
replaced every 12 hours. If rodents were found in the traps at inspection
times, they were sedated until death. These capture activities took place
over a period of nine months. The traps were placed along a line of 10
points with approximately two traps per point; traps were laid on the
Table 2 shows the urban conditions in which they live. They have poor
sanitation and these areas that are liable to flooding favor the proliferation
of rats. An aggravating factor for the risk of spurious infections is the
frequent ingestion of game meat and viscera by residents.
Table 2
Socio-economic, cultural and urban characteristics of the study area. Tucumanzal District, Porto Velho, Rondônia, 2012
Subareas
A0
A1
A2
A3
A4
Total inhabitants
323
546
442
301
737
Up to 1.5
< 1.0
< 1.0
Up to 1.0
Up to 1.0
Wastewater discharged to the stream
Per capita income (MW)
No
Yes
Yes
No
Yes
Igarapé (tributary river)
No
Yes
Yes
No
Yes
Households with river water catchment and/or sewage (%)
75
60
20
90
95
2
Households with running water (CAERD) (%)
95
85
60
95
90
Paved roads (%)
95
90
75
100
100
3 times
3 to 4 times
3 times 3
3 times
3 to 5 times
35
35
41.1
27.2
34.4
Weekly garbage collection1
Game meat consumption among residents (%)
1
- Selective garbage collection was not mentioned. 2 - Refers to inundation during rainfall. 3 Garbage collection is not performed on some streets. MW: minimum wages.
41
Material collected from rodents: Table 3 summarizes the results
of anatomopathological studies of the livers of the 50 captured rats; the
data are categorized by subareas.
Table 3
Anatomopathological analysis of the rat livers. Tucumanzal District, Porto
Velho, Rondônia, 2012
Total/Subarea
Necrosis
Septal
Fibrosis
Parasites
and/or eggs
A0
07
Zero
Zero
Zero
A1
11
03 (27.3%)
02 (18.8%)
Zero
A2
05
Zero
Zero
Zero
A3
15
04 (26.6%)
Zero
Zero
A4
12
04 (33.3%)
02 (16.6%)
01 (8.3%)
Total
50
11 (22%)
04 (8%)
01 (2%)
Subareas
In addition to being inspected for eggs and worms (which would
confirm the infection beyond any doubt), tissue samples were also
inspected for the presence of septal fibrosis (probable infection), focal
fibrosis, necrosis and inflammation (suspected infection).
Several anatomopathological studies of the livers of rats infected by
C. hepatica have shown that while the observed presence of parasite eggs
and/or worms definitively confirms the infection, the presence of hepatic
septal fibrosis is also an important characteristic that appears after the death
and degeneration of worms in the presence of eggs14,37. Such a finding,
though non-conclusive, would strongly suggest infection by C. hepatica.
Material collected from humans: Serology (IIF) of blood samples
from the 490 residents of the selected area of Porto Velho was performed
to quantify the prevalence of C. hepatica infection.
As observed in Table 4, nine of the 490 human blood samples were
positive by IIF for C. hepatica at the 1:150 dilution (1.8%); four of these
also reacted positively at the 1:400 dilution (0.8%) (two in subarea A1
and two in subarea A3).
An equal number of men and women tested positive by IIF at the
1:400 dilution; these individuals were aged 10-19 (one case), 20-29 (two
cases) and above 50 (one case).
Table 4
Serology (IIF) results by subarea, gender and serum dilution. Tucumanzal
District, Porto Velho, 2012
Number of
Individuals
Positive at
1:150 dilution
Positive at
1:400 dilution
Zero
66
1
Zero
A1
117
4
2
A2
90
Zero
Zero
Subarea
A3
66
4
2
A4
151
Zero
Zero
490 (100%)
9 (1.8%)
4 (0.8%)
Total
42
The following laboratory tests were further performed on the
individuals with positive serology at the 1:400 dilution: complete blood
count and measurement of levels of aspartate aminotransferase (ATS),
alanine aminotransferase (ALT), bilirubin total and fractions. As observed
in Table 5, only one of the four residents with positive serology at the
1:400 dilution displayed altered laboratory parameters, namely, a mild
to moderate increase in her total and indirect bilirubin levels. All of these
individuals underwent a liver ultrasound, but none of them displayed
any abnormalities.
DISCUSSION
Although previously published studies show that the prevalence of
C. hepatica in certain rodent species, tends to be high (between 56.5 and
89.3%)5,7,20,31, the prevalence of C. hepatica infection appears to be low
among residents of the studied area and among rodents that live close
to their dwellings.
To ascertain whether a rodent is infected with C. hepatica, eggs
or worms must be found in its liver. In the present study, eggs and
worms were detected in the liver of only one of the animals captured in
subarea A4, which corresponds to a prevalence of 2%. This animal also
presented with septal fibrosis. Studies have shown that septal fibrosis
occurs in practically all rats infected with C. hepatica5,14,22,37. FERREIRA
& ANDRADE14 stated that experimental C. hepatica infection in rats
represents a good model for the study of liver septal fibrosis because
all rats in their experiments developed septal fibrosis after the 40th day
of parasite infection. A similar result was found by SANTOS et al.37.
In the study, three other animals had septal fibrosis, which could
raise the prevalence among rodents to 8%; however, 8% is still lower
than the values encountered in other studies7. Seventeen animals in the
present study also had non-specific inflammation, and eleven animals
had marked necrotic areas that can be associated with infection by the
parasite, although not a specific manifestation.
The low prevalence of infection in rodents in the study area (Table
4) is consistent with the low prevalence encountered among humans. As
observed by SPRATT & SINGLETON41, most C. hepatica infections
in non-rodent mammals occur when these animals live near infected R.
norvegicus populations5.
It is likely that the reduced size of the rat sample did not allow the
present study to demonstrate the association between the presence of
infected rodents and a high prevalence of parasite infection. It is important
to try to understand why the prevalence of C. hepatica in rats in the urban
region of Porto Velho was so low. Infection with this parasite occurs
after the infected animal dies and its liver decays in the environment,
thus releasing the eggs that become infective after a certain period of
time. Once ingested by other animals, these eggs cause infection in a
wide variety of species, including humans. The ingestion of eggs, even
directly from the livers of infected animals, does not necessarily lead
to infection, as the eggs must travel undamaged through the digestive
tract, be released in the feces and reach the soil, where their maturation
cycle is completed. The city of Porto Velho is located in a very rainy
region with long daily episodes of high-volume rainfall. The site where
the present study was conducted has streams that usually overflow, thus
“washing out” the site and carrying much of the accumulated garbage to
Table 5
Results of C. hepatica positive serology (IIF) residents (1:400). Tucumanzal District, Porto Velho, Rondônia, 2012
Resident
Laboratory test
Reference Values
1
2
3
4
Red blood cells 106/mm3
4.85
4.81
5.24
5.18
4.1 - 6.0
Hemoglobin g/dL
13.9
14.3
13.6
15.6
12 - 18
Hematocrit %
41.7
43.8
42.3
45.8
37 - 54
Leukocytes 103/mm3
5.320
6.75
9.09
6.44
4 - 10
Band cells %
2
2
2
1
0-4
Segmented neutrophils %
46
65
63
50
45 - 70
Eosinophil’s %
5
1
1
2
1-5
Basophils %
0
0
0
0
0-2
Typical lymphocytes %
38
29
29
37
20 - 40
Atypical lymphocytes %
0
0
0
0
0-2
Monocytes %
9
5
5
10
2 - 10
Platelets 10 /mm
235
197
323
336
150 - 450
ATS U/L
13
10
21
17
10 - 39
ALT U/L
12
11
23
13
10 - 37
Total bilirubin mg/dL
1
1.1
0.9
1.5
1.2
Direct bilirubin mg/dL
0.2
0.2
0.2
0.2
0.4
Indirect bilirubin mg/dL
0.8
0.9
0.7
1.3
0.8
3
3
the Madeira River channel. Therefore, animal carcasses do not remain in
the environment; as a result, the spread of eggs and their ability to mature
in an adequate environment is limited. It is possible that the study area
represents a region where the disease has only recently been introduced
instead of an endemic region where a high prevalence in rats was detected.
When the results obtained in the present study are compared with
those of CAMARGO et al.4 in riverine populations of the state of
Rondônia, it becomes clear that the riverine population experiences
much higher rates of contamination than the urban population, but only
with regard to the spurious infections. When the spurious infection rates
(1:150 dilution) of both populations are compared (x2 =153.6 and p <
0.0000001), it appears likely that the riverine population experiences
much higher rates of spurious contamination (34.1%) than the urban
population (1.8%), perhaps due to the ingestion of wild animal viscera.
However, when the seroprevalence is analyzed at higher dilutions that
are indicative of true infection (1:500 in the study of CAMARGO et al.
and 1:400 in the present study), similar rates of approximately 0.8% are
found in both populations (x2 = 0.2289 and p = 0.2161).
In reality, the data presented in this study corroborate the results of
CAMARGO et al.4. Because the riverine population is characterized by
low socio-economic conditions, people from this area tend to frequently
eat game meat and use the viscera to prepare ‘farofa,’ a dish made of
manioc flour mixed with giblets. The consumption of viscera greatly
increases the likelihood of ingesting non-embryonated C. hepatica
eggs, which may not cause infection but does result in enough antigen
stimulation to trigger seropositivity at a 1:150 dilution. As reported in
the present study (Table 2), the consumption of game meat is much
less common in the urban population than in the riverine population.
According to CAMARGO et al.4, 91.7% of the riverine population had
ingested viscera in the 15 days prior to the survey. In contrast, 27.2 to
41.1% of the urban population had ingested game meat in the 15 days
preceding the study. This is the most likely explanation for the different
rates of spurious infection (positive serology up to 1:150) between the
riverine and urban populations.
Hepatic capillariasis is a disease with potentially high rates of
contamination, low infectivity and low pathogenicity4. The low prevalence
among humans can be explained not only by the low prevalence among
rodents but also by the presence of running water and septic tanks in most
dwellings, which prevent the exposure of humans to embryonated eggs.
The physical examination of the four residents with positive
serology (IIF) for C. hepatica at the 1:400 dilution found no significant
physiological alterations, such as jaundice, pain or visceromegaly. None
of these four individuals had any abnormal laboratory findings (Table 5);
similarly, results of their liver and bile duct ultrasound exams appeared
to be within normal limits.
Abdominal pain and jaundice associated with hepatitis are clinical
signs typically observed in humans infected with C. hepatica. CHOE
et al.8 reported the case of a female 14-month-old child who presented
with the triad of persistent fever, hepatomegaly and leukocytosis with
eosinophilia. SAWAMURA et al.39 reported three cases of children
aged between 18 and 36 months infected with the parasite; their disease
43
progressed to include symptoms of pain, choluria, jaundice, weight
loss, fever and night sweats that only disappeared after antiparasitic
treatments were administered. For these reasons, a C. hepatica infection
must be considered as a differential diagnosis in cases of suspected
hepatitis A, B or C and leptospirosis, which are frequent in the studied
region.
hepatica in the laboratory for the IIF tests. JCDeS participated in field
work, data collection and analysis. LPB participated in field work, data
collection and analysis. ESR participated in field work, rat capture and
preparation of the specimens (liver) in paraffin blocks. All authors read
and approved the final manuscript.
AUTHORS’ INFORMATION
Future studies in other districts or older cities in the state may help
answer these questions and better understand this zoonosis.
CONCLUSIONS
• Anatomopathological analyses of the livers of 50 rats captured in
the periphery of dwellings in the study area and serological tests of
490 individuals in the same area using IIF were performed. A lower
prevalence of C. hepatica infection was found in rodents in this study
than in other studies in the literature. However infection of humans
lies close to that found in other studies.
• Spurious infections characterized by positive serology (IIF) at a low
serum dilution (i.e., 1:150) were detected among the residents. This
finding suggests that humans have contact with C. hepatica eggs.
This prevalence is much lower than the prevalence reported in the
riverine population (x2 = 154.6, p < 0.0000001), most likely because
the riverine population habitually consumes wild game viscera
infected with C. hepatica, while consumption in the studied urban
population is much lower.
• A low prevalence (approximately 1%) of true infections was
encountered. Infection with C. hepatica should be included in
differential diagnosis lists along with viral hepatitis and leptospirosis.
Importantly, the immediate treatment of capillariasis reduces its
lethality8,39.
• Even though the prevalence of C. hepatica is low, the municipality
should improve its urban areas to diminish the rat population.
• To better understand the situation encountered in this study (namely,
the low prevalence of C. hepatica infection in an urban area of Porto
Velho, the authors propose to 1) expand the study area, performing
new assessments in other districts of the city or in Guarajá-Mirim,
a nearby city as old as Porto Velho, and 2) increase the sample size
by capturing and analyzing more rats.
COMPETING INTERESTS
All the authors declare no conflict of interest.
AUTHORS’ CONTRIBUTIONS
EJGdR coordinated the research and wrote the article. LMAC:
supervised the research and made the statistical analysis. SAB participated
in the assessments and medical follow-ups of the residents during the data
collection stage. MMdS participated in the supervision of the serological
tests for the blood samples of residents. ERH performed the serological
tests (IIF) for the serum blood samples of residents. MBdC performed
the anatomopathological tests of the rat livers. EMC prepared the slides
with the liver sections from rats experimentally infected by Capillaria
44
EJGdR: Physician, first author of the study. Professor of Pediatrics,
preceptor of the Internship in Outpatient Pediatrics, São Lucas Medical
School in Porto Velho, state of Rondônia and Master’s candidate at the
Institute of Biomedical Sciences, University of São Paulo (Universidade
de São Paulo - USP). LMAC: Physician. Researcher, PhD, Coordinator
and Professor at the Institute of Biomedical Sciences-USP and at the
São Lucas Medical School in Porto Velho. SAB: Physician. Professor
of Medicine and preceptor of the course of Basic Health Care at São
Lucas Medical School in Porto Velho, RO. PhD candidate at the Institute
of Biomedical Sciences-USP. MMdS: Biologist. PhD and researcher at
Fiocruz, state of Bahia. ERH: Pharmacist. PhD and researcher at the
Central Laboratory for Public Health of Rondônia. MBdC: Veterinarian.
PhD and professor at the Federal University of Brasília. EMC:
Veterinarian. Professor and PhD Researcher at the Federal University of
Mato Grosso. JCDeS: Student of the Scientific Initiation Program of the
Graduate Program of Medicine at São Lucas Medical School, holder of a
CNPq (National Council of Technological and Scientific Development)]
grant. LPB: Scientific Initiation student of the Graduate Program of
Medicine at São Lucas Medical School, holder of a CNPq grant. ESR:
Scientific Initiation student of the Graduate Program of Biology at São
Lucas Medical School, holder of a CNPq grant.
LIST OF ABBREVIATIONS USED
C. hepatica - Capillaria hepatica; ELISA - Enzyme-Linked
Immunosorbent Assay; IBGE - Brazilian Institute of Geography and
Statistics; IIF - Indirect immunofluorescence; N - sample size; PBS
- Phosphate Buffered Saline; PVC- Polyvinyl chloride; R. norvegicus Rattus norvegicus; R. rattus - Rattus rattus; RPM - rotations per minute.
RESUMO
Estudo da prevalência da Capillaria hepatica em humanos e
roedores em área urbana da cidade de Porto Velho, Rondônia,
Brasil
Introdução: Capilaríase hepática é causada pela Capillaria
hepatica (syn. Calodium hepaticum) (Bancroft, 1893), Travassos, 1915
(Nematoda, Trichinelloidea, Capillariidae), sendo uma zoonose comum
entre roedores, porém rara em humanos. Setenta e dois casos humanos
foram relatados na literatura mundial desde o primeiro caso descrito
por MACARTHUR em 192417,27. O objetivo desse estudo é determinar
a prevalência da Capillaria hepatica em humanos e roedores de área
urbana da cidade de Porto Velho, capital de Rondônia, Brasil. Método:
Após realizar um censo da área, 490 moradores foram aleatoriamente
selecionados e assinaram termo de consentimento, foram colhidas
amostras de sangue para testar anticorpos anti-Capillaria hepatica.
Simultaneamente, ratos foram capturados para determinação da
prevalência deste parasita através do exame histopatológico em cortes
de fígado. Resultados: Foi encontrada entre humanos prevalência de
1,8% de positividade para anticorpos específicos em diluição de 1:150,
indicando exposição aos ovos do parasito; 0,8% desses também deram
testes positivos quando seus soros sofreram diluição de 1:400, indicando
infecção verdadeira. Nos ratos, a prevalência foi de 2%. Conclusão:
A prevalência encontrada para o parasito entre homens e roedores foi
baixa. Enquanto a prevalência encontrada entre humanos esteve dentro
dos limites encontrados na literatura, a prevalência entre roedores foi
bem menor.
ACKNOWLEDGEMENTS
To Prof. Doctor Luis Marcelo Aranha Camargo for his availability
and patience as supervisor of this study. For his generosity in sharing with
me his knowledge and experience in the field of Medicine and Medical
Research, and for always seeking to help me, guide me, and clarify any
questions that arose - and they were many - during the conduction of
the study.
To all colleagues who participated in the study, contributing with
their knowledge and willingness to help and often giving up their time
to support me. Without them, it would have been impossible to conduct
this study. To the Institute of Biomedical Sciences-USP, one of the most
well-renowned education institutions in Latin America, for allowing
me to enroll as a Master’s candidate. To the Faculdade de Medicina São
Lucas for providing the necessary support for developing this work. To
the students of the medicine program and related programs in the field
of health who rolled up their sleeves and helped with the field work by
collecting data and blood from the residents, capturing rats at the research
site, preparing material from the rats for anatomopathological tests and
preparing slides for serological tests. Without them, this research study
would have taken much longer and all planned objectives may not have
been reached. To the inhabitants of the district of Tucumanzal for their
collaboration.
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http://dx.doi.org/10.1590/S0036-46652015000100007
SEROPOSITIVITY FOR ASCARIOSIS AND TOXOCARIOSIS AND CYTOKINE EXPRESSION AMONG
THE INDIGENOUS PEOPLE IN THE VENEZUELAN DELTA REGION
Zaida ARAUJO(1), Sietze BRANDES(2), Elena PINELLI(2), María A. BOCHICHIO(1), Andrea PALACIOS(1),
Albina WIDE(3), Bruno RIVAS-SANTIAGO(4) & Juan Carlos JIMÉNEZ(5)
SUMMARY
The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/
secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification
of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were
adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the
other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively.
While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The
percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera
were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized.
When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and
193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly
observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 expressions showed that
there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara
seropositivity was prevalent among Warao indigenous.
KEYWORDS: Zoonoses; Ascaris suum; Toxocara canis; Warao.
INTRODUCTION
Infections with gastrointestinal nematode parasites are widespread
and contribute significantly to both morbidity and mortality among
humans, and livestock in developing countries45. The most prevalent
parasitic helminth in humans, Ascaris lumbricoides, is estimated to
infect 1.5 billion people globally4. Toxocara canis and Ascaris suum
are roundworms of dogs and pigs, respectively; these are the causative
agents of important zoonoses such as toxocariosis and ascariosis2,4.
Humans may accidentally become infected with T. canis or A. suum
after ingestion of embryonated eggs present in soil contaminated with
dog or pig feces or after consumption of infected raw or undercooked
meat2,45. In the accidental hosts, the T. canis larvae do not develop to the
adult stage but persist in tissues as the larval stage for many years33,34.
Once the infective eggs are ingested the larvae hatch, penetrate the
small intestine and migrate to different tissues in the body inducing
inflammatory responses. Migration of larvae can lead to a syndrome
known as Visceral Larva Migrans (VLM). Symptoms of VLM include
fever, hepatosplenomegaly and respiratory distress such as wheezing,
coughing and episodic airflow obstruction34,38. Other symptoms include
eosinophilic pneumonia (Loeffler’s pneumonia) that bears a clinical
resemblance to the pulmonary inflammatory responses observed in
asthmatic patients. Immunological features of these zoonoses include
eosinophilia and increased serum IgE levels30,36,40.
Diagnosis of these zoonoses depends mostly on serological tests
because the eggs are not passed in the feces of the host and biopsies
to detect the larvae are usually negative11,12. Since the studies made
by SAVIGNY37, the antigens mostly used for the immunodiagnostic
tests are excreted products derived from larvae cultivated in vitro and
are referred to as Toxocara excretory/secretory (TES) antigens35,37.
Nematode excretory/secretory (E/S) antigens are not species or genus
specific and serum samples from patients with ascariosis, filariosis
and strongyloidiosis show cross-reactivity with ES from T. canis and
A. suum antigens when using enzyme-linked immune assay (ELISA),
immunoprecipitation and Western blotting3,5,18,20,21,29,37. A major concern
is the specificity of the ELISA and WB diagnosis of T. canis and A. suum
in areas where gastrointestinal nematode infections of humans also exist.
In this context, in most areas in which A. lumbricoides is endemic, which
is a common intestinal nematode of children, exposure to A. suum and
(1) Laboratorio de Inmunología de Enfermedades Infecciosas, Instituto de Biomedicina, Universidad Central de Venezuela, Caracas, Venezuela.
(2) Centre for Infectious Diseases Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
(3) Laboratorio de Biotecnología, Instituto de Medicina Tropical, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela.
(4) Unidad de Investigación Médica Zacatecas, Instituto Mexicano del Seguro Social, Zacatecas, México.
(5) Laboratorio de Bioquímica, Instituto de Inmunología, Universidad Central de Venezuela.
Correspondence to: Dr. Zaida Araujo, Laboratorio de Inmunología de Enfermedades Infecciosas, Instituto de Biomedicina, Universidad Central de Venezuela. Caracas 1043, Venezuela.
ARAUJO, Z.; BRANDES, S.; PINELLI, E.; BOCHICHIO, M.A.; PALACIOS, A.; WIDE, A.; RIVAS-SANTIAGO, B. & JIMÉNEZ, J.C. - Seropositivity for ascariosis and toxocariosis and
cytokine expression among the indigenous people in the Venezuelan Delta region.. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 47-55, 2015.
T. canis is likely to be sufficiently common to confuse serodiagnosis. In
Venezuela, diagnosis of zoonotic infections with A. suum and T. canis
is routinely made by ELISA after absorption of serum samples with A.
lumbricoides antigens, a nematode antigenically related with A. suum
and T. canis6,22.
Although parasitic helminth infections generally do not lead to
mortality; chronic infections can lead to considerable morbidity25,31.
Chronic helminth infections are characterized by skewing towards a T
helper 2 type response as well as regulatory responses8,43. The regulatory
network is thought to prevent strong immune responses against parasitic
worms, allowing their long-term survival and restricting pathology. A
number of parasitic nematodes have also been reported to exert potent
immunomodulatory effects also suppressing immune responses to
non-parasite antigens and to other infectious agents in a nonspecific
manner30,41. PATERSON et al. have reported that the body fluid from the
adult Ascaris suum (ABF) has potent immunomodulatory activity and
that the effects observed are consistent with skewing towards Th2-type
response32,40. In addition, the induction of interleukin-10 by ABF also
suggests that T regulatory cells may play a role in immunomodulation
of immune responses by parasitic helminths32.
The present study aimed to measure anti-Ascaris suum and antiToxocara canis antibodies in sera through ELISA and Western blotting
techniques. In addition parasitological examination from stool samples
and the cytokine gene expression of pro-inflammatory and Th1/Th2-type
cytokines in blood of Warao indigenous, a population that live closely
with pigs and dogs, were performed.
The study was conducted in Warao indigenous communities from
the Antonio Díaz and Pedernales municipalities, in the Venezuelan
Delta, two parasitic-endemic rural regions of the Delta Amacuro State.
The Warao indigenous communities are settled in remote rural areas.
The houses are constructed on wooden stilts on the River Orinoco. Most
inhabitants do not have access to reliable potable water for drinking
nor adequate sanitation systems. Within this indigenous population is
not easy to carry out research projects because invasive procedures
cannot be used to take samples due to ethical considerations. Adults
and children aged 15 to 70 and four to 14, respectively, were studied,
of whom according to ELISA reactivity were grouped as: 1) Sera
positive for the excretory/secretory (E/S) antigens of Ascaris suum
(AES), 2) Sera positive for the E/S antigens of Toxocara canis (TES),
3) Sera positive for both, AES and TES antigens and 4) Sera negative
for both AES and TES antigens which were used as a negative reference
(control group).
Blood samples were collected in vacutainers with and without EDTA
as anticoagulant. Serum was separated and stored at -20 °C until use.
Individuals were included in the study taking into account inclusion and
non inclusion criteria. Inclusion criteria: 1) The volunteers are healthy
individuals without evidence of clinical symptoms suggesting pulmonary
infection. Non inclusion criteria: 1) Individuals who were HIV positive,
2) Patients taking immunosuppressive drugs (e.g., corticosteroids,
azathioprine and cyclophosphamide), 3) Participants who did not
sign an informed consent agreement. This study was approved by the
Ethical Committee of the Biomedicine Institute (protocol number PG48
09-8007/2011). All the inhabitants who participated were included after
obtaining a free and informed consent statement from them.
The excretory-secretory (E/S) antigen derived from Toxocara canis
and Ascaris suum was prepared as previously described33. Briefly, adults
of T. canis and A. suum worms were collected from the feces of naturally
infected dogs and pigs, after routine deworming using antihelminthic
treatment. Eggs were collected from the uteri of female worms and were
allowed to embryonate in 0.05 M H2SO4 in the dark at room temperature
for 4-6 weeks. Embryonated eggs were stored in 0.05 M H2SO4 at 4 °C
until use. Larvae were freed from the egg shells and allowed to migrate
through cotton wool contained in Pasteur pipettes that were placed in
tubes filled with medium at 37 °C overnight. The migrating larvae were
collected and counted. A suspension of 150 larvae per mL medium
was incubated at 37 °C. Fresh medium was added and after a week, the
harvested medium was used as the E/S antigens.
Detection of anti-Toxocara and anti-Ascaris IgG antibodies was
performed using an ELISA and the excretory/secretory (E/S) antigens
derived either from T. canis or A. suum larvae as previously reported33.
Medium binding ELISA microtiter plates (Nunc, Roskilde, Denmark)
were used for the Toxocara ELISA and high binding plates (Greiner,
Frickenhausen, Germany) were used for the Ascaris ELISA. The plates
were coated with E/S antigens (10 μg/mL) diluted in 0.1 M sodium
carbonate (Na2CO3), pH 9.6. The plates were incubated overnight (without
lids) at 37 °C to allow the E/S antigens to dry onto the wells. They were
then washed three times with phosphate-buffered saline (pH 7.2) containing
0.05% v/v Tween-20 (PBS/Tween). For the Ascaris ELISA an additional
blocking step was performed by adding 2% bovine serum albumin (BSA)
(Boehringer Mannheim, GmbH, Germany) solution in PBS/Tween to
every well. The plates were incubated for 30 min at 37 °C and thereafter
washed three times with PBS/Tween. Serum samples were diluted 1:40 in
2% BSA/PBS/Tween and added to the plates. After one hour incubation at
37 °C, the plates were washed and anti-human IgG conjugated to alkaline
phosphatase (DAKO, Glostrup, Denmark) diluted in 4% BSA/PBS/Tween
was added for one hour at 37 °C. After the plates were washed, substrate,
H2O2, 0.05% and 5-ASA was added for one hour at room temperature
after which the absorbance was read at 450 nm. The extinction value of
the tested serum and of the cut-off serum was used to calculate a ratio. A
ratio higher or equal to 1.0 was considered positive. The cut-off value was
defined as the mean absorbance of 20 serum samples from healthy blood
donors plus three times the standard deviation33.
Electrophoresis procedure was performed; TES and AES antigens
were fractionated by polyacrylamide gel electrophoresis with dodecil
sulfate (SDS-PAGE) according to LAEMMLI17 10% running gel and a
4% acrylamide stacking gel were used. 1.2 µg of TES or AES antigens
was mixed with sample buffer (Tris 0.5 M, pH 6.8; 10% SDS, 0.4 mL of
2-mercaptoethanol, 0.3% bromophenol blue, 2 mL of glycerol) for one
min and applied to a polyacrylamide gel. The sample was electrophoresed
with constant voltage (100 V) until bromophenol blue had entered the
running gel when it was increased to 120 V. Transfer buffer, pH 8.3,
contained three g of Tris base, 14.4 g of glycine and one g of SDS.
Molecular weight standards (Sigma SDS-200) were included to calculate
molecular weights.
Transfer was performed according to TOWBIN et al.42 in a Miniprotean II cell (Bio-Rad Laboratories, CS, US) using 180 mAmp
applied over two hours to a nitrocellulose membrane in transfer buffer.
Nitrocellulose strips containing transferred proteins were rinsed with PBS
and incubated for one hour with PBS-Tween and 5% skimmed milk to
block remaining free sites and test sera diluted 1:100 overnight. Following
three washes with PBS-Tween to remove unbound antibody, strips were
incubated for one hour in anti-human IgG conjugated with peroxidase
diluted 1:2000 (Vector lab, Inc). After three washes with PBS-Tween and
5% skimmed milk, TMB substrate KIT containing two drops of buffer
stock solution, three drops of tetramethylbenzidine (TMB), two drops
of stabilization solution and two drops of hydrogen peroxide was added
and bands were visible within 5-15 min (peroxidase substrate kit TMB
SK-4400, Vector lab., Inc). To prevent cross-reactions, sera tested in the
Toxocara-ELISA and Ascaris-ELISA were pre-absorbed with A. suum
or A. lumbricoides extracts diluted 1:50 in PBS with 5% skimmed milk,
at room temperature.
For coproparasitological tests, two stool samples per individual
were taken and collected fresh or preserved in Railliet-Henry solution
at room temperature. Stool samples collected were analyzed using the
gravitational sedimentation, Kato-Katz techniques and two fecal smears
per individual were analyzed by direct microscopic observation to detect
eggs of helminth and also protozoan cysts. The results of the stool
examinations were provided to patients and the parents, and appropriate
treatment for parasite infections was given.
As regards relative quantification of cytokine expressions, total
RNA was extracted from peripheral blood cells by using a Total RNA
Isolation System kit (Promega Corporation, WI, US) following the
instructions of the supplier, and the RNA content was measured in a
spectrophotometer at 260 nm. cDNA was made from five micrograms
RNA using a Reverses Transcription System kit (Promega Corporation,
WI US). The RNA was incubated with a one µL of oligo dT primer
(50 µM), made up to 12 µL with sterile and RNaseOut-free water, and
incubated at 70 ºC for 10 min, after which it was quickly cooled on ice.
A total of two µL 10X first-strand buffer (100 mMTris-HCl, pH 8.8 at
25 ºC; 500 mMKCl; 1% Triton X-100), two µL MgCl2 (25 mM), two
µL deoxynucleoside trisphosphate mix (10 mM of each dATP, dGTP,
dCTP and dTTP) and one µL of RNaseOutRNase inhibitor (40 U/µL)
were added. The mix was incubated at 42 ºC for two min after which
it was further incubated at 42 ºC for 50 min and 70 ºC for 10 min
with one µL of AMV Reverse Transcriptase. The cDNA content was
measured in a spectrophotometer at 280/260 nm. The cDNA samples
were stored at -80 °C until use.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was
performed, 25 nanograms of cDNA generate as above was amplified and
made up to 10.5 µL with sterile and nuclease-free water. Twelve and a
half µL of the master-mix containing the PCR buffer (50 mMTris-HCl,
pH 9; 50 mMNaCl; 5 mM MgCl2; 200 µM of each deoxynucleoside
trisphosphate, dATP, dGTP, dCTP and dTTP) and the Taq DNA
polymerase (50 U/µL) and one µL of each primer (five mM) was added
in a final volume of 25 µL. Sequences of the used primer pair are shown
in Figure 1. Mixtures with cDNA were placed in a MJ mini Personal
Thermal Cycler (BioRad Laboratories, CA, US) preheated to 95 ºC for
10 min. Cycling parameters were 40 cycles of denaturation at 95 ºC for
15 sec, annealing at 60 ºC for one min, extension at 70ºC for one min
and a final extension for seven min at 72 ºC. For IL-2 and β-actin the
annealing was carried out at 58 ºC and 55 ºC respectively. Amplified
products were separated by 2% agarose gel electrophoresis, stained with
SYBR Green I (Sigma-Aldrich Co, St. Louis MO, US) and visualized by
a Benchtop UV transilluminator, MultiDoc-It Digital Imaging System
camera combination.
IL-2
Forward: 5'-AAGTTTTACATGCCCAAGAAGG-3'
Reverse: 5'-AAGTGAAGTTTTTGCTTTGAGC-3'
IL-4
Forward: 5'-CACCGAGTTGACCGTAACAG-3'
Reverse: 5'-GCCCTGCAGAAGGTTTCC-3'
IL-6
Forward, 5'-ATGTAGCCGCCCACACAGA-3'
Reverse, 5'-CATCCATCTTTTTCAGCCAT-3'
IL-10
Forward, 5'-ACAGGGAAGAAATCGATGACA-3'
Reverse, 5'-TGGGGGAGAACCTGAAGAC-3'
IL-12p35
Forward, 5'-CACTCCCAAAACCTGCTGAG-3'
Reverse, 5'-TCTCTTCAGAAGTGCAAGGGTA-3'
IFN-γ
Forward, 5'-TTTGGATGCTCTGGTCATCTT-3'
Reverse, 5'-TTTGGATGCTCTGGTCATCTT-3'
TGF-β
Forward, 5'-CAGCCGGTTGCTGAGGTA-3'
Reverse, 5'-GCAGCACGTGGAGCTGTA-3'
TNF-α
Forward, 5'-GCCAGAGGGCTGATTAGAGA-3'
Reverse, 5'-CAGCCTCTTCTCCTTCCTGAT-3'
β-actin
Forward, 5'-GTGGGGCGCCCCAGGCACCA-3'
Reverse, 5'-CTCCTTAATGTCACGCACGATTTC-3'
Fig. 1 - Primers sequences. The following primer pairs were used: IL-2, IL-4, IL-6, IL-10,
IL-12p35, IFN-γ, TGF-β, TNF-α and β-actin as internal control.
Statistical analysis was carried out using the software Epi-Info 6.0.
Chi-square test was used to compare the significance of the differences
according to the percentage values of seropositivities for infection by
Ascaris suum (AES) and Toxocara canis (TES). A logistical regression
method was used to compare independent variables including cytokine
expression in peripheral blood cells from Warao indigenous with or
without antibodies against the AES and TES antigens. A probability
value p < 0.05 was considered statistically significant.
RESULTS
Study population. Forty three adults aged 40 ± 16 years old (25
females and 18 males) and seven children aged 9 ± 2.6 years old (four
females and three males) were studied (data not shown).
Comparable seropositivity for ascariosis and toxocariasis. The
percentage of individuals with AES and TES seropositivity is shown in
Table 1. Findings indicate that in adults, 19/43 (44.1%) were seropositive
for both parasites, whereas children had only seropositivity for one or the
other helminth, 0/7 (0%), there was significant difference between adult
and children groups, p < 0.03 (Table 1). For ascariosis, the percentage
of AES seropositivity in adults and children was 10/43 (23.3%) and
4/7 (57.1%), respectively (Table 1). While that for toxocariasis, the
percentage of TES seropositivity in adults and children was low; 4/43
(9.3%) and 1/7 (14.3%), respectively, there was no significant difference
among groups. The percentage of seronegativity was comparable for
AES and TES antigens in adults 12/43 (27.9%) and children 2/7 (28.6%)
(Table 1).
49
Table 1
Comparable seropositivity for Toxocara canis and Ascaris suum
Female (%)
57.1
20.0
42.1
80.0(c)
Male (%)
42.9
80.0
57.9
20.0(d)
Adults (%)
23.3
9.3
44.1
27.9
shown). Western blotting procedures showing the obtained bands and
the WB profile according to AES seropositivity are shown in Figure 2.
Western blotting analysis using AES antigens showed the AES pattern
composed by bands ranging from 52 kDa to 206.9 kDa (some of the most
frequently recognized bands are shown in Figure 2A). The WB profile
could be divided in the following groups according IgG reactivity to
AES antigens; the group composed of bands between 50 kDa and 100
kDa was the most reactive (50%), followed by 151-207 kDas (37.5%)
and 101-150 kDas (12.5%) (Fig. 2B).
Children (%)
57.1
14.3
0
28.6
As regards T. canis, the immunoblotting showed a pattern of bands,
Marker
A. suum
Positive
T. canis A.suum/T. canis A.suum/T. canis
Positive
Positive
Negative
(a)
(b)
For the AES and TES seropositivity group there was significant difference between
(a) and (b), p < 0.03. Within the seronegativity group there was significant
difference between females (c) and males (d), p < 0.002.
Gender difference in ascariosis and toxocariosis. When the
possible gender effect on seropositivity for A. suum and T. canis was
analyzed, findings showed that gender has no influence on either AES
seropositivity; 8/14 (57.1%) and 6/14 (42.9%) for females and males,
respectively, or AES/TES seropositivity; 8/19 (42.1%) and 11/19
(57.9%) for females and males, respectively (Table 1). In contrast, a
high percentage of males showed TES seropositivity 4/5 (80.0%) as
compared to females 1/5 (20.0%), there was not a significant difference,
probably due to the low number of individuals that composed these
groups (Table 1). Within the seronegative group there was a significant
difference between females 12/15 (80.0%) and males 3/15 (20.0%), p
< 0.002 (Table 1).
Age distribution and seropositivity for Ascaris and Toxocara. The
possible age effect on seropositivity for A. suum and T. canis was also
analyzed. The distribution of the age group was: 0-10, 11-20, 21-40 and
41-60 years old and the age group of 61 years and older. The percentage
of A. suum seropositivity ranged from 7.7% for the age group of 0-10
years old to 30.8% for the age group of 21-40 years old. The latter showed
a higher percentage of A. suum seropositivity statistically significant as
compared to the age groups of 0-10 (7.7%), 11-20 (10.3%) years old
and 61 years and older (7.9%), 0.01 < p < 0.04. There was no significant
difference between the age groups of 21-40 (30.8%) and 41-60 (17.9%)
years old (data not shown).
The percentage of T. canis seropositivity ranged from 2.6% for the
age group 0-10 years old to 25.6% for the age group of 21-40 years
old. The latter showed a higher percentage of T. canis seropositivity
statistically significant as compared to the age groups of 0-10 (2.6%),
11-20 (5.1%) years old and the age group of 61 (6.7%) years and older,
0.006 < p < 0.02. There was no significant difference between the age
groups of 21-40 (25.6%) and 41-60 (10.3%) years old. The seropositivity
thereafter clearly decreases for the age group of 61 years and older (data
not shown).
Western blotting and band patterns. In order to identify the pattern
of bands displayed by sera from Warao; AES and TES antigens were
analyzed by Western blotting technique. For A. suum, the immunoblotting
showed a pattern of bands ranging from 52 to 206.9 kDas, 12 bands of
206.9, 200.2, 193.6, 149, 114.6, 97.2, 94.1, 82.5, 67.8, 65.6, 55.6 and 52.2,
kDas from A. suum were detected; of these, three bands of 193.6 kDa
(33.3%), 200.2 kDa and 97.2 kDa (22.2%) were principally recognized.
Sera from healthy individuals did not show any IgG reactivity (data not
50
Fig. 2 - Western blotting of Ascaris suum antigens. Western blotting procedures showing
some of the most frequently recognized bands obtained according to AES seropositivity
(2A). Tested sera were assayed in order to identify the bands of A. suum. WM: Molecular
weight marker (2A). Percentage of individuals who composed the groups of bands according
to anti-AES IgG antibodies (2B): Group of bands lower than 50 kDa ( ), group of bands
between 50 and 100 kDa ( ), group of bands between 101-150 kDa ( ), and group of bands
between 151- 207 kDa ( ).
which was ranging from 22.9 kDa to 223 kDas; among these 11 major
bands were principally identified; 84.9 kDa (37.7%), 52.2 kDa (33.3%),
131.3 kDa (31.1%), 98.2 kDa (28.8%), 119.1 kDa (26.6%), 41 kDa
(26.2%), 184.4 kDa (24.4%), 175.6 and 47.4 kDas (22.2%), 193.6 kDa
(15.5%) and 50 kDa (11.9%). Sera from healthy individuals did not show
any reactivity (data not shown). Western blotting procedures showing the
obtained bands and the WB profile according to TES seropositivity are
shown in Figure 3. Western blotting analysis using TES antigens showed
the TES pattern composed by bands ranging from 22.9 kDa to 223 kDa
(some of the most frequently recognized bands are shown in Figure 3A).
The WB profile according to TES seropositivity could be divided into
the following groups: the group composed of bands lower than 50 kDa
(42.1%) was the most reactive; followed by the group of bands between
50 kDa and 100 kDa (28%), 151 kDa and 207 kDa (18.4%) and 101-150
kDas (11.4%) (Fig. 3B).
Coproparasitological tests and intestinal parasites. The stool
examinations showed that intestinal parasites are significantly frequent
among Warao indigenous. Among these, protozoas (80.0%) were more
frequent than helminths (20.0%). For the children group; Blastocystis
hominis and Iodamoeba butschlii were the most prevalent parasites
(85.7%), followed by Entamoeba coli (71.4%), Entamoeba histolytica
and Hymenolepis nana (28.5%) and Giardia duodenalis, Trichuris
trichiura and Ascaris lumbricoides (14.2%). For the adult group; E. coli
was the most prevalent parasite (35.8%), followed by B. hominis (28.3%),
I. butschlii (10.8%), Endolimax nana (7.4%), H. nana and T. trichiura
(4.4%), Chilomastix mesnili (2.1%) and Ascaris lumbricoides (1.5%).
Cytokine expressions and seropositivity for Ascaris and Toxocara.
Figure 4 shows the cytokine expressions when RT-PCR assays were
used for the relative quantification of mRNA encoding for IL-2, IL-4,
IL-6, IL-10, IL-12p35 (Fig. 4A), and IFN-γ, TGF-β and TNF-α (Fig.
4B) in peripheral blood cells from Warao indigenous. Results indicated
that the largest expression of transcript was for IFN-γ (100%) followed
by TNF-α (90%), IL-2 (85%), IL-4 (67.5%), IL-12p35 (55%), TGF-β
(50%), IL-6 and IL-10 (17.5%) (Fig. 5). When a logistic regression
method was used to compare independent variables including cytokine
expression in peripheral blood cells from Warao indigenous that had
or did not have antibodies against the AES and TES antigens showed
that there was only a significant increased expression of the IL-4 in
individuals with TES seropositivity (p < 0.002), whereas, the expression
of all cytokines transcripts were not different to that of individuals with
AES seropositivity (Fig. 5).
DISCUSSION
A preliminary assessment of zoonotic helminths such as A. suum
and T. canis among Warao indigenous offered an opportunity to study
these two zoonotic infections, namely ascariosis and toxocariosis in
some communities that live closely with pigs and dogs. During infection
with A. suum and T. canis, both cellular and humoral immune responses
develop. In humans, several studies on the epidemiology, pathology
and diagnosis of toxocariasis are available6,11,28,41 however, much less is
known about human infections with A. suum. Few studies have reported
on Ascaris suum being able to mature to the adult stage in the human
host31,38. NEJSUM et al. refer to this infection as zoonotic ascariasis and
have recently reported that A. suum can also mature to the adult stages
in chimpanzees28. There are however other studies reporting VLM cases
Fig. 3 - Western blotting of Toxocara canis antigens. Western blotting procedures
showing some of the most frequently obtained bands according to TES seropositivity
(3A). Tested sera were assayed in order to identify the bands of T. canis. WM:
Molecular weight marker. Band (3A). Percentage of individuals who composed
the groups according to anti-TES IgG antibodies (3B): Group of bands lower than
50 kDa ( ), group of bands between 50 and 100 kDa ( ), group of bands between
101‑150 kDa ( ), and group of bands between 151- 207 kDa ( ).
that are suspected to be caused by Ascaris suum in which pulmonary
and liver lesions have been described31. As suggested by ARIZONO et
al. the pathogenic or physiogenetic factors that determine the course of
human infection with pig-derived Ascaris remain to be elucidated2. In the
present study, seropositivity associated with ascariasis and toxocariasis
using the indirect-ELISA IgG was evidenced. The findings indicated
that both adults and children showed high AES seropositivity, 23.3%
and 57.1%, respectively as compared to TES seropositivity, 9.3% and
14.3%, respectively. In the past, antigenic preparations of T. canis adults
or larvae were used for the immunodiagnosis of toxocariosis24,39; however
51
the latter was improved by SAVIGNY, who obtained secreted antigens
called Toxocara excretory-secretory antigens (TES-Ag) and used them
together with ELISA (TES-ELISA)37.
Fig. 4 - Profile of cytokine expression. RT-PCR assays were used for the quantification of
mRNA encoding for IL-2, IL-4, IL-6, IL-10, IL-12p35 (3A). 1 = Molecular weight markers
(50pb), 2-5 = IL-2 (2: sample positive, 3: sample negative, 4: positive control and 5: negative
control), 6-9 = IL-4 (6: sample positive, 7: sample negative, 8: positive control and 9: negative
control), 10-13 = IL6 (10: sample positive, 11: sample negative, 12: positive control and
13: negative control), 14-17 = IL-10 (14: sample positive, 15: sample negative, 16: control
positive and 17: control negative), 18-21 = IL-12 (18: sample positive, 19: sample negative, 20:
control positive and 21: control negative), 22 = Molecular weight markers (100pb). RT-PCR
assays were also used for the quantification of mRNA encoding for IFN-γ, TNF-α, TGF-β
and β-actin (3B). 1: Molecular weight markers (50pb), 2-4 = IFN-γ, (2: sample positive, 3:
control positive and 4: control negative), 5-8 = TNF-α (5: sample positive, 6: sample negative,
7: control positive and 8: control negative), 9-12 = TGF-β (9: sample positive, 10: sample
negative, 11: control positive and 12: control negative), 13-15 = β-actin (13: sample positive,
14: control positive and 15: control negative), 16 = Molecular weight markers (100pb).
Fig. 5 - Percentage of individuals showing positive expression of cytokine. The percentage
of Warao indigenous with positive expression of cytokine: IL-2 ( ), IL-4 ( ), IL-6 ( ),
IL-10 ( ), IL-12 ( ), IFN-γ( ), TGF-β ( ) and TNF-α ( ). (*)There was a significant
increased expression of the IL-4 in individuals with reactivity IgG to TES antigens (p < 0.002).
52
A serological follow up was carried out in 27 children with
toxocariasis, the results showed, that the highest sensitivity of 100%
was reached when the avidity of T. canis antibodies IgG was evaluated
using ELISA and WB12,20,23,42. In addition, a study determined the T. canis
(TES) seropositivity rate among healthy people with eosinophilia over
10%; the results showed that 67% were positive to bands sizes of 66, 56,
32 and 13 kDas by WB; while that in ELISA, 65% of sera were positive
to TES antigen15. In the present study, 22.2% and 8.8% of serum were
positive to bands size of 32 kDa and 66 kDa, respectively by Western Blot,
while that the TES seropositivity obtained by using indirect-ELISA IgG,
shows 9.3% and 14.3% in indigenous adults and children, respectively.
Several reports describe high and low sensitivity of anti-TES IgG method;
these different findings can be explained since it has been reported that
serum immunoglobulin G antibodies are produced against a variety of
epitopes on the antigen surface and also the number and the species of
serologically reactive antigens varied greatly from individual to individual
or population to population and the level of specific antibodies could also
vary with the age of individuals6,15,33,39. In this context, the possible age
effect on seropositivity for both of these zoonotic infections was studied.
The findings in relation to the age distribution according A. suum and T.
canis seropositivity showed that the trend of seropositivity is similar for
both pathogens; however, the A. suum seropositivity is slightly higher
(30.8%) than T. canis seropositivity (25.6%), especially for the age group
of 21-40 years old, both seropositivities were significantly increased as
compared to the age groups of 0-10, 11-20 years old and 61 years and
older, it was observed that the seropositivity thereafter clearly increases
with age, but it decreased for the age group of 61 years and older. Whether
A. suum and T. canis transmission for this population is by direct ingestion
of contaminated soil remains to be investigated.
On the other hand, in the adult group, 44.1% were seropositive
for both parasites; these results suggest that infection by Toxocara is
essentially as common as that by Ascaris; however AES seropositivity
in adults and children was shown to be higher; 23.3% and 57.1%, than
TES seropositivity, 9.3% and 14.3%, respectively. A high Toxocara
seropositivity in slum areas of Caracas and El Mojan, Venezuela10,21,22 has
been reported. Since it is more common to see pigs than dogs in these
indigenous communities, a low exposure of children to T. canis, could
explain the present findings.
As mentioned above, the ELISA based on the use of excretory/
secretory antigens produced by the larvae of the A. suum and T. canis is
the most common approach for serodiagnosis; however, the specificity
of which can be inadequate in regions of endemic helminthiasis14,32.
In this context, it was reported that reactivity of sera to AES antigens
using the ELISA test was reduced by pre-absorption with extracts of A.
lumbricoides, a nematode antigenically related to A. suum and although
this topic is still controversial, the most recent research reports that
molecular biology has shown it to be a single species, authors concluded
that A. lumbricoides and A. suum are a single species and that the name
A. lumbricoides Linnaeus 1758 has taxonomic priority; therefore A.
suum Goeze 1782 should be considered a synonym of A. lumbricoides19,
the latter is a common intestinal nematode of Venezuelan children10,21.
Since Western blotting has been proposed as a confirmatory test for the
diagnosis of toxocariosis20, this method was performed using AES and
TES antigens. For AES antigens, the immunoblotting showed a pattern of
bands ranging from 52 to 206.9 kDas, with the predominance of the three
of these; while that for TES antigens; this pattern of bands was ranging
from 22.9 to 223 kDas; among these 11 major bands were principally
identified. It has been reported that bands of high molecular weight
principally are responsible for the cross-reactivity between T. canis and
A. suum; however, authors reported that a band with molecular weight
around 55 to 66 kDa is also, at least, responsible for the cross-reactivity
between both parasites17,28. Concerning, the existence of cross-reactivity
among A. suum and T. canis, Western blotting was performed using serum
samples from patients with positive result by ELISA. The findings showed
the existence of cross-reactivity among A. suum and T. canis, four major
bands were principally identified 52.2, 94.1 kDa, 149 kDa, and 193.6 kDa.
In experimental animals, cross-reactivity between T. canis and A. suum
has also been reported, so the Western blotting showed that the rat IgG
recognized three proteins of 190, 160 and 33 kDas in the antigens from
F. hepatica, T. canis and A. suum; the author suggests that the existence
of cross-reactivity among these antigens seems to also demonstrate the
presence of structural similarities, such as tegumental proteins35.
Findings about pre-absorption treatment performing Western
blotting technique and using serum samples from individuals with A.
suum seropositivity showed five bands principally identified; 159.4,
137.8, 125.1, 29.2, and 24 kDas; whereas when using serum from
individuals with T. canis seropositivity, four bands were principally
identified; 203.1, 152, 144.7, and 131.3 kDas. The available
seroprevalence data about AES and TES antigens here analyzed and
results about the microscopic examination of stool samples showing
that a high prevalence of intestinal parasites among indigenous exist,
especially parasites such as Blastocystis hominis, Hymenolepis nana
and Entamoeba coli suggesting that further studies must be performed
to improve the sensitivity and specificity of the Ascaris and Toxocara
ELISA test by pre-absorption with extracts of prevalent parasites among
Warao indigenous and more serum samples.
On the other hand, relative quantification of cytokine expressions,
Th1 and Th2 in peripheral blood cells from indigenous studied was also
evidenced. In Venezuela there are 28 different ethnic groups, Warao are
one of them (no persons of mixed race or indigenous), whose socioeconomic status is low; they do not have access to health care compared
with the Creole people from the urban areas, Warao individuals also have
recurrent or overwhelming parasite infections1. Helminth infections are
among the most potent stimulators of Th2-type immune responses and
have been widely demonstrated to modify responsiveness to both non
parasite antigens and other infectious agents in a nonspecific manner
in infected animals; the balance of Th1 and Th2 immune responses is
known to be crucial for determining both the protective and pathological
responses to infections with a variety of pathogens13,16,8. In the case of
a number of gastrointestinal nematode infections, Th2 responses are
generally associated with protection, while Th1 responses are associated
with susceptibility8,43.
A study showed that A. lumbricoides infections in endemic regions
are associated with a highly polarized type 2 cytokine response8. In
addition, a significant association between intestinal helminthic infections
and mycobacterial diseases, such as pulmonary tuberculosis and multibacillary leprosy, has been demonstrated by several authors1,7. It has been
reported that concomitant helminthic infection in patients with diagnosed
tuberculosis skews their cytokine profile toward a T helper 2 response7.
Since that, official data on the tuberculosis situation in Venezuela showed
that between 1997 and 2001 the tuberculosis rate was between 93.2 and
81.0 among Warao indigenous population26,27. Based on this notion and
the significant association between intestinal helminthic infections and
mycobacterial diseases mentioned above, studies of relative quantification
of cytokine expression were performed; the findings showed that there
was only a significant increased expression of the IL-4 in individuals with
TES seropositivity (p < 0.002), whereas, the expression of the IFN-γ,
IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 transcripts were not different
to that of individuals with AES seropositivity. The results of the present
study suggest that T. canis antigens have a potent immunomodulatory
activity and that the effects observed are consistent with skewing towards
a Th2-type response rather than induction of Th1-type response. The
latter probably due to two syndromes that have been identified, which
remain for a long time in the host, the visceral larva migrans syndrome
(VLM) and the ocular larva migrans syndrome (OLM)9. Importantly, the
induction of interleukin-4 by T. canis antigens also suggests that T cells
may play a role in immunomodulation of immune responses by parasitic
helminths that result in a dominant Th2 type of the immune response.
While this presumably promotes parasite survival, it may markedly impair
protective immune responses to Mycobacterium tuberculosis infection.
Further studies are needed to understand the association between
helminthic infections such as A. suum and T. canis and a dominant Th2
cytokine profile in Warao indigenous, which could favor persistent M.
tuberculosis infection in this population. Finally, the immunoblotting
and ELISA techniques may constitute useful methods for the diagnosis
of the zoonoses infections like ascariosis or toxocariosis, which are
prevalent among Warao indigenous. In addition, measures to control
these helminthic infections are recommended.
RESUMEN
Seropositividad para ascariosis y toxocariosis y expresión de
citocinas entre la población indígena de la región del delta
Venezolano
El objetivo del presente estudio fue determinar la seropositividad de
infección por Ascaris suum y Toxocara canis, utilizando antígenos de
excreción/secreción (E/S) de Ascaris suum (AES) y Toxocara canis (TES)
en una población indígena. Adicionalmente, se cuantificó la expresión de
citocinas a partir de células de sangre periférica. Un total de 50 indígenas
Warao se incluyeron en el estudio; 43 fueron adultos y 7 niños. Entre los
adultos, 44,1% fueron seropositivos para ambos parásitos; mientras que
los niños sólo mostraron seropositividad a uno u otro de los helmintos.
Para ascariosis, el porcentaje de seropositividad para los antígenos AES
fue alto tanto en adultos como en niños; 23,3% y 57,1%, respectivamente.
Para toxocariosis, el porcentaje de seropositividad para los antígenos TES
fue bajo en adultos así como en niños; 9,3% y 14,3%, respectivamente.
El porcentaje de seronegatividad fue similar tanto para los antígenos
AES como para TES en adultos (27,9%) y niños (28,6%). Cuando la
seropositividad fue analizada a través de la técnica de Western blotting
utilizando los antígenos AES; 3 bandas de 97,2, 193,6 y 200,2 kDas
fueron principalmente reconocidas. Para los antígenos TES, 9 bandas
fueron mayormente identificadas; 47,4, 52,2, 84,9, 98,2, 119,1, 131,3,
175,6, 184,4 y 193,6 kDas. Los análisis coproparasitológicos mostraron
que los parásitos Blastocystis hominis, Hymenolepis nana y Entamoeba
coli fueron los parásitos intestinales más comúnmente observados. La
cuantificación de la expresión de las citocinas IFN-γ, IL-2, IL-6, TGF-β,
TNF-α, IL-10 e IL-4 mostró que hubo un significante incremento de
la expresión de IL-4 entre los indígenas con seropositividad para los
53
antígenos TES (p < 0.002). La seropositividad para Ascaris y Toxocara
fue prevalente entre los indígenas Warao.
17.Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 1970;227:680-5.
ACKNOWLEDGEMENTS
18.Lescano SA, Nakhle MC, Ribeiro MC, Chieffi PP. IgG antibody responses in mice
coinfected with Toxocara canis and other helminthes or protozoan parasites. Rev
Inst Med Trop Sao Paulo. 2012;54:145-52.
This study was supported by Consejo de Desarrollo Humanístico y
Científico de la Universidad Central de Venezuela, Projects No. CDCH/
UCV-PG 09-8007-11/AIA-09-8397-12).
19.Leles D, Gardner SL, Reinhard K, Iñiguez A, Araujo A. Are Ascaris lumbricoides and
Ascaris suum a single species? Parasit Vectors. 2012;5:42-9.
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55
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http://dx.doi.org/10.1590/S0036-46652015000100008
TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH
LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS
Cristiane Beatriz de OLIVEIRA(1), Cídia VASCONCELLOS(1), Neusa Y. SAKAI-VALENTE(2), Mirian Nacagami SOTTO(1), Fernanda Guedes LUIZ(3),
Walter BELDA JÚNIOR(1), Maria da Gloria Teixeira de SOUSA(2), Gil BENARD(2) & Paulo Ricardo CRIADO(1)
SUMMARY
There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the
involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum.
Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments
were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4
were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both
LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three
groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression
likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2
expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute
to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.
KEYWORDS: Toll like receptor 2; Toll like receptor 4; Dermatophytosis; Trichophyton rubrum.
INTRODUCTION
Dermatophytosis is the infection of keratinized structures caused
by members of the fungi of the genera Trichophyton, Epidermophyton
and Microsporum. These fungi are adapted to infect keratinized tissues
by virtue of their ability to utilize keratin as a nutrient source. Sites of
infection include hair, nails, and the stratum corneum of the skin24,26. The
clinical presentation of dermatophytosis depends on several factors: (i)
the site of infection, (ii) the immunological response of the host, and (iii)
the species of infecting fungus20. More than 40 dermatophyte species
that infect humans (anthropophilic), animals (zoophilic) or are present
in soil (geophilic) have been identified20. The infections caused by the
anthropophilic species tend to be chronic but the resultant inflammation
is minimal20,21.
About 90% of chronic dermatophyte infections are caused by T.
rubrum and T. mentagrophytes8,9, possibly because these organisms may
suppress inflammation and cell-mediated immunity7.
Keratinocyte is the predominant cell type in the epidermis,
comprising over 90% of the cells5. However, during an inflammatory
process the innate immune network of the epidermis consists of not
only the pre-existing keratinocytes, but also of rapidly mobilized host
defense cellular components such as neutrophils, mast cells, eosinophils,
and macrophages25. Recognition of pathogens by innate immune cells
is mediated by pattern recognition receptors (PRRs) that recognize
conserved pathogen-associated molecular patterns (PAMPs). Toll-like
receptors (TLRs) are a family of PRRs that have recently been identified
as crucial signaling receptors mediating the innate immune recognition,
and comprise a family of 10 receptors with distinct recognition profiles
in humans17. Human keratinocytes are known to express TLRs 1 to 6
and TLR93,10,11,14,15,19,22.
Although it is known if certain elements of the fungal wall increase
TLR expression upon recognition by these receptors, a previous in vitro
study demonstrated that whole Trichophyton rubrum conidia could
diminish TLR expression on a keratinocyte cell line7. However, there are
few studies on human immune response to dermatophytes. In this study
it is demonstrated that TLR4 expression is lower on keratinocytes of
patients with either localized or disseminated dermatophytosis compared
with normal skin.
PATIENTS AND METHODS
Two groups of patients with dermatophytosis were evaluated:
seven patients with disseminated dermatophytosis (involving at least
(1) Department of Dermatology, Medical School, University of Sao Paulo, São Paulo, SP, Brazil.
(2) Laboratory of Medical Investigation Unit 53, Division of Clinical Dermatology, Medical School, University of Sao Paulo, São Paulo, SP, Brazil.
(3) Instituto Pasteur de São Paulo, Laboratório de Diagnóstico da Raiva, São Paulo, SP, Brazil.
Correspondence to: Maria da Gloria T. de Sousa, Instituto de Medicina Tropical, Laboratório de Micologia, Av. Dr. Eneas de Carvalho Aguiar 500, 05403-000 São Paulo, SP, Brasil. Phone:
+55 11 30617499, Fax: +55 11 30817190. E-mail: [email protected]
OLIVEIRA, C.B.; VASCONCELLOS, C.; SAKAI-VALENTE, N.Y.; SOTTO, M.N.; LUIZ, F.G.; BELDA JÚNIOR, W.; SOUSA, M.G.T.; BENARD, G. & CRIADO, P.R. - Toll-like receptors
(TLR) 2 and 4 expression of keratinocytes from patients with localized and disseminated dermatophytosis. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 57-61, 2015.
three distinct body parts), and eight with localized dermatophytosis
(involvement of only one body part). All of the patients were recruited
from the Mycology Outpatient Clinic of the Division of Clinical
Dermatology, Clinics Hospital, University of São Paulo. Twenty skin
samples from healthy individuals undergoing plastic surgery were
included as controls. The following inclusion criteria were used: (i)
the subjects had dermatophytosis but did not present any comorbidity
affecting the immune response or predisposing them to dermatophytosis
(e.g., primary or secondary immunosuppression, diabetes mellitus,
Cushing’s disease, transplant recipients); (ii) T. rubrum was the fungal
agent; and (iii) the subjects had not used topical or systemic treatments
in the last month. Patients who were under 18 years of age or pregnant
were excluded. All patients signed an informed consent form previous to
inclusion in the study. The study was approved by the Ethics Committee of
the Clinics Hospital, University of São Paulo Medical School (#673/06).
Identification of T. rubrum was done by microscopic examination of
samples obtained from lesions that had been cultured in Agar Sabouraud13
(Becton, Dickinson and Company, Heidelberg/Germany).
Each measurement unit contained 3.11 pixels. The image analysis
system measured the mean color density of the immunohistochemical
staining in the epidermis, which represents the mean intensity of the
staining within the positive area (range, 0-255)18. The expression of
TLRs 2 and 4 in the epithelium was calculated as the product of the area
of positive staining and mean density, normalized by the corresponding
epithelial basement membrane length18. This index of protein expression
takes into account both the intensity and the area of staining. A simple
average of the values for each photographic field on the same slide was
used.
Immunohistochemistry of the biopsies of the lesions: Biopsies
were taken with a standard dermatological biopsy punch. For the patients
with dermatophytosis, two biopsies were taken: one on the border of the
active lesion and another from unaffected skin on the same body part at
least 4 cm away from the affected area. Twenty controls were obtained
from cosmetic surgery.
The average age for the groups were as follows: 35 (range: 19-51)
years for the patients with DD, 39 (range: 18-62) years for those with LD,
and 57 (range: 46-72) for the controls. The average disease progression
time was 16 (range: 2-60) months in the patients with DD and 19 (range:
1-60) months in the patients with LD.
Slides were dewaxed in xylene and hydrated through a graded
series of ethanol. Endogenous peroxidase was blocked with 3%
hydrogen peroxide. Antigen retrieval for TLR2 and TLR4 analysis
was performed by incubation of slides in retrieval solution pH 9.0
(S2368, DakoCytomation, Carpinteria, CA, E.U.A) in a water bath
for 25 min at 95 ºC. They were then incubated overnight at 4 ºC
in the presence of a 1:50 dilution of anti-TLR2 primary antibody
(sc-10739, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A) and
a 1:400 dilution of anti-TLR4 primary antibody (ab47093, Abcam,
Cambridge, MA, U.S.A). The specific antigen-antibody reactions were
detected with an alkaline-phosphatase-based system (EnVisionTM G2
system/AP; DakoCytomation, Carpinteria, CA, U.S.A.) for TLR2 and a
streptavidin-biotin-peroxidase-based system for TLR4, according to the
manufacturer’s instructions (LSABTM+ system-HRP; DakoCytomation,
Carpinteria, CA, U.S.A.). The reactions for TLR2 were visualized
using Liquid Permanent Red chromogen (DakoCytomation) and the
reactions for TLR4 were visualized using the 3,3’-diaminobenzidinetetrahydrochloride (DAB) chromogen (Sigma) and counterstained with
Carazzi haematoxylin.
Statistical analysis: The number of positive cells of the three groups
of tissue reaction was compared using Graph Pad Prism version 5.00
for Windows (Graph Pad software, San Diego, CA, USA) to perform a
Kruskal Wallis and Dunn’s post test with the level for significance set
at 95%.
RESULTS
Hematoxilin staining did not reveal any inflammatory infiltrate in
the epidermis of the patients (data not shown), which was unremarkable
when compared with that of the healthy skin biopsies.
All reactions were performed with positive and negative controls.
The latter comprised omission of the primary antibody.
Quantification of immunostained cells was performed using
AxioVision 4.8.2 software (Zeiss).
Image analysis: The epidermis was photographed along its entire
length under a 10X eyepiece and a 20X objective coupled to a Carl Zeiss
AxioCam MR3 camera mounted on a Zeiss Axiophot optical microscope.
The images were analyzed using the Image Pro Plus program. Prior to
quantification, the epidermis was divided into upper epidermis (surface)
and lower epidermis (deep tissue) at 50% of its thickness.
58
Fig. 1 - TLR2 expression in unaffected (a) and affected (b) epidermis from an individual
with localized dermatophytosis and in unaffected (c) and affected (d) epidermis from an
individual with disseminated dermatophytosis. (e) shows TLR2 expression in the epidermis
of a healthy individual. Magnification: x200
Fig. 2 - TLR4 expression in unaffected (a) and affected (b) epidermis from an individual
with localized dermatophytosis and in unaffected (c) and affected (d) epidermis from an
individual with disseminated dermatophytosis. (e) shows TLR4 expression in the epidermis
of a healthy individual. Magnification: x200
TLR4 expression was reduced in the upper epidermis of either LD
or DD dermatophytosis patients, as compared with the control group
(Fig. 3A). The TLR4 staining optical density index was 145 ± 22 in the
controls, compared with 109 ± 9 in biopsies of LD patients and 110 ±
15 of the DD patients (p < 0.001). Interestingly, TLR4 expression in
unaffected skin in both dermatophytosis groups was similar to that in the
respective affected areas (LD: 109 ± 9; DD: 110 ± 7), and significantly
reduced compared with the control group (p < 0.001) (Fig. 3A). There
were no differences between individuals with LD and DD (Fig. 2a, 2b,
2c, 2d and 2e).
No differences were observed in TLR4 expression between
unaffected and affected skin samples in the two patient groups. In
addition, there were no differences between individuals with LD and
DD (Fig. 2a, 2b, 2c and 2d).
In the lower epidermis, TLR4 expression was also lower in patients
with dermatophytosis than in controls (Fig. 3A). The optical density index
was 145 ± 17 in controls and 111 ± 7 and 111 ± 13 respectively in the
unaffected and affected areas of DD patients (Fig. 3A). In patients with
LD, the optical density indices were respectively 111 ± 8 and 110 ± 5.
As in the upper epidermis, there were no differences between unaffected
and affected skin areas (Fig. 2a, 2b, 2c and 2d).
TLR2 expression was diffusely detected in the upper and lower
epidermis of both patients and controls (Fig. 1 and 3B). In contrast to
the TLR4 findings, no differences were detected in TLR2 expression in
the upper and lower epidermis in the LD and DD when compared with
Fig. 3 - (A) Comparison of TLR4 expression in unaffected and affected skin of patients with
disseminated (DD) and localized dermatophytosis (LD), and in skin of healthy controls. (B)
A comparison of TLR2 expression in unaffected and affected skin of patients with DD or
LD and in skin of healthy controls. TLR4 and TLR2 expression were quantified based on the
optical density index (ODI) of the lower and upper epidermis layers. Results are presented
as mean ± SEM. * p = 0.01.
the control group. The intensity of expression of TLRs 2 and 4 in the
epidermis was apparently comparable, although this comparison should
be regarded with caution since the chromogens used for each staining
differed.
DISCUSSION
T. rubrum infections are often chronic and result in minor
inflammation. Additionally, these infections are marked by polarization
during the immediate immune response and the later inadequate cellular
response26. The patients in this study had chronic disease; patients with
both the disseminated and localized forms had an average duration
of more than one year. Accordingly, no inflammatory infiltrate in the
epidermis of the infected skin areas was observed. The reasons for such
lack of inflammatory response are not yet known.
59
Recognition of pathogens by innate immune cells is mediated
by PRRs that recognize PAMPs. TLRs (comprising a family of 10
receptors with distinct recognition profiles in humans) act as pattern
recognition receptors and these receptors are able to recognize fungi
and polarize the immune response1,2. Various cell subtypes express
these receptors, including human keratinocytes, which express TLRs
1 to 6, and TLR96,23. Moreover, TLR2 is highly expressed in normal
skin, particularly in proliferating basal keratinocytes, but TLR4 is less
detectable by immunostaining3,19. Constitutive expression of mRNA
specific for both TLR2 and TLR4 has recently been demonstrated in
cultured human keratinocytes and gingival epithelial cells19. A report
showed that TLR2 mRNA was more prominently upregulated in normal
human keratinocytes than TLR4 mRNA19. Thus keratinocytes may act
both as a mechanical and an immunological barrier to pathogens, the
latter by triggering an innate immune response to control the infectious
process3,5.
Among TLR family members, both TLR2 and TLR4 have been shown
to recognize bacterial and fungal components and mediate the production
of the cytokines required for the development of an effective immunity.
TLR2 has been implicated in the recognition of Gram-positive bacteria
components, bacterial lipoproteins, and zymosan while TLR4 recognizes
lipopolysaccharide (LPS) and O-linked mannans1.
Here, marked TLR2 and TLR4 expression was observed across the
entire thickness of the epidermis of patients with LD and DD, confirming
previously published studies on the expression of TLR2 and TLR4 on
keratinocytes3,10,11
No significant differences were observed between TLR2 and TLR4
expression levels in both the upper epidermis and lower epidermis when
dermatophytosis patients and controls were compared, as has been
described in normal epidermis3,11,15,19,22. However, reduced TLR4 was
detected, but not TLR2 expression, in the upper and lower epidermis
layer in both DD and LD patients when compared with the control group.
This reduced expression was equally observed in patients with LD and
DD in the sample group of this study.
The expression of TLRs 2 and 4 in the biopsies of affected
(dermatophytosis) and adjacent unaffected (healthy skin) areas of
the patients were also compared. There were no differences in the
expression between unaffected and affected areas. As a constitutive
defect in TLR4 seems unlikely, it is conceivable that the down
modulation present in the affected skin spreads to adjacent, seemingly
clinically healthy areas.
Another important observation is that there was no difference in the
expression of the TLR between individuals with LD or DD. Therefore,
this suggests that TLR2 and TLR4 expression levels do not explain the
more extensive lesions. GARCÍA-MADRID et al. (2011) showed that
keratinocytes can recognize and respond to cell wall components of
T. rubrum. Viable intact conidia inhibited TLR2 and TLR6 expression
by cultured human keratinocytes while conidial homogenate from
T. rubrum increased the expression of TLR2, TLR4 and TLR6 7.
Recently, YUKI et al.27 demonstrated that TLR2 activation enhances
tight junctions among keratinocytes, a crucial step in maintaining
the functional activity of epidermal barriers against infective agents,
such as bacteria or fungi. TLR2 inhibition increased the permeability
60
of the cutaneous barrier, thereby decreasing the cohesion between
keratinocytes and facilitating fungal invasion27. Thus, the appropriate
TLR2 expression in the upper epidermis layer would represent a
protective mechanism inhibiting the fungal invasion and dissemination
into the epidermis while limiting the inflammatory process and reducing
tissue damage.
Contrary to this protective effect of TLR2 on keratinocytes tight
junctions, there is evidence that TLR4 induces the production of
inflammatory cytokines and neutrophils recruitment. Furthermore, TLR2
has been shown to induce a Th-2 response10,11. Thus, predominance of
TLR2-induced Th-2 response would not be appropriate for the elimination
of the invading dermatophytes, explaining at least in part the chronic
nature and extension of the lesions. The association with the decreased
expression in superficial epidermis of TLR4, and the consequent decrease
in pro-inflammatory cytokines production and neutrophil recruitment
would, in turn, explain the limited inflammatory response in the lesions
caused by T. rubrum. In addition, Th2 responses can also inhibit Th17
responses, which are potent inducers of neutrophil infiltration and
production of pro-inflammatory cytokines4,12,14,16.
In conclusion, these results demonstrate reduced expression
of TLR4, a signaling receptor that recognizes important bacterial
(lipopolysaccharide) and fungal (O-linked mannans) components,
in the epidermis of dermatophotysis lesions. This down modulation
may represent a mechanism underlying the reduced inflammatory
response observed in this cutaneous fungal infection. Elucidation of the
mechanisms by which T. rubrum interferes with the role of keratinocytes
in the initial stage of the innate immune response is essential to the
development of better therapeutic strategies.
RESUMO
Expressão de receptores do tipo Toll 2 e 4 nos queratinócitos de
pacientes com dermatofitose localizada e disseminada
A literatura sobre o papel da resposta imune inata em dermatofitose é
escassa. Este estudo se propôs a investigar a participação dos receptores
do tipo Toll 2 e 4 (TLRs) 2 e 4 em pacientes com dermatofitose
localizada (LD) e disseminada (DD, definida como lesões em pelo
menos três segmentos corpóreos distintos), causadas por Trichophyton
rubrum. Foram analisados cortes histológicos de 15 pacientes recémdiagnosticados, oito com LD e sete com DD. O grupo controle foi
composto por 20 amostras de pele de indivíduos saudáveis submetidos
a cirurgia plástica. TLR-2 e TLR-4 foram quantificados em lesões
cutâneas por imunohistoquímica. Encontramos uma expressão reduzida
de TLR-4 na epiderme superior e inferior nos dois grupos, LD e DD,
quando comparados com o grupo controle; a expressão de TLR-2 foi
preservada na epiderme superior e inferior de todos os três grupos.
Como a sinalização por TLR-4 induz produção de citocinas inflamatórias
e recrutamento de neutrófilos, a menor expressão desta molécula
provavelmente contribui para a não resolução da infecção e conseqüente
natureza persistente da dermatofitose. Como a sinalização via TLR-2
tem sido descrita como fator de regulação do processo inflamatório e de
preservação da estrutura epidérmica, a sua expressão inalterada nas lesões
dos pacientes com DD e DL pode contribuir também para a persistência
da infecção e do reduzido processo inflamatório que são característicos
das infecções por dermatófitos.
ACKNOWLEDGMENTS
13. Lacaz CS, Porto E, Martins JEC, Heins-Vaccari EM, Takahashi de Melo N. Tratado
de micologia médica. 9 ed. São Paulo: Sarvier; 2002.
Financial support: This work was supported by Fundação de
Amparo à Pesquisa do Estado de São Paulo (contracts #11/51001-1 and
#2010/19369-6). GB is a senior researcher of the Conselho Nacional
para o Desenvolvimento Científico e Tecnológico.
14. Mempel M, Voelcker V, Kollisch G, Plank C, Rad R, Gerhard M, et al. Toll-like
receptor expression in human keratinocytes: nuclear factor kappaB controlled gene
activation by Staphylococcus aureus is Toll-like receptor 2 but not Toll-like receptor 4
or platelet activating factor receptor dependent. J Invest Dermatol. 2003;121:1389-96.
CONFLICTS OF INTEREST
15. Miller LS, Modlin RL. Toll-like receptors in the skin. Semin Immunopathol.
2007;29:15-26.
The authors declare no conflicts of interest.
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Accepted: 6 August 2014
61
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http://dx.doi.org/10.1590/S0036-46652015000100009
BAT-BORNE RABIES IN LATIN AMERICA
Luis E. ESCOBAR(1,2), A. Townsend PETERSON(3), Myriam FAVI(4), Verónica YUNG(4) & Gonzalo MEDINA-VOGEL(1)
SUMMARY
The situation of rabies in America is complex: rabies in dogs has decreased dramatically, but bats are increasingly recognized as
natural reservoirs of other rabies variants. Here, bat species known to be rabies-positive with different antigenic variants, are summarized
in relation to bat conservation status across Latin America. Rabies virus is widespread in Latin American bat species, 22.5%75 of bat
species have been confirmed as rabies-positive. Most bat species found rabies positive are classified by the International Union for
Conservation of Nature as “Least Concern”. According to diet type, insectivorous bats had the most species known as rabies reservoirs,
while in proportion hematophagous bats were the most important. Research at coarse spatial scales must strive to understand rabies
ecology; basic information on distribution and population dynamics of many Latin American and Caribbean bat species is needed;
and detailed information on effects of landscape change in driving bat-borne rabies outbreaks remains unassessed. Finally, integrated
approaches including public health, ecology, and conservation biology are needed to understand and prevent emergent diseases in bats.
KEYWORDS: Rabies virus; Bats; Geographic distribution; Biodiversity.
INTRODUCTION
Bats offer diverse cultural and economic contributions to human
situations, such as ecotourism, vector control, guano, medicinal
products, and religious significance, among others42. Bat diets include
insects, fruits, leaves, flowers, nectar, pollen, fish, other vertebrates, and
blood41. Insectivorous bats consume large quantities of insects and other
arthropods under natural conditions or related to anthropogenic activities,
controlling important agricultural pests and potential disease vectors39,40,42.
Nectarivorous bats help to maintain diversity in forests through dispersal
of seeds and pollen, essential to many plant species with high economic,
biological, and cultural value42. With around 1230 species, bats are the
second most diverse mammal order (after rodents), with an impressively
broad ecological and geographic distribution41,42.
Rabies virus is the most important virus in the genus Lyssavirus
because, from a global perspective, its distribution, human cases (> 55,000
deaths per year), wide range of potential reservoirs, and veterinary and
economic cost implications make it the most important viral zoonosis73.
Rabies transmission cycles in wild and domestic carnivores have existed
almost worldwide, whereas bat-mediated transmission of rabies virus
occurs only in North, Central, and South America; in Europe, Africa, Asia,
and Australia, bats are reservoirs of different Lyssavirus species44,55,72,87.
In America, bats now constitute the principal rabies reservoir73,74, rabies
is thought to have occurred in tropical America since pre-Hispanic times,
being transmitted predominantly by hematophagous vampire bats3,
although recent phylogenetic reconstructions suggest that rabies virus
in the Americas is unlikely to have originated from vampire bats46. The
first scientific report of rabies in America was by CARINI (1911), in São
Paulo, Brazil7. Advances in diagnostic techniques have now contributed
to an understanding of bat-rabies dynamics83.
In Latin America, human rabies cases have decreased in recent
decades57-61, with mortality rates estimated at 0.01-0.60 per 100,000
individuals29,37. Between 1993 and 2002, annual incidence of human
rabies in Latin America was 105 cases, ranging 0.00-0.09 per 100,000
individuals in South America, 0.00-0.10 in Central America, and 0.000.06 in the Caribbean9. Brazil, Peru, Mexico, and Colombia are the
countries with most human cases of rabies in the region80, although on
a per capita basis Peru and Colombia dominate.
In fact, by 2013, human and canine rabies rates in Latin America had
decreased by 95% compared to previous years (Fig. 1). Epidemiological
surveillance is considered to have been essential for control of rabies in
Latin America79. However, while reports of rabid dogs in Latin America
have declined, the number of bat rabies cases appears stable (Fig. 1).
Although further data compilation is needed for a clearer picture of this
phenomenon, in Latin America, data on rabies are woefully limited and
biased by uneven surveillance effort.
Antigenic variants of rabies (AgV) can be identified by monoclonal
antibody techniques29. Dog rabies (variants 1 and 2) has decreased
(1) Facultad de Ecología y Recursos Naturales, Universidad Andres Bello, Av. República 440, Santiago Centro, Chile.
(2) Center for Global Health and Translational Science, State University of New York Upstate Medical University, Syracuse, New York, USA.
(3) Biodiversity Institute, University of Kansas, Lawrence 66045, USA.
(4) Sección Rabia, Subdepartamento de Virología, Instituto de Salud Pública de Chile, Av. Marathon 1000, Ñuñoa, Santiago, Chile.
Correspondence to: Gonzalo Medina-Vogel. E-mail: [email protected]
ESCOBAR, L.E.; PETERSON, A.T.; FAVI, M.; YUNG, V. & MEDINA-VOGEL, G. - Bat-borne rabies in Latin America. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 63-72, 2015.
Fig. 1 - Dog (blue line) and bat (red line) rabies cases during 2003-2013, based on samples from Latin American and Caribbean countries considered in this study. Belize, Costa Rica, Ecuador,
Guatemala, Guyana, French Guyana, and Haiti did not have reports for this period. Notice the linear trend (black line) for each host group. Proportion of positive bat (green dash line) and dog
samples (purple dash line) is shown. Source: SIEPI-PANAFTOSA/PAHO-WHO, data available on http://siepi.panaftosa.org.br/
dramatically (Fig. 1), and now occurs only in circumscribed areas of
Latin America. Hence, according to current epidemiological reports,
bats now constitute the principal reservoir in Latin America73,74. Crossspecies spillover is well appreciated in bat-borne rabies19. Since 1975, at
least 500 bat-associated cases of human rabies have been reported from
across Latin America2. In 2004, the Regional Program for the Elimination
of Rabies of the Pan American Health Organization (PAHO) reported
for the first time more human cases of rabies derived from wild animals
(bats, other small mammals) than from dogs78: for example, in 2005, 13
cases of human rabies derived from dogs were reported, compared with
60 human cases derived from bats80. Indeed, even in Latin American
countries considered “dog rabies free,” human cases caused by bats
have been reported4,21.
Both vampire and non-vampire bats have been involved in these
events4,21. Hence, after vampire bats, insectivorous bats have assumed a
greater role as sources of the virus in Latin America10,26,38,75,78,90. In spite
of the significant economic, ecological, and cultural stigmas and fears
associated with this disease9, rabies surveillance in bats is limited in
developing countries44. Consequently, the aim of this article is to review
rabies occurrence in bats, evaluate geographic patterns in species richness
of potential bat rabies reservoirs, and summarize knowledge of antigenic
variants, ecology, food habits, and conservation status in key bat species.
This article aims to characterize potential bat rabies reservoirs and guide
new steps in research.
METHODS
For information on bat species (geographic distribution, diet,
conservation status), data from the current, online IUCN database
(www.iucn.org; accessed 13 Jan 2013) were used. To identify potential
bat rabies reservoirs, summaries were made of bat species reported
rabies-positive by country (i.e., Argentina, Belize, Bolivia, Brazil,
Chile, Colombia, Costa Rica, Cuba, Dominican Republic, Ecuador,
El Salvador, Guatemala, French Guyana, Guyana, Haiti, Honduras,
Mexico, Nicaragua, Panama, Paraguay, Peru, Suriname, Trinidad
and Tobago, Uruguay, and Venezuela). First, the Web of Science was
64
searched for articles related to “bat rabies” in Latin American countries
between 1953 and 2012 in English and Spanish, a number of articles
from this search were used as search effort in posterior analysis. Because
several articles from Latin American journals were not available via
Web of Science, Google Scholar was searched for articles, theses, and
official sources available online using the same criteria. Publications
including rabies diagnosis based on histopathology, direct fluorescent
antibody tests, or molecular techniques were included. When multiple
manuscripts source the same bat species or antigenic variants from
the same country, only the older such reference was cited (Table 1).
To date, the most valuable compilation of rabies-positive bat species
in Latin America was published by CONSTANTINE (2009), so part
of this article’s analysis is based on his data. For preliminary bat
distributional information, vector-format based maps (shapefiles) from
IUCN36 were used; maps were handled using ArcGIS 9.3 (ESRI). Chisquare tests were used to evaluate associations (α = 0.05) between the
response variable (i.e., number of rabies-positive species) and factors
such as bat family, diet, and conservation status. Linear regressions
were conducted to evaluate association between bat species (richness)
with rabies-positive species and the number of manuscripts from the
Web of Science (i.e., research effort) by country and rabies antigenic
variants with bat species rabies positive by country. Statistical analyses
were carried out in R71.
RESULTS
Bat species richness patterns: In all, 333 bat species were
documented from 24 Latin American and Caribbean countries36. The
countries with the highest species richness were Colombia (172 species),
Brazil (155 species), and Venezuela (152 species; Fig. 2). Fifty-two
species were endemic to single countries: Mexico had 17, and Brazil
and Peru had nine each. None of these single-country endemic species
were reported as rabies-positive. The number of species by family was
Phyllostomidae (168 species), Vespertilionidae (82 species), Molossidae
(38 species), Emballonuridae (21 species), Mormoopidae (nine species),
Natalidae (seven species), Thyropteridae (four species), and Noctilionidae
and Furipteridae (two species each).
Table 1.
Bat species known to be rabies-positive in Latin America and the Caribbean
Insectivorous
Argentina
Eumops auripendulus†12
Eumops patagonicus†10
Histiotus montanus*10
Myotis sp*10
Myotis nigricans*10
Tadarida brasiliensis†10,33
Eptesicus furinalis*69
Molossus molossus†69
Lasiurus blossevillii*69
Lasiurus cinereus*33,69
Lasiurus ega*69
Belize
Myotis fortidens*12
Myotis nigricans*12
Molossus sinaloae†12
Bolivia
Frugivorous
Nectarivorous
Omnivorous
Carnivorous
Artibeus lituratus•15
Artibeus jamaicensis•12
Phyllostomus
discolor•12
Brazil
Cynomops abrasus†82
Cynomops planirostris†82
Eptesicus diminutus*82
Eptesicus furinalis*82
Eptesicus brasiliensis*82
Eumops glaucinus†82
Eumops perotis†82
Eumops auripendulus†82
Histiotus velatus*82
Lasiurus cinereus*82
Lasiurus ega*82
Lasiurus egregius*82
Lonchorhina aurita•82
Lophostoma brasiliense•82
Micronycteris megalotis•82
Molossops neglectus†82
Molossus rufus†82
Myotis albescens*82
Myotis levis*82
Myotis nigricans*82
Myotis riparius*82
Nyctinomops laticaudatus†82
Nyctinomops macrotis†82
Promops nasutus†12
Tadarida brasiliensis†82
Colombia
Eptesicus brasiliensis*65
Artibeus planirostris•82
Anoura caudifer•82
Carollia perspicillata•82
Platyrrhinus lineatus•82
Anoura geoffroyi•82
Sturnira lilium•12
Glossophaga soricina•82
•82
Uroderma bilobatum
Vampyrodes caraccioli•12
Phyllostomus
hastatus•82
Chrotopterus
auritus•82
Trachops
cirrhosus•82
Hematophagous
AgV
Desmodus
rotundus•69
V369
V433,69
V633,69
E69
H69
M69
Desmodus
rotundus•12
-
Desmodus
rotundus•12
V322
V522
Desmodus
rotundus•82
Diaemus
youngi•82
Diphylla
ecaudata•82
V326,35
V426,35
V526
V626,35
E26
H26
Eu26
N26
Lb26
Desmodus
rotundus•68
V368
V468
65
Table 1.
Bat species known to be rabies-positive in Latin America and the Caribbean (cont.)
Insectivorous
Frugivorous
Nectarivorous
Omnivorous
Carnivorous
Hematophagous
AgV
Desmodus
rotundus•4
V34
Costa Rica
Cuba
Eptesicus fuscus*12
Eumops glaucinus†54
Chile
Histiotus macrotus*23,24
Histiotus montanus*23,24
Lasiurus borealis*23,24
Lasiurus cinereus*23,24
Myotis chiloensis*23,24
Tadarida brasiliensis†23,24
Ecuador
-
M20,23,24,92
V420,23,24,92
V620,23,24,92
H20,23,24,92
Desmodus
rotundus•27
V327
Desmodus
rotundus•12
-
Desmodus
rotundus•12
-
Desmodus
rotundus•52
V352
Desmodus
rotundus•13
-
Desmodus
rotundus•90
Diaemus
youngi•12
Diphylla
ecaudata•1
V317,43,50,89
V417,43,50,89
V517,43,50,89
V617,43,50,89
V817,43,50,89
V917,43,50,89
V1117,43,50,89
Desmodus
rotundus•12
-
El Salvador
Guatemala
Myotis fortidens*12
French Guyana
Phyllostomus
discolor•12
Honduras
Mexico
Antrozous pallidus*12
Eptesicus fuscus*12
Lasiurus ega*12
Lasiurus intermedius*12
Lasiurus seminolus*12
Macrotus waterhousii•12
Molossus rufus†12
Mormoops megalophylla₤1
Myotis velifer*12
Nyctinomops laticaudatus†12
Nyctinomops macrotis†12
Pteronotus personatus₤12
Pipistrellus subflavus*12
Pteronotus parnellii₤12
Pteronotus davyi₤12
Rhogeessa parvula*1
Rhogeessa tumida*12
Nicaragua
66
Carollia subrufa•12
Glossophaga soricina
Leptonycteris nivalis•1
Leptonycteris
yerbabuenae•12
•1
Phyllostomus
discolor•12
Noctilio
leporinus‡1
Table 1.
Bat species known to be rabies-positive in Latin America and the Caribbean (cont.)
Insectivorous
Frugivorous
Panama
Cynomops planirostris†1
Molossus coibensis†1
Molossus currentium†12
Myotis nigricans*1
Paraguay
Lasiurus ega*81
Peru
Myotis nigricans*12
Nectarivorous
Omnivorous
Uroderma bilobatum•1
Carnivorous
Hematophagous
-
Noctilio sp.‡12
Artibeus sp.•75
Artibeus concolor•12
Carollia spp.•75
Glossophaga soricina•12
•12
Platyrhinus sp.
Platyrrhinus lineatus•12
Uroderma sp.•75
Phyllostomus
hastatus•12
Phyllostomus
elongatus•12
AgV
Desmodus
rotundus•12
V681
V364
Desmodus
rotundus•75
V391
Dominican Republic
Trinidad and Tobago
Diclidurus albusʃ31
Pteronotus davyi₤31
Pteronotus parnellii₤12
Uruguay
Lasiurus ega*69
Myotis spp.* 69
Venezuela
Desmodus
rotundus•31
Diaemus
youngi•31
-
Desmodus
rotundus•69
V469
V332
Diphylla
M16
ecaudata•1
V316
Molossus rufus
Desmodus
16
V5
rotundus•16
Family: *Vespertilionidae; •Phyllostomidae; †Molossidae; ₤Mormoopidae; ‡Noctilionidae; ʃEmballonuridae. AgV: Antigenic variants by country. E:
Antigenic variant for Eptesicus spp.; Eu: Eumops; H: Antigenic variant for Histiotus spp.; Lb: Lasiurus borealis; M: Antigenic variant for Myotis
spp.; N: Nyctinomops; V3, V5, V8, V11: Antigenic variant for D. rotundus; V4, V9: T. brasiliensis; V6: Lasiurus spp.
†16
The largest host geographic distributions were for Lasiurus
cinereus (39.2 x 106 km2), L. blossevillii (22.6 x 106 km2), and Tadarida
brasiliensis (17.7 x 106 km2), all insectivorous. Considering other diets,
the species with the largest distributions were Sturnira lilium 16.4 x 106
km2 (frugivorous), Glossophaga soricina 15.7 x 106 km2 (nectarivorous),
Noctilio leporinus 15.5 x 106 km2 (carnivorous), and Desmodus rotundus
19.3 x 106 km2 (hematophagous).
In all, 75 (22.5%) Latin American bat species have been confirmed as
rabies-positive, at least as incidental records (see Table 1). The countries
with more bat species rabies-positive reports were Brazil (43), Mexico (31),
and Argentina (13; Fig. 3). Only Guyana, Suriname, and Haiti are countries
lacking bat-rabies records. It was found that the number of rabies-positive
species is not related to number of bat species (richness) reported per
country (r2 = 0.1238, df = 24, p = 0.078). From the first search of articles
(i.e., Web of Science), no association was found (r = 0.2768, df = 7, P =
0.4708) between the number of bat species and publications by country;
for example, Chile, with the fewest bat species, has nine publications about
bat-borne rabies while Colombia with the highest number of bat species
has only four publications. An association was found between number of
67
44% (four), Molossidae 42% (16), Phyllostomidae with 17% (29), and
Emballonuridae 5% (one species; see Table 1). Considering diet type,
significant effects of diet on rabies positivity were found (X2 = 23.29,
p = 0.0002): the highest proportions of species rabies-positive were
hematophagous 100% (three), carnivorous 60% (three), insectivorous
27% (50), followed by nectarivorous 19% (five), frugivorous 13% (10
species), and omnivorous 11% (four).
Antigenic variants: Only 13 (60%) countries with rabies-positive bats
reported information on antigenic variants (Fig. 2; Table 1). Significant
relationships were found between the number of rabies-positive species
and the number of antigenic variants reported by countries (r2 = 0.83,
P < 0.001; Fig. 3). Brazil had the highest number of rabies-positive bat
species (43 species), with nine antigenic variants; in contrast, Mexico
had fewer rabies-positive bat species, but an impressive number (seven)
of antigenic variants. Indeed, in Mexico, four variants are in vampire
bats and three in non-hematophagous bats, primarily insectivores (Fig.
3). Chile is the Latin American country with the fewest bat species,
but four viral variants are known (Fig. 3); this number is impressive in
comparison with Argentina and Mexico, which are known to have six
and seven variants, respectively, but with much greater bat diversity
(Fig. 2). The most frequent variants reported by country were AgV3
(12 countries), found mainly in D. rotundus; AgV4 (six countries), in T.
brasiliensis; and AgV6 (five countries), in Lasiurus spp.
Fig. 2 - Bat richness showing the number of bat species (rabies positive or not) present in Latin
America (colored shading) and number of antigenic variants of bat rabies reported (gray bars).
publications and rabies AgV by country (r = 0.775, df = 7, p = 0.0142), as
well as an association between the number of publications and the number
of bat species rabies-positive by country (r = 0.883, df = 7, p = 0.001).
In terms of numbers of species known to be rabies-positive by
family, significant effects of family were found (X2 = 24.29, p = 0.001);
the most consistently rabies-positive family was Vespertilionidae 64%
(25 species), followed by Noctilionidae 50% (one), Mormoopidae
Conservation of bats in Latin America: Only one species from the
rabies-positive group had increasing populations (Eptesicus fuscus); most
(90%) rabies-positive species are considered as Least Concern (Fig. 4).
Indeed, rabies-positive species are more likely to be classed as Least
Concern when compared with species where rabies virus has not been
detected (X2 = 41.13, p < 0.001). Bat species rabies-positive in Latin
American and the Caribbean include one endangered species (Leptonycteris
nivalis), and three species (L. yerbabuenae, Eumops perotis, Mormoops
megalophylla) that have decreasing populations36. According to IUCN
(2012), information was insufficient to classify the conservation threat
status for 44 (13%) bat species reported in Latin America.
Fig. 4 - Conservation status for all bat species and rabies positive bat species in Latin America
and the Caribbean. CR: Critically Endangered, EN: Endangered, VU: Vulnerable, NT: Near
Threatened, LC: Least Concern, DD: Data Deficient.
DISCUSSION
Fig. 3 - Numbers of rabies-positive species and antigenic variants of rabies reported by
country (Table 1). Ven. = Venezuela.
68
Bat-borne rabies in Latin America and the Caribbean presents a
complex and incompletely understood situation. Across the region, bats
of all diet types have been found infected with rabies, but insectivorous
bats include the highest number of rabies-positive species (184 species),
but the lowest proportion of species diversity (27%); for hematophagous
and carnivorous species high proportions of rabies-positive species were
found (100% and 60% respectively), but numbers of species for these
diets were low. Because only three hematophagous bat species are known,
and only three carnivorous species were reported as rabies-positive,
results from these chi-square tests must be considered with caution, as
the low numbers of observations may render the results unreliable. In
light of frequent commensalism with humans, insectivorous bats present
risk of rabies transmission to humans63, as in the case of the insectivorous
bat T. brasiliensis, found abundantly in urban environments from Mexico
to Argentina and Chile10,25,76,90.
Hematophagous bats include only three species, but a significant role in
numerous rabies outbreaks in humans and livestock has been attributed to
D. rotundus populations, possibly in light of their ecological plasticity and
wide geographic distribution47. The diet and cryptic behavior of vampire
bats represent an overt source of human and animal bite contact, compared
to other bat diet types88. Viral characterization using monoclonal antibodies
gives clues about the mammal reservoir involved4,21,69, but, considering the
high diversity of viral lineages in Latin America, molecular genetic tools are
often used for confirmation25,32,67,69,90,92. The number of bat species rabiespositive and rabies AgV by country appear to be linked to research effort,
but not to bat species richness by country. More antigenic variants were
reported in countries where more bat species rabies-positive are found (Fig.
3). This close association between amount of rabies-positive species and
number of antigenic variants is strong evidence that more lineages could
be found if countries with high bat biodiversity increase research effort.
For example, a report was found of T. brasiliensis as rabies-positive for
Dominican Republic, but no reports were found for Haiti, even though the
two countries share a single island62.
However, substantial gaps exist in the knowledge of bat-rabies
ecology, such as how the virus spreads among populations86. Seasonal
migrations of species of bats in the genus Lasiurus may link to the spread
of rabies virus over thousands of kilometers along migration routes43.
Nevertheless, rabies virus variants linked to this genus have not been
reported in all Latin American and Caribbean countries where the species
is present. Geographic origins of rabies in the Americas remain unclear,
but recent evidence indicates that vampire strains may not be the source
of bat-borne rabies in the Americas46.
Antigenic variants differ among bat species and geographic locations.
For instance, T. brasiliensis is widely distributed in Latin America,
and across its distribution, diverse rabies antigenic variants have been
reported44. In Mexico, T. brasiliensis is the main reservoir of AgV9, but
in South America the same species carries AgV489. Lasiurus spp., on
the other hand, carry AgV6 across their broad geographic distribution43,
although with some exceptions26 and rabies lineages from other bat
species have been found in Lasiurus genus, suggesting cross-species
transmission70,93, contrasting with a report from North America, where
Lasiurus are more likely to be donors than recipients of spillover 83.
These differences in the distribution of virus variants may result from
geographic isolation and host behavior55, showing the complex dynamics
of rabies in bat populations90. Bat rabies antigenic variants have also
been found in skunks (Mephitis mephitis) and gray foxes (Urocyon
cinereoargenteus) of North America, demonstrating successful bat-borne
rabies host shift events to novel host species with viral persistence and
adaptation for transmission45,48. In Latin America, bat-borne antigenic
variants of rabies have been found in domestic carnivores (dogs and cats)
in Mexico, Costa Rica, Colombia, Brazil, Argentina, and Chile4,65,69,76,90,92.
Bat rabies outbreaks have been associated with habitat disturbance
and ecosystem alteration44, with some historic and current evidence in
Latin America5,14,32,49,51,56,77; a recent key article highlighted the need to
understand how anthropogenic perturbation triggers outbreaks of batborne diseases34, and this phenomenon demands deeper study.
The rabies literature presently focuses largely on disease diagnosis
and detection of rabies; few studies have sought to understand host-virus
dynamics or the ecology of these interactions18,28,83-85. An understanding of
virus and host ecology is fundamental, however, to preventing outbreaks
in humans and animals. Indeed, a series of significant research gaps,
were found as follows: 1) Relatively few countries report antigenic
variant identifications. As a result, virus variant distributions are poorly
characterized geographically. To date, the most relevant and complete
phylogenetic studies of bat-borne rabies have not included spatial
analyses11,83; detailed geographic and environmental characterization
of bat rabies could enhance future phylogeographic research. Better
characterization of rabies lineages in Latin America brings the opportunity
to identify bat-borne rabies in humans and understand how climate is
linked to rabies lineage distributions in the Americas. STREICKER et al.
(2012b), found effects of climate on viral evolution of bat rabies across
temperate and tropical regions, although more detailed analysis is needed
for tropical lineages. 2) Little is known about the ecology of rabies-bat
dynamics. In Latin America, few ecological studies have been undertaken
regarding rabies persistence mechanisms (but see BLACKWOOD et al.,
2013); further research should focus on longitudinal serologic studies
to understand temporal and spatial infection dynamics of rabies in bat
populations30,34. 3) Bat species carrying rabies are not reported in all
countries: such epidemiological gaps delay human rabies diagnosis
and prevention4. 4) Latin American bat species population status is
frequently poorly known. Understanding of bat population dynamics is
indispensable in comprehending the ecology of this and other infectious
diseases34. Finally, 5) effects of habitat fragmentation on virus occurrence
in bats and transmission to humans are poorly studied: although land-use
change has been suggested as related to rabies outbreaks, no scientific
quantification of this phenomenon exists34.
Density of mammals in human settlements (mainly cats and dogs) may
prove more important than just bat presence in determining transmission
risk of non-hematophagous bat rabies to people4,22,45,65,66,68,92, in view of
low prevalence in bat colonies24. Considering that bats are natural rabies
hosts, an integrated approach should seek equilibrium among public
health, agriculture, and biodiversity conservation interests. Public health
agencies should include bat ecologists in their teams, to understand bat
population dynamics for rabies prevention34; unfortunately, such links are
still missing. A strategic opportunity to reduce the gap between ecology
and public health is the Red Latinoamericana para la Conservación de
Murciélagos (Latin American Network for Bat Conservation; www.
relcomlatinoamerica.net). On the other hand, present laboratory-based
rabies surveillance in Latin America has been advancing programs to
eliminate dog rabies, a valuable source of data for bat-borne rabies
studies34. Finally, bat conservation has become a significant concern
in recent years72, but an important number of species in the region are
deficient in data to ascertain their conservation status.
69
RESUMEN
Rabia transmitida por murciélagos en Latino América
La situación de rabia en América es compleja: la rabia en perros ha
disminuido drásticamente pero los murciélagos están siendo reconocidos
cada vez más como reservorios naturales de otras variantes de rabia. Aquí
compilamos las especies de murciélagos reconocidas como positivas
a rabia con diferentes variantes antigénicas, así como su relación con
el estado de conservación de los murciélagos a lo largo de América
Latina. El virus de rabia está ampliamente distribuido en las especies
de murciélagos de América Latina, 22.5% (75) de las especies de
murciélagos conocidas han sido confirmadas como especies positivas
a rabia. La mayoría de las especies de murciélagos reportadas como
positivas a rabia son clasificadas por la Unión Internacional para la
Conservación de la Naturaleza como “Preocupación Menor”. De acuerdo
al tipo de dieta, los murciélagos insectívoros tuvieron la mayor cantidad
de especies reconocidas como reservorio del virus rabia, mientras en
proporción los hematófagos fueron los más importantes. Investigaciones
a escala gruesa deben buscar entender aspectos de ecología de la rabia;
es necesaria la información básica sobre la distribución y dinámica
poblacional para muchas especies de murciélagos de América Latina y
el Caribe; y el efecto del cambio del paisaje en la generación de brotes
de rabia transmitida por murciélagos permanece sin ser evaluado. Por
último, para entender y prevenir enfermedades emergentes a partir de los
murciélagos es necesario un enfoque integral incluyendo salud pública,
ecología y biología de la conservación.
ACKNOWLEDGMENTS
Special thanks to the Programa para la Conservación de los
Murciélagos de Chile (PCMCH) for promoting the development of
this review, and Emma Stapleton who donated the ArcGIS license.
Thanks also to Ruben Barquéz, who reviewed an earlier version of this
manuscript, and to Valeska Rodriguez for assistance in data compilation.
Universidad Andres Bello provided the grant DI-412-13/I. LEE is student
in the Conservation Medicine Program at the Universidad Andres Bello,
this manuscript is part of the fulfillment of his PhD degree.
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Received: 2 August 2013
http://dx.doi.org/10.1590/S0036-46652015000100010
ASSESSMENT OF THE PRESENCE OF Toxocara EGGS IN SOILS OF AN ARID AREA
IN CENTRAL-WESTERN ARGENTINA
María Viviana BOJANICH(1), José Mario ALONSO(2), Nadina Ayelén CARABALLO(3), Mercedes Itatí SCHÖLLER(3),
María de los Ángeles LÓPEZ(2), Leandro Martín GARCÍA(3) & Juan Ángel BASUALDO(4)
SUMMARY
With the aim of studying the contamination of soils with eggs of Toxocara spp. in an arid area in the central-western region
of Argentina, 76 soil samples were collected from 18 towns belonging to six provinces of central-western Argentina. They were
processed by the centrifugal flotation method. No eggs of Toxocara spp. were found. It can be concluded that the negative results are
directly related to the characteristics of the environment and climate present in the studied area. The finding of eggs in soils depends
on several factors: the presence of canine or feline feces, the hygienic behavior of pet owners, the presence of stray animals without
veterinary supervision, the weather and environmental conditions, and laboratory techniques used; and all these circumstances must
be considered when comparing the results found in different geographical regions. In order to accurately define the importance of
public spaces in the transmission of infection to humans, it is important to consider the role of backyards or green spaces around
housing in small towns, where the population is not used to walking pets in public spaces, and in such cases a significant fraction of
the population may acquire the infection within households.
KEYWORDS: Toxocariosis; Soil contamination; Zoonotic; Environment; Laboratory method.
INTRODUCTION
Toxocariosis is the most frequently reported zoonotic geohelminthic
infection worldwide and is caused by Toxocara canis or Toxocara cati.
Although it has not yet been clearly demonstrated which one of these
species is the most relevant in the epidemiology of the infection, due to the
inability of routine procedures to distinguish one species from the other3,9.
Study Area: Soils from the central-western region of Argentina were
studied. This is an extensive area located between the parallels 28 and 32
of SL and the meridians 64 and 68 of WL. The climate in this region is dry,
arid, with cold winters (an average annual temperature of less than 12 °C)
and warm summers, with an important daily temperature amplitude, with
little snow and/or rainfall (< 200 mm per year), dry winds and scarce
vegetation. Historical records of temperature and precipitation averages
for the month of September in this region are: minimum temperature
7 °C, average temperature 15 °C, maximum temperature 23 °C and
average rainfall 12 mm16.
The life cycle of Toxocara shows the importance of soil in the
transmission of infection to humans. The eggs expelled in pets’ feces
complete their maturation in the soil, until full development of infective
larvae, and thus, they contaminate the surface of the soil18. Public places
like parks, sidewalks, beaches etc are commonly shared by people and
dogs as places for recreation and transit, and, therefore, in order to
understand the epidemiological pathways of various zoonoses in relation
with the environment, the rate of parasitic intestinal infestation of pets
is usually studied and related to soil contamination13.
Although Toxocara spp. is described as an agent present worldwide,
no reports exist regarding the contamination of recreational public areas
in the central-western region of Argentina; for this reason, the aim of
this work is to assess the contamination of soils with eggs of Toxocara
spp. in that area.
Children’s recreational sites were selected in the following locations:
the city of Mendoza and the towns of La Paz and Uspallata in the province
of Mendoza; the towns of Caucete and St. Lucia in the province of San
Juan; the city of Merlo in the province of San Luis; the city of La Rioja
and the towns of Patquía, Chepes and Anillaco in the province of La Rioja;
the city of San Fernando and the town of Valle Viejo in the province of
Catamarca; and the towns San Antonio de Arredondo, El Condor, Nono,
Las Rabonas, San Javier and Luyaba in the western region of the province
of Cordoba (Fig. 1).
(1) Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Corrientes, Corrientes, Argentina. E-mail: [email protected]
(2) Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina. E-mails: [email protected], [email protected]
(3) Becaria Secretaría General de Ciencia y Técnica, Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina. E-mails: [email protected],
[email protected], [email protected]
(4) Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina. E-mail: [email protected]
Correspondence to: María Viviana Bojanich, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Corrientes, Corrientes, Argentina. E-mails:
[email protected]
BOJANICH, M.V.; ALONSO, J.M.; CARABALLO, N.A.; SCHÖLLER, M.I.; LÓPEZ, M.A.; GARCÍA, L.M. & BASUALDO, J.A. - Assessment of the presence of Toxocara eggs in soils of
an arid area in central-western Argentina. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 73-6, 2015
factors, such as environmental conditions (including light, temperature,
humidity and air quality)4, the sampling site, the laboratory method
employed etc21. Toxocara spp. eggs require a period of time under
appropriate environmental conditions to become infective to definitive
and paratenic hosts. Temperature and humidity are important factors
known to affect the levels of development in soil10. MIZGAJSKA stated
that, in arid conditions, with exposure to sunlight and temperatures below
10 °C, Toxocara eggs die. It should be noted that sampling of this work
was carried out in late winter15.
In the environment, feces carrying hundreds of thousands of eggs
are dispersed by the physical action of: trampling, rain, wind, vectors
etc., spreading the eggs’ parasitic forms. The presence of Toxocara eggs
is an indicator of canine fecal contamination above ground, resulting in
the exposure of every human in the local population regardless of sex,
age or socioeconomic status8.
Fig. 1 - Map of Argentina showing the provinces composing the central-western region.
Sampling: In September 2012, 76 soil samples were collected from
35 different sites in 18 towns belonging to six provinces of the centralwestern region of Argentina.
Samples were taken from sandboxes and children’s playgrounds
in squares and parks, and from other sites where recreational use was
evident. Samples consisted of 200-250g of dry soil collected from every
square meter of the site. Bare ground with little drainage and no evidence
of fouling by dogs or cats were selected. Samples were kept in the dark
at room temperature until processing2.
Methodology: Soil samples were carefully mixed and sieved through a
4 mm2 mesh to remove large stones and plant residues and were divided into
four aliquots of 5 g each. The aliquots were washed with tap water twice,
centrifuged and the supernatants were discarded. Pellets were suspended in
two different flotation solutions: two in saturated ClNa solution (δ 1.205)17
and two in saturated sucrose solution (δ 1.27)23. Each suspension was mixed
and centrifuged at 2000 g for 10 min, and then completed with solution
to the formation of meniscus and covered with a cover slip. After 20 min
they were observed under light microscopy. No egg count was performed
and the data were recorded as presence/absence of eggs1.
RESULTS AND DISCUSSION
No eggs of Toxocara spp. were found in any of the 76 samples studied.
The recovery of T. canis eggs from soil samples depends on several
74
In Argentina, urban contamination by Toxocara eggs has been
extensively described in the central areas of large and medium-size cities
due to the poor standards of care and irresponsible pet ownership (Table
1). The following surveys should also be mentioned: MINVIELLE et al.
on public parks and sidewalks in the city of La Plata14; FONROUGE et
al.8 and CÓRDOBA et al.6, who also studied public walks in La Plata,
RUBEL & WISNIVESKY in Buenos Aires19; MARTIN & DEMONTE
in Santa Fe11; SORIANO et al. in the city of Neuquén22; and SÁNCHEZ
THEVENET et al. on the contamination of canine feces found on
sidewalks in the cities of Comodoro Rivadavia (Province of Chubut)20.
There are also many reports regarding the environmental pollution in
Northeastern Argentina, a subtropical region with high temperatures and
high levels of humidity during most of the year. Other noteworthy surveys
include those conducted in the city of Corrientes by MILANO et al.12,13
and by ALONSO et al. who evaluated the contamination in Resistencia
(Province of Chaco) in 20011 and in 20062.
Contrastingly, there are few reports on soil contamination in rural
settlements of the country. However, the reports of CHIODO et al. on the
presence of Toxocara spp. eggs in soil samples from General Mansilla, a
small rural location in northern Buenos Aires Province5, and the findings
of FILLAUX et al. in some locations in the provinces of Chubut, Neuquén
and Río Negro7 can be cited.
It may be concluded that the negative results found in this work were
directly related to the environmental conditions present in the studied
area. Temperature is responsible for the rate of embryonation while
moisture is essential for encouraging development and maintaining egg
viability in general10. Regions with high thermal amplitude, low moisture,
strong sunlight and soils with little vegetation provide unfavorable
conditions for the viability of the eggs of Toxocara spp.
Discovery of eggs in the soil of public areas depends on the
presence of canine or feline feces, the hygienic behavior of pet owners,
the presence of stray animals without veterinary supervision, weather
conditions, laboratory techniques employed in the survey etc, and all these
circumstances must be considered when comparing the results found in
different geographical regions. Moreover, in order to accurately define
the importance of public spaces in the transmission of the infection to
humans, it is important to consider the role of backyards or green spaces
around housing in small towns, where the population is not used to
TABLE 1
Surveys performed on environmental contamination with Toxocara eggs in public locations in Argentina
Location (publication year)
Frequency of positive T. canis eggs in soils
and dog feces samples
Alonso et al.
Resistencia (2006)
20.6-33.3% (n=612)
Alonso et al.
Resistencia (2001)
3.4% (n=333)
La Plata (2002)
12.1% (n=140)
Chubut, Neuquén, Río Negro (2007)
35.1% (n=114)
Authors
Córdoba et al.
Fillaux et al.
Fonrouge et al.
La Plata (2000)
13.2% (n= 242)
Martin & Demonte.
Santa Fe (2008)
25.7% (n=393)*
Milano & Oscherov
Corrientes (2002)
0.3 (n=324)
Milano & Oscherov
Corrientes (2005)
16% (n=362)*
La Plata (1993)
10.7-13% (n=351)*
Minvielle et al.
Rubel & Wisnivesky
Sánchez Thevenet et al.
Soriano et al.
Buenos Aires (2005)
9%-17% (n=2417)*
Comodoro Rivadavia (2003)
17.7% (n=163)*
Neuquén (2010)
16.35% (n=1944)*
n = total number of soil samples; n)* = total number of dog feces samples.
walking pets in public spaces, and in such cases a significant fraction of
the population may acquire the infection within households.
These features justify the need for an international consensus to
achieve agreements, in order to standardize methods for soil sampling
and laboratory processing, and thus, make results more comparable
among the surveys conducted in various regions of the world. This could
eventually redefine the epidemiologic importance of public spaces in the
transmission of geohelminthic infections to humans.
RESUMEN
Evaluación de la presencia de huevos de Toxocara en suelos de una
zona árida en la región centro-oeste Argentina
Con el objetivo de estudiar la contaminación de los suelos con
huevos de Toxocara spp, se obtuvieron 76 muestras de suelo de 18
pueblos pertenecientes a 6 provincias del centro-oeste de Argentina. Las
muestras fueron procesadas por el método de centrifugación-flotación.
No se encontraron huevos de Toxocara spp. en ninguna de las muestras
de suelo. Llegamos a la conclusión de que los resultados negativos
podrían estar en relación directa con las características ambientales y
climáticas presentes en el área estudiada. El hallazgo o no de huevos
en el suelo depende de varios factores: la presencia de heces de caninos
o felinos, el comportamiento de los dueños de mascotas, la presencia
de animales abandonados y sin control veterinario, las condiciones
climáticas y ambientales y las técnicas de laboratorio utilizadas. Todas
estas circunstancias deben ser consideradas cuando se comparan los
resultados encontrados en diferentes regiones geográficas. Con el fin de
definir la importancia que tienen los espacios públicos en la transmisión
de la infección a los humanos, se debería considerar el papel que tienen
los patios y veredas de las viviendas en las ciudades pequeñas, donde la
población no acostumbra a pasear mascotas en parques y plazas, y en
esos casos, la población puede adquirir la infección dentro de los hogares.
ACKNOWLEDGEMENTS
To the Secretaría General de Ciencia y Técnica of Univesidad
Nacional del Nordeste -Argentina for funding this work, and to Mrs.
Mariana Climent for the translation of the manuscript.
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19.Rubel D, Wisnivesky C. Magnitude and distribution of canine fecal contamination
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Argentina. Vet Parasitol. 2005;133:339-47.
11. Martin U, Demonte M. Urban contamination with zoonotic parasites in the central region
of Argentina. Medicina (B Aires). 2008;68:363-6.
12.Milano AMF, Oscherov EB. Contaminación por parásitos caninos de importancia
zoonótica en playas de la ciudad de Corrientes, Argentina. Parasitol Latinoam.
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76
http://dx.doi.org/10.1590/S0036-46652015000100011
BRIEF COMMUNICATION
MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF
HUMAN STRONGYLOIDIASIS
Marcelo Andreetta CORRAL(1), Fabiana Martins PAULA(2), Maiara GOTTARDI(1), Dirce Mary Correia Lima MEISEL(2),
Pedro Paulo CHIEFFI(1,2,3) & Ronaldo César Borges GRYSCHEK(1,2)
SUMMARY
Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological
diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human
strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl
(TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis
patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were
analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9%
specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest
the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.
KEYWORDS: Strongyloides venezuelensis; Parasitological diagnosis; Immunological diagnosis; Membrane fractions.
Strongyloidiasis is a human intestinal infection caused by
Strongyloides stercoralis, which affects between 30-100 million people
in the world2, and is endemic in tropical and subtropical regions15. In
immunocompetent hosts it is often an asymptomatic infection. However,
immunocompromised hosts may develop a fatal hyperinfection syndrome
or disseminated strongyloidiasis9,17.
Strongyloidiasis diagnosis usually depends on the identification of
larvae in stool samples9. However, parasitological diagnosis presents
low sensitivity, due to an intermittent larval shedding19. Immunological
methods, such as enzyme-linked immunosorbent assay (ELISA), are
an alternative for the diagnosis of strongyloidiasis3, especially using
heterologous antigenic extracts from Strongyloides venezuelensis4,5,6.
The development of more accurate immunological methods, using
purified parasitic antigens, can improve the diagnostic sensitivity and
specificity4,5,7. The surface of parasitic nematodes has been shown to be
antigenic in many infected hosts12, but membrane antigens have been
underexplored. The aim of this study was to verify the use of soluble
and membrane antigen extracts obtained from filariform larvae of
S. venezuelensis for human strongyloidiasis immunodiagnosis.
Serum samples were obtained at the Hospital das Clínicas da
Faculdade de Medicina da Universidade de São Paulo (HCFMUSP)
from 92 individuals: 20 patients harboring S. stercoralis larvae (Group
I); 32 patients with other parasites (Group II) [Schistosoma mansoni
(n = 9), hookworm (n = 4), Ascaris lumbricoides (n = 2), Hymenolepis
nana (n = 1), Enterobius vermicularis (n = 1), Giardia lamblia (n = 3);
Endolimax nana (n = 3), Blastocystis sp. (n = 3) and six poly-infected
samples (S. mansoni, A. lumbricoides, E. coli, Blastocystis sp. and
E. nana/hookworm and H. nana/hookworm, S. mansoni, E. coli and
E. histolytica/E.dispar/G. intestinalis and E. nana/A. lumbricoides
and Blastocystis sp./S. mansoni, E. nana and Blastocystis sp.)]; and
40 apparently healthy individuals based on their clinical observation,
without evidence of contact with S. stercoralis infection or previous
history of strongyloidiasis (Group III). All feces samples were analyzed
by the LUTZ method11, a gravity sedimentation technique, the RUGAI
method18, based on positive larval termo-hydrotropism, and agar plate
culture method16, by the observation of larvae tracks over the agar. Due
to the difficulty of obtaining three or more stool samples, a decision was
made to analyze a single sample using more sensitive techniques for
the detection of S. stercoralis larvae, such as Rugai and culture on an
agar plate. It has been shown that the combination of these two methods
facilitates detection of 95% of infections caused by S. stercoralis when
only one sample is analyzed16. The study received approval from the
Research Ethics Committee of Universidade de São Paulo, state of São
Paulo, Brazil (protocol 266.046).
(1) Instituto de Medicina Tropical de São Paulo, USP, São Paulo, SP, Brazil.
(2) Laboratório de Investigação Médica (LIM-06), Hospital das Clínicas da Faculdade de Medicina, USP, São Paulo, SP, Brazil.
(3) Faculdade de Ciências Médicas, Santa Casa, São Paulo, SP, Brazil.
Correspondence to: Ronaldo César Borges Gryschek. Laboratório de Investigação Médica (LIM-06), Hospital das Clínicas da Faculdade de Medicina, USP, São Paulo. Instituto de Medicina
Tropical, prédio II, 2º andar, Av. Dr. Enéas de Carvalho Aguiar 470, 05403-000 São Paulo, SP, Brasil. Tel: +55 11 3061-8220. E-mail: [email protected]
CORRAL, M.A.; PAULA, F.M.; GOTTARDI, M.; MEISEL, D.M.C.L.; CHIEFFI, P.P. & GRYSCHEK, R.C.B. - Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis
of human strongyloidiasis. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 77-80, 2015.
For antigenic extraction, S. venezuelensis filariform larvae (L3) were
obtained from charcoal cultures of feces of experimentally infected
Rattus norvegicus (Wistar), protocol (CPE-IMT 2011/126). L3 PBS
(phosphate-buffered saline, 0.01M pH 7.2) or Tris-HCl 25mM pH
7.5, containing protease inhibitors (Sigma-Aldrisch, St. Louis, MO,
USA) were added to 400,000 samples and disrupted in an ice bath
using a tissue homogenizer for five cycles of five minutes each. The
suspensions were centrifuged at 12,400g for 30 minutes at 4 ºC and
the supernatant was collected (soluble fractions SS and TS, PBS and
Tris-HCl, respectively). SS pellets were resuspended in 1% SDS, heated
to 100 ºC for five minutes, centrifuged at 12,400g for 30 minutes at
4 ºC and the supernatant was collected (membrane fraction SM). ST
pellets were resuspended in 5M urea, 2M thiourea and 4% CHAPS;
disrupted in an ice bath using a tissue homogenizer for five cycles of
five minutes; centrifuged at 12,400g for 30 minutes at 4 ºC and the
supernatant was collected (membrane fraction TM). All fractions were
analyzed for protein content according to LOWRY et al.10, subdivided
into aliquots and stored at -20 ºC until use.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described by LAEMMLI8. Fractions were
submitted to electrophoresis using a 12% acrylamide separation gel
analyzed by silver nitrate.
ELISA was performed according to COSTA-CRUZ et al.3 with
modifications using the four obtained fractions. Briefly, polystyrene
microplates were coated with each of the antigenic fractions (SS, TS,
SM and TM) at concentrations of 10 µg/mL in carbonate-bicarbonate
buffer (0.06 mmol/L, pH 9.6), incubated overnight at 4 ºC. Afterwards, the
microplates were blocked with PBS containing 0.05% Tween 20 plus 3%
non-fat milk (PBS-TM) for 45 minutes at 37 ºC. Serum samples diluted
1:200 in PBS-TM and enzyme-conjugated peroxidase-goat anti-human
IgG Fc specific (Sigma-Aldrisch, St. Louis, MO, USA) was then added
at 1:30,000 in PBS-TM. The assay was developed by adding the enzyme
substrate consisting of hydrogen peroxide and orthophenylenediamine to
0.1 mol/L citrate phosphate buffer pH 5.5 for 15 minutes. The reaction
was interrupted with H2SO4 (2N). Optical densities (OD) were determined
at 492 nm in an ELISA reader (Thermo Fischer Scientific, Waltham,
MA, USA).
Statistical analyses were performed using the GraphPad Prism
software, version 5.0 (Graph Pad Software Inc. San Diego, USA). The
cut-off value, sensitivity and specificity were established by receiver
operating characteristic (ROC) curve analysis using Groups II and III
as a negative control. The concordance was carried out by analysis of
the Kappa coefficient (κ). Statistical significance was set at p < 0.05.
The protein concentrations were 1.3-1.8 mg/mL and 6.2-6.9 mg/mL
for soluble and membrane fractions, respectively. Electrophoretical
profiles of each antigenic fraction after 12% SDS-PAGE are shown
in Figure 1. Extract preparations showed several proteic compounds
with molecular weights ranging from < 15 to 150 KDa. In membrane
preparation, SM and TM fractions of 50, 70 and 120KDa were evident.
The diagnostic parameters (sensibility and specificity) and diagnostic
efficiency of ELISA in the detection of IgG anti S. stercoralis are shown in
Table 1. Analysis of the ROC (Fig. 2) showed that SM and TM efficiently
distinguished patients from Group I and controls (Groups II and III).
78
Fig. 1 - Electrophoretic profiles of antigenic fractions SS, SM and TS, TM, soluble and
membrane fractions from phosphate saline and Tris-HCl, respectively; 12% SDS-PAGE
stained by silver nitrate. To each antigenic fraction were used 10 µg/mL.
Table 1
Diagnostic parameters of ELISA in detection of IgG anti-S.stercoralis using
soluble fractions and membrane antigens
Antigens
Se (%)
Sp (%)
κ
DA (CI 95%) (%)
SS
90
88.9
0.71
89.2 (82.9-95.5)
TS
90
88.9
0.71
89.2 (82.9-95.5)
SM
95
94.4
0.85
94.6 (90.0-99.2)
TM
95
94.4
0.85
94.6 (90.0-99.2)
Se = Sensitivity; Sp = Specificity; κ = Kappa Index; DA = Diagnostic Accuracy;
p < 0.05.
Cross-reactivity in Group II was observed in serum samples from
patients infected with S. mansoni (1/9 in SS, SM and TM; 2/9 in ST),
hookworms (1/4 in SS and ST) and those polyinfected (1/6 ST).
Considering the difficulties of obtaining more specific antigenic
fractions for strongyloidiasis immunodiagnosis, efforts to achieve a
reliable diagnostic test are needed. The present study was conducted
to verify the use of membrane fractions as a source of antigens for
the serological diagnosis of strongyloidiasis. The surface of parasitic
nematodes has been shown to be antigenic in many infected hosts11.
Several studies have demonstrated fractionation of Strongyloides
antigenic extracts 4,5,7. There is a high concern about the antigen
preparations used in the tests, particularly in the evaluation of buffers
for the extraction of proteins, constituting an essential stage in obtaining
antigens. The buffer utilized in the extraction of antigenic fractions is
phosphate buffer3,5,6,7. However, the use of Tris-HCl buffer has been
reported in an attempt to study the Strongyloides13,14, but not in direct
application to immunodiagnosis.
imunológico da estrongiloidíase humana. O objetivo deste estudo foi
avaliar frações de membrana de S. venezuelensis para o imunodiagnóstico
da estrongiloidíase humana. Para tanto, frações solúveis e de membrana
foram obtidas em solução salina fosfato (SS e MS) e Tris-HCl (ST e
MT) de larvas filarioides de S. venezuelensis. Amostras de soro de 92
indivíduos, sendo 20 com estrongiloidíase (Grupo I); 32 com outras
parasitoses (Grupo II), e 40 indivíduos saudáveis (Grupo III), foram
analisadas pelo teste Imunoenzimático (ELISA). As frações solúveis
(SS e ST) apresentaram 90,0% e 88,9%, enquanto que as frações de
membrana (MS e MT) demonstraram 95,0% e 94,4%, de sensibilidade e
especificidade, respectivamente. Os resultados obtidos permitem indicar
as frações de membranas como antígeno alternativo para o diagnóstico
da estrongiloidíase humana.
ACKNOWLEDGMENTS
The authors would like to thank the Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP 2013/04236-9)
REFERENCES
Fig. 2 - Receiver-operating characteristic curve (ROC-AUC) analysis indicating the optimum
point of reactions (cut-off); LR = Likelihood ratio; AUC = Area under curve.
Sensitivity and specificity using the SS fraction have been described
in the literature. Compared to the present work, GONZAGA et al.6 and
FELICIANO et al.4 showed similar values of sensitivity and specificity
using saline extract of the S. venezuelensis. On the other hand, INÊS
et al.7 showed lower values of sensitivity (76.6%) and high specificity
(92.9%). The comparison of techniques for the immunodiagnosis of
human strongyloidiasis by BISOFFI et al. has recently been reported1.
This study showed sensitivity varying from 75.4 to 93.9% and specificity
values varying from 94.8 to 100%. In the present study 95% sensitivity
and 94.4% specificity were obtained. Then, to obtain membrane antigens,
the pellets were treated with detergent, which is a relatively simple and
easy procedure, and does not require specialized apparatus, thus reducing
the cost of production in the laboratory.
The results showed high sensitivity and specificity of the membrane
antigens compared to soluble preparations. In soluble antigen preparation,
large amounts of protein present in the pellets are usually discarded.
This is not the case when membrane antigens are prepared, so the pellet
proteins are not discarded. Besides this, there is the difficulty of obtaining
antigen for the diagnosis of human strongyloidiasis. So, it is essential to
search for ways to obtain larger amounts of antigen, that can bring results
with high sensitivity and specificity in the diagnosis of strongyloidiasis.
In conclusion, membrane fractions of S. venezuelensis can be alternative
antigens for immunodiagnosis of human strongyloidiasis.
RESUMO
Frações de membrana de Strongyloides venezuelensis para o
imunodiagnóstico da estrongiloidíase humana
Strongyloides venezuelensis é um nematódeo parasita de roedores,
frequentemente usado como antígeno heterólogo para o diagnóstico
1. Bisoffi Z, Buonfrate D, Sequi M, Mejia R, Cimino RO, Krolewiecki AJ, et al. Diagnostic
accuracy of five serologic tests for Strongyloides stercoralis infection. PLoS Negl
Trop Dis. 2013;8:e-2640.
2.Concha R, Harrington W Jr, Rogers AI. Intestinal strongyloidiasis: recognition,
management and determinants of outcome. J Clin Gastroenterol. 2005;39:203-11.
3.Costa-Cruz JM, Madalena J, Silva DA, Sopelete MC, Campos DMB, Taketomi EA.
Heterologous antigen extract in the ELISA for the detections of human IgE antiStrongyloides stercoralis. Rev Inst Med Trop Sao Paulo. 2003;45:265-8.
4.Feliciano ND, Gonzaga HT, Gonçalves-Pires MRF, Gonçalves ALR, Rodrigues RM,
Ueta MT, et al. Hydrophobic fractions from Strongyloides venezuelensis for use
in the human immunodiagnosis of strongyloidiasis. Diagn Microbiol Infect Dis.
2010;67:153-61.
5.Gonzaga HT, Ribeiro VS, Cunha-Júnior JP, Ueta MT, Costa-Cruz JM. Usefulness of
concanavalin-A non-binding fraction of Strongyloides venezuelensis larvae to detect
IgG and IgA in human strongyloidiasis. Diagn Microbiol Infect Dis. 2011;70:78-84.
6.Gonzaga HT, Vila-Verde C, Nunes DS, Ribeiro VS, Cunha-Júnior JP, Costa-Cruz JM.
Ion-exchange protocol to obtain antigenic fractions with potential for serodiagnosis
of strongyloidiasis. Parasitology. 2012;140:69-75.
7.Inês E de J, Silva MLS, Souza JN, Teixeira MCA, Soares NM. The role glycosylated
epitopes in the serodiagnosis of Strongyloides stercoralis infection. Diagn Microbiol
Infect Dis. 2013;76:31-5.
8.Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 1970;227:680-5.
9. Liu LX, Weller PF. Strongylodiasis and other intestinal nematode infections. Infect Dis
Clin North Am. 1993;37:655-82.
10.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin
phenol reagent. J Biol Chem. 1951;93:265-75.
11. Lutz A. O Schistosomum mansoni e a schistosomatose, segundo observações feitas no
Brasil. Mem Inst Oswaldo Cruz. 1919,11:121-55.
12.Northern C, Grove DI, Warton A, Lovegrove FT. Surface labelling of Strongyloides
ratti: stage-specificity and cross-reactivity with S. stercoralis. Clin Exp Immunol.
1989;75:487-92.
79
13.Northern C, Grove DI. Strongyloides stercoralis: antigenic analysis of infective larvae
and adult worms. Int J Parasitol. 1990;30:381-7.
14.Paula FM, Castro-Borges W, Júnior OS, de Souza Gomes M, Ueta MT, Rodrigues V.
The ubiquitin-proteasome system in Strongyloididae. Biochemical evidence for
developmentally regulated proteolysis in Strongyloides venezuelensis. Parasitol Res.
2009;105:567-76.
15.Paula FM, Costa-Cruz JM. Epidemiological aspects of strongyloidiasis in Brazil.
Parasitology. 2011;138:1331-40.
16.Paula FM, Gottardi M, Corral MA, Chieffi PP, Gryschek RC. Is the agar plate culture
a good tool for the diagnosis of Strongyloides stercoralis in candidates for
transplantation? Rev Inst Med Trop Sao Paulo. 2013;55:291.
80
17.Requena-Méndez A, Chiodini P, Bisoffi Z, Buonfrate D, Gotuzzo E, Muñoz J. The
laboratory diagnosis and follow up of strongyloidiasis: a systematic review. PLOS
Negl Trop Dis. 2013;7:e-2002.
18. Rugai E, Mattos T, Brisola AP. Nova técnica para isolar larvas de nematoides das fezes:
modificação do método de Baermann. Rev Inst Adolfo Lutz. 1954;14:5-8.
19. Uparanukraw P, Phongsri S, Morakote N. Fluctuations of larval excretion in Strongyloides
stercoralis infection. Am J Trop Med Hyg.1999;60:967-73.
Accepted: 6 August 2014
http://dx.doi.org/10.1590/S0036-46652015000100012
BRIEF COMMUNICATION
IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS
IN ISOLATES OF Acanthamoeba
Vinicius José MASCHIO, Gertrudes CORÇÃO & Marilise Brittes ROTT
SUMMARY
Acanthamoeba is a ‘‘Trojan horse’’ of the microbial world. The aim of this study was to identify the presence of Pseudomonas
as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoebaresistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.
KEYWORDS: Acanthamoeba; Pseudomonas; Amoeba-resistant microorganism.
Acanthamoeba is an opportunistic human pathogen that is
ubiquitously distributed in the environment13. It is a causative agent of
cutaneous lesions, sinus infections, vision threatening keratitis and rare
but fatal encephalitis, known as granulomatous amoebic encephalitis.
In addition, it has the ability to act as a host/reservoir for microbial
pathogens10,16.
a reservoir for some amoeba-resistant strains of Pseudomonas, similar
to what was shown for Legionella spp.6. This is important, given the
role of Pseudomonas aeruginosa as a causative agent of pneumonia5.
Acanthamoeba has been isolated from contact lens care systems
contaminated with Gram-negative bacteria, including Pseudomonas
aeruginosa6.
Free-living amoebae feed by phagocytosis mainly on bacteria,
fungi, and algae, and digestion occurs within phagolysosomes. Some
microorganisms have evolved and have become resistant to predation
by protists, since they are not internalized or are able to survive, grow,
and exit free-living amoebae after internalization. Acanthamoeba is
shown to be host/reservoir for numerous bacteria, including the genus
Pseudomonas spp., among other bacterial pathogens13.
Many studies have evaluated the interaction between Acanthamoeba
spp. and Pseudomonas spp., as well as investigated the presence of these
bacterial genera as amoeba-resistant bacteria3,8,11.
Pseudomonas spp. are highly adaptable bacteria that can colonize
various environmental niches, including soil and marine habitats,
plants and animals. Pseudomonas spp. are also opportunistic human
pathogens, causing infection of the eyes, ears, skin, urethra and
respiratory tract in cystic fibrosis (CF) in burned patients, as well as
other immunocompromised individuals15.
In nature, free-living amoebae of the genus Acanthamoeba feed by
Pseudomonas spp., which are widely distributed in the environment. Their
encounter may be facilitated through better adherence of Pseudomonas
spp. (than E. coli) to Acanthamoeba 2. However, some Pseudomonas
spp. have evolved to become resistant to predation by amoebae, as
demonstrated by the isolation of Acanthamoeba naturally infected with
P. aeruginosa6,13. Hence, free-living amoebae might also play a role as
In this study, the conventional technique of Polymerase Chain
Reaction (PCR) was used, in order to identify the presence of the genus
Pseudomonas spp. as amoeba-resistant microorganisms in isolates of
Acanthamoeba.
A total of 12 environmental samples existing in the laboratory were
used in this study: seven isolates from air-conditioning units identified
as Acanthamoeba A2, A3, A4, A5, A7, A8 and A10, and five isolates
from contact lens cases, Acanthamoeba A1, A6, A9, A11 and A12. The
isolates were cultured in PYG media at 30 ºC (2% protease peptone,
0.2% yeast extract, and 1.5% glucose) supplemented with penicillin
and streptomycin (Life Technologies). The total DNA in the sample was
extracted, as described by ALJANABI & MARTINEZ1. The fresh culture
containing 106 trophozoites was homogenized in 400 µL of sterile salt
homogenizing buffer (0.4 M NaCl 10 mM Tris–HCl pH 8.0 and 2 mM
EDTA pH 8.0), then, 40 µL of 20% SDS (2% final concentration) and
8 µL of 20 mg/mL protenase K (400 µg/mL final concentration) were
added and mixed well. The samples were incubated at 65 ºC for, at
Departamento de Microbiologia, Imunologia e Parasitologia. Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Correspondence to: Marilise Brittes Rott. Fax: +55 51 3308 3445. E-mail: [email protected]
MASCHIO, V.J.; CORÇÃO, G. & ROTT, M.B. - Identification of Pseudomonas spp. as amoeba-resistant microorganisms in isolates of Acanthamoeba. Rev. Inst. Med. Trop. Sao Paulo,
57(1): 81-3, 2015.
least, one h, after which 300 µL of 6 M NaCl (NaCl saturated H2O)
was added to each sample. Samples were vortexed for 30s at maximum
speed, and tubes spun down for 30 min at 10,000 x g. The supernatant
was transferred to fresh tubes. An equal volume of isopropanol was
added to each sample and samples were incubated at -20 ºC for one h.
Samples were then centrifuged for 20 min, at 4 ºC and at 10,000 x g.
The pellet was washed with 70% ethanol, dried and finally resuspended
in 100 µL sterile dH2O.
After extraction, the isolates were screened for the presence of bacterial
endosymbiont - Bacteria domain - through the 16S rDNA gene amplified
by PCR, using primers fD1 (5’-AGAGTTTGATCCTGGCTCAG-3’) and
rP2 (5’-ACGGCTACCTTGTTACGACTT-3’) that amplify 1500 bp in
size, described by WEISBURG et al.17, under the following conditions:
five min at 94 °C, followed by 35 cycles of one min at 94 °C, one min
at 55 °C and one min at 72 °C.
The identification of the presence of Pseudomonas genus
DNA occurred using the primers described by SPILKER et al.14
PA-GS-F (5’- GACGGGTGAGTAATGCCTA-3’) and PA-GS-R
(5’-CACTGGTGTTCCTTCCTATA-3’) that amplifies 618 pb in size.
Amplification was performed in a total volume of 25 μL containing 30
ng DNA, 10 pmol each primer, 5 pmol dNTP, reaction buffer (50 mM
KCl2, 10 mM Tris–HCl), 1.5 mM MgCl2, and 1 U of Platinum Taq DNA
Polymerase (InvitrogenTM). The amplification reaction was carried out
in a PTC-150 Minicycler MJ Research thermocycler, under the following
conditions: five min at 94 °C, followed by 35 cycles of one min at 94 °C,
one min at 58 °C and one min at 72 °C.
The amplification product was separated in 1% agarose gel,
stained with 0.5 µM/mL ethidium bromide and observed under a UVlight transilluminator. PCR products were purified using a QIAquick
purification kit (QIAGEN GmbH, Hilden, Germany) according to
the manufacturer’s instructions, and resolved with a MegaBace 1000
automated sequencer. Analysis of the DNA sequences was performed
with the Chromas Lite program and compared to those present in
GenBank (http://blast.ncbi.nlm.nih.gov/).
In the present study, all isolates of Acanthamoeba showed
internalized bacteria when primers are used to amplify the Bacteria
domain and all isolates showed the genus Pseudomonas spp. as amoebaresistant microorganisms (Fig. 1). A total of six PCR products (Ap1
to Ap6) were sent for sequencing (Table 1) and all were confirmed as
Pseudomonas spp.
Fig. 1 - Samples of Acanthamoeba A1, A6, A9, A11 and A12 isolated from contact lens cases,
and A2, A3, A4, A5, A7, A8, and A10 isolated from air conditioning units. Positive control
(PC) strain of Pseudomonas aeruginosa ATCC 278532.
CALVO et al.3 analyzed Acanthamoeba spp. originated from
natural and anthropogenic environments and recorded the presence of
Pseudomonas spp. as amoeba-resistant microorganisms in 26.1% of the
isolates. GARCIA et al. (4) evaluated isolates from water coming from
reservoirs and obtained 32.6% positive for Pseudomonas spp. In a study
on clinical isolates of Acanthamoeba spp., IOVIENO et al.8 observed that
Pseudomonas spp. were present as amoeba-resistant microorganisms in
59% of the isolates studied.
Pseudomonas spp. have also been reported to be involved in keratitis
and fatal pneumonia7, among other diseases. Their presence may have
a great impact on immune-suppressed individuals, since around 96%
of the Pseudomonas spp. isolated from hot tubs and indoor swimming
pools in a surveillance study display antimicrobial resistance9. Therefore,
their prevalence in the environment, not only in recreational water but
as part of biofilms in systems of distribution of drinking water, as well
as their relevance in human pathogenicity led researchers to seek for its
occurrence in amoeba hosts3.
Table 1
Percentage of similarity and access number compared to GenBank sequences of identified bacteria in this study
Fragment from the gel
(GenBank accession)
Similarity
BLAST
Access GenBank (number for access)
A1
Ap1 (KF160336)
98%
Pseudomonas sp. c145(2012) 16S ribosomal RNA gene, partial sequence
(JQ781629.1)
A3
Ap2 (KF160337)
96%
Uncultured Pseudomonas sp. clone 3F10 16S ribosomal RNA gene, partial
sequence (HM438578.1)
A4
Ap3 (KF160338)
99%
Pseudomonas sp. CJ-S-R2A3 16S ribosomal RNA gene, partial sequence
(HM584286.1)
A6
Ap4 (KF160339)
99%
(JQ781629.1)
A10
Ap5 (KF160340)
99%
Pseudomonas fluorescens strain C-D-TSA4 16S ribosomal RNA gene, partial
sequence (HM755599.1)
A12
Ap6 (KF160341)
97%
(JQ781629.1)
Acanthamoeba
82
MASCHIO, V.J.; CORÇÃO, G. & ROTT, M.B. - Identification of Pseudomonas spp. as amoeba-resistant microorganisms in isolates of Acanthamoeba. Rev. Inst. Med. Trop. Sao Paulo,
57(1): 81-3, 2015.
The possible role of Acanthamoeba as an evolutionary precursor of
pathogenicity in microbial pathogens has been suggested12. Bacteria or
other microbial endosymbiont may also enhance the pathogenicity of
Acanthamoeba12. However, the results have been inconclusive. There are
a few reports suggesting that amoeba-resistant microorganisms enhance
the virulence of Acanthamoeba6.
In addition to the bacteria identified in this work, the presence of other
pathogenic amoeba-resistant microorganisms in the water samples tested
cannot be discarded. Acanthamoeba spp. are also potential reservoirs of
Mycobacterium spp.3 and Legionella spp., among others microorganisms3.
5.Garau J, Gomez L. Pseudomonas aeruginosa pneumonia. Curr Opin Infect Dis.
2003;16:135-43.
6.Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol
Rev. 2004;17:413-33.
7.Huhulescu S, Simon M, Lubnow M, Kaase M, Wewalka G, Pietzka AT, et al. Fatal
Pseudomonas aeruginosa pneumonia in a previously healthy woman was most likely
associated with a contaminated hot tub. Infection. 2011;39:265-9.
8.Iovieno A, Ledee DR, Miller D, Alfonso EC. Detection of bacterial endosymbionts in
clinical Acanthamoeba isolates. Ophthalmology. 2010;17:445-52.
RESUMO
9. Lutz JK, Lee J. Prevalence and antimicrobial-resistance of Pseudomonas aeruginosa in
swimming pools and hot tubs. Int J Environ Res Public Health. 2011;8:554-64.
Identificação de Pseudomonas spp. como microrganismo resistente
a ameba em isolados de Acanthamoeba
10. Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin
Microbiol Rev. 2003;16:273-307.
Acanthamoeba é um ‘’ Cavalo de Tróia’’ do mundo microbiano. Este
estudo teve como objetivo identificar a presença de Pseudomonas como
microrganismo resistente a ameba em 12 isolados de Acanthamoeba.
Todos os isolados apresentaram o gênero Pseudomonas spp. como um
microrganismo resistente a ameba. Assim, podemos ver que os isolados
de Acanthamoeba estudados são hospedeiros de Pseudomonas.
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aeruginosa. Environ Microbiol. 2012;4:1349-62.
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Received: 20 September 2013
Accepted: 28 March 2014
83
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http://dx.doi.org/10.1590/S0036-46652015000100013
BRIEF COMMUNICATION
ELEVATED TRANS-MAMMARY TRANSMISSION OF Toxocara canis LARVAE IN BALB/c MICE
Paula de Lima TELMO(1), Luciana Farias da Costa de AVILA(2), Cristina Araújo dos SANTOS(3), Patrícia de Souza de AGUIAR(1),
Lourdes Helena Rodrigues MARTINS(3), Maria Elisabeth Aires BERNE(2) & Carlos James SCAINI(1)
SUMMARY
Toxocariasis is a widespread zoonosis and is considered an important worldwide public health problem. The aim of this study was
to investigate the frequency of trans-mammary Toxocara canis infection in newborn BALB/c mice nursed by females experimentally
infected with 1,200 eggs after delivery. After 50 days of age, the presence of larvae in different organs of the offspring was investigated.
Trans-mammary infection was confirmed in 73.9% of the mice that had been nursed by infected females. These data show a high
trans-mammary transmission of T. canis and confirm the significance of this transmission route in paratenic hosts.
KEYWORDS: Toxocariasis; Lactation; Breast-feed; Paratenic host.
The enzootic cycle of the nematode Toxocara canis in dogs, definitive
hosts, is assured by congenital transmission. Quiescent larvae in pregnant
female tissues are stimulated, most likely by hormonal mechanisms; the
larvae then cross the placenta and migrate to the fetus. Moreover, the dogs
can also become infected by ingesting larvae present in the colostrum
or milk during the first weeks of life3. Infection in paratenic hosts,
including humans, occurs mainly through the ingestion of embryonated
Toxocara eggs present in contaminated soil7 and may also occur through
the ingestion of larvae present in undercooked meats or viscera of birds
and mammals9,20. Although most cases are attributed to T. canis, there is
evidence that T. cati might cause the disease in humans6.
In addition to these well-documented transmission routes, in the
last 50 years, several studies in experimental models have confirmed the
vertical transmission of T. canis larvae11,17,18,19 and one study evaluated
and confirmed the trans-mammary transmission in ICR mice10. Almost
two decades ago, ANDERSON (1996) had already warned about of
the possibility of T. canis larvae be transmitted to the fetus when the
mother acquires an infection during pregnancy; this event could lead
to the development of the neurologic form in the affected child. More
recently, a case of congenital newborn T. canis infection was recorded
in Argentina13. Although less frequent, vertical transmission in paratenic
host was also reported by T. cati16. Due to occurrence of trans-mammary
transmission in ICR mice10 and the variation between the intensity of the
infection in different species of experimental models, this study aimed to
investigate the frequency of trans-mammary infection of T. canis larvae
in newborn BALB/c mice nursed by females that were experimentally
infected after delivery.
T. canis eggs were collected from the uterine tubes of adult female
parasites obtained after the treatment of young dogs with pyrantel
pamoate (15 mg/kg). Unembryonated T. canis eggs were incubated
in 2% formalin at 28 ºC with daily airings for a period of 30 days4.
Simultaneously, three female and three male mice were mated. After
giving birth, the females were intragastrically inoculated with 1200
embryonated T. canis eggs19. The animals were weaned after 21 days
and were kept in their cages until they reached an age of 50 days. After
this period, the presence of T. canis larvae in the organs of the dams and
their offspring was investigated. Three females that were mated in the
same period but not experimentally infected were used as controls. The
animals were kept in an acclimatized environment at 22 ºC (± 2 ºC) with
a light-dark cycle of 12 h and food and water available ad libitum. This
study was approved by the Ethics Committee in Research at the Federal
University of Rio Grande (CEPAS No. 098/2009). All the experiments
were carried out following the Federal Government legislation on animal
care. All of the mice were euthanized by cervical dislocation, according
to animal ethics guidelines (CFMV Resolution No. 1000).
Tissue digestion was performed according to the methodology
described by HAVASIOVÁ-REITEROVÁ et al. (1995), with modifications,
for the detection of larvae in the liver, lungs, heart, kidneys, eyes, and
skeletal muscles. The organs were macerated, added to a solution of
0.2% pepsin and 0.26% hydrochloric acid in Milli-Q water, and kept
in an incubator shaker of 37 ºC with constant agitation overnight. The
material was then centrifuged at 2000 × g for four minutes, and the pellet
was examined under microscope at (100×) for larvae recovery from the
organs and skeletal muscles of mice. To investigate the central nervous
(1) Post-Graduate Program in Health Sciences - Parasitology Laboratory, Universidade Federal do Rio Grande (FURG), Academic Area of the University Hospital, FURG.
(2) Post-Graduate Program in Parasitology, Universidade Federal de Pelotas (UFPEL). Pelotas, RS, Brazil.
(3) Parasitology Laboratory, Universidade Federal do Rio Grande, FURG. Rio Grande, RS, Brazil.
Correspondence to: Paula de Lima Telmo. Rua General Osório S/N, 96200-190, Centro, Rio Grande, RS, Brazil. E-mail: [email protected]
TELMO, P.L.; AVILA, L.F.C.; SANTOS, C.A.; AGUIAR, P.S.; MARTINS, L.H.R.; BERNE, M.E.A. & SCAINI, C.J. - Elevated trans-mammary transmission of Toxocara canis larvae in
BALB/c mice. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 85-7, 2015.
system infection, brain fragments from the offspring were compressed
between glass slides (optical microscopy) (100×). Maternal infection
was confirmed by the identification of larvae in the brain using the same
methodology. The occurrence of breast transmission was calculated along
with the frequency. The frequency of breast transmission and the number
of larvae per fragment were calculated.
for serological monitoring in women before and during pregnancy.
However, to better understand the importance of this transmission route,
further studies should be conducted with different stages of infection and
different species of paratenic hosts.
Trans-mammary transmission of T. canis larvae was confirmed in all
litters that were nursed by the experimentally infected females. All the
larvae were recovered from the brain and the parasite was not detected
in other organs examined. Among all the mice nursed by the three
experimentally infected females, 73.9% (17/23) had T. canis larvae in
their brains. The transmission of T. canis to all three litters analyzed was
confirmed; two litters exhibited 100% transmission, whereas the third
litter exhibited 25% transmission (Table 1). Infection was confirmed in
all the lactating animals, and no larvae were recovered from the control
group.
Elevada transmissão transmamária de larvas de Toxocara canis
em camundongos BALB/c
Table 1
Number of T. canis-positive offspring and total larvae recovered from the
brains
Number of
offspring
Positive
offspring
Total larvae
Litter 1
8
8
45
Litter 2
7
7
41
Litter 3
8
2
5
Total
23
17 (73.9%)
91
In recent decades, several studies have been conducted to evaluate
the vertical transmission of T. canis larvae in paratenic hosts11,17,18,19. The
confirmation of T. canis trans-mammary transmission came only a few
years ago with the observation of larvae in the brain of ICR mice nursed
by females that had recently been infected with 300 eggs10. In the present
study, trans-mammary infection was observed in mice at 50 days of age,
demonstrating that the larvae are retained in the host brain during the
chronic phase of the disease.
The accumulation of T. canis larvae in the brain favors the vertical
transmission of the parasite because the larvae may remain viable in this
tissue for months or even years5. This phenomenon is important because
of the physiological immunosuppression that occurs during pregnancy
and lactation12. The increase of T reg cells during pregnancy appears to
play an important role in blocking maternal effector T cells1. Moreover,
the hormonal fluctuation of progesterone and prolactin promotes
attenuation of the inflammatory responses during lactation14. Thus, these
factors could facilitate the transmission of larvae from the female’s brain
to the offspring. However, according to MOR & CARDENAS (2010),
the effects of pregnancy and lactation on the female immunosuppression
are misleading since the immune system is modulated, but not fully
suppressed.
Because congenital T. canis infection is known to occur in humans13,
and high levels of trans-mammary transmission of T. canis larvae have
been observed in experimental models, such as this study, greater
attention should be paid to infection in pregnant women and to the need
86
RESUMO
A toxocaríase é zoonose amplamente difundida e considerada
importante problema de saúde pública. O objetivo deste estudo
foi avaliar a frequência da transmissão transmamária de Toxocara
canis em camundongos BALB/c neonatos amamentados por fêmeas
experimentalmente infectadas com 1.200 ovos logo após o parto. Após
50 dias de idade, foi avaliada a presença de larvas em diferentes órgãos
dos neonatos. A infecção por via transmamária foi confirmada em
73,9% dos camundongos amamentados por fêmeas infectadas. Estes
dados demonstram elevada transmissão transmamária de T. canis e
confirmam a importância desta via de transmissão em hospedeiros
paratênicos.
CONFLICT-OF-INTEREST
None.
ACKNOWLEDGMENTS
Thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for their financial support.
REFERENCES
1. Alijotas-Reig J, Llurba E, Gris JM. Potentiating maternal immune tolerance in pregnancy:
a new challenging role for regulatory T cells. Placenta. 2014;35:241-8.
2.Anderson BC. Warning about potential for congenital neural larva migrans. J Am Vet
Med Assoc. 1996;208:185.
3.Burke TM, Roberson EL. Prenatal and lactational transmission of Toxocara canis and
Ancylostoma caninun: experimental infection of the bitch at midpregnancy and at
parturition. Int J Parasitol. 1985;15:485-90.
4.Da Costa de Avila LF, da Fonseca JS, Dutra GF, de Lima Telmo P, Silva AM, Berne
ME, et al. Evaluation of the immunosuppressive effect of cyclophosphamide and
dexamethasone in mice with visceral toxocariasis. Parasitol Res. 2012;110:443-7.
5.Dunsmore JD, Thompson RCA, Bates IA. The accumulation of Toxocara canis larvae
in the brains of mice. Int J Parasitol. 1983;13:517-21.
6. Fisher M. Toxocara cati: an underestimated zoonotic agent. Trends Parasitol. 2003;19:16770.
7.Glickman LT, Schantz PM. Epidemiology and pathogenesis of zoonotic toxocariasis.
Epidemiol Rev. 1981;3:230-50.
8. Havasiová-Reiterová K, Tomasovicová O, Dubinský P. Effect of various doses of infective
Toxocara canis and Toxocara cati eggs on the humoral response and distribution of
larvae in mice. Parasitol Res. 1995;81:13-7.
9.Hoffmeister B, Glaeser S, Flick H, Pornschlegel S, Suttorp N, Bergmann F. Cerebral
toxocariasis after consumption of raw duck liver. Am J Trop Med Hyg. 2007;76:600-2.
TELMO, P.L.; AVILA, L.F.C.; SANTOS, C.A.; AGUIAR, P.S.; MARTINS, L.H.R.; BERNE, M.E.A. & SCAINI, C.J. - Elevated trans-mammary transmission of Toxocara canis larvae in
BALB/c mice. Rev. Inst. Med. Trop. Sao Paulo, 57(1): 85-7, 2015.
10. Jin Z, Akao N, Ohta N. Prolactin evokes lactational transmission of larvae in mice infected
with Toxocara canis. Parasitol Int. 2008;57:495-8.
17.Oshima T. Influence of pregnancy and lactation on migration of the larvae of Toxocara
canis in mice. J Parasitol. 1961;47:657-60.
11.Lee K, Min HK, Soh CT. Transplacental migration of Toxocara canis larvae in
experimentally infected mice. J Parasitol. 1976;62:460-5.
18. Reiterová K, Tomasovicová O, Dubinský P. Influence of maternal infection on offspring
immune response in murine larval toxocariasis. Parasite Immunol. 2003;25:361-8.
12. Luppi P. How immune mechanisms are affected by pregnancy. Vaccine. 2003;21:3352-7.
19.Schoenardie ER, Scaini CJ, Pepe MS, Borsuk S, de Avila LF, Villela M, et al. Vertical
transmission of Toxocara canis in successive generations of mice. Rev Bras Parasitol
Vet. 2013;22:623-6.
13.Maffrand R, Avila-Vázquez M, Princich D, Alasia P. Toxocariasis ocular congénita en
un recién nacido premáturo. An Pediatr (Barc). 2006;64:595-604.
14.Monasterio N, Vergara E, Morales T. Hormonal influences on neuroimmune responses
in the CNS of females. Front Integr Neurosci. 2014;7:110.
20. Yoshikawa M, Nishiofuku M, Moriya K, Ouji Y, Ishizaka S, Kasahara K, et al. A familial
case of visceral toxocariasis due to consumption of raw bovine liver. Parasitol Int.
2008;57:525-9.
15. Mor G, Cardenas I. The immune system in pregnancy: a unique complexity. Am J Reprod
Immunol. 2010;63:425-33.
Received: 20 February 2014
16.Moura JVLM, Santos SV, Castro JM, Chieffi PP. Estudo experimental acerca da
transmissão vertical de Toxocara cati em Mus musculus. Arq Med Hosp Fac Ci Med
Santa Casa Sao Paulo. 2011;56:138-40.
87
57(1):88, January-February, 2015
http://dx.doi.org/10.1590/S0036-46652015000100014
CORRESPONDENCE
NEUROCYSTICERCOSIS AND AFEBRILE SEIZURE
Sir,
The report on “neurocysticercosis and afebrile seizure3” is very interesting. SAHU et al. noted that “neurocysticercosis should be suspected in
every child presenting with afebrile seizure especially with a radio-imaging supportive diagnosis in tropical developing countries or areas endemic for
teniasis/cysticercosis3”. In fact, neurocysticercosis is a very important tropical neurological infection. This disease can be seen worldwide. The MRI
finding and immune response in neurocysticercosis is very interesting. KISHORE et al. found that “immune response was sub-optimal even in MRI
positive cases and conversely, few MRI negative cases were seropositive1.” Hence, to diagnose, both investigations are necessary1. Furthermore, there
are many concerns on treatment of the neurocysticercosis presenting with afebrile seizure. First, the efficacy of antiparasitic drug might be reduced
due to drug interaction with antiepileptic drug2. To manage the case of neurocysticercosis, searching for possible infestation in other organs of the
patient is suggested since disseminated infestation can be expected. Also, other concomitant parasitic infestation must be searched for.
Viroj WIWANITKIT
Visiting professor, Hainan Medical University, China;
visiting professor, Faculty of Medicine, University of Nis, Serbia;
adjunct professor, Joseph Ayobabalola University, Nigeria;
professor, senior expert, Surin Rajabhat, Thailand.
Correspondence to: Professor Viroj Wiwanitkit
Wiwanitkit House, Bangkhae, Bangkok Thailand 10160.
REFERENCES
1.Kishore J, Mukhopadhyay C, Pradhan S, Ayyagari A, Gupta RK. Neurocysticercosis in clinically suspected and MRI proven cases: evidence of sub-optimal antibody response. Indian J
Pathol Microbiol. 2004;47:290-4.
2.Na-Bangchang K, Vanijanonta S, Karbwang J. Plasma concentrations of praziquantel during the therapy of neurocysticerosis with praziquantel, in the presence of antiepileptics and
dexamethasone. Southeast Asian J Trop Med Public Health. 1995;26:120-3.
3.Sahu PS, Seepana J, Padela S, Sahu AK, Subbarayudu S, Barua A. Neurocysticercosis in children presenting with afebrile seizure: clinical profile, imaging and serodiagnosis. Rev Inst
Med Trop Sao Paulo. 2014;56:253-8.
http://dx.doi.org/10.1590/S0036-46652015000100015
LOOSE AND COMPACT AGGLOMERATES OF 50 NM MICROVESICLES DERIVED FROM
GOLGI AND ENDOPLASMIC RETICULUM MEMBRANES IN PRE- AND IN -APOPTOTIC
MYCOPLASMA INFECTED HELA CELLS: HOST-PARASITE INTERACTIONS
UNDER THE TRANSMISSION ELECTRON MICROSCOPE
São Paulo, November 17, 2014
Dear Editor
The fine structure of apoptotic HeLa cells from cultures contaminated
with mycoplasma in early and in advanced stages of the cell demise
process differs from those so far described in apoptotic cells.
The observed changes are enhanced after exposure of the cells to
staurosporine. At low microscopic magnifications cells that have apparent
normal cytoplasm and nuclei, actually may be harbouring cystic-like
profile(s) of parasitic origin in an altered cytoplasm. The membranes
of the transitional elements of the endoplasmic reticulum (TER) appear
fragmented in irregular branching stripes of the smooth component of
the TER (Fig. 1, white asterisks in L delimited area). The concentration
of the rough endoplasmic reticulum (RER) membranes is less than in
normal HeLa cells. Near to the smooth ER tubule-saccular elements lie
groups of 50 nm microvesicles aside stacked, thin, various sized profiles
of Golgi saccules ( ] ). The 50 nm microvesicles bud off mainly from the
periphery of the stacked Golgi elements (Fig 1 thin arrow heads inside
line U) and also from the extremities of smooth ER tubules (Fig. 1 small
arrows). Small groups of compacted microvesicles are noted in cells still
maintaining normal nuclear appearance (not shown). With the start of
chromatin condensation progressively larger compact microvesicular
clusters are formed. These attain sizes larger (Fig. 2) than those of
the clusters of microvesicles derived from the fragmentation of Golgi
saccules seen in mitotic (LUCOCQ et al., 1989; SESSO et al., 1999) and
in apoptotic (SESSO et al., 1999) cells. Contemporaneously two major
cytoplasmic alterations may be noted in contaminated cells namely when
treated with staurosporine. Occasionally, both deformations appear in
the same cell. One, is progressive cytoplasmic loss by formation at the
cell periphery of blebs that separate from the inner cytoplasm (Figs. 3
and 4) or by localized detachment of sectors of the peripheral cytoplasm
with various forms and sizes (not shown). In some cells, the remainder
thin, cytoplasm with few mitochondria and rough ER profiles surrounds
the nucleus in a ring-like form. Such small cells are noted in heavily
contaminated samples. Some of the cells exhibit sectors of the cytoplasm
with a reticulated appearance. Such net-like regions are composed by
various sized tubular and ellipsoidal, apparently empty profiles. It is
unclear if the smooth membranes that compose these regions with
reticulated aspect, may have derived from the Golgi apparatus. The
shape and size of the empty spaces correspond to those from villus-like
formations seen free close to and emerging from the cell surface. In
contaminated cells namely after staurosporine treatment the free villus like forms are seen sprouting from the cell surface and also free nearby.
Spheroidal 50-100nm (thin arrow in Fig. 5) profiles with inner structure
identical to that of villus-like elements are consistently proximate to the
fake villi. Vestiges of what can be remnants of the villus-simile structures
and/or of the parasite itself are seen in these spaces (Figs. 6 and 7).
It is yet undetermined whether the early structural changes expressed
by foci of assembled microvesicles at the transitional endoplasmic
reticulum-Golgi interface is an exclusive type of membranal alteration
preceding overt apoptosis in mycoplasma infected cells.
All cytoplasmic membrane bound organelles as peroxisomes,
lysosome-endosomes and the Golgi apparatus derive from microvesicles
that bud off from the ER. The ER is also mobilized by promoters
of cellular stress (references in DOLAI & ADAK, 2014). The here
described structural deviation of the ER-Golgi interface from the
normal condition may represent more than only a mycoplasma induced
alteration of the programmed cell death mechanism. It is speculative,
whether this initial accumulation of microvesicles in cells with
typical normal nuclei is part of a general forewarning mechanism of
cell defence. In a less intense cell stress than that occurring here the
observed changes of the TER could eventually pass undetected under
the transmission electron microscope.
The mycoplasmas fine structures of our samples are identical (NIRPAZ et al., 2002; KORNSPAN et al., 2010;) and similar (EDWARDS
& FOGH, 1960; HUMMELER et al., 1965; TAYLOR-ROBINSON et
al., 1991) to those from various mycoplasmas strains seen in cultures
and in infected cells.
Antonio SESSO(1),
Edite Hatsumi YAMASHIRO-KANASHIRO(2)
Noemia Mie ORII(3)
Noemi Nosomi TANIWAKI(4)
Joyce KAWAKAMI(5)
Sylvia Mendes CARNEIRO(6)
(1) Laboratório de Imunopatologia, Instituto de Medicina Tropical
(IMT) de São Paulo
(2) Laboratório de Soroepidemiologia do Instituto de Medicina
Tropical de São Paulo
(3) Laboratório de Investigação em Dermatologia e Imunodeficiência
IMT de São Paulo
(4) Laboratório de Microscopia Eletrônica, Instituto Adolfo Lutz de
São Paulo
(5) Setor de Estudo da Inflamação, Instituto do Coração da
Universidade de São Paulo
(6) Laboratório de Biologia Celular, Instituto Butantan de São Paulo
Sponsored by FAPESP (Proc. 2013/22.816-2)
Correspondence to: Prof. Antonio Sesso,
Lab. Imunopatologia, Inst. Med. Tropical de S.Paulo,
Av. Dr. Enéas de Carvalho Aguiar 470, 05403-000 São Paulo, SP, Brasil
SESSO, A.; YAMASHIRO-KANASHIRO, E.H.; ORII, N.M.; TANIWAKI, N.N.; KAWAKAMI, J. & CARNEIRO, S.M. - Loose and compact agglomerates of 50 nm microvesicles derived
from Golgi and endoplasmic reticulum membranes in pre- and in -apoptotic mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope.
Rev. Inst. Med. Trop. Sao Paulo, 57(1): 89-91, 2015.
Figs. 1-7 are a synopsis of fine structural observations in some 50 samples of staurosporine (0.1 µM - 2.0 µM for 2 - 24h) and 10 control samples of HeLa cell cultures contaminated with
mycoplasma. Fig. 1 is from an apparent normal cell at low microscopic exam. Figs 2, 6 and 7 and 3 and 4 are from apoptotic cells exposed to staurosporine 0.5 µM/3h and 0.1 µM/6h, respectively.
Fig. 5 is from a non apoptotic cell exposed to staurosporine 0.1 µM/4h. Bars -1 µM. Fig. 1 - HeLa cell-from a heavily contaminated cell culture devoid of staurosporine where apoptosis occurred.
Normal nucleus (N). Smooth, branched ER profiles (extremities of thin white lines departing from the central white asterisks in the area delimited by the lower (L) dotted line. Conglomerates
of 50 nm (lower and upper right part of the figure). Part of the microvesicles bud off from smooth ER membranes (arrow head) and from tubular elements (small arrows). The majority of
90
SESSO, A.; YAMASHIRO-KANASHIRO, E.H.; ORII, N.M.; TANIWAKI, N.N.; KAWAKAMI, J. & CARNEIRO, S.M. - Loose and compact agglomerates of 50 nm microvesicles derived
from Golgi and endoplasmic reticulum membranes in pre- and in -apoptotic mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope.
Rev. Inst. Med. Trop. Sao Paulo, 57(1): 89-91, 2015.
the grouped microvesicles pinch off from the extremities of stacked Golgi saccules (thin arrow heads at the periphery of the frontally exposed dense ellipsoidal-like profile delimited by U)].
Stacked, thin Golgi saccular profiles of various lengths that often appear dense ( ] ). Fig. 2 - Large, compact conglomeration of various sized microvesicles with predominance of the ones with
50 nm. Groups of former dilated stacked Golgi saccules (black asterisks) are interspersed among the microvesicles. A large such dilated saccule contains remnants of a parasite or of material
of parasitic origin (MPO) (empty triangle). Fig. 3 - The cytoplasm of this apoptotic cell is delimited in two concentric major regions. The peripheral one is partitioned into adjacent blebs
containing predominantly membranes of the ER. The inner part of the cytoplasm contains clustered, swollen mitochondria. Fig. 4 - Dismantling of the peripheral apoptotic cytoplasm by a
contemporaneous detachment of the previously formed bleb regions. Fig. 5 - Non apoptotic infected (cysts of parasitic origin, upper arrows) cell, with an abnormally elongated thin cytoplasm.
The free villus-like structures often appear curved aside spheroidal 50-100 nm (thin arrow) elements. They assemble in various degrees in the sectors of the contaminated cells apoptotic or not
undergoing progressive dismantling of the peripheral cytoplasm. Fig. 6 - Most of the cytoplasm above the apoptotic nucleus is occupied by membrane bound void spaces of various sizes and
forms. The mitochondria are unusually dense, a common occurrence in contaminated cells, apoptotic or not. Adherent to the limiting membrane of the space indicated (open triangle) a fluffy
material possibly derived from MPO. Fig. 7 - Sector of an apoptotic cytoplasm with net-like membranal arrangement as in Fig. 6. Compact mass of parasitic material in a cystic-like form
(open triangle). The upper, middle and vertical white lines indicate remnants of parasitic origin that were not completely removed by the processing of the cells. Part of an elongate membrane
bound space possibly still fully occupied by MPO is indicated by the lower line that branches in two.
REFERENCES
1. Dolai S, Adak S. Endoplasmic reticulum stress responses in Leishmania. Mol Biochem Parasitol. 2014;197(1-2):1-8.
2. Edwards GA, Fogh J. Fine structure of pleuropneumonia-like organisms in pure culture and in infected tissue culture cells. J Bacteriol. 1960;79:267-76.
3. Hummeler K, Armstrong D, Tomassini N. Cytopathogenic mycoplasmas associated with two human tumors. II. Morphological aspects. J Bacteriol. 1965;90;511-6.
4. Kornspan JD, Tarshis M, Rottem S. Invasion of melanoma cells by Mycoplasma hyorhinis: enhancement by protease treatment. Infect Immun. 2010;78:611-7.
5. Lucocq JM, Berger EG, Warren G. Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J Cell Biol. 1989;109;463-74.
6. Nir-Paz R, Prévost MC, Nicolas P, Blanchard A, Wróblewski H. Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane-active peptides and enrofloxacin
in human tissue cell cultures. Antimicrob Agents Chemother. 2002;46:1218-25.
7. Sesso A, Fujiwara DT, Jaeger M, Jaeger R, Li TC, Monteiro MM, et al. Structural elements common to mitosis and apoptosis. Tissue Cell. 1999;31:357-71.
8. Taylor-Robinson D, Davies HA, Sarathchandra P, Furr PM. Intracellular location of mycoplasmas in cultured cells demonstrated by immunocytochemistry and electron microscopy.
Int J Exp Pathol. 1991;72:705-14.
91
57(1):92, January-February, 2015
http://dx.doi.org/10.1590/S0036-46652015000100016
INFLUENZA VIRUS SURVEILLANCE BY THE INSTITUTO ADOLFO LUTZ,
INFLUENZA SEASON 2014: ANTIVIRAL RESISTANCE
São Paulo, August 4, 2014
Dear Sir,
Neuraminidase (NA) inhibitors (NAIs) are the only antivirals that
are effective for prophylaxis and the treatment of seasonal influenza
A and B infections. There are currently two NAIs approved in most
countries: oseltamivir (Tamiflu; F. Hoffmann - La Roche) and zanamivir
(Relenza; GlaxoSmithKline plc.). The development of drug resistance
is a major drawback for any antiviral therapy, and these specific antiinfluenza drugs are not excluded from this rule. Thus, the proper use of
NAIs and worldwide monitoring for the presence and spread of drug
resistant influenza viruses are of the utmost importance. The existence
of a global surveillance network for influenza, underpinning vaccine
strain selection, is a valuable asset when seeking to track the emergence
of antiviral resistance.
The Instituto Adolfo Lutz, São Paulo, SP, Brazil, plays a role
in national and global influenza surveillance. The objective of the
present study was to monitor antiviral resistance to assist public
health authorities with decisions regarding prophylaxis and treatment
strategies.
Using the real time polymerase chain reaction assay (rRT-PCR),
influenza viruses of type A, subtype H1N1pdm09 and H3N2, as well
as type B viruses, were identified and antiviral resistance testing
was conducted using pyrosequencing2 and Sanger dideoxy sequence
analysis5.
Prior to the emergence of the pandemic virus in 2009, the
presence of the oseltamivir resistance-conferring marker, H275Y, was
identified in seasonal influenza A (H1N1). In 2014, influenza virus
surveillance identified the same marker, H275Y, in an influenza A
(H1N1) pdm09 strain isolated from a 20 year-old pregnant woman
living in Mato Grosso/Cuiabá, the Midwest region of Brazil. The virus
was collected in March 2014. In addition, two permissive secondary
NA mutations; V241I and N369K were detected in the virus isolated
in the Midwest region of Brazil1. These mutations are known to
negate the impact of the NA H275Y oseltamivir resistance mutation
on viral replicative fitness. This patient was treated with oseltamivir,
rocephin, azithromycin and made a full recovery from the respiratory
disease.
The choice of assay for assessing the susceptibility of the influenza
virus to NAIs depends on factors pertaining to appropriateness of the
setting, cost, sustainability, speed in obtaining valid results, reliability
in terms of predictive values, and accessibility. The high sensitivity
of genotypic assays allows for testing of clinical specimens, thus
eliminating the need for virus propagation in cell culture. In addition,
rapid genotypic testing facilitates more appropriate patient management
and can significantly advance and assist in large-scale epidemiological
studies of drug-resistant variants4.
Katia Corrêa de Oliveira SANTOS (1)
Daniela Bernardes Borges da SILVA (1)
Margarete Aparecida BENEGA(1)
Renato de Sousa PAULINO(1)
Elian Reis E SILVA Jr(1)
Dejanira dos Santos PEREIRA(2)
Aparecida Duarte Hg MUSSI(2)
Valéria Cristina da SILVA(1,3)
Larissa V. GUBAREVA(4)
Terezinha Maria de PAIVA(1)
(1) Laboratory of Respiratory Viruses NIC/WHO,
Instituto Adolfo Lutz, São Paulo, SP, Brazil.
(2) Central Laboratory of Public Health of the
Mato Grosso State, Cuiabá, Brazil
(3) Epidemiologic Surveillance Center of the
Mato Grosso State, Cuiabá, Brazil.
(4) Molecular Epidemiology Team Virus Surveillance
and Diagnosis Branch Influenza Division,
Centers for Disease Control and Prevention, Atlanta, GA, USA
Correspondence to: Terezinha M. de Paiva, Instituto Adolfo Lutz,
Nucleo de Doencas Respiratorias
Av. Dr. Arnaldo 355, 01246-902 Sao Paulo, SP, Brasil
Phone: 55 11 30682913, Fax: 55 11 30853505
REFERENCES
1. Butler J, Hooper KA, Petrie S, Lee R, Maurer-Stroh S, Reh L, et al. Estimating
the fitness advantage conferred by permissive neuraminidase mutations in
recent oseltamivir - resistant A (H1N1) pdm09 influenza viruses. PLOS Pathog.
2014;10(4):e1004065.
2. Deyde VM, Gubareva LV. Influenza genome analysis using pyrosequencing method:
current applications for a moving target. Expert Rev Mol Diagn. 2009;9:493-509.
3. Marx C, Gregianini TS, Lehmann FK, Lunge VR, Carli SD, Dambrós BP, et al.
Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil. Mem Inst
Oswaldo Cruz. 2013;108:392-4.
4. Okomo-Adhiambo M, Sheu TG, Gubareva LV. Assay for monitoring susceptibility
of influenza viruses to neuraminidase inhibitors. Influenza Other Respir Viruses.
2013;7(Suppl 1):44-9.
5. Sheu TG, Deyde VM, Okomo-Adhiambo M, Garten RJ, Xu X, Bright RA, et al.
Surveillance for neuraminidase inhibitor resistance among human influenza A and
B viruses circulating worldwide from 2004 to 2008. Antimicrob Agents Chemother.
2008;52:3284-92.
6. Souza TM, Resende PC, Fintelman-Rodrigues N, Gregianini TS, Ikuta N, Fernandes
SB, et al. Detection of oseltamivir-resistant pandemic influenza A(H1N1)pdm2009
in Brazil: can community transmission be ruled out? PLOS One. 2013;8(11):e80081.

1 - Instituto de Medicina Tropical de São Paulo

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