3 - 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
EDITORS
EMERITUS EDITORS
Prof. Dr. Thales F. de Brito Prof. Dr. Luis Rey (Founding Editor)
Prof. Dr. Thelma S. Okay
Prof. Dr. Carlos da Silva Lacaz
Associate Editor: Prof. Dr. Pedro Paulo Chieffi EDITORIAL BOARD
Alan L. de Melo (Belo Horizonte, MG)
Alberto Duarte (S. Paulo, SP)
Angela Restrepo M. (Medellin, Colombia)
Anna Sara S. Levin (S. Paulo, SP)
Antonio A. Barone (S. Paulo, SP)
Antonio Carlos Nicodemo (S. Paulo, SP)
Antonio Sesso (S. Paulo, SP)
Antonio W. Ferreira (S. Paulo, SP)
Barnett L. Cline (New Orleans, USA)
Carlos F. S. Amaral (Belo Horizonte, MG)
Celso Granato (S. Paulo, SP)
Cesar A. Cuba Cuba (Brasília, DF)
César Naquira V. (Lima, Peru)
Clarisse M. Machado (S. Paulo, SP)
Claudio S. Pannuti (S. Paulo, SP)
Dalton L. F. Alves (Belo Horizonte, MG)
Eridan Coutinho (Recife, PE)
Ernesto Hofer (Rio de Janeiro, RJ)
Euclides A. Castilho (S. Paulo, SP)
Eufrosina S. Umezawa (S. Paulo, SP)
Expedito J. A. Luna (S. Paulo, SP)
Fan Hui Wen (S. Paulo, SP)
Fernando A. Corrêa (S. Paulo, SP)
Fernando Montero‑Gei (San José, Costa Rica)
Flair J. Carrilho (S. Paulo, SP)
Gil Benard (S. Paulo, SP)
Gioconda San-Blas (Caracas, Venezuela)
Govinda Visvesvara (Atlanta, USA)
Heitor F. Andrade Jr. (S. Paulo, SP)
Hiro Goto (S. Paulo, SP)
Ises A. Abrahamsohn (S. Paulo, SP)
João Carlos Pinto Dias (Belo Horizonte, MG)
João Renato Rebello Pinho (Sao Paulo, SP)
José Ângelo A. Lindoso (S. Paulo, SP)
José Eduardo Levi (S. Paulo, SP)
José M. R. Zeitune (Campinas, SP)
Julia Maria Costa-Cruz (Uberlândia, MG)
Julio Litvoc (S. Paulo, SP)
Luiz Carlos Severo (P. Alegre, RS)
Luiz T. M. Figueiredo (Rib. Preto, SP)
Lygia B. Iversson (S. Paulo, SP)
Marcello Fabiano de Franco (S. Paulo, SP)
Marcos Boulos (S. Paulo, SP)
M. A. Shikanai‑Yasuda (S. Paulo, SP)
Maria I. S. Duarte (S. Paulo, SP)
Maria L. Higuchi (S. Paulo, SP)
Mario Mariano (S. Paulo, SP)
Mirian N. Sotto (S. Paulo, SP)
Moisés Goldbaum (S. Paulo, SP)
Moysés Mincis (S. Paulo, SP)
Moysés Sadigursky (Salvador, BA)
Myrthes T. Barros (S. Paulo, SP)
Nilma Cintra Leal (Recife, PE)
Paulo C. Cotrim (São Paulo, SP)
Paulo M. Z. Coelho (Belo Horizonte, MG)
Regina Abdulkader (S. Paulo, SP)
Ricardo Negroni (B. Aires, Argentina)
Robert H. Gilman (Baltimore, USA)
Roberto Martinez (Rib. Preto, SP)
Ronaldo Cesar B. Gryschek (S. Paulo, SP)
Semíramis Guimarães F. Viana (Botucatu, SP)
Silvio Alencar Marques (Botucatu, SP)
Tsutomu Takeuchi (Tokyo, Japan)
Venâncio A. F. Alves (S. Paulo, SP)
Vicente Amato Neto (S. Paulo, SP)
Zilton A. Andrade (Salvador, BA)
Executive Board: Maria do Carmo Berthe Rosa. Librarians: Maria Ângela de Castro Fígaro Pinca; Carlos José Quinteiro
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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.
TO WHOM IT MAY CONCERN
From 2016 on, the REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE
SÃO PAULO (Journal of the São Paulo Institute of Tropical Medicine) will be
published only on line, free access.
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 (1-3)
ISSN 0036-4665
II
Impact Factor: 1.007
5-year Impact Factor: 1.088
ISSN0036-4665
Rev. Inst. Med. Trop. Sao Paulo
Vol. 57
No. 3
P. 185-276
May-June, 2015
CONTENTS
MYCOLOGY
185 Frequency of Candida species in a tertiary care hospital in Triangulo
Mineiro, Minas Gerais State, Brazil
R.P. MENEZES, J.C. FERREIRA, W.M. SÁ, T.A. MOREIRA, L.D.S. MALVINO, L.B.
ARAUJO, D.V.D.B. RÖDER, M.P.A. PENATTI, R.C. CANDIDO & R.S. PEDROSO
BACTERIOLOGY
193Raw tropical oysters as vehicles for multidrug-resistant Vibrio
parahaemolyticus
R.A. COSTA, R.L. ARAÚJO & R.H.S.F. VIEIRA
PARASITOLOGY
197 Anthelmintic activity of lapachol, β-lapachone and its derivatives
against Toxocara canis larvae
T. MATA-SANTOS, N.F. PINTO, H.A. MATA-SANTOS, K.G. DE MOURA, P.F.
CARNEIRO, T.S. CARVALHO, K.P. DEL RIO, M.C.F.R. PINTO, L.R. MARTINS,
J.M. FENALTI, P.E.A. DA SILVA & C.J. SCAINI
205 Molecular characterization and sequence phylogenetic analysis of
surface antigen 3 (SAG3) gene of local Indian isolates (Chennai
and Izatnagar) of Toxoplasma gondii
V. SUDAN, A.K. TEWARI & H. SINGH
211 Occurrence of Blastocystis spp. in Uberaba, Minas Gerais, Brazil
M. CABRINE-SANTOS, E.N. CINTRA, R.A. CARMO, G.A.N. NASCENTES, A.L.
PEDROSA, D. CORREIA & M.B. OLIVEIRA-SILVA.
VIROLOGY
215Saint Louis encephalitis virus in Mato Grosso, Central-Western
Brazil
L.B.S. HEINEN, N. ZUCHI, O.P. SERRA, B.F. CARDOSO, B.H.F. GONDIM,
M.A.M. SANTOS, F.J.D. SOUTO, D.A.J. PAULA, V. DUTRA & R. DEZENGRINISLHESSARENKO
221Lack of association between herpesvirus detection in saliva and
gingivitis in HIV-infected children
R.A. OTERO, F.N.N. NASCIMENTO, I.P.R. SOUZA, R.C. SILVA, R.S. LIMA, T.F.
ROBAINA, F.P. CÂMARA, N. SANTOS & G.F. CASTRO
ENTOMOLOGY
227 Inventory of mosquitoes (Diptera: Culicidae) in conservation units
in Brazilian tropical dry forests
C.F. SANTOS, A.C. SILVA, R.A. RODRIGUES, J.S.R. JESUS & M.A.Z. BORGES
233 Phlebotomine fauna (Diptera: Psychodidae) in an area of fishing
tourism in Central-Western Brazil
A.F. BRILHANTE, M.E.M.C. DORVAL, E.A.B. GALATI, H.C. ROCHA, G.
CRISTALDO & V.L.B. NUNES
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]
EPIDEMIOLOGY
239Head lice in hair samples from youths, adults and the elderly in
Manaus, Amazonas State, Brazil
S.C.B. NUNES, R.B. MORONI, J. MENDES, S.C.B. JUSTINIANO & F.T. MORONI
CHAGAS DISEASE
245 Effects of vitamin C supplementation on the chronic phase of Chagas
disease
R.G. MARIM, A.S. GUSMÃO, R.E.P. CASTANHO, R. DEMINICE, A.L.S. THEREZO,
A.A. JORDÃO JÚNIOR, M.R. ASSIS, E.F. TAIPEIRO & L.P.A. MARTINS
NOCARDIOSIS
251 Molecular identification and antimicrobial resistance pattern of
seven clinical isolates of Nocardia spp. in Brazil
L.A.Z. CONDAS, M.G. RIBEIRO, M.D. MURO, A.P.C. VARGAS, T. MATSUZAWA,
K. YAZAWA, A.K. SIQUEIRA, T. SALERNO, G.H.B. LARA, R.M. RISSETI, K.S.
FERREIRA & T. GONOI
LEISHMANIASIS
257Genotype characterization of Leishmania (Viannia) braziliensis
isolated from human and canine biopsies with American cutaneous
leishmaniasis
L.T. FERREIRA, A.H.S. GOMES & V.L. PEREIRA-CHIOCCOLA
MALARIA
263 Seasonal distribution of malaria vectors (Diptera: Culicidae) in rural
localities of Porto Velho, Rondonia, Brazilian Amazon
L.H.S. GIL, M.S. RODRIGUES, A.A. LIMA & T.H. KATSURAGAWA
BRIEF COMMUNICATION
269Chicken coops, Triatoma dimidiata infestation and its infection
with Trypanosoma cruzi in a rural village of Yucatan, Mexico
E. KOYOC-CARDEÑA, A. MEDINA-BARREIRO,, F.J. ESCOBEDO-ORTEGÓN,
J.C. RODRÍGUEZ-BUENFIL, M. BARRERA-PÉREZ, E. REYES-NOVELO,
J. CHABLÉ-SANTOS, C. SELEM-SALAS, G. VAZQUEZ-PROKOPEC & P.
MANRIQUE-SAIDE
CASE REPORT
273 Tuberculosis infection might increase the risk of invasive candidiasis
in an immunocompetent patient
X.-H. CHEN, Y.-C. GAO, Y. ZHANG, Z.-H. TANG, Y.-S. YU & G.-Q. ZANG.
LETTER TO THE EDITOR
276 West Nile fever in Brazil: sporadic case, silent endemic disease or
epidemic in its initial stages?
M.A.C.S.VIEIRA, A.A.X. AGUIAR, A.S.BORBA, H.C.L. GUIMARÃES, K.D.
EULÁLIO, L.L. ALBUQUERQUE-NETO, M.A. SALMITO & O.B.LIMA
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. 3
P. 185-276
Maio-Junho, 2015
CONTEÚDO
MICOLOGIA
185 Frequência de espécies de Candida em hospital terciário do Triângulo Mineiro, Minas Gerais, Brasil
R.P. MENEZES, J.C. FERREIRA, W.M. SÁ, T.A. MOREIRA, L.D.S. MALVINO, L.B.
ARAUJO, D.V.D.B. RÖDER, M.P.A. PENATTI, R.C. CANDIDO & R.S. PEDROSO
239 Pediculose da cabeça em amostras de cabelos de jovens, adultos e
idosos em Manaus, estado do Amazonas, Brasil
S.C.B. NUNES, R.B. MORONI, J. MENDES, S.C.B. JUSTINIANO & F.T. MORONI
DOENÇA DE CHAGAS
BACTERIOLOGIA
193Ostras tropicais cruas como fonte de Vibrio parahaemolyticus
multirresistentes
R.A. COSTA, R.L. ARAÚJO & R.H.S.F. VIEIRA
PARASITOLOGIA
197 Atividade anti-helmíntica do lapachol, β-lapachona e derivados
contra larvas de Toxocara canis
T. MATA-SANTOS, N.F. PINTO, H.A. MATA-SANTOS, K.G. DE MOURA, P.F.
CARNEIRO, T.S. CARVALHO, K.P. DEL RIO, M.C.F.R. PINTO, L.R. MARTINS,
J.M. FENALTI, P.E.A. DA SILVA & C.J. SCAINI
205 Caracterização molecular e análise filogenética de sequências do
antígeno de superfície 3 (SAG3) em isolados indianos (Chennai e
Izatnagar) de Toxoplasma gondii
V. SUDAN, A.K. TEWARI & H. SINGH
245 Efeitos da suplementação de vitamina C na fase crônica da doença
de Chagas
R.G. MARIM, A.S. GUSMÃO, R.E.P. CASTANHO, R. DEMINICE, A.L.S. THEREZO,
A.A. JORDÃO JÚNIOR, M.R. ASSIS, E.F. TAIPEIRO & L.P.A. MARTINS
NOCARDIOSE
251 Identificação molecular e perfil de sensibilidade a antimicrobianos
de sete isolados clínicos de Nocardia spp. no Brasil
L.A.Z. CONDAS, M.G. RIBEIRO, M.D. MURO, A.P.C. VARGAS, T. MATSUZAWA,
K. YAZAWA, A.K. SIQUEIRA, T. SALERNO, G.H.B. LARA, R.M. RISSETI, K.S.
FERREIRA & T. GONOI
LEISHMANIOSE
257Caracterização genotípica de isolados de Leishmania (Viannia)
braziliensis provenientes de biopsias de humanos e cães com
leishmaniose tegumentar americana
L.T. FERREIRA, A.H.S. GOMES & V.L. PEREIRA-CHIOCCOLA
211 Ocorrência de Blastocystis spp. em Uberaba, Minas Gerais, Brasil
M. CABRINE-SANTOS, E.N. CINTRA, R.A. CARMO, G.A.N. NASCENTES, A.L.
PEDROSA, D. CORREIA & M.B. OLIVEIRA-SILVA
VIROLOGIA
215 Vírus da encefalite de Saint Louis em Mato Grosso, Centro-Oeste,
Brasil
L.B.S. HEINEN, N. ZUCHI, O.P. SERRA, B.F. CARDOSO, B.H.F. GONDIM,
M.A.M. SANTOS, F.J.D. SOUTO, D.A.J. PAULA, V. DUTRA & R. DEZENGRINISLHESSARENKO
221 Ausência de associação entre a detecção de herpesvírus na saliva
e gengivite em crianças infectadas pelo HIV
R.A. OTERO, F.N.N. NASCIMENTO, I.P.R. SOUZA, R.C. SILVA, R.S. LIMA, T.F.
ROBAINA, F.P. CÂMARA, N. SANTOS & G.F. CASTRO
ENTOMOLOGIA
227 Inventário de mosquitos (Diptera: Culicidae) em unidades de conservação em florestas tropicais secas brasileiras
C.F. SANTOS, A.C. SILVA, R.A. RODRIGUES, J.S.R. JESUS & M.A.Z. BORGES
233 Fauna flebotomínea (Diptera: Psychodidae) em área de turismo
pesqueiro no Centro-Oeste do Brasil
A.F. BRILHANTE, M.E.M.C. DORVAL, E.A.B. GALATI, H.C. ROCHA, G.
CRISTALDO & V.L.B. NUNES
IV
EPIDEMIOLOGIA
MALARIA
263 Distribuição sazonal de vetores da malária (Diptera: Culicidae) em
localidades rurais de Porto Velho, Rondônia, Amazônia Brasileira
L.H.S. GIL, M.S. RODRIGUES, A.A. LIMA & T.H. KATSURAGAWA
COMUNICAÇÃO BREVE
269Gallineros, la infestación por Triatoma dimidiata y su infección
con Trypanosoma cruzi en una localidad rural de Yucatán, México
E. KOYOC-CARDEÑA, A. MEDINA-BARREIRO,, F.J. ESCOBEDO-ORTEGÓN,
J.C. RODRÍGUEZ-BUENFIL, M. BARRERA-PÉREZ, E. REYES-NOVELO,
J. CHABLÉ-SANTOS, C. SELEM-SALAS, G. VAZQUEZ-PROKOPEC & P.
MANRIQUE-SAIDE
RELATO DE CASO
273Tuberculose pode aumentar o risco de candidíase invasiva em
paciente imunocompetente
X.-H. CHEN, Y.-C. GAO, Y. ZHANG, Z.-H. TANG, Y.-S. YU & G.-Q. ZANG.
CARTA AO EDITOR
276 West Nile fever in Brazil: sporadic case, silent endemic disease or
epidemic in its initial stages?
M.A.C.S.VIEIRA, A.A.X. AGUIAR, A.S.BORBA, H.C.L. GUIMARÃES, K.D.
EULÁLIO, L.L. ALBUQUERQUE-NETO, M.A. SALMITO & O.B.LIMA
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(3):185-191, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300001
FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO,
MINAS GERAIS STATE, BRAZIL
Ralciane de Paula MENEZES(1), Joseane Cristina FERREIRA(2), Walkiria Machado de SÁ(3), Tomaz de Aquino MOREIRA(3), Lucivânia Duarte Silva MALVINO(3),
Lucio Borges de ARAUJO(4), Denise Von Dolinger de Brito RÖDER(1,5), Mario Paulo Amante PENATTI(6), Regina Celia CANDIDO(2) & Reginaldo dos Santos PEDROSO(1,6)
SUMMARY
Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients.
The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated
from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures,
abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of
Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The
RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%)
were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%)
C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates.
In vitro resistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish
different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the
OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the
C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B,
flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex
species, mainly with the OPA09 marker.
KEYWORDS: Antifungal susceptibility; Candida species; Candidemia; Genotyping.
INTRODUCTION
In recent decades, candidemia has increased significantly worldwide
due to increased lifespans of immunosuppressed patients or transplant and
HIV/AIDS patients7,10,15,44. In many countries, the invasive infection of
Candida yeast is a considerable public health problem, due to its severity,
cause of increased hospital stays, cost, and contribution to high indexes
of morbimortality. Some reports note that the mortality index caused
by candidemia may reach 40-60% of hospital-admitted patients10,18,39,48.
reported to be predominant, especially in hospital environments28,39,44.
According to some investigators, this is due to the selective pressure
from the prophylactic use of fluconazole in patients at risk of developing
invasive fungal infections18,41. Variable frequencies of different species
of Candida are identified depending on the hospital complexity and/or
geographic region13.
Invasive candidiasis is related to several factors that compromise
patient conditions, such as neutropenia, organ transplantations, previous
colonization by Candida species, prolonged use of antibiotics, presence
of catheters for nasogastric feeding, use of urinary or parenteral probes for
hemodialysis or mechanical ventilation, neoplasia, immunosuppressive
diseases, drugs, and gastrointestinal surgeries30.
The choice of treatment for candidemia or invasive candidiasis is
mainly based on two factors: Candida species and the condition of the
host immune system. Depending on the protocol of the institution and
the availability of antifungal agents, azoles (fluconazole, voriconazole,
and posaconazole), polyene (amphotericin B), and/or echinocandins
(caspofungin, anidulafungin, and micafungin) are used for the treatment.
Echinocandins are recommended for prophylaxia and for the treatment
of different groups of patients due to their efficacy and low toxicity in
critical patients compared to other azoles and amphotericin B11,31,49.
For many years, C. albicans was regarded as the main cause of
invasive fungal infections, but lately, non-C. albicans species have been
Candidiasis epidemiology has been studied by genotypic analysis,
which employs molecular tools with high discriminating power to
(1) Post-Graduation Program, FAMED, Federal University of Uberlândia (UFU), Uberlândia, Minas Gerais, Brazil.
(2) Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil.
(3) Clinical Hospital of Uberlândia, UFU, Uberlândia, Minas Gerais, Brazil.
(4) Faculty of Mathematics, UFU, Uberlândia, Minas Gerais, Brazil.
(5) Institute of Biomedical Sciences, UFU, Uberlândia, Minas Gerais, Brazil.
(6) Technical School of Health, UFU, Uberlândia, Minas Gerais, Brazil.
Correspondence to: Reginaldo dos Santos Pedroso, Av. Amazonas s/nº, Block 4K, Campus Umuarama, 38400-902 Uberlandia, MG, Brasil. Phone: +55 (34) 3225-8459.
E-mail: [email protected]
MENEZES R.P.; FERREIRA J.C.; SÁ W.M.; MOREIRA, T.A.; MALVINO, L.D.S.; ARAUJO, L.B.; RÖDER, D.V.D.B.; PENATTI, M.P.A.; CANDIDO, R.C. & PEDROSO, R.S. - Frequency
of Candida species in a tertiary care hospital in Triangulo Mineiro, Minas Gerais State, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 185-91, 2015.
distinguish different isolates, and thus allowing for improved accuracy
in clinical and epidemiological studies29,34. These studies attempt to
relate the genotypes of isolates with pathogenicity and epidemiology.
Genotypes with varying degrees of heterogeneity were found in different
anatomical sites among various population groups, including patients and
healthy individuals, and in different geographical areas4,33,37.
The most commonly used molecular methods include polymorphism
detection in the length of restriction fragments (RFLP) with hybridization
(Southern blot) or amplification (AFLP), karyotyping in pulsed-field
gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and
other techniques based on polymerase chain reaction (PCR) of random
amplified polymorphic DNA (RAPD-PCR)2,29,34.
Regional peculiarities and hospital complexity services may influence
the predominance of Candida species. These emphasize the need for
studies on epidemiology, prevalence, and resistance to antifungals.
This study aims to evaluate the frequency, in addition to testing the
susceptibility to antifungals as well as genetic polymorphisms, of
Candida species isolated from samples of blood, CVC, and abdominal
fluids of hospitalized patients in a tertiary hospital in the Triangulo
Mineiro region, Minas Gerais State, Brazil.
MATERIAL AND METHODS
Isolates in the study: Candida samples included in the study were
obtained from patients admitted to the Clinical Hospital of the Federal
University of Uberlândia (UFU) in the city of Uberlândia located in
the Triangulo Mineiro region, Minas Gerais State, Brazil, during the
period of July 2010-June 2011. The isolates were from blood cultures,
CVC, and abdominal fluids. Chromogenic agar (BD CHROMagar®
Candida, France) and Sabouraud dextrose agar were used to isolate
the yeasts, which were identified by classical methods21 and confirmed
by the Auxacolor2® system (Bio-Rad, France). Candida albicans and
C. dubliniensis were differentiated by PCR utilizing specific primers,
according to the technique described by ESTRADA-BARRAZA et al.14.
Samples were stored in BHI-glycerol broth at -20 oC. Experiments were
conducted after sample activation and incubation at 35 oC for 24-48 h.
Antifungal susceptibility tests: The broth microdilution method
described in document M27-A3, Clinical Laboratory Standard Institute
(CLSI)8, was used for the tests. Antifungals amphotericin B (Fungizon,
Bristol Myers Squibb, Brazil), fluconazole (Pfizer, Sandwich, UK),
and itraconazole (Janssen, Beerse, Belgium) were tested in culture
plates of RPMI-1640 medium containing glutamine, without sodium
bicarbonate, and buffered using pH-7.0 MOPS with glucose (18g/L).
The final concentrations of the antifungal agents were 0.03-16 µg/mL
for amphotericin B and itraconazole and 0.25-64 µg/mL for fluconazole.
Briefly, yeasts were inoculated in Sabouraud dextrose agar and incubated
at 35 oC for 24 h. Culture suspensions adjusted to 1-5×106cells/mL were
prepared in sterilized saline. Susceptibility tests were made in duplicates
and the microdilution plates were incubated at 35 oC for 48 h. Control
strains were C. parapsilosis ATCC 22019 and C. krusei ATCC 6258.
The minimum inhibitory concentration (MIC) was determined visually.
For the azoles, MICs corresponded to the concentration inhibiting
around 50% of growth for each microorganism compared to the control
well (without antifungal); for amphotericin B, the MIC was the smaller
drug concentration that inhibited 100% of yeast growth7. For azoles,
186
breakpoints were as indicated in CLSI8,43, and, for amphotericin B, due to
lack of consensus, the values suggested by NGUYEN et al.29 were used.
Molecular typing: DNA extraction was performed according to
the method of BOLANO et al.4. The RAPD-PCR tests were performed
with initiator oligonucleotides OPA09 (5’GGGTAACGCC3’), OPB11
(5’GTAGACCCGT3’), and OPE06 (5’AAGACCCCTC3’) (Invitrogen,
São Paulo, Brazil). The reaction final volume was 25 µL and contained
2 µL DNA (60 ng/mL), 0.25 mmol of each deoxynucleotide (dATP,
dCTP, dTTP, and dGTP) (Invitrogen), 1U Taq polymerase (Invitrogen),
2.5mM MgCl2, and 2.5mM initiator nucleotide. All amplifications
were conducted in a thermalcycler (Eppendorf, Mastercycle Gradient,
USA), consisting of an initial amplification cycle of four min at 92 oC
followed by 40 cycles of 40 s at 92 oC, 40 oC for 1.5 min, and 72 oC
for two min, and finally followed by five min at 72oC. Amplification
fragments were separated by agarose gel (1.4%) electrophoresis for
three h at 80V and 100mA. The gels were stained with ethidium bromide
and visualized under UV light and the images were captured by a
photo documentation system. Profiles for each sample were analyzed
visually, and bands were classified as present (1) or absent (0). Genetic
relationships (similarity coefficients) were calculated by the Jaccard
coefficient equation (Sj) based on the position of fragments using the
equation Sj = nAB/(nAB+a+b),where nAB is the number of bands shared
by two samples: a, the number of exclusive bands for the first sample
and b, for the second sample40. Values of Sj from 0.99-1.00 represent
the same genotype, values from 0.800-0.99 represent clonally related
samples (strongly similar but not identical), and values less than
0.800 indicate distinct samples. Dendrograms based on Sj values were
generated for comparison by the unweighted pair group method with
the arithmetical averages (UPGMA) method utilizing the multivariate
statistical package program (MVSP).
Ethical committee: This study was approved by the Ethical
Committee for Human Research of the Federal University of Uberlândia
(UFU) under the number 317/10.
Statistical analysis: Qualitative variables were compared using the
chi-square test, and the G test was used for quantitative results. In both
tests, statistical significance was considered when p < 0.05.
RESULTS
During the study period, 63 cultures of body fluids from individuals
with suspected systemic candidiasis were positive for Candida spp., of
which 47 were in blood, nine were in CVC, and seven in abdominal
fluids, all obtained from 58 hospitalized patients at the Clinical Hospital
of Federal University of Uberlândia. Thirty-four were from males and
24 from females. Ages of the patients ranged from one day to 94 years,
with a mean age of 42 years. Most patients who developed systemic
candidiasis and who had a positive culture were older than or equal to
21 years (Fig. 1).
Of the 63 Candida isolates, 18 (28.5%) were identified as C. albicans
and 45 (72.5%) as non-C. albicans, distributed as follows: 20 (31.7%) C.
parapsilosis complex species; 14 (22.2%) C. tropicalis; four (6.4%) C.
glabrata; four (6.4%) C. krusei; two (3.3%) C. kefyr; and one (1.6%) C.
lusitaniae. Candida dubliniensis was not identified by PCR. Except for
C. albicans (p = 0.050), the distribution of species between males and
Molecular analyses by RAPD-PCR with primers OPA09 and OPB11
produced different molecular profiles. Primer OPE06 did not amplify any
genome fragments of the isolates included in the study. Analysis of the
dendrogram generated from band profiles among isolates of the same
species showed isolate groups with Sj = 1.00 (identical isolates with the
same profile) and Sj < 0.80 (distinct samples). Table 3 shows frequencies
of profiles generated with each primer for the most frequent species. Each
profile relates isolates showing the same genotypes (Sj = 1.00). Candida
parapsilosis complex strains showed five (A-E) and four (A-D) profiles
with OPA09 and OPB11, respectively.
Note: Other species: C. krusei (4); C. glabrata (4); C. lusitaniae (1) and C. kefyr (2).
Fig. 1 - Distribution of Candida spp. according to age of hospitalized patients who developed
systemic candidiasis during the period of July 2010-June 2011.
females was not statistically different for members of the C. parapsilosis
complex (p = 0.057), C. tropicalis (p = 0.4497), and other species (p =
0.2008). However, there was a predominant tendency of C. albicans and
C. parapsilosis complex species to affect males, once 55.5% and 65% of
isolates, respectively, were obtained from male patients.
As shown in Table 1, the highest frequency of Candida isolates was
from blood cultures (55.6%), CVC (14.3%), simultaneous isolations
from blood-CVC (19%) and abdominal fluids (11.1%).
In vitro resistance to amphotericin B was observed in one isolate
of C. albicans, in one of the C. parapsilosis complex, and in six other
species - three C. krusei, two C. glabrata, and one C. kefyr - all with a
MIC of 2 µg/mL. None of the isolates, except C. krusei, was resistant
to azoles in vitro.
Dose-dependent susceptibility to itraconazole was detected in one
isolate of C. glabrata. Table 2 shows the MIC ranges of antifungals tested
against Candida species.
Profile A of the each primer was composed of the higher number
of isolates. Candida albicans isolates presented six and two profiles,
respectively, with primers OPA09 and OPB11 (Table 3). Candida
tropicalis isolates produced only one profile with OPA09 and two
unrelated ones (Sj < 0.8) with OPB11 (A-B) (Table 3). Two C. kefyr
strains were demonstrated to be distinct strains with both primers (Sj <
0.80). Candida krusei showed two profiles with OPA09, each one with
two isolates with similarity indexes that the indicated strains were clonally
related (0.99 > Sj > 0.80); OPB11 produced only one profile, with 100%
similarity among isolates. Candida glabrata produced two profiles with
each one of the primers; OPA09 and OPB11 grouped three isolates in
profile A and another isolate in profile B, with A and B being unrelated
(Sj < 0.80) for both primers.
DISCUSSION
The predominance of Candida species non-C. albicans observed
in this study confirms results reported in other studies from different
Brazilian regions12,23,28. The C. parapsilosis complex occurred at the
highest frequency compared to other species, including C. albicans.
Observations from other Latin American countries and Tunisia show that
C. parapsilosis-induced infections increased significantly in the past two
decades9,22. Candida albicans, C. tropicalis, and C. parapsilosis complex
species are the most frequent species isolated in candidemia cases and
constitute 82.5% as a whole of the isolates in this study and, in some
other instances, represent more than 90% of etiologies30.
Candida parapsilosis has been reported as the second or third most
frequent Candida species in candidemias9-12,15,24,27,35,38. In fact, in 2005,
the C. parapsilosis complex was reclassified into three species: C.
parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis42. These
three species may exhibit, according to some researchers, differences in
Table 1
Frequency of Candida species isolated from clinical specimens of patients from the Clinical Hospital of Federal University of Uberlândia who developed systemic
candidiasis during the period of July 2010-June 2011
Clinical specimens
C. parapsilosis
C. albicans
C. tropicalis
Others*
Total
Blood
11 (17.4%)
9 (14.3%)
10 (15.9%)
5 (7.9%)
35 (55.6%)
CVC
6 (9.5%)
1 (1.6%)
1 (1.6%)
1 (1.6%)
9 (14.3%)
Blood + CVC
3 (4.8%)
5 (7.9%)
2 (3.2%)
2 (3.2%)
12 (19.0%)
Abdominal fluids
0 (0.0%)
3 (4.8%)
1 (1.6%)
3 (4.8%)
7 (11.1%)
20 (31.7%)
18 (28.6%)
14 (22.2%)
11 (17.4%)
63 (100%)
Total isolates
*Other species: C. krusei (4); C. glabrata (4); C. lusitaniae (1); and C. kefyr (2). CVC = central venous catheter.
187
Table 2
In-vitro susceptibility of Candida species to three antifungal agents
Species
(n)
MIC (µg/mL)
Antifungal agents
C. parapsilosis
(20)
C. albicans
(18)
C. tropicalis
(14)
C. krusei
(4)
C. glabrata
(4)
C. kefyr
(2)
C. lusitaniae
(1)
Range
MIC50
MIC90
Resistant
n (%)
Amphotericin B
0.5-2.0
1.0
1.0
1 (5%)
Fluconazole
0.125-1.0
0.5
0.5
0
Itraconazole
0.03-0.125
0.03
0.03
0
Amphotericin B
0.5-2.0
0.5
1.0
1 (5.6%)
Fluconazole
0.125-0.5
0.125
0.5
0
Itraconazole
0.03
0.03
0.03
0
Amphotericin B
0.5-1.0
1.0
1.0
0
0
Fluconazole
0.125-0.5
0.25
0.5
Itraconazole
0.03-0.06
0.03
0.06
0
Amphotericin B
1.0-2.0
-
-
3 (75%)
Fluconazole*
-
-
-
4 (100%)
Itraconazole
0.03-0.12
-
-
0
Amphotericin B
1.0-2.0
-
-
2 (50%)
Fluconazole
0.5-4.0
-
-
0
Itraconazole
0.25-0.3
-
-
0
Amphotericin B
0.5-2.0
-
-
1 (50%)
Fluconazole
0.125-0.5
-
-
0
Itraconazole
0.06-0.125
-
-
0
Amphotericin B
1
-
-
0
Fluconazole
0.25
-
-
0
Itraconazole
0.03
-
-
0
*C. krusei is intrinsically resistant to fluconazole.
Table 3
Frequency of cluster profiles and isolates per cluster with primers
OPA09 and OPB11
OPA09
Species(n)
OPB11
Molecular Frequency of Molecular Frequency of
profile*
isolates
profile*
isolates
Candida
parapsilosis
(20)
A
B
C
D
E
10
3
3
3
1
A
B
C
D
13
4
1
1
7
4
4
1
1
1
A
B
14
4
Candida
albicans (18)
A
B
C
D
E
F
A
14
A
B
13
1
Candida
tropicalis
(14)
*A cluster was considered when it grouped isolates with 100% similarity.
188
patterns of susceptibility to antifungal and biofilm production11. Of all
the Candida isolates, they were detected in 55.6% of samples from blood
cultures, 14.3% from CVC, 11.1% from abdominal fluids, and 19% from
blood and CVC simultaneously. Positive results in blood cultures are
considered the main indicators of invasive infections. Although cultures
of samples obtained from other organic sites may be secondary in the
diagnostics of hospital infection, these Candida isolates may have a
predictive value for the occurrence of candidemias1,50.
Similar to what happened with bacteria, the indiscriminate use
of antifungal drugs has stimulated the occurrence of fungi with
decreased susceptibility or even in vitro resistance, especially
among Candida species6. In this study, the susceptibility of isolates
in relation to fluconazole, itraconazole, and amphotericin B, which
were the antifungals used for treatment of invasive candidiasis in
the service during the period studied, was analyzed. However, recent
studies have pointed primarily to the use of echinocandins26,31,49. Most
isolates were susceptible to the three antifungals evaluated. Candida
krusei and C. glabrata are known to be resistant and less susceptible
to fluconazole, respectively31,32,34,45,49. In vitro resistance of Candida
species, notably non-C. albicans, to fluconazole has been reported in
different hospital studies13,16,31,32,36,39. Itraconazole has been recently
utilized in the treatment of candidemia in neutropenic patients because
it is less toxic than amphotericin B, as well as having shown a similar
effectiveness to that presented by other azoles17. One isolate (25%) of
C. glabrata showed a dose-dependent susceptibility to itraconazole,
while NEUFELD et al.28 reported a dose-dependent susceptibility in
only 3.4% of the isolates in their studies.
Resistance to amphotericin B has not been reported among
isolates of different regions20,27,35,39. In this study, a MIC of 2 µg/mL
was determined for some isolates, especially non-C. albicans ones,
characterizing in vitro resistance. Data on the clinical outcomes of
patients were not generated in this study, as in vitro results do not mean
in vivo resistance, due to the fact that the cut-off point for amphotericin
B is not established by the standardization committee due to technical
difficulties related to the antifungal and culture media, as reported in
the literature8,23,26,28,29. The results should be considered an alert and they
should emphasize the importance of continuous surveillance to detect
occasional isolates that are resistant to one or more antifungals. Future
vigilance studies, including monitoring of patients, on antimicrobial
resistance will show if these results were occasional or common
occurrences.
The genetic variability of clinical isolates has been used to
demonstrate cases of cross infection that occur in health care, but also
to determine if the isolates of one anatomical site are identical to isolates
from other sites of the same patient19,37.
In this study, the RAPD methodology was utilized in an attempt to
reveal molecular variants of Candida spp. Based on the gel patterns and
on the dendrograms obtained (data not shown), six profiles (A-F) were
determined with primer OPA09 while OPB11 allowed only two (A-B)
for isolates of C. albicans. Primer OPA09 had a higher discriminatory
power especially for C. albicans and C. parapsilosis complex (Table
3). Neither of the two primers was able to discriminate isolates of C.
tropicalis. Isolates of other species occurred in small numbers, so it is not
possible to discuss this. Several studies have shown the discriminatory
power of different primers and have suggested the use of multiple primers
to improve the sensitivity of the results25,37,46,47.
This study identified a variety of strains in the patients involved,
especially for isolates C. albicans and C. parapsilosis complex. However,
it was not possible to show a cross infection at all. However, in 12
patients who had blood and CVC, positive cultures were isolated to the
same species, and these exhibited the same genotype when blood and
CVC isolates were compared. This might be evidence of hematological
dissemination of this particular microorganism from the CVC, but also
blood-to-CVC. Identifying the source of infection is an important way
to prevent infection. However, it suggests that prospective studies,
including clinical data of patients and correlating these data with the
microbiological characteristics of isolated samples may provide important
insights for Candida spp. epidemiology in inpatients.
In conclusion, of the Candida species isolated during the study period,
the most frequent were C. parapsilosis complex species followed by C.
albicans and C. tropicalis. Most samples were susceptible to antifungals
fluconazole, itraconazole, and amphotericin B. The genotypic markers
seemed efficient at discriminating the isolates of C. albicans and C.
parapsilosis; high genetic polymorphism was observed for isolates
of C. albicans and C. parapsilosis complex species, mainly with the
OPA09 marker.
RESUMO
Frequência de espécies de Candida em hospital terciário do
Triângulo Mineiro, Minas Gerais, Brasil
As infecções causadas por espécies de Candida são problema
de grande impacto para a saúde pública, devido à alta incidência em
pacientes hospitalizados e como causa de mortalidade. O presente estudo
teve como objetivo avaliar a frequência de Candida spp. isoladas de
pacientes hospitalizados, assim como a sensibilidade aos antifúngicos e
o polimorfismo genético por RAPD-PCR. Os microrganismos incluíram
isolados de hemocultura, líquido abdominal e ponta de cateter venoso
central de pacientes internados no Hospital de Clínicas da Universidade
Federal de Uberlândia, região do Triângulo Mineiro, Minas Gerais, Brasil,
no período de julho de 2010-junho de 2011. Os testes de sensibilidade
aos antifúngicos foram realizados por microdiluição em caldo e na
análise por RAPD-PCR foram utilizados os oligonucleotídeos OPA09,
OPB11, e OPE06. Dos 63 isolados, 18 (28,5%) foram C. albicans, 20
(31,7%) C. parapsilosis, 14 (22,2%) C. tropicalis, quatro (6,4%) C.
glabrata, quatro (6,4%) C. krusei, dois (3,3%) C. kefyr, e um (1,6%) C.
lusitaniae. Resistência in-vitro à anfotericina B foi observada em 12,7%
dos isolados. Não foi observada resistência in-vitro aos azólicos, exceto
para os isolados de C. krusei. Os oligonucleotídeos OPA09 e OPB11
possibilitaram distinguir diferentes espécies. Isolados de C. albicans
apresentaram seis clusters e o complexo C. parapsilosis, cinco clusters,
com o iniciador OPA09, por RAPD-PCR, mostrando a variabilidade
genética daquelas espécies. Conclui-se que o complexo C. parapsilosis
foi a espécie mais frequente, e a maioria dos isolados foi sensível in vitro
aos antifúngicos testados. Alto polimorfismo genético foi observado para
os isolados de C. albicans e complexo C. parapsilosis, principalmente
com o oligonucleotídeo OPA09.
ACKNOWLEDGMENTS
The authors are grateful to the National Research Council (CNPq) for
the Scientific Initiation Fellowship awarded to R. P. Menezes, the Dean of
the Undergraduate Federal University of Uberlândia (PROGRAD-UFU;
edict 05/2010), to the Foundation for Research Support of Minas Gerais
(FAPEMIG; process nº. APQ-00464-11), and to the Dean of Research
and Graduate of the Federal University of Uberlândia (PROPP-UFU,
Edict 04/2011) for financial support. They are also grateful to Lorraine
Cristina Ribeiro Silva, Roterdan Martins Rosa, and Adriano Gonçalves
Martins for technical assistance with some tests.
CONFLICT OF INTEREST
The authors have no conﬂict of interest to declare.
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34. Pfaller MA, Diekema DJ, Rinaldi MG, Barnes R, Hu B, Veselov AV. Results from
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35. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public
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36. Pfaller MA, Messer SA, Moet GJ, Jones RN, Castanheira M. Candida bloodstream
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37. Pinto PM, Resende MA, Koga-Ito CY, Tendler M. Genetic variability analysis among
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38. Pires RH, Santos JM, Zaia JE, Martins CHG, Mendes-Giannini MJS. Candida
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39. Sellami A, Sellami H, Neji S, Makni F, Abbes S, Cheikhrouhou F, et al. Antifungal
susceptibility of bloodstream Candida isolates in Sfax Hospital,Tunisia.
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40. Soll DR. The ins and outs of DNA fingerprinting the infections fungi. Clin Microbiol
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41. Talarmin JP, Boutoille D, Tattevin P, Dargère S, Weinbreck P, Ansart S, et al.
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Received: 3 March 2014
Accepted: 5 August 2014
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57(3):193-196, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300002
RAW TROPICAL OYSTERS AS VEHICLES FOR MULTIDRUG-RESISTANT Vibrio parahaemolyticus
Renata Albuquerque COSTA(1,2), Rayza Lima ARAÚJO(1,2) & Regine Helena Silva dos Fernandes VIEIRA(1,2)
SUMMARY
The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and
frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics:
gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg),
nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant
to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46).
Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers
is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.
KEYWORDS: Vibrio parahaemolyticus; Crassostrea rhizophorae; Antimicrobial resistance.
INTRODUCTION
The consumption of raw oysters has been constantly associated with
bacterial etiology outbreaks, and Vibrio parahaemolyticus has been
highlighted as one of the main species responsible for this phenomenon5.
This species, frequently present in marine and estuarine environments, is
part of the indigenous microbiota of aquatic organisms16,17 and its ability
to cause diseases seems to be related to virulence factors, such as the
presence of tdh and trh genes18.
Strains origin: 87 V. parahaemolyticus strains - isolated from soft
tissues with the intervalvar liquids of C. rhizophorae oysters - were taken
from the bacterial collection of the Environmental and Fish Microbiology
Laboratory at the Institute of Marine Sciences (LABOMAR-UFC). The
study was based on 15 samples of fresh (sold at room temperature) and
15 samples of frozen (sold at -4 °C) oysters obtained from two restaurants
in Fortaleza-Brazil in 2010. Each sample consisted of 10 specimens,
for a total of 300 specimens examined. For isolation and purification of
the strains, 50 g of the intervalvar tissues and fluid was taken from each
sample of 10 specimens and added to 450 mL alkaline peptone water
(1% NaCl). The homogenate (which corresponded to a 10-1 dilution) was
used to make serial decimal dilutions from 10-2 to 10-4. Thus, 0.2 mL
aliquots of each dilution were spread plated on thiosulfate-citrate-bile
salt-sucrose agar (TCBS-Difco) and incubated at 35 °C for 18h. Three
blue-green colonies for each sample were randomly selected and cultured
in tryptone soy agar (TSA-Difco) (1% NaCl).
Oyster-associated outbreaks caused by V. parahaemolyticus are
well documented9,15,7,10, and represent a worldwide problem. In the
United States, McLAUGHLIN et al.11 reported a large outbreak of
gastroenteritis - involving episodes of watery diarrhea - associated with
V. parahaemolyticus serotype O6:K18.
According to DANIELS & SHAFAIE3, V. parahaemolyticus strains
responsible for cases of gastroenteritis are usually sensitive to antibiotics
commonly used in the treatment of enteric infections. However, for
patients with V. parahaemolyticus wound infections and septicemia,
the treatment - intravenous antimicrobial agents - is similar to that for
patients with V. vulnificus infection. Thus, besides virulence, the threat
of antimicrobial-resistant vibrios is also worth mentioning6.
Considering the risk that the consumption of oysters may pose to
human health, the following study aimed to determine the antimicrobial
susceptibility profile of Vibrio parahaemolyticus strains from fresh and
frozen oysters Crassostrea rhyzophorae sold in Fortaleza-Brazil.
Biochemical characterization of the strains: All colonies (n = 37
from fresh oysters, and n = 48 from frozen oysters) were submitted to
biochemical identification using the key developed by NOGUEROLA
& BLANCH13. The strains presented the following phenotypic profile:
(1) Gram-negative curved rods, (2) oxidase (+) in oxidase strips
(Laborclin), (3) sucrose (-) in Basal Media for Carbohydrate containing
0.5% (w/v) of sucrose (35 ºC for five days), (4) indol (+) in Sulfide-IndoleMotility Agar (35 ºC for 48 h), (5) ortho-Nitrophenyl-β-galactosideONPG (-) in saline solution with a drop of toluene and buffered solution
(1) Sea Science Institute, Federal University of Ceará, Av. Abolição 3207, 60165-081 Fortaleza, Ceará, Brazil.
(2) Engineering Fishing Department, Campus do Pici, Federal University of Ceará, blocks 825, 827 and 840, 60356-000 Fortaleza, Ceará, Brazil.
Correspondence to: Renata Albuquerque Costa. E-mail: [email protected]
COSTA, R.A.; ARAÚJO, R.L. & VIEIRA, R.H.S.F. - Raw tropical oysters as vehicles for multidrug-resistant Vibrio parahaemolyticus. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 193-6, 2015.
of ONPG 13.3 mM (37 ºC for 24 h), (6) mannitol acid (+) in Basal
Media for Carbohydrate containing 0.5% (w/v) of mannitol (35ºC for
5 days), (7) Voges-Proskauer (-) in MRVP broth (35ºC for 96 h), (8)
D-glucosamine cs (+) in Basal Media for Carbohydrate containing 0.5%
(w/v) of D-glucosamine (35 ºC for five days), (9) growth at 0% (-) and 8%
(+) NaCl in Alkaline Peptone Water (35 ºC for 24 h), and (10) arginine
dihydrolase (-), lysine decarboxylase (+), ornithine decarboxylase (+) in
basal media (0.02 g of bromocresol purple, 5 g of peptone, 3 g of extract
yeast, 10 g of sodium chloride and 1 g of glucose in one liter of distilled
water, pH 8,5) containing 0.125% (w/v) of arginine, lysine and ornithine,
respectively, with incubation at 35 ºC for seven days.
Antibiogram: The antimicrobial susceptibility pattern was carried
out by disk diffusion method1, with Muller-Hinton Agar (MH) containing
1% NaCl. Nine antibiotics were tested for each strain: gentamicin (Gen 10
µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5
µg), Chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline
(Tet 30 µg), vancomycin (Van 30 µg) and Erythromycin (Ery 15 µg).
Zones of inhibition were measured using a digital caliper (Digimess) and
each strain behavior was classified as sensitive, intermediate or resistant,
according to CLSI1 recommendations.
Plasmid curing: Strains that showed resistance to at least one
antimicrobial underwent plasmid curing in broth Luria Bertani
supplemented with acridine orange (SIGMA A-6014) at 0.1 mg mL-1
12
. After the curing procedure, the strains were again subjected to
antibiotic susceptibility testing (described above). Thus, the resistance
was considered chromosomal when observed after the curing procedure;
otherwise it was characterized as plasmid.
RESULTS
Table 1
Multiple antimicrobial resistance in Vibrio parahaemolyticus strains isolated
from samples of fresh and frozen oysters
Profile
Fresh
Frozen
MAR
Van+Pen+Amp+Ery
2
16
0.4
Van+Pen+Amp
16
30
0.3
Van+Pen+Ery
4
1
0.3
Van+Pen
13
1
0.2
Van+Amp
1
-
0.2
Van+Ery
1
-
0.2
Total
37 (94.9%) 48 (100%)
*VAN: vancomycin 30 µg; PEN: penicillin 10U; AMP: ampicillin 10 µg; ERY:
erythromycin 15 µg; MAR: multiple antibiotic resistance.
Table 2
Profile of chromosomal and plasmid resistance to antibiotics in Vibrio
parahaemolyticus strains isolated from samples of fresh and frozen oysters
Antibiotics
Van
Pen
Amp
Ery
Fresh oysters
Number of resistant strains
38
36
Chromosomal resistance
38
33
9
-
-
3
10
6
Plasmid resistance
19
6
Frozen oysters
From the 87 V. parahaemolyticus isolates tested, more than 96.5%
were resistant to vancomycin and penicillin, and 74.7% showed resistance
to ampicillin. Resistance to erythromycin was observed in 74.7% of the
isolates. In contrast, all strains were sensitive to chloramphenicol, and
more than 95.4% were sensitive to gentamicin, ciprofloxacin, tetracycline,
nalidixic acid and gentamicin.
Isolates from fresh oysters showed resistance rates to the following
antibiotics: Van (n = 38; 97.4%), Pen (n = 36; 92.3%), Amp (n = 19;
48.6%), Ery (n = 6, 15.4%). Resistance rates for the frozen oysters isolates
were: Van (n = 48; 100%), Pen (n = 48; 100%), Amp (n = 46; 95.8%),
Ery (n = 16, 33.3%).
A high rate of multiple resistance was observed in strains isolated
from fresh (94.9%) and frozen (100%) oysters. The most recurrent multiresistant profile in both fresh and frozen sources was Van+Pen+Amp
(Table 1). V. parahaemolyticus strains isolated from both types presented
a MAR oscillating from 0.2 to 0.4.
Plasmid curing indicated a chromosomal resistance profile in 100%
of Van-resistant strains. Isolates with a plasmid resistance profile were
more frequent in strains extracted from fresh oysters (Table 2).
DISCUSSION
The occurrence of antimicrobial-resistant vibrios in oysters poses
194
Number of resistant strains
48
48
46
16
Chromosomal resistance
48
47
45
11
-
1
1
5
Plasmid resistance
*VAN: vancomycin 30 µg; PEN: penicillin 10U; AMP: ampicillin 10 µg; ERY:
erythromycin 15 µg.
a threat to their consumers. HAN et al.6 investigated the susceptibility
of vibrios isolated from oysters and reported a high rate of penicillinresistant V. parahaemolyticus. This finding is similar to the results
obtained in the present study, since the resistance to Amp was found in
isolates from both types of oysters (Table 1).
DARAMOLA et al.2 determined the antimicrobial resistance profiles
of V. parahaemolyticus strains isolated from water samples, sediments
and mussels from the Humber River estuary in the U.K. - an area where
shellfish harvest and mussel culture occurs. The authors reported that
all isolates were sensitive to chloramphenicol, presented a low level
resistance to vancomycin (3.9%), ampicillin (1.3%), and high rates
(73.7%) of resistance to gentamicin. In the present research, a large
number of Van and Amp-resistant strains was detected; in contrast,
sensitivity to Gen and Chlo were observed. Comparing the results to those
of DARAMOLA et al.2, it is possible to suggest that the mechanisms
of antimicrobial resistance in the same bacterial species undergo a
differentiation process according to the region.
OTTAVIANI et al.14, in a study on the susceptibility of Vibrio
(including V. parahaemolyticus) isolated from fresh and frozen sold
seafood (shellfish, shrimp, squid and cod), found Vibrio strains without
resistance mechanisms to ciprofloxacin and nalidixic acid, as well as
isolates with multiple resistance profiles to different combinations of
antimicrobials, including ampicillin and penicillin, as in the present study.
The authors suggest that for the plasmid role in Vibrio, multiple resistance
to antibiotics must be investigated, even though most of the studies until
that moment indicated that this characteristic is inherent to that genus.
The high rate of multiple resistance observed in this study raises
questions as to the effectiveness of antimicrobial agents commonly
used in the treatment of gastroenteritis caused by Vibrio. It is possible
to consider that Chl, Nal, Cip, Tet and Gen should be selected to treat
diseases caused by V. parahaemolyticus, as has been reported in the
literature. KHAN et al.8 determined the susceptibility of 27 strains of
the same species isolated from cultured shrimp in Khulna (Bangladesh),
and suggested that the tetracycline and gentamicin were the best
choice for controlling diseases caused by enteric bacteria, including
V. parahaemolyticus. Thus, it is necessary to establish therapy with
appropriate antimicrobials for a more effective treatment of infections
caused by V. parahaemolyticus, V. vulnificus, and others19. The authors
above suggest that the antimicrobial ciprofloxacin is effective in these
cases, in accordance with the findings of this study.
In accordance with the findings in this study, ZULKIFLI et al.20
investigated the resistance of V. parahaemolyticus strains isolated from
cockles in Indonesia, and reported rates of resistance to penicillin and
ampicillin higher than 50%, as well as a 100% sensitivity to gentamicin.
LOZANO-LEÓN et al.10 investigated an outbreak of gastroenteritis
involving 64 people in Spain and revealed the presence of V.
parahaemolyticus in fecal samples of all patients involved. Symptoms
appeared within 12 to 24 hours after the consumption of raw oysters at a
street market in the city of Vigo (Galicia, Spain). The study also revealed
that 100% of the isolates were resistant to ampicillin, erythromycin
and vancomycin, antibiotics commonly used in the treatment of
gastroenteritis. The strains used in this study showed a similar resistance
profile to those responsible for the aforementioned outbreak, a fact which
confirms that the consumption of raw oysters poses a potential risk to
human health.
The results of the susceptibility test after the plasmid curing suggest
that the antimicrobial resistant profile from V. parahaemolyticus strains
isolated from oysters are linked to chromosomal genes, in accordance with
the literature. DEVI et al.4, in a study on the antimicrobial susceptibility
in strains from the same species isolated from shrimps cultivated in the
southeast of India, also found low rates of plasmid resistance, reporting
that there were no modifications in the resistance to chloramphenicol,
oxytetracycline and trimethoprim before and after plasmid curing.
Considering the high rates of resistance, especially multiple
resistance, the findings of this study support the assertion that oysters
may serve as hosts to vibrios which are resistant to drugs used in the
gastroenteritis treatment in human beings. Thus, the ingestion of those
bivalve mollusks without prior cooking is strongly unadvisable.
RESUMO
Ostras tropicais cruas como fonte de Vibrio parahaemolyticus
multirresistentes
O presente estudo objetivou determinar o perfil de suscetibilidade a
antimicrobianos de cepas de Vibrio parahaemolyticus oriundas de ostras
“in natura” e congeladas comercializadas em Fortaleza-Brasil. Oitenta e
sete (87) cepas foram submetidas ao antibiograma com emprego de nove
antibióticos: gentamicina (Gen 10 µg), ampicilina (Amp 10 µg), penicilina
G (Pen 10U), ciprofloxacin (Cip 5 µg), cloranfenicol (Clo 30 µg), ácido
nalidíxico (Nal 30 µg), tetraciclina (Tet 30 µg), vancomicina (Van 30 µg)
e eritromicina (Eri 15 µg). Todas as cepas mostram-se resistentes a pelo
menos um antibiótico, e 85 (97,7%) apresentaram multirresistência, com
predomínio do perfil Van+ Pen+Amp (n = 46). Foi detectada resistência
plasmidial a Pen, Amp e Eri. Dessa forma, o risco que o consumo de ostras
cruas representa para a saúde dos consumidores merece ser destacado,
uma vez que esses bivalves podem ser veículos de transmissão de micro
organismos multirresistentes a fármacos antibacterianos.
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antibiotic susceptibility of Vibrio parahaemolyticus from shrimp at Khulna region
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9.Lipp EK, Rose JB. The role of seafood in foodborne diseases in the United States of
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Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption
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11.McLaughlin JB, DePaola A, Bopp CA, Martinek KA, Napolilli NP, Allison CG, et al.
Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters.
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13. Noguerola I, Blanch AR. Identification of Vibrio spp. with a set of dichotomous keys. J
Appl Microbiol. 2008;105:175-85.
14. Ottaviani D, Bacchiocchi I, Masini L, Leoni F, Carraturo A, Giammarioli M, et al.
Antimicrobial susceptibility of potentially pathogenic halophilic vibrios isolated
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15.Pollack CV Jr, Fuller J. Update on emerging infections from the Centers for Disease
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Received: 14 May 2014
57(3):197-204, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300003
ANTHELMINTIC ACTIVITY OF LAPACHOL, β-LAPACHONE AND ITS DERIVATIVES AGAINST
Toxocara canis LARVAE
Taís MATA-SANTOS(1), Nitza França PINTO(1), Hilton Antônio MATA-SANTOS(2), Kelly Gallan DE MOURA(3), Paula Fernandes CARNEIRO(3),
Tatiane dos Santos CARVALHO(3), Karina Pena DEL RIO(3), Maria do Carmo Freire Ribeiro PINTO(3), Lourdes Rodrigues MARTINS(1),
Juliana Montelli FENALTI(1), Pedro Eduardo Almeida DA SILVA(4) & Carlos James SCAINI(1)
SUMMARY
Anthelmintics used for intestinal helminthiasis treatment are generally effective; however, their effectiveness in tissue parasitosis (i.e.
visceral toxocariasis) is moderate. The aim of this study was to evaluate the in vitro activity of lapachol, β-lapachone and phenazines
in relation to the viability of Toxocara canis larvae. A concentration of 2 mg/mL (in duplicate) of the compounds was tested using
microculture plates containing Toxocara canis larvae in an RPMI-1640 environment, incubated at 37 °C in 5% CO2 tension for 48
hours. In the 2 mg/mL concentration, four phenazines, lapachol and three of its derivatives presented a larvicide/larvistatic activity
of 100%. Then, the minimum larvicide/larvistatic concentration (MLC) test was conducted. The compounds that presented the best
results were nor-lapachol (MLC, 1 mg/mL), lapachol (MLC 0.5 mg/mL), β-lapachone, and β-C-allyl-lawsone (MLC, 0.25 mg/mL).
The larvae exposed to the compounds, at best MLC with 100% in vitro activity larvicide, were inoculated into healthy BALB/c mice
and were not capable of causing infection, confirming the larvicide potential in vitro of these compounds.
KEYWORDS: Toxocara canis; Quinones; Chemotherapy; Anthelmintics.
INTRODUCTION
Human visceral toxocariasis is a neglected zoonotic infection
caused by the larvae of Toxocara canis and, less frequently, Toxocara
cati31. According to recent reports, their prevalence seems to be
underestimated mainly because of the difficulties of diagnosis and
non-specific symptomatology36. The symptoms of this parasitic disease
are characterized by cutaneous reactions, extensive eosinophilia,
hepatomegaly, myocarditis, pulmonary infiltrates, and nodules
accompanied by cough and fever13,18. The severity of symptoms depends
on the location of the larvae and the number of larvae housed in tissues,
which induces mechanical damage and, in turn, results in an immunemediated inflammatory response26. Therefore, death is frequently
associated with inflammatory granulomatous reactions around the
larvae15, which may persist for a long time and, with it, reactivated larval
migration into the eye or the brain may occur at any time40. The long-term
survival of T. canis larvae has been attributed to molecular strategies
evolved by the parasite26.
Generally, the drugs used to treat this disease have limited
effectiveness, such as diethylcarbamazine and thiabendazole faced
with poor tolerability and the need for prolonged use30. The low water
solubility of benzimidazole compounds appears to collaborate with the
low bioavailability of compounds in this group, such as albendazole38,
the drug of choice in the treatment of visceral toxocariasis9. Nevertheless,
albendazole is the drug that crosses the blood brain barrier34 and shows
results superior to thiabendazole37 and diethylcarbamazine, because
it does not reduce the levels of specific IgE and produces side effects
in treated patients25. Therefore, an effective drug for treating human
infections caused by T. canis is still needed28.
Among the possibilities of assisting in the treatment of visceral
toxocariasis, natural and synthetic products33 stand out. Plant extracts
are important sources of biologically active natural products and may
be a model for the development of new drugs12,32.
Lapachol, an important representative of the quinone group, is
isolated from plants of the Bignoniaceae family19. It performs biological
activities against several pathogens, especially anti-parasitic activities
against Trypanosoma cruzi, Schistosoma mansoni, Leishmania
amazonensis and L. braziliensis7,23,24.
β-lapachone is an ortho-naphthoquinone, a natural derivative of
lapachol, present in small quantities in the woods of Tabebuia spp
(Bignoniaceae). β-lapachone is easily synthesized by sulfuric acid
treatment of lapachol16 and has a wide range of biological activities,
including trypanocidal, antibacterial, anti-inflammatory, and anticancer
activity2,3,4,7,29.
(1) Universidade Federal do Rio Grande, Faculdade de Medicina, Área Interdisciplinar em Ciências Biomédicas, Laboratório de Parasitologia. Rio Grande, RS, Brazil.
(2) Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, Departamento de Análises Clínicas e Toxicológicas. Rio de Janeiro, RJ, Brazil.
(3) Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, Núcleo de Pesquisa de Produtos Naturais. Rio de Janeiro, RJ, Brazil.
(4) Universidade Federal do Rio Grande, Faculdade de Medicina, Área Interdisciplinar em Ciências Biomédicas, Laboratório de Micobactérias. Rio Grande, RS, Brazil.
Correspondence to: Taís Mata dos Santos, Universidade Federal do Rio Grande, Faculdade de Medicina, Área Interdisciplinar em Ciências Biomédicas, Laboratório de Parasitologia, R.
General Osório s/n, Área Acadêmica do Hospital Universitário, 96200-190 Rio Grande, RS, Brasil. Tel.: +55.53.32338871. E-mail: [email protected]
MATA-SANTOS, T.; PINTO, N.F.; MATA-SANTOS, H.A.; DE MOURA, K.G.; CARNEIRO, P.F.; CARVALHO, T.S.; DEL RIO, K.P.; PINTO, M.C.F.R.; MARTINS, L.R.; FENALTI, J.M.; DA
SILVA, P.E.A. & SCAINI, C.J. - Anthelmintic activity of lapachol, β-lapachone and its derivatives against Toxocara canis larvae. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 197-204, 2015.
Several heterocyclic compounds were synthesized from β-lapachone
(i.e., phenazines) and have attracted considerable attention due to
their biological activities, including antimalarial5, antimycobacterial2,
antitumor, and antiparasitic21 ones. Therefore, the use of this group of
compounds as pharmacophores for the development of new drugs has
consequently been investigated.
In this study, lapachol, β-lapachone and three of its derivatives, and
17 phenazines synthesized from β-lapachone analogues were tested
against T. canis larvae.
MATERIALS AND METHODS
Synthesis: Lapachol was extracted from the heartwood of Tabebuia
spp (Tecoma) and purified by recrystallization from ethanol, following
a previously described procedure11. Nor-lapachol was synthesized from
lapachol through Hooker oxidation14.
β-lapachone, nor-β-lapachone, and β-C-allyl-lawsone were obtained
through the cyclisation of the prenyl side chain of lapachol, nor-lapachol
and C-allyl-lawsone, respectively. 10 mmol of the naphthoquinone were
solubilized in 15mL of sulfuric acid and mixed for several minutes. The
reaction was poured over cold water. The red solid was filtered, washed
with cold water (3 × 100 mL) and purified by recrystallization using a
mixture of acetone/hexane17.
The phenazines were prepared by the reaction of the naphthoquinone
(1.00 mmol), o-phenylenediamine (1.10 mmol) and sodium acetate
(1.30 mmol) in glacial acetic acid (50 mL). The reaction was maintained
under reflux for two hours and monitored by TLC. After the reaction, the
mixture was poured over ice and left to incubate overnight. The yellow
precipitate was filtered through a Buchner funnel, washed with cold
water (3 × 100 mL), and the phenazine was isolated. All phenazines
were synthesized with > 95% yield35.
Test compounds: All synthesized compounds were solubilized
in DMSO at 2.5% (Sigma®) and in sterile distilled water to obtain a
concentration of 2 mg/mL33.
Preparation of T. canis larvae: T. canis eggs were initially collected
directly from the uterine tubes of female adult parasites following the
treatment of young dogs with pyrantel pamoate (15 mg/kg). Afterwards,
the eggs were incubated in a 2% formalin solution at 28 °C for 30 days
in a humidity of > 90%27. By using a 5% sodium hypochlorite solution
(Vetec), the eggs’ protein cover was dissolved and the hatched T. canis
larvae were collected in sterile tubes for cultivation with a (Gibco) RPMI1640 medium supplemented with (Sigma) 25mM HEPES, 1% glucose,
(Gibco) PSF antibiotic-antimycotic solution, and 0.4 µg/mL ofloxacin.
Samples were maintained at 37 °C strain with 5% CO2.
Larvicidal/larvistatic activity test: A microplate was used to
measure the activity of substances at a concentration of 2 mg/mL. The
tests were conducted in duplicate. 100 T. canis larvae, 200 µL of RPMI1640 medium, and 100 µL of the test substances were added in each well.
The larvae were then maintained at 37 °C for 48 hours with 5% CO2.
The activity was tested in vitro and after exposure to the test
compound the larval mobility was tested by the state of the larvae (i.e.,
198
motile, immobile but not dead, or dead). Cell viability was tested by
using a 0.4% trypan blue indicator.
The substances that showed larvicidal activity in 100% of larvae with
the in vitro test at concentrations of 2 mg/mL were re-tested at lower
concentrations (MLC) (i.e., 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL
and 0.05 mg/mL). Afterwards, the substances with larvicidal/larvistatic
activity at the lowest concentrations were assessed for their viability of
infection in mice. In order to assess their viability, the content of each
microplate well was inoculated into 5-week-old BALB/c female mice by
intraperitoneal injection. All mice were given food without antibiotics and
had free access to water. The mice were kept on a 12 hour light to 12 hour
dark cycle at a 22 °C (± 2 °C) room temperature.
Furthermore, a control group of live larvae (100 larvae/well) in mice
was used to confirm the viability of larvae that were not exposed to
the substances. A single mouse was used for each compound and each
control. Mice were euthanized after 30 days of inoculation. The animals
were examined for larvae by having their carcass, brain, liver, lungs,
kidneys, heart, eyes, and spleen digested in a solution of 1% hydrochloric
acid and 1% pepsin39.
RESULTS
Lapachol, β-lapachone and three of its derivatives, and 17 phenazines
were tested against T. canis larvae.
β-lapachone and β-C-allyl-lawsone showed the highest activity
(MLC = 0.25 mg/mL), followed by lapachol (MLC = 0.5 mg/mL) and
nor-lapachol (MLC = 1 mg/mL) (Table 1).
Out of the 17 phenazines tested on T. canis larvae, four compounds
(i.e., compounds 1, 2, 3, and 4) showed 100% activity at a concentration
of 2 mg/mL. Additionally, three compounds (i.e., compounds 5, 16, and
17) showed a larvicidal activity of 78.6-98.4% at the same concentration.
The other phenazines showed < 14% activity (Table 2).
The larvae exposed to the compounds with 100% activity in vitro were
not viable and, therefore, were not able to infect the mice. The control
group consisted of live larvae and caused infection when inoculated into
the mice, which validates the in vitro evaluation criteria used in this study.
DISCUSSION
The search for new therapeutic prototypes with effectiveness against
T. canis larvae housed in human tissues is relevant for the efficacy of
visceral toxocariasis treatment. The new drugs should eradicate all
larvae housed in the tissues, not only decrease the intensity of infection
as it was noted in the administration of albendazole1,6,32,33, ivermectin,
mebendazole, and thiabendazole22 in mice.
In this study, the possible effect of lapachol and β-lapachone and its
derivatives against T. canis larvae was tested. Among all the synthetic
compounds tested, β-lapachone and β-C-allyl-lawsone showed the best
anthelmintic activity in vitro. Although these results are relevant, the
quinones present significant toxicity, possibly due to the redox potential.
This toxicity may cause cell damage due to oxidative stress, which could
result in undesirable side effects10.
Table 1
Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with lapachol and derivatives (n = 5)
Nº
Chemical structure
Chemical compound
Activity
Standart
deviation
MLC
Larvae viability in
mice
Lapachol
C15H14O3
100%
Zero
≤ 500 µg/mL
Negative
β- lapachone
C15H14O3
100%
Zero
≤ 250 µg/mL
Negative
Nor-lapachol
C14H12O3
100%
Zero
≤ 1,000 µg/mL
Negative
Nor-β-lapachone
C14H12O3
11.9%
0.8
-
-
β-C-allyl-lawsone
C13H10O3
100%
Zero
≤ 250 µg/mL
Negative
4.2%
0.4
-
Positive
O
OH
1
O
O
O
2
O
O
OH
3
O
O
O
4
O
O
O
5
O
Control
Live larvae (no compound)
Negative to detection of T. canis larvae in mice tissues; Positive to detection of T. canis larvae in mice tissues.
Nevertheless, due to the presence of larvicidal activity and by the
easy access of quinones to natural sources from Brazilian flora7, justify
the utilization of these compounds as a pharmacophore to develop
heterocyclic derivatives more active and less toxic.
This approach was previously used to synthesize trypanocidal
naphthoimidazoles from β-lapachone and to demonstrate that
naphthoimidazoles were more active and less toxic than β-lapachone8.
The larvicidal potential of in vitro tests and the capacity to inhibit
viability of infection in the mice, demonstrated by quinones, indicated
the relevance of studies in this area. Furthermore, motivates realize
cytotoxicity studies, for further evidence of the biological activity of
these compounds, in preclinical trials in experimental models, aiming
the development of prototype compound with anthelmintic activity which
could be used in the treatment of visceral toxocariasis.
Four phenazines (i.e., compounds 1, 2, 3, and 4) out of the 17,
showed 100% activity at a concentration of 2 mg/mL. However, these
phenazines did not present satisfactory results when exposed to low
concentrations; similar results were obtained with the same phenazines
against Plasmodium falciparum, P. berghei5, and Mycobacterium
tuberculosis2. In these studies, the compounds showed 50% antimalarial
activity in vitro, and only one-fourth of the phenazines tested against M.
tuberculosis demonstrated strong antimycobacterial activity (minimum
inhibitory concentration = 0.78 µg/mL). A significant antimalarial
activity in vitro was also shown in the other phenazines synthesized
from naphthols that were assayed against P. falciparum strains resistant
to chloroquine. However, they are not able to promote an effective cure
when tested against P. berghei in vivo20.
199
Table 2
Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17)
No.
Chemical structure
N
1
N
Chemical compound
Activity
Standart
deviation
MLC
Larvae viability in
mice
C21H22N2O
100%
Zero
2,000 µg/mL
Negative
C36H42N2O4
100%
Zero
2,000 µg/mL
Negative
C19H16N2O
100%
Zero
2,000 µg/mL
Negative
C19H18N2O
100%
Zero
2,000 µg/mL
Negative
C20H22N2O
78.6%
7.1
-
-
C36H34N2O4
1.76%
0.03
-
-
O
O
N
O
N
2
O
O
N
N
3
OH
N
N
4
O
N
N
5
OH
O
6
O
200
O
O
N
N
Table 2
Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17) (cont.)
No.
Chemical compound
Activity
Standart
deviation
MLC
Larvae viability in
mice
N
C34H30N2O4
1.0%
0.1
-
-
N
C34H38N2O4
3.8%
18.0
-
-
N
C20H18N2O
4.2%
8.8
-
-
N
C20H16N2O
1.3%
4.5
-
-
N
C32H22N2O4
6.5%
2.2
N
C32H26N2O4
1.5%
1.1
-
-
Chemical structure
O
N
O
7
O
O
O
N
O
8
O
O
N
9
OH
N
10
OH
O
11
O
O
12
O
O
N
O
O
N
O
201
Table 2
Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17) (cont.)
No.
Chemical structure
Chemical compound
Activity
Standart
deviation
MLC
Larvae viability in
mice
C19H20N2O
14.0%
47.4
-
-
C20H20N2O
2.5%
1.4
-
-
C21H22N2O
4.0%
0.7
-
-
C36H30N2O4
94.5%
3.5
-
-
-
98.4%
0.4
-
-
No compound
1.3%
0.4
-
Positive
N
N
13
OH
N
N
14
O
N
15
N
O
O
O
16
N
N
O
O
H3C
CH3
17
N
O
CH3
N
CT
CT: Control; Negative to detection of T. canis larvae in mice tissues; Positive to detection of T. canis larvae in mice tissues.
Structural changes that arose in other phenazines (i.e., compound
5-17) tested in this study did not increase the specific activity of
the molecules. The lower activity of compounds 5-17, compared to
202
compounds 1-4, indicates that new modifications to these molecules are
necessary to promote effective action against T. canis larvae.
ACKNOWLEDGEMENTS
The authors are grateful to Antônio Ventura Pinto and Núcleo de
Pesquisa de Produtos Naturais (UFRJ) for the compounds.
FINANCIAL SUPPORT
This work was supported by Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior. Authors declare no conflict of interests.
RESUMO
Atividade anti-helmíntica do lapachol, β-lapachona e derivados
contra larvas de Toxocara canis
Os anti-helmínticos empregados no tratamento das helmintoses
intestinais, de modo geral, são eficazes, porém nas parasitoses teciduais,
como é o caso da toxocaríase visceral, a eficácia é moderada. Este estudo
teve como objetivo avaliar in vitro a atividade do lapachol, β-lapachona
e fenazinas derivadas da β-lapachona sobre a viabilidade de larvas de
Toxocara canis. Os compostos foram testados na concentração de 2 mg/mL
(em duplicata) em placas de microcultivo, contendo larvas de T. canis em
meio RPMI-1640, sendo incubados, a 37 °C, em tensão de CO2 de 5%,
por 48 horas. Na concentração de 2 mg/mL, quatro fenazinas, o lapachol
e três derivados, apresentaram atividade larvicida/larvostática de 100%.
A seguir, foi realizado o teste de concentração larvicida/larvostártica
mínima (CLM). Os compostos que apresentaram os melhores resultados
foram o nor-lapachol (CLM, 1 mg/mL), lapachol (CLM, 0,5 mg/mL), a
β-lapachona e a β-C-alil-lausona (CLM, 0,25 mg/mL). As larvas expostas
aos compostos, na melhor CLM 100% in vitro foram inoculadas em
camundongos BALB/c saudáveis não sendo capazes de causar infecção,
confirmando o potencial larvicida in vitro desses compostos.
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Received: 21 July 2014
Accepted: 22 September 2014
204
57(3):205-209, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300004
MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC
ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES
(CHENNAI AND IZATNAGAR) OF Toxoplasma gondii
Vikrant SUDAN(1), Anup Kumar TEWARI(2) & Harkirat SINGH(3)
SUMMARY
Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess
the homologous variations between the different loci of various strains of parasites. Design and setting: The present communication
deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3)
of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI. Method: The surface antigen
3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.
Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The
strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain
(both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them. Conclusion:
Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain
and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary
between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as
SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed
studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are
important from the point of view of molecular phylogeny.
KEYWORDS: Indian isolates; Molecular characterization; SAG3; Toxoplasma gondii.
INTRODUCTION
Toxoplasma gondii, an obligate intracellular coccidian parasite, has
acquired utmost zoonotic relevance in the current scenario around the
globe, accounting for abortions, stillbirths, and neonatal complications
in livestock, especially in sheep, goats and pigs9,16,30. The condition leads
to life-threatening consequences both in immunocompromised human
patients suffering from acquired immune deficiency syndrome (AIDS)
and those with organ transplants2. In India, the condition has exhibited
itself as acquired ocular toxoplasmosis4, in immunocompetent patients,
bringing about possible similarities with South American strains which
are known to exhibit a high rate of ocular involvement20. A third of the
world’s total population is thought to be at risk of infection22. Of late,
different strains of Toxoplasma gondii are known to induce different
cytokine responses 5 and thereby vary in their pathogenesis. The
surface antigens of T. gondii are the major targets as key molecules for
immunodiagnosis as well as immunoprophylaxis because of their initial
presentation to the host immune system. Surface antigen 3 (SAG3), an
under-reported 43kDa glycoaminoglycan-binding protein associated with
binding of host heparin sulfate proteoglycans (HSPGs)18, shares primary
structure similarity with another proven Surface antigen 1 (SAG1)7
protein. It was considered interesting to carry out the primer-directed
amplification of the open reading frame (ORF) of surface antigen 3
(SAG3) gene of Indian isolates of T. gondii viz. Chennei (CHEN) and
Izatnagar (IZN) isolates, maintaining them at the IVRI and cloning them
in a heterologous prokaryotic system. Moreover, the two Indian isolates
used in the present study are known to vary between themselves as far
as homologies related to other gene loci like GRA 526, MIC 323 and SAG
227 are concerned, but there is no literature available as far as SAG3
homologies are concerned. In the present study, the cloned genes were
custom sequenced and the information was compared with the available
sequences of the same gene in the GenBank in order to establish the
phylogenetic identity of the SAG3 gene among the various isolates.
METHODS
Propagation of T. gondii tachyzoites: Inbred Swiss albino adult
mice, maintained on standard feed (pellets) and water ad libitum, were
(1) Assistant Professor, Department of Parasitology, College of Veterinary Sciences & Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam
Go Anusandhan Sansthan (DUVASU), Mathura - 281001, India.
(2) Principal Scientist, Division of Parasitology, IVRI, Izatnagar, India.
(3) Assistant Professor, Department of Parasitology, GADVASU, Ludhiana, India.
Correspondence to: Vikrant Sudan, Assistant Professor, Department of Parasitology, College of Veterinary Sciences & Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa
Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura - 281001, India; Email: [email protected]
SUDAN, V.; TEWARI, A.K. & SINGH, H. - Molecular characterization and sequence phylogenetic analysis of surface antigen 3 (SAG-3) gene of local Indian isolates (Chennai and Izatnagar)
of Toxoplasma gondii. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 205-9, 2015.
intraperitoneally infected with 100 mouse-adapted Chennei and Izatnagar
T. gondii tachyzoite isolates that were cryopreserved and maintained at
a divisional laboratory, IVRI. These two Indian isolates were originally
isolated from the tested-positive blood, heart and brain tissues of freerange chickens (Gallus domesticus) naturally infected by T. gondii25 and
isolated after Cat inoculation assays. The infected mice were monitored
daily for the development of signs of infection. Infected mice exhibiting
peritonitis were euthanized and peritoneal lavage was aspirated following
inoculation of 5 mL of sterile phosphate buffered saline (PBS, pH 7.2)
in the peritoneal cavity with due care in avoiding injury to visceral
organs. The contents were washed thrice with PBS (pH 7.2) and the live
tachyzoites were counted.
Separation of host cell-free tachyzoites: The host cell-free
tachyzoites were separated using standard protocol 15. Briefly, the
peritoneal fluid containing free tachyzoites and tachyzoite infected
macrophages was collected in PBS (pH 7.4) and washed thrice in
PBS (pH 7.4) while repeatedly centrifuging at 5000 rpm for 10 min.
Following this, a final pellet was re-suspended in 5 mL of PBS (pH
7.4). The intracellular tachyzoites were separated and made free from
the macrophages by passing the contents repeatedly through a 27g
needle fitted in a 10 mL sterile syringe. The host cell-free tachyzoite
suspension was washed with 20 mL of PBS (pH 7.4), debris was allowed
to settle down in the centrifuge tube for 10 min and the supernatant was
collected and, following this, passed through a pre-wetted (with PBS
pH 7.4) polycarbonate membrane filter of 3 µm pore size slowly (at the
rate of one mL per 2-3 min). The filtrate was centrifuged (3000 rpm for
10 min) and the tachyzoites in sediment were re-suspended in one mL
of PBS (pH 7.4).
Isolation of total RNA of T. gondii: Total RNA was extracted
directly from the purified tachyzoites using Trizol® reagent (Gibco BRL)
while following the manufacturer’s protocol. Briefly, one mL of Trizol
was added to the suspension containing 5-10x106 tachyzoites, repeatedly
pipetted to kill the tachyzoites and following this, incubated at 30 oC for
five min to dissociate nucleoprotein complexes. The suspension was
vigorously shaken for 15 sec after adding 0.2 mL of chloroform and then
centrifuged at 12,000g for 15 min at 4 oC. This facilitates the separation
into lower organic phase and upper aqueous phase. The aqueous phase
was transferred to a fresh tube, 0.5mL of the isopropyl alcohol was poured
into the tube and the RNA was allowed to precipitate while keeping the
tube at 15-30 oC for 10 min. The tube was centrifuged at 12,000g for
10 min at 4 oC. The RNA pellet was washed once with one mL of 75%
ethanol prepared using 0.01% of diethylpyrocarbonate (DEPC) treated
water. The sample was mixed by vortexing and centrifuged at 7,500 x g
for five min at 4 oC. The RNA pellet was air-dried, reconstituted in 100
µL of RNA storage buffer (Ambion) and stored at -20 oC until further
use. Purity and concentration of total RNA was checked by ethidium
bromide stained agarose gel electrophoresis, performed at 2-3 volts/cm2.
Synthesis of complimentary DNA (cDNA) by reverse transcription:
cDNA was synthesized from the total RNA isolated from the T. gondii
tachyzoites of both the isolates, using oligo dT primer while following
the standard protocol23. The cDNA, thus synthesized, was quantified
using a spectrophotometer (Nanodrop®, USA).
Polymerase chain reaction-based (PCR) amplification of the
SAG3 gene of Indian isolates: The entire open reading frame (ORF) of
206
the SAG3 gene of T. gondii (CHN and IZN isolates) was PCR amplified
using a pair of specific primers as described by SUDAN et al. 201228
(forward primer (TS3F) 5’-ATGCAGCTGTGGCGGCGCAG-3’ and
reverse (TS3R) 5’-TTAGGCAGCCACATGCACAAG-3’). The PCR
reactions were carried out in a standard 25 µL reaction volume with initial
denaturation of DNA strands at 95 oC for five min followed by 32 cycles
of denaturation at 95 oC for 50 sec, primer annealing at 62 oC for 75 sec
and strand elongation at 72 oC for 50 sec. Thereafter one cycle of final
extension of the strands was carried out at 72 oC for 12 min. The PCR
amplifications were confirmed by visualization of the product on 1.5%
agarose gel stained with ethidium bromide following electrophoresis.
Molecular cloning and characterization of the SAG3 gene of
Indian isolates: The amplified ORF of the SAG3 genes of Indian isolates
of T. gondii were purified using a Qiagen Mini elute gel extraction kit
(Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s
protocol. Following this, competent Escherichia coli DH5α cells were
prepared following the standard calcium chloride treatment method23.
Ligation reaction for the cloning of SAG3 (amplified from T. gondii
Indian isolates) into InsTAclone PCR cloning vector (Qiagen, Germany)
as well as transformation of DH5α cells was carried out as per the
company’s protocol. The positive clones were identified by blue-white
colony screening method. Further confirmation was carried out by
restriction analysis of the plasmid DNA isolated from the white colonies
with PstI and EcoRI as well as by colony PCR following standard
protocol24. The restriction digestion reaction was carried out at 37 oC
for four h. The digested product as well, as the colony PCR amplified
products, was visualized in the ethidium bromide-stained agarose gel
following electrophoresis. The subcultures of a positive clone harboring
the desired SAG3 genes of both the Indian isolates were custom DNA
sequenced from the Department of Biochemistry, Delhi University.
Data analysis: The sequence information received was analyzed
using DNASTAR and GeneTool software. The sequences, hence
received sequence submitted to GenBank (Accession No.: HQ291783 &
HQ291784 for Chennei and Izatnagar isolates, respectively). Moreover,
these two sequences were compared with an earlier sequenced RH strain
sequence (Accession No.: FJ825705) from the department along with
other published sequences viz., CEP (Accession No.: AF340229); P-Br
(Accession No.: AY187280) and PRU (Accession No.: AF340228) from
across the world through the GenBank using online Nucleotide BLAST
Softwares (http://blast.ncbi.nlm.nih.gov/).
RESULTS
Viability of cryopreserved T. gondii: All the infected mice started
showing characteristic signs of the disease from Day-7 Post Infection (PI).
The clinical signs included raised & rough fur coat, pendulous abdomen,
severe ascites, dullness, tachypnoea marked by resting on either the
walls of the cages, on the nozzle of water bottle or on other resting mice
with their forelegs. Microscopically, a large number of tachyzoites were
detectable (either free or within the peritoneal macrophages suspended
in the aspirated peritoneal fluid).
PCR amplification, molecular cloning and molecular
characterization of the SAG3 gene of Indian isolates: The whole ORF
of the SAG3 gene was amplified from the cDNA of Indian isolates of
T. gondii using the specific forward and reverse primers. The amplicons
were resolved as a single band of 1158 bp (Fig. 1). It was further purified
for ligation in InsTAclone PCR cloning vector. The selection of positive
colonies was performed by colony PCR using the specific primers and
also by restriction enzyme digestion of the recombinant plasmids with
PstI and EcoRI for the release of insert. The results of restriction enzyme
digestion (Fig. 2) as well as colony PCR (Fig. 3) were checked by agarose
gel electrophoresis.
Fig. 3 - Colony PCR confirming the amplifications of 1158 bp specific SAG3 amplicons
of Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus; Lane
C: Positive control DNA of T. gondii; Lane IZN 1,2: Amplicon of 1158 bp from T. gondii
Izatnagar isolate; Lane CHEN 1,2: Amplicon of 1158 bp from T. gondii Chennai isolate.
Fig. 1 - Specific PCR amplification of ORF of SAG3 gene of Indian isolates of T. gondii
on 1.5% agarose gel. Lane CHEN: Amplicon of 1158 bp from T. gondii Chennai isolate;
Lane M: Marker 100 bp DNA ladder plus; Lane IZN: Amplicon of 1158 bp from T. gondii
Izatnagar isolate.
that of the earlier sequenced RH strain sequence. A comparison of the
nucleotide sequence of T. gondii Indian isolates revealed 100% homology
between the Chennei and the Izatnagar isolates. Furthermore, there is a
99.3% identity with P-Br and the CEP SAG3 sequence and 98.4% with
PRU. A phylogenetic association, for analyzing the identity between
strains and testing the robustness of the association, was done using the
online bootstrap method (http://blast.ncbi.nlm.nih.gov/) to delineate its
relationship with other referral stains (Fig. 5).
Fig. 4 - Sequence pair distances of SAG3 Clustal V (weighted).
Fig. 5 - Phylogenetic tree of nucleotide sequence of SAG3 Clustal V (weighted).
Fig. 2 - Release of SAG3 insert by restriction digestion of insTA cloning vector of the
two Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus (MBI
Fermentas); Lane IZN: Insert release after PstI and EcoRI digestion of vector containing
Izatnagar isolate; Lane CHEN: Insert release after PstI and EcoRI digestion of vector
containing Chennai isolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning
vector.
Data analysis: The nucleotide sequence revealed 99.9% (Fig.4)
sequence homology of SAG3 ORF between the Indian isolates with
The Adenine and Thymine (A+T) content of the SAG3 gene of both
the Indian isolates was found to be 42.57%, whereas the Guanine and
Cytosine (G+C) content was 57.43%. The nucleotide homology was
found to be 99.9% with the earlier sequenced RH strain. There was a
substitution of a single nucleotide of A instead of G at the 397th position of
the SAG3 nucleotide sequence of both the Indian isolates. The nucleotide
substitution resulted in the change of a single nucleotide residue in the
deduced amino acid sequence at the 133rd position as asparagine (N)
instead of aspartic acid (D). As a whole, Indian isolates were closer to
the RH strain than to the P-Br strain (Brazilian strain) and CEP strain and
PRU strains (USA), with respect to the nucleotide homology.
207
DISCUSSION
CONCLUSION
The significance of toxoplasmosis has increased particularly in
immune compromised and/or HIV/AIDS patients, with an alarming
prevalence in developing countries such as India. The presence of
brain cysts is often associated with various psychiatric disorders and
behavioral alterations29 such as schizophrenia8, 32 alongside other brain
pathologies and ocular involvements25 in both immunocompromised
and immunocompetent individuals1,11. In order to precisely define the
magnitude of the disease, it was of interest to investigate the genetic
diversity of the pathogen among the T. gondii strains using advanced
biotechnological approaches.
In the present study, the SAG3 gene of T.gondii was cloned,
sequenced and aligned, before being compared with various published
strains and the homologies between the two Indian isolates were found
both with one another and with other strains across the globe. The two
Indian isolates used in the present study are known to vary between
themselves as far as homologies related to other genes are concerned
but they were found to be 100% homologous as far as SAG3 locus is
concerned. This could be attributed to the fact that this SAG3 might be
a conserved locus and therefore, further detailed studies are thereby
warranted to exploit the use of this particular molecule in diagnostics
and immunoprophylactics. The findings are important from the point of
view of molecular phylogeny.
Surface antigen 3 (SAG3), a 43kDa glycoprotein, is a
glycosylphosphatidylinisotol-anchored (GPI) membrane-bound protein
in the developmental stages of the pathogen (tachyzoites & bradyzoites)
parasite6,19. The protein was earlier identified as P43. It was cloned and
sequenced for the first time by CESBRON-DELAUW et al. in 19947
followed by FUX et al. in 200313. SAG3 has primary structure similarity
with Surface antigen 1 (SAG1)7. SAG3 is a glycoaminoglycan-binding
protein associated with binding of host heparin sulfate proteoglycans
(HSPGs)18. The SAG3-HSPGs interaction facilitates the parasite’s
attachment to target cells. Furthermore, it has been shown that targeted
disruption of the GPI-anchored surface antigen SAG3 gene in T. gondii
resulted in decreased host cell adhesion and virulence of the parasite for
mice10. In immunoprophylactic application, rSAG3 conferred partial
protection in mice, which was mediated through Th1 type immune
response21. However, molecular characterization of the SAG3 gene of T.
gondii of Indian isolates has not been attempted so far. The present study
reports the molecular characterization of the surface antigen 3 (SAG3) gene
of T. gondii of Indian isolates and ascertains its molecular homology with
some other strains of the same parasites that are prevalent across the globe.
Worldwide, only one valid species of Toxoplasma exists. However,
based on molecular genotyping studies, varied fundamental clonal
population isolates of T. gondii have been recognized. The molecular
diversity in the distinct and/or related Toxoplasma stabilates is routinely
evaluated by sequence-based analysis among the different isolates.
Recently, different strains of Toxoplasma gondii have been known
to induce varying levels of cytokine responses5 and thereby vary in
their pathogenesis, hence the study of the phylogeny has gained ultra
importance owing to the variation in pathogenesis at the strain levels.
Moreover, the two Indian isolates used in the present study are known to
vary between themselves as far as homologies related to other gene loci
like GRA 526, MIC 323 and SAG 227 are concerned but they were found
to be 100% homologous as far as SAG3 locus is concerned. This could
be attributed to the fact that this SAG3 might be a conserved locus and
thereby, further detailed studies are thereby warranted to exploit the use
of this particular molecule in diagnostics and immunoprophylactics.
Interestingly, differences at the lineages sequence level of DNA
among the predominant clones are less than 2%14. Transmission of the
parasite through carnivorism and scavenging among intermediate hosts,
bypassing sexual recombination events in the definitive host, i.e. cats17, 30,
parthenogenetic formation of oocysts by many unfertilized macrogametes
of the parasite in the small intestine of cats12, and simultaneous infection
with different strains of T. gondii are some plausible reasons that can
explain the existence of the clonal population structure in T. gondii3.
208
RESUMO
Caracterização molecular e análise filogenética de sequências do
antígeno de superfície 3 (SAG3) em isolados indianos (CHENNAI
E IZATNAGAR) de Toxoplasma gondii
Contexto e objetivo. A caracterização molecular de isolados
indianos de Toxoplasma gondii é importante para a investigação de
variações genéticas existentes entre cepas do parasito em diferentes
locos gênicos. Delineamento e disposição. A presente comunicação
realizou a clonagem e o sequenciamento dos 1158 pares de base
correspondendo à totalidade do quadro de leitura do antígeno de
superfície 3 (SAG3) de Toxoplasma gondii em dois isolados indianos
(Chennai e Izatnagar) mantidos em um biorrepositório localizado em
IVRI. Método. As sequências do SAG3 dos dois isolados indianos foram
clonadas, sequenciadas e posteriormente comparadas com sequências
SAG3 de Toxoplasma gondii disponíveis em publicações. Resultados.
A comparação das sequências revelou 99,9% de homologia com a cepa
RH padrão; 99,3% de homologia com as cepas P-Br e CEP; e 98,4%
de homologia com a cepa PRU. Os dois isolados indianos eram 100%
idênticos no que diz respeito à sequência SAG3. Conclusão. Concluiu-se
que os isolados indianos são filogeneticamente mais próximos da cepa
RH em relação à cepa brasileira P-Br, ou às cepas CEP e PRU (USA).
No entanto, a análise de outros genes de Toxoplasma gondii destes dois
isolados indianos mostrou diferenças na composição de nucleotídeos,
ao contrário do que foi encontrado para o locus SAG3. Estes resultados
poderiam ser atribuídos ao fato do locus SAG3 ser altamente conservado,
necessitando de estudos adicionais para determinar se SAG3 poderia ser
utilizado no diagnóstico da toxoplasmose. No entanto, estes resultados
são importantes do ponto de vista da filogenia molecular.
ACKNOWLEDGMENTS
The authors are thankful to the Director, IVRI for providing the
facilities and to the ICAR for the fellowship awarded to the first author
during the perusal of his master’s programme. The authors declare that
there is no conflict of interest.
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209
LIBRARY OF THE
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57(3):211-215, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300005
OCCURRENCE OF Blastocystis spp. IN UBERABA, MINAS GERAIS, BRAZIL
Marlene CABRINE-SANTOS(1), Eduardo do Nascimento CINTRA(1), Rafaela Andrade do CARMO(1), Gabriel Antônio Nogueira NASCENTES(2),
André Luiz PEDROSA(3), Dalmo CORREIA(4) & Márcia Benedita de OLIVEIRA-SILVA(3)
SUMMARY
Intestinal parasites are a problem for public health all over the world. The infection with Blastocystis, a protozoan of controversial
pathogenicity, is one of the most common among them all. In this study, the occurrence of intestinal parasites, with emphasis on
Blastocystis, in patients at the Universidade Federal do Triângulo Mineiro was investigated in Uberaba (MG) through microscopy of
direct smears and fecal concentrates using Ritchie’s method. Feces of 1,323 patients were examined from April 2011 to May 2012.
In 28.7% of them at least one intestinal parasite was identified, and the most frequent organisms were Blastocystis spp. (17.8%)
and Giardia intestinalis (7.4%). The occurrence of parasitism was higher in children aged 6 -10 years old, and the infection with
Blastocystis spp. was higher above the age of six (p < 0.001). The exclusive presence of G. intestinalis and of Blastocystis spp. was
observed in 5.4% and 12.2% of the patients, respectively. Regarding patients with diarrheic feces, 8% revealed unique parasitism
of Blastocystis spp. Other intestinal parasites observed in children were Ascaris lumbricoides (0.3%) and Entamoeba histolytica/
dispar/moshkovskii (1.4%). The Ritchie’s method was more sensitive (92.8%) when compared to direct microscopy (89.8%), with
high agreement between them (97.7%, kappa = 0.92). In conclusion, the occurrence of Blastocystis spp. in Uberaba is high and the
presence of diarrheic feces with exclusive presence of the parasite of Blastocystis spp. was observed.
KEYWORDS: Blastocystis spp.; Intestinal parasites; Stools; Uberaba (MG).
INTRODUCTION
Intestinal infections by protozoa are frequent all over the world,
being most prominent in developing countries, since the majority of
the infections are generally acquired by the ingestion of foods or water
contaminated by human and/or animal feces, generally caused by the
lack of basic sanitation and conditions of hygiene4,6,12. In this context
the infection with Blastocystis spp., an anaerobic intestinal protozoan is
one of the most prevalent6,9,12, occurring in approximately 1.5% to 10%
of the population in developed countries and 30% to 60% in developing
countries19. However, these data are underestimated, since laboratory
technicians are generally not sufficiently trained to detect it or simply
do not report their findings. Moreover, routine techniques for stool
analysis such as the water spontaneous sedimentation (HOFFMANPONS-JANER)8 which leads to the breakage of the vacuolar stage of the
parasite, is one of the mostly detected stages in the stool examination,
leading to the false negative results14.
Although the infection with Blastocystis spp. is one of the most
prevalent amongst the intestinal parasites, its impact on public health
is not known, since its pathogenicity has been noted as controversial
by several authors6,9,10,21. However, in spite of the controversial issue
that Blastocystis pathogenesis represents, there are no explanations for
patients who present symptoms like diarrhea, fever, vomit, abdominal
pain, and nauseas in the absence of any other parasite but Blastocystis6,9,10.
In addition to this, studies have shown that stress conditions can lead to
increased susceptibility and pathogenicity of Blastocystis, it is also an
opportunistic parasite in immunocompromised patients5,16. There is a
huge lack of information regarding the pathogenesis, the diagnosis and
the epidemiology of this protozoan20. In this study, it is shown that the
occurrence of Blastocystis in Uberaba is high, followed by the infection
of Giardia intestinalis, and that direct methods, especially Ritchie’s,
are suitable for the diagnosis of the parasite. Moreover, the presence of
diarrheal stools with unique parasitism by Blastocystis spp. was observed.
The present paper is a cross-sectional study with a non-probability
sample of patients who were treated at the Universidade Federal do
Triângulo Mineiro Hospital, between April 2011 and May 2012. All
patients referred to carry out a stool test suffered from acute or chronic
diarrhea or complaints of constant abdominal pain and/or weakness
was included. Age, presence of underlying diseases, HIV/AIDS or
gastrointestinal symptoms were not considered as exclusion criteria.
The specimens were examined by the microscopy direct of smears and
fecal concentrates by Ritchie’method18. Briefly, the examination by direct
(1) Instituto de Ciências da Saúde, Universidade Federal do Triângulo Mineiro, Uberaba/MG, Brazil.
(2) Disciplina de Microbiologia e Imunologia, Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro (IFTM), Uberaba/MG, Brazil.
(3) Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba/MG, Brazil.
(4) Disciplina de Doenças Infecciosas e Parasitárias, Universidade Federal do Triângulo Mineiro, R. Frei Paulino 30, Abadia, Uberaba, Minas Gerais, Brazil.
Correspondence to: Marlene Cabrine-Santos, Universidade Federal do Triângulo Mineiro, Av. Getúlio Guaritá, s/n, Abadia, 38025-440 Uberaba, Minas Gerais, Brasil. Tel.: +55 3433185542.
Fax: +55 3433185462. E-mail: [email protected]
CABRINE-SANTOS, M.; CINTRA, E.N.; CARMO, R.A.; NASCENTES, G.A.N.; PEDROSA, A.L.; CORREIA, D. & OLIVEIRA-SILVA, M.B. - Occurrence of Blastocystis spp. in Uberaba,
Minas Gerais, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 211-4, 2015.
microscopy was conducted with an amount of stool placed in a drop of
Lugol solution in a slide/coverslip and observed by optical microscopy
with a 400× objective lense. Ritchie’s method was performed by keeping
the feces in 3.7% formaldehyde, adding ethyl ether and then centrifuging
the mixture at 1,200×g/5min. The sediment was observed with the 400×
objective. The statistical software Statistica 10.0 (Statsoft, Tulsa, OK,
2011) was used to perform the statistical analysis.
The association between risk factors and presence of Blastocystis spp.
was verified by the chi-squared classic or, whenever necessary by the
chi-square test with Yates correction and Fisher’s exact test. Moreover,
the association force was measured by the calculation of the Odds ratio
with confidence intervals of 95%. The agreement between the microscopy
direct and the Ritchie’s method was evaluated by means of Kappa
coefficient. Results which demonstrate a level of significance lower than
0.05 (p < 0.05) were considered significant. This study was approved
by the ethics committee in research at the UFTM under number 1804.
RESULTS
Stool specimens from 1,323 patients were examined, with 44.1%
male and 55.9% female. From the analyzed samples, 28.7% presented
an intestinal pathogenic parasite or not, being Blastocystis spp. (17.8%)
and G. intestinalis (7.4%) the most observed (Table 1). The known
pathogenic parasites analysis showed a positivity of 10.4% (138/1323),
with the highest occurrence detected by G. intestinalis. The presence
of non-pathogenic parasites occurred in 7.3% of the samples (Table 1).
Table 1
Occurrence of intestinal parasites in stool samples of patients from the Clinical
Hospital at the Universidade Federal do Triângulo Mineiro, Uberaba, MG
Positivity of the diagnostic tests
n
%
Intestinal parasites
379
28.65
Blastocystis sp.
235
17.76
Giardia intestinalis
98
7.41
Entamoeba coli
59
4.46
Endolimax nana
36
2.72
Entamoeba histolytica/dispar/moshkovskii
26
1.97
Taenia sp.
6
0.45
Ascaris lumbricoides
2
0.15
Isospora belli
1
0.08
Strongyloides stercoralis
2
0.15
Chilomastix mesnili
1
0.08
Hookworms
1
0.08
Enterobius vermicularis
1
0.08
1
0.08
Exclusive presence of Blastocystis
161
12.17
Exclusive presence of Giardia
71
5.37
Hymenolepis nana
From the evaluated samples, 33.47% (442) were from children aged
0-5 years and 11.04% (146) were from children aged between 6-10 years
212
(Table 2), and in 27.8% and 44.1 % of them, respectively, the presence
of parasitism by at least one organism was observed, whether pathogenic
or not. G. intestinalis infection occurred in 86.7% of cases (85/98) in
patients aged between 0-10 years (Table 2). Ascaris lumbricoides (3,
0.3%) and Entamoeba histolytica/dispar/moshkovskii (13, 0.7%) were
also observed in children.
Table 2
Parasitism by Giardia intestinalis and Blastocystis spp. according to the age
group of patients treated at the Clinical Hospital, Universidade Federal do
Triângulo Mineiro, Uberaba (MG)
No. of patients
Giardia intestinalis
(n/%)
Blastocystis
spp.
(n/%)
0-5
442
70 (15.8)
51 (1.5)
6-10
146
15 (10.2)
44 (30.1)
11-20
103
6 (5.8)
25 (24.1)
21-50
407
5 (1.2)
70 (17.2)
> 50
225
2 (0.8)
45 (20.0)
Total
1,323
98
235
Age (years)
Overall, parasitism was higher in male patients (32.6%) than in
females (25.5%) (95% CI = 1.1 to 1.79, p < 0.005) and higher in the
range age of 6-10 years (95% IC = 1.48-3.60; p < 0.001). Interestingly,
parasitism of Blastocystis spp. was significantly higher in patients
presenting over six years of age (p < 0.001, Table 2). The analysis of
parasitism by G. intestinalis or Blastocystis spp. by age in relation to
gender showed no significant difference.
The unique presence of Blastocystis in feces occurred in 161/1,323
samples (12.1%). The analysis of the consistency of feces at the moment
of the examination showed that, among the solid samples, softened and
liquid, 12.0% (107/892), 15.8% (34/215) and 8.0% (6/75), respectively,
were positive exclusively for Blastocystis spp., showing no statistical
difference (p = 0.152). The information of the consistency of 14 stool
samples with exclusively positivites for Blastocystis sp was not taken.
The analysis by Ritchie’s method was more sensitive for the diagnosis
of Blastocystis (92.8%) than that by direct microscopy (89.8%), with a
ratio of 97.7% agreement (Kappa = 0.92).
DISCUSSION AND CONCLUSIONS
In this study the occurrence of intestinal infections by protozoa and/
or helminths in Uberaba (MG) was of 28.7%. However, only 10.4% of
the stool samples presented some pathogenic parasite, in which 7.4%
corresponded to the infection by Giardia that occurred mainly in children
between 0-10 years of age. These data are in accordance with other
studies carried out in several regions of Brazil11,14,17. Regarding age and
gender, the presence of intestinal parasites was higher in male children
aged between the ages of six and 10 years. In relation to parasitism by
Blastocystis (17.8%) it was higher in patients over six years of age and
had no direct relation to gender. G. intestinalis infection presented no
correlation with gender either. Regarding age, the data agrees with other
studies19 and differs from some authors which showed that Blastocystis
infection was higher in children than in adults9,14,15. In relation to gender,
there is no agreement which indicates that gender shows the highest
occurrence of Blastocystis spp.14,19.
The occurrence of Blastocystis spp. infection was higher when
compared with all other parasites, an observation that corroborates
other studies4,6,9,12,16. The exclusive occurrence of Blastocystis in 8% of
diarrheal stools suggests that it may have a pathogenic character, as some
authors agree19,21. Some authors observed the presence of Blastocystis
in stool samples from HIV-infected, homosexuals, travelers, day care
children, animal handlers, and mentally handicapped individuals2,16.
Besides, in immunocompromised patients, the parasite must be
considered pathogenic and patients should be treated accordingly for
Blastocystis if no other pathogens are detected2,16. According to them,
the pathogenicity of Blastocystis is possibly associated with low host
immunity, modified intestinal microbiota, and concomitant presence
of irritable bowel syndrome and the virulence of the parasite strain.
According to CHANDRAMATHI et al. (2014), pathogenicity may also
be host stress dependent, which would lead to a suppression of both
immune responses and to the oxidant-antioxidant regulatory system.
However, more studies are needed to exclude other possible causes of
diarrhea, such as rotavirus infection or metabolic disorders.
In the literature, several authors suggest that the search of
this protozoan via the direct method6,9,12, trichrome staining and
cultivation1,10,21, states that the concentration methods should not be
employed for observation of B. hominis as they destroy cell morphology.
In this study, both methods, direct and Ritchie’s showed higher sensitivity
to 89.8%, with a high agreement percentage (97.7%, kappa = 0.92),
being appropriate to the diagnosis of Blastocystis. Although the culture
is efficient, its cost is higher than the direct method, which has good
sensitivity for detecting Blastocystis, since the vacuolar shapes of this
parasite are usually released in large amounts in feces. In the authors’
experience and unlike that of other authors1, staining of fecal smears for
direct identification of Blastocystis from feces is not easy to analyze,
as the microscopist needs experience to obtain a good result. Thus, the
Ritchie’s method is a good concentration method, as it is fast and effective,
as demonstrated by other authors14. The HPJ method is also effective if
used to dilute 3.7% of formaldehyde stools, since the water breaks the
vacuolar, granular and amoeboid shapes of the parasite.
Infection with non-pathogenic parasites (Endolimax nana,
Entamoeba coli, Chilomastix mesnilli) occurred in 7.3% of the samples
(Table 1). Human infection by non-pathogenic protozoa has been reported
by several authors in Brazil7,13,14,22 and it highlights the need of their
own reports in laboratory reports, therefore it should be considered as
an indicator of fecal contamination of food and water consumed by the
population.
In conclusion, the occurrence of Blastocystis spp. in Uberaba (MG)
was high, this scenario indicates the importance of investigating the
main route of parasite transmission and their association with the clinical
symptoms manifestation. Furthermore, this study showed that the direct
and Ritchie’s method were effective for the diagnosis of Blastocystis
spp. and that there is a need for the description of commensal protozoa
in laboratory reports and for the training of laboratory technicians to
improve in order to detect it.
RESUMO
Ocorrência de Blastocystis spp. em Uberaba, Minas Gerais, Brasil
Parasitos intestinais são um problema de saúde pública no mundo e
a infecção por Blastocystis, protozoário de patogenicidade controversa,
é uma das mais frequentes. Nesse estudo foi investigada a ocorrência
de parasitos intestinais em pacientes atendidos na Universidade Federal
do Triângulo Mineiro, em Uberaba (MG), com ênfase em Blastocystis,
pelos métodos parasitológicos direto e de Ritchie. Foram examinadas
fezes de 1.323 pacientes de abril/2011 a maio/2012. Em 28,7% deles
foi identificado um parasito intestinal, sendo Blastocystis spp. (17,8%)
e Giardia intestinalis (7,4%) os mais frequentes. A ocorrência de
parasitismo foi maior em crianças de 6-10 anos e a infecção por
Blastocystis spp. foi maior acima de seis anos (p < 0,001). Presença
exclusiva de G. intestinalis e de Blastocystis spp. foi observada em 5,4%
e 12,2% dos pacientes, respectivamente, sendo que dos pacientes com
fezes diarreicas, 8% apresentavam parasitismo exclusivo por Blastocystis
spp. Outros parasitos intestinais observados em crianças foram Ascaris
lumbricoides (0,3%) e Entamoeba histolytica/dispar/moshkovskii
(1,4%). O método de Ritchie foi mais sensível (92,8%) que o direto
(89,8%), com alta concordância entre eles (97,7%, kappa = 0,92). Em
conclusão, a ocorrência de Blastocystis spp. em Uberaba é elevada e foi
observada a presença de fezes diarreicas com parasitismo exclusivo por
Blastocystis spp.
ACKNOWLEDGMENTS
The authors would like to thank Oberdan Ricardo Ribeiro, the
laboratory technician at Hospital de Clínicas at UFTM for his assistance
in the collection and processing of samples.
Financial support: FAPEMIG- Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (APQ04094-10).
AUTHOR’S CONTRIBUTIONS
MCS and ALP were responsible for the experimental design of the
study; ENC and RAC were responsible for the execution techniques and
parasitological examination of stools along with MCS and MBOs. GANN
was responsible for the statistical analysis and DC for the attending and
for the referral of the patients. All authors reviewed and contributed to
the writing of this manuscript. MCS is responsible for the manuscript.
CONFLICT OF INTERESTS
No conflict of interests was declared.
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57(3):215-220, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300006
SAINT LOUIS ENCEPHALITIS VIRUS IN MATO GROSSO, CENTRAL-WESTERN BRAZIL
Letícia Borges da Silva HEINEN(1), Nayara ZUCHI(1), Otacília Pereira SERRA(1), Belgath Fernandes CARDOSO(1),
Breno Herman Ferreira GONDIM(2), Marcelo Adriano Mendes dos SANTOS(3), Francisco José Dutra SOUTO(1),
Daphine Ariadne Jesus de PAULA(1), Valéria DUTRA(1) & Renata DEZENGRINI-SLHESSARENKO(1)
SUMMARY
The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible
for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial
diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may
eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely
dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum
samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012.
Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools
containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV
was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple coinfection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might
have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was
positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates
both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state
of Pará. This is the first report of SLEV molecular identification in MT.
KEYWORDS: Arbovirus; Molecular epidemiology; SLEV; Dengue virus; DENV; Virological surveillance; Tropical diseases.
INTRODUCTION
Saint Louis encephalitis virus (SLEV) is a recognized human
pathogen classified in the Japanese encephalitis virus complex, Flavivirus
genus, Flaviviridae family, circulating in the Americas. SLEV is an
arbovirus maintained by zoonotic cycles involving Culex (Cx.) spp.
and other mosquitoes as vectors; birds as amplifiers; humans and other
animals as accidental final hosts7. Most human infections are subclinical.
Some are unspecific acute febrile infections rarely accompanied by
meningoencephalitis with increased severity and fatality in the elderly24.
Reports of human infection in Brazil are scarce. The first report
of human infection in Brazil was evidenced in Pará, in 197016. In the
1990’s, detection of anti-SLEV antibodies, including seroconversion, was
reported in residents of an ecological reserve in Vale do Ribeira, SP19.
A few human cases have been reported more recently: in a woman from
the city of São Paulo, SP, 200418 and in 20 patients from São José do Rio
Preto (SP), two years later12,13,23. One case was identified in a suspected
dengue fever patient from Ribeirão Preto (SP) in 20149. SLEV infection
might not be rare in humans, but instead is often mistaken with dengue
virus (DENV) and goes largely undiagnosed in Brazil.
SLEV is widely dispersed throughout the New World, from Canada
to Argentina. However, clinical infection has become more frequent in
the United States of America (USA) and, to a lesser extent, in Central and
South America25. The virus was first reported during a human encephalitis
outbreak in Saint Louis, USA, in 193326. SLEV was first identified in
Brazil in the 1960’s in Sabethes belisarioi pools from the state of Pará
(PA), in the northern region of the country16,24. Later, between 1967 and
1969, it was detected in sentinel mice, sylvatic rodents and birds in the
state of São Paulo (SP)8.
Serological studies to estimate SLEV prevalence in the population of
Central Brazil are limited in number. Often, antigenic similarity between
DENV, SLEV and other flaviviruses compromises seroprevalence studies
due to cross-reactions. A few reports indicate 5% seroprevalence in
northern and southeastern Brazil20. Seroprevalence studies with SLEV
demonstrate that prevalence ranges from 3-43% within the Brazilian
population9. Acute clinical infections are rarely reported in Brazil,
possibly because humans are accidental or final hosts, and infections
are frequently mild or unapparent, accompanied by transient low-titer
(1) Programa de Pós-Graduação em Ciências da Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso (FM/UFMT), Cuiabá, Mato Grosso, Brazil.
(2) Curso de Graduação em Medicina, Faculdade de Medicina, Universidade Federal de Mato Grosso (FM/UFMT), Cuiabá, Mato Grosso, Brazil.
(3) Laboratório Central de Saúde Pública do Mato Grosso, MT-Laboratório, Secretaria Estadual de Saúde, Cuiabá, Mato Grosso, Brazil.
Correspondence to: Renata Dezengrini Slhessarenko, Departamento de Ciências Básicas em Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso, Av Fernando Correa da
Costa 2367, CCBS-I, sala 82, 78060-900 Cuiabá, Mato Grosso, Brasil. Tel: +55-65-9213-7333; Fax: +55-65-3615-8863. E-mail: [email protected]
HEINEN, L.B.S.; ZUCHI, N.; SERRA, O.P.; CARDOSO, B.F.; GONDIM, B.H.F.; SANTOS, M.A.M.; SOUTO, F.J.D.; PAULA, D.A.J.; DUTRA, V. & DEZENGRINI-SLHESSARENKO,
R. - Saint Louis encephalitis virus in Mato Grosso, Central-Western Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 215-20, 2015.
viremia, and because routine differential diagnosis is not available12.
Despite these difficulties, molecular approaches are important tools for
population screening and the monitoring of flaviviruses12.
Among domestic animals, serologic evidence of SLEV circulation
in horses has been reported in different states across Brazil, including
Minas Gerais (MG), Rio de Janeiro (RJ), Mato Grosso do Sul (MS),
Paraíba (PB), São Paulo (SP) and Pará (PA)15,17,20,21. Infected horses
rarely develop clinical symptoms. However, one fatal neurological case
of SLEV in a horse was recently described in MG20. Detection of SLEV
in birds and serological evidence in horses from the state of Mato Grosso
(MT) has also been reported14,18. Concerning vector species, SLEV is
frequently identified in arthropods in the Amazon18 and other regions
of the country. In 1993, SLEV was identified in Anopheles triannulatus
and Culex spp. in northwestern São Paulo19. Usually, mosquito species
serving as SLEV vectors vary according to geographical region. Cx.
pipiens, Cx. quinquefasciatus and Cx. negripalpus are the most frequently
involved in SLEV transmission in the Americas. The virus persists in
Culex spp. and these mosquitoes have been thought of responsible for
SLEV maintenance between seasons10.
DENV is the most common flavivirus worldwide, representing an
important health public problem. Reports of dengue fever outbreaks
are frequent in Brazil. During epidemics, laboratory diagnosis of nonspecific acute febrile illnesses has been directed towards this flavivirus
infection, hindering the detection of other arboviruses. The aim of this
study was to investigate, using molecular approaches, other flaviviruses
possibly circulating in MT.
Human and arthropod sampling: After receiving approval from the
institutional Ethics Committee (CEP/HUJM/100/2011), serum samples
and epidemiological data were obtained from 604 patients in 20 cities
across MT who sought medical care between October, 2011 and July,
2012 for acute febrile illnesses lasting less than five days. Also, 3,433
female Culex spp. were captured with Nasci aspirators from 184 censitary
sectors of Cuiabá between January and May, 2013, identified using GPS
locators. Three places were sampled at each sector. These Culicidae were
identified according to dichotomy keys3,4 and a nested-PCR for Culex
quinquefasciatus22 and allocated to 409 pools of between one and ten
mosquitoes; 403 with 3,425 Cx. quinquefasciatus, five with seven Cx.
bidens or Cx. interfor and one with one female of Culex spinosus.
Flaviviruses detection: Viral RNA from patient serum and total
RNA from mosquito pools were extracted according to manufacturers’
instructions (QIAamp Viral RNA Mini Kit, Qiagen and Trizol, Invitrogen,
respectively). Extracted RNA was subject to a multiplex semi-nested
reverse transcription PCR (RT-PCR) for a nucleotide region of flaviviruses
NS5 gene (958 bp), followed by a species-specific secondary reaction
differentiating 11 flaviviruses, as previously described 1. Flaviviruspositive samples were confirmed by at least two independent single
reactions with the same forward and species-specific reverse primer. PCR
products were then submitted to nucleotide sequencing (3500 Genetic
Analyzer, Applied Biossystems, USA). RNA from the SLEV strain
genotype V-B BeH 355964 and no template were included as controls
in all the reactions. Nucleotide sequences obtained from the positive
control were analyzed to exclude contamination.
216
The minimum infection rate (MIR) was calculated with the formula
([number of positive pools / total specimens tested] x 1000), considering
the total of Culex quinquefasciatus specimens tested (3,433 mosquitoes).
SLEV-positive samples were subjected to inoculation in C6/36 cells16.
Nucleotide and amino acid sequence analysis of an envelope gene
region from SLEV: A region of the SLEV envelope gene (477 bp) was
amplified in positive samples via semi-nested RT - PCR and sequenced
for phylogenetic analysis6. A phylogenetic tree was constructed with the
neighbor-joining method, based on the Tamura-Nei distance model and
1,000 bootstrap replicates (Geneious R7 7.1.7, USA) using reference
SLEV sequences from the GenBank database (PubMed, NCBI, USA).
Deduced amino acid sequences were also analyzed (Geneious R7 version
7.1.7; Molecular Evolutionary Genetics Analysis version 5.05, USA),
including residues present at specific positions characteristic of SLEV
lineages10.
Nucleotide sequences obtained in this study were deposited
at GenBank, pubMed (accession numbers: KJ699354; KJ957827;
KJ847419; KJ801827).
RESULTS
Clinical and epidemiological findings: During this transversal
observational study, three patients from the metropolitan area of Cuiabá
who tested positive for DENV without neurological or hemorrhagic
manifestations were also positive for SLEV. The first patient, a 47-yearold woman working in general services, sought medical care at a local
hospital in May, 2012 with hyperthermia and posterior neck pain. She
was also positive for DENV-1 and DENV-4 by RT-PCR, constituting a
triple co-infection. DENV-4 was isolated from the serum of this patient.
The second patient, a 55-year-old male civil engineer, was treated at
the same hospital on the same day for hyperthermia, headache, emesis
and epigastric pain. The third patient was a 10-year-old male school pupil
who sought medical care in the city of Várzea Grande in March 2012.
Though clinical information was not available in his case, both patients
were also positive for DENV-4 by RT-PCR.
All patients were urban residents without history of traveling or visits
to sylvatic or rural areas. None of the patients reported previous cases of
a similar disease. Attempts to recover SLEV from the co-infected serum
were unsuccessful.
One pool containing a single Cx. quinquefasciatus female was
positive for SLEV in the metropolitan area of Cuiabá, with a MIR of
0.29 per 1000 specimens of this species.
The vast majority of the patients included in this study were positive
for DENV serotypes (331/604 patients, 54.8%) and were admitted during
an epidemic coinciding with the introduction of the DENV-4 serotype
in MT. The hyperendemicity of the four serotypes in Cuiabá, MT, nine
co-infections between DENV-1/DENV-4 and one between DENV-2/
DENV-4 are going to be discussed separately.
Analysis of a partial sequence of the envelope gene: Phylogenetic
analysis of an envelope glycoprotein gene region with 477 bp showed that
SLEV identified in a female Cx. quinquefasciatus mosquito (SLEV_BR/
MT-CbaAr499/2013) and in human (SLEV_BR/MT-CbaH364/2012)
samples in the present study belong to genotype V-A.
Envelope amino acid sequences from SLEV genotypes II, V and VIII
retrieved from GenBank were compared with the human SLEV_BR/
MT-CbaH364/2012 and the mosquito SLEV_BR/MT-CbaAr499/2013
amino acid sequences. Variations in amino acid residues previously
described as specific for strains belonging to lineages II, V and VIII were
not present in the partial envelope gene sequence obtained here10. None
of the variations described for other lineages in positions present in the
partial envelope amino acid sequences analyzed were observed in the MT
samples. A high homology was observed between the partial envelope
amino acid sequences obtained from the female Cx sp. SLEV_BR/
MT-CbaAr499/2013 and the human SLEV_BR/MT-CbaH364/2012.
However, the human SLEV_BR/MT-CbaH364/2012 presented a leucine
residue at the position 96, whereas all the other amino acid sequences
from genotypes II, V and VII presented a proline residue. The SLEV_BR/
MT-CbaAr499/2013 obtained from mosquito exhibited an amino acid
substitution for asparagine at position 45, whereas all the other SLEV
strains in this study, including the human SLEV_BR/MT-CbaH364/2012,
have a lysine residue at the same position (Fig 3).
be circulating in MT. Although under-notification is common, data
from the Notifiable Diseases Information System in 2012 included
44,814 notifications of dengue fever cases in MT, 10,742 of them in
Cuiabá and 3,133 in Várzea Grande, attributed to DENV-4 (96.2 %)
and DENV-1 (3.8 %)2,11.
DISCUSSION
Dengue fever outbreaks occur frequently in Brazil, including in
MT. However, testing samples from febrile patients solely for DENV
and the yellow fever virus could mean that other flaviviruses silently cocirculating in the region may go undetected. For this reason, differential
diagnosis during dengue outbreaks should be performed routinely.
To the authors’ knowledge, this is the first report of SLEV and DENV4 co-infections in Brazilian patients, including a DENV-1, DENV-4 and
SLEV triple infection. Clinical complications were not identified at the
time of sample collection. Therefore, it was not possible to determine
the etiology of the acute febrile illness. DENV-3 and SLEV co-infection,
accompanied by hemorrhagic manifestations without increased severity,
was previously reported during a dengue fever epidemic in the city of
São José do Rio Preto, SP, southeastern Brazil12.
Fig 1 - Distribution of patients with acute febrile illness in the state of Mato Grosso between
2011 and 2012 tested for flaviviruses species by multiplex semi-nested RT-PCR. Cities with
patients who tested positive for dengue virus (DENV) serotypes 1 and 4 and Saint Louis
encephalitis virus (SLEV) are identified.
SLEV infection may be underestimated in MT. Only patients with
acute febrile illness for less than five days were included in the present
study. Further studies to estimate seroprevalence in the population, as
well as to identify the vector species transmitting the virus, are a matter
to be addressed shortly.
SLEV is currently classified by eight lineages, 15 subtypes, based
on envelope gene or genome sequences. These lineages correlate with
the geographical distribution of the virus. The Brazilian strains reported
so far belong to SLEV genotypes II, III, V, and VIII (subtypes A and
B), with V and VIII being the most prevalent in the Amazon region18.
One Cx. quinquefasciatus female captured in the Bela Vista
neighborhood of Cuiabá was positive for SLEV (MIR = 0.29). Aedes and
Culex spp. are involved in DENV and SLEV transmission, respectively24.
The co-infections described here likely resulted from exposure to both
infected mosquito species. However, several other mosquito species have
been described as SLEV vectors.
Phylogenetic analysis shows that SLEV identified in the present
study within humans (SLEV_BR/MT-CbaH364/2012) and mosquitoes
(SLEV_BR/MT-CbaAr499/2013) belong to genotype V-A, closely related
to isolates from animals in the Amazon region, in the state of Pará (Fig.
2). Previously, genotype VIII-B was isolated from birds in the Amazon
in MT in 197418.
Although SLEV has been detected in both urban and sylvatic
environments of Brazil and that Amazônia and Pantanal constitute the
majority of territory in Mato Grosso, the three human cases reported
here were detected within a large metropolitan area, located in the
Cerrado biome (Fig 1). These cases were reported during a large
DENV-4 outbreak, indicating that flaviviruses besides DENV may
The phylogenetic tree, constructed with strains belonging to the
eight lineages of SLEV, demonstrates a common ancestry between
the human SLEV_BR/MT-CbaH364/2012, mosquito SLEV_BR/MTCbaAr499/2013 and SLEV isolates from PA and Argentina belonging to
genotype V. The MT SLEV samples clustered with a bootstrap value of
98%, originating a clade in genotype V. They also indicated a homology
217
Fig 2 - Phylogenetic tree of envelope gene sequences from SLEV_BR/MT-CbaH364/2012, SLEV_BR/MT-CbaAr499/2013 and Saint Louis encephalitis virus (SLEV) genotypes deposited
at GenBank (NCBI), using the neighbor-joining method, Tamura-Nei distance model and 1,000 bootstrap replicates. Outgroups included Japanese encephalitis virus (JEV), West Nile virus
(WNV), and dengue virus 1 (DENV-1).
of 99% in the nucleotide sequence, suggesting that the same virus may
be circulating in vectors and hosts. Although in the present study (Fig.
2), the cluster within lineage V is not supported by a high bootstrap value
(65.9%), similar results were described by others when analyzing partial
envelope sequences of SLEV strains belonging to the same lineage10,23.
The distance between the isolates in the phylogenetic tree belonging
to lineage V ranged from 0.008% between the two samples from MT,
to 0.060% among the BeAn288398 and BeAn203235 strains, from
genotypes VA and VB, respectively.
Central and South American strains and some North American isolates
from California and West Texas10. Genotype V-A, dispersed throughout
the Americas, was already reported in Brazil, Argentina, Peru and
Trinidad & Tobago in vertebrate hosts and arthropod vectors. In Brazil,
this genotype has been detected in different cities of PA and the state
of Rondônia. Genotype V-B was only reported in PA18. Genotype VIII
subtypes A and B are frequent in the Brazilian Amazon and, genotype
VIII-B was already isolated from Amazon region birds of MT in 1974
and from one horse with a neurological disease from MG in 201318,20.
The SLEV samples circulating in MT demonstrated a greater
similarity to the BRA PA BeAn259507 isolate. This isolate belongs to
genotype V-A, obtained from domestic birds in Altamira, PA. SLEV
strains obtained from animals in Belém (BR PA An BeAn248398, BR
PA An BeAn246407 and BRA PA An BeAn259507) and from Culex
spp. in Argentina (ARG Ar 78v6507) are allocated in the same branch
as they belong to the same genotype.
The analysis of the envelope amino acid sequences revealed a high
homology between the human SLEV_BR/MT-CbaH364/2012 and
the mosquito SLEV_BR/MT-CbaAr499/2013. None of the variations
described for other lineages in positions that were present in the
analyzed envelope amino acid partial sequence were observed in the MT
samples10. The leucine residue at position 96 in the human SLEV_BR/
MT-CbaH364/2012, whereas all the other studied amino acid sequences
from genotypes II, V and VII presented a proline residue, has already
been described in the human SLEV sample from São José do Rio
Preto, SP23. Additionally, the SLEV_BR/MT-CbaAr499/2013 obtained
from mosquitoes showed an asparagine at position 45, whereas all the
other SLEV strains analyzed in the present study, including the human
SLEV_BR/MT-CbaH364/2012, have a lysine residue at the same position
(Fig. 3). The homology between the human and arthropod SLEV samples,
identified in MT, indicates that the same virus is perhaps circulating in
both vector and vertebrate host populations.
Although SLEV circulates between arthropod vectors, mammalian
and avian hosts, isolates of the virus generally do not exhibit a high
level of genetic diversity; indeed, the most diverse isolates have a 10.1%
nucleotide divergence and, strains within each lineage show less than
5.5% nucleotide divergence10. In this regard, the sequences included
in the study from genotypes VA and VB indicate nucleotide similarity
between 96.1 and 99%.
The most prevalent genotypes in Brazil are V and VIII, existing
throughout the Amazon basin, mainly in PA18. Lineage V is composed of
218
Birds are believed to carry SLEV to different regions, and may be
Fig 3 - Alignment of deduced amino acid envelope protein (partial sequence) of Saint Louis encephalitis virus (SLEV) obtained from a human with acute febrile illness (SLEV_BR/MTCbaH364/2012) and a Culex quinquefasciatus female (SLEV_BR/MT-CbaAr499/2013) compared to reference SLEV strains available at GenBank database. Amino acid substitutions unique
to the samples from Mato Grosso are in bold.
responsible for introducing SLEV V-A in MT, due to migratory routes
from the Amazon to Pantanal. Culex mosquitoes are abundant in MT,
and most likely maintain viral transmission between birds as well as
sporadic transmission to horses and humans.
SLEV infections in humans may occur sporadically in MT and
be more frequent than observed in this study, conducted only with
patients who sought medical care during a dengue outbreak. The
absence of routine differential diagnosis may contribute to the lack
of previous reports. Therefore, these findings indicate the necessity
for broad-spectrum clinical-epidemiological investigations during
dengue outbreaks. Active surveillance of arboviral circulation should
be routinely performed in MT in the imminence of introduction
or reintroduction of these viruses. Additional studies involving
other animal species, birds and vector mosquitoes are necessary
to comprehend the epidemiological cycle and magnitude of SLEV
circulation in MT.
RESUMO
Vírus da encefalite de Saint Louis em Mato Grosso, Centro-Oeste,
Brasil
O vírus da dengue (DENV), frequentemente envolvido em
epidemias de grande proporção, e o vírus da febre amarela (YFV),
responsável por surtos silvestres esporádicos, são considerados os
flavivírus circulantes mais importantes no Brasil. Por este motivo, o
diagnóstico laboratorial de doença febril aguda indiferenciada durante
períodos epidêmicos é frequentemente direcionado para dengue e
febre amarela no país, dificultando a detecção de outros arbovírus
possivelmente circulantes, incluindo o vírus da encefalite de Saint
Louis (SLEV), que é amplamente disperso nas Américas. O objetivo
deste estudo foi investigar molecularmente a presença de 11 flavivírus
no soro de 604 pacientes durante grande epidemia de dengue no
estado de Mato Grosso (MT), Centro-Oeste do Brasil, entre 20112012. Concomitantemente, 3.433 fêmeas de Culex spp. capturadas
com aspirador de Nasci na cidade de Cuiabá, MT e alocadas em 409
pools com 1-10 mosquitos em 2013 foram testadas por multiplex seminested RT-PCR para os mesmos flavivírus. O SLEV foi detectado em
três pacientes co-infectados com o DENV-4 das cidades de Cuiabá e
Várzea Grande, MT. Um dos pacientes apresentava tripla co-infecção
com DENV-1. Nenhum paciente referiu histórico recente de viagem
ou acesso a áreas rurais/silvestres. Um pool contendo uma fêmea de
Culex quinquefasciatus foi positivo para o SLEV, apresentando taxa de
infecção mínima (MIR) de 0,29 por 1000 espécimes desta espécie. A
análise filogenética indica que ambas as amostras formam um cluster
com isolados do genótipo V-A do SLEV obtidos de animais na região
amazônica do estado do Pará. Este é o primeiro relato de identificação
molecular do SLEV no MT.
ACKNOWLEDGMENTS
The authors thank Ana E. Viniski, Sumako U. Kinoshita (LACEN/
MT, SES, Cuiabá), Liliana V. A. Correa, (FM, UFMT, Cuiabá) for
their assistance. In addition, they thank Fernanda C. Pereira, a medical
graduate student, for her scientific training, Roberta V. M. Bronzoni
(UFMT Sinop) for providing RNA of the SLEV positive control and
Mauricio L. Nogueira (FAMERP) for training and discussion of the
results.
FINANCIAL SUPPORT
This study was supported by the National Council for Scientific and
Technological Development (CNPq; grant 472890/2011-5). NZ, LBSH,
OPS and BFC were recipients of the Coordination for the Improvement
of Higher Education Personnel (CAPES) scholarships; FCP and BHFG
were recipients of the UFMT scientific initiation scholarships.
219
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57(3):221-225, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300007
LACK OF ASSOCIATION BETWEEN HERPESVIRUS DETECTION IN SALIVA AND GINGIVITIS IN
HIV‑INFECTED CHILDREN
Renata A. OTERO(1), Flávia N.N. NASCIMENTO(1), Ivete P.R. SOUZA(1), Raquel C. SILVA(2), Rodrigo S. LIMA(2), Tatiana F. ROBAINA(2),
Fernando P. CÂMARA(2), Norma SANTOS(2) & Gloria F. CASTRO(1)
SUMMARY
The aims of this study were to compare the detection of human herpesviruses (HHVs) in the saliva of HIV-infected and healthy
control children, and to evaluate associations between viral infection and gingivitis and immunodeficiency. Saliva samples were
collected from 48 HIV-infected and 48 healthy control children. Clinical and laboratory data were collected during dental visits and
from medical records. A trained dentist determined gingival indices and extension of gingivitis. Saliva samples were tested for herpes
simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV)
by nested polymerase chain reaction assays. Thirty-five HIV-infected and 16 control children had gingivitis. Seventeen (35.4%) HIVinfected children and 13 (27%) control children were positive for HHVs. CMV was the most commonly detected HHV in both groups
(HIV-infected, 25%; control, 12.5%), followed by HSV-1 (6.2% in both groups) and HSV-2 (HIV-infected, 4.2%; control, 8.3%).
The presence of HHVs in saliva was not associated with the presence of gingivitis in HIV-1-infected children (p = 0.104) or healthy
control children (p = 0.251), or with immunosuppression in HIV-infected individuals (p = 0.447). Gingivitis was correlated with HIV
infection (p = 0.0001). These results suggest that asymptomatic salivary detection of HHVs is common in HIV-infected and healthy
children, and that it is not associated with gingivitis.
KEYWORDS: HIV infection; Herpesvirus; Periodontitis; Gingivitis; Children.
INTRODUCTION
Herpesviruses are large DNA-enveloped viruses belonging to
the Herpesviridae family. Herpesviruses are highly disseminated in
nature. Of more than 200 known, eight are human pathogens: herpes
simplex virus 1 (HSV-1), herpes simplex 2 (HSV-2), varicella zoster
virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV),
and human herpesviruses 6, 7 and 8 (HHV-6, -7, -8)23. Transmission
occurs by contact, and primary infections generally occur early in life,
followed by persistence of the virus in the organism. Herpesvirus diseases
occur primarily in immunosuppressed individuals; fatal infections in
immunocompetent hosts are rare23.
Several studies have implicated herpesviruses in the etiology of
periodontitis26-29. Apparently, periodontal tissue breakdown occurs
more frequently and progresses more rapidly in herpesvirus-infected
than in herpesvirus-free periodontal sites 26-29. Herpesviruses may
cause periodontal pathosis as a direct result of virus infection and
replication, or as a consequence of virally induced impairment of
periodontal immune defenses, resulting in heightened virulence of
resident bacterial pathogens8,26. The herpesviral-bacterial hypothesis
of periodontitis development proposes that active herpesvirus infection
initiates periodontal tissue breakdown and that host immune responses
against the herpesvirus infection are important components of the
etiopathogeny of the disease28. The herpesvirus infection triggers the
release of proinflammatory cytokines, which have the potential to activate
osteoclasts and matrix metalloproteinases and to impair antibacterial
immune mechanisms, causing an upgrowth of periodontopathic bacteria28.
High frequencies of EBV and CMV genomes have been noted
in adults with progressive periodontitis, in localized and generalized
aggressive (juvenile) periodontitis, HIV-associated periodontitis, acute
necrotizing ulcerative gingivitis, periodontal abscesses, and some rare
types of advanced periodontitis associated with medical disorders26.
Other herpesviruses such as HHV-6, HHV-7, HHV-8, and HSV-1, have
also been associated with periodontitis4,15,19. In contrast, HSV-2 appears
to be uncommon at periodontal sites7,32. However, the pathogenesis of
herpesviruses in periodontitis has not yet been fully elucidated.
Human herpesviruses (HHVs) have often been detected in the saliva
(1) Department of Pediatric Dentistry, School of Dentistry, Universidade Federal do Rio de Janeiro, RJ, Brazil.
(2) Department of Virology, Microbiology Institute, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Correspondence to: Norma Santos, Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS - Bl. I, Ilha do Fundão, 21941902 Rio de Janeiro, RJ, Brasil. Phone: 55 21 2560-8344 extension 165, Fax: 55 21 2560-8028. E-mail: [email protected]
OTERO, R.A.; NASCIMENTO, F.N.N.; SOUZA, I.P.R.; SILVA, R.C.; LIMA, R.S.; ROBAINA, T.F.; CÂMARA, F.P.; SANTOS, N. & CASTRO, G.F. - Lack of association between herpesvirus
detection in saliva and gingivitis in HIV-infected children. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 221-5, 2015.
of HIV-infected individuals4,5,7,11,12,15,18, and several studies have shown
that highly active antiretroviral therapy (HAART) does not significantly
reduce the prevalence or the load of HHVs in saliva11,17,22,32. The elevated
frequency of HHVs infections in association with periodontitis in HIVinfected individuals7,8,11,12 suggests that these viruses play a role in the
disease. In children, the prevalence of some oral manifestations associated
with HIV infection was reduced after HAART initiation. However, other
lesions emerged24, as these individuals are prone to develop opportunistic
viral infections, especially those caused by Herpesviridae family
members, in the oral mucosa. Little information is available on HHVs
co-infection in the saliva of HIV-infected children.
periodontal probe. Gingivitis was considered to be present when gingival
bleeding occurred on probing1. The extension of gingivitis was classified
according to the System for the Classification of Periodontal Diseases
and Conditions2; patients with gingivitis at <30% of sites surveyed were
classified as having localized gingivitis, and those with >30% of surveyed
sites affected were classified as having generalized gingivitis.
The aims of this study were to detect HHVs in the saliva of HIV1-infected children in comparison with healthy control children, and to
evaluate possible associations between viral infection and gingivitis and
immunodeficiency stage.
Sample processing: The saliva samples were centrifuged and
pelleted, and DNA was extracted using the Wizard Genomic DNA
purification kit (Promega, Madison, WI, USA) according to the
manufacturer’s instructions.
Virus detection: All samples were subjected to human β-globin
gene amplification to determine the integrity and quality of extracted
DNA and to avoid false-negative results3. Specimens were analyzed
using conventional polymerase chain reaction (PCR) assays, as described
previously, to detect the presence of HSV-1/2, VZV, EBV, and CMV30.
PCR products were detected using 1.2% agarose gel electrophoresis and
staining with ethidium bromide.
Samples: The ethics committees of the Hospital Universitário
Clementino Fraga Filho and Institute of Pediatrics and Childcare
Martagão Gesteira, Universidade Federal do Rio de Janeiro (UFRJ),
Brazil, approved the study protocol. The parents of all children involved
in the study provided written informed consent in accordance with
Resolution 196/96 of the Brazilian Ministry of Health.
The study population was composed of patients attending the UFRJ
School of Dentistry between August 2009 and July 2010. Participants
were selected by convenience sampling during initial appointments for
dental treatment. The HIV-1-infected group was made up of 48 children
of both sexes, ranging from six to 12 years old, who were patients at
the Institute of Pediatrics and Childcare Martagão Gesteira, UFRJ, with
definitive diagnoses of HIV infection. The following medical history data
were extracted from their medical records: diagnosis of HIV infection,
results of most recent (closest to the day of saliva sample collection;
maximum interval, three months) laboratory tests (viral load, CD4 and
CD8 counts, and CD4/CD8 ratio) and use of anti-retroviral agents (at
the time of saliva sample collection). The immunodeficiency stages of
HIV-infected individuals were defined using CD4 counts, according to
the classification of the Centers for Disease Control and Prevention6.
The control group consisted of 48 healthy children, ranging from
seven to 12 years old, who attended the UFRJ Pediatric Dentistry
Clinic and showed no clinical evidence of systemic or chronic disease.
They were considered clinically healthy because they were receiving
no medical treatment for any disease and showed no clinical sign of
immunosuppression, systemic disease, and/or had no history of a risk
factor for HIV infection. These data were collected through medical
anamnesis with the patients’ parents and the attending physician. Children
in the control group did not undergo testing to confirm serological HIV
negativity because there was no reason to justify this procedure, which
the local ethics committees therefore disallowed.
Prior to saliva sample collection, all children in the HIV-1-infected
and control groups underwent oral and oropharyngeal examinations
by a trained and calibrated dentist to identify oral manifestations such
as gum bleeding, mouth ulcers, oral mucosal lesions, and cervical
lymphadenopathy. The gingival index was assessed using a sterile
222
Individuals were not allowed to brush their teeth or eat for one h
before providing saliva samples. Five milliliters of paraffin-stimulated
saliva were collected in a sterile container. The samples were kept in
an ice-filled cooler and submitted for laboratory analysis within two h.
First-round PCR reactions consisted of the addition of 5 µL of
extracted DNA to 20 µL of PCR mix containing 0.5 µM of each of
the primers HHV-F1 and HHV-R1, 0.125 µM of each of the primers
VZV-F1 and VZV-R1, and 1x PCR buffer; 1.5 mM MgCl2 and 0.2 mM of
deoxyribonucleotide triphosphates; and 2.5 U of GoTaq DNA polymerase
(Promega). First-round PCR was carried out as follows: one cycle at 94
°C for three min, followed by 35 cycles at 94 °C for 45 s, 65.5 °C for one
min, 72 °C for one min, and final extension at 72 °C for seven min. For
nested PCR, 0.5 µL of first-round product was transferred to 25 µL PCR
mix similar to that described above, but containing second-round primers
(HHV-F2, HHV-R2, VZV-F2, and VZV-R2), at the same concentrations
as in the first round. PCR conditions were the same as in the first round,
except that the annealing temperature was changed to 63 °C. Positive and
negative controls were included in each run. Infected cell cultures were
used as positive controls for HSV-1 and HSV-2 (Vero cells), EBV (Daudi
cells), and CMV (MRC-5 cells). For VZV, clinical samples obtained from
patients with varicella diagnoses confirmed by PCR amplification and
sequencing analysis were used as positive controls. Negative controls
consisted of saliva samples previously demonstrated to be HHVs. The
expected sizes of the PCR products for first-round and nested PCRs,
respectively, were: HSV-1/2, 742 and 493 pb; VZV, 650 and 356 pb;
EBV, 748 and 499 pb; and CMV, 817 and 565 pb.
Because some PCR products had very similar sizes, sequencing
analysis was used to confirm their specificity and to differentiate HSV-1 and
HSV-2. Amplified DNA from all HSV-positive samples and three CMVpositive samples was purified using the Wizard SV gel and PCR clean-up
system kit (Promega), and sequences were determined using the BigDye
terminator cycle sequencing kit and the ABI PRISM 3100 automated DNA
sequencer (Applied Biosystems, Foster City, CA, USA) using the same
PCR primers. DNA sequences were edited using the Chromas software
(Technelysium Pty. Ltd., Brisbane, QLD, Australia) and compared with
the DNA sequences available in GenBank (http://www.ncbi.nlm.nih.gov)
using the BLAST tool (http://www.ncbi.nlm.gov/BLAST).
Statistical analysis: Using the Epi-Info statistical program (version
3.5.1., CDC, Atlanta, GA, USA), data from the two groups were
compared using the Mann-Whitney test for means of continuous variables
(age) and the χ2 test and Fischer’s exact tests for categorical variables (sex,
presence of HV types in saliva, and gingivitis). Within the HIV-infected
group, the χ2 and Fischer’s exact tests were used to verify correlations
between the presence of HVs in saliva and immunosuppression, HAART,
and the presence of oral manifestations.
RESULTS
the saliva of HIV-1-infected and healthy children (p = 0.167); however,
HSV-2 was more common in the control group and CMV infection
was more common in immunocompromised HIV-1-infected children.
Sequence analysis confirmed the PCR results and allowed differentiation
between HSV-1 and HSV-2 strains.
Table 2
Herpesviruses detected in saliva from HIV-1–infected and healthy control
children
Virus1
The mean age of the 48 HIV-1-infected children was 9.58 years;
45.8% of these subjects were male, 70.8% were receiving HAART, and
52.1% had no immunosuppression. The mean age of healthy control
children (47.9% male) was 9.04 years. There groups did no differ in
terms of age or sex (p > 0.05). Other clinical and medical data from the
study subjects are shown in Table 1.
HSV-1
Variable
Age (years), mean
(range)
Sex
HAART
HIV-1-infected
subjects
Control subjects
9.58 (6-12)
9.04 (7-12)
Male 22 (45.8%)
Male 23 (47.9%)
Female 26 (54.2%)
Female 25 (52.1%)
Yes, 34 (70.8%)
No, 14 (29.2%)
<200; 9 (18.7%)
CD4+ count (cells/µL)
200-499; 14 (29.2%)
>500; 25 (52.1%)
Gingivitis
Yes, 35 (72.9%)
Yes, 16 (33.3%)
No, 13(27.1%)
No, 32 (66.7%)
Candidosis
n=4
-1
Linear gingival
erythema
n=5
-
Angular cheilitis
n=1
-
Oral ulcer
n=1
Oral findings
-
HAART = highly active antiretroviral therapy; -: absence of symptoms.
1
Seventeen (35.4%) of the 48 HIV-1-infected children were positive
for HHVs: 6.2% (3/48) were positive for HSV-1, 4.2% (2/48) for HSV-2,
and 25.0% (12/48) were positive for CMV. In the control group, 13/48
(27.0%) children were positive for HHVs: 6.2% were positive for HSV-1,
8.3% (4/48) for HSV-2, and 12.50% (6/48) were positive for CMV. No
VZV, EBV or co-infection with those viruses was detected in either group
(Table 2). No significant difference was observed in HHVs detection in
3 (6.2)
3 (6.2)
p
1.00
HSV-2
2 (4.2)
4 (8.3)
0.458
CMV
12 (25.0)
6 (12.5)
0.117
Total
17 (35.4)
13 (27.0)
0.167
1
Table 1
Demographic, clinical, and immunological characteristics of children in the
HIV-1–infected and control groups
HIV-1-infected Control subjects
subjects (%)
(%)
N = 48
N = 48
EBV and VZV were not detected.
HIV-1-infected individuals were classified into three immunologic
categories: no evidence of suppression (CD4+ > 500 cells/µL; CD4+ % >
25), moderate suppression (CD4+ = 200-499 cells/µL; CD4+ % [15-24),
and severe suppression (CD4+ < 200 cells/µL; CD4+ % <15)19 (Table 1).
Twelve of 25 (48.0%) children with no evidence of immunosuppression,
2/14 (14.3%) children with moderate immunosuppression, and 3/9
(33.3%) children with severe immunosuppression were HHVs positive.
No correlation was found between HHV infection and the degree of
immunosuppression (p = 0.447).
Eleven of 34 (32.4%) individuals undergoing HAART and 6/14
(42.8%) children not receiving HAART were HHVs positive. However,
no significant correlation between HHVs detection in saliva and receipt
of HAART was observed (p = 0.489).
Thirty-five of 48 (72.9%) HIV-1-infected children had gingivitis
at the time of sample collection; 10 (28.6%) were positive for HHVs
infection. In the control group, six of 16 (37.5%) children with gingivitis
were HHVs positive. No significant correlation between the presence of
HHVs in saliva and the presence and extension of gingivitis was observed
within each group, HIV-1-infected children (p = 0.104) and healthy
control children (p = 0.251), or when the HIV-1-infected group was
compared with the control group (p = 0.491). However, HIV infection
was strongly correlated with gingivitis (p = 0.0001).
Four (8.3%) HIV-1-infected children had candidosis, five (10.4%)
had linear gingival erythema (LGE), one (2.1%) had an oral ulcer, and
one (2.1%) had angular cheilitis (Table 1). One subject with candidosis
and LGE and one subject with angular cheilitis were HSV-1 positive;
one subject with LGE was CMV positive. HHV detection in saliva was
not correlated with any oral symptom.
DISCUSSION
Herpesviruses, most commonly CMV, EBV, and HSV-1, have been
detected in oral samples from immunosuppressed and immunocompetent
individuals with gingivitis7,9,11–13,15,16,18,25,33.
223
Median CMV detection rates in healthy periodontium and in
individuals with gingivitis are about 8% and 33%, respectively27. CMV
has been detected in 25-49% of immunocompetent individuals and
40-62% of HIV-infected individuals with gingivitis11-13,16,33. Previous
studies have not been able to demonstrate a clear association between the
presence of CMV and gingivitis because the virus was detected at high
frequencies in control groups. The present study found CMV in the saliva
of 25% of HIV-1-infected children and 12.5% of healthy control children.
Although CMV was detected more often in immunocompromised
children, CMV could not be clearly associated with gingivitis.
HSV-1 is detected less frequently than CMV and EBV in the saliva of
individuals with periodontitis12,13,25, but its detection has been described in
patients with gingivitis11,16. The present study found HSV-1 in the saliva
of 6.2% (3/48) of subjects from both groups.
HSV-2 is rarely detected in saliva7,20,27,31,32, but it was detected in
both groups in the present study. Two HIV-1-infected boys aged 10
and 11 years were HSV-2 positive; one of these subjects, a severely
immunosuppressed (CD4+ count = 149 cells/µL [11.27%]) boy who
was not undergoing HAART, had gingivitis. The other HIV-1-infected,
HSV-2-positive child had no evidence of immunosuppression (CD4+
count = 724 cells/µL [29%]) and was receiving HAART. Four children
(aged 7-9 years) in the control group were HSV-2 positive; three of them
had gingivitis.
A recent review of HHVs in periodontitis showed that EBV is
detected in association with gingivitis in 20% of cases and with healthy
periodontium in 8% of cases27. Several studies have described EBV
detection rates of 48-90% in the saliva of HIV-infected individuals9,14,16,17
and 17-40% in the saliva of healthy individuals5,11,14-16,22. Surprisingly, the
present study did not detect EBV in HIV-1-infected or healthy children
with or without gingivitis.
Few studies have reported VZV excretion in saliva. Such excretion
is usually observed in stressed individuals20 or those with herpes zoster
manifestations10,21. WANG et al.32 detected VZV DNA in the saliva of
5.1% (3/59) of HIV-positive subjects and 1.9% (1.53) of healthy control
subjects. They detected VZV in individuals undergoing HAART and
concluded that such an event is infrequent in the saliva of asymptomatic
HIV-positive persons and that HAART does not reduce the risk of
asymptomatic VZV excretion 32. Accordingly, VZV has not been
associated with periodontal disease27. Consistent with these findings, the
present study did not detect VZV in its study population.
In this study, HHVs detection in the saliva of HIV-1-infected and
healthy children with and without gingivitis was compared. Although
sample size potentially limits the statistical power of the results, the
study’s findings are comparable to those reported in the literature.
CMV was the most prevalent virus detected in both groups, followed
by HSV-1 and HSV-2. EBV and VZV were not detected in either group.
No association was demonstrated between HHV detection in saliva and
the presence of gingivitis. No association between the detection of HV
DNA in saliva and the level of immunosuppression in HIV-1-infected
children was observed. Moreover, HAART did not seem to reduce virus
shedding. However, a strong correlation between HIV infection and
gingivitis was confirmed.
224
RESUMO
Ausência de associação entre a detecção de herpesvírus na saliva e
gengivite em crianças infectadas pelo HIV
Os objetivos deste estudo foram detectar a presença de herpesvírus
humanos (HHVs) na saliva de crianças infectadas pelo HIV, em
comparação com controles saudáveis e avaliar a associação entre
infecção viral, gengivite e imunodeficiência. Para este fim, foram colhidas
amostras de saliva de 48 crianças HIV-positivas e 48 controles saudáveis.
O índice gengival e extensão de gengivite foram determinados por um
dentista treinado. Informações clínicas e laboratoriais foram obtidas
durante a consulta odontológica e dos registros médicos. As amostras
de saliva foram testadas para detecção de vírus herpes simplex tipos 1 e
2 (HSV-1 e HSV-2), vírus da varicela-zoster (VVZ), vírus Epistein-Barr
(EBV) e citomegalovírus (CMV) através de nested-PCR. Trinta e cinco
crianças HIV-positivas e 16 crianças do grupo controle apresentavam
gengivite. Dezessete (35,4%) crianças HIV-positivas e 13 (27%) crianças
controle testaram positivo para a presença de HHVs. CMV foi o vírus
mais comum detectado em ambos os grupos (25% HIV-positivas e 12,5%
de controle), seguido por HSV-1 (6,2% de ambos os grupos) e HSV-2
(4,2% HIV-positivas e 8,3% de controle). Não houve associação entre
a detecção de HHVs na saliva e a presença de gengivite em ciranças
HIV-positivas (p = 0.104) ou crianças saudáveis (p = 0,251), ou com
imunossupressão em indivíduos HIV-positivos (p = 0,447). Foi observada
uma correlação entre a infecção por HIV e a presença de gengivite (p
= 0,0001). Os resultados sugerem que a detecção salivar assintomática
de HHVs é comum entre crianças HIV-positivas e crianças saudáveis, e
não está associada à gengivite.
ACKNOWLEDGMENTS
This study was supported in part by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação
Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ), Brazil.
The authors thank Soluza dos Santos Gonçalves for technical
assistance.
The authors declare that they have no conflict of interest.
AUTHORS’ CONTRIBUTIONS
GFC, IPRS, and NS conceived and designed the study and analyzed
the data. RAO, FNNN, RCS, and RSL were responsible for data and
sample collection and PCR analysis. All authors have read and approved
the final manuscript.
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225
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57(3):227-232, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300008
INVENTORY OF MOSQUITOES (DIPTERA: CULICIDAE) IN CONSERVATION UNITS IN BRAZILIAN
TROPICAL DRY FORESTS
Cleandson Ferreira SANTOS(1), Alex Chavier SILVA(1), Raquel Andrade RODRIGUES(1), Jamilli Sanndy Ramos de JESUS(1) & Magno Augusto Zazá BORGES(1)
SUMMARY
In Brazil, most studies of the Culicidae family are concentrated in rainforest regions. As such, there is a lack of knowledge regarding
the diversity of Culicidae in regions with different climatic and vegetational characteristics. The aim of this study was to compile an
inventory of Culicidae in protected areas of the semi-arid region of the state of Minas Gerais, Brazil, in order to better understand
the diversity of the family within this region. The study was conducted across four protected areas in the northern region of the state,
in tropical dry forest (TDF) fragments. Sampling methods included Shannon trap and CDC light trap, as well as active collection. A
total of 11,219 mosquito specimens were collected between August 2008 and July 2012, belonging to 11 genera and 45 species; 15
new records for the state of Minas Gerais were registered, as well as 26 new records for semi-arid regions within the state. The high
number of new Culicidae records in this region demonstrates the importance of inventory studies for increasing the knowledge of
culicid biodiversity in Minas Gerais, and in particular within semi-arid regions of the state.
KEYWORDS: Culicidae; Tropical dry forest (TDF); Conservation unit; Semi-arid; Minas Gerais.
INTRODUCTION
Mosquitoes (Diptera: Culicidae) are a group of insects that in their
early stages develop in a variety of aquatic habitats, including permanent
(i.e. rivers and lakes) and transient; transient habitats can include any
receptacle that accumulates water, such as hollow trees, bromeliad tanks,
fallen plant material, and even animal tracks21.
Studies of Culicidae diversity in Brazil were mainly focused on
rainforests in the southeastern and southern regions of the country,
which coincide with the location of major national research centers. The
Amazon rainforest is another important, well-studied region, primarily
because of its significance for the transmission of several diseases, such
as malaria and wild-type yellow fever7. However, the authors remain
unsure of the diversity of mosquitoes in Brazilian regions with different
climatic characteristics and forms of vegetation.
Despite the high diversity of plant and animal species in other
biomes, such as the Cerrado (Savanna) and Caatinga (Semi-arid forest),
there are very few studies of Culicidae diversity in these areas, and in
particular, few in the transition zones between these biomes in northern
Minas Gerais (MG). This region is primarily tropical dry forest (TDF),
characterized by deciduous forest vegetation and a semi-arid climate,
due to low humidity and low rainfall.
The last major survey of Culicidae in Minas Gerais was conducted
in 1962 by MACIEL16. The author compiled his own data with data
from literature, as well as from the former Department of Rural
Endemic Diseases. With this, he created a list of the Culicidae in Minas
Gerais and the municipalities where they were found, as well as the
coordinates of the collection sites. Overall, 119 species of Culicidae
were reported as occurring in 168 municipalities. The upper-middle
area of the São Francisco region appears in this report due to a study
in 1960 by ANDRADE & LEAL1 on Anopheles in the São Francisco
river, which contains two surveys done in the city of Manga in 1947 and
1954. Thereafter, the only published work in northern Minas Gerais was
by GAMA et al.13, in which the authors present a list of Anophelines
collected in the municipality of Varzelândia.
The present study aims to conduct an inventory of the Culicid fauna
in conservation units within a semi-arid region of the state of Minas
Gerais, Brazil, in order to better understand the diversity of Culicidae
in this region.
Samples were collected within four conservation units administered
by the State Forestry Institute (Instituto Estadual de Florestas - IEF).
These areas are in the northern region of Minas Gerais, in the mid-São
Francisco Valley, and are as follows: (1) the Mata Seca State Park MSSP
(Parque Estadual da Mata Seca - PEMS) (14o48’36’’S - 43o55’12’’),
located in the municipality of Manga; (2) the Lagoa do Cajueiro State
(1) Laboratório de Ecologia e Controle Biológico de Insetos, Departamento de Biologia Geral, Universidade Estadual de Montes Claros, Montes Claros, MG, Brazil.
Correspondence to: Cleandson Ferreira Santos. E-mail: [email protected]
SANTOS, C.F.; SILVA, A.C.; RODRIGUES, R.A.; JESUS, J.S.R. & BORGES, M.A.Z. - Inventory of mosquitoes (Diptera: Culicidae) in conservation units in Brazilian tropical dry forests.
Rev. Inst. Med. Trop. Sao Paulo, 57(3): 227-32, 2015.
Park - LCSP (Parque Estadual Lagoa do Cajueiro - PELC) (14o55’08’’S
- 43o56’23’’W) and (3) the Jaíba Biological Reserve - JBR (Reserva
Biológica de Jaíba) (15º3’57.81”S - 43º45’45.03”W), both located in
the municipality of Matias Cardoso; and (4) the Serra Azul Biological
Reserve - SABR (Reserva Biológica de Serra Azul) (15º11’32.20”S 43º54’41.1”W), located in the municipality of Jaíba (Fig. 1).
As the study areas are located within a Caatinga-Cerrado transition
zone, they contain fragments of tropical dry forest (TDF). These
formations are broadly defined as having a vegetation type typically
dominated by deciduous trees (at least 50% deciduousness during the
dry season), with an average annual temperature ≥ 25 °C, total annual
precipitation between 700 and 2,000 mm, and three or more dry months
per year (precipitation < 100 mm/month22). According to the Köppen
classification, regions with TDFs have a seasonal tropical climate (Aw)
with an average annual temperature of 24.4 ºC and an average annual
precipitation of 871 mm2.
trapping utilized two sampling methods, both beginning at dusk: one
Shannon-type light trap exposed for a period of two hours and two CDC
light traps exposed for a period of 12 hours per plot. A third sampling
method consisted of “active collections” used to sample mosquitoes
with daytime activity, and was performed once at each sample point
for 45 minutes. Briefly, active collections consisted of using a manual
vacuum to collect all mosquitoes landing on researcher’s bodies prior
to the attempted blood meal. Transportation and mounting techniques
for mosquitoes were based on previous reports by FORATTINI10 and
CONSOLI & OLIVEIRA6. Specimens were taxonomically identified
and incorporated into the entomological collection of the Laboratory of
Ecology and Biological Control of Insects (Laboratório de Ecologia e
Controle Biológico de Insetos - LECBI) at Montes Claros State University
(Universidade Estadual de Montes Claros - Unimontes). Species
identification was carried out using dichotomous keys by CONSOLI &
OLIVEIRA6, FARAN9, FORATTINI10 and LANE15.
RESULTS
The Culicidae collections were carried out in 20 x 50 m plots, located
within tropical dry forest fragments during the dry and rainy seasons
between August 2008 and July 2012, on a total of 18 nights and across
504 hours of collections in the dry seasons, with the same sampling
effort taking place in the wet seasons during the study period. Night
During the study period, a total of 11,219 mosquitoes were collected
(11 genera and 45 species). There were 15 new records for Minas Gerais
overall, and 26 new records for the semi-arid region of Minas Gerais
(Table 1).
Fig. 1 - Map of the conservation units located in the northern region of the state of Minas Gerais, Brazil, where Culicidae were sampled in the period between August 2008 and July 2012
(215 × 279 mm; 300 × 300 DPI)
228
Table 1
Culicidae species sampled in the dry and wet seasons in the period between August 2008 and July 2012 in Mata Seca State Park (MSSP), Lagoa do Cajueiro State
Park (LCSP), Jaiba Biological Reserve (JBR) and Serra Azul Biological Reserve (SABR), in the northern region of the state of Minas Gerais, Brazil
SPECIES
Anophelinae
Anopheles (Nys.) albitarsis Lynch Arribalzaga, 1878
An. (Nys.) argyritarsis Robineau-Desvoidy, 1827
An. (Nys.) braziliensis (Chagas, 1907)
An. (Nys.) darlingi Root, 1926
An. (Nys.) deaneorum Rosa-Freitas, 1989 +
An. (Nys.) evansae (Brethes, 1926) +
An. (Nys.) triannulatus triannulatus (Neiva & Pinto, 1922)
An. (Nys.) Albimanus section/Oswaldoi Subgroup
Culicinae
Tribe Aedomyiini
Aedeomyia (Ady.) squamipennis (Lynch Arribalzaga, 1878)+
Tribe Aedini
Aedes (How.) fulvithorax (Lutz, 1904)+
Ae. (Och.) fulvus (Wiedemann, 1828)
Ae. (Och.) hastatus Dyar 1922*+
Ae. (Och.) scapularis (Rondani 1848)
Ae. (Och.) serratus (Theobald 1901)
Ae. (Och.) stigmaticus (Edwards 1922)*+
Ae. (Och.) taeniorhynchus (Wiedemann 1821)
Ae. (Stg.) aegypti (Linnaeus 1762)
Haemagogus (Con.) leucocelaenus (Dyar & Shannon, 1924)+
Hg. (Hag.) janthinomys Dyar, 1921+
Hg. (Hag.) spegazzinii Brethés, 1912
Psorophora (Gra.) cingulata Fabricius, 1805
Ps. (Jan.) albigenu (Peryassu, 1908)*+
Ps. (Jan.) discrucians (Walker, 1856)*+
Ps. (Jan.) ferox (Von Humboldt, 1819)
Tribe Culicini
Culex (Cux.) ameliae Casal, 1967*+
Cx. (Cux.) bidens Dyar, 1922*+
Cx. (Cux.) habilitator Dyar & Knab, 1906*+
Cx. (Cux.) restuans Theobald, 1901*+
Cx. (Cux.) salinarius Coquillett, 1904*+
Cx. (Cux.) saltanensis Dyar, 1928*+
Cx. (Cux.) scimitar Branch & Seabrook, 1959*+
Cx. (Mel.) complexo Vomerifer
Cx. (Mel.) group Atratus
Cx. (Mel.) section Melanoconion
Tribe Mansoniini
Coquillettidia (Rhy.) albicosta (Peryassú, 1908)+
Cq. (Rhy.) hermanoi (Lane & Coutinho, 1940)*+
Cq. (Rhy.) juxtamansonia (Chagas, 1907)
Cq. (Rhy.) lynchi Shannon 1931*+
Dry
MSSP
Wet
LCSP
Dry
Wet
19
23
0
53
0
0
33
4
4
37
2
76
0
0
27
5
17
79
0
8
4
0
2
0
12
31
0
0
0
25
0
0
0
0
0
0
0
0
0
0
0
JBR
SABR
Dry
Wet
Total
Dry
Wet
9
13
1
16
2
1
3
1
0
0
0
0
0
0
0
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
50
153
3
153
6
2
65
12
4
4
0
0
0
0
51
0
2
1
393
0
134
0
1
0
2
2
0
7
30
6
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
1
13
3
526
3
207
0
0
2
2
0
0
30
557
63
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
176
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
802
1
0
1
0
0
0
2
2
0
9
3
1
16
4
1,929
4
342
1
1
2
4
4
2
37
596
72
1
0
0
0
0
0
0
2
1
0
0
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
7
0
0
0
1
1
0
0
0
0
0
1
1
1
2
1
1
3
2
1
7
2
0
0
0
332
10
0
0
0
1
0
0
0
181
1
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
334
192
1
2
229
Table 1
Culicidae species sampled in the dry and wet seasons in the period between August 2008 and July 2012 in Mata Seca State Park (MSSP), Lagoa do Cajueiro State
Park (LCSP), Jaiba Biological Reserve (JBR) and Serra Azul Biological Reserve (SABR), in the northern region of the state of Minas Gerais, Brazil (cont.)
SPECIES
Cq. (Rhy.) nigricans (Coquillett, 1904)
Cq. (Rhy.) venezuelensis (Theobald, 1912)
Mansonia (Man.) humeralis Dyar & Knab 1916+
Ma. (Man.) indubitans Dyar & Shannon 1925+
Ma. (Man.) pseudotitillans (Theobald, 1901)+
Ma. (Man.) titillans (Walker, 1848)
Tribe Uranotaeniini
Uranotaenia (Ura.) geometrica Theobald, 1901
Ur. (Ura.) lowii Theobald 1901*+
Ur. (Ura.) pulcherrima Lynch Arribalzaga 1891*+
Tribe Sabethini
Limatus paraensis (Theobald 1903)
Sabethes (Pey.) undosus (Coquillett, 1906)+
Total
MSSP
Dry
Wet
7
810
9
598
65
400
3
26
24
401
161
1,352
LCSP
Dry
Wet
0
38
0
116
228
510
730
113
0
9
508
1,010
JBR
Dry
0
0
0
0
0
3
Wet
0
0
2
1
0
0
SABR
Dry
Wet
0
0
0
1
0
1
0
2
0
0
0
6
Total
855
724
1,206
875
434
3,040
0
0
0
0
0
0
0
1
0
2
0
2
0
0
0
0
0
0
0
0
1
0
1
0
2
2
3
0
0
444
0
1
4,693
0
0
1,589
17
0
3,459
0
0
6
0
0
185
0
0
8
0
0
835
17
1
11,219
* New record to the Minas Gerais State. +new record to the semi-arid region of Minas Gerais.
DISCUSSION
Of all the collected specimens, 8,170 (73%) were characterized
by their use of permanent breeding habitats (e.g. ponds, marshes, river
backwaters and puddles) for their larval and pupal (immature) stages;
these individuals represented Anophelinae subfamily and Mansoniini,
Aedomyiini tribes and, in some cases, Culicini. The remaining 27%
(3,040 specimens) were characterized by their use of temporary
breeding habitats (e.g., puddles, hollow bamboo, bromeliads and other
phytotelmata) in their larval and pupal stages, representing primarily
Aedini, Uranotaeniini and Sabethini tribes.
Mosquito species belonging to the Mansoniini tribe were the
most abundant (68.3%), of which the species Mansonia titillans alone
accounted for 27.10% of all mosquitoes sampled in the study. Mosquitoes
of the Aedini tribe were the second most abundant group of all mosquitoes
collected (26.87%), with Aedes scapularis as the dominant species
within the tribe (17.19%). Among the Anopheles species collected,
Anopheles darlingi was the most abundant and amounted to 1.36% of
all mosquitoes sampled.
The large percentage of mosquito species using permanent reservoirs
might be related to the relatively long dry periods, which are characteristic
of the study area. Prolonged droughts can have a damaging effect on
the viability of Aedini mosquitoes’ eggs24 and can negatively affect the
nutritional quality of the detritus found in temporary breeding habitats4.
Despite the long dry periods, the community of mosquitoes manages to
survive, mainly using the vegetation surrounding the ponds located in
PEMS and PELC.
The large abundance of mosquitoes within the Mansoniini tribe can
be explained by the influence of ponds located on the banks of the São
Francisco River, located in the MSSP and LCSP. Even in dry seasons,
230
these ponds act as major breeding grounds for Mansoniini mosquitoes
in the larval and pupal stages as they contain lots of aquatic vegetation,
the aerenchyma of the roots providing the mosquitoes with oxygen10.
Some Mansoniini mosquitoes, such as Coquillettidia venezuelensis,
are involved in the transmission of arboviruses, such as Eastern equine
encephalitis virus (actual vectors) and Oropouche virus (potential
vectors)10. In addition, Ma. titillans have been found to be naturally
infected with the Venezuelan equine encephalitis virus. Thus, the large
abundance of mosquitoes of the genus Mansonia in the conservation
units sampled could potentially impact wild bird conservation, as these
mosquitoes are ornithophilic and can transmit avian malaria14,26.
The high abundance of Aedes scapularis was probably related to
the vegetational structure of the study area, which is in the process of
natural regeneration from successive anthropogenic pressures, such as
agriculture and livestock farming17. These environments provide ideal
conditions for the establishment of Ae. scapularis populations, as these
mosquitoes have a marked tendency to invade artificially modified
environments8,11,12. Furthermore, the larval and pupal stages of Ae.
scapularis develop in temporary ground pools formed by rainfall, and
are comparable to those known to exist in environments in the initial
stages of natural regeneration5,10.
At least 15 viruses have been isolated from Ae. scapularis, including
the Rocio virus, Yellow fever virus, and Venezuelan equine encephalitis
virus; this species may also be a vector of Bancroftian filariasis18,20.
VASCONCELOS et al.27 isolated a strain of Yellow fever virus from
field-captured Ae. scapularis. Previously, only experimental laboratory
infections had been reported in this species. Considering the ecological
and epidemiological characteristics reported for this species, these
mosquitoes can be a potential bridge between wild arboviruses and
human populations in this region, given the current state of anthropogenic
modifications of the study region.
Mosquitoes of the Psorophora genus were the most abundant Aedini
after Ae. scapularis; this might be explained by the fact that these types of
mosquitoes share the same breeding habitats3,19,25. Although Psorophora
have been found to carry some types of infection in nature, mosquitoes
of this genus are not considered epidemiologically significant vectors.
These mosquitoes are, however, treated as potential incidental vectors of
disease due to some of their behavioral characteristics, such as eclecticism
in the choice of blood host and exophilic behavior10.
The abundance of An. darlingi recorded deserves particular attention,
as this species is the main vector of malaria parasites in Brazil and is
widely distributed across South America23; additionally, these mosquitoes
have an increased capacity for taking blood meals within and around
residential regions6. Although Anopheles argyritarsis and Anopheles
triannulatus are not the primary vectors of the Plasmodium species
responsible for malaria, these species are of great epidemiological interest
because of their high abundance and anthropophilic nature6.
The abundance of new Culicid records for Minas Gerais State, and
for the semi-arid region of the state, indicates that studies of mosquito
communities in forest remnants are still required, especially with regards
to the development and maintenance of support programs aimed at the
prevention of disease transmission to humans and other animals.
RESUMO
Inventário de mosquitos (Diptera: Culicidae) em unidades de
conservação em florestas tropicais secas brasileiras
No Brasil, a maior parte dos estudos relacionados à família
Culicidae se concentram em regiões de florestas úmidas, existindo uma
lacuna no conhecimento da diversidade destes mosquitos em regiões
com características climáticas e vegetacionais diferentes. O objetivo
desse trabalho foi inventariar a fauna de culicídeos em unidades de
conservação do semi-árido de Minas Gerais, visando assim contribuir
para o conhecimento da diversidade de Culicidae desta região. O
estudo foi realizado em quatro unidades de conservação localizadas
na região norte do estado de Minas Gerais, Brasil, área representada
por fragmentos de Floresta Tropical Seca (FTS). Foram utilizados três
métodos de coleta: armadilha do tipo Shannon, armadilha luminosa do
tipo CDC e coleta ativa. Durante o período de agosto de 2008 a julho
de 2012 foi coletado um total de 11.219 espécimes de mosquitos,
distribuídos em 11 gêneros e 45 espécies. Foram registrados 15 novos
registros de mosquitos para o estado de Minas Gerais e 26 novos
registros para a região do semi-árido de Minas Gerais. O elevado
número de novos registros de Culicidae na região demonstra a
importância de estudos de inventário para o aumento do conhecimento
da biodiversidade de culicídeos em Minas Gerais, e em particular a
região do semi-árido do estado.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the staff of the Instituto Estadual
de Florestas (IEF-MG), for allowing them to work and stay at Mata
Seca State Park (MSSP), Lagoa do Cajueiro State Park (LCSP), Jaiba
Biological Reserve (JBR) and Serra Azul Biological Reserve (SABR)
for logistical support. This work was carried out with the aid of a grant
from Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq -563304/2010-3 and 562955/2010-0), Fundação de Amparo à
Pesquisa de Minas Gerais (FAPEMIG CRA - APQ-00001-11) and the
Inter-American Institute for Global Change Research (IAI-CRN II-021).
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Received: 20 December 2013
57(3):233-238, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300009
PHLEBOTOMINE FAUNA (DIPTERA: PSYCHODIDAE) IN AN AREA OF FISHING TOURISM IN
CENTRAL-WESTERN BRAZIL
Andreia Fernandes BRILHANTE(1), Maria Elizabeth Moraes Cavalheiros DORVAL(2), Eunice Aparecida Bianchi GALATI(1),
Hilda Carlos da ROCHA(2), Geucira CRISTALDO(2) & Vânia Lúcia Brandão NUNES(3)
SUMMARY
The aim of this study was to identify behavioral aspects of the sandfly fauna of a fishing tourism area in the municipality of Bonito
(MS). Monthly captures were undertaken from December 2009 to November 2010, using automatic CDC type light traps, from 18h00
to 06h00, in a forested area, a savannah area, peridomiciles and animal shelters near peridomiciliary areas. Nyssomyia whitmani was
the most frequent out of a total of 6,699 specimens collected, belonging to 16 species, followed by Psathyromyia bigeniculata and
Lutzomyia longipalpis, found in all the environments investigated, though in their greatest numbers in the animal shelters. Ny. whitmani
exhibited its highest frequencies during the dry months, coincident with the fishing season, when the risk of transmission of cutaneous
leishmaniasis for tourists and inhabitants increases. Noteworthy was the finding of two species naturally infected by flagellates: Ny.
whitmani and Pa. bigeniculata. The local population and visiting tourists should be warned of the threat posed by leishmaniasis and
the health authorities alerted to the need for adopting environmental sanitary measures, especially regarding such animal shelters as
they seem to provide favorable conditions to the proliferation, maintenance and breeding opportunities of phlebotomines.
KEYWORDS: Sandflies; Leishmaniasis; Natural infection; Animal’s shelters; Vectors; Ecotourism.
INTRODUCTION
American visceral leishmaniasis (AVL) has been recorded in
increasing numbers of human and canine cases in the state of Mato
Grosso do Sul (MS), which is one of the states with the greatest incidences
in the central-western region of Brazil4,6,26. Meanwhile, American
cutaneous leishmaniasis (ACL) has been recorded in the majority of
municipalities12,17. However, despite the wide distribution and growing
number of human cases, epidemiological studies on leishmaniasis in MS
are few and far between.
In Bonito (MS), which is a municipality classified at a moderate
transmission level of leishmaniasis, studies have indicated the occurrence
of both human and canine cases of AVL and ACL, and these diseases
are spreading due to the implementation of ecotourism and livestock
activities in the area4,16,24.
Three species of Leishmania (Ross) have already been reported
in MS: Leishmania (Leishmania) infantum chagasi Cunha & Chagas,
Leishmania (Leishmania) amazonensis Lainson & Shaw and Leishmania
(Viannia) braziliensis Vianna and their respective vectors, Lutzomyia
longipalpis (Lutz & Neiva), Bichromomyia flaviscuttelata (Mangabeira)
and Nyssomyia whitmani (Antunes & Coutinho), all of which are found
in both rural and urban areas3,11,12,17,24,25.
The Águas do Miranda district, has fishing tourism as its main
economic source and presents socio-economic and environmental
conditions favorable to the transmission of the endemic diseases under
consideration. These facts together with the results of research into the
canine population of the district, which have shown 40% out of the 92
animals as seropositive for Leishmania (VLB Nunes, unpublished data),
have motivated the present study for the purpose to identify behavioral
aspects of the sandfly fauna, including its species abundance, diversity,
evenness, monthly distribution and natural infection by flagellates, to
identify potential vectors of Leishmania spp.
Study locality: Águas do Miranda District (20o 45’ 44.4”S, 56º 05’
42.8”W) is 75 km from the municipality of Bonito and 180 km from
Campo Grande, the capital of the state of MS. The permanent human
population consists of 450 inhabitants, which may rise to as many as
10,000 in the fishing season, from March to October. The local economy
is based mainly on fishing and the tourist trade29.
The prevalent vegetation belongs to the great savannah (“cerrado”)
(1) Universidade de São Paulo, Faculdade de Saúde Pública, Av. Dr. Arnaldo 715, 01246-904 São Paulo, SP, Brazil. E-mails: [email protected], [email protected]
(2) Universidade Federal de Mato Grosso do Sul, Laboratório de Parasitologia Humana, Centro de Ciências Biológicas e da Saúde, Cidade Universitária, s/n, 79070-900 Campo Grande, MS,
Brazil. E-mails: [email protected], [email protected], [email protected]
(3) Universidade Anhanguera, Uniderp, Laboratório de Parasitologia Humana, Rua Alexandre Herculano 1400, Parque dos Poderes, 79037-280 Campo Grande, MS, Brazil.
Correspondence to: Andreia Brilhante. E-mail: [email protected]
BRILHANTE, A.F.; DORVAL, M.E.M.C.; GALATI, E.A.B.; ROCHA, H.C.; CRISTALDO, G. & NUNES, V.L.B. - Phlebotomine fauna (Diptera: Psychodidae) in an area of fishing tourism
in Central-Western Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 233-8, 2015.
domain; however, it presents particularities associated with local
environmental conditions, such as forest patches in areas with more
fertile soil and a more plentiful supply of water. Noteworthy is the
vegetation cover of the Bodoquena range, a typical forest associated with
calcareous rocks known as Dry Forest or Submontane Semideciduous
Seasonal Tropical Forest. The climate is tropical with an annual average
temperature of 22 ºC29,38.
Capture sites:A total of nine sites were sampled in different
environments (Fig. 1): peridomiciliary areas near fruit trees, native
grass and tuberous vegetables (A1), native species of trees, fruit trees
and bamboos (A2) and bordered by a stream, within a bamboo grove,
near fruit and ornamental trees (A3); area of savannah with selective
extraction of timber and native species of trees (A4); gallery forests with
some secondary vegetation which grew after the selective extraction of
timber (A5, A6) and animal shelters such as a pigsty (A7), a henhouse
(A8) and a perch (A9), near peridomiciliary areas.
The flagellates found in the gut of the dissected females of two sand
fly species were inoculated intradermally, in the hind legs of hamsters
(Mesocricetus auratus). The animals were observed weekly during
12 months for checking the appearance of lesions. After euthanasia,
the spleens of these animals were removed and inoculated into NNN
culture medium (blood agar) for isolation of the parasites. Cultures were
maintained at 25 °C and examined weekly for one month to observe if
there was proliferation of flagellates.
The pluviometric data used in the analysis was obtained from the
Aquidauna meteorological station, the nearest to Águas do Miranda
district, at about 30 km away.
Data analysis: Species abundance was calculated for all the ecotopes
investigated in accordance with ROBERTS & HSI (1979)31. Initially the
Index of Species Abundance (ISA) was obtained by the application of the
formula: ISA = a + Rj/k; where: a = number of ecotopes investigated
in which the given species was not present, multiplied by c; c being
obtained as follows: a ranking of the species was established, ranging
from 1.0 to N (attributing the value of 1.0 to the most abundant species),
for each ecotope. The highest value obtained in the ranking of the species
(taking all the ecotopes into consideration) + 1.0 = c; Rj = the sum of the
positions in the ranking of a particular species in all the ecotopes and k
= the number of ecotopes sampled.
The Standard Index of Species Abundance (SISA) was used to convert
ISA into a scale of 0 to 1.0. According to this, the most abundant species
are those which are closer to 1.0. The formula used for the calculation
is: SISA = c-ISA/c-1.
The diversity and evenness were obtained, respectively, by using
Shannon’s Diversity Index (H) and that of Pielou (J). In accordance with
HAYEK & BUZAS (1997)19, the respective formulae are:
H = - Σ p (ln p); p: frequency of each species in a particular ecotope;
J = H/ln s; s: number of species in each ecotope.
Fig. 1 - Distribution of capture sites in the district of Águas do Miranda, in the municipality
of Bonito, Mato Grosso do Sul, from December 2009 to November 2010, Brazil 04/30/2012.
Source: Google Earth.
Methodology: The phlebotomines were captured on three consecutive
nights, once a month, during the period from December 2009 to November
2010 using modified automatic light CDC traps23 from 18h00 to 06h00.
One trap was installed per night in each of the nine sites sampled.
The insects captured with the CDCs were transferred to nylon cages.
The females were recaptured with polyethylene tubes, in which they
were anaesthetized with sulfuric ether. Then, after dissection to expose
the gut and spermathecae, under stereomicroscopy, they were examined
under an optical microscope (400x) for identification of the phlebotomine
species and investigation of flagellates. The male insects were kept in
Petri dishes under refrigeration until their clarification in accordance with
the technique given by FORATTINI (1973)13. Species identification was
undertaken in accordance with GALATI (2003)18 and the abbreviation
of the species names follows MARCONDES (2007)22.
234
The project was submitted to the Ethics Committee on Animal Use
in Research (CEUA) Anhanguera-UNIDERP University and approved
according to opinion No. 63-006/09.
RESULTS
A total of 6,699 phlebotomine specimens were captured, Brumptomyia
avellari (Costa Lima), Br. brumpti (Larrousse), Brumptomyia sp.,
Evandromyia sp. (Cortelezzii complex), Ev. lenti (Mangabeira), Ev.
termitophila (Martins, Falcão & Silva), Lutzomyia longipalpis (Lutz &
Neiva), Sciopemyia sordellii (Shannon & Del Ponte), Nyssomyia neivai
(Pinto), Ny. whitmani (Antunes & Coutinho), Psathyromyia aragaoi
(Costa Lima), Pa. campograndensis Oliveira, Andrade Filho, Falcão
& Brazil, Pa. hermalenti (Martins, Silva & Falcão), Pa. bigeniculata
(Floch & Abonnenc), Pa. punctigeniculata (Floch & Abonnenc) and
Micropygomyia quinquefer (Dyar) (Table 1).
The greatest phlebotomine species richness (15) and frequency
(95.35%) occurred in the henhouse (A8), representing almost the totality
of the specimens captured during the period studied. On the other hand,
the lowest species richness (3) occurred in the pigsty (A7) and the lowest
Table 1
Phlebotomines of both sexes captured with light traps in distinct environments, in the district of Águas do Miranda, Bonito municipality, Mato Grosso do Sul, Brazil,
December 2009 to November 2010
Environments
Peridomiciles
Forest and savannah
Animal shelters
Total
%
SISA
-
2
0.03
0.164
-
3
0.04
0.107
-
15
0.22
0.342
1
128
2.06
0.678
-
38
0.57
0.357
1
-
1
0.01
0.014
8
885
1
936
13.80
0.853
-
4
-
4
0.07
0.043
25
-
323
23
373
6.44
0.457
9
9
6
3270
7
3342
49.26
0.830
-
-
-
2
-
11
0.18
0.107
-
-
-
-
4
-
6
0.10
0.135
3
1
-
-
4
-
8
0.12
0.164
-
1
-
-
8
-
9
0.13
0.114
9
19
10
2
1773
4
1820
26.90
0.764
0.107
Species ecotopes
A1
A2
A3
A4
A5
A6
A7
A8
A9
Brumptomyia sp.
1
-
1
-
-
-
-
-
Br. avellari
-
-
-
1
1
-
-
1
Br. brumpti
-
1
1
3
2
-
-
8
Cortelezzii complex
1
2
2
1
24
2
-
95
Ev. lenti
-
3
-
23
3
-
-
9
Ev. termitophila
-
-
-
-
-
-
-
Lu. longipalpis
4
5
1
18
5
9
Mi. quinquefer
-
-
-
-
-
-
Ny. neivai
-
1
1
-
-
Ny. whitmani
3
-
4
34
Pa. aragaoi
-
-
-
9
Pa. campograndensis
-
-
2
Pa. hermanlenti
-
-
-
Pa. punctigeniculata
-
-
-
Pa. bigenicutala
-
1
2
Sc. sordellii
-
-
-
1
1
-
-
1
-
3
0.06
Total
9
13
14
102
66
55
16
6388
36
6699
100
%
0.13
0.19
0.21
1.52
1.00
0.82
0.24
95.35
0.54
Shannon (H)
1.21
1.58
1.95
1.78
1.69
1.38
0.97
1.23
1.05
1.30
Pielou (J)
0.87
0.88
0.94
0.77
0.73
0.85
0.89
0.45
0.65
0.47
100
A1, A2, A3: peridomiciliary areas, A4: savannah, A5: gallery forest, A6: gallery forest, A7: pigsty, A8: henhouse, A9: perch. H: Shannon’s diversity; J: Pielou’s evenness; %: percentage, SISA: standardized index of species abundance.
frequencies in the peridomiciles (Table 1).
The highest diversity indices were recorded in peridomicile (A3) and
in a savannah area (A4) and the lowest in the pigsty (A7). The highest
values of the indices were low. The henhouse (A8) had the greatest species
richness, but the evenness index was the lowest (Table 1).
Lu. longipalpis was the most abundant species, followed by Ny.
whitmani, Pa. bigeniculata, and the complex species cortelezzii and Ny.
neivai (Table 1).
The distribution of the three most abundant species captured in all
the ecotopes sampled is shown in Figure 2. Lu. longipalpis presented
peaks in January and November, Ny. whitmani in July, August, October
and November and Pa. bigeniculatus in November.
The monthly distribution of rainfall, average temperature and relative
humidity is shown in Figure 3.
The rates of natural infection detected by optical microscopy were
Fig. 2 - Number of specimens of both sexes collected per month of the species Lu. Longipalpis,
Ny. whitmani and Pa. bigeniculata, in the district of Águas do Miranda, Bonito, Mato Grosso
do Sul, December 2009 to November 2010.
0.07% (1/1418) for Ny. whitmani and 0.23% (1/408) for Pa. bigeniculata.
The flagellates were observed in the hindgut and midgut.
The animals inoculated with the gut Ny. whitmani and Pa.
bigeniculata containing flagellates did not develop a lesion during the
235
municipality (MS) where a rate of 0.16% was recorded in 613 dissected
females. Although both these rates are low, which is usually the case when
only optical microscopy is used, the numbers of the infected sources of
the parasite in the areas of the present study seem to be much lower than
those at Corguinho, which may be explained as due to the higher level of
anthropic activity in Águas do Miranda. Low frequencies of this species
in the other animal shelters (pigsty and perch) corroborate the results
found by NUNES (2008)24 and GALATI et al. (2003)16.
Fig. 3 - Monthly distribution of rainfall (mm), temperature (ºC) and relative humidity (%) in
the municipality of Bonito, from December 2009 to November 2010.
observation period and in the cultures in the hamsters’ spleens showed
no growth of flagellated form.
DISCUSSION
The greatest frequency and species richness of the phlebotomines
captured occurred in anthropic environments, probably attracted to the
peridomicile due to blood meal sources represented by domestic and
synanthropic animals. The predominance of Ny. whitmani in a henhouse
(A8) near native forest suggests that this species has a close relationship
with both wild and anthropic environments, in which the forest serves
as a shelter and breeding place for adults, as do the shaded areas in the
peridomicile7. Furthermore, the walls and roof of the henhouse, as well as
the chickens can serve as substrates for males waiting for the opportunity
to mate with the females seeking blood meal sources in this ecotope,
since the males of hematophagous insects, dispersed throughout their
habitats, may obtain a mating advantage by staying near the host and
waiting for the females to arrive1. It is noteworthy that one specimen
of this species, naturally infected by flagellates, was captured in this
henhouse, suggesting that this ecotope is attractive to the synanthropic
animals which constitute the Leishmania reservoir or that the infected
sand fly had moved from the forest to the henhouse .
The highest diversity and evenness indices in Águas do Miranda,
especially in the peridomiciliary environment, may demonstrate the
presence of these insects in areas of preserved forests and anthropic
action in these locations. The findings of this study differed from those
of NUNES et al. (2008)24 and ANDRADE et al. (2009)4, which found
lower values in urban areas of the municipality of Bonito. The highest
frequency of Ny. whitmani (49.26%) in the henhouse (A8) may explain
the lowest evenness and diversity despite being the ecotope with the
highest species richness.
The most abundant species calculated by SISA were Lu. longipalpis
and Ny. whitmani, which showed a wide distribution of these species in the
ecotopes sampled, this indicates that these species may be participating
in the cycle of transmission of leishmaniasis agents in the area, also
observed in others areas of the municipality of Bonito and the state of
Mato Grosso do Sul4,17,24,25.
The natural infection rate found for Ny. whitmani (0.07%) was
lower than that recorded by GALATI et al. (1996)17 in the Corguinho
236
In this study, Ny. whitmani was more abundant in the cold, dry period,
a result similar to that found by GALATI et al. (1996)17 in MS, and also in
the state of Rio de Janeiro (RJ) by SOUZA et al. (2002)36. It is important
to note that the greater part of this dry, cold period coincides with the
fishing season (March to October), thus indicating the concomitant risk of
the transmission of cutaneous leishmaniasis to both the local population
and visiting tourists.
Lu. longipalpis was found in anthropic environments in which
animals are reared, but is also habitual in other environments, both
rural and urban, where AVL and canine visceral leishmaniasis (CVL)
occur4,24,25, which suggests that this species may be the vector responsible
for the transmission of Le. i. chagasi to the canine population of the
district, which, in a research project undertaken in 2009, presented 40%
seropositive dogs for Le. i. chagasi in a population of 92; the parasite
isolated by the Polymerase Chain Reaction (PCR) technique being Le. i.
chagasi (VLB Nunes, unpublished data). These observations have been
found in other areas of the country and the public health authorities should
be alerted, since that CLV cases precede human AVL and the dog has a
fundamental role in the domestic transmission8, 20.
Pa. bigeniculata, considered for long time as a junior synonym of Pa.
shannoni, recently had its status of species resurrected. The difference
between these two species is mainly the thoracic coloration, i.e. while Pa.
shannoni presents pronotum and paratergite straw, prescutum, scutum,
and scutellum brown, pleurae off-white, Pa. bigeniculata presents
pronotum, paratergite, prescutum, scutum, and scutellum brown, upper
anepisternum straw and the other pleural off-white sclerites33.
Pa. shannoni is considered in the United States an important arbovirus
vector9 and has been captured naturally infected by Leishmania mexicana
in peri-urban areas of Mexico34, by Leishmania sp. in Guatemala32
and also developed experimental infection with L. i. chagasi when
feeding on infected dog37. Ps. shannoni s. lat has been associated with
the transmission of Endotrypanum schaudinni, a trypanosomatid of
sloths14, and was found naturally infected by Leishmania sp. in Serra
do Baturité, in the northeastern region of Brazil30. The finding of Pa.
bigeniculata naturally infected by flagellates, in a henhouse close to a
forest, demonstrates the need to clarify its epidemiological significance in
relation to anthropophily and the transmission of the leishmaniasis agent,
especially because it presents close affinity with Pa. shannoni for which
there are records of its infection, being either natural or experimental,
by Leishmania spp.32,37.
Ny. neivai presents no widespread geographical distribution in the
state of Mato Grosso do Sul, being found mainly in the southeastern
and eastern mesoregions2,5. It is worth highlighting the considerable
abundance of the species found in this study, especially in animal shelters,
because it has been reported as naturally infected by Leishmania and is
suspected of involvement in the transmission of cutaneous leishmaniasis
by Le. braziliensis in such Brazilian states15,22,27,28 and also in neighboring
countries10.
support and to Bonito’s municipal government for their logistic support.
The Cortelezzii complex includes the species Ev. cortelezzii, Ev.
sallesi and Ev. corumbaensis, which are all found in MS2,3,17. The only
possible way to distinguish them with confidence is by using males.
As only females were identified in this study (data not given), it was
impossible to identify the species of this complex. Recent studies
have reported the natural infection of Ev. cortelezzii and Ev. sallesi by
Leishmania3,35, thus calling for studies on their vectorial capacity, since
many of the localities where they have been captured are endemic for
leishmaniasis, as in the area covered by the present study.
1. Alexander B, de Carvalho RL, McCallum H, Pereira MH. Role of the domestic chicken
(Gallus gallus) in the epidemiology of urban visceral leishmaniasis in Brazil. Emerg
Infect Dis. 2002;8:1480-5.
The District of Águas do Miranda revealed a diverse sandfly fauna,
with 16 species, some of them proven vectors of leishmaniasis agents
in Brazil and others that have been described as naturally infected and
which may, therefore, act as potential vectors. In light of the above, the
local population and visiting tourists should be warned of the threat
posed by leishmaniasis and the health authorities alerted of the need for
adopting environmental sanitary measures, especially regarding such
animal shelters, as they seem to provide favorable conditions to the
proliferation, maintenance and breeding opportunities of phlebotomines.
RESUMO
Fauna flebotomínea (Diptera: Psychodidae) em área de turismo
pesqueiro no Centro-Oeste do Brasil
O objetivo deste estudo foi identificar aspectos do comportamento da
fauna flebotomínea de área de turismo pesqueiro localizada no município
de Bonito (MS). Foram realizadas capturas mensais no período de
dezembro de 2009 a novembro de 2010, utilizando armadilhas luminosas
automáticas do tipo CDC das 18:00h às 6:00h, em matas, área de cerrado,
peridomicílios e abrigos de animais próximos a áreas peridomiciliares.
De um total de 6.699 espécimes coletados, pertencentes a 16 espécies,
Nyssomyia whitmani foi a mais frequente, seguida de Psathyromyia
bigeniculata e Lutzomyia longipalpis, encontradas em todos os tipos
de ambientes, porém com maior expressão em abrigos de animais. Ny.
whitmani apresentou frequências mais elevadas nos meses mais secos,
coincidentes com a estação da pesca, o que eleva o risco de transmissão
da leishmaniose tegumentar a turistas e moradores da área. Importante
ressaltar o encontro de duas espécies naturalmente infectadas por
flagelados: Ny. whitmani e Pa. bigeniculata. A população local e turistas
devem ser advertidos da ameaça que representam as leishmanioses e as
autoridades de saúde alertadas para adoção de medidas de saneamento
ambiental, principalmente com relação aos abrigos de animais, que
parecem fornecer condições favoráveis para a proliferação, manutenção
e reprodução de flebotomíneos.
The authors declare there is no conflict of interests.
ACKNOWLEDGEMENTS
The authors wish to express their thanks to the Universidade
Anhanguera - Uniderp, to the Brazilian agency CAPES for their financial
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infection of Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae, Phlebotominae)
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Received: 16 December 2013
57(3):239-244, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300010
HEAD LICE IN HAIR SAMPLES FROM YOUTHS, ADULTS AND THE ELDERLY
IN MANAUS, AMAZONAS STATE, BRAZIL
Suellen Cristina Barbosa NUNES(1), Raquel Borges MORONI(2), Júlio MENDES(3), Sílvia Cássia Brandão JUSTINIANO(4) & Fábio Tonissi MORONI(5)
SUMMARY
A study of head lice infestations among young people, adults and elderly individuals was conducted from August 2010 to July 2013
in Manaus, AM, Northern Brazil. Hair samples collected from 1,860 individuals in 18 barber shops and beauty parlors were examined
for the ectoparasite. The occurrence of pediculosis and its association with factors, such as sex, age, ethnicity, hair characteristics and
the socioeconomic profile of salon customers, salon location and seasonal variation were determined. The overall occurrence rate
was 2.84%. Occurrence was higher in hair samples from non-blacks and the elderly. Higher occurrence was also observed during
kindergarten, elementary and junior education school holidays. The results indicate that the occurrence of head lice among young
people, adults and the elderly in Manaus is relatively low compared to that determined in children and in other regions of the country.
After children, the elderly were the most affected. The study also indicated the need to adopt additional procedures to improve surveys
among the population with low or no purchasing power, which is usually the most affected by this ectoparasitic disease.
KEYWORDS: Head lice; Hair samples; Pediculus capitis; Epidemiology; Manaus.
INTRODUCTION
Pediculosis, infestation by Pediculus capitis De Geer (head lice),
has worldwide distribution, including Brazil11, and is observed in all
age groups, though particularly among children4,23,28. It is considered
one of several ectoparasites neglected by the scientific community and
healthcare authorities12.
Infestation is characterized by intense itching, secondary infection
and anemia in cases of severe infestation and inadequate diet 23.
Severe infestations are associated with low socioeconomic status, hair
characteristics, parasite resistance to insecticides, genetic factors, and
cultural habits6,8,10,24.
Besides the physical symptoms described above, it can cause
embarrassment among children. Certain population groups, particularly
adults, often resist having their heads examined for the diagnosis of the
parasite, which is a fairly sensitive diagnostic method3,16. Given this
restriction, researchers have sought alternative methods of assessment
regarding the degree of importance of this disease in certain populations.
Analysis of samples of hair cut in salons and similar establishments is one
such technique7,9,16,18. Although it does not present the same sensitivity
as direct scalp examination3, it is an alternative that permits verification
of the epidemiological aspects of this parasitosis in population groups
that have such restrictions.
In Brazil, epidemiological studies concerning this ectoparasitosis
are concentrated in the southeastern region, while information from
the northern and northeastern regions regarding infestation remains
scarce1,4,13, except for a recent study involving school-age children in
urban areas of Manaus4 and another one from the state of Acre concerning
infant dermatitis, which included head lice1.
Epidemiological features associated with head lice infestation
among young people and adults were studied using analysis of hair cut
in salons and similar establishments, located within the urban area of
Manaus1,13. This study provided information concerning the occurrence,
its monthly distribution and possible associations of several factors
which, according to the literature3,4, can influence its occurrence, such
as hair characteristics, ethnicity, age, socioeconomic status, the location
of sampling, and seasonal variation.
Manaus, in the Amazonas State, occupies an area of 11,401,092 km²
(4,401,986 m2) and is located at 60°01’30” W and 03°06’07” S. The city
has 1,982,179 inhabitants and is divided into six administrative zones
comprising 63 neighborhoods15. The study was conducted by obtaining
and examining hair samples from customers of 18 beauty parlors and
barber shops located in five of the six administrative zones of this city.
The criteria for choosing the establishments investigated were: random
(1) PPGIBA/Universidade Federal do Amazonas, Manaus, Amazonas, Brazil.
(2,3) ICBIM/Universidade Federal de Uberlândia, Minas Gerais, Brazil.
(4) Pesquisa e Pós-graduação, Universidade Nilton Lins, Manaus, Amazonas, Brazil.
(5) ICB/Universidade Federal do Amazonas, Manaus, Amazonas, Brazil.
Correspondence to: Dra. Raquel B. Moroni. Parasitologia/ICBIM/Universidade Federal de Uberlândia. Av. Pará 1720, Bloco 4C, Campus Umuarama, 38408-100 Uberlândia, MG, Brasil.
NUNES, S.C.B.; MORONI, R.B.; MENDES, J.; JUSTINIANO, S.C.B. & MORONI, F.T - Head lice in hair samples from youths, adults and the elderly in Manaus, Amazonas State, Brazil.
Rev. Inst. Med. Trop. S. Paulo, 57(3): 239-44, 2015.
selection, their location according to zone and the acceptance of their
owners/managers for inclusion in the study. Two to four establishments
were sampled in each administrative zone. Each establishment was
visited around 15 times, with each visit on a different day to obtain hair
samples, between August 2010 and July 2013. Customers who attended
the establishments and appeared to be between 15 and 65 years of age
were included in the study.
The owners or managers of the institutions signed a term of free and
informed consent. Details on the ethical procedures adopted are described
in the project approved by the Research Ethics Committee of the Federal
University of Amazonas, under protocol no. CAAE 0099.0.115.000-09.
Hair samples were collected after each hair cut and individually
placed in properly labeled plastic bags. Information concerning their
origin and the physical appearance of the customers was recorded
on a form. The samples were sent to the laboratory and analyzed
with a magnifying glass and microscope to check the condition and
characteristics of the hair18 (size, type, color, and thickness). Samples
considered positive were those on which any of the developmental stages
of lice (eggs, nymphs or adults) were identified, irrespective of whether
they were viable at the time of examination.
Hair characteristics were determined according to BORGES &
MENDES2, following training with their method prior to evaluation. The
hair samples were classified according to the following characteristics:
length, color and thickness. Hair of up to 3 cm in length was considered
short, medium length was from 3 to 10 cm, and long was over 10 cm,
according to the parameters stipulated by19. Hair color was grouped into
four categories: light (blonde and red), dark (brown and black), gray, and
dyed. Regarding thickness, there were two categories: thin and thick2.
The ethnicity, sex and age group of the clients whose hair was
sampled were determined based on observations regarding the physical
appearance of the individuals during their haircuts. Regarding ethnicity,
the individuals were divided into blacks and non-blacks. Age group was
established according to WHO30: young people, those who appeared to
be 15-29 years old; adults, 30-59 years old; and elderly, those over 60
years old2.
The socioeconomic profile of the clients was inferred according to
the value of the haircuts charged by the establishments. Thus, customers
who sought a haircut in an establishment that charged R$ 5.00 to R$
10.00 were considered to be of low socioeconomic status, R$ 15.00 of
medium status and $ 20.00 to R$ 30.00 of high status.
The monthly distribution of infestation was monitored for three
years. School age children seemed to act as reservoirs of this disease4
and studies have demonstrated that significant variation in occurrence
appears among children between the academic and vacation periods in
Minas Gerais3, 20.
Thus, this work also investigated whether there was any relation
between infestations in children and individuals of other age groups by
comparing prevalence rates obtained in these two periods of the year, i.e.
the occurrence determined during months that composed the academic
periods of kindergarten, elementary and junior education and the months
these children were on vacation.
The χ2 statistical test was used to compare two or more proportions.
In cases where significant differences between more than two proportions
were observed, the data were submitted to angular transformation (p’ =
arc. sen √p’), followed by multiple comparisons using the Tukey range
test4,29. A 5% level of significance was adopted29. Confidence intervals
(95%) were also calculated for the occurrence and prevalence ratios
determined.
RESULTS
An occurrence of 2.84% was detected for the 1,860 hair samples
obtained from the 18 establishments surveyed. The highest occurrence
rates were found in the south-central (3.5%) and west-central areas
(3.47%). However, the differences between these rates and those
obtained for other areas, where the rates were lower, were not significant
(χ20.05,4 = 1.67, p > 0.75) (Table 1).
Of the 53 positive samples, 37 had non-viable lice eggs, three had
adult lice and 13 had viable eggs.
In general, the hair characteristics, length, color and thickness,
did not significantly influence the prevalence rates obtained (Table 3).
However, ethnicity and age had a significant influence on distribution,
Table 1
Occurrence of head lice among clients of barber shops and beauty parlors according to their location in the different zones
of the city of Manaus, Amazonas State, Brazil
Location
No. of samples
examined
No. of samples
positives
Occurrence rate (%)
95% confidence interval
Prevalence ratio
South zone
400
9
2.25 (0.8 - 3.7) Aa
-
V to VIII
East zone
400
10
2.5 (1.22 - 4.03) A
1.1 (0.73 - 1.64)
IX to XII
North zone
400
11
2.75 (1.15 - 4.35) A
1.22 (0.51 - 2.88)
XIII to XVI
West-central zone
460
16
3.47 (1.8 - 5.14) A
1.54 (0.68 - 3.45)
XVII and XVIII
South-central zone
200
7
3.5 (0.96 - 6.04) A
1.55 (0.58 - 4.11)
-
1860
53
-
-
Establishments
I to IV
Total
a
= occurrence rates with different letters are statistically different from each other by the Tukey test at a 5% level of significance.
240
such that non-black individuals and the elderly had higher occurrence
rates (χ20.05,1 = 5.05, p = 0.025; χ20.05,2 = 7.65, p < 0.025) (Table 2). The
socioeconomic profiles of the clients, inferred from the price of a haircut
charged by the establishment, did not significantly influence the rate of
head lice infestation either (χ20.05,2 = 2.16, p > 0.25) (Table 2).
The results gathered here, together with those obtained for school
children in the same city4, reflect the degree of importance of this
ectoparasitosis for the population. However, it should be noted that
given the lower sensitivity of this technique in relation to direct scalp
examination, the real rate of occurrence of the study population is likely
to be greater than that found. The differences in sensitivity are partly
due to factors like the length of the hair that is cut18 and the number of
people aware that they have head lice, who would not choose to cut their
hair in such establishments. An additional limitation of this study is the
fact that the establishments were not sampled every month of the year.
Another fact that influences the monthly head lice distribution is that
part of the inactive infestation results from active infestations acquired
in the months prior to those in which the hair samples were obtained.
However, considering that numerous establishments were sampled
throughout the study period, the data obtained over the months were
grouped into the academic and vacation periods of the year, while the
number of hair samples obtained were satisfactory from a statistical point
of view; possible bias resulting from this sampling procedure does not
significantly influence the statistical analysis.
Analysis of the monthly distribution of the occurrence rates revealed
that the highest rate was obtained in July, a school holiday period (Fig. 1).
The rates obtained for the months that composed the academic periods
and the months that composed the school holiday period of school-age
children were grouped and compared; the rate determined for the holiday
period was significantly higher (χ20.05,1 = 13.28, p < 0.01) (Table 2).
DISCUSSION
The use of hair samples collected from barbershops for estimating
the prevalence of pediculosis capitis was adopted for the first time in
Brazil by18. The overall prevalence of head lice infestation in this study
was relatively low, compared with other studies conducted in the state
of Minas Gerais using this same technique3. However, the data obtained
corroborate the results of recent research among school children in
Manaus using the head inspection technique4, in which an occurrence
rate of 18% was obtained in children, a rate that is also considered low
for this age group compared with studies conducted in other regions of
the country13,23.
It is also worth highlighting, particularly in this study, that the portion
of the general population having a less favorable socioeconomic profile
may not be able to easily afford a haircut, and thus this population group
may not have been adequately sampled. The fact that no significant
differences were observed in comparisons between the occurrence
Table 2
Occurrence of head lice among several groups of clients of barber shops and beauty parlors, in the city of Manaus, Amazonas State, Brazil
No. of samples examined
No. of samples positives
Occurrence rate (%)
Prevalence ratio
Male
1433
39
2.72 (2.22 - 3.8) A
-
Female
465
14
3.01 (2.46 - 2.98) A
1.10 (0.59 - 2.05)
Black
656
11
1.67 (0.69 - 2.65) Aa
-
Non-black
1204
42
3.48 (2.45 - 4.51) B
2.08 (1.07 - 4.01)
Youth
652
24
3.68 (2.24 - 5.12) B
1.93 (1.06 - 3.50)
Adult
998
19
1.90 (1.06 - 2.74) B
-
Elderly
210
10
a
4.76 (1.89 - 7.63) A
2.50 (1.18 - 5.30)
Sex
Ethnicity
Age group
Socioeconomic profile
b
Low
730
20
2.73 (1.56 - 3.9) A
-
Medium
400
13
3.25 (1.52 - 4.98) A
1.19 (1.67 - 2.36)
High
530
20
3.77 (2.15 - 5.39) A
1.38 (1.33 - 2.54)
No
1437
30
2.08 (1.34 - 2.82)Aa
-
Yes
423
23
5.43(3.33 - 7.59)B
2.61 (1.53 - 4.44)
School holidays
a
= occurrence rates with different letters are statistically different from each other by the Tukey test at a 5% level of significance. b = inference based on the price charged
to customers by the establishment providing the service.
241
Table 3
Occurrence of head lice according to the hair characteristics of clients of barber shops and beauty parlors, in the city of Manaus, Amazonas State, Brazil
No. of samples examined
No. of samples positives
Occurrence rate (%)
Prevalence ratio
Short
378
11
2.91 (1.22 - 4.60) A
1.62 (0.68 - 3.88)
Medium
502
9
1.79 (0.64 - 2.94) A
-
Long
980
33
3.36 (2.24 - 4.48) A
1.87 (1.49 - 2.36)
Hair length
Type of hair
Curly
164
6
3.65 (0.78 - 6.52) A
2.06 (0.76 - 5.59)
Wavy
564
10
1.77 (0.69 - 2.85) A
-
Straight
1132
37
3.26 (2.23 - 4.29) A
1.84 (1.29 - 2.62)
197
2
1.01 (0 - 2.4) Aa
-
Hair color
Dyed
Light
122
3
2.45 ( 0 - 5.19) B. A
2.42 (0.40 - 14.33)
Dark
1466
44
3.00 (2.13 - 3.87) B. A
2.97 (0.72 - 12.09)
Gray
90
4
4.44 (2.27 - 6.61) C. B
4.39 (1.06 - 18.09)
Thin
1067
29
2.71 (1.74 - 3.68) A
-
Thick
793
24
3.02 (1.83 - 4.21) A
1.14 (0.68 - 1.92)
Hair thickness
a
= occurrence rates with different letters are statistically different from each other by the Tukey test at a 5% level of significance.
25
% of posi v e samples
20
15
10
5
0
jan
feb
mar
apr
*Number of samples examined, N = 1860.
may
jun
jul
aug
sep
oct
nov
dec
months
Fig. 1 - Monthly distribution of samples examined and samples positive for head lice, collected
from 18 barber shops and beauty parlors, in Manaus, Amazonas State, Northern Brazil, from
August 2010 to July 2013. School holiday period: Jan, Jun, Jul and Dec. Academic periods:
Feb, Mar, Apr, May, Aug, Sep, Oct and Nov.
rates obtained for the different socioeconomic statuses, according to
the price of a haircut charged by the establishment, seems to strengthen
this hypothesis. Thus, procedures must be adopted to obtain more
representative samples of this population group. One procedure adopted
by BORGES et al.3 to sample this population group was the examination
of samples of free haircuts during events promoted by charities and
242
similar institutions in Uberlândia, MG. This should provide a clearer
understanding of the real situation of this ectoparasitic disease in various
age groups of the population group that are generally most affected12,21.
Analysis of the monthly variations of infestation and comparison with
the data obtained for schoolchildren in Manaus and other Brazilian
cities4, 13 indicated that the profile of temporal variation of infestation in
adults is different to the temporal variation in children. The profile of
infestations in adults appears to be influenced by the difference in time
spent together between these two age groups during the two periods of
the year, the academic year and school vacations. Studies conducted
in Brazil have shown that school-age children have the highest rates
of head lice infestation2,13,20 and peak rates are observed in the months
comprising the academic periods of the year4, when children spend a
lot of the time in school and less time with the other members of their
respective families. The greater interaction between children and their
families during the vacations partially explains the increase in prevalence
in other age groups during this period of the year.
Several authors mention the influence of temperature and humidity
on the temporal profile of infestation13,22. The North and Northeastern
regions of Brazil are subject to less annual variations in temperature.
However, humidity has greater variation and is considered relatively
low in some months, in which the lowest occurrence was observed. This
result is consistent with findings reported by authors who also performed
studies in the northeastern region13.
A higher occurrence rate among the elderly was also observed
in Minas Gerais3. The data obtained herein, together with those just
described, reinforce the hypothesis that the elderly are the second most
commonly affected age group in some cities. This is probably due,
at least in part, to the following factors: they spend more time with
children than other family members, and a number of the elderly are
dependent on caregiver and live in or attend nursing homes or similar
collective environments, under conditions conducive to the transmission
of ectoparasitic diseases.
The higher occurrence among non-blacks verified in this study was
also observed in American schools that attend a multiracial population
of children14. In contrast, several studies have reported higher prevalence
among black individuals2,4,6. These divergent reports could be related to
several factors that have comparative influence on head lice infestation,
such as different forms of hair among different ethnic groups, variations
in cultural habits and different socioeconomic conditions5,17.
It should be emphasized that scalp examination is a more sensitive
technique for diagnosing this parasitosis than the examination of cut hair
samples. Thus, scalp examination should be used whenever possible.
Despite their limitations, the diagnostic technique and sampling system
adopted allowed for the accomplishment of the main objectives of the
study, i.e. obtain information concerning the degree of importance of
this ectoparasitic disease and the factors that most strongly influence
the occurrence of head lice in the study population. However, additional
procedures that would make the technique more sensitive while
maintaining the ease of its application among the target population
are required. Measures that improve sampling among the section of
the population that finds it difficult or impossible to pay for a haircut
would grant a more accurate view of the degree of importance of this
ectoparasitic disease in the population group with low or no purchasing
power, which is usually the group most affected by this parasitosis.
RESUMO
Pediculose da cabeça em amostras de cabelos de jovens, adultos e
idosos em Manaus, Estado do Amazonas, Brasil
Estudo sobre a pediculose do couro cabeludo em jovens, adultos
e idosos foi realizado de agosto de 2010 a julho de 2013 em Manaus
- AM, região norte do Brasil. Amostras de cabelos obtidas de 1.860
indivíduos em 18 barbearias e salões de beleza foram examinadas à
procura do parasito. Procurou-se verificar a ocorrência da pediculose e
sua associação com fatores tais como: sexo, idade, etnia, características
dos cabelos e perfil socioeconômico dos clientes dos salões, localização
dos salões e variação sazonal. A taxa de ocorrência encontrada foi
2,84%. Ela foi maior em amostras de cabelos de não negros e dos idosos.
Também se observou maior prevalência no período de férias escolares
do ensino fundamental e médio. Os resultados indicam que a ocorrência
da pediculose em jovens, adultos e idosos em Manaus é relativamente
baixa em comparação com as encontradas em crianças e as encontradas
em outras regiões do país. Depois das crianças, os idosos seriam os
mais acometidos. O estudo também indica a necessidade de adotar
procedimentos adicionais para melhor amostrar a faixa da população
com menor ou com nenhum poder aquisitivo, a qual geralmente é a mais
acometida por esta ectoparasitose.
ACKNOWLEDGMENTS
The authors would like to thank the establishments involved in
this work and the National Council of Scientific and Technological
Development (CNPq) and the Amazon Research Foundation (FAPEAM)
for providing study grants.
FINANCIAL SUPPORT
The National Council of Scientific and Technological Development
(CNPq/MCT-Amazônia 2006/2008) provided support for this study.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
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Received: 15 January 2014
Accepted: 30 July 2014
57(3):245-250, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300011
EFFECTS OF VITAMIN C SUPPLEMENTATION ON THE CHRONIC PHASE OF CHAGAS DISEASE
Ricardo Guimarães MARIM(1), Alex Silva de GUSMÃO(1), Roberto Esteves Pires CASTANHO(1), Rafael DEMINICE(3), Altino Luiz Silva THEREZO(2),
Alceu Afonso JORDÃO JÚNIOR(3), Marcos Renato de ASSIS(4), Elane de Fátima TAIPEIRO(5) & Luciamare Perinetti Alves MARTINS(1)
SUMMARY
Introduction: In order to examine the effectiveness of vitamin C (ascorbic acid) in combating the oxidative insult caused by
Trypanosoma cruzi during the development of the chronic phase of Chagas disease, Swiss mice were infected intraperitoneally with
5.0 × 104 trypomastigotes of T. cruzi QM1strain. Methods: Mice were given supplements of two different doses of vitamin C for
180 days. Levels of lipid oxidation (as indicated by thiobarbituric acid reactive substances-TBARS), total peroxide, vitamin C, and
reduced glutathione were measured in the plasma, TBARS, total peroxide and vitamin C were measured in the myocardium and
histopathologic analysis was undertaken in heart, colon and skeletal muscle. Results: Animals that received a dose equivalent to 500
mg of vitamin C daily showed increased production of ROS in plasma and myocardium and a greater degree of inflammation and
necrosis in skeletal muscles than those that received a lower dose or no vitamin C whatsoever. Conclusion: Although some research
has shown the antioxidant effect of vitamin C, the results showed that animals subject to a 500 mg dose of vitamin C showed greater
tissue damage in the chronic phase of Chagas disease, probably due to the paradoxical actions of the substance, which in this pathology,
will have acted as a pro-oxidant or pro-inflammatory.
KEYWORDS: Lipid peroxidation biomarkers; Chagas disease; Parasitemia; Inflammation; Ascorbic acid; ROS; RNS
INTRODUCTION
Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi,
a flagellate protozoan9 that infects approximately 12–14 million people in
Latin America15. After contact with the parasite, patients develop the acute
phase of the illness, which may be asymptomatic in some individuals or
may show non-specific symptoms, such as fever, tachycardia, weakness,
and lymphadenopathy. This evolves into an indeterminate phase, with
no clinical signs, but with subclinical pathologies17. Many years after
infection, about 30% of the patients develop the chronic phase of the
disease, which has characteristic signs including megaesophagus,
megacolon and cardiomegaly24. During the chronic phase, the presence
of microfoci parasites leads to the constant production of interleukin-12,
which activates macrophages and generates a Th1 immune response
consistent with delayed hypersensitivity, which leads to tissue damage14,24.
Much of the damage to the host in Chagas disease is caused by an
excess of free radicals, more specifically by the metabolism of reactive
oxygen species (ROS) and reactive nitrogen species (RNS). These are
produced by phagocytic cells stimulated by inflammatory mediators in
tissues parasitized by T. cruzi. They may harm any cellular component,
but the components that are affected most strongly are the cellular
membranes18,23,42,43.
To combat the formation of free radicals and neutralize them before
they cause damage, biological systems use molecules and enzymes such
as reduced glutathione (GSH), superoxide dismutase (SOD), catalase,
glutathione peroxidase (GSH-Px), and vitamin E. Others, such as vitamin
C (ascorbic acid), glutathione reductase (GSH-Rd), and glutathione
peroxidase (GSH-Px) are used to repair damage that has already been
done42.
A study by WEYERS et al.44 showed that appropriate doses of
vitamin C can have a preventive effect against the lipid peroxidation
induced by free radicals; both those formed naturally and those caused
by exogenous compounds in mice experimentally dosed with the
antibacterial drug ciprofloxacin. Vitamin C decreased the oxidative
stress on the liver and lipid peroxidation in the mouse kidney caused
by this drug43. According to MAY33, ascorbic acid’s ability to reduce
oxidative stress would depend on its concentration and its potential for
recycling in blood and endothelium.
However researches of CHEN et al.11 showed that pharmacological
doses of ascorbic acid favored the cytotoxicity of tumor cells by formation
of hydrogen peroxide (H2O2), leading to the decreased growth of tumors
in mice due to pro-oxidant action. For LEVINE et al.29 ascorbate could be
used as a treatment for viral and bacterial infections and in other human
(1) Department of Parasitology of Marília Medical School (FAMEMA), R. Dona Maria Feres 165, 17519-070 Marília, São Paulo, Brazil.
(2) Department of Pathology of Marília Medical School (FAMEMA), Av. Monte Carmelo 800, 17519-030 Marília, São Paulo, Brazil.
(3) Department of Medical Clinic, Division of Nutrition and Metabolism of Ribeirão Preto Medical School (FMRP/USP), Av. Bandeirantes 3900, annex A, 14049-900 Ribeirão Preto, São
Paulo, Brazil.
(4) Department of Rheumatology of Marília Medical School (FAMEMA), Av. Monte Carmelo 800, 17519-030 Marília, São Paulo, Brazil.
(5) Department of Biochemisty of Marília Medical School (FAMEMA), Av. Monte Carmelo 800, 17519-030 Marília, São Paulo, Brazil.
Correspondence to: Luciamáre Perinetti Alves Martins, Laboratório de Parasitologia, FAMEMA, R. Dona Maria Feres 165, 17519-070 Marília, São Paulo, Brasil. E-mail: [email protected]
MARIM, R.G.; GUSMÃO, A.S.; CASTANHO, R.E.P.; DEMINICE, R.; THEREZO, A.L.S.; JORDÃO JÚNIOR, A.A.; ASSIS, M.R.; TAIPEIRO, E.F. & MARTINS, L.P.A. - Effects of vitamin
C supplementation on the chronic phase of Chagas disease. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 245-50, 2015.
pathogens, where the production of H2O2 and ROS could be beneficial
in disease progression.
A study by MAÇAO et al.30 showed that oxidative stress was
controlled in the myocardium of patients in the chronic phase of Chagas
disease when 500 mg of vitamin C and 800 UI of vitamin E were
administered daily for a six-month period, probably as a consequence of
vitamin E consumption and antioxidant protection. According to KOURY
& DONANGELO28, vitamins C and E are important “street sweepers”
of free radicals.
Nonetheless, CHEN10 noted that with a lower concentration of
vitamin E, the administration of high doses of vitamin C appears to
promote lipid peroxidation and significantly decreases the animals’
antioxidant potential.
Based on these studies, the authors decided to investigate the
effectiveness of vitamin C in combating the oxidative insult caused by
progressive T. cruzi inflammation during the evolution of Chagas disease in
its chronic phase, by administering two different daily dosages of vitamin
C to mice experimentally infected with the T. cruzi QM1strain, since the
common sense of its role in antioxidant defense makes its use indiscriminate
among people, including chronic carriers of Chagas disease.
day after infection for histopathologic analysis after CO2 euthanasia22.
The tissues were embedded in paraffin and 5 μm sections were stained
with hematoxylin-eosin and examined under a light microscope at
a magnification of 400×. Five sequential histological sections were
examined for each fragment, and the fragments were analyzed and graded
for inflammation and amastigote nests for a total of 10 high magnification
fields for each type of tissue. The investigator examining the sections
was unaware of the grouping of the mice.
A semi-quantitative scale of one to three plus signs was used in order
to grade the inflammatory process and the number of amastigote nests.
This way, inflammation and/or necrosis were examined separately to the
number of amastigotes for each animal: “+” signified mild inflammation,
necrosis and rare amastigote nests; “++” moderate inflammation,
necrosis and moderate number of amastigote nests; and “+++”, intense
inflammation, necrosis and frequent amastigote nests.
Biochemistry: Blood samples were collected on the 180th day after
infection. The mice were exposed to CO2 (100% CO2) for a few seconds
as a form of anesthetizing them, and then the blood was collected by
cardiac puncture into heparin. This method of euthanasia was chosen
as studies conducted by GODIN & GARNETT22 and BILLERT &
DROBNICK5 show that exposure to this gas may not increase oxidative
damage to tissues.
Infection of mice: Thirty 20-day-old male “Swiss” mice, weighing
on average 13 grams, were intraperitoneally infected with 5.0 × 104
trypomastigotes of T. cruzi QM1 strain32 via blood from other previouslyinfected mice, which was obtained by cardiac puncture into heparin. The
trypomastigotes were counted following the BRENER6 method and the
intraperitoneal infection was performed by inoculating 0.1 mL of blood
using an Injex® insulin syringe. Following this procedure, three groups
of 10 mice were chosen at random and named P (placebo), Vit C 60 (mice
that received the dose of vitamin C (D60); see next section for the doses
given), and Vit C 500 (mice that received a dosage of vitamin C (D500)).
The animals were kept in individual cages to facilitate handling and fed
with the standard Nuvilab CR-1® diet, which is composed of: ground
whole corn, soybean meal, wheat bran, calcium carbonate, calcium
phosphate, sodium chloride, vegetable fat, mineral vitamin premix, amino
acid, and water ad libitum.
Calculation of vitamin C dosage and treatment: Vit C 60 group
(a D60 dose) was given a daily dose of 60 mg of vitamin C, equal to the
average weight of the mice × (8.6 × 10–4 mg per gram of weight), diluted
in 10 μL of human mineral water (Soft®). Vit C 500 group (a D500 dose)
was given a daily dose of 500 mg of vitamin C, equal to the average
weight of the mice × (7.14 × 10–3 mg per gram of weight) diluted in 10
μL of human mineral water (Soft®). The P group was a placebo group:
each animal received 10 μL of human mineral water (Soft®) daily. Every
morning, all mice were treated orally with 10 μL of vitamin C (D60 or
D500) in mineral water or mineral water (Soft®) alone, using a Gilson
automatic pipette. The three groups were treated for 180 days, starting
from the infection date. Vitamin C used in this research was Cewin® (in
drops doses) from Sanifi-Aventis Laboratory.
Histopathologic analysis: A fragment of heart, colon and skeletal
thigh muscle from all mice that survived was collected on the 180th
246
Blood was centrifuged at 1500 g immediately after collection and the
plasma stored at -80 ºC. For the measurement of vitamin C, 200 µL plasma
samples were immediately acidified with 800 µL 5% trichloroacetic acid
and stored at -80 ºC for later analysis. One fragment of heart muscle from
each mouse was immediately frozen in liquid nitrogen and subsequently
stored at -80 °C. Oxidative stress markers were determined around 3 - 5
weeks after sampling. All samples were stored at -80 ºC before analysis. Thiobarbituric acid reactive substances (TBARS) were used as
biomarkers of lipid peroxidation in plasma and heart muscle and
measured using a method adapted from COSTA et al.13. The total
concentration of TBARS was determined by the difference in absorbance
between samples and in a standard solution of malondialdehyde. The
total peroxide in the cardiac muscle and plasma was determined using
the FOX method, as described by SÖDERGREN et al.38. This method
uses a comparison with the standard curve of H2O2. Quantification of
plasma glutathione was performed by adapting the method described by
COSTA et al.13 using the standard curve of GSH.
The vitamin C concentration in plasma and heart muscle was
determined according to BESSEY’s4 method, 100 µL of a solution
containing 2,4-dinitrophenylhydrazine (2%), thiourea (5%), and copper
sulfate (0.6%) in sulfuric acid (25%) added to 300 µL of acidified
plasma. After a 4-hour incubation in a 37 °C water bath, 200 µL sulfuric
acid (65%) was added and the solution was incubated for 20 min at
room temperature. Reading was performed on a spectrophotometer
(Spectramax M5, Molecular Devices) at 520 nm and compared with the
standard curve of vitamin C.
Statistics: The results were analyzed by Normality Test (ShapiroWilks) to verify that the data followed a normal distribution and
homogeneity of variances in the groups of Levene’s test. Statistical
inference was performed using ANOVA one-way and Post-hoc analyses
were performed with the Tukey (HSD) test1. The significance level used
was 5%.
(lipid oxidation levels) in the myocardium showed no statistically
significant differences between the dosages.
This study was approved by the Ethics Committee of the Faculty
of Medicine of Marília (FAMEMA) under number 133/10. The manner
of treatment, care, and euthanasia of mice followed the standards set
by Colégio Brasileiro de Experimentação Animal/Brazilian College of
Animal Experimentation (Cobea)39.
The vitamin C results showed no statistical differences in plasma and
myocardium between the three groups with p > 0.05 for all comparisons.
RESULTS
After 180 days of treatment two animals had died in the placebo
group, three had died in group Vit C 60, and four had died in group Vit
C 500. The study was therefore performed on eight animals in group P,
seven in group Vit C 60, and six in group Vit C 500. Group Vit C 500
suffered greater damage to the skeletal muscle, showing a greater degree
of inflammation than group Vit C 60 and a greater degree of necrosis
than group P (Table 1). With regards to cardiac muscle and the colon, all
animals in group Vit C 500 had inflammation, with mice of this group
showing a greater level of damage.
An analysis of TBARS, total peroxide, vitamin C, and plasma GSH is
shown in Table 2 and an analysis of TBARS, total peroxide and vitamin C
in the myocardium is shown in Table 3. Group P had lower total peroxide
concentrations in plasma than group Vit C 500 (p = 0.021, Table 2). The
TBARS analysis in plasma showed a statistically significant difference
between group P and group Vit C 500 (p = 0.021, Table 2), and between
group Vit C 60 and group Vit C 500 (p = 0.022, Table 2). There was no
statistically significant difference between the groups in the plasma GSH
(p > 0.05, Table 2).
There was a higher total production of peroxides in the myocardium
of group Vit C 500 than in group P (p = 0.050, Table 3). TBARS analysis
DISCUSSION
To date, no research has been done to test the effectiveness of vitamin
C in combating ROS and RNS produced during the evolution of Chagas
disease. This study has shown that oral administration of 500 mg of
vitamin C, per 180 days, in mice infected with T. cruzi can be harmful
to the host, leading to increased total peroxide and TBARS, which may
increase the severity of lesions, characteristic of chronic phase of this
disease.
In this study, the vitamin C doses of 60 mg and 500 mg provided
to groups Vit C 60 and Vit C 500 resulted in no statistically significant
differences in myocardial and plasma vitamin C concentrations relative
to group P (p > 0.05 in all comparisons). The absence of differences
between experimental groups in these results at the time of plasma and
tissue collection could be explained by the physiological mechanism of
control of concentrations of vitamin C, where ascorbate concentrations
in plasma and tissue are tightly controlled. According to LEVINE et al.29,
the vitamin C concentration was tightly controlled by absorption, tissue
transport and renal reabsorption and excretion.
Surprisingly, the dosage of 500 mg caused detrimental repercussions
to the host. This is confirmed by the study’s results regarding the number
of mice that survived the treatment and the histopathologic findings,
in which group Vit C 500 was shown to have suffered greater skeletal,
cardiac muscle and colonic tissue damage, causing the death of a large
number of animals.
Table 1
Degree of histopathologic lesions and parasitism in mice infected by T. cruzi QM1 and treated with two different doses of vitamin C (VitC60= D60; VitC500= D500)
and placebo (P): (+) mild inflammation, necrosis and rare amastigote nests; (++) moderate inflammation, necrosis and moderate number of amastigote nests; (+++)
intense inflammation, necrosis and frequent amastigote nests. * “%” indicates the percentage of infected mice affected by necrosis or inflammation in each group,
and the absolute number observed is given in parentheses
P
Skeletal
muscle
Cardiac
muscle
Colon
Inflammation
Amastigote
nests
VitC60
Necrosis
% (8)*
Inflammation
Amastigote
nests
% (7)
% (7)
— — 42.6% (3) —
—
14.3% (1)
— — — 85.7% (6)
—
—
—
—
—
— — — % (8)
% (8)
+
12.5% (1)
—
++
—
—
—
+++
— — — +
50.0% (4)
12.5% (1)
—
++
25.0% (2)
—
—
+++
— — — +
62.5% (5)
—
—
42.9% (3)
28.6% (2)
++
25.0% (2)
—
—
28.6% (2)
+++
— — — — 50.0% (4) VitC500
Necrosis
% (7)
Inflammation
Amastigote
nests
Necrosis
% (6)
% (6)
% (6)
50.0% (3)
— 100% (6)
—
—
—
— —
— 50.0% (3)
—
—
50.0% (3)
—
—
— — — —
50.0% (3)
—
—
—
—
50.0% (3)
—
—
— — — — —
247
Table 2
Mean values and standard deviation of thiobarbituric acid reactive substances (TBARS), total peroxide, vitamin C and reduced glutathione (GSH) in plasma of mice
treated with two different doses of vitamin C (VitC60= D60; VitC500= D500) and placebo (P) during the chronic phase of experimental infection by T. cruzi QM1.
(SD = standard deviation)
Variable
P
VitC60
VitC500
Compared Groups
TBARS (µmol/L)
34.8 (SD = 2.5)
34.8 (SD = 2.8)
41.0 (SD = 6.1)
P & VitC60
P & VitC500*
VitC60 &VitC500*
Total peroxide (µmol H2O2
equivalent/L)
88.4 (SD = 35.2)
134.3 (SD = 38.1)
148.6 (SD = 40.0)
P & VitC60
P & VitC500*
VitC60 & VitC500
Vitamin C (mg/dL)
0.28 (SD = 0.07)
0.29 (SD = 0.07)
0.33 (SD = 0.04)
P & VitC60
P & VitC500
VitC60 & VitC500
237.13 (SD = 29.45)
203.30 (SD = 46.77)
183.32 (SD = 25.59)
P & VitC60
P & VitC500
VitC60 & VitC500
GSH (mmol/L)
* Groups differ by Tukey test at 5% probability.
Table 3
Mean values and standard deviation of thiobarbituric acid reactive substances (TBARS), total peroxide and vitamin C in the myocardium of mice treated with two
different doses of vitamin C (VitC60= D60; VitC500= D500) and placebo (P) during the chronic phase of experimental infection by T. cruzi QM1. (SD = standard
deviation)
Variables
P
VitC60
VitC500
Compared Groups
TBARS (µmol/g protein)
82.4 (SD = 20.3)
73.6 (SD = 14.3)
100.4 (SD = 27.1)
P & VitC60
P & VitC500
VitC60 &VitC500
Total peroxide (µmol H2O2
equivalent/g protein)
161.4 (SD = 82.3)
304.1 (SD = 172.7)
368.4 (SD = 188.6)
P & VitC60
P & VitC500*
VitC60 &VitC500
Vitamin C (μg/g prot)
6.61 (SD = 1.13)
6.76 (SD = 0.74)
6.97 (SD = 0.76)
P & VitC60
P & VitC500
VitC60 &VitC500
* Groups differ by Tukey test at 5% probability.
Corroborating the histopathologic findings, a greater total peroxide
production in group Vit C 500 than in group P was observed, both in
plasma (p = 0.021) and in the myocardium (p = 0.050), as well as an
increase in plasma TBARS concentrations in group Vit C 500 (p = 0.021).
Although a tendency of decrease in GSH concentration of group Vit C
500 was observed, suggesting that antioxidant response was activated to
neutralize ROS and RNS during disease progression, these results were
not statistically significant.
Researches of MAÇAO et al.30 demonstrated a decrease in TBARS and
increase in GSH when chronic chagasic patients were supplemented daily
with 500 mg vitamin C and 800 IU vitamin E. The effectiveness of these
antioxidants has been studied by various authors3,46, who indicated that both
vitamins C and E operate in the context of an integrated system in which the
overall antioxidant status is under homeostatic control, so that the change
in status of a single antioxidant may affect the status of the other in a way
that the ratio C/E can be of central importance to antioxidant protection21.
248
Nevertheless, the aim of this study was to evaluate the effects
of the isolated uses of ascorbic acid. These results show that daily
supplementation, only with the equivalent of 500 mg of vitamin C, is
coupled with a statistically significant increase in production of free
radicals and lipid peroxidation between Vit C 60 Vit C 500 (p = 0.022),
as shown by the result.
Although the antioxidant properties of vitamin C are well
established2,20,26, it is still debatable that it beholds a possible prooxidant8,16,37 and an anti / pro-inflammatory effect27,34.
In vitro studies performed by CLEMENT et al.12, showed that, in
high concentrations, vitamin C can exert a pro-oxidant effect before
antioxidant, but in vivo the biological relevance of these events remains
controversial8.
However, in case of an injury inflicted by these tissues, the possibility
of free transition metal Fe3+ and Cu2+ increases, which may be reduced
by ascorbate7. In vitro, the interaction of these reduced metals with
H2O2 produce hydroxyl radicals and lipid alkoxyl radicals (LO.), causing
oxidative damage, and the ascorbate would then act as pro oxidant.
Therefore, it is possible that a high intake of vitamin C could
be harmful to persons who have chronic inflammatory conditions19,
because it would cause the interaction of these catalytically active
metals with ascorbate, as in the chronic phase of Chagas disease. This
would also cause damage to surrounding tissues, which contribute to the
inflammatory process, besides the fact that their own ROS amplify the
release of pro-inflammatory cytokines.
Although several studies in humans and other species delineate the
functional significance of vitamin C in resistance to infection40,41, the
effects of a high dose on the activity of the immune system is still scarce.
Studies of HORNIG et al.25 reported the increased bioavailability of
nitric oxide in patients with chronic heart failure after arterial and oral
administration of vitamin C and NOH et al.36, discovered that megadoses
of vitamin C increased the production of interferon, IL-2 and TNF-α,
showing a positive correlation between serum levels of vitamin C and pro
inflammatory cytokines. Moreover, MIKIROVA et al.34 discovered that
high doses of vitamin C reduces inflammation in patients with cancer.
These results showed an increase in the inflammatory process with
the presence of vitamin C and, in a high dosage, an exacerbation of
tissue damage. This infers that, in the chronic phase of Chagas disease,
the administration of 500 mg of vitamin C provides a pro-oxidant and
pro-inflammatory medium to these animals. Since the effects were not
observed in shorter periods of supplementation31, these results may indicate
the potential risk of for patients at the chronic phase of Chagas disease in
ingesting high vitamin C concentrations for a long period of time. These
results will be elucidated through future research on the technique of
immunohistochemistry and may highlight whether the exacerbation of the
inflammatory process is associated with the presence of the parasite or its
cell debris, or if it occurred due to the actions of vitamin C.
Therefore, these results indicate the need for further investigation
to elucidate the mechanisms whereby the high dosage of vitamin C
may operate in the parasite35,45 or in the harmful events identified by
this research.
RESUMO
Efeitos da suplementação de vitamina C na fase crônica da doença
de Chagas
Introdução: Para verificar a eficácia da vitamina C em combater o
insulto oxidativo causado pelo Trypanosoma cruzi durante a evolução
da fase crônica da doença de Chagas, camundongos Swiss foram
previamente infectados via intraperitoneal com 5.0 × 104 tripomastigotas
da cepa QM1 de T. cruzi. Métodos: Camundongos foram suplementados
com duas diferentes doses de vitamina C por 180 dias. Foram mensurados
os níveis de peroxidação lipídica (indicado por substâncias reativas ao
ácido tiobarbitúrico-TBARS), peróxido total, vitamina C, e glutationa
reduzida no plasma e TBARS, peróxido total e vitamina C no miocárdio,
e foi realizado o estudo histopatológico em coração, cólon e músculo
esquelético. Resultados: Animais que receberam diariamente uma
dosagem equivalente a 500 mg de vitamina C apresentaram aumento
na produção de ROS e RNS no plasma e no miocárdio e maior grau de
inflamação e necrose em músculo esquelético em comparação àqueles que
receberam doses menores ou nenhuma vitamina C. Conclusão: Embora
muitas pesquisas tenham mostrado o efeito antioxidante da vitamina C,
nossos resultados mostraram que os animais que foram expostos a 500 mg
de vitamina C apresentaram maior dano tecidual na fase crônica da doença
de Chagas, provavelmente devido a ações paradoxais desta substância,
onde nesta patologia, poderá agir como pró-oxidante ou pró-inflamatória.
ACKNOWLEDGEMENTS
Statistical assistance made through the Extension Project “Statistical
advice to students and teachers of undergraduate and graduate programs
of FFC-CM, UNESP and external researchers,” coordinated by Prof.
Dr. Sebastião Marcos Ribeiro de Carvalho, Department of Educational
Psychology, the FFC - Campus Marília, UNESP.
The authors of “Effects of vitamin C supplementation on the chronic
phase of Chagas disease” declare no conflicts of interest in developing
this study.
FINANCIAL SUPPORT
The authors thank the São Paulo State Foundation for Research
Support (FAPESP) for financial support for this project.
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57(3):251-256, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300012
MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL
ISOLATES OF Nocardia spp. IN BRAZIL
Larissa Anuska Zeni CONDAS(1), Márcio Garcia RIBEIRO(1), Marisol Domingues MURO(2), Agueda Palmira Castagna de VARGAS(3),
Tetsuhiro MATSUZAWA(4), Katsukiyo YAZAWA(4), Amanda Keller SIQUEIRA(1), Tatiana SALERNO(1), Gustavo Henrique Batista LARA(1),
Rafaela Mastrangelo RISSETI(1), Karen Spadari FERREIRA(5) & Tohru GONOI(4)
SUMMARY
Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals.
The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This
report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion
method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of
which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases
were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp) identified four
isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two
cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia
asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials
were amikacin (100%), amoxicillin/clavulanate (100%), cephalexin (100%) and ceftiofur (100%), while isolates had presented most
resistance to gentamicin (43%), sulfamethoxazole/trimethoprim (43%) and ampicillin (29%). However, on the inhibitory minimal
concentration test (MIC test), only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.
KEYWORDS: Nocardiosis; Nocardia; Opportunistic disease; Antimicrobial susceptibility test.
INTRODUCTION
Nocardiosis is a chronic and severe pyogranulomatous disease
caused by the environmentally ubiquitous actinomycete of the Nocardia
genus1. Nocardiosis is an emerging disease in humans and animals
worldwide2,13,26,32,34. According to BAIO et al. (2013) nocardiosis
is a neglected disease, particularly in patients with some degree
of immunosuppression1. For many years, since Edmond Nocard’s
first description of the pathogen in 1888, its diagnosis was based on
phenotypic methods17. To date, 102 species of Nocardia have been
discovered using molecular methods, of which seven have been
reclassified and 90 currently stand on the list of prokaryotic names with
standing nomenclature24. Among these species, at least 25 are pathogenic
to humans and animals8,13,17,29. N. asteroides, N. brasiliensis, N. farcinica,
N. nova, N. cyriacigeorgica and N. veterana are the main species related
to nocardiosis in humans1,11,14,17.
Either tegumentary injury or the inhalation of bacteria is considered
the most common route of transmission of Nocardia spp. in humans7.
Clinically, the main manifestations of human nocardiosis are pneumonia,
encephalitis, lymphadenitis, lymphangitis and cutaneous tissue lesions33.
Therapy consists of a prolonged course, its success depending
on the species of bacteria, the virulence of the strain, the organs
affected, the time of evolution and the health status of the susceptible
individual(s)1. Nocardia spp. is refractory to conventional antimicrobial
therapy. Sulfonamides potentiated by trimethoprim, minocycline,
aminoglycosides (amikacin, gentamicin) and cephalosporins (ceftiofur,
ceftriaxone, cephalexin) alone or in combination, based on in vitro tests3,
are the choices of treatment for human and animal nocardiosis26,28,31.
The purpose of this case report is to present the species of Nocardia
involved in human nocardiosis in Brazil, and their respective drug
susceptibility pattern.
Isolates. Seven strains of Nocardia spp., identified in three Hospitals
from different states of Brazil (one in Rio Grande do Sul, one in São Paulo
and five in Paraná), were isolated from clinical cases of nocardiosis. The
strains were isolated from different specimens (bronchial wash, cutaneous
and organ fragments) in defibrinated sheep blood agar (5%), Sabouraud
or Lowenstein agar, and maintained aerobically at 37 ºC for three to
(1) Department Veterinary Hygiene and Public Health, Universidade Estadual Paulista “Júlio de Mesquita Filho”, FMVZ/UNESP Botucatu, Sao Paulo, Brazil.
(2) Universidade Federal do Parana, Clinical Hospital, Curitiba, PR, Brazil.
(3) Department of Preventive Veterinary Medicine, Universidade Federal de Santa Maria, UFSM, Rio Grande do Sul, Brazil.
(4) Medical Mycology Research Centre of Chiba University, Chiba, Japan.
(5) Department of Biological Science, Microbiology, Immunology and Parasitology Sector, Universidade Federal de São Paulo, UNIFESP, São Paulo, SP, Brazil.
Correspondence to: Márcio Garcia Ribeiro, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Medicina Veterinária, Caixa Postal 560, Distrito de Rubião Júnior, 18618970 Botucatu, São Paulo, Brasil. Fax: +55 (14) 3881-6579, Phone: +55 (14) 3881- 6270. E-mail: [email protected]
CONDAS, L.A.Z.; RIBEIRO, M.G.; MURO, M.D.; VARGAS, A.P.C.; MATSUZAWA, T.; YAZAWA, K.; SIQUEIRA, A.K.; SALERNO, T.; LARA, G.H.B.; RISSETI, R.M.; FERREIRA, K.S.
& GONOI, T. - Molecular identification and antimicrobial resistance pattern of seven clinical isolates of Nocardia spp. in Brazil. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 251-6, 2015.
10 days. Colonies suggestive of the genus Nocardia were evaluated by
dry, convex, adherent, and white to orange color aspects. After 48 to 72
hours post-inoculation, colonies were submitted to Gram and Kinyoun
stain8,27. Gram-positive, filamentous to cocobacillary, partially acid-fast
organisms were identified as Nocardia.
Molecular identification. Molecular analysis was carried out in
the Medical Mycology Research Center, Chiba, Japan. Genomic DNA
for sequencing was performed according to KAGEYAMA et al., 2004.
The 16S rRNA gene was amplified by PCR using a DNA thermal cycler
(TaKaRa Bio Inc., Chiba, Japan) under the following conditions: 35
cycles at 94 ºC for 60 seconds for denaturation, 60 ºC for 60 seconds
for primer annealing, and 72 ºC for 120 seconds for primer extension.
PCR primers were the prokaryotic 16S rRNA universal primer pairs,
8F and 691R, 520F and 1100R, and 926F and 1542R. DNA sequences
were determined with an automatic sequence analyzer (ABI PRISMTM
3130; Applied Biosystems, Japan), using the same primers and a dye
terminator cycle sequencing kit (Applied Biosystems). Near-complete
16S rRNA gene sequences consisting of approximately 1400 bases pairs
were obtained. The sequence of the 16S rRNA gene was compared to the
GenBank database using BLAST, and 16S rRNA sequences of related
Nocardia-type strains were retrieved from the database. These sequences
were submitted to GenBank/JJBJ/EMBL.
In vitro drug susceptibility tests. All strains were examined using
the disk-diffusion test and minimum inhibitory concentration test (MIC
test) based on E-test™ (E-test™, AB biodisk, BioMérieux, Dalvägen,
Sweden), according to the procedures described by GLUPCKZYNSKI
et al. 2006 and NCCLS 2006. Isolates resistant to three or more
antimicrobials were considered multi-resistant23.
The study of drug susceptibility in Actinomycetes is more laborious
because this group of bacteria usually grows in clumps. Due to this
adherent characteristic during bacterial growth, the observed irregular
broth turbidity makes it difficult to measure the optimum inoculum
concentration. Naturally, the precise concentration of unit colony
formation is essential for the correct interpretation of both antimicrobial
tests11,20.
The isolates were briefly subcultured twice in blood agar (5%) to
ensure their purity. After 48 hours, they were inoculated in brain-heartinfusion at 37 ºC for another 48 hours. Sterile glass beads were added at
the time the strains were vortexed to decrease the formation of clumps
and subsequently obtain a more accurate optical density (OD) equivalent
to a 0.5 McFarland standard to disk-diffusion test and 1.0 McFarland
standard for E-test.
All of the strains were submitted to a disk-diffusion test and the
inhibition zones interpreted following standards according to BAUER
et al., 1966 and AMBAYE et al. 1997. The antibiotics selected were
amikacin (30 μg), ampicillin (10 μg), amoxicillin/clavulanate (20
μg/10 μg), ceftiofur (30 μg), cefoperazone (75 μg), ceftriaxone (30 μg),
cephalexin (30 μg), cefuroxime (30 μg), cefalonium (30 μg), imipenem
(10 μg), gentamicin (10 μg), mynocicline (30 μg) and sulfamethoxazole/
trimethoprim (25 μg).
In the MIC test, a maximum of five E-test strips were attached
to Mueller Hinton agar and were then incubated at 37 ºC. The
252
results were recorded after 48 - 72 hours because of the organisms’
fastidious growth3,20. The following antibiotics were used: amikacin
(0.016 - 256 µg/mL), ampicillin (0.016 - 256 µg/mL), amoxicillin/
clavulanate (0.016 - 256 µg/mL), ceftriaxone (0.016 - 256 µg/mL),
gentamicin, sulfamethoxazole-trimethoprim (0.002 - 32 µg/mL) and
imipenem (0.002 - 32 µg/mL).
The similarities between the results from the disk-diffusion test and
E-test were analyzed using the Kappa agreement index. According to
the values obtained, the agreement analysis between the tests followed
the criteria established by THRUSFIELD (1995). All statistical analysis
was done using Bioestat v.5.0 and SPSS v.14 packages. The Chi-squared
and Fisher’s exact tests were used to analyze whether the resistance
percentages of Nocardia spp. were normally distributed between strains
for each antibiotic tested with the disk-diffusion method. The level of
significance for this test was p < 0.0533.
RESULTS
All strains were taken from young adult males between 28 and
35 years of age. Three patients displayed cutaneous manifestations
(exhibiting fistulous mycetomas due to traumatic inoculation), while two
showed pulmonary, one neurological and one systemic. Two individuals
with pulmonary symptoms, one with neurological, and one with
systemic were co-infected with immunosuppressive diseases, or were
undergoing/had undergone prolonged treatments with chemotherapy or
corticotherapy. Of all the patients, four died due to the severity of their
disease (these were pulmonary, neurological and systemic cases), two
recovered (cutaneous manifestations), and one case had no documented
outcome (cutaneous manifestation) [Table 1].
The microbiological culture of samples showed dry, convex, strongly
adherent, whitish to orange-brown colonies with a powdery surface
after 48-96 hours of incubation at 37 ºC. Gram and modified ZiehlNeelsen stains showed thin, branched filaments sometimes fragmented
in cocobacillary forms, suggestive of the Nocardia genus.
The sequence of the 16S rDNA gene enabled the identification of
four strains - N. farcinica, two N. nova, and one N. asiatica - based on
its 99.6% or higher sequence similarity to the reference sequence in
GenBank (DDBJ/GenBank/EMBL), using BLAST as recommended
by CLSI, 2008. The access number for the isolates in GenBank is as
follows: IFM 11128/ AB 633331 - N. nova; IFM 11096/ AB 630965 N. farcinica; IFM 11231/ AB 636474 - N. farcinica; IFM 11232/ AB
636475 - N. farcinica; IFM 11099/ AB 630966 - N. nova; IFM 11100/
AB 630967 - N. asiatica; IFM 11285/ AB 638765 - N. farcinica.
In this study, N. farcinica was observed in two cutaneous, one
systemic and other pulmonary cases; N. nova was present in one
neurological manifestation and one pulmonary; and Nocardia asiatica
in one cutaneous case (Table 1).
The in vitro drug susceptibility test, based on disk-diffusion, is
presented in Table 2. Based on the disk-diffusion test, Nocardia spp.
isolates were sensitive to amikacin, amoxicillin/clavulanate, cephalexin,
cefalonium, ceftiofur, ceftriaxone and minocycline. However, around
50% of the Nocardia spp. isolates were resistant to ampicillin, gentamicin
and sulfamethoxazole/trimethoprim. Of the seven isolates, two were
Table 1
Summary of clinical presentation and outcomes of seven patients with nocardiosis. Santa Maria, RS; Curitiba, PR and Botucatu, SP, Brazil
Nocardia species
N. nova
N. farcinica
N. asiatica
Clinical
presentation
Site of isolate
Age/sex
Underlying
condition
Neurologic
Liquor
32/male
Pulmonary
Sputum
33/male
Dermatologic
Biopsy
Dermatologic
Biopsy
Pulmonary
Broncoalveolar wash
Systemic
Liquor
Dermatologic
Biopsy
35/male
Treatment
Outcome
Chemotherapy
NA*1
Death
HIV
4 drugs*2
Death
30/male
None
NA
Recovery
28/male
None
NA
Recovery
35/male
HIV
NA
Death
34/male
Chemoterapy
NA
Death
NA
NA
NA
*1 NA = not available; *2 Patient came in on advanced stage of disease, he was treated with clindamycin, sulfamethoxazole/trimethoprim, imipenem and amphotericin
before death.
multi-resistant to three or more antimicrobials. One isolate of N. farcinica
was resistant to ampicillin, cefoperazone and gentamicin; and the other
isolate of N. farcinica was resistant to ampicillin, sulfamethoxazole/
trimethoprim, gentamicin and cefuroxime.
The minimum inhibitory concentration of tested antimicrobials
showed suitable breakpoints, with all the isolates being susceptible to
Table 2
Standard inhibition zone diameter and percentage of susceptibility of 7
Nocardia spp. isolates to selected antimicrobials in disk-diffusion test. UNESP,
Botucatu, SP, Brazil
Zone diameter (mm)a
the tested antimicrobials (Table 3) except for one isolate of Nocardia
farcinica, which was resistant to sulfamethoxazole/trimethoprim.
Table 3
Minimum inhibitory concentrations (µg/mL) and susceptibility proportion
estimates of Nocardia spp. isolated from seven case reports. UNESP, Botucatu,
SP, Brazil
MIC50a
(µg/mL)
MIC90a
(µg/mL)
%Susceptible
(Overall n=7)
Amikacin
0.20
0.40
100
Amoxicillin/ clavulanate
0.5
3
100
Antimicrobials
0.25
1
100
I
S
% Susceptible
Ampicillin
R
Cephalexin
1
2
100
Amikacin
≤14
15-16
≥17
100
Ceftriaxone
1.5
2
100
Amoxicillin/
clavulanate
≤13
14-17
≤13
100
Gentamicin
1
2
100
Imipenem
0.75
1,5
100
Ampicillin
≤13
14-16
≥17
29
1.5
3
86
Cephalexin
≤14
15-17
≥18
100
Sulfamethoxazole/
trimethoprim
Cephalonium
≤14
15-17
≥18
100
Cefoperazone
≤15
16-20
≥21
43
Ceftiofur
≤17
18-20
≥21
100
Ceftriaxone
≤13
14-20
≥21
86
Cefuroxime
≤14
15-17
≥18
72
Imipenem
≤13
14-15
≥16
72
Gentamicin
≤10
11-14
≥15
57
Mynocicline
≤14
15-19
≥19
100
Sulfamethoxazole/
trimethoprim
≤10
11-15
≥16
43
Antimicrobial
a
Zone of inhibition diameter (mm) by disk diffusion method susceptibility interpretative guidelines based on NCCLS (2006), AMBAYE et al. (1997), and BAUER
et al. (1966). R = resistant, I = intermediate, S = susceptible.
MIC50 and MIC90 : values are concentrations at which ≥ 50% and ≥90% of isolates
are inhibited by antimicrobials.
a
The agreement between the tests was considered low by the statistical
analysis.
DISCUSSION
These findings reinforce that molecular techniques are a reliable,
suitable and quick method for the diagnosis of species of Nocardia genus
taken from a human origin. Phenotypic evaluations could be performed
to identify the Nocardia species, combining tests such as the hydrolysis
of organic compound (adenine, xanthine, hypoxanthine, casein, esculin
and tyrosine), carbohydrate assimilation, antimicrobial susceptibility
pattern, citrate utilization and acetamid and arylsulfatase utilization,
among others8,27,36. However, they are usually laborious, time-consuming,
and require experience in evaluating the results. For this reason, molecular
methods emerged as an alternative that can be used on several different
253
body fluids and tissue samples, and can also be used for identifying strains
that are difficult to grow in a conventional medium8,29.
The 16S rRNA gene is highly conserved with constant regions and, to
date, its complete sequence of approximately 1400 bp has a considerably
large database on GenBank, which allows for the identification of
most Nocardia species29. However, because 16S rRNA shows minimal
variation and is also present in different numbers of copy variants, this
gene should be sequenced in its total 1400 bps and follow the standards
of CLSI, which recommends a similarity higher than 99.6%. Nowadays,
other researchers are reporting on the sequencing of housekeeping genes
with a better discriminatory power, even with partial gene sequencing16,29.
The incidence of human nocardiosis has increased in the last two
decades across several countries, particularly in patients affected by
immunosuppressive therapies or diseases1,8. In Brazil, the 22 cases of
human nocardiosis, predominantly displaying pulmonary symptoms,
were most prevalent in adult males (59.2%) between the ages of 21 and 84.
Most cases were related to immunosuppressed conditions (69.9%), such
as transplants, corticotherapy and being HIV-positive30. These findings
agree with similar observations that described immunocompromised
conditions in humans affected by nocardiosis10,19,21,30, reinforcing the
opportunistic behavior of the Nocardia genus7. However, it is important to
stress that cutaneous presentations are not always associated to a previous
health condition, indicating possible transmission through cutaneous
trauma, as suggested by other studies4. In this study, nocardiosis only
affected men between the ages of 28 and 35. This is consistent with the
results of other studies, which have also identified similar occurrences
of nocardiosis in young adult males, indicating the occupational risk of
human infection by Nocardia species in mainly immunocompromised
patients exposed to the agent within their environment10,21,34.
The clinical picture of human nocardiosis is diverse, though
cutaneous and pulmonary manifestations are present in the majority of
cases4,5. In the last few years, N. farcinica has been the most isolated
species in reviews of different kinds of patient infection, whether with or
without immunosuppression, representing around 22% and 45% of cases
respectively19,26,32,34. Evidence shows that N. farcinica is widely distributed
in the environment, has a high potential of virulence and is closely
associated to a great number of fatal nocardiosis cases, including those
with systemic dissemination8. Cases related to N. farcinica confirmed
the severity of the disease in immunosuppressed patients.
In other reports, N. nova has been associated to approximately 20%
of isolates in the United States and was the most pathogenic species in
Canada until 200834,35. N. nova has been attributed to different clinical
presentations in humans, mainly related to immunosuppressive conditions
or trauma4,35. These findings reinforce this species’ pathogenic potential
for humans, and the risks of developing systemic nocardiosis, resulting
in death.
N. asiatica was recently identified in human nocardiosis in Brazil, and
this is one of few reports worldwide5,21,22 which reinforces the necessity
of identifying more isolates and understanding their epidemiology.
The lower efficacy of human strains to gentamicin and ampicillin in the
disk- diffusion test is probably related to the pattern of low susceptibility
of N. farcinica resistance, indicating that these are not recommended
254
as therapy20,31. However, the high efficacy of cephalosporins against N.
farcinica isolates and the high success rate of amoxicillin/clavulanate
against N. nova could be justified by the variability in β-lactamase
activity present in the bacteria’s cell wall3,20. In agreement with previous
results, minocycline appeared to be highly effective as in vitro against
the Nocardia species isolated in these reports3,31, however MUÑOZ et al.
(2007) observed resistance to this drug in N. nova and N. cyriacigeorgica.
Sulfamethoxazole/trimethoprim presented a lower efficacy in
disk-diffusion tests against the isolates and are perhaps, therefore, not
appropriate for therapy. Similar observations with a larger number of
clinical isolates were made by TREMBLAY et al. (2011), informing
of the importance of establishing treatment protocols for in vitro tests.
However, despite the worldwide resistance pattern to sulfonamides
recorded in clinical isolates, few patients failed therapy with these drugs,
suggesting that the inhibition breakpoint for sulfonamides still poses a
challenge and varies among different laboratories9. This can result from
the methodology suggested by CLSI in which sulfonamide breakpoints
are based on 80% of inhibition of growth endpoint compared to the
100% inhibition of growth by other drugs. In future research, more
isolates should have the antimicrobial profile analyzed using the same
CLSI specifications, as performed in this study, and new breakpoints
for Nocardia species should perhaps be considered. Moreover, the
indiscriminate use of the drug for comorbidities can induce resistance
of Nocardia to sulfonamides and molecular mechanisms, thus it should
be analyzed8,9,26.
Some studies showed the applicability of the E-test for antimicrobial
susceptibility testing for the analysis of actinomycete resistance20.
However, disagreement between MIC and disk diffusion tests has been
indicated in other papers3,25. These differences probably occur due to
colony lumps formed during culture growth, which makes it difficult to
obtain a precise McFarland scale, or an ideal count of colony-forming
units25. So far, for Nocardia the broth dilution method is still favorable,
in comparison to the E-test, in order to assure the significance of the
minimum inhibitory concentration method.
Despite the small number of isolates, it was possible to notice
that nocardiosis in Brazil mainly affects men and immunosuppressed
patients with localized or disseminated infection. Clinical manifestations
could vary depending on the species, virulence of isolates, and
immunocompromised factors of the patients. The cases reported in this
study were seen in patients with a higher comorbidity, predominantly
associated with pulmonary or disseminated forms. Antimicrobial
resistances of isolates reinforce the importance of prior in vitro tests
before initiating therapy. Further investigation with a larger number of
cases and isolates is necessary.
RESUMO
Identificação molecular e perfil de sensibilidade a antimicrobianos
de sete isolados clínicos de Nocardia spp. no Brasil
Nocardia é um microorganismo ubiquitário relacionado a infecções
piogranulomatosas, com difícil resolução tecidual em humanos e animais.
A doença é mundialmente emergente devido ao aumento de doenças
e tratamentos imunossupressores. Este relato de casos ocorridos no
Brasil visa apresentar a identificação molecular dos isolados e o padrão
de sensibilidade a antimicrobianos por disco-difusão e concentração
inibitória mínima (CIM) através de fitas E-test®. Os casos ocorreram
em homens, em idade adulta. Três quadros foram cutâneos, dois
pulmonares, um neurológico e um sistêmico. O quadro respiratório,
o neurológico e um sistêmico estavam associados à doença ou terapia
imunossupressoras. O sequenciamento do gene 16S rRNA (1491pb)
possibilitou a identificação de quatro isolados de Nocardia farcinica, dois
de Nocardia nova e um de Nocardia asiatica. N. farcinica foi observada
em dois casos dermatológicos, um pulmonar e um quadro sistêmico, N.
nova foi isolada de um caso neurológico e outro pulmonar; e N. asiatica
em um caso dermatológico. O teste de disco-difusão mostrou que
amicacina (100%), amoxicilina/clavulanato (100%), cefalexina (100%)
e ceftiofur (100%) foram mais efetivos; enquanto gentamicina (43%),
sulfametoxazol/trimetoprim (43%) e ampicilina (29%) foram menos
efetivos. No entanto, no teste de concentração inibitória mínima (CIM),
apenas um dos quatro isolados da espécie Nocardia farcinica mostrou-se
resistente a sulfametoxazole-trimetropina.
ACKNOWLEDGMENTS
To FAPESP Funding (Protocols 2009/56037-1 and 2010/53494-5)
and Mycology Research Center of University of Chiba - Japan.
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Accepted: 23 Septembeer 2014
57(3):257-262, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300013
GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN
AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS
Lasaro Teixeira FERREIRA(1), Aparecida Helena de Souza GOMES(2) & Vera Lucia PEREIRA-CHIOCCOLA(1)
SUMMARY
Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The
evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.)
braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the
genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods:
Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil
(36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from
biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus. Results:
Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results
showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among
14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three
samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest
that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of
Brazil, showing great variability among the natural populations of endemic foci.
KEYWORDS: American cutaneous leishmaniasis; Leishmania (Viannia) braziliensis; RFLP-PCR; Polymorphism.
INTRODUCTION
The Leishmania genus causes leishmaniasis, which constitutes a
variety of chronic diseases. There is a wide spectrum of clinical forms,
including those affecting the skin, mucosa, or internal organs16,18.
The subgenera Leishmania Viannia is the causative agent of newworld cutaneous leishmaniasis, comprising the species L. (V.) braziliensis,
L. (V.) panamensis and L. (V.) guyanesis, among others18,26. Infections
by these species cause three clinical types of American tegumentary
leishmaniasis (ATL): localized cutaneous, mucosal, and disseminated
leishmaniasis. Cutaneous lesions are restricted to the entry site of the
parasites, whereas the mucosal strain is defined by its spreading to the
mucosal surfaces of the upper digestive and airway tracts. Disseminated
leishmaniasis is characterized by large-scale spreading to distant
cutaneous sites2,14,15,24.
Despite the fact that cutaneous leishmaniasis is caused by at least
seven different Leishmania species in Brazil, the vast majority of cases
are caused by the L. (V.) braziliensis sub-genera, which can be transmitted
by different phlebotomine sandfly vectors via animal reservoirs across a
wide geographic distribution1,7,16,18,28.
ATL is widely distributed across the Americas. Between 2001 and
2011, around 270,500 cases were reported, with an average of 27,500
new cases/year. Around 3 - 5% of patients who develop cutaneous
lesions are also susceptible to mucosal leishmaniasis23,30. In the state
of São Paulo there are approximately 400 new cases per year. Another
substantial problem is the urbanization of the infection. Autochthonous
cases have been reported in urban areas. The incidence of peri-urban and
urban cases has been increasing. Approximately 10% of the population
living in endemic areas is at risk of acquiring the infection29. ATL is also
considered one of the most common dermatological syndromes diagnosed
in travelers (or tourists) who have visited endemic areas15.
The life cycle of L. (V.) braziliensis includes different reservoirs, such
as humans and wild and domestic mammals, as well as various vector
species. Therefore, Leishmania strains can be maintained in both rural and
urban settings, thereby affecting the epidemiology of the infection. Due
to the proximity of dogs and humans, studies have shown the important
role of domestic dogs in ATL19,21. Studies using molecular techniques to
characterize L. (V.) braziliensis populations have contributed to a better
(1) Laboratório de Biologia Molecular de Parasitas, Instituto Adolfo Lutz, São Paulo, SP, Brazil.
(2) Laboratório Regional de Sorocaba, Instituto Adolfo Lutz, Sorocaba, SP, Brazil.
Correspondence to: Vera Lucia Pereira-Chioccola, Laboratório de Biologia Molecular de Parasitas, Instituto Adolfo Lutz, Av. Dr Arnaldo 351, 8 andar, 01246-000 São Paulo, SP, Brasil.
Phone: +55.11.3068-2991. Fax +55.11.3068-2890. E-mail: [email protected]
FERREIRA, L.T.; GOMES, A.H.S. & PEREIRA-CHIOCCOLA, V.L. - Genotype characterization of Leishmania (Viannia) braziliensis isolated from human and canine biopsies with American
cutaneous leishmaniasis. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 257-262, 2015.
understanding of the abilities of these parasites and their vectors in
adapting to changes in their original forest habitats, and the consequent
public health implications13.
twice in phosphate-buffered saline (pH 7.2) at 1,000g for 10 min. The
parasite pellets were used for DNA extraction. L. (V.) braziliensis strain
DNA also was used in reactions as a positive control.
Despite the significance of ATL to the Brazilian public health
system, the genetic diversity of L. (V.) braziliensis in some endemic
areas of Brazil has been poorly researched, as in the state of São Paulo.
Therefore, this study aims to analyze the genetic diversity of a L. (V.)
braziliensis population collected from patients and dogs in the state of
São Paulo with cutaneous lesions, avoiding in vitro cultivation. The
reason for evaluating polymorphism in humans and dogs was due to the
importance of both species within the parasite's life cycle. The results
indicate a high variability in isolates collected in patients and dogs from
the state of São Paulo. Additionally, this study has shown the possibility
of performing genotyping directly on clinical samples without having
to isolate the parasite.
DNA purification: Before performing DNA extraction, clinical
samples and WHO Leishmania reference strains were crushed and
digested in a lysis buffer until tissue lysis was complete, (This-HCl,
10 mM, pH 8.0; EDTA 10mM; SDS, 0,5%; N-laurilsarcozil, 0.01%;
proteinase K, 100 µg/mL) by incubation in water bath at 56 °C. Then,
DNA molecules were extracted by a QIAamp DNA Mini Kit (Qiagen),
according to the manufacturer’s instructions. DNA concentration and
purity was determined by the ratio of O. D. at 260 and 280 nm in a
NanoDrop ND1000 (Thermo Scientific).
Human and dog samples: The selection of positive samples was
made in biopsies received by an in-house Laboratory over a period of
nine years (2003 - 2012). The biopsies were collected by medical or
veterinary health services. The human or canine lesions were cleansed
with antiseptics after the administration of a local anesthetic. The borders
of the lesions were scraped or smears of material were obtained by a
punch biopsy of the lesions and immediately added to tubes containing
1-2 mL of a sterile 0.85% NaCl and 200 µg/mL gentamicin solution,
sent to the laboratory within 48 hours and promptly processed to
confirm clinical diagnosis. All biopsies recorded were from patients
with the cutaneous clinical form. Samples were tested by routine
diagnosis, which included molecular and parasitological methods. The
methodologies applied were PCR, using two different sets of primers, and
a parasitological method (microscopic observation). These DNA samples
were from patients and dogs living in 36 different municipalities and
endemic areas for ATL in the state of São Paulo, Brazil (Alumínio, Aruja,
Avaré, Bauru, Bragança Paulista, Cajamar, Campinas, Caraguatatuba,
Cerquilho, Conhal, Cubatão, Guapiara, Guarulhos, Ibirá, Ilha Bela,
Indaiatuba, Iperó, Iporanga, Itapera, Itupeva, Jaboticabal, Jundiaí,
Mairiporã, Marília, Miracatu, Mirandópolis, Mogi Guaçu, Monte Mor,
Pilar do Sul, Ribeira, Salto, São Paulo, Sorocaba, Suzano, Tatuí, Tietê).
Epidemiological registers of the different Public Dermatology Clinics
or Centers for Zoonosis Control were analyzed to determine the locality
of the Leishmania infection of each patient (or dog).
Ethical considerations: This study was performed according to the
recommendations of the Human Ethics Committee (CONEP-IAL) and
“Sociedade Brasileira de Ciência em Animais de Laboratório/Colégio
Brasileiro de Experimentação Animal” (SBCAL/COBEA). Both Ethic
Committees of Instituto Adolfo Lutz have approved of this study.
Leishmania strains: For genotype standardization, the following
WHO standard Leishmania strains were used: L. (V.) guyanensis
(MHOM/BR/1975/M4147), L. (L.) amazonensis (IFLA/BR/1967/PH8),
L. (L.) major (MHOM/SU/1973/5-ASKH), L. (L.) infantum (MHOM/
BR/1974/PP75), and L. (V.) braziliensis (MHOM/BR/1975/M2903). The
Leishmania strains were maintained by serial passages and grown at 24
ºC in M-199 medium, supplemented with 10% calf serum and 0.25%
hemin25. In the log phase, 1 x 108 parasites were harvested and washed
258
Routine Leishmania diagnosis
Parasitological diagnosis: Skin biopsy imprints were plated onto a
glass slide, fixed with methanol and stained with Giemsa11. The presence
of amastigotes was observed microscopically with an immersion objective
(×1,000).
PCR targets for Leishmania and internal controls: The Leishmania
genus was identified by a 120-bp PCR product, amplified from a
conserved region of kDNA minicircles of Leishmania spp., using the
primer set 150/15223. L. (V.) braziliensis was identified by an amplified
fragment of 146-149 bp from the multicopy spliced leader (SL) RNA
gene using the primer set LU-5A/LB-3C, which amplifies a 146-149 bp
sequence from the SL12,17. These tests were carried out under the same
aforementioned conditions11,12. To check PCR inhibitors, canine and human
samples were assayed using a reference gene, whose primer sets were
GAPDH4F/GAPDH4R and β1-β2, respectively, in the same conditions
as previously described3,11. After the thermal cycles, PCR products were
electrophoresed in 2% agarose gel and stained with ethidium bromide.
DNA fragments were made visible under UV illumination.
L. (V.) braziliensis genotyping by RFLP-PCR (restriction
fragment length polymorphism-PCR): Originally, 132 DNA extracts
from biopsies, positive for L. (V.) braziliensis, were analyzed for
genotype determination. PCR was used for diagnosis and genotyping
directly from clinical DNA samples. Each test was performed by
adding 5 µL from each DNA template and 25 pmol from each primer
for a final volume of 25 µL. The amplifications were carried out with
a kit purchased from Promega (Go Taq Green Master Mix). The PCR
mix (12.5 μL) was composed of one unit of Taq DNA polymerase,
10 mM Tris-HCl, pH 8.5; 50 mM KCl; 1.5 mM MgCl2; and 200 mM
of each dNTP. In genotype reactions the primer set used was IR1/
IR2 (5’-GCTGTAGGTGAACCTGCAGCAGCTGGATCATT-3’ and
5’-GCGGGTAGTCCTGCCAAACACTCAGGTCTG-3’), which
amplified a 1-1.2-kb sequence from the ITS region between the small and
large subunits of the rDNA locus in a temperature annealing at 56 ºC5.
PCR-amplified products were digested with a HhaI restriction enzyme,
which were separated by electrophoresis in an 8% polyacrylamide gel
and stained with ethidium bromide. DNA fragments were made visible
under UV illumination. The images from reactions for diagnosis and
genotyping were analyzed by a MiniBIS Gel Imager and Documentation
system (BioSystematica). The size of the fragments was based on a
comparison with molecular-weight size markers. In genotyping reactions,
the banding patterns were used to group the isolates into genotypes with
the same banding pattern for the restriction enzyme.
Quality assurance: Each DNA extraction batch included a DNA
extraction from Leishmania-free eukaryotic samples as a negative
control. In each reaction, a tube containing nuclease-free water and PCR
mix was used as a blank control. Separate rooms were used for i. DNA
extraction, ii. PCR mix and primer preparation, iii. the adding of DNA
from clinical samples and positive control; and iv. post-PCR agarose-gel
electrophoresis analysis. DNA samples were assayed in duplicate and
at least twice.
RESULTS
The first experiments were conducted using the DNA extracted from
WHO reference strains to establish the genotype by RFLP-PCR, using
the primer set IR1/IR2 and additional treatment with HhaI enzymes.
Figure 1 shows the restriction patterns of the six WHO reference strains.
L. (V.) guyanensis and L. (V.) braziliensis showed the same restriction
profile. On the other hand, L. (L.) amazonensis, L. (L.) major and L. (L.)
infantum had specific restriction profiles.
Fig. 1 - Restriction patterns of PCR products digested with HhaI in DNA extracted from
standard Leishmania strains include the following: L. (V.) guyanensis (MHOM/BR/1975/
M4147) (1), L. (L.) amazonensis (IFLA/BR/1967/PH8) (2), L. (L.) major (MHOM/SU/1973/5ASKH) (3), L. (L.) infantum (MHOM/BR/1974/PP75) (4), and L. (V.) braziliensis (MHOM/
BR/1975/M2903) (5). Digested products were resolved in 2% agarose gel stained with
ethidium bromide. MM, 50-bp ladder.
Next, genotype experiments were conducted on the 132 DNA
samples taken from biopsies with a positive parasitological and molecular
diagnosis. All samples also tested positive for L. (V.) braziliensis (in
PCR), which was previously determined by the LU-5A/LB-3C primer
set, whose products range in size from 146 to 149 bp9,12,17. According the
epidemiological registers of the Public Dermatology Clinics and Centers
for Zoonosis Control, all samples analyzed were from patients or dogs
with an autochthonous Leishmania infection (in the same locality as the
biopsy collection).
Of the 132 DNA samples, only 52 (40%) were successfully
genotyped, as 1 - 1.2 kb products were amplified by the IR1/IR2 primer
set. The other 80 samples were not genotyped, as PCR products were not
amplified by this primer set. As expected, no amplification was detected
in DNA extracted from DNA as a negative control and PCR products
were obtained for all positive controls.
Table 1 shows the specification of the 52 genotyped samples in
detail, which included the collection date of the biopsies (2003 - 2012),
as well as the host (human or canine) and locality within the state of
São Paulo. The 52 samples were distributed in nine distinct patterns, as
shown in Figure 2.
Pattern 1 was identical to those found in L. (V.) guyanensis and L.
(V.) braziliensis WHO reference strains (Fig. 1). Furthermore, this L. (V.)
braziliensis pattern was the most common, since out of the 52 genotyped
samples, 29 (56%) belonged to pattern 1 and were distributed across
11 different municipalities. Pattern 2 was recurrent in nine samples
distributed across three municipalities. The other patterns (3 - 9) were
uncommon and found in few samples: Pattern 3 (three municipalities),
4 (two municipalities), 5 (one municipality), 6 (one municipality),
7 (one municipality), 8 (two municipalities), 9 (two municipalities),
respectively. The details and distribution of the clinical samples from
the 44 human patients and eight dogs for each L. (V.) braziliensis isolate
are shown in Table 2 and Figure 3. The majority (30) of the samples
were from Sorocaba. The others (22) were distributed across the other
14 municipalities.
DISCUSSION
ATL has been growing worldwide in both incidence and range,
principally due the increase in human migration. This mobility contributes
Fig. 2 - Amplified products (1-1.2 kb) of clinical samples of the ITS region between the small and large subunits of rDNA locus from L. (V.) braziliensis were digested with HhaI (RFLP
patterns). Among the 52 clinical samples, nine restriction patterns were shown. Digested products were resolved in 8% polyacrylamide gels stained with ethidium bromide. MM, 50-bp ladder.
259
Table 1
Clinical samples genotyped by RFLP-PCR in this study
Sample code-month/year
Host
Municipality
07-09/2003
Human
Sorocaba
18-09/2003
Human
20-09/2003
Canine
26-10/2003
Canine
64-09/2004
65-09/2004
Sample code-month/year
Host
Municipality
1063-07/2008
Human
Sorocaba
Sorocaba
1153-11/2008
Human
Campinas
Sorocaba
1324-02/2009
Human
Sorocaba
Marilia
1622-08/2009
Human
Sorocaba
Human
Sorocaba
1758-02/2010
Human
Jundiai
Human
Sorocaba
1945-06/2010
Human
Jundiai
84-11/2004
Canine
Ilha Bela
1946-10/2010
Human
Jundiai
115-03/2005
Human
Sorocaba
1985-11/2010
Human
Bauru
125-03/2005
Human
Sorocaba
2001-12/2010
Human
Sorocaba
157-06/2005
Human
Itupeva
2036-02/2011
Canine
Iporanga
194-08/2005
Human
Sorocaba
2037-02/2011
Canine
Iporanga
253-11/2005
Human
Sorocaba
2038-02/2011
Canine
Iporanga
274-05/2006
Human
Mairiporã
2072-04/2011
Human
Iperó
275-05/2006
Human
Cajamar
2098-06/2011
Human
Sorocaba
279-05/2006
Human
Itapera
2135-09/2011
Human
Sorocaba
281-05/2006
Human
Sorocaba
2136-09/2011
Human
Sorocaba
282-05/2006
Human
Sorocaba
2150-09/2011
Human
Sorocaba
288-06/2006
Human
Itupeva
2151-09/2011
Human
Sorocaba
304-08/2006
Human
Sorocaba
2152-09/2011
Human
Sorocaba
327-08/2006
Canine
Avaré
2163-10/2011
Human
Sorocaba
354-10/2006
Human
Sorocaba
2302-01/2012
Human
Sorocaba
504-09/2007
Human
Sorocaba
2538-05/2012
Human
Sorocaba
560-12/2007
Human
Sorocaba
2656-07/2012
Human
Ribeira
684-03/2008
Human
Sorocaba
2657-07/2012
Human
Ribeira
829-05/2008
Human
Sorocaba
2658-07/2012
Human
Ribeira
832-05/2008
Canine
Caraguatatuba 2883-12/2012
Human
Iperó
to the emergence of leishmanial infection in low or non-endemic areas13.
To prevent new cases in these areas, epidemiological strategies must be
implemented, such as rapid diagnosis, treatment and vector control. The
importance of the study of genetic variability of Leishmania is mainly
due to its correlation with the epidemiological aspects of the disease,
such as geographic location, clinical forms, virulence, pathogenicity,
drug resistance and antigenic variation, among others6,13.
Species belonging to the L. (V.) braziliensis sub-genera are highly
prevalent in patients with ATL in Brazil. Other Brazilian studies have
shown the genetic variability of these parasites, which would explain their
adaptation to changes in diverse environmental conditions4,6. Thus, with
such resilience, these parasites are more likely to infect multiple hosts.
Although different genetic studies have analyzed L. (V.) braziliensis
isolates from other Brazilian regions4,6,10,11,21,27, none have been conducted
in the state of São Paulo.
260
The idea of conducting this study in the state of São Paulo was
motivated by the state's increase of ATL incidences for the last 20 years.
Currently, 147 municipalities have already recorded transmission. Thus,
in this study, biopsies from 24.5% of these municipalities (36) were
investigated. However, due to the low sensitivity of the IR1/IR2 primer
set, samples from only 15 municipalities were genotyped.
One of the methods used to evaluate the genetic polymorphism of
L. (V.) braziliensis isolates in different Brazilian regions is the analysis
of RFLP in the internal transcribed spacers (ITS) of the ribosomal DNA
(rDNA) locus. These studies have shown that molecular markers are
suitable for population genetics and epidemiological studies4,5,6.
Despite the low sensitivity of the IR1/IR2 primer set and that the
clinical samples presented a low quantity of parasites in comparison
with culture isolates, 40% (52/132) of them were genotyped. Similar data
Table 2
Distribution of the nine L. (V.) braziliensis profiles isolated from human and
canine clinical samples in 15 municipalities of the state of São Paulo
L. (V.) braziliensis
genotyping (n)
Pattern 1 (29 samples)
Pattern 5 (1 sample)
Total of samples
Municipality
(n=15)
Bauru
Cajamar
Caraguatatuba
Iperó
Iporanga
Itapera
Itupeva
Jundiai
Mairiporã
Ribeira
Sorocaba
Jundiai
Ribeira
Sorocaba
Avaré
Marilia
Sorocaba
Campinas
Sorocaba
Sorocaba
Sorocaba
Sorocaba
Iporanga
Sorocaba
Ilha Bela
Sorocaba
Human
Canine
1
1
2
1
2
2
1
2
13
1
1
7
1
1
1
1
2
2
1
1
44
1
2
1
1
1
1
1
8
Fig. 3 - Map of South America and Brazil (A) indicating the location of the state of São Paulo
(A). Map of the state of São Paulo (B) indicating the municipalities studied and geographical
distribution of the L. (V.) braziliensis patterns: 1 (red), 2 (yellow), 3 (green), 4 (blue), 5
(purple), 6 (orange), 7 (pink), 8 (light blue) and 9 (gray).
has previously shown20 the possibility of performing RFLP-PCR using
small amounts of Leishmania DNA from host tissues. Consequently, it is
possible to genotype Leishmania populations with the analysis of DNA
extracted directly from clinical samples. This information is important,
because in many laboratories there are no conditions in which to isolate
and culture parasites from clinical samples20. Another interesting finding
was the fact that DNA samples isolated 11 years ago (2003) were of
good quality and could be used to genotype L. (V.) braziliensis isolates,
as shown in Table 1.
Results showed that L (V.) braziliensis seems to be a species with
great genetic diversity, as nine different patterns were observed in 52
different DNA samples from 15 municipalities using PCR-RFLP. As
shown in Figure 3, the different L (V.) braziliensis patterns were spread
throughout the regions. This genetic variability has already been shown in
other Brazilian studies4,10,11,21,27. Additionally, the parasite polymorphism
was correlated with different clinical forms of the disease, effectiveness
of treatment and cytokines expression10,21,27.
According to other studies4,6,23,29, genotypic variations exhibited by
L (V.) braziliensis could be explained by the adaption of parasites to
changes in the transmission process, as originally, the biological cycle was
restricted to forest environments. Similar to in other Brazilian regions,
the gradual removal of vegetation has also occurred in São Paulo in
recent years29. As a result, these parasites have adapted to infect a wider
diversity of sand flies and reservoirs.
RESUMO
Caracterização genotípica de isolados de Leishmania (Viannia)
braziliensis provenientes de biopsias de humanos e cães com
leishmaniose tegumentar americana
Introdução: A leishmaniose tegumentar americana (LTA) é causada
pelo sub-gênero Leishmania (Viannia) braziliensis. A evolução da LTA
resulta com a evolução das lesões iniciais. A diversidade genética de
L. (V.) braziliensis em algumas áreas endêmicas brasileiras, como no
estado de São Paulo, é pouco conhecida. Assim, este estudo teve como
objetivo analisar a variabilidade genética de isolados de L. (V.) braziliensis
coletados de biopsias de pacientes e cães com LTA no estado de São
Paulo. Métodos: O diagnóstico da leishmaniose foi realizado por PCR.
As 132 biópsias analisadas foram coletadas em diferentes regiões do
Estado de São Paulo, Brasil (36 municípios). A caracterização genética
de L. (V.) braziliensis foi realizada por RFLP-PCR utilizando DNA
extraído das biopsias. O conjunto de iniciadores utilizado amplificou a
região ITS de Leishmania. Resultados: Das 132 amostras analisadas,
52 (40%) foram completamente genotipadas por RFLP-PCR (44 de
pacientes e oito de cães). Os resultados mostraram nove padrões distintos.
A maioria das amostras genotipadas foi de Sorocaba (30), e as demais
foram distribuídas entre 14 outros municípios. O primeiro padrão foi
mais frequente (29 amostras), seguido pelo padrão 2 (nove amostras),
padrão 3 (três amostras). Padrões 4, 6, 7, 8 e 9 foram compostos de
duas amostras de cada um e o padrão 5, com uma amostra. Conclusão:
Estes resultados sugerem que cepas polimórficas de L. (V.) braziliensis
circulam no estado de São Paulo. Estes dados são concordantes com
estudos em outras regiões do Brasil, mostrando grande variabilidade
destas populações naturais de focos endêmicos.
261
ACKNOWLEDGMENTS
This study was supported by grants from the FAPESP (Fundação
de Amparo à Pesquisa do Estado de São Paulo, Brazil). Proc2011/13939-8. L.T.F. was supported by a fellowship from CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,
Brazil). V.L.P.C. was supported by a fellowship from CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico, Brazil)
Produtividade em Pesquisa, Proc. 303489/2012-0. Jim Hesson of
AcademicEnglishSolutions.com proofread the English.
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57(3):263-267, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300014
SEASONAL DISTRIBUTION OF MALARIA VECTORS (DIPTERA: CULICIDAE) IN RURAL LOCALITIES
OF PORTO VELHO, RONDÔNIA, BRAZILIAN AMAZON
Luiz Herman Soares GIL, Moreno de Souza RODRIGUES, Alzemar Alves de LIMA & Tony Hiroshi KATSURAGAWA
SUMMARY
We conducted a survey of the malaria vectors in an area where a power line had been constructed, between the municipalities of
Porto Velho and Rio Branco, in the states of Rondônia and Acre, respectively. The present paper relates to the results of the survey
of Anopheles fauna conducted in the state of Rondônia. Mosquito field collections were performed in six villages along the federal
highway BR 364 in the municipality of Porto Velho, namely Porto Velho, Jaci Paraná, Mutum Paraná, Vila Abunã, Vista Alegre do
Abunã, and Extrema. Mosquito captures were performed at three distinct sites in each locality during the months of February, July,
and October 2011 using a protected human-landing catch method; outdoor and indoor captures were conducted simultaneously at each
site for six hours. In the six sampled areas, we captured 2,185 mosquitoes belonging to seven Anopheles species. Of these specimens,
95.1% consisted of Anopheles darlingi, 1.8% An. triannulatus l.s., 1.7% An. deaneorum, 0.8% An. konderi l.s., 0.4 An. braziliensis,
0.1% An. albitarsis l.s., and 0.1% An. benarrochi. An. darlingi was the only species found in all localities; the remaining species
occurred in sites with specific characteristics.
KEYWORDS: Anopheles; Rondônia; HBR; Hydroelectric power plant.
INTRODUCTION
Although cases of malaria have decreased in Brazil since 2005, the
disease remains an important public health problem in the country. In
2012 alone, approximately 240,000 cases were recorded here, of which
more than 90% occurred in the Amazon region3.
Anopheline mosquitoes are the only vectors of Plasmodium spp.
parasites to humans. These mosquitoes are found in tropical and
neotropical regions. As of 2004, 476 species had been recorded26, of
which approximately 100 are considered vectors or potential vectors
of Plasmodium spp. to humans22,38. In the Amazon region, the main
disease vector is Anopheles darlingi, although other species such
as An. deaneorum, An. triannulatus, and An. nuneztovari may play
important roles in the epidemiology of the disease35,36,43. The occurrence
of an anopheline species in an endemic area is associated with the
environmental characteristics of the region.
South America is currently among the regions most affected by
environmental changes, mainly because of the great pressure caused
by humans in the area. The construction of hydroelectric power plants,
family farming, and selective logging, among many other activities, has
become common in the region44. The changes caused by these activities
can drastically influence the population dynamics of malaria vectors,
thereby increasing malaria cases in the region. Thus, the assessment of
mosquito population density and species occurrence in areas where these
activities take place or will take place is essential for evaluating the risk
of malaria transmission and for designing and implementing effective
measures of control and/or prevention.
Therefore, our study aimed to assess the changes in the number and
composition of anopheline species along the construction corridor of a
power transmission line.
Mosquito capture was conducted along a 380 km stretch of the
BR 364 highway, between Porto Velho (RO) and Rio Branco (AC).
Six distinct localities with the highest population densities along the
stretch were selected. These localities were Extrema (09°45'29.1"S
66°21'34.3"W), Vista Alegre (09°39'39.8"S 65°43'57.2"W), Abunã
(09°41'48.9"S 65°22'15.8"W), Mutum Paraná (09°37'01.3"S
64°56'24.4"W), Jaci Paraná (09°15'37.2"S 64°23'44.1"W), and Porto
Velho (08°48'35.5"S 63°56'30.3"W) (Fig. 1). Vegetation in these areas
predominantly consists of open rain forest with a high degree of human
disturbance, as evidenced by the removal of native vegetation and its
replacement by pasture.
Fundação Oswaldo Cruz (FIOCRUZ Rondônia), Rua da Beira 7671, Bairro Lagoa, 76812-245 Porto Velho, RO, Brazil.
Correspondence to: Tony Hiroshi Katsuragawa, Tel.: +55 69 3219-6012, Fax: +55 69 3216-5442. E-mail: [email protected]
GIL, L.H.S.; RODRIGUES M.S.; LIMA, A.A. & KATSURAGAWA, T.H. - Seasonal distribution of malaria vectors (Diptera: Culicidae) in rural localities of Porto Velho, Rondônia, Brazilian
Amazon. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 263-7, 2015.
Table 1
Number and distribution of collected mosquitoes
Species
Anopheles albitarsis l.s.
Anopheles benarrochi
Anopheles braziliensis
Anopheles darlingi
Fig. 1 - Locations of capture sites. (A) State of Rondônia location in Brazil. (B) Magnified
view of Porto Velho municipality and localities of anopheline mosquito capture sites (circles)
and hydroelectric power plants (triangle) location.
Captures were performed at three sites in each locality during
the months of February, July, and October 2011. Three dwellings
were selected at each site, and protected human landing catches
were performed simultaneously inside and outside of each dwelling.
The collection sites remained the same throughout the whole study
for posterior comparison. Mosquitoes were stored in plastic cups
and identified using dichotomous keys for females6,13. The species
compositions of the distinct areas were compared using PERMANOVA,
followed by PERMDISP and scored on NMDS. For these analyses,
the vegan package was used in R37. The comparison of An. darlingi
density among localities and between indoor and outdoor dwellings was
performed using ANOVA, and the negative binomial error distribution
was adjusted to correct overdispersion problems. All analyses were
performed at a significance level of 5%.
Anopheles deaneorum
Anopheles konderi l.s.
Anopheles triannulatus l.s.
264
Frequency
0.09
0.09
0.37
95.10
38
17
40
1.74
0.78
1.83
Area
AB; MP
JP
MP; PVH
PVH; EX; VA;
AB; MP; JP
EX; VA
JP; PVH
PVH; JP; MP
AB = Abunã; EX = Extrema; JP = Jaci Paraná; MP = Mutum Paraná; PVH =
Porto Velho; VA = Vista Alegre Abunã.
Fig. 2 - Scoring of the areas according to the composition of anopheline species.
Table 2
Number and distribution of Anopheles darlingi mosquitoes
collected by locality
RESULTS
We collected 2,185 anopheline mosquitoes belonging to seven
species in the six localities. Of the mosquitoes collected, 95.1% were
identified as An. darlingi, 1.8% as An. triannulatus l.s., 1.7% as An.
deaneorum, 0.8% as An. konderi l.s., 0.4% as An. braziliensis, 0.1% as
An. albitarsis l.s., and 0.1% as An. benarrochi l.s. Anopheles darlingi
was collected in all localities. The remaining species were found in sites
with unique characteristics (e.g., An. braziliensis was predominantly
found in areas of secondary growth, such as forest clearings and
pastures) (Table 1). Mutum Paraná, Porto Velho, and Jaci Paraná
exhibited four of the seven collected species, whereas only two were
found in Extrema, Vista Alegre do Abunã, and Vila Abunã. Moreover,
although these areas had the same number of species, their species
composition (i.e., species found in the area) significantly differed
(PERMANOVA S.S = 1.37; Pseudo-F < 0.001). However, no significant
differences in Anopheles spp. community homogeneity were observed
among the study areas (Permdisp p = 0.34; Fig. 2). Analysis of the
distribution patterns of the main malaria vectors showed that the number
of An. darlingi differed significantly among localities (ANOVA χ2 =
44.35, p < 0.001), with Porto Velho exhibiting the highest mean number
of individuals of this species (144.55 ± 59.81) and Vila Abunã exhibiting
the lowest (5.66 ± 1.04). The mean distribution of An. darlingi among
the localities is summarized in Table 2. With the exception of Vista
Alegre do Abunã, An. darlingi numbers were significantly higher
in outdoor than indoor dwellings (ANOVA χ2 = 27.79, p < 0.001;
Fig. 3).
Total
2
2
8
2,078
Locality
Porto Velho
Jaci Paraná
Extrema
Mutum Paraná
Vila Abunã
Vista Alegre do Abunã
Mean ± SD
144.55 ± 59.81
37.22 ± 10.40
26.55 ± 12.27
23.77 ± 2.78
7.11 ± 1.04
5.66 ± 2.71
Contrast
analysis
a
b
b
b
c
c
p value
< 0.001
0.30
0.63
SD: standard deviance; columns followed by different letters indicate statistical
difference.
Fig. 3 - Mean of Anopheles darlingi collected per night in and around dwellings of various
localities under study. AB = Abunã; EX = Extrema; JP = Jaci Paraná; MP = Mutum Paraná;
PVH = Porto Velho; VA = Vista Alegre Abunã.
DISCUSSION
In Brazil, six species of anopheline mosquitoes play significant
roles in the transmission of Plasmodium spp. parasites to humans.
Three of these species were collected in our study: An. darlingi, An.
triannulatus, and An. braziliensis. Other species such as An. albitarsis
l.s. may be secondary or specific vectors in particular areas29,35,36,43. The
prevalence of An. darlingi has remained consistently higher than that
of other species since the first studies of malaria vectors in Rondônia,
even amid the environmental changes that have taken place over the
last few decades5,7,10,11,12,16,17,18,32. The high prevalence of Anopheles
darlingi in this study was expected, as this species presents the most
anthropophilic behavior among the sample collected. Furthermore, many
of the environmental changes in this region (e.g., flooding of areas and
the creation of water reservoirs for human use) have increased the number
of breeding sites for this species33.
The peak number of species occurred in July. Anopheles darlingi
showed two distinct population peaks, the first in March/May (during
the rainy season) and the second in August/September (end of the dry
season)18. The peak in July may be explained by the environmental
changes in the area due to the implementation of two hydroelectric power
plants, or a delay in the response of An. darlingi to the rainy season.
The susceptibility of anopheline mosquitoes to infection by the
predominant Plasmodium species recorded in the region, P. falciparum
and P. vivax, has been previously reported9,27,28,31. Although the highest
number of mosquitoes occurred around dwellings, the results highlight
that An. darlingi exhibits highly anthropophilic behavior. Moreover,
although we did not find a higher number of mosquitoes inside dwellings,
these species are highly endophilic15,21,30, making the study region a highrisk area for the transmission and prevalence of malaria.
New farming frontiers in areas where malaria is endemic require more
public policies for mosquito control8,34,41. Climate change, urbanization,
and new settlements for agriculture and the rearing of livestock are among
the factors that can lead to epidemics of malaria and other vector-borne
diseases41.
As the localities of the present study are situated within the area
of influence of two hydroelectric power plants in the Madeira River,
with intense anthropization and increased water surface, and the results
obtained indicate high vector densities in urbanized areas, the current
malaria situation requires attention. The Madeira River carries a large
quantity of suspended sediment, which favors the predominance of
mosquitoes of the Mansonia genus. The river water does not exhibit
this characteristic, however, the conditions in this water promote the
proliferation of An. darlingi39. Therefore, despite the decreased number
of malaria cases over the last few years in Brazil3, urban expansion in
the proximity of these new water reservoirs increases the risk of malaria
transmission14,40.
Factors such as the creation of water reservoirs and deforestation
caused by the construction of hydroelectric power plants and power
lines, as well as peri-urban transmission of disease18,42, have increased
the density of malaria vectors1,2,4,5,23,24,25, thereby elevating the risk of
epidemics within the next several decades19,20.
Because anopheline mosquitoes are key determinants in the
transmission of Plasmodium, which can infect and cause malaria in
humans, the regular monitoring of the disease transmission of these
vectors in relation to regional climate cycles is of paramount importance.
Moreover, scientifically-based joint and coordinated action between
public authorities and construction entrepreneurs should be conducted
to control malaria transmission in the Brazilian Amazon.
RESUMO
Distribuição sazonal de vetores da malária (Diptera: Culicidae)
em localidades rurais de Porto Velho, Rondônia, Amazônia
brasileira
Foi realizado levantamento de vetores de malária na área que
compreende a construção da linha de transmissão entre os municípios de
Porto Velho e Rio Branco, estados de Rondônia e Acre, respectivamente.
Os dados aqui apresentados mostram os resultados do levantamento
da fauna dos Anopheles realizado em Rondônia. As capturas foram
realizadas no município de Porto Velho em seis aglomerados
populacionais ao longo da rodovia federal BR 364, denominados Porto
Velho, Jaci Paraná, Mutum Paraná, Vila Abunã, Vista Alegre do Abunã
e Extrema. As capturas ocorreram em três diferentes pontos de cada
uma das localidades nos meses de fevereiro, julho e outubro de 2011,
seguindo a metodologia de coleta por atração humana protegida em dois
ambientes, sendo no intradomicílio e no peridomicílio simultaneamente
com duração de seis horas. Nas áreas amostradas foram capturados 2.185
anofelinos pertencentes a sete espécies de Anopheles sp. dos quais 95,1%
foram identificados como Anophels darlingi, 1,8% An. triannulatus l.s.,
1,7% An. deaneorum, 0,8% An. konderi l.s., 0,4 An. braziliensis, 0,1%
An. albitarsis l.s., e 0,1% An. benarrochi. Anopheles darlingi foi a única
espécie amostrada em todas as localidades enquanto as demais espécies,
ocorreram em locais com características singulares.
FINANCIAL SUPPORT
This study received financial and logistical support from Cepemar
Serviços de Consultoria em Meio Ambiente Ltda.
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57(3):269-272, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300015
BRIEF COMMUNICATION
CHICKEN COOPS, Triatoma dimidiata INFESTATION AND ITS INFECTION WITH Trypanosoma cruzi
IN A RURAL VILLAGE OF YUCATAN, MEXICO
Edgar KOYOC-CARDEÑA(1), Anuar MEDINA-BARREIRO(1), Francisco Javier ESCOBEDO-ORTEGÓN(2), Jorge Carlos RODRÍGUEZ-BUENFIL(3),
Mario BARRERA-PÉREZ(2), Enrique REYES-NOVELO(2), Juan CHABLÉ-SANTOS(1), Celia SELEM-SALAS(1),
Gonzalo VAZQUEZ-PROKOPEC(4) & Pablo MANRIQUE-SAIDE(1)
SUMMARY
This study longitudinally investigated the association between Triatoma dimidiata infestation, triatomine infection with Trypanosoma
cruzi and household/backyard environmental characteristics in 101 homesteads in Molas and Yucatan, Mexico, between November
2009 (rainy season) and May 2010 (dry season). Logistic regression models tested the associations between insect infestation/infection
and potential household-level risk factors. A total of 200 T. dimidiata were collected from 35.6% of the homesteads, mostly (73%)
from the peridomicile. Of all the insects collected, 48% were infected with T. cruzi. Infected insects were collected in 31.6% of the
homesteads (54.1% and 45.9% intra- and peridomiciliary, respectively). Approximately 30% of all triatomines collected were found
in chicken coops. The presence of a chicken coop in the backyard of a homestead was significantly associated with both the odds of
finding T. dimidiata (OR = 4.10, CI 95% = 1.61-10.43, p = 0.003) and the presence of triatomines infected with T. cruzi (OR = 3.37,
CI 95% = 1.36-8.33, p = 0.006). The results of this study emphasize the relevance of chicken coops as a putative source of T. dimidiata
populations and a potential risk for T. cruzi transmission.
KEYWORDS: Peridomicile; Triatoma dimidiata; Trypanosoma cruzi; Chagas disease.
In the Mexican state of Yucatan, Chagas disease is an endemic
zoonosis transmitted domestically by Triatoma dimidiata Latreille 1811
(Hemiptera: Reduviidae), the only locally proven vector. T. dimidiata can
be collected in domestic, peridomestic and sylvatic habitats of Yucatan.
House infestation is described as seasonal, occurring mainly due to the
dispersal of adult insects from peridomestic and sylvatic habitats during
the late dry season with non-apparent or limited colonization2,5.
in a house and keeping chickens in a corral were strong determinants
for house infestation in rural communities. Such findings from Yucatan
agree with reports of house infestation and colonization by T. dimidiata
in Guatemala11. This study confirms the significance of the peridomicile
environment, and particularly of chicken coops, as a source of T. dimidiata
populations and a potential risk factor for T. cruzi transmission in a rural
village in Yucatan, Mexico.
Few studies in Yucatan have examined the importance of household
and backyard characteristics in the prevalence of triatomine infestations
and their infection rates with T. cruzi in and around houses. GUZMÁNMARÍN et al. 8 reported that household triatomine infestation in
rural communities was associated with the type/quality of housing,
e.g. construction with natural materials, thatched roofs, unplastered
walls or walls with adobe plastering and the lack of cemented floors.
However, other studies have reported that the location of a house within
a community (especially if located on the periphery) is a significant risk
factor for infestation and the invasion of dispersing adult insects2,5,13.
REYES-NOVELO et al.14 showed that T. dimidiata did colonize animal
shelters (e. g. chicken and dove coops, dog houses and opossum nests).
More recently, DUMONTEIL et al.5 quantified that the number of dogs
Fieldwork was carried out between November 2009 and May 2010 in
a sample of 101 homesteads (each homestead including the house and all
peridomestic structures found in the front and backyard) from Molas, a
rural village located in the Southeast of Mexico (20° 48’58’’ N and 089°
37’54’ W). The community has a population of 2,014 inhabitants, living
in 553 houses and surrounded by a subtropical deciduous forest within the
Cuxtal ecological reserve. Altitude is 10 m. a. s. l. Climate is characterized
by an average annual temperature of 25.9 °C, with an annual rainfall of 8001000 mm, occurring mainly between June and November. Molas is located
within the highest risk area for Chagas disease in the state of Yucatan2,3.
Homestead infestation with triatomines was evaluated through:
i) active collections both intra- and peridomiciliary and; ii) householders’
(1) Departamento de Zoología, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, México.
(2) Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, México.
(3) Departamento de Epidemiología, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, México.
(4) Department of Environmental Sciences, Emory University, Atlanta, Georgia, United States of America.
Correspondence to: Enrique Reyes-Novelo, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Av. Itzaes No. 490 por 59 Col. Centro, Mérida,
Yucatán, México. C. P. 97000. E-mail: [email protected]
KOYOC-CARDEÑA, E.; MEDINA-BARREIRO, A.; ESCOBEDO-ORTEGÓN, F.J.; RODRÍGUEZ-BUENFIL, J.C.; BARRERA-PÉREZ, M.; REYES-NOVELO, E.; CHABLÉ-SANTOS,
J.; SELEM-SALAS, C.; VAZQUEZ-PROKOPEC, G. & MANRIQUE-SAIDE, P. - Chicken coops, Triatoma dimidiata infestation and its infection with Trypanosoma cruzi in a rural
village of Yucatan, Mexico. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 269-72, 2015.
participatory collections within the houses. Two cross-sectional timed
manual active searches for triatomines (described by Gürtler et al. 1999)
were performed, one during the 2009 rainy season (November) and the
other one during the 2010 dry season (May).
Collections were performed inside houses (interdomiciliary) and
in front/backyards (peridomiciliary) by teams of two trained research
personnel (30 min in each ecotope to complete one man/hour/homestead
between 8:00 a.m. and 1:00 p.m.). Intradomiciliary searches included
inside walls, the base of the roof and furniture. Peridomiciliary searches
focused on animal housing, rock and woodpiles, tree trunks, and any
other potential triatomine refuges. In addition, householders were invited
to take part in a six-month participatory vector surveillance strategy4
between December and March, 2010.
Trypanosoma cruzi DNA extraction from individual triatomines
and PCR amplification of T. cruzi kinetoplast DNA were performed
as described by REYES-NOVELO et al.14 based on the EDWARDS
et al. 6 and MOSER et al. 12 protocols. The primers used were
TcZ1: 5’-CGAGCTCTTGCCCACACGGGTGCT-3’- and TcZ2
5’-CCTCCAAGCAGCGGATAGTTCAGG-3’. Amplification
was performed in a Techne TC132 (Barloworld Scientific LTD,
Staffordshire, UK) thermal cycler. A 188bp fragment identified the
presence of T. cruzi DNA following the electrophoresis of a percentage
of PCR product in a 2% agarose-TBE stained with ethidium bromide (10
µg/mL) and further documentation in an EDAS 290 gel documentation
system (Kodak, Rochester, USA). Local strains of T. cruzi were used
as positive controls, whereas the whole mixture minus DNA was used
as a negative control.
A household survey was performed to investigate a range of
household/backyard characteristics previously reported as significant in
the infestation of T. dimidiata and other triatomines1,10,15 - type/material
of the house (roof, walls, floor); use of window screening; presence of
rubbish, rock/wood piles, stone walls, abandoned lots on the sides; the
presence of domestic animals e. g. dogs, cats, poultry, horses, sheep,
cattle, and the presence of animal housing structures (organized by
species) e. g. chicken coops, pig corrals, house stables and kennels.
Using a Fisher’s exact test, statistical analyses compared the sex
and stage of development of T. dimidiata between locations (intra- and
peridomicile). Comparisons of infection by sex between seasons were
not performed because of the low number of insects collected. Tests
were carried out to study the association between triatomine infestation
(positive homesteads) and T. cruzi infection with household-level
potential risk factors. Variables of interest were analyzed using c2 to
reduce the model, by comparing percentages in contingency tables. Those
with p < 0.25 were included in a logistic regression analysis. Adjusted
Odds Ratio and Confidence Intervals (α = 0.05) were calculated with
SPSS® (v17.0).
A total of 200 T. dimidiata specimens were collected from 35.6%
(36/101) homesteads throughout the study period (Table 1). Overall, a
greater number of adults were collected than nymphs (p < 0.05), with a
higher male:female abundance ratio (p < 0.05) between ecotopes (Table
1). The majority of specimens (73%) - both adults and nymphs - were
collected in the peridomicile environment; nevertheless, 22.5% of the
adults and 4.5% of the nymphs collected were reported to have been
found intradomiciliary.
Collection methods were complementary. Active collections yielded
more specimens (65%) than participatory collections. 130 T. dimidiata
specimens were captured by active collection, mostly peridomiciliary
(97.7%), with a sample composed by adults and nymphs in a similar
ratio. 70 specimens of T. dimidiata were captured through householders’
collections, mostly reported as intradomiciliary (74.3%) and consisting
mostly of adult triatomines (77%).
Overall, 48% (96/200) of the T. dimidiata specimens collected
tested positive for T. cruzi (Table 1) and were found in 31.6% (32/101)
of homesteads. The infected specimens were mostly adults (p < 0.05);
but the proportion of nymphs infected was high (37.5%). Slightly more
infected T. dimidiata were found intradomiciliary (54.1%) than in the
peridomicile environment (45.9%). Infection prevalence detection was
higher in participatory collections (59/70) than in active collections
(37/130) (p < 0.05).
Table 1
Triatoma dimidiata infestation (by stage of development, sex and location) and infection with Trypanosoma cruzi in homesteads in the rural community of Molas,
Yucatan, Mexico
Total (%)
Nymphs (%)
Adults (%)
♂ (%)
♀ (%)
Intradomiciliary
54 (27.0)
9 (16.7)
45 (83.3)
19 (35.2)
26 (48.1)
Peridomiciliary
146 (73.0)
79 (54.1)
67 (45.9)
42 (28.8)
25 (17.1)
Total
200 (100)
88 (44.0)
112 (56.0)*
61 (30.5)
51 (25.5)*
Intradomiciliary
52(54.1)
9 (17.3)
43 (82.7)
18 (34.6)
25 (48.1)
Peridomiciliary
44 (45.9)
27 (61.4)
17 (38.6)
8 (18.2)
9 (20.4)
Total
96 (100)
36 (37.5)
60 (62.5)*
26 (27.1)
34 (35.4)
Infestation
Infection
* Significant statistical difference in the frequencies of developmental stage and sex between locations, as given by Fisher’s exact test (p < 0.05). Statistical tests regarding
infestation and infection were performed separately.
270
Table 2
Active collection and Trypanosoma cruzi infection of Triatoma dimidiata from peridomicilary chicken coops in homesteads in the rural community of Molas,
Yucatan, Mexico
Total
Nymphs (%)
Adults (%)
♂ (%)
♀ (%)
Infestation
41
23 (56.1)
18 (43.9)
15 (36.6)*
3 (7.3)
Infection
20
19 (95)*
1 (5)
0 (0)
1 (5)
Infestation
26
13 (50.0)
13 (50.0)
5 (19.2)
8 (30.8)
Infection
10
4 (40)
6 (60)
3 (30)
3 (30)
1st active collection
Rainy season
2nd active collection
Dry season
*Significant statistical difference in frequencies of developmental stage and sex between seasons, as given by Fisher’s exact test (p < 0.05). Statistical tests regarding
infestation and infection were performed separately.
Three housing/backyard characteristics initially had p < 0.25: the
presence of a chicken coop in the backyard, the type of walls in the
house and wood storage; but only the presence of a chicken coop was
significantly and positively associated with the presence of T. dimidiata
(OR = 4.10, p = 0.003; 95% CI = 1.61-10.43) in the final model. The
presence of a chicken coop was also positively associated with T.
dimidiata infected with T. cruzi (OR = 3.37, p = 0.006; 95% CI = 1.368.33).
Fifty-four homesteads were found in the area with at least one
chicken coop. The general structure of local chicken coops consists of
cages of 1.30 - 1.50m in height, square in shape, and with sides of 2 - 3m
in length. Coops are built on a 4-log base, one on each corner, holding
a roof made of either zinc or cardboard and surrounded by a chicken
wire fence. The ground is commonly covered with compacted dirt and
small stones. Approximately 26% (14/54) of the chicken coops had
T. dimidiata, and 64.3% (9/14) had triatomines infected with T. cruzi.
Of all peridomestic triatomines, 45.9% (67/146) were collected from
chicken coops, with nymphs and adults found in a similar ratio (36:31,
respectively. 44.8% of all specimens collected from chicken coops were
infected (30/67), with a higher percentage of infected nymphs (76.7%
compared to 23.3% in adults).
rabbit hutch had a large population of triatomines, this type of refuge
was not as commonly found in the peridomiciles as chicken coops. Piles
of rocks and wood were quite common, but only one was found to be
infested with triatomines.
While the debate concerning whether house infestation by
triatomines is influenced by the peridomicile and/or the sylvatic habitats
continues4,13,14, the results of this study expose the significance of chicken
coops located in the peridomicile as a potential source of T. dimidiata
populations. Preceding studies in Yucatan report that T. dimidiata
infestations occur seasonally but transiently, i.e. with a limited capacity
for colonizing households in Yucatan2,7. These findings indicate the
existence of peri- and intradomiciliary infestation and the high prevalence
of infected triatomines not only during the dry season, but also in the
rainy season. Colonization in houses (based on the collection of nymphs)
is indeed uncommon during the rainy season, but increases during the
dry season.
This study shows that chicken coops are a risk factor for insect
infestation and parasite infection. Nonetheless these findings should be
re-evaluated in other communities infested by T. dimidiata.
RESUMEN
Triatomine specimens collected from chicken coops were obtained
exclusively by active collection and were found on the floor and under
stones. During the sectional-active collection in the rainy season, 9.3%
(5/54) of homesteads with chicken coops were positive for triatomines.
During the second active collection in the following dry season, 16.7%
(9/54) of homesteads with chicken coops were positive for triatomines
(the majority identified positive for the first time and only two were
consistent from the collection four months earlier (Table 2).
Chicken coops are known to play an important role in the maintenance
of T. dimidiata populations, both as a refuge for invading insects11,16 and as
a primary source of blood for triatomines9. All coops that tested positive
in Molas had chickens, except for one, where chickens were removed
two weeks before the survey. Among the total homesteads sampled in
the locality, only two other sites were found to be used by triatomines
as refuges: a rabbit hutch and a pile of rocks and wood. Although the
Gallineros, la infestación por Triatoma dimidiata y su infección con
Trypanosoma cruzi en una localidad rural de Yucatán, México
Investigamos longitudinalmente la asociación entre la infestación
por Triatoma dimidiata, su infección con Trypanosoma cruzi y las
características ambientales de los domicilios/peridomicilios en 101
viviendas de Molas, Yucatán, México entre Noviembre de 2009
(temporada lluviosa) y Mayo de 2010 (temporada seca). Mediante
modelos de regresión logística se probaron asociaciones entre la
infestación/infección de T. dimidiata y factores de riesgo potenciales
a nivel de las viviendas. Se colectó un total de 200 individuos de T.
dimidiata en el 35.6% de las viviendas, mayormente del peridomicilio
(73%). De todos los triatominos colectados el 48% se encontraron
infectados con T. cruzi. Los triatomas infectados fueron colectados
en el 31.6% de las viviendas (54.1% y 45.9% en intra y peridomicilio
271
respectivamente). Aproximadamente el 30% de todos los triatominos
colectados, fueron encontrados en gallineros. La presencia de un gallinero
en el peridomicilio de una vivienda se asoció significativamante tanto
con las posibilidades de encontrar T. dimidiata (OR = 4.10, CI 95% =
1.61-10.43, p = 0.003) como con la presencia de triatominos infectados
con T. cruzi (OR = 3.37, CI 95% = 1.36-8.33, p = 0.006). Los resultados
de este estudio enfatizan la relevancia de los gallineros como fuente
putativa de poblaciones de T. dimidiata y como una fuente potencial de
riesgo de transmisión de T. cruzi.
ACKNOWLEDGEMENTS
This study was funded by the project “Estudio multidisciplinario para
la identificación de variables asociadas a la transmisión de enfermedades
zoonóticas y enfermedades transmitidas por vector en Yucatán” of the
Red epidemiológica de Enfermedades Zoonóticas y Transmitidas por
Vector (ETV’s) de Importancia en Salud Pública (PROMEP 2008103.5/09/12.58. SISTPROY CIRB-2009-0006).
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of plant genomic DNA for PCR analysis. Nucleic Acid Res. 1991;19:1349.
7.Gourbiere S, Dumonteil E, Rabinovich JE, Minkoue R, Menu F. Demographic and
dispersal constraints for domestic infestation by non-domicilated Chagas disease
vectors in the Yucatan Peninsula, Mexico. Am J Trop Med Hyg. 2008;78:133-9.
8. Guzmán-Marín E, Barrera-Perez M, Rodríguez-Felix ME, Escobedo-Ortegon F, ZavalaVelázquez J. Índices entomológicos de Triatoma dimidiata en el estado de Yucatán.
Rev Biomed. 1991;2:20-9.
9. Guzmán-Marín E, Barrera-Perez M, Rodríguez-Félix ME, Zavala-Velázquez J. Hábitos
biológicos de Triatoma dimidiata en Yucatán, México. Rev Bioméd. 1992;3:125-31.
10.Guzmán-Tapía Y, Ramírez-Sierra M, Dumonteil E. Urban infestation of Triatoma
dimidiata in the city of Mérida, Yucatán, México. Vector Borne Zoonotic Dis.
2007;7:597-606.
11. Monroy CM, Bustamante DM, Rodas A, Enriquez ME, Rosales R. Habitats, dispersion
and invasion of sylvatic Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae)
in Peten, Guatemala. J Med Entomol. 2003;40:800-6.
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12.Moser D, Kirchoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA
amplification using the polymerase chain reaction. J Clin Microbiol. 1989;27:1477-82.
1. Cohen JM, Wilson ML, Cruz-Celis A, Ordoñez R, Ramsey JM. Infestation by Triatoma
pallidipennis (Hemiptera: Reduviidae: Triatominae) is associated with housing
characteristics in rural Mexico. J Med Entomol. 2006;43:1252-60.
13.Ramírez-Sierra MJ, Herrera-Aguilar M, Gourbiere S, Dumonteil E. Patterns of house
infestation dynamics by non-domiciliated Triatoma dimidiata reveal a spatial gradient
of infestation in rural villages and potential insect manipulation by Trypanosoma
cruzi. Trop Med Int Health. 2010;15:77-86.
2.Dumonteil E, Gourbiere S, Barrera-Pérez M, Rodríguez-Félix E, Ruíz-Piña H, BañosLópez O, et al. Geographic distribution of Triatoma dimidiata and transmission
dynamics of Trypanosoma cruzi in the Yucatan peninsula of Mexico. Am J Trop Med
Hyg. 2002;67:176-83.
3. Dumonteil E, Gourbiere S. Predicting Triatoma dimidiata abundance and infection rate:
a risk map for natural transmission of Chagas disease in the Yucatan peninsula of
Mexico. Am J Trop Med Hyg. 2004;70:514-9.
4. Dumonteil E, Ramírez-Sierra MJ, Ferral J, Euán-Garcia M, Chavez-Nuñez L. Usefulness
of community participation for the fine temporal monitoring of house infestation by
non-domiciliated triatomines. J Parasitol. 2009;95:469-71.
5.Dumonteil E, Nouvellet P, Rosecrans K, Ramírez-Sierra MJ, Gamboa-León MR,
Cruz-Chan JV, et al. Eco-Bio-Social determinants for house infestation by nondomiciliated Triatoma dimidiata in the Yucatan peninsula, Mexico. PLOS Negl Trop
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14. Reyes-Novelo E, Ruiz-Piña HA, Escobedo-Ortegon J, Barrera-Perez M, Manrique-Saide
P, Rodríguez-Vivas RI. Triatoma dimidiata (Latreille) abundance and infection with
Trypanosoma cruzi in a rural community of Yucatan, Mexico. Neotrop Entomol.
2013;42:317-24.
15.Starr MD, Rojas JC, Zeledón R, Hird DW, Carpenter TE. Chagas’ disease: risk factors
for house infestation by Triatoma dimidiata, the major vector of Trypanosoma cruzi
in Costa Rica. Am J Epidemiol. 1991;133:740-7.
16. Zeledón R, Montenegro VM, Zeledón O. Evidence of colonization of man-made ecotopes
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57(3):273-275, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300016
CASE REPORT
TUBERCULOSIS INFECTION MIGHT INCREASE THE RISK OF INVASIVE CANDIDIASIS IN AN
IMMUNOCOMPETENT PATIENT
Xiao-Hua CHEN(1), Yun-Chao GAO(2), Yi ZHANG(1), Zheng-Hao TANG(1), Yong-Sheng YU(1) & Guo-Qing ZANG(1)
SUMMARY
Deep Candida infections commonly occur in immunosuppressed patients. A rare case of a multiple deep organ infection with
Candida albicans and spinal tuberculosis was reported in a healthy young man. The 19-year-old man complained of month-long fever
and lower back pain. He also had a history of scalded mouth syndrome. Coinfection with Mycobacterium tuberculosis and Candida
albicans was diagnosed using the culture of aspirates from different regions. Symptoms improved considerably after antifungal and
antituberculous therapy. This case illustrates that infection with tuberculosis might impair the host’s immune system and increase the
risk of invasive candidiasis in an immunocompetent patient.
KEYWORDS: Invasive candidiasis; Spine tuberculosis; Coinfection; Immunocompetent patient.
INTRODUCTION
Invasive candidiasis is a clinical condition that generally occurs in
immunosuppressive patients and those with general defects in the immune
system. Candida albicans is the most common fungal pathogen capable
of causing infections, ranging from slight mucocutaneous disorders to
invasive diseases which affect multiple organs3. Deep Candida infections
rarely occur in healthy hosts, but the incidence is greatly increased
in immunosuppressive patients. Tuberculosis remains a major global
health problem and has become significantly more prevalent in the past
decade. Moreover, spinal tuberculosis may not display typical symptoms
and, sometimes, show predominant extrapulmonary manifestations that
result in delayed or missed diagnosis. Furthermore, multiple deep organ
infections caused by Candida albicans and spinal tuberculosis occurring
simultaneously in the same patient are very rare. In view of this, the
following report presents the rare case of multiple deep abscesses caused
by Candida albicans with simultaneously occurring spinal tuberculosis
in an immunocompetent patient.
CASE REPORT
A 19-year-old man complained of recurrent fever and lower back pain
for a month. He had a history of scalded mouth syndrome with no regular
antibiotic or antifungal drug treatment six months prior to hospitalization.
He had neither history of taking immunosuppressors, nor of any disease
indicative of immunodeficiency. He had, however, received the Bacillus
Calmette-Guérin (BCG) vaccine during childhood. On admission, his
vital signs included an oral temperature of 39 ºC, heart rate of 92 beats
per minute, respiratory rate of 20 breaths per minute and blood pressure
of 130/80 mmHg. Physical examination revealed a 9×8cm mass in the
right of the patient’s neck, but no lesions were found on the oral mucosa.
The remainder of the systemic examination was unremarkable.
Laboratory results included a leukocyte count of 14800×103/mm3,
serum glucose level of 143 mg/dL, blood urea nitrogen of 40 mg/
dL and creatinine level of 1.7 mg/dL. Inflammatory markers were
elevated with an erythrocyte sedimentation rate (ESR) of 120 mm/h
and C-reactive protein (CRP) level of 11.1 mg/L. Both a tuberculin
skin test and human immunodeficiency virus (HIV) antibody exam
were negative. Comprehensive immunological studies, including
serum immunoglobulins and complement levels, tests for cell-mediated
immunity (NK, CD3, CD4, CD8, CD4/CD8 and CD19) and autoantibody
tests, were normal. 1, 3-β-D-glucan assay levels and the galactomannan
test were normal. Blood cultures were also negative. Whole-body positron
emission tomography/computed tomography (PET/CT) revealed multiple
abscesses in the right of the patient’s neck, liver and right psoas major
area respectively (Fig.1A); and there was a raised uptake of [18F] FDG
in vertebral bodies of T11, T12 and L1. Consequently, percutaneous
abscess drainage was conducted on the upper body using B-mode
ultrasonography and drained brown fluids (30 mL, 280 mL and 130 mL,
respectively, Fig.1B) were sent to the microbiology lab. Also, drainage
tubes were inserted into abscesses in the liver and psoas major, but were
removed after no drainage took place. Results of the Chromagar Candida
Medium (Chromagar, France) cultures were positive for Candida albicans
(Fig. 1C), but no acid-fast bacilli were detected. The germ tube test was
positive and the documented diagnosis using API 20C Aux systems
(BioMeriux, France) was Candida albicans, which was sensitive to
amphotericin B, fluconazole, itraconazole, voriconazole, caspofungin
(1) Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China.
(2) Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China.
Correspondence to: Guo-Qing Zang, Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, 200233 Shanghai, China. Phone:
+ 86 21 24058673. Fax: + 86 21 24058384. E-mail: [email protected]
CHEN, X.-H.; GAO, Y.-C.; ZHANG, Y.; TANG, Z.-H.; YU, Y.-S. & ZANG, G.-Q. - Tuberculosis infection might increase the risk of invasive candidiasis in an immunocompetent patient.
and 5-fluorocytosine. Based on antifungal guidelines and susceptibility
tests, he was treated with intravenous 35 mg amphotericin B daily (0.5
mg/kg per day). The patient responded to antifungal therapy and his
fever abated after ten days of treatment. Abscess detection using B-mode
ultrasonography revealed that the extent of infection in the upper body
had greatly decreased at the end of three-week antifungal therapy.
Fig. 2 - A. MRI showing altered signal intensity in T11, T12, L1 and unique osteolytic lesions
in the above vertebral bodies (arrow). B, C. Histopathologic examination of aspirates showing
caseous material and acid fast bacilli (arrow), respectively.
Fig. 1 - A. Whole-Body positron emission tomography/computed tomography (PET/CT)
showing increased uptake of [18F]FDG appear in right neck, liver, right psoas major area
respectively (arrows). B. The brown fluids were drained from the liver under B-mode
ultrasonography inducted. C. Microphotography of Candida albicans Gram staining 1000×.
However, there was no improvement in the patient’s lower back
pain. Spinal magnetic resonance imaging (MRI) showed altered signal
intensity in T11, T12, L1 and unique osteolytic lesions in upper vertebral
bodies (Fig. 2A). Subsequently, computed tomography (CT) guided
percutaneous vertebral biopsy specimens from the T12 vertebrae revealed
caseous material and the acid fast bacilli were identified (Fig.2B, C).
Also, the aspirate culture showed growth of the M. tuberculosis. Chest
CT, urine and sputum examinations were normal. Taking histological and
microbiological findings into consideration, the patient was treated with
antitubercular therapy (rifampicin, isoniazid, pyrazinamide, ethambutol)
for 12 months. In addition, he was treated with a total dose of 1500
mg of amphotericin B, while oral fluconazole and 5-flucytosine were
subsequently prescribed for six months. By his 2-year follow up, there
were complete resolutions of the lesions in the upper vertebral bodies
and no evidence of new abscesses.
DISCUSSION
Candida is an opportunistic fungal pathogen which generally
forms part of the normal flora of the oropharynx, gastrointestinal tract,
and urinary tract in the healthy human. Candida albicans is the most
common pathogenic cause of fungal infections in humans, which can
involve multiple organs, including the brain, mediastinum, kidney, heart,
lung, pancreas, liver and peritoneum. Invasive candidiasis (IC) involves
the infection and spread of Candida via the bloodstream and normally
affects the immunocompromised or immunodeficient, such as those
with diabetes, neutropenia or burns; people undergoing hemodialysis,
abdominal surgery or total parenteral nutrition; and those undergoing long
274
term therapy with broad-spectrum antibiotics or corticosteroids11. The
incidence of IC in the US between 1996 and 2003 was 19-24 infections
per 10,000 annual hospital discharges12.
As one knows, microscopic examination and the cultivation of
clinical samples for the diagnosis of fungal infection are gold-standard
methods. Diagnosis of invasive candidiasis remains difficult and is
generally confirmed by the direct microscopic examination of the fluid
drained during percutaneous or surgical abscess drainage. Candida
infection in patients is clinically very difficult to recognize and its
diagnosis is frequently missed because of non-specific clinical features,
poor diagnostic yield of traditional microbiological techniques and the
non-specificity of radiological imaging9. The results of blood cultures
are frequently negative, as was the case for this patient. In addition, 1,
3-β-D-glucan assay levels and galactomannan tests were normal in this
case. Recently, some studies testified the importance of [18F] FDG-PET/
CT in detecting non-central nervous system invasive fungal infection
at an early stage and assessing the efficiency of antifungal therapy5.
With the help of whole-body PET/CT scans, the patient’s multiple
deep abscesses were found in time. Although the whole-body PET/CT
provides a new, more sensitive way of observing deep organ infections,
its high costs mean that it cannot be widely used in clinical examinations.
According to the clinical practice guidelines for the management of
candidiasis by the Infectious Diseases Society of America and antifungal
susceptibility testing, amphotericin B is the current antifungal treatment
of choice10. The patient responded to antifungal therapy and the number
of abscesses was greatly decreased. In spite of the high death rates of
invasive candidiasis, the patient survived due to the combination of early
diagnosis, percutaneous drainage and antifungal therapy.
Tuberculosis (TB), a disease more common in immunocompromised
persons, is a major global problem still prevalent in developing countries,
despite the availability of highly effective treatment for decades. China
has the second highest level of tuberculosis in the world, registering
12% of global cases (0.9 million - 1.1 million) in 201214. Tuberculosis’
CHEN, X.-H.; GAO, Y.-C.; ZHANG, Y.; TANG, Z.-H.; YU, Y.-S. & ZANG, G.-Q. - Tuberculosis infection might increase the risk of invasive candidiasis in an immunocompetent patient.
insidious nature often leads to delayed or missed diagnosis, sometimes
with devastating consequences for the patient. In this patient, Candida
spondylitis was initially thought of as the cause of his back pain, yet it
showed no improvement following antifungal treatment. However, the
patient’s history of BCG vaccination, negative skin tests and normal chest
scans were not disregarded when considering the diagnosis of tuberculosis.
Percutaneous vertebral biopsy was very useful in confirming diagnosis and
could help avoid open surgical biopsy in future patients. The mainstay in
the treatment of spinal tuberculosis is the conservative management of
antituberculous drugs. Surgery is needed only if there is neurological deficit
or spinal instability. In the management of osteoarticular tuberculosis,
chemotherapy including isoniazid, rifampin, ethambutol, and pyrazinamide
is recommended for approximately 12 - 18 months8. The patient was
administered antituberculous treatment with the above four drugs for a
12-month period. At the final 2-year follow-up, the patient was free of
symptoms, though there was little change in the radiological picture except
for increased sclerosis at the margin of the lesion.
Deep Candida infections generally occur in immunosuppressive
patients, especially those who have been treated with immunosuppressors.
Recent studies showed that M. tuberculosis promoted down-modulatory
immune mediators to counteract Th1-type cells and patients’ innate
immunity, and might have suppressive effects on the host’s immune
system1,2. Also, levels of FoxP3 gene expression and IL-10 secretion
were raised in both pulmonary TB and extra-pulmonary TB7. The
secretion of IL-10 by regulatory T cells can account for the inhibition
of T cell responses and increase the risk of tuberculosis reactivation6.
A case of coinfection with M. tuberculosis and Cryptococcus gattii in
a healthy young woman indicated that infection with TB predisposed
to infection with Cryptococcus by down-regulate immune system and
altered host defenses13. In the case featured in this report, the patient was
immunocompetent and had no history of using immunosuppressive drugs
or having any recent major surgery. It is likely that Candida albicans
invaded his bloodstream through impaired oral mucosa membranes and
stuck to the endothelial cells of his blood vessels, in turn transmigrating
into the tissue4. This indicated that TB infection may impair the host’s
immune system and that the spread of Candida albicans maybe ultimately
lead to the occurrence of multiple deep organ infections in the healthy
young man.
In conclusion, multiple deep organ infections with Candida albicans
and spinal tuberculosis are rare in healthy young men. Moreover, as it
is always difficult to identify M. tuberculosis, early diagnosis should be
considered in patients with clinical and radiological findings suggesting
tuberculosis to avoid missing diagnosis, particularly in developing
countries where tuberculosis is endemic. Furthermore, the present case
report suggests that tuberculosis infection might increase the risk of
invasive candidiasis in patients without significant immunodeficiency.
This hypothesis needs to be confirmed by prospective cohorts with
sufficiently large sample sizes.
RESUMO
Tuberculose pode aumentar o risco de candidíase invasiva em
paciente imunocompetente
As infecções profundas por Candida ocorrem geralmente em
pacientes imunossuprimidos. Relatamos caso raro de infecções profundas
em múltiplos órgãos por Candida albicans e neuro tuberculose em
homem jovem saudável. Um jovem de 19 anos de idade queixou-se
de febre e lombalgia há um mês. Relatava ainda histórico de síndrome
da boca escaldada. Foi diagnosticada co-infecção por Mycobacterium
tuberculosis e Candida albicans em cultura do aspirado de diferentes
regiões do organismo. Os sintomas melhoraram significativamente após
a terapia antifúngica e antituberculosa. Este caso é apresentado para
mostrar que a tuberculose pode prejudicar o sistema imune do hospedeiro
e aumentar o risco de candidíase invasiva em paciente imunocompetente.
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57(3):276, May-June, 2015
http://dx.doi.org/10.1590/S0036-46652015000300017
LETTER TO THE EDITOR
WEST NILE FEVER IN BRAZIL: SPORADIC CASE, SILENT ENDEMIC DISEASE OR EPIDEMIC IN ITS
INITIAL STAGES?
May 21, 2015
Dear Editor
During the initial reports in some countries of Africa and Asia and until the 1990s,
West Nile fever (WNF) was only considered a “minor” public health problem. The disease
gained awareness after outbreaks occurred in Israel, Australia and European countries and,
in particular, due to the large number of people and animals affected in the United States of
America between the end of the 1990s and the beginning of the new century. Thereafter, signs
of West Nile virus (WNV) circulation were detected in the Cayman Islands, El Salvador,
Guatemala, Belize, Colombia, Venezuela, and Argentina. However, viral isolation was rarely
achieved, and records of human, equine and avian morbidity in Latin America are lacking.
Moreover, for unclear reasons, there was no correspondence between the expansion of the
geographic range of viral circulation and the occurrence of significant animal or human
morbidity by WNF in these regions7. Given the evidence of WNV circulation in South
American countries beginning in 2003, the Brazilian Ministry of Health adopted the reporting
of suspected human cases of WNF7. In parallel with the implementation of surveillance
strategies for monitoring the introduction of the virus into the country, Brazilian researchers
posed the question: “West Nile Encephalitis: our next epidemic?”3. Between 2002 and 2013,
serological evidence of viral circulation was found in horses and birds in the Amazon and
Pantanal regions4,6,8. Serological surveys conducted in the states of Rio Grande do Sul (2002)
and Rio Grande do Norte (2003), which included a significant number of birds of various
species, found no evidence of WNV circulation in the country1.
In 2010, despite the negative results of the study “Is West Nile virus a potential cause of
central nervous system infection in Brazil?”, SOARES et al.10 concluded that “With the recent
activity in Argentina, it is fundamental to continue to monitor for this virus as an emerging
cause of neurological disease in South America”. Similarly, in early 2014 FIGUEIREDO &
FIGUEIREDO2 advised: “It is necessary to improve the surveillance of SLEV, ROCV, and
WNV in Brazil. Therefore, doctors must include flaviviruses (not only dengue) and other
arboviruses in their differential diagnosis of acute febrile disease and of meningoencephalitis.
In fact, if the doctors do not think on these pathogens, it will perpetuate the mistaken idea
that these diseases do not exist here”.
In August 2014, a ranch worker from a rural area of Aroeiras do Itaim municipality (Piauí
State, Brazil) was admitted to the Natan Portella Institute for Tropical Diseases (Teresina,
Piauí State, Brazil) with clinical symptoms of acute encephalitis. Since June 2013, a sentinel
surveillance program of viral encephalitis has been instituted by the Municipal Health
Department of Teresina. A research protocol established in partnership with the Evandro
Chagas Institute (Ananindeua, Pará State, Brazil) enabled the shipment of blood, cerebrospinal
fluid and fecal samples in an attempt to isolate and molecularly and serologically detect herpes
viruses, enteroviruses and arboviruses. From the start of the program until the admission of
this patient, samples from 36 patients had been examined. In the second half of November
2014, the Evandro Chagas Institute released the results of the examinations that undoubtedly
confirmed that Piauí had recorded the first human case of WNF in Brazil12.
At the announcement of the first confirmed case of WNF in Brazil, the Ministry of
Health released the following statement: “It is noteworthy that this case was an isolated
event; the chain of transmission was not identified, and an in-depth investigation is being
performed to clarify the mode of transmission. It does not have epidemiological significance
to Brazil nor does it represent a risk to the public health of Piauí or Brazil”5. Thereafter,
the Brazilian Society of Tropical Medicine expressed concern about the circumstances in
which the diagnosis of the first case of WNF in Brazil was conducted: “Even though this
was an isolated case, the situation is worrisome. Its absence in Brazil, until this case, was
enigmatic. After all, why would a virus disseminated throughout North America and the
Caribbean not enter into Brazil where its insect vectors and animal reservoirs are present?
Then, suddenly it appears in the State of Piauí? (...) This question is even more troublesome
because the virus was identified just after routine surveillance was instituted in a referral
hospital. The conclusion is obvious: the virus has already been circulating undetected in
Brazil for some time”11.
The epidemiology news portal from the International Society for Infectious Diseases,
ProMED-mail, also expressed moderate concern following the disclosure of the diagnosis:
“Vector? Isn’t missing ... There: all the elements of a transmission cycle are present. To deny
that the risks of dissemination and, eventually, that outbreaks are a tangible reality would
be naive, reckless and, to some degree, irresponsible”9.
The detection of the first case of WNF in Brazil may have distinct epidemiological
meaning expressed by the assumptions that the encephalitis surveillance system was able to
detect: (1) the initial phase of an outbreak or an epidemic of WNF in the State of Piauí; (2)
a case of an already endemic disease at low levels that so far had an unknown occurrence
or was undetected in the region or in the country or (3) a sporadic case that emerged under
exceptional circumstances that is so far unexplained.
The capacity of Brazilian biomes to provide an ecological niche conducive to the spread
of WNV remains enigmatic. To date, there have been no other cases with a confirmed diagnosis
of WNF in the country. The team involved in the surveillance of viral encephalitis in Teresina
is advocating the hypothesis of a silent endemic (at low levels) of WNF in Piauí and, perhaps,
in Brazil. The manifestations of encephalitis caused by various viruses (and even by non-viral
agents) have a significant amount of coincident signs and symptoms. The low specificity of
clinical, cerebrospinal fluid data, radiological and electroencephalographic “patterns” of
WNV encephalitis hinders its recognition. Thus, the reporting of suspected cases, which is
an essential step for the national reference laboratories to perform specific diagnostic tests, is
fairly limited. The classical assumption of the herpetic nature of viral encephalitis, the lack of
specific therapies against most viruses and the lack of diagnostic methods in most Brazilian
hospitals are factors that combined lead to the non-recognition of the etiologic agents involved
in central nervous system infections. These assertions may indicate that other cases of WNV
encephalitis may have occurred without clinical recognition (which is admittedly difficult) of
the disease and without performing the tests necessary for an etiological diagnosis. Brazilian
clinicians, researchers and epidemiologists have a challenge ahead, given that the clarification
of the current status of WNV circulation does have an epidemiological relevance to Teresina
municipality, for Piauí State and to Brazil.
Marcelo A. Cunha e Silva VIEIRA(1), Aline de Almeida Xavier AGUIAR(2), Amaríles
de Souza BORBA(3), Herlon Clístenes Lima GUIMARÃES(4), Kelsen Dantas
EULÁLIO(5), Linduarte Leitão de ALBUQUERQUE-NETO(5), Maria do Amparo
SALMITO(3) & Oriana Bezerra LIMA(3)
(1) Natan Portella Institute of Tropical Medicine, Dept. Neurology,
R. Governador Artur de Vasconcelos 151, 64001-450 Teresina, PI, Brazil.
Telephone: +55 86 3221 3413. E-mail: [email protected].
(2) Municipal Health Dept., Zenon Rocha Emergency Hospital, Teresina, PI, Brazil.
(3) Municipal Health Dept., Surveillance Secretariat, Teresina, PI, Brazil.
(4) State Health Dept., Surveillance Secretariat, PI, Brazil.
(5) Natan Portella Inst. of Tropical Medicine, Dept. Infectious Diseases, Teresina, PI, Brazil.
Correspondence to: Marcelo A.C.S. VIEIRA
Funding: This research received no grant from any funding agency in the public,
commercial or not-for-profit sectors.
REFERENCES
1. Araújo FAA, Vianna RST, Wada MY, Silva EV, Doretto L, Cavalcante GC, et al.
Inquérito sorológico em aves migratórias e residentes de Galinhos/RN para detecção
do vírus do Nilo Ocidental e outros vírus. Bol Eletrôn Epidemiol. 2004;4(2):1-12.
2. de Figueiredo ML, Figueiredo LT. Review on infections of the central nervous system
by St. Louis encephalitis, Rocio and West Nile flaviviruses in Brazil, 2004-2014. Adv
Microbiol. 2014;955-61.
3. Luna EJA, Pereira LE, Souza RP. Encefalite do Nilo Ocidental, nossa próxima
epidemia? Epidemiol Serv Saúde. 2003;12(1):7-19.
4. Melandri V, Guimarães AÉ, Komar N, Nogueira ML, Mondini A, Fernandez-Sesma
A, et al. Serological detection of West Nile virus in horses and chicken from Pantanal,
Brazil. Mem Inst Oswaldo Cruz. 2012;107:1073-5.
5. Ministério da Saúde, Brasil, 2014. Saúde confirma primeiro caso de Febre do Nilo.
(cited 2015 Mar 30). Available from: http://portalsaude.saude.gov.br/index.php/
cidadao/principal/agencia-saude/15962-saude-confirma-primeiro-caso-de-febre-donilo
6. Ometto T, Durigon EL, de Araujo J, Aprelon R, de Aguiar DM, Cavalcante GT, et
al. West Nile virus surveillance, Brazil, 2008-2010. Trans R Soc Trop Med Hyg.
2013;107:723-30.
7. Pauvolid-Corrêa A, Varella RB. Aspectos epidemiológicos da febre do oeste do Nilo.
Rev Bras Epidemiol. 2008;11:463-72.
8. Pauvolid-Corrêa A, Campos Z, Juliano R, Velez J, Nogueira RM, Komar N. Serological
evidence of widespread circulation of West Nile virus and other flaviviruses in equines
of the Pantanal, Brazil. PLOS Negl Trop Dis. 2014;8:e2706.
9. ProMED-mail. Febre do Nilo Ocidental: Brazil (PI), humanos, casos suspeitos,
surto? International Society for Infectious Diseases. 2014. Archive number
20141224.3052950. (cited 2015 Mar 30). Available from: http://promedmail.org/
direct.php?id=3052950
10. Soares CN, Castro MJ, Peralta JM, Freitas MR, Puccioni-Sohler M. Is West Nile virus
a potential cause of central nervous system infection in Brazil? Arq Neuropsiquiatr.
2010;68:761-3.
11. Sociedade Brasileira de Medicina Tropical. Caso de febre do Nilo Ocidental no Piauí
é preocupante, alertam especialistas. Brasília: Newsletter SMBT; 2014. Edição 41.
15 de dezembro de 2014. (cited 2015 Mar 30). Available from: http://sbmt.org.br/
portal/caso-de-febre-do-nilo-ocidental-no-piaui-e-preocupante-alertam-especialistas
12. Vieira MA, Romano AP, Borba AS, Silva EV, Chiang JO, Eulalio KD, et al. West
Nile virus encephalitis: the first human case recorded in Brazil. Am J Trop Med Hyg.
2015;pii:15-0170. (Epub ahead of print).

3 - Instituto de Medicina Tropical de São Paulo

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