ISSN 0036-4665 ISSN 1678-9946 on Line EDITORS EMERITUS EDITORS Prof

Established: 1959. The year 2015 is the 57th anniversary of continuous publication

ISSN 0036-4665 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) Fernando A. Corrêa (S. Paulo, SP) Maria I. S. Duarte (S. Paulo, SP) Alberto Duarte (S. Paulo, SP) Fernando Montero‑Gei (San José, Costa Rica) Maria L. Higuchi (S. Paulo, SP) Angela Restrepo M. (Medellin, Colombia) Flair J. Carrilho (S. Paulo, SP) Mario Mariano (S. Paulo, SP) Anna Sara S. Levin (S. Paulo, SP) Gil Benard (S. Paulo, SP) Mirian N. Sotto (S. Paulo, SP) Antonio A. Barone (S. Paulo, SP) Gioconda San-Blas (Caracas, Venezuela) Moisés Goldbaum (S. Paulo, SP) Antonio Carlos Nicodemo (S. Paulo, SP) Govinda Visvesvara (Atlanta, USA) Moysés Mincis (S. Paulo, SP) Antonio Sesso (S. Paulo, SP) Heitor F. Andrade Jr. (S. Paulo, SP) Moysés Sadigursky (Salvador, BA) Antonio W. Ferreira (S. Paulo, SP) Hiro Goto (S. Paulo, SP) Myrthes T. Barros (S. Paulo, SP) Barnett L. Cline (New Orleans, USA) Ises A. Abrahamsohn (S. Paulo, SP) Nilma Cintra Leal (Recife, PE) Carlos F. S. Amaral (Belo Horizonte, MG) João Carlos Pinto Dias (Belo Horizonte, MG) Paulo C. Cotrim (São Paulo, SP) Celso Granato (S. Paulo, SP) João Renato Rebello Pinho (Sao Paulo, SP) Paulo M. Z. Coelho (Belo Horizonte, MG) Cesar A. Cuba Cuba (Brasília, DF) José Ângelo A. Lindoso (S. Paulo, SP) Regina Abdulkader (S. Paulo, SP) César Naquira V. (Lima, Peru) José Eduardo Levi (S. Paulo, SP) Ricardo Negroni (B. Aires, Argentina) Clarisse M. Machado (S. Paulo, SP) José M. R. Zeitune (Campinas, SP) Robert H. Gilman (Baltimore, USA) Claudio S. Pannuti (S. Paulo, SP) Julia Maria Costa-Cruz (Uberlândia, MG) Roberto Martinez (Rib. Preto, SP) Dalton L. F. Alves (Belo Horizonte, MG) Julio Litvoc (S. Paulo, SP) Ronaldo Cesar B. Gryschek (S. Paulo, SP) Eridan Coutinho (Recife, PE) Luiz Carlos Severo (P. Alegre, RS) Semíramis Guimarães F. Viana (Botucatu, SP) Ernesto Hofer (Rio de Janeiro, RJ) Luiz T. M. Figueiredo (Rib. Preto, SP) Silvio Alencar Marques (Botucatu, SP) Euclides A. Castilho (S. Paulo, SP) Lygia B. Iversson (S. Paulo, SP) Tsutomu Takeuchi (Tokyo, Japan) Eufrosina S. Umezawa (S. Paulo, SP) Marcello Fabiano de Franco (S. Paulo, SP) Venâncio A. F. Alves (S. Paulo, SP) Expedito J. A. Luna (S. Paulo, SP) Marcos Boulos (S. Paulo, SP) Vicente Amato Neto (S. Paulo, SP) Fan Hui Wen (S. Paulo, SP) M. A. Shikanai‑Yasuda (S. Paulo, SP) Zilton A. Andrade (Salvador, BA)

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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 ISSN 1678-9946 on line

II Impact Factor: 1.007 5-year Impact Factor: 1.088

ISSN 0036-4665 ISSN 1678-9946 on line

Rev. Inst. Med. Trop. Sao Paulo Vol. 57 No. 3 P. 185-276 May-June, 2015

CONTENTS

MYCOLOGY EPIDEMIOLOGY 185 Frequency of Candida species in a tertiary care hospital in Triangulo 239 Head lice in hair samples from youths, adults and the elderly in Mineiro, Minas Gerais State, Brazil Manaus, Amazonas State, Brazil R.P. MENEZES, J.C. FERREIRA, W.M. SÁ, T.A. MOREIRA, L.D.S. MALVINO, L.B. S.C.B. NUNES, R.B. MORONI, J. MENDES, S.C.B. JUSTINIANO & F.T. MORONI ARAUJO, D.V.D.B. RÖDER, M.P.A. PENATTI, R.C. CANDIDO & R.S. PEDROSO CHAGAS DISEASE BACTERIOLOGY 245 Effects of vitamin C supplementation on the chronic phase of Chagas 193 Raw tropical oysters as vehicles for multidrug-resistant Vibrio disease parahaemolyticus R.G. MARIM, A.S. GUSMÃO, R.E.P. CASTANHO, R. DEMINICE, A.L.S. THEREZO, R.A. COSTA, R.L. ARAÚJO & R.H.S.F. VIEIRA A.A. JORDÃO JÚNIOR, M.R. ASSIS, E.F. TAIPEIRO & L.P.A. MARTINS

PARASITOLOGY NOCARDIOSIS 197 Anthelmintic activity of lapachol, β-lapachone and its derivatives 251 Molecular identification and antimicrobial resistance pattern of against Toxocara canis larvae seven clinical isolates of Nocardia spp. in Brazil T. MATA-SANTOS, N.F. PINTO, H.A. MATA-SANTOS, K.G. DE MOURA, P.F. L.A.Z. CONDAS, M.G. RIBEIRO, M.D. MURO, A.P.C. VARGAS, T. MATSUZAWA, CARNEIRO, T.S. CARVALHO, K.P. DEL RIO, M.C.F.R. PINTO, L.R. MARTINS, K. YAZAWA, A.K. SIQUEIRA, T. SALERNO, G.H.B. LARA, R.M. RISSETI, K.S. J.M. FENALTI, P.E.A. DA SILVA & C.J. SCAINI FERREIRA & T. GONOI

205 Molecular characterization and sequence phylogenetic analysis of LEISHMANIASIS surface antigen 3 (SAG3) gene of local Indian isolates (Chennai 257 Genotype characterization of Leishmania (Viannia) braziliensis and Izatnagar) of Toxoplasma gondii isolated from human and canine biopsies with American cutaneous V. SUDAN, A.K. TEWARI & H. SINGH leishmaniasis L.T. FERREIRA, A.H.S. GOMES & V.L. PEREIRA-CHIOCCOLA 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. MALARIA PEDROSA, D. CORREIA & M.B. OLIVEIRA-SILVA. 263 Seasonal distribution of malaria vectors (Diptera: Culicidae) in rural localities of Porto Velho, Rondonia, Brazilian Amazon VIROLOGY L.H.S. GIL, M.S. RODRIGUES, A.A. LIMA & T.H. KATSURAGAWA 215 Saint Louis encephalitis virus in Mato Grosso, Central-Western Brazil BRIEF COMMUNICATION L.B.S. HEINEN, N. ZUCHI, O.P. SERRA, B.F. CARDOSO, B.H.F. GONDIM, 269 Chicken coops, Triatoma dimidiata infestation and its infection M.A.M. SANTOS, F.J.D. SOUTO, D.A.J. PAULA, V. DUTRA & R. DEZENGRINI- with Trypanosoma cruzi in a rural village of Yucatan, Mexico SLHESSARENKO E. KOYOC-CARDEÑA, A. MEDINA-BARREIRO,, F.J. ESCOBEDO-ORTEGÓN, J.C. RODRÍGUEZ-BUENFIL, M. BARRERA-PÉREZ, E. REYES-NOVELO, 221 Lack of association between herpesvirus detection in saliva and J. CHABLÉ-SANTOS, C. SELEM-SALAS, G. VAZQUEZ-PROKOPEC & P. gingivitis in HIV-infected children MANRIQUE-SAIDE 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 CASE REPORT 273 Tuberculosis infection might increase the risk of invasive candidiasis ENTOMOLOGY in an immunocompetent patient 227 Inventory of mosquitoes (Diptera: Culicidae) in conservation units X.-H. CHEN, Y.-C. GAO, Y. ZHANG, Z.-H. TANG, Y.-S. YU & G.-Q. ZANG. in Brazilian tropical dry forests C.F. SANTOS, A.C. SILVA, R.A. RODRIGUES, J.S.R. JESUS & M.A.Z. BORGES LETTER TO THE EDITOR 276 West Nile fever in Brazil: sporadic case, silent endemic disease or 233 Phlebotomine fauna (Diptera: Psychodidae) in an area of fishing epidemic in its initial stages? M.A.C.S.VIEIRA, A.A.X. AGUIAR, A.S.BORBA, H.C.L. GUIMARÃES, K.D. tourism in Central-Western Brazil EULÁLIO, L.L. ALBUQUERQUE-NETO, M.A. SALMITO & O.B.LIMA A.F. BRILHANTE, M.E.M.C. DORVAL, E.A.B. GALATI, H.C. ROCHA, G. CRISTALDO & V.L.B. NUNES

ADDRESS SUBSCRIPTIONS INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO FOREIGN COUNTRIES Av. Dr. Enéas de Carvalho Aguiar, 470 One year (six issues)...... U$ 200.00 05403-000 São Paulo, SP - Brazil Single issue...... U$ 50.00 Phone/Fax: 55.11.3062.2174; 55.11.3061-7005 e-mail: [email protected] III Impact Factor: 0.907 5-year Impact Factor: 1.213

ISSN 0036-4665 ISSN 1678-9946 on line

Rev. Inst. Med. Trop. Sao Paulo Vol. 57 No. 3 P. 185-276 Maio-Junho, 2015

CONTEÚDO

MICOLOGIA EPIDEMIOLOGIA 185 Frequência de espécies de Candida em hospital terciário do Triân- 239 Pediculose da cabeça em amostras de cabelos de jovens, adultos e gulo Mineiro, Minas Gerais, Brasil idosos em Manaus, estado do Amazonas, Brasil R.P. MENEZES, J.C. FERREIRA, W.M. SÁ, T.A. MOREIRA, L.D.S. MALVINO, L.B. S.C.B. NUNES, R.B. MORONI, J. MENDES, S.C.B. JUSTINIANO & F.T. MORONI ARAUJO, D.V.D.B. RÖDER, M.P.A. PENATTI, R.C. CANDIDO & R.S. PEDROSO DOENÇA DE CHAGAS BACTERIOLOGIA 245 Efeitos da suplementação de vitamina C na fase crônica da doença 193 Ostras tropicais cruas como fonte de Vibrio parahaemolyticus de Chagas multirresistentes R.G. MARIM, A.S. GUSMÃO, R.E.P. CASTANHO, R. DEMINICE, A.L.S. THEREZO, R.A. COSTA, R.L. ARAÚJO & R.H.S.F. VIEIRA A.A. JORDÃO JÚNIOR, M.R. ASSIS, E.F. TAIPEIRO & L.P.A. MARTINS

PARASITOLOGIA NOCARDIOSE 197 Atividade anti-helmíntica do lapachol, β-lapachona e derivados 251 Identificação molecular e perfil de sensibilidade a antimicrobianos contra larvas de Toxocara canis de sete isolados clínicos de Nocardia spp. no Brasil T. MATA-SANTOS, N.F. PINTO, H.A. MATA-SANTOS, K.G. DE MOURA, P.F. L.A.Z. CONDAS, M.G. RIBEIRO, M.D. MURO, A.P.C. VARGAS, T. MATSUZAWA, CARNEIRO, T.S. CARVALHO, K.P. DEL RIO, M.C.F.R. PINTO, L.R. MARTINS, K. YAZAWA, A.K. SIQUEIRA, T. SALERNO, G.H.B. LARA, R.M. RISSETI, K.S. J.M. FENALTI, P.E.A. DA SILVA & C.J. SCAINI FERREIRA & T. GONOI

205 Caracterização molecular e análise filogenética de sequências do LEISHMANIOSE antígeno de superfície 3 (SAG3) em isolados indianos (Chennai e 257 Caracterização genotípica de isolados de Leishmania (Viannia) Izatnagar) de Toxoplasma gondii braziliensis provenientes de biopsias de humanos e cães com V. SUDAN, A.K. TEWARI & H. SINGH 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. MALARIA PEDROSA, D. CORREIA & M.B. OLIVEIRA-SILVA 263 Distribuição sazonal de vetores da malária (Diptera: Culicidae) em localidades rurais de Porto Velho, Rondônia, Amazônia Brasileira VIROLOGIA L.H.S. GIL, M.S. RODRIGUES, A.A. LIMA & T.H. KATSURAGAWA 215 Vírus da encefalite de Saint Louis em Mato Grosso, Centro-Oeste, Brasil COMUNICAÇÃO BREVE L.B.S. HEINEN, N. ZUCHI, O.P. SERRA, B.F. CARDOSO, B.H.F. GONDIM, 269 Gallineros, la infestación por Triatoma dimidiata y su infección M.A.M. SANTOS, F.J.D. SOUTO, D.A.J. PAULA, V. DUTRA & R. DEZENGRINI- con Trypanosoma cruzi en una localidad rural de Yucatán, México SLHESSARENKO E. KOYOC-CARDEÑA, A. MEDINA-BARREIRO,, F.J. ESCOBEDO-ORTEGÓN, J.C. RODRÍGUEZ-BUENFIL, M. BARRERA-PÉREZ, E. REYES-NOVELO, 221 Ausência de associação entre a detecção de herpesvírus na saliva J. CHABLÉ-SANTOS, C. SELEM-SALAS, G. VAZQUEZ-PROKOPEC & P. e gengivite em crianças infectadas pelo HIV MANRIQUE-SAIDE 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 RELATO DE CASO 273 Tuberculose pode aumentar o risco de candidíase invasiva em ENTOMOLOGIA paciente imunocompetente 227 Inventário de mosquitos (Diptera: Culicidae) em unidades de con- X.-H. CHEN, Y.-C. GAO, Y. ZHANG, Z.-H. TANG, Y.-S. YU & G.-Q. ZANG. servação em florestas tropicais secas brasileiras C.F. SANTOS, A.C. SILVA, R.A. RODRIGUES, J.S.R. JESUS & M.A.Z. BORGES CARTA AO EDITOR 276 West Nile fever in Brazil: sporadic case, silent endemic disease or 233 Fauna flebotomínea (Diptera: Psychodidae) em área de turismo epidemic in its initial stages? M.A.C.S.VIEIRA, A.A.X. AGUIAR, A.S.BORBA, H.C.L. GUIMARÃES, K.D. pesqueiro no Centro-Oeste do Brasil EULÁLIO, L.L. ALBUQUERQUE-NETO, M.A. SALMITO & O.B.LIMA A.F. BRILHANTE, M.E.M.C. DORVAL, E.A.B. GALATI, H.C. ROCHA, G. CRISTALDO & V.L.B. NUNES

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 IV e-mail: [email protected] Rev. Inst. Med. Trop. Sao Paulo 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 reported to be predominant, especially in hospital environments28,39,44. According to some investigators, this is due to the selective pressure In recent decades, candidemia has increased significantly worldwide from the prophylactic use of fluconazole in patients at risk of developing due to increased lifespans of immunosuppressed patients or transplant and invasive fungal infections18,41. Variable frequencies of different species HIV/AIDS patients7,10,15,44. In many countries, the invasive infection of of Candida are identified depending on the hospital complexity and/or Candida yeast is a considerable public health problem, due to its severity, geographic region13. cause of increased hospital stays, cost, and contribution to high indexes of morbimortality. Some reports note that the mortality index caused The choice of treatment for candidemia or invasive candidiasis is by candidemia may reach 40-60% of hospital-admitted patients10,18,39,48. mainly based on two factors: Candida species and the condition of the host immune system. Depending on the protocol of the institution and Invasive candidiasis is related to several factors that compromise the availability of antifungal agents, azoles (fluconazole, voriconazole, patient conditions, such as neutropenia, organ transplantations, previous and posaconazole), polyene (amphotericin B), and/or echinocandins colonization by Candida species, prolonged use of antibiotics, presence (caspofungin, anidulafungin, and micafungin) are used for the treatment. of catheters for nasogastric feeding, use of urinary or parenteral probes for Echinocandins are recommended for prophylaxia and for the treatment hemodialysis or mechanical ventilation, neoplasia, immunosuppressive of different groups of patients due to their efficacy and low toxicity in diseases, drugs, and gastrointestinal surgeries30. critical patients compared to other azoles and amphotericin B11,31,49.

For many years, C. albicans was regarded as the main cause of Candidiasis epidemiology has been studied by genotypic analysis, invasive fungal infections, but lately, non-C. albicans species have been 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 breakpoints were as indicated in CLSI8,43, and, for amphotericin B, due to in clinical and epidemiological studies29,34. These studies attempt to lack of consensus, the values suggested by NGUYEN et al.29 were used. relate the genotypes of isolates with pathogenicity and epidemiology. Genotypes with varying degrees of heterogeneity were found in different Molecular typing: DNA extraction was performed according to anatomical sites among various population groups, including patients and the method of BOLANO et al.4. The RAPD-PCR tests were performed healthy individuals, and in different geographical areas4,33,37. with initiator oligonucleotides OPA09 (5’GGGTAACGCC3’), OPB11 (5’GTAGACCCGT3’), and OPE06 (5’AAGACCCCTC3’) (Invitrogen, The most commonly used molecular methods include polymorphism São Paulo, Brazil). The reaction final volume was 25 µL and contained detection in the length of restriction fragments (RFLP) with hybridization 2 µL DNA (60 ng/mL), 0.25 mmol of each deoxynucleotide (dATP, (Southern blot) or amplification (AFLP), karyotyping in pulsed-field dCTP, dTTP, and dGTP) (Invitrogen), 1U Taq polymerase (Invitrogen), gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and 2.5mM MgCl2, and 2.5mM initiator nucleotide. All amplifications other techniques based on polymerase chain reaction (PCR) of random were conducted in a thermalcycler (Eppendorf, Mastercycle Gradient, amplified polymorphic DNA (RAPD-PCR)2,29,34. USA), consisting of an initial amplification cycle of four min at 92o C followed by 40 cycles of 40 s at 92 oC, 40 oC for 1.5 min, and 72 oC Regional peculiarities and hospital complexity services may influence for two min, and finally followed by five min at 72oC. Amplification the predominance of Candida species. These emphasize the need for fragments were separated by agarose gel (1.4%) electrophoresis for studies on epidemiology, prevalence, and resistance to antifungals. three h at 80V and 100mA. The gels were stained with ethidium bromide This study aims to evaluate the frequency, in addition to testing the and visualized under UV light and the images were captured by a susceptibility to antifungals as well as genetic polymorphisms, of photo documentation system. Profiles for each sample were analyzed Candida species isolated from samples of blood, CVC, and abdominal visually, and bands were classified as present (1) or absent (0). Genetic fluids of hospitalized patients in a tertiary hospital in the Triangulo relationships (similarity coefficients) were calculated by the Jaccard Mineiro region, Minas Gerais State, Brazil. 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 MATERIAL AND METHODS 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 Isolates in the study: Candida samples included in the study were the same genotype, values from 0.800-0.99 represent clonally related obtained from patients admitted to the Clinical Hospital of the Federal samples (strongly similar but not identical), and values less than University of Uberlândia (UFU) in the city of Uberlândia located in 0.800 indicate distinct samples. Dendrograms based on Sj values were the Triangulo Mineiro region, Minas Gerais State, Brazil, during the generated for comparison by the unweighted pair group method with period of July 2010-June 2011. The isolates were from blood cultures, the arithmetical averages (UPGMA) method utilizing the multivariate CVC, and abdominal fluids. Chromogenic agar (BD CHROMagar® statistical package program (MVSP). Candida, France) and Sabouraud dextrose agar were used to isolate the yeasts, which were identified by classical methods21 and confirmed Ethical committee: This study was approved by the Ethical by the Auxacolor2® system (Bio-Rad, France). Candida albicans and Committee for Human Research of the Federal University of Uberlândia C. dubliniensis were differentiated by PCR utilizing specific primers, (UFU) under the number 317/10. according to the technique described by ESTRADA-BARRAZA et al.14. Samples were stored in BHI-glycerol broth at -20 oC. Experiments were Statistical analysis: Qualitative variables were compared using the conducted after sample activation and incubation at 35 oC for 24-48 h. chi-square test, and the G test was used for quantitative results. In both tests, statistical significance was considered when p < 0.05. Antifungal susceptibility tests: The broth microdilution method described in document M27-A3, Clinical Laboratory Standard Institute RESULTS (CLSI)8, was used for the tests. Antifungals amphotericin B (Fungizon, Bristol Myers Squibb, Brazil), fluconazole (Pfizer, Sandwich, UK), During the study period, 63 cultures of body fluids from individuals and itraconazole (Janssen, Beerse, Belgium) were tested in culture with suspected systemic candidiasis were positive for Candida spp., of plates of RPMI-1640 medium containing glutamine, without sodium which 47 were in blood, nine were in CVC, and seven in abdominal bicarbonate, and buffered using pH-7.0 MOPS with glucose (18g/L). fluids, all obtained from 58 hospitalized patients at the Clinical Hospital The final concentrations of the antifungal agents were 0.03-16 µg/mL of Federal University of Uberlândia. Thirty-four were from males and for amphotericin B and itraconazole and 0.25-64 µg/mL for fluconazole. 24 from females. Ages of the patients ranged from one day to 94 years, Briefly, yeasts were inoculated in Sabouraud dextrose agar and incubated with a mean age of 42 years. Most patients who developed systemic at 35 oC for 24 h. Culture suspensions adjusted to 1-5×106cells/mL were candidiasis and who had a positive culture were older than or equal to prepared in sterilized saline. Susceptibility tests were made in duplicates 21 years (Fig. 1). and the microdilution plates were incubated at 35 oC for 48 h. Control strains were C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. Of the 63 Candida isolates, 18 (28.5%) were identified as C. albicans The minimum inhibitory concentration (MIC) was determined visually. and 45 (72.5%) as non-C. albicans, distributed as follows: 20 (31.7%) C. For the azoles, MICs corresponded to the concentration inhibiting parapsilosis complex species; 14 (22.2%) C. tropicalis; four (6.4%) C. around 50% of growth for each microorganism compared to the control glabrata; four (6.4%) C. krusei; two (3.3%) C. kefyr; and one (1.6%) C. well (without antifungal); for amphotericin B, the MIC was the smaller lusitaniae. Candida dubliniensis was not identified by PCR. Except for drug concentration that inhibited 100% of yeast growth7. For azoles, C. albicans (p = 0.050), the distribution of species between males and

186 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.

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.

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 Note: Other species: C. krusei (4); C. glabrata (4); C. lusitaniae (1) and C. kefyr (2). related (0.99 > Sj > 0.80); OPB11 produced only one profile, with 100% similarity among isolates. Candida glabrata produced two profiles with Fig. 1 - Distribution of Candida spp. according to age of hospitalized patients who developed each one of the primers; OPA09 and OPB11 grouped three isolates in systemic candidiasis during the period of July 2010-June 2011. profile A and another isolate in profile B, with A and B being unrelated (Sj < 0.80) for both primers. females was not statistically different for members of the C. parapsilosis complex (p = 0.057), C. tropicalis (p = 0.4497), and other species (p = DISCUSSION 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 The predominance of Candida species non-C. albicans observed isolates, respectively, were obtained from male patients. in this study confirms results reported in other studies from different Brazilian regions12,23,28. The C. parapsilosis complex occurred at the As shown in Table 1, the highest frequency of Candida isolates was highest frequency compared to other species, including C. albicans. from blood cultures (55.6%), CVC (14.3%), simultaneous isolations Observations from other Latin American countries and Tunisia show that from blood-CVC (19%) and abdominal fluids (11.1%). C. parapsilosis-induced infections increased significantly in the past two decades9,22. Candida albicans, C. tropicalis, and C. parapsilosis complex In vitro resistance to amphotericin B was observed in one isolate species are the most frequent species isolated in candidemia cases and of C. albicans, in one of the C. parapsilosis complex, and in six other constitute 82.5% as a whole of the isolates in this study and, in some species - three C. krusei, two C. glabrata, and one C. kefyr - all with a other instances, represent more than 90% of etiologies30. MIC of 2 µg/mL. None of the isolates, except C. krusei, was resistant to azoles in vitro. 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, Dose-dependent susceptibility to itraconazole was detected in one the C. parapsilosis complex was reclassified into three species: C. isolate of C. glabrata. Table 2 shows the MIC ranges of antifungals tested parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis42. These against Candida species. 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%) Total isolates 20 (31.7%) 18 (28.6%) 14 (22.2%) 11 (17.4%) 63 (100%) *Other species: C. krusei (4); C. glabrata (4); C. lusitaniae (1); and C. kefyr (2). CVC = central venous catheter.

187 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.

Table 2 In-vitro susceptibility of Candida species to three antifungal agents

Species MIC (µg/mL) Resistant Antifungal agents (n) n (%) Range MIC50 MIC90 Amphotericin B 0.5-2.0 1.0 1.0 1 (5%) C. parapsilosis Fluconazole 0.125-1.0 0.5 0.5 0 (20) Itraconazole 0.03-0.125 0.03 0.03 0 Amphotericin B 0.5-2.0 0.5 1.0 1 (5.6%) C. albicans Fluconazole 0.125-0.5 0.125 0.5 0 (18) Itraconazole 0.03 0.03 0.03 0 Amphotericin B 0.5-1.0 1.0 1.0 0 C. tropicalis Fluconazole 0.125-0.5 0.25 0.5 0 (14) Itraconazole 0.03-0.06 0.03 0.06 0 Amphotericin B 1.0-2.0 - - 3 (75%) C. krusei Fluconazole* - - - 4 (100%) (4) Itraconazole 0.03-0.12 - - 0 Amphotericin B 1.0-2.0 - - 2 (50%) C. glabrata Fluconazole 0.5-4.0 - - 0 (4) Itraconazole 0.25-0.3 - - 0 Amphotericin B 0.5-2.0 - - 1 (50%) C. kefyr Fluconazole 0.125-0.5 - - 0 (2) Itraconazole 0.06-0.125 - - 0 Amphotericin B 1 - - 0 C. lusitaniae Fluconazole 0.25 - - 0 (1) Itraconazole 0.03 - - 0 *C. krusei is intrinsically resistant to fluconazole.

Table 3 patterns of susceptibility to antifungal and biofilm production11. Of all Frequency of cluster profiles and isolates per cluster with primers the Candida isolates, they were detected in 55.6% of samples from blood OPA09 and OPB11 cultures, 14.3% from CVC, 11.1% from abdominal fluids, and 19% from blood and CVC simultaneously. Positive results in blood cultures are OPA09 OPB11 considered the main indicators of invasive infections. Although cultures of samples obtained from other organic sites may be secondary in the Species(n) Molecular Frequency of Molecular Frequency of diagnostics of hospital infection, these Candida isolates may have a profile* isolates profile* isolates predictive value for the occurrence of candidemias1,50. A 10 A 13 Candida B 3 B 4 Similar to what happened with bacteria, the indiscriminate use parapsilosis C 3 C 1 of antifungal drugs has stimulated the occurrence of fungi with (20) D 3 D 1 decreased susceptibility or even in vitro resistance, especially E 1 among Candida species6. In this study, the susceptibility of isolates A 7 A 14 in relation to fluconazole, itraconazole, and amphotericin B, which B 4 B 4 were the antifungals used for treatment of invasive candidiasis in Candida C 4 the service during the period studied, was analyzed. However, recent 26,31,49 albicans (18) D 1 studies have pointed primarily to the use of echinocandins . Most E 1 isolates were susceptible to the three antifungals evaluated. Candida F 1 krusei and C. glabrata are known to be resistant and less susceptible to fluconazole, respectively31,32,34,45,49. In vitro resistance of Candida Candida A 14 A 13 species, notably non-C. albicans, to fluconazole has been reported in tropicalis B 1 different hospital studies13,16,31,32,36,39. Itraconazole has been recently (14) utilized in the treatment of candidemia in neutropenic patients because *A cluster was considered when it grouped isolates with 100% similarity. it is less toxic than amphotericin B, as well as having shown a similar

188 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.

effectiveness to that presented by other azoles17. One isolate (25%) of RESUMO C. glabrata showed a dose-dependent susceptibility to itraconazole, while NEUFELD et al.28 reported a dose-dependent susceptibility in Frequência de espécies de Candida em hospital terciário do only 3.4% of the isolates in their studies. Triângulo Mineiro, Minas Gerais, Brasil

Resistance to amphotericin B has not been reported among As infecções causadas por espécies de Candida são problema isolates of different regions20,27,35,39. In this study, a MIC of 2 µg/mL de grande impacto para a saúde pública, devido à alta incidência em was determined for some isolates, especially non-C. albicans ones, pacientes hospitalizados e como causa de mortalidade. O presente estudo characterizing in vitro resistance. Data on the clinical outcomes of teve como objetivo avaliar a frequência de Candida spp. isoladas de patients were not generated in this study, as in vitro results do not mean pacientes hospitalizados, assim como a sensibilidade aos antifúngicos e in vivo resistance, due to the fact that the cut-off point for amphotericin o polimorfismo genético por RAPD-PCR. Os microrganismos incluíram B is not established by the standardization committee due to technical isolados de hemocultura, líquido abdominal e ponta de cateter venoso difficulties related to the antifungal and culture media, as reported in central de pacientes internados no Hospital de Clínicas da Universidade the literature8,23,26,28,29. The results should be considered an alert and they Federal de Uberlândia, região do Triângulo Mineiro, Minas Gerais, Brasil, should emphasize the importance of continuous surveillance to detect no período de julho de 2010-junho de 2011. Os testes de sensibilidade occasional isolates that are resistant to one or more antifungals. Future aos antifúngicos foram realizados por microdiluição em caldo e na vigilance studies, including monitoring of patients, on antimicrobial análise por RAPD-PCR foram utilizados os oligonucleotídeos OPA09, resistance will show if these results were occasional or common OPB11, e OPE06. Dos 63 isolados, 18 (28,5%) foram C. albicans, 20 occurrences. (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. The genetic variability of clinical isolates has been used to lusitaniae. Resistência in-vitro à anfotericina B foi observada em 12,7% demonstrate cases of cross infection that occur in health care, but also dos isolados. Não foi observada resistência in-vitro aos azólicos, exceto to determine if the isolates of one anatomical site are identical to isolates para os isolados de C. krusei. Os oligonucleotídeos OPA09 e OPB11 from other sites of the same patient19,37. possibilitaram distinguir diferentes espécies. Isolados de C. albicans apresentaram seis clusters e o complexo C. parapsilosis, cinco clusters, In this study, the RAPD methodology was utilized in an attempt to com o iniciador OPA09, por RAPD-PCR, mostrando a variabilidade reveal molecular variants of Candida spp. Based on the gel patterns and genética daquelas espécies. Conclui-se que o complexo C. parapsilosis on the dendrograms obtained (data not shown), six profiles (A-F) were foi a espécie mais frequente, e a maioria dos isolados foi sensível in vitro determined with primer OPA09 while OPB11 allowed only two (A-B) aos antifúngicos testados. Alto polimorfismo genético foi observado para for isolates of C. albicans. Primer OPA09 had a higher discriminatory os isolados de C. albicans e complexo C. parapsilosis, principalmente power especially for C. albicans and C. parapsilosis complex (Table com o oligonucleotídeo OPA09. 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 ACKNOWLEDGMENTS possible to discuss this. Several studies have shown the discriminatory power of different primers and have suggested the use of multiple primers The authors are grateful to the National Research Council (CNPq) for to improve the sensitivity of the results25,37,46,47. the Scientific Initiation Fellowship awarded to R. P. Menezes, the Dean of the Undergraduate Federal University of Uberlândia (PROGRAD-UFU; This study identified a variety of strains in the patients involved, edict 05/2010), to the Foundation for Research Support of Minas Gerais especially for isolates C. albicans and C. parapsilosis complex. However, (FAPEMIG; process nº. APQ-00464-11), and to the Dean of Research it was not possible to show a cross infection at all. However, in 12 and Graduate of the Federal University of Uberlândia (PROPP-UFU, patients who had blood and CVC, positive cultures were isolated to the Edict 04/2011) for financial support. They are also grateful to Lorraine same species, and these exhibited the same genotype when blood and Cristina Ribeiro Silva, Roterdan Martins Rosa, and Adriano Gonçalves CVC isolates were compared. This might be evidence of hematological Martins for technical assistance with some tests. dissemination of this particular microorganism from the CVC, but also blood-to-CVC. Identifying the source of infection is an important way CONFLICT OF INTEREST to prevent infection. However, it suggests that prospective studies, including clinical data of patients and correlating these data with the The authors have no conflict of interest to declare. microbiological characteristics of isolated samples may provide important insights for Candida spp. epidemiology in inpatients. REFERENCES

In conclusion, of the Candida species isolated during the study period, 1. Agvald-Ohman C, Klingspor L, Hjelmqvist H, Edlund C. Invasive candidiasis in the most frequent were C. parapsilosis complex species followed by C. long-term patients at a multidisciplinary intensive care unit: Candida colonization index, risk factors, treatment, and outcome. Scand J Infect Dis. 2008;40:145-53. albicans and C. tropicalis. Most samples were susceptible to antifungals fluconazole, itraconazole, and amphotericin B. The genotypic markers 2. Bacelo KL, Costa KRC, Ferreira JC, Candido RC. Biotype stability of Candida seemed efficient at discriminating the isolates of C. albicans and C. albicans isolates after culture storage determined by randomly amplified polymorphic parapsilosis; high genetic polymorphism was observed for isolates DNA and phenotypic methods. Mycoses. 2010;53:468-74. of C. albicans and C. parapsilosis complex species, mainly with the 3. Balajee SA, Sigler L, Brandt ME. DNA and the classical way: identification of OPA09 marker. medically important molds in the 21st century. Med Mycol. 2007;45:475-90.

189 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.

4. Barros LM, Boriollo MFG, Alves ACBA, Klein MI, Gonçalves RB, Höfling JF. 21. Lacaz CS, Porto E, Martins JEC, Heins Vaccari EM, Melo NT. Tratado de micologia Genetic diversity and exoenzyme activities of Candida albicans and C. dubliniensis médica. São Paulo: Sarvier; 2002. isolated from the oral cavity of Brazilian periodontal patients. Arch Oral Biol. 2008;53:1172-8. 22. Makni F, Sellami A, Trabelsi H, Sellami H, Cheikh-Rouhou F, Neji S, et al. Evolution de la flore des levures isolées au CHU de Sfax, Tunisie. J Mycol Med. 2010;20:42-7. 5. Bolano A, Stinchi S, Preziosi R, Bistoni F, Allegrucci M, Baldelli F, et al. Rapid methods to extract DNA and RNA from Cryptococcus neoformans. FEMS Yeast 23. Matta DA, Almeida LP, Machado AM, Azevedo AC, Kusano EJU, Travassos NF, et Res. 2001;1:221-4. al. Antifungal susceptibility of 1,000 Candida bloodstream isolates to five antifungal drugs: results of a multicenter study conducted in São Paulo, Brazil, 1995-2003. 6. Brion LP, Uko SE, Goldman DL. Risk of resistance associated with fluconazole Diagn Microbiol Infect Dis. 2007;57:300-404. prophylaxis: systematic review. J Infect. 2007;54:521-9. 24. Menezes EA, Vasconcelos Júnior AA, Cunha FA, Cunha MCSO, Braz BHL, 7. Chang MR, Correia FP, Costa LC, Xavier PCN, Palhares DB, Taira DL, et al. Candida Capelo LG, et al. Identificação molecular e suscetibilidade antifúngica de Candida bloodstream infection: data from a teaching hospital in Mato Grosso do Sul, Brazil. parapsilosis isoladas no Ceará, Brasil. J Bras Patol Med Lab. 2012;48:415-20. Rev Inst Med Trop Sao Paulo. 2008;50:265-8. 25. Miranda LN, van der Heijden IM, Costa SF, Sousa AP, Sienra RA, Gobara S, et al. 8. Clinical and Laboratory Standards Institute (CLSI). Method for broth dilution Candida colonisation as a source for candidaemia. J Hosp Infect. 2009;72:9-16. antifungal susceptibility testing of yeasts: approved standard. 3rd ed. Wayne: Clinical and Laboratory Standard Institute; 2008. (CLSI document M27-A3). 26. Moen MD, Lyseng-Williamson KA, Scott LJ. Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive 9. Colombo AL, Nucci M, Salomão R, Branchini ML, Richtmann R, Derossi A, et al. fungal infections. Drugs. 2009;69:361-92. High rate of non-albicans candidemia in Brazilian tertiary care hospitals. Diagn Microbiol Infect Dis. 1999;34:281-6. 27. Motta AL, Almeida GM, Almeida JN, Burattini MN, Rossi F. Candidemia epidemiology and susceptibility profile in the largest Brazilian teaching hospital 10. Colombo AL, Guimarães T. Epidemiologia das infecções hematogênicas por Candida complex. Braz J Infect Dis. 2010;14:441-8. spp. Rev Soc Bras Med Trop. 2003;36:599-607. 28. Neufeld PM, Santos LH, Ribeiro MD, Silva MF, Rocha ACM, Silva M, et al. 11. Colombo AL, Guimarães T, Camargo LF, Richtmann R, Queiroz-Telles FD, Salles Prevalência e susceptibilidade in-vitro a itraconazol e anfotericida B de isolados MJ, et al. Brazilian guidelines for the management of candidiasis: a joint meeting clínicos de Candida. Rev Bras Anal Clin. 2009;41:119-25. report of three medical societies: Brazilian Society of Infectious Diseases, Infectious Diseases Society of Paulista, and Brazilian Society of Tropical Medicine. Braz J 29. Nguyen MH, Clancy CJ, Yu VL, Yu YC, Morris AJ, Snydam DR, et al. Do in-vitro Infect Dis. 2013;17:283-312. susceptibility data predict microbiologic response to amphotericin B? Results of a prospective study of patients with Candida fungemia. J Infect Dis. 1998;177:425-30. 12. Colombo AL, Guimarães T, Silva LR, Monfardini LPA, Cunha AK, Rady P, et al. Prospective observational study of candidemia in São Paulo, Brazil: incidence 30. Nucci M, Queiroz-Telles F, Tobón AM, Restrepo A, Colombo AL. Epidemiology of rate, epidemiology, and predictors of mortality. Infect Control Hosp Epidemiol. opportunistic fungal infections in Latin America. Clin Infect Dis. 2010;51:561-70. 2007;28:570-6. 31. Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr, 13. Colombo AL, Garnica M, Aranha Camargo LF, Da Cunha CA, Bandeira AC, Borghi D, et al. Clinical practice guidelines for the management of candidiasis: 2009 update et al. Candida glabrata: an emerging pathogen in Brazilian tertiary care hospitals. by the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:503-35. Med Mycol. 2013;51:39-44. 32. Pereira JM, Paiva JA. Tratamento da candidíase invasiva no doente crítico. Rev Port 14. Estrada-Barraza D, Dávalos- Martínez A, Flores-Padilla L, Mendoza-Elias R, Sánchez- Med Int. 2010;17:23-30. Vargas LO. Comparación entre métodos convencionales CHROMagar Candida® y el método de la PCR para la identificación de especies de Candida en aislamientos 33. Pfaller MA, Cabezudo I, Hollis R, Huston B, Wenzel RP. The use of biotyping and clínicos. Rev Iberoam Micol. 2011;28:36-42. DNA fingerprinting in typing Candida albicans from hospitalized patients. Diagn Microbiol Infect Dis. 1990;13:481-9. 15. Falagas ME, Roussous N, Vardakas KZ. Relative frequency of albicans and the various non-albicans Candida spp. among candidemia isolates from inpatients in 34. Pfaller MA, Diekema DJ, Rinaldi MG, Barnes R, Hu B, Veselov AV. Results from various parts of the world: a systematic review. Int J Infect Dis. 2010;14:e954-e66. the ARTEMIS DISK global antifungal surveillance study: a 6.5-year analysis of susceptibilities of Candida and other yeast species to fluconazole and voriconazole 16. Furlaneto MC, Rota JF, Quesada RM, Furlaneto-Maia L, Rodrigues R, Oda S, et by standardized disk diffusion testing. J Clin Microbiol. 2005;43:5848-59. al. Species distribution and in-vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a three-year period. Rev Soc Bras 35. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public Med Trop. 2011;44:595-9. health problem. Clin Microbiol Rev. 2007;20:133-63.

17. Gafter-Gvili A, Vidal L, Goldberg E, Leibovici L, Paul M. Treatment of invasive 36. Pfaller MA, Messer SA, Moet GJ, Jones RN, Castanheira M. Candida bloodstream candidal infections: systematic review and meta-analysis. Mayo Clin Proc. infections: comparison of species distribution and resistance to echinocandin and 2008;83:1011-21. azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009). Int J Antimicrob Agents. 18. Giolo MP, Svidzinski TIE. Fisiopatogenia, epidemiologia e diagnóstico laboratorial 2011;38:65-9. da candidemia. J Bras Patol Med Lab. 2010;46:225-34. 37. Pinto PM, Resende MA, Koga-Ito CY, Tendler M. Genetic variability analysis among 19. Gurbuz M, Kaleli I. Molecular analysis of Candida albicans isolates from clinical clinical Candida spp. isolates using random amplified polymorphic DNA. Mem Inst specimens. Mycopathologia. 2010;169:261-7. Oswaldo Cruz. 2004;99:147-52.

20. Junqueira JC, Vilela SF, Rossoni RD, Barbosa JO, Costa AC, Rasteiro VM, et al. 38. Pires RH, Santos JM, Zaia JE, Martins CHG, Mendes-Giannini MJS. Candida Oral colonization by yeasts in HIV-positive patients in Brazil. Rev Inst Med Trop parapsilosis complex water isolates from a hemodialysis unit: biofilm production Sao Paulo. 2012;54:17-24. and in-vitro evaluation of the use of clinical antifungals. Mem Inst Oswaldo Cruz. 2011;106:646-54.

190 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.

39. Sellami A, Sellami H, Neji S, Makni F, Abbes S, Cheikhrouhou F, et al. Antifungal 45. Tortorano AM, Kibbler C, Peman J, Bernhardt H, Klingspor L, Grillot R. Candidaemia susceptibility of bloodstream Candida isolates in Sfax Hospital,Tunisia. in Europe: epidemiology and resistance. Int J Antimicrob Agents. 2006;27:359-66. Mycopathologia. 2011;171:417-22. 46. Trost A, Graf B, Eucker J, Sezer O, Possinger K, Göbel UB, et al. Identification of 40. Soll DR. The ins and outs of DNA fingerprinting the infections fungi. Clin Microbiol clinically relevant yeasts by PCR/RFLP. J Microbiol Methods. 2004;56:201-11. Rev. 2000;13:332-70. 47. Valerio HM, Weibert-Oliveira RCB, Resende MA. Differentiation of Candida species 41. Talarmin JP, Boutoille D, Tattevin P, Dargère S, Weinbreck P, Ansart S, et al. obtained from nosocomial candidemia using RAPD-PCR technique. Rev Soc Bras Epidémiologie des candidérmies; étude observationnelle prospective d’un an dans Med Trop. 2006;39:174-8. l’Ouest de la France. Med Mal Infect. 2009;39:877-85. 48. Viudes A, Permán J, Cantón E, Ubeda P, López-Ribot JL, Gobernado M. Candidemia 42. Tavanti A, Davidson AD, Gow NAR, Maiden MCJ, Odds FC. Candida orthopsilosis at a tertiary care hospital: epidemiology, treatment, clinical outcome, and risk factors and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and for death. Eur J Microbiol Infect Dis. 2002;21:767-74. III. J Clin Microbiol. 2005;43:284-92. 49. Walsh TJ, Gamaletsou MN. Treatment of fungal disease in the setting of neutropenia. 43. The European Committee on Antimicrobial Susceptibility Testing, Subcommittee Hematology Am Soc Hematol Educ Program. 2013;2013:423-7. on Antifungal Susceptibility Testing (EUCAST-AFST). EUCAST technical note on fluconazole. Clin Microbiol Infect. 2008;14:193-5. 50. Wang JL, Chang SC, Hsueh PR, Chen YC. Species distribution and fluconazole susceptibility of Candida clinical isolates in a medical center in 2002. J Microbiol 44. Tortorano AM, Peman J, Bernhardt H, Klingspor L, Kibbler CC, Faure O, et Immunol Infect. 2004;37:236-41. al. Epidemiology of candidaemia in Europe: results of a 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study. Received: 3 March 2014 Eur J Clin Microbiol Infect Dis. 2004;23:317-22. Accepted: 5 August 2014

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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 MATERIAL AND METHODS

The consumption of raw oysters has been constantly associated with Strains origin: 87 V. parahaemolyticus strains - isolated from soft bacterial etiology outbreaks, and Vibrio parahaemolyticus has been tissues with the intervalvar liquids of C. rhizophorae oysters - were taken highlighted as one of the main species responsible for this phenomenon5. from the bacterial collection of the Environmental and Fish Microbiology This species, frequently present in marine and estuarine environments, is Laboratory at the Institute of Marine Sciences (LABOMAR-UFC). The part of the indigenous microbiota of aquatic organisms16,17 and its ability study was based on 15 samples of fresh (sold at room temperature) and to cause diseases seems to be related to virulence factors, such as the 15 samples of frozen (sold at -4 °C) oysters obtained from two restaurants presence of tdh and trh genes18. in Fortaleza-Brazil in 2010. Each sample consisted of 10 specimens, for a total of 300 specimens examined. For isolation and purification of Oyster-associated outbreaks caused by V. parahaemolyticus are the strains, 50 g of the intervalvar tissues and fluid was taken from each well documented9,15,7,10, and represent a worldwide problem. In the sample of 10 specimens and added to 450 mL alkaline peptone water United States, McLAUGHLIN et al.11 reported a large outbreak of (1% NaCl). The homogenate (which corresponded to a 10-1 dilution) was gastroenteritis - involving episodes of watery diarrhea - associated with used to make serial decimal dilutions from 10-2 to 10-4. Thus, 0.2 mL V. parahaemolyticus serotype O6:K18. aliquots of each dilution were spread plated on thiosulfate-citrate-bile salt-sucrose agar (TCBS-Difco) and incubated at 35 °C for 18h. Three According to DANIELS & SHAFAIE3, V. parahaemolyticus strains blue-green colonies for each sample were randomly selected and cultured responsible for cases of gastroenteritis are usually sensitive to antibiotics in tryptone soy agar (TSA-Difco) (1% NaCl). commonly used in the treatment of enteric infections. However, for patients with V. parahaemolyticus wound infections and septicemia, Biochemical characterization of the strains: All colonies (n = 37 the treatment - intravenous antimicrobial agents - is similar to that for from fresh oysters, and n = 48 from frozen oysters) were submitted to patients with V. vulnificus infection. Thus, besides virulence, the threat biochemical identification using the key developed by NOGUEROLA of antimicrobial-resistant vibrios is also worth mentioning6. & BLANCH13. The strains presented the following phenotypic profile: (1) Gram-negative curved rods, (2) oxidase (+) in oxidase strips Considering the risk that the consumption of oysters may pose to (Laborclin), (3) sucrose (-) in Basal Media for Carbohydrate containing human health, the following study aimed to determine the antimicrobial 0.5% (w/v) of sucrose (35 ºC for five days), (4) indol (+) in Sulfide-Indole- susceptibility profile of Vibrio parahaemolyticus strains from fresh and Motility Agar (35 ºC for 48 h), (5) ortho-Nitrophenyl-β-galactoside- frozen oysters Crassostrea rhyzophorae sold in Fortaleza-Brazil. ONPG (-) 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 Table 1 Media for Carbohydrate containing 0.5% (w/v) of mannitol (35ºC for Multiple antimicrobial resistance in Vibrio parahaemolyticus strains isolated 5 days), (7) Voges-Proskauer (-) in MRVP broth (35ºC for 96 h), (8) from samples of fresh and frozen oysters 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% Profile Fresh Frozen MAR (+) NaCl in Alkaline Peptone Water (35 ºC for 24 h), and (10) arginine Van+Pen+Amp+Ery 2 16 0.4 dihydrolase (-), lysine decarboxylase (+), ornithine decarboxylase (+) in basal media (0.02 g of bromocresol purple, 5 g of peptone, 3 g of extract Van+Pen+Amp 16 30 0.3 yeast, 10 g of sodium chloride and 1 g of glucose in one liter of distilled Van+Pen+Ery 4 1 0.3 water, pH 8,5) containing 0.125% (w/v) of arginine, lysine and ornithine, respectively, with incubation at 35 ºC for seven days. Van+Pen 13 1 0.2 Van+Amp 1 - 0.2 Antibiogram: The antimicrobial susceptibility pattern was carried Van+Ery 1 - 0.2 out by disk diffusion method1, with Muller-Hinton Agar (MH) containing 1% NaCl. Nine antibiotics were tested for each strain: gentamicin (Gen 10 Total 37 (94.9%) 48 (100%) µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 *VAN: vancomycin 30 µg; PEN: penicillin 10U; AMP: ampicillin 10 µg; ERY: µg), Chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline erythromycin 15 µg; MAR: multiple antibiotic resistance. (Tet 30 µg), vancomycin (Van 30 µg) and Erythromycin (Ery 15 µg). Zones of inhibition were measured using a digital caliper (Digimess) and Table 2 each strain behavior was classified as sensitive, intermediate or resistant, Profile of chromosomal and plasmid resistance to antibiotics in Vibrio 1 according to CLSI recommendations. parahaemolyticus strains isolated from samples of fresh and frozen oysters

Plasmid curing: Strains that showed resistance to at least one antimicrobial underwent plasmid curing in broth Luria Bertani Antibiotics supplemented with acridine orange (SIGMA A-6014) at 0.1 mg mL-1 Van Pen Amp Ery 12. After the curing procedure, the strains were again subjected to Fresh oysters antibiotic susceptibility testing (described above). Thus, the resistance was considered chromosomal when observed after the curing procedure; Number of resistant strains 38 36 19 6 otherwise it was characterized as plasmid. Chromosomal resistance 38 33 9 -

RESULTS Plasmid resistance - 3 10 6 Frozen oysters From the 87 V. parahaemolyticus isolates tested, more than 96.5% Number of resistant strains 48 48 46 16 were resistant to vancomycin and penicillin, and 74.7% showed resistance to ampicillin. Resistance to erythromycin was observed in 74.7% of the Chromosomal resistance 48 47 45 11 isolates. In contrast, all strains were sensitive to chloramphenicol, and Plasmid resistance - 1 1 5 more than 95.4% were sensitive to gentamicin, ciprofloxacin, tetracycline, *VAN: vancomycin 30 µg; PEN: penicillin 10U; AMP: ampicillin 10 µg; ERY: nalidixic acid and gentamicin. erythromycin 15 µg. Isolates from fresh oysters showed resistance rates to the following antibiotics: Van (n = 38; 97.4%), Pen (n = 36; 92.3%), Amp (n = 19; a threat to their consumers. HAN et al.6 investigated the susceptibility 48.6%), Ery (n = 6, 15.4%). Resistance rates for the frozen oysters isolates of vibrios isolated from oysters and reported a high rate of penicillin- were: Van (n = 48; 100%), Pen (n = 48; 100%), Amp (n = 46; 95.8%), resistant V. parahaemolyticus. This finding is similar to the results Ery (n = 16, 33.3%). obtained in the present study, since the resistance to Amp was found in isolates from both types of oysters (Table 1). A high rate of multiple resistance was observed in strains isolated from fresh (94.9%) and frozen (100%) oysters. The most recurrent multi- DARAMOLA et al.2 determined the antimicrobial resistance profiles resistant profile in both fresh and frozen sources was Van+Pen+Amp of V. parahaemolyticus strains isolated from water samples, sediments (Table 1). V. parahaemolyticus strains isolated from both types presented and mussels from the Humber River estuary in the U.K. - an area where a MAR oscillating from 0.2 to 0.4. shellfish harvest and mussel culture occurs. The authors reported that all isolates were sensitive to chloramphenicol, presented a low level Plasmid curing indicated a chromosomal resistance profile in 100% resistance to vancomycin (3.9%), ampicillin (1.3%), and high rates of Van-resistant strains. Isolates with a plasmid resistance profile were (73.7%) of resistance to gentamicin. In the present research, a large more frequent in strains extracted from fresh oysters (Table 2). number of Van and Amp-resistant strains was detected; in contrast, sensitivity to Gen and Chlo were observed. Comparing the results to those DISCUSSION of DARAMOLA et al.2, it is possible to suggest that the mechanisms of antimicrobial resistance in the same bacterial species undergo a The occurrence of antimicrobial-resistant vibrios in oysters poses differentiation process according to the region.

194 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.

OTTAVIANI et al.14, in a study on the susceptibility of Vibrio RESUMO (including V. parahaemolyticus) isolated from fresh and frozen sold seafood (shellfish, shrimp, squid and cod), found Vibrio strains without Ostras tropicais cruas como fonte de Vibrio parahaemolyticus resistance mechanisms to ciprofloxacin and nalidixic acid, as well as multirresistentes isolates with multiple resistance profiles to different combinations of antimicrobials, including ampicillin and penicillin, as in the present study. O presente estudo objetivou determinar o perfil de suscetibilidade a The authors suggest that for the plasmid role in Vibrio, multiple resistance antimicrobianos de cepas de Vibrio parahaemolyticus oriundas de ostras to antibiotics must be investigated, even though most of the studies until “in natura” e congeladas comercializadas em Fortaleza-Brasil. Oitenta e that moment indicated that this characteristic is inherent to that genus. sete (87) cepas foram submetidas ao antibiograma com emprego de nove antibióticos: gentamicina (Gen 10 µg), ampicilina (Amp 10 µg), penicilina The high rate of multiple resistance observed in this study raises G (Pen 10U), ciprofloxacin (Cip 5 µg), cloranfenicol (Clo 30 µg), ácido questions as to the effectiveness of antimicrobial agents commonly nalidíxico (Nal 30 µg), tetraciclina (Tet 30 µg), vancomicina (Van 30 µg) used in the treatment of gastroenteritis caused by Vibrio. It is possible e eritromicina (Eri 15 µg). Todas as cepas mostram-se resistentes a pelo to consider that Chl, Nal, Cip, Tet and Gen should be selected to treat menos um antibiótico, e 85 (97,7%) apresentaram multirresistência, com diseases caused by V. parahaemolyticus, as has been reported in the predomínio do perfil Van+ Pen+Amp (n = 46). Foi detectada resistência literature. KHAN et al.8 determined the susceptibility of 27 strains of plasmidial a Pen, Amp e Eri. Dessa forma, o risco que o consumo de ostras the same species isolated from cultured shrimp in Khulna (Bangladesh), cruas representa para a saúde dos consumidores merece ser destacado, and suggested that the tetracycline and gentamicin were the best uma vez que esses bivalves podem ser veículos de transmissão de micro choice for controlling diseases caused by enteric bacteria, including organismos multirresistentes a fármacos antibacterianos. V. parahaemolyticus. Thus, it is necessary to establish therapy with appropriate antimicrobials for a more effective treatment of infections REFERENCES caused by V. parahaemolyticus, V. vulnificus, and others19. The authors above suggest that the antimicrobial ciprofloxacin is effective in these 1. Clinical and Laboratory Standards Institute (CLSI). Performance standards for cases, in accordance with the findings of this study. antimicrobial susceptibility testing. Wayne: CLSI; 2010. (Supplement M100-S20). 2. Daramola BA, Williams R, Dixon RA. In vitro antibiotic susceptibility of Vibrio 20 In accordance with the findings in this study, ZULKIFLI et al. parahaemolyticus from environmental sources in northern England. Int J Antimicrob investigated the resistance of V. parahaemolyticus strains isolated from Agents. 2009;34:499-500. cockles in Indonesia, and reported rates of resistance to penicillin and ampicillin higher than 50%, as well as a 100% sensitivity to gentamicin. 3. Daniels NA, Shafaie A. A review of pathogenic Vibrio infections for clinicians. Infect Med. 2000;17:665-85.

10 LOZANO-LEÓN et al. investigated an outbreak of gastroenteritis 4. Devi R, Surendran PK, Chakraborty K. Antibiotic resistance and plasmid profiling of involving 64 people in Spain and revealed the presence of V. Vibrio parahaemolyticus isolated from shrimp farms along the southwest coast of parahaemolyticus in fecal samples of all patients involved. Symptoms India. World J Microbiol Biotechnol. 2009;25:2005-12. appeared within 12 to 24 hours after the consumption of raw oysters at a 5. DePaola A, Jones JL, Woods J, Burkhardt W 3RD, Calci KR, Krantz JA, et al. Bacterial street market in the city of Vigo (Galicia, Spain). The study also revealed and viral pathogens in live oysters: 2007 United States market survey. Appl Environ that 100% of the isolates were resistant to ampicillin, erythromycin Microbiol. 2010;76:2754-68. and vancomycin, antibiotics commonly used in the treatment of gastroenteritis. The strains used in this study showed a similar resistance 6. Han F, Walker RD, Janes ME, Prinyawiwatkul W, Ge B. Antimicrobial susceptibilities profile to those responsible for the aforementioned outbreak, a fact which of Vibrio parahaemolyticus and Vibrio vulnificusisolates from Louisiana Gulf and retail raw oysters. Appl Environ Microbiol. 2007;73:7096-8. confirms that the consumption of raw oysters poses a potential risk to human health. 7. Kaufman GE, Myers ML, Pass CL, Bej AK, Kaysner CA. Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific The results of the susceptibility test after the plasmid curing suggest north-west outbreaks. Lett Appl Microbiol. 2002;34:155-61. that the antimicrobial resistant profile from V. parahaemolyticus strains 8. Khan AW, Hossain SJ, Uddin SN. Isolation, identification and determination of isolated from oysters are linked to chromosomal genes, in accordance with antibiotic susceptibility of Vibrio parahaemolyticus from shrimp at Khulna region 4 the literature. DEVI et al. , in a study on the antimicrobial susceptibility of Bangladesh. Res J Microbiol. 2007;2:216-27. in strains from the same species isolated from shrimps cultivated in the southeast of India, also found low rates of plasmid resistance, reporting 9. Lipp EK, Rose JB. The role of seafood in foodborne diseases in the United States of that there were no modifications in the resistance to chloramphenicol, America. Rev Sci Tech 1997;16:620-40 oxytetracycline and trimethoprim before and after plasmid curing. 10. Lozano-León A, Torres J, Osorio CR, Martínez-Urtaza J. Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption Considering the high rates of resistance, especially multiple in Spain. FEMS Microbiol Lett. 2003;226:281-4. 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 11. McLaughlin JB, DePaola A, Bopp CA, Martinek KA, Napolilli NP, Allison CG, et al. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. gastroenteritis treatment in human beings. Thus, the ingestion of those N Engl J Med. 2005;353:1463-70. bivalve mollusks without prior cooking is strongly unadvisable.

195 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.

12. Molina-Aja A, García-Casca A, Abreu-Grobois A, Bolán-Mejía C, Roque A, Gomez-Gil 17. Su YC, Liu C. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. B. Plasmid profiling and antibiotic resistance of Vibrio strains isolated from cultured 2007;24:549-58. penaeid shrimp. FEMS Microbiol Lett. 2002;213:7-12. 18. Vieira RHSF, Costa RA, Menezes FGR, Silva GC, Theophilo GND, Rodrigues DP, et 13. Noguerola I, Blanch AR. Identification of Vibrio spp. with a set of dichotomous keys. J al. Kanagawa-negative, tdh- and trh-positive Vibrio parahaemolyticus isolated from Appl Microbiol. 2008;105:175-85. fresh oysters marketed in Fortaleza, Brazil. Curr Microbiol. 2011;63:126-30.

14. Ottaviani D, Bacchiocchi I, Masini L, Leoni F, Carraturo A, Giammarioli M, et al. 19. Zanetti S, Spanu T, Deriu A, Romano L, Sechi LA, Fadda G. In vitro susceptibility of Antimicrobial susceptibility of potentially pathogenic halophilic vibrios isolated Vibrio spp. isolated from the environment. Int J Antimicrob Agents. 2001;17:407-9. from seafood. Int J Antimicrob Agents. 2001;18:135-40. 20. Zulkifli Y, Alitheen NB, Raha AR, Yeap SK, Marlina, Son R, et al. Antibiotic resistance 15. Pollack CV Jr, Fuller J. Update on emerging infections from the Centers for Disease and plasmid profiling of Vibrio parahaemolyticus isolated from cockles in Padang, Control and Prevention. Outbreak of Vibrio parahaemolyticus infection associated Indonesia. Int Food Res J. 2009;16:53-8. with eating raw oysters and clams harvested from Long Island Sound-Connecticut, New Jersey, and New York, 1998. Ann Emerg Med. 1999;34:679-80. Received: 14 May 2014 Accepted: 8 August 2014 16. Shimohata T, Takahashi A. Diarrhea induced by infection of Vibrio parahaemolyticus. J Med Invest. 2010;57:179-82.

196 Rev. Inst. Med. Trop. Sao Paulo 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 the drug of choice in the treatment of visceral toxocariasis9. Nevertheless, albendazole is the drug that crosses the blood brain barrier34 and shows Human visceral toxocariasis is a neglected zoonotic infection results superior to thiabendazole37 and diethylcarbamazine, because caused by the larvae of Toxocara canis and, less frequently, Toxocara it does not reduce the levels of specific IgE and produces side effects cati31. According to recent reports, their prevalence seems to be in treated patients25. Therefore, an effective drug for treating human underestimated mainly because of the difficulties of diagnosis and infections caused by T. canis is still needed28. non-specific symptomatology36. The symptoms of this parasitic disease are characterized by cutaneous reactions, extensive eosinophilia, Among the possibilities of assisting in the treatment of visceral hepatomegaly, myocarditis, pulmonary infiltrates, and nodules toxocariasis, natural and synthetic products33 stand out. Plant extracts accompanied by cough and fever13,18. The severity of symptoms depends are important sources of biologically active natural products and may on the location of the larvae and the number of larvae housed in tissues, be a model for the development of new drugs12,32. which induces mechanical damage and, in turn, results in an immune- mediated inflammatory response26. Therefore, death is frequently Lapachol, an important representative of the quinone group, is associated with inflammatory granulomatous reactions around the isolated from plants of the Bignoniaceae family19. It performs biological larvae15, which may persist for a long time and, with it, reactivated larval activities against several pathogens, especially anti-parasitic activities migration into the eye or the brain may occur at any time40. The long-term against Trypanosoma cruzi, Schistosoma mansoni, Leishmania survival of T. canis larvae has been attributed to molecular strategies amazonensis and L. braziliensis7,23,24. evolved by the parasite26. β-lapachone is an ortho-naphthoquinone, a natural derivative of Generally, the drugs used to treat this disease have limited lapachol, present in small quantities in the woods of Tabebuia spp effectiveness, such as diethylcarbamazine and thiabendazole faced (Bignoniaceae). β-lapachone is easily synthesized by sulfuric acid with poor tolerability and the need for prolonged use30. The low water treatment of lapachol16 and has a wide range of biological activities, solubility of benzimidazole compounds appears to collaborate with the including trypanocidal, antibacterial, anti-inflammatory, and anticancer low bioavailability of compounds in this group, such as albendazole38, 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 motile, immobile but not dead, or dead). Cell viability was tested by (i.e., phenazines) and have attracted considerable attention due to using a 0.4% trypan blue indicator. their biological activities, including antimalarial5, antimycobacterial2, antitumor, and antiparasitic21 ones. Therefore, the use of this group of The substances that showed larvicidal activity in 100% of larvae with compounds as pharmacophores for the development of new drugs has the in vitro test at concentrations of 2 mg/mL were re-tested at lower consequently been investigated. 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 In this study, lapachol, β-lapachone and three of its derivatives, and activity at the lowest concentrations were assessed for their viability of 17 phenazines synthesized from β-lapachone analogues were tested infection in mice. In order to assess their viability, the content of each against T. canis larvae. microplate well was inoculated into 5-week-old BALB/c female mice by intraperitoneal injection. All mice were given food without antibiotics and MATERIALS AND METHODS 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. Synthesis: Lapachol was extracted from the heartwood of Tabebuia spp (Tecoma) and purified by recrystallization from ethanol, following Furthermore, a control group of live larvae (100 larvae/well) in mice a previously described procedure11. Nor-lapachol was synthesized from was used to confirm the viability of larvae that were not exposed to lapachol through Hooker oxidation14. the substances. A single mouse was used for each compound and each control. Mice were euthanized after 30 days of inoculation. The animals β-lapachone, nor-β-lapachone, and β-C-allyl-lawsone were obtained were examined for larvae by having their carcass, brain, liver, lungs, through the cyclisation of the prenyl side chain of lapachol, nor-lapachol kidneys, heart, eyes, and spleen digested in a solution of 1% hydrochloric and C-allyl-lawsone, respectively. 10 mmol of the naphthoquinone were acid and 1% pepsin39. solubilized in 15mL of sulfuric acid and mixed for several minutes. The reaction was poured over cold water. The red solid was filtered, washed RESULTS with cold water (3 × 100 mL) and purified by recrystallization using a mixture of acetone/hexane17. Lapachol, β-lapachone and three of its derivatives, and 17 phenazines were tested against T. canis larvae. The phenazines were prepared by the reaction of the naphthoquinone (1.00 mmol), o-phenylenediamine (1.10 mmol) and sodium acetate β-lapachone and β-C-allyl-lawsone showed the highest activity (1.30 mmol) in glacial acetic acid (50 mL). The reaction was maintained (MLC = 0.25 mg/mL), followed by lapachol (MLC = 0.5 mg/mL) and under reflux for two hours and monitored by TLC. After the reaction, the nor-lapachol (MLC = 1 mg/mL) (Table 1). mixture was poured over ice and left to incubate overnight. The yellow precipitate was filtered through a Buchner funnel, washed with cold Out of the 17 phenazines tested on T. canis larvae, four compounds water (3 × 100 mL), and the phenazine was isolated. All phenazines (i.e., compounds 1, 2, 3, and 4) showed 100% activity at a concentration were synthesized with > 95% yield35. 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. Test compounds: All synthesized compounds were solubilized The other phenazines showed < 14% activity (Table 2). in DMSO at 2.5% (Sigma®) and in sterile distilled water to obtain a concentration of 2 mg/mL33. 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 Preparation of T. canis larvae: T. canis eggs were initially collected group consisted of live larvae and caused infection when inoculated into directly from the uterine tubes of female adult parasites following the the mice, which validates the in vitro evaluation criteria used in this study. 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 DISCUSSION 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 The search for new therapeutic prototypes with effectiveness against larvae were collected in sterile tubes for cultivation with a (Gibco) RPMI- T. canis larvae housed in human tissues is relevant for the efficacy of 1640 medium supplemented with (Sigma) 25mM HEPES, 1% glucose, visceral toxocariasis treatment. The new drugs should eradicate all (Gibco) PSF antibiotic-antimycotic solution, and 0.4 µg/mL ofloxacin. larvae housed in the tissues, not only decrease the intensity of infection 1,6,32,33 Samples were maintained at 37 °C strain with 5% CO2. as it was noted in the administration of albendazole , ivermectin, mebendazole, and thiabendazole22 in mice. Larvicidal/larvistatic activity test: A microplate was used to measure the activity of substances at a concentration of 2 mg/mL. The In this study, the possible effect of lapachol and β-lapachone and its tests were conducted in duplicate. 100 T. canis larvae, 200 µL of RPMI- derivatives against T. canis larvae was tested. Among all the synthetic 1640 medium, and 100 µL of the test substances were added in each well. compounds tested, β-lapachone and β-C-allyl-lawsone showed the best

The larvae were then maintained at 37 °C for 48 hours with 5% CO2. anthelmintic activity in vitro. Although these results are relevant, the quinones present significant toxicity, possibly due to the redox potential. The activity was tested in vitro and after exposure to the test This toxicity may cause cell damage due to oxidative stress, which could compound the larval mobility was tested by the state of the larvae (i.e., result in undesirable side effects10.

198 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.

Table 1 Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with lapachol and derivatives (n = 5)

Standart Larvae viability in Nº Chemical structure Chemical compound Activity MLC deviation mice

OH Lapachol 1 100% Zero ≤ 500 µg/mL Negative C15H14O3

O β- lapachone 2 100% Zero ≤ 250 µg/mL Negative C15H14O3 O

OH Nor-lapachol 3 100% Zero ≤ 1,000 µg/mL Negative C14H12O3

O Nor-β-lapachone 4 11.9% 0.8 - - C14H12O3 O

O β-C-allyl-lawsone 5 100% Zero ≤ 250 µg/mL Negative C13H10O3 O

Control Live larvae (no compound) 4.2% 0.4 - Positive

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 could be used in the treatment of visceral toxocariasis. easy access of quinones to natural sources from Brazilian flora7, justify the utilization of these compounds as a pharmacophore to develop Four phenazines (i.e., compounds 1, 2, 3, and 4) out of the 17, heterocyclic derivatives more active and less toxic. showed 100% activity at a concentration of 2 mg/mL. However, these phenazines did not present satisfactory results when exposed to low This approach was previously used to synthesize trypanocidal concentrations; similar results were obtained with the same phenazines naphthoimidazoles from β-lapachone and to demonstrate that against Plasmodium falciparum, P. berghei5, and Mycobacterium naphthoimidazoles were more active and less toxic than β-lapachone8. tuberculosis2. In these studies, the compounds showed 50% antimalarial activity in vitro, and only one-fourth of the phenazines tested against M. The larvicidal potential of in vitro tests and the capacity to inhibit tuberculosis demonstrated strong antimycobacterial activity (minimum viability of infection in the mice, demonstrated by quinones, indicated inhibitory concentration = 0.78 µg/mL). A significant antimalarial the relevance of studies in this area. Furthermore, motivates realize activity in vitro was also shown in the other phenazines synthesized cytotoxicity studies, for further evidence of the biological activity of from naphthols that were assayed against P. falciparum strains resistant these compounds, in preclinical trials in experimental models, aiming to chloroquine. However, they are not able to promote an effective cure the development of prototype compound with anthelmintic activity which when tested against P. berghei in vivo20.

199 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.

Table 2 Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17)

Standart Larvae viability in No. Chemical structure Chemical compound Activity MLC deviation mice

N N 1 C21H22N2O 100% Zero 2,000 µg/mL Negative

O N O N

2 C36H42N2O4 100% Zero 2,000 µg/mL Negative O O

3 N C19H16N2O 100% Zero 2,000 µg/mL Negative

N N 4 C19H18N2O 100% Zero 2,000 µg/mL Negative

N N 5 C20H22N2O 78.6% 7.1 - -

O N O N 6 C36H34N2O4 1.76% 0.03 - - O O

200 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.

Table 2 Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17) (cont.)

Standart Larvae viability in No. Chemical structure Chemical compound Activity MLC deviation mice

O N O N 7 C34H30N2O4 1.0% 0.1 - -

O O

O N O N 8 C34H38N2O4 3.8% 18.0 - -

O O

N N 9 C20H18N2O 4.2% 8.8 - -

10 N C20H16N2O 1.3% 4.5 - -

O N O N 11 C32H22N2O4 6.5% 2.2

O O

O N O N 12 C32H26N2O4 1.5% 1.1 - -

O O

201 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.

Table 2 Larvicide/larvistatic activity, MLC and in vivo viability of the T. canis larvae treated with phenazines (n = 17) (cont.)

Standart Larvae viability in No. Chemical structure Chemical compound Activity MLC deviation mice

13 N C19H20N2O 14.0% 47.4 - -

N N 14 C20H20N2O 2.5% 1.4 - -

N N 15 C21H22N2O 4.0% 0.7 - -

O N O N 16 C36H30N2O4 94.5% 3.5 - - O O

H3C

CH3

CH3 17 N O - 98.4% 0.4 - -

CT No compound 1.3% 0.4 - Positive

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 compounds 1-4, indicates that new modifications to these molecules are 5-17) tested in this study did not increase the specific activity of necessary to promote effective action against T. canis larvae. the molecules. The lower activity of compounds 5-17, compared to

202 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.

ACKNOWLEDGEMENTS 8. De Moura KCG, Salomão K, Menna-Barreto RF, Emery FS, Pinto MC, Pinto AV, et al. Studies on the trypanocidal activity of semi-synthetic pyran[b-4,3]naphtho[1,2-d] imidazoles from -lapachone. Eur J Med Chem. 2004;39:639-45. The authors are grateful to Antônio Ventura Pinto and Núcleo de b Pesquisa de Produtos Naturais (UFRJ) for the compounds. 9. Despommier D. Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects. Clin Microbiol Rev. 2003;16:265-72. FINANCIAL SUPPORT 10. Dubin M, Fernandez-Villamil SH, Stoppani AO. Cytotoxicity of β-lapachone, an o-naphthoquinone with possible therapeutic use. Medicina (B Aires). 2001;61:343-50. This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Authors declare no conflict of interests. 11. Ettlinger MG. Hydroxynaphthoquinones. III. The structure of lapachol peroxide. J Am Chem Soc. 1950;72:3472-4. RESUMO 12. Falkenberg, MB. Quinonas. In: Simões CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR, editors. Farmacognosia: da planta ao medicamento. Porto Atividade anti-helmíntica do lapachol, β-lapachona e derivados Alegre: Ed. UFRGS; 2004. p. 1102. contra larvas de Toxocara canis 13. Fan CK, Liao CW, Cheng YC. Factors affecting disease manifestation of toxocarosis in Os anti-helmínticos empregados no tratamento das helmintoses humans: genetics and environment. Vet Parasitol. 2013;193:342-52. intestinais, de modo geral, são eficazes, porém nas parasitoses teciduais, como é o caso da toxocaríase visceral, a eficácia é moderada. Este estudo 14. Fieser LF, Fieser M. Naphthoquinone antimalarials. XII. The hooker oxidation reaction. J Am Chem Soc. 1948;70:3215-22. teve como objetivo avaliar in vitro a atividade do lapachol, β-lapachona e fenazinas derivadas da β-lapachona sobre a viabilidade de larvas de 15. Glickman LT, Schantz PM, Cypess RH. Canine and human toxocariasis: review of Toxocara canis. Os compostos foram testados na concentração de 2 mg/mL transmission, pathogenesis, and clinical disease. J Am Vet Med Assoc. 1979;175:1265- (em duplicata) em placas de microcultivo, contendo larvas de T. canis em 9. meio RPMI-1640, sendo incubados, a 37 °C, em tensão de CO2 de 5%, 16. Hooker SC. Lomatiol. Part II. Its occurrence, constitution, relation to and conversion into por 48 horas. Na concentração de 2 mg/mL, quatro fenazinas, o lapachol lapachol. Also a synthesis of lapachol. J Am Chem Soc. 1936;58:1181-90. e três derivados, apresentaram atividade larvicida/larvostática de 100%. A seguir, foi realizado o teste de concentração larvicida/larvostártica 17. Hooker SC. The constitution of lapachol and its derivatives. Part IV. Oxidation with mínima (CLM). Os compostos que apresentaram os melhores resultados potassium permanganate. J Am Chem Soc. 1936;58:1168-73. foram o nor-lapachol (CLM, 1 mg/mL), lapachol (CLM, 0,5 mg/mL), a 18. Hotez PJ, Wilkins PP. Toxocariasis: America’s most common neglected infection of β-lapachona e a β-C-alil-lausona (CLM, 0,25 mg/mL). As larvas expostas poverty and a helminthiasis of global importance? PLOS Negl Trop Dis. 2009;3:400. aos compostos, na melhor CLM 100% in vitro foram inoculadas em camundongos BALB/c saudáveis não sendo capazes de causar infecção, 19. Hussain H, Krohn K, Ahmad VU, Miana GA, Green IR. Lapachol: an overview. Arkivoc. confirmando o potencial larvicida in vitro desses compostos. 2007;2:145-71.

20. Hussain H, Specht S, Sarite SR, Saeftel M, Hoerauf A, Schulz B, et al. A new class of REFERENCES phenazines with activity against a chloroquine resistant Plasmodium falciparum strain and antimicrobial activity. J Med Chem. 2011;54:4913-7. 1. Abo-Shehada MN, Herbert IV. The migration of larval Toxocara canis in mice. II. Post- intestinal migration in primary infections. Vet Parasitol. 1984;17:75-83. 21. Laursen JB, Nielsen J. Phenazine natural products: biosynthesis, synthetic analogues and biological activity. Chem Rev. 2004;104:1663-85. 2. Coelho TS, Silva RS, Pinto AV, Pinto MC, Scaini CJ, Moura KC, et al. Activity of β-lapachone derivatives against rifampicin-susceptible and–resistant strains of 22. Lescano SZ, Chieffi PP, Amato Neto V, Ikai DK, Ribeiro MCSA. Anthelmintics in Mycobacterium tuberculosis. Tuberculosis (Edinb). 2010;90:293-7. experimental toxocariasis: effects on larval recovery of Toxocara canis and on immune response. J Bras Patol Med Lab. 2005;41:21-4. 3. Da Silva MN, Ferreira VF, De Souza MCBV. Um panorama atual da química e da farmacologia de naftoquinonas, com ênfase na β-Lapachona e derivados. Química 23. Lima NMF, Correia CS, Leon LL, Machado GMC, Madeira MF, Santana AEG, et Nova. 2003;26:407-16. al. Antileishmanial activity of lapachol analogues. Mem Inst Oswaldo Cruz. 2004;99:757-61. 4. De Almeida ER, da Silva Filho AA, dos Santos ER, Lopes CA. Antiinflammatory action of lapachol. J Ethnopharmacol. 1990;29:239-41. 24. Lopes JN, Cruz FS, Docampo R, Vasconcellos ME, Sampaio MC, Pinto AV, Gilbert B. In vitro and in vivo evaluation of the toxicity of 1,4-naphthoquinone and 5. De Andrade-Neto VF, Goulart MOF, da Silva Filho JF, da Silva MJ, Pinto MC, Pinto 1,2-naphthoquinone derivatives against Trypanosoma cruzi. Ann Trop Med Parasitol. AV, et al. Antimalarial activity of phenazines from lapachol, beta-lapachone and its 1978;72:523-31. derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. Bioorg Med Chem Lett. 2004;14:1145-9. 25. Magnaval JF. Comparative efficacy of diethylcarbamazine and mebendazole for the treatment of human toxocariasis. Parasitology. 1995;110:529-33. 6. Delgado O, Botto C, Mattei R, Escalante A. Effect of albendazole in experimental toxocariasis of mice. Ann Trop Med Parasitol. 1989;83:621-4. 26. Maizels RM. Toxocara canis: molecular basis of immune recognition and evasion. Vet Parasitol. 2013;193:365-74. 7. De Moura KCG, Emery FS, Neves-Pinto C, Pinto MCFR, Dantas AP, Salomão K, et al. Trypanocidal activity of isolated naphthoquinones from Tabebuia and some 27. Nunes CM. Imunodiagnóstico da larva migrans visceral através de um método de ELISA heterocyclic derivatives: a review from an interdisciplinary study. J Braz Chem Soc. indireto competitivo. [Tese]. São Paulo: Universidade de São Paulo, Faculdade de 2001;12:325-38. Medicina Veterinária e Zootecnia; 1996.

203 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.

28. Othman AA. Therapeutic battle against larval toxocariasis: are we still far behind? Acta 35. Silva RSF. Síntese de fenazinas e derivados lactônicos e halogenados a partir de Trop. 2012;124:171-8. naftoquinonas naturais com atividade contra Mycobacterium tuberculosis. [Tese]. Rio de Janeiro: Universidade Federal do Rio de Janeiro, Núcleo de Pesquisa de Produtos 29. Pardee AB, Li YZ, Li CJ. Cancer therapy with beta-lapachone. Current Cancer Drug Naturais; 2009. Targets. 2002;2:227-42. 36. Smith H, Holland C, Taylor M, Magnaval JF, Schantz P, Maizels R. How common is human 30. Pawlowski Z. Toxocariasis in humans: clinical expression and treatment dilemma. J toxocariasis? Towards standardizing our knowledge. Trends Parasitol. 2009;25:182-8. Helminthol. 2001;75:299-305. 37. Stürchler D, Schubarth P, Gualzata M, Gottstein B, Oettli A. Thiabendazole vs. albendazole 31. Quattrocchi G, Nicoletti A, Marin B, Bruno E, Druet-Cabanac M, Preux PM. in treatment of toxocariasis: a clinical trial. Ann Trop Med Parasitol. 1989;83:473-8. Toxocariasis and epilepsy: systematic review and meta-analysis. PLOS Negl Trop Dis. 2012;6:1775. 38. Torrado S, Torrado S, Cadorniga R, Torrado JJ. Formulation parameters of albendazole solution. Int J Pharm. 1996;140:45-50. 32. Reis M, Trinca A, Ferreira MJ, Monsalve-Puello AR, Grácio MAA. Toxocara canis: potential activity of natural products against second-stage larvae in vitro and in vivo. 39. Wang GX, Luo ZJ. A novel method for the recovery of Toxocara canis in mice. J Exp Parasitol. 2010;126:191-7. Helminthol. 1998;72:183-4.

33. Satou T, Horiuchi A, Akao N, Koike K, Futja K, Nikaido T. Toxocara canis: search for 40. Wisniewska-Ligier M, Wozniakowska-Gesicka T, Sobolewska-Drylariska J, Markiewicz- a potential drug amongst β-carboline alkaloids – in vivo and mouse studies. Exp Jógwiak A, Wieczorek M. Analysis of the course and treatment of toxocariasis in Parasitol. 2005;110:134-9. children: a long term observation. Parasitol Res. 2012;110:2363-71.

34. Schneier AJ, Durand ML. Ocular toxocariasis: advances in diagnosis and treatment. Int Received: 21 July 2014 Ophthalmol Clin. 2011;51:135-44. Accepted: 22 September 2014

204 Rev. Inst. Med. Trop. Sao Paulo 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 binding of host heparin sulfate proteoglycans (HSPGs)18, shares primary structure similarity with another proven Surface antigen 1 (SAG1)7 Toxoplasma gondii, an obligate intracellular coccidian parasite, has protein. It was considered interesting to carry out the primer-directed acquired utmost zoonotic relevance in the current scenario around the amplification of the open reading frame (ORF) of surface antigen 3 globe, accounting for abortions, stillbirths, and neonatal complications (SAG3) gene of Indian isolates of T. gondii viz. Chennei (CHEN) and in livestock, especially in sheep, goats and pigs9,16,30. The condition leads Izatnagar (IZN) isolates, maintaining them at the IVRI and cloning them to life-threatening consequences both in immunocompromised human in a heterologous prokaryotic system. Moreover, the two Indian isolates patients suffering from acquired immune deficiency syndrome (AIDS) used in the present study are known to vary between themselves as far and those with organ transplants2. In India, the condition has exhibited as homologies related to other gene loci like GRA 526, MIC 323 and SAG itself as acquired ocular toxoplasmosis4, in immunocompetent patients, 227 are concerned, but there is no literature available as far as SAG3 bringing about possible similarities with South American strains which homologies are concerned. In the present study, the cloned genes were are known to exhibit a high rate of ocular involvement20. A third of the custom sequenced and the information was compared with the available world’s total population is thought to be at risk of infection22. Of late, sequences of the same gene in the GenBank in order to establish the different strains of Toxoplasma gondii are known to induce different phylogenetic identity of the SAG3 gene among the various isolates. cytokine responses5 and thereby vary in their pathogenesis. The surface antigens of T. gondii are the major targets as key molecules for METHODS immunodiagnosis as well as immunoprophylaxis because of their initial presentation to the host immune system. Surface antigen 3 (SAG3), an Propagation of T. gondii tachyzoites: Inbred Swiss albino adult under-reported 43kDa glycoaminoglycan-binding protein associated with 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 the SAG3 gene of T. gondii (CHN and IZN isolates) was PCR amplified T. gondii tachyzoite isolates that were cryopreserved and maintained at using a pair of specific primers as described by SUDAN et al. 201228 a divisional laboratory, IVRI. These two Indian isolates were originally (forward primer (TS3F) 5’-ATGCAGCTGTGGCGGCGCAG-3’ and isolated from the tested-positive blood, heart and brain tissues of free- reverse (TS3R) 5’-TTAGGCAGCCACATGCACAAG-3’). The PCR range chickens (Gallus domesticus) naturally infected by T. gondii25 and reactions were carried out in a standard 25 µL reaction volume with initial isolated after Cat inoculation assays. The infected mice were monitored denaturation of DNA strands at 95 oC for five min followed by 32 cycles daily for the development of signs of infection. Infected mice exhibiting of denaturation at 95 oC for 50 sec, primer annealing at 62 oC for 75 sec peritonitis were euthanized and peritoneal lavage was aspirated following and strand elongation at 72 oC for 50 sec. Thereafter one cycle of final inoculation of 5 mL of sterile phosphate buffered saline (PBS, pH 7.2) extension of the strands was carried out at 72 oC for 12 min. The PCR in the peritoneal cavity with due care in avoiding injury to visceral amplifications were confirmed by visualization of the product on 1.5% organs. The contents were washed thrice with PBS (pH 7.2) and the live agarose gel stained with ethidium bromide following electrophoresis. tachyzoites were counted. Molecular cloning and characterization of the SAG3 gene of Separation of host cell-free tachyzoites: The host cell-free Indian isolates: The amplified ORF of the SAG3 genes of Indian isolates tachyzoites were separated using standard protocol15. Briefly, the of T. gondii were purified using a Qiagen Mini elute gel extraction kit peritoneal fluid containing free tachyzoites and tachyzoite infected (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s macrophages was collected in PBS (pH 7.4) and washed thrice in protocol. Following this, competent Escherichia coli DH5α cells were PBS (pH 7.4) while repeatedly centrifuging at 5000 rpm for 10 min. prepared following the standard calcium chloride treatment method23. Following this, a final pellet was re-suspended in 5 mL of PBS (pH Ligation reaction for the cloning of SAG3 (amplified fromT. gondii 7.4). The intracellular tachyzoites were separated and made free from Indian isolates) into InsTAclone PCR cloning vector (Qiagen, Germany) the macrophages by passing the contents repeatedly through a 27g as well as transformation of DH5α cells was carried out as per the needle fitted in a 10 mL sterile syringe. The host cell-free tachyzoite company’s protocol. The positive clones were identified by blue-white suspension was washed with 20 mL of PBS (pH 7.4), debris was allowed colony screening method. Further confirmation was carried out by to settle down in the centrifuge tube for 10 min and the supernatant was restriction analysis of the plasmid DNA isolated from the white colonies collected and, following this, passed through a pre-wetted (with PBS with PstI and EcoRI as well as by colony PCR following standard pH 7.4) polycarbonate membrane filter of 3 µm pore size slowly (at the protocol24. The restriction digestion reaction was carried out at 37 oC rate of one mL per 2-3 min). The filtrate was centrifuged (3000 rpm for for four h. The digested product as well, as the colony PCR amplified 10 min) and the tachyzoites in sediment were re-suspended in one mL products, was visualized in the ethidium bromide-stained agarose gel of PBS (pH 7.4). following electrophoresis. The subcultures of a positive clone harboring the desired SAG3 genes of both the Indian isolates were custom DNA Isolation of total RNA of T. gondii: Total RNA was extracted sequenced from the Department of Biochemistry, Delhi University. directly from the purified tachyzoites using Trizol® reagent (Gibco BRL) while following the manufacturer’s protocol. Briefly, one mL of Trizol Data analysis: The sequence information received was analyzed was added to the suspension containing 5-10x106 tachyzoites, repeatedly using DNASTAR and GeneTool software. The sequences, hence pipetted to kill the tachyzoites and following this, incubated at 30 oC for received sequence submitted to GenBank (Accession No.: HQ291783 & five min to dissociate nucleoprotein complexes. The suspension was HQ291784 for Chennei and Izatnagar isolates, respectively). Moreover, vigorously shaken for 15 sec after adding 0.2 mL of chloroform and then these two sequences were compared with an earlier sequenced RH strain centrifuged at 12,000g for 15 min at 4 oC. This facilitates the separation sequence (Accession No.: FJ825705) from the department along with into lower organic phase and upper aqueous phase. The aqueous phase other published sequences viz., CEP (Accession No.: AF340229); P-Br was transferred to a fresh tube, 0.5mL of the isopropyl alcohol was poured (Accession No.: AY187280) and PRU (Accession No.: AF340228) from into the tube and the RNA was allowed to precipitate while keeping the across the world through the GenBank using online Nucleotide BLAST tube at 15-30 oC for 10 min. The tube was centrifuged at 12,000g for Softwares (http://blast.ncbi.nlm.nih.gov/). 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 RESULTS 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 Viability of cryopreserved T. gondii: All the infected mice started µL of RNA storage buffer (Ambion) and stored at -20 oC until further showing characteristic signs of the disease from Day-7 Post Infection (PI). use. Purity and concentration of total RNA was checked by ethidium The clinical signs included raised & rough fur coat, pendulous abdomen, bromide stained agarose gel electrophoresis, performed at 2-3 volts/cm2. 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 Synthesis of complimentary DNA (cDNA) by reverse transcription: with their forelegs. Microscopically, a large number of tachyzoites were cDNA was synthesized from the total RNA isolated from the T. gondii detectable (either free or within the peritoneal macrophages suspended tachyzoites of both the isolates, using oligo dT primer while following in the aspirated peritoneal fluid). the standard protocol23. The cDNA, thus synthesized, was quantified using a spectrophotometer (Nanodrop®, USA). PCR amplification, molecular cloning and molecular characterization of the SAG3 gene of Indian isolates: The whole ORF Polymerase chain reaction-based (PCR) amplification of the of the SAG3 gene was amplified from the cDNA of Indian isolates of SAG3 gene of Indian isolates: The entire open reading frame (ORF) of T. gondii using the specific forward and reverse primers. The amplicons

206 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.

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.

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. 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.

Fig. 4 - Sequence pair distances of SAG3 Clustal V (weighted).

Fig. 5 - Phylogenetic tree of nucleotide sequence of SAG3 Clustal V (weighted). 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 Fig. 2 - Release of SAG3 insert by restriction digestion of insTA cloning vector of the substitution of a single nucleotide of A instead of G at the 397th position of 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 the SAG3 nucleotide sequence of both the Indian isolates. The nucleotide Izatnagar isolate; Lane CHEN: Insert release after PstI and EcoRI digestion of vector substitution resulted in the change of a single nucleotide residue in the rd containing Chennai isolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning deduced amino acid sequence at the 133 position as asparagine (N) vector. 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 Data analysis: The nucleotide sequence revealed 99.9% (Fig.4) PRU strains (USA), with respect to the nucleotide homology. sequence homology of SAG3 ORF between the Indian isolates with

207 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.

DISCUSSION CONCLUSION

The significance of toxoplasmosis has increased particularly in In the present study, the SAG3 gene of T.gondii was cloned, immune compromised and/or HIV/AIDS patients, with an alarming sequenced and aligned, before being compared with various published prevalence in developing countries such as India. The presence of strains and the homologies between the two Indian isolates were found brain cysts is often associated with various psychiatric disorders and both with one another and with other strains across the globe. The two behavioral alterations29 such as schizophrenia8, 32 alongside other brain Indian isolates used in the present study are known to vary between pathologies and ocular involvements25 in both immunocompromised themselves as far as homologies related to other genes are concerned and immunocompetent individuals1,11. In order to precisely define the but they were found to be 100% homologous as far as SAG3 locus is magnitude of the disease, it was of interest to investigate the genetic concerned. This could be attributed to the fact that this SAG3 might be diversity of the pathogen among the T. gondii strains using advanced a conserved locus and therefore, further detailed studies are thereby biotechnological approaches. warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of Surface antigen 3 (SAG3), a 43kDa glycoprotein, is a view of molecular phylogeny. glycosylphosphatidylinisotol-anchored (GPI) membrane-bound protein in the developmental stages of the pathogen (tachyzoites & bradyzoites) RESUMO 6,19 parasite . The protein was earlier identified as 43P . It was cloned and sequenced for the first time by CESBRON-DELAUW et al. in 19947 Caracterização molecular e análise filogenética de sequências do followed by FUX et al. in 200313. SAG3 has primary structure similarity antígeno de superfície 3 (SAG3) em isolados indianos (CHENNAI with Surface antigen 1 (SAG1)7. SAG3 is a glycoaminoglycan-binding E IZATNAGAR) de Toxoplasma gondii protein associated with binding of host heparin sulfate proteoglycans (HSPGs)18. The SAG3-HSPGs interaction facilitates the parasite’s Contexto e objetivo. A caracterização molecular de isolados attachment to target cells. Furthermore, it has been shown that targeted indianos de Toxoplasma gondii é importante para a investigação de disruption of the GPI-anchored surface antigen SAG3 gene in T. gondii variações genéticas existentes entre cepas do parasito em diferentes resulted in decreased host cell adhesion and virulence of the parasite for locos gênicos. Delineamento e disposição. A presente comunicação mice10. In immunoprophylactic application, rSAG3 conferred partial realizou a clonagem e o sequenciamento dos 1158 pares de base protection in mice, which was mediated through Th1 type immune correspondendo à totalidade do quadro de leitura do antígeno de response21. However, molecular characterization of the SAG3 gene of T. superfície 3 (SAG3) de Toxoplasma gondii em dois isolados indianos gondii of Indian isolates has not been attempted so far. The present study (Chennai e Izatnagar) mantidos em um biorrepositório localizado em reports the molecular characterization of the surface antigen 3 (SAG3) gene IVRI. Método. As sequências do SAG3 dos dois isolados indianos foram of T. gondii of Indian isolates and ascertains its molecular homology with clonadas, sequenciadas e posteriormente comparadas com sequências some other strains of the same parasites that are prevalent across the globe. SAG3 de Toxoplasma gondii disponíveis em publicações. Resultados. A comparação das sequências revelou 99,9% de homologia com a cepa Worldwide, only one valid species of Toxoplasma exists. However, RH padrão; 99,3% de homologia com as cepas P-Br e CEP; e 98,4% based on molecular genotyping studies, varied fundamental clonal de homologia com a cepa PRU. Os dois isolados indianos eram 100% population isolates of T. gondii have been recognized. The molecular idênticos no que diz respeito à sequência SAG3. Conclusão. Concluiu-se diversity in the distinct and/or related Toxoplasma stabilates is routinely que os isolados indianos são filogeneticamente mais próximos da cepa evaluated by sequence-based analysis among the different isolates. RH em relação à cepa brasileira P-Br, ou às cepas CEP e PRU (USA). Recently, different strains of Toxoplasma gondii have been known No entanto, a análise de outros genes de Toxoplasma gondii destes dois to induce varying levels of cytokine responses5 and thereby vary in isolados indianos mostrou diferenças na composição de nucleotídeos, their pathogenesis, hence the study of the phylogeny has gained ultra ao contrário do que foi encontrado para o locus SAG3. Estes resultados importance owing to the variation in pathogenesis at the strain levels. poderiam ser atribuídos ao fato do locus SAG3 ser altamente conservado, Moreover, the two Indian isolates used in the present study are known to necessitando de estudos adicionais para determinar se SAG3 poderia ser vary between themselves as far as homologies related to other gene loci utilizado no diagnóstico da toxoplasmose. No entanto, estes resultados like GRA 526, MIC 323 and SAG 227 are concerned but they were found são importantes do ponto de vista da filogenia molecular. 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 ACKNOWLEDGMENTS thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The authors are thankful to the Director, IVRI for providing the facilities and to the ICAR for the fellowship awarded to the first author Interestingly, differences at the lineages sequence level of DNA during the perusal of his master’s programme. The authors declare that among the predominant clones are less than 2%14. Transmission of the there is no conflict of interest. parasite through carnivorism and scavenging among intermediate hosts, bypassing sexual recombination events in the definitive host, i.e. cats17, 30, REFERENCES parthenogenetic formation of oocysts by many unfertilized macrogametes of the parasite in the small intestine of cats12, and simultaneous infection 1. Alvarado-Esquivel C, Alanis-Quiñones OP, Arreola-Valenzuela MA, Rodríguez- Briones with different strains of T. gondii are some plausible reasons that can A, Piedra-Nevarez IJ, Duran-Morales E, et al. Seroepidemiology of Toxoplasma 3 gondii infection in psychiatric inpatients in a northern Mexican city. BMC Infect explain the existence of the clonal population structure in T. gondii . Dis. 2006;6:178.

208 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.

2. Angel SO, Matrajt M, Margarit J, Nigro N, Illescas E, Pszenny V, et al. Screening of 19. Kazemi B, Maghen L, Bandehpour M, Shahabi S, Haghighi A. Gene cloning of P43 active Toxoplasma in patients by DNA hybridization with ABGTg7 probe in blood surface protein of Toxoplasma gondii tachyzoite and bradyzoite (SAG3). Gene Ther samples. J Clin Microbiol. 1997;35:591-5. Mol Biol. 2007;11:113-6.

3. Ajzenberg D, Banuls AL, Su C, Dumetre A, Demar M, Carme B, et al. Genetic diversity, 20. Khan A, Jordan C, Muccioli C, Vallochi AL, Rizzo LV, Belfort JR, et al. Genetic divergence clonality and sexuality in Toxoplasma gondii. Int J Parasitol. 2004;34:1185-96. of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil. Emerg Infect Dis. 2006;12:942-9. 4. Balasundaram MB, Andavar R, Palaniswamy M, Venkatapathy N. Outbreak of acquired ocular toxoplasmosis involving 248 patients. Arch Ophthalmol. 2010;128:28-32. 21. Lee YH, Shin DW, Lee JH, Nam HW, Ahn MH. Vaccination against murine toxoplasmosis using recombinant Toxoplasma gondii SAG3 antigen alone or in combination with 5. Beaman MH, Wong SY, Remington JS. Cytokines, Toxoplasma and intracellular Quil A. Yonsei Med J. 2007;48:396-404. parasitism. Immunol Rev. 1992;127:97-117. 22. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965-76. 6. Burg JL, Perelman D, Kasper IH, Ware PL, Boothroyd JC. Molecular analysis for gene encoding the major surface antigens of Toxoplasma gondii. J Immunol. 23. Praveen K, Bansal GC, Vikram K, Zahid AK, Saravanan BC, Tewari AK, et al. Cloning 1988;141:3584-91. and molecular characterization of microneme protein-3 (MIC3) gene of Izatnagar and Chennai isolates of Toxoplama gondii. J Vet Parasitol. 2012;26:31-4. 7. Cesbron-Delauw MF, Tomavo S, Beauchamps P, Fourmaux MP, Camus D, Capron A, et al. Similarities between the primary structures of two distinct major surface protein 24. Sambrook J, Fritch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New of Toxoplasma gondii. J Biol Chem. 1994;269:16217-22. York: Cold Spring Harbor; 1989.

8. Daryani A, Sharif M, Hosseini SH, Karimi SA, Gholami S. Serological survey of 25. Sreekumar C, Graham DH, Dahl E, Lehmann T, Raman M, Bhalerao DP, et al. Genotyping Toxoplasma gondii in schizophrenia patients referred to psychiatric hospital, Sari of Toxoplasma gondii isolates from chickens from India. Vet Parasitol. 2003;118(3- city, Iran. Trop Biomed. 2010;27:476-86. 4):187-94.

9. Dubey JP. Toxoplasmosis: a waterborne zoonosis. Vet Parasitol. 2004;126:57-72. 26. Singh H, Tewari AK, Mishra AK, Maharana BR, Rao JR, Raina OK. Molecular cloning, comparative sequence analysis and prokaryotic expression of GRA5 protein of 10. Dzierszinski F, Mortuaire M, Cesbron-Delauw MF, Tomavo S. Targeted disruption of the Toxoplasma gondii. Indian J Anim Sci. 2011a;81:209-15. glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice. Mol 27. Singh H, Tewari AK, Mishra AK, Maharana BR, Rao JR, Raina OK. Molecular cloning and Microbiol. 2000;37:574-82. comparative sequence analysis of open reading frame of SAG2 gene of Toxoplasma gondii. J Vet Parasitol. 2011b;25:107-12. 11. Fekadu A, Shibre T, Cleare AJ. Toxoplasmosis as a cause for behavior disorders - overview of evidence and mechanisms. Folia Parasitol (Praha). 2010;57:105-13. 28. Sudan V, Tewari AK, Singh H, Saravanan BC, Sankar M. Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain. J Parasit Dis. 12. Ferguson DJ. Toxoplasma gondii and sex: essential or optional extra? Trends Parasitol. 2012;36:210-14. 2002;18:355-89. 29. Sudan V, Tewari AK, Singh H. An insight into the behavior, course and kinetics of acute 13. Fux B, Rodrigues CV, Portela RW, Silva NM, Su C, Sibley D, et al. Role of cytokines infection of Toxoplasma gondii human RH strain in experimentally infected murine and major histocompatibility complex restriction in mouse resistance to infection model. Iranian J Parasitol. 2014;9:114-9. with a natural recombinant strain (Type I–III) of Toxoplasma gondii. Infect Immun. 2003;71:6392-401. 30. Su C, Evans D, Cole RH, Kissinger JC, Ajioka JW, Sibley LD. Recent expansion of Toxoplasma through enhanced oral transmission. Science. 2003;299(5605):414-6. 14. Grigg ME, Bonnefoy S, Hehl AB, Suzuki Y, Boothroyd JC. Success and virulence in Toxoplasma as the result of sexual recombination between two distinct ancestries. 31. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int Science. 2001;294:161-5. J Parasit. 2000;30(12-13):1217-58.

15. Gross U, Muller WA, Knapp S, Heesemann J. Identification of a virulence-associated 32. Webster JP, Lamberton PHL, Donnelly CA, Torrey EF. Parasites as causative agents of antigen of Toxoplasma gondii by a mouse monoclonal antibody. Infect Immun. human affective disorders? The impact of anti-psychotic, mood- stabilizer and anti- 1991;59:4511-6. parasite medication on Toxoplasma gondii’s ability to alter host behavior. Proc Biol Soc. 2006;273(1589):1023-30. 16. Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect. 2002;8:634-40. Received: 8 April 2014 Accepted: 8 August 2014 17. Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis. 1993;172:1561-66.

18. Jacquet A, Coulon L, De Nève J, Daminet V, Haumont M, Garcia L, et al. The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans. Mol Biochem Parasitol. 2001;116:35-44.

209 LIBRARY OF THE SÃO PAULO INSTITUTE OF TROPICAL MEDICINE

Website: http://www.imt.usp.br/sobre-o-imtsp/biblioteca Address: Biblioteca do Instituto de Medicina Tropical de São Paulo da Universidade de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470. 05403-000 - São Paulo - SP - Brazil. Telephone: 5511 3061-7003

The Library of the São Paulo Institute of Tropical Medicine (IMTSP Library) was created on January 15, 1959 in order to serve all those who are interested in tropical diseases.

The IMTSP Library has a collection consisting of books, theses, annals of congresses, journals, and reference works.

The collection of the Library can be searched through the USP Bibliographic Database – Dedalus at the URL http://200.144.190.234/F Rev. Inst. Med. Trop. Sao Paulo 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 patients who present symptoms like diarrhea, fever, vomit, abdominal pain, and nauseas in the absence of any other parasite but Blastocystis6,9,10. Intestinal infections by protozoa are frequent all over the world, In addition to this, studies have shown that stress conditions can lead to being most prominent in developing countries, since the majority of increased susceptibility and pathogenicity of Blastocystis, it is also an the infections are generally acquired by the ingestion of foods or water opportunistic parasite in immunocompromised patients5,16. There is a contaminated by human and/or animal feces, generally caused by the huge lack of information regarding the pathogenesis, the diagnosis and lack of basic sanitation and conditions of hygiene4,6,12. In this context the epidemiology of this protozoan20. In this study, it is shown that the the infection with Blastocystis spp., an anaerobic intestinal protozoan is occurrence of Blastocystis in Uberaba is high, followed by the infection one of the most prevalent6,9,12, occurring in approximately 1.5% to 10% of Giardia intestinalis, and that direct methods, especially Ritchie’s, of the population in developed countries and 30% to 60% in developing are suitable for the diagnosis of the parasite. Moreover, the presence of countries19. However, these data are underestimated, since laboratory diarrheal stools with unique parasitism by Blastocystis spp. was observed. technicians are generally not sufficiently trained to detect it or simply do not report their findings. Moreover, routine techniques for stool MATERIAL AND METHODS analysis such as the water spontaneous sedimentation (HOFFMAN- PONS-JANER)8 which leads to the breakage of the vacuolar stage of the The present paper is a cross-sectional study with a non-probability parasite, is one of the mostly detected stages in the stool examination, sample of patients who were treated at the Universidade Federal do leading to the false negative results14. Triângulo Mineiro Hospital, between April 2011 and May 2012. All patients referred to carry out a stool test suffered from acute or chronic Although the infection with Blastocystis spp. is one of the most diarrhea or complaints of constant abdominal pain and/or weakness prevalent amongst the intestinal parasites, its impact on public health was included. Age, presence of underlying diseases, HIV/AIDS or is not known, since its pathogenicity has been noted as controversial gastrointestinal symptoms were not considered as exclusion criteria. by several authors6,9,10,21. However, in spite of the controversial issue The specimens were examined by the microscopy direct of smears and that Blastocystis pathogenesis represents, there are no explanations for 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 (Table 2), and in 27.8% and 44.1 % of them, respectively, the presence Lugol solution in a slide/coverslip and observed by optical microscopy of parasitism by at least one organism was observed, whether pathogenic with a 400× objective lense. Ritchie’s method was performed by keeping or not. G. intestinalis infection occurred in 86.7% of cases (85/98) in the feces in 3.7% formaldehyde, adding ethyl ether and then centrifuging patients aged between 0-10 years (Table 2). Ascaris lumbricoides (3, the mixture at 1,200×g/5min. The sediment was observed with the 400× 0.3%) and Entamoeba histolytica/dispar/moshkovskii (13, 0.7%) were objective. The statistical software Statistica 10.0 (Statsoft, Tulsa, OK, also observed in children. 2011) was used to perform the statistical analysis. Table 2 The association between risk factors and presence of Blastocystis spp. Parasitism by Giardia intestinalis and Blastocystis spp. according to the age was verified by the chi-squared classic or, whenever necessary by the group of patients treated at the Clinical Hospital, Universidade Federal do chi-square test with Yates correction and Fisher’s exact test. Moreover, Triângulo Mineiro, Uberaba (MG) the association force was measured by the calculation of the Odds ratio with confidence intervals of 95%. The agreement between the microscopy Giardia intes- Blastocystis direct and the Ritchie’s method was evaluated by means of Kappa Age (years) No. of patients tinalis spp. coefficient. Results which demonstrate a level of significance lower than (n/%) (n/%) 0.05 (p < 0.05) were considered significant. This study was approved 0-5 442 70 (15.8) 51 (1.5) by the ethics committee in research at the UFTM under number 1804. 6-10 146 15 (10.2) 44 (30.1) RESULTS 11-20 103 6 (5.8) 25 (24.1) 21-50 407 5 (1.2) 70 (17.2) Stool specimens from 1,323 patients were examined, with 44.1% male and 55.9% female. From the analyzed samples, 28.7% presented > 50 225 2 (0.8) 45 (20.0) an intestinal pathogenic parasite or not, being Blastocystis spp. (17.8%) Total 1,323 98 235 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 Overall, parasitism was higher in male patients (32.6%) than in of non-pathogenic parasites occurred in 7.3% of the samples (Table 1). 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, Table 1 parasitism of Blastocystis spp. was significantly higher in patients Occurrence of intestinal parasites in stool samples of patients from the Clinical presenting over six years of age (p < 0.001, Table 2). The analysis of Hospital at the Universidade Federal do Triângulo Mineiro, Uberaba, MG parasitism by G. intestinalis or Blastocystis spp. by age in relation to gender showed no significant difference. Positivity of the diagnostic tests n % Intestinal parasites 379 28.65 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 Blastocystis sp. 235 17.76 of the examination showed that, among the solid samples, softened and Giardia intestinalis 98 7.41 liquid, 12.0% (107/892), 15.8% (34/215) and 8.0% (6/75), respectively, Entamoeba coli 59 4.46 were positive exclusively for Blastocystis spp., showing no statistical difference (p = 0.152). The information of the consistency of 14 stool Endolimax nana 36 2.72 samples with exclusively positivites for Blastocystis sp was not taken. Entamoeba histolytica/dispar/moshkovskii 26 1.97 The analysis by Ritchie’s method was more sensitive for the diagnosis Taenia sp. 6 0.45 of Blastocystis (92.8%) than that by direct microscopy (89.8%), with a Ascaris lumbricoides 2 0.15 ratio of 97.7% agreement (Kappa = 0.92). Isospora belli 1 0.08 DISCUSSION AND CONCLUSIONS Strongyloides stercoralis 2 0.15 Chilomastix mesnili 1 0.08 In this study the occurrence of intestinal infections by protozoa and/ Hookworms 1 0.08 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% Enterobius vermicularis 1 0.08 corresponded to the infection by Giardia that occurred mainly in children Hymenolepis nana 1 0.08 between 0-10 years of age. These data are in accordance with other Exclusive presence of Blastocystis 161 12.17 studies carried out in several regions of Brazil11,14,17. Regarding age and gender, the presence of intestinal parasites was higher in male children Exclusive presence of Giardia 71 5.37 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 From the evaluated samples, 33.47% (442) were from children aged had no direct relation to gender. G. intestinalis infection presented no 0-5 years and 11.04% (146) were from children aged between 6-10 years correlation with gender either. Regarding age, the data agrees with other

212 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.

studies19 and differs from some authors which showed that Blastocystis RESUMO 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 Ocorrência de Blastocystis spp. em Uberaba, Minas Gerais, Brasil occurrence of Blastocystis spp.14,19. Parasitos intestinais são um problema de saúde pública no mundo e The occurrence of Blastocystis spp. infection was higher when a infecção por Blastocystis, protozoário de patogenicidade controversa, compared with all other parasites, an observation that corroborates é uma das mais frequentes. Nesse estudo foi investigada a ocorrência other studies4,6,9,12,16. The exclusive occurrence of Blastocystis in 8% of de parasitos intestinais em pacientes atendidos na Universidade Federal diarrheal stools suggests that it may have a pathogenic character, as some do Triângulo Mineiro, em Uberaba (MG), com ênfase em Blastocystis, authors agree19,21. Some authors observed the presence of Blastocystis pelos métodos parasitológicos direto e de Ritchie. Foram examinadas in stool samples from HIV-infected, homosexuals, travelers, day care fezes de 1.323 pacientes de abril/2011 a maio/2012. Em 28,7% deles children, animal handlers, and mentally handicapped individuals2,16. foi identificado um parasito intestinal, sendo Blastocystis spp. (17,8%) Besides, in immunocompromised patients, the parasite must be e Giardia intestinalis (7,4%) os mais frequentes. A ocorrência de considered pathogenic and patients should be treated accordingly for parasitismo foi maior em crianças de 6-10 anos e a infecção por Blastocystis if no other pathogens are detected2,16. According to them, Blastocystis spp. foi maior acima de seis anos (p < 0,001). Presença the pathogenicity of Blastocystis is possibly associated with low host exclusiva de G. intestinalis e de Blastocystis spp. foi observada em 5,4% immunity, modified intestinal microbiota, and concomitant presence e 12,2% dos pacientes, respectivamente, sendo que dos pacientes com of irritable bowel syndrome and the virulence of the parasite strain. fezes diarreicas, 8% apresentavam parasitismo exclusivo por Blastocystis According to CHANDRAMATHI et al. (2014), pathogenicity may also spp. Outros parasitos intestinais observados em crianças foram Ascaris be host stress dependent, which would lead to a suppression of both lumbricoides (0,3%) e Entamoeba histolytica/dispar/moshkovskii immune responses and to the oxidant-antioxidant regulatory system. (1,4%). O método de Ritchie foi mais sensível (92,8%) que o direto However, more studies are needed to exclude other possible causes of (89,8%), com alta concordância entre eles (97,7%, kappa = 0,92). Em diarrhea, such as rotavirus infection or metabolic disorders. conclusão, a ocorrência de Blastocystis spp. em Uberaba é elevada e foi observada a presença de fezes diarreicas com parasitismo exclusivo por In the literature, several authors suggest that the search of Blastocystis spp. this protozoan via the direct method6,9,12, trichrome staining and cultivation1,10,21, states that the concentration methods should not be ACKNOWLEDGMENTS employed for observation of B. hominis as they destroy cell morphology. In this study, both methods, direct and Ritchie’s showed higher sensitivity The authors would like to thank Oberdan Ricardo Ribeiro, the to 89.8%, with a high agreement percentage (97.7%, kappa = 0.92), laboratory technician at Hospital de Clínicas at UFTM for his assistance being appropriate to the diagnosis of Blastocystis. Although the culture in the collection and processing of samples. is efficient, its cost is higher than the direct method, which has good sensitivity for detecting Blastocystis, since the vacuolar shapes of this Financial support: FAPEMIG- Fundação de Amparo à Pesquisa do parasite are usually released in large amounts in feces. In the authors’ Estado de Minas Gerais (APQ04094-10). experience and unlike that of other authors1, staining of fecal smears for direct identification of Blastocystis from feces is not easy to analyze, AUTHOR’S CONTRIBUTIONS 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, MCS and ALP were responsible for the experimental design of the as demonstrated by other authors14. The HPJ method is also effective if study; ENC and RAC were responsible for the execution techniques and used to dilute 3.7% of formaldehyde stools, since the water breaks the parasitological examination of stools along with MCS and MBOs. GANN vacuolar, granular and amoeboid shapes of the parasite. was responsible for the statistical analysis and DC for the attending and for the referral of the patients. All authors reviewed and contributed to Infection with non-pathogenic parasites (Endolimax nana, the writing of this manuscript. MCS is responsible for the manuscript. Entamoeba coli, Chilomastix mesnilli) occurred in 7.3% of the samples (Table 1). Human infection by non-pathogenic protozoa has been reported CONFLICT OF INTERESTS 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 No conflict of interests was declared. an indicator of fecal contamination of food and water consumed by the population. REFERENCES

In conclusion, the occurrence of Blastocystis spp. in Uberaba (MG) 1. Amato Neto V, Rodríguez Alarcón RS, Gakiya E, Bezerra, RC, Ferreira CS, Braz LM. was high, this scenario indicates the importance of investigating the Blastocistose: controvérsias e indefinições. Rev Soc Bras Med Trop. 2003;36:515-7. main route of parasite transmission and their association with the clinical 2. Basak S, Rajurkar MN, Mallick SK. Detection of Blastocystis hominis: a controversial symptoms manifestation. Furthermore, this study showed that the direct human pathogen. Parasitol Res. 2014;113:261-5. and Ritchie’s method were effective for the diagnosis of Blastocystis spp. and that there is a need for the description of commensal protozoa 3. Boreham PF, Stenzel DJ. The current status of Blastocystis hominis. Parasitol Today. in laboratory reports and for the training of laboratory technicians to 1993;9:251. improve in order to detect it.

213 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.

4. Borges JD, Alarcón RSR, Amato Neto V, Gakiya E. Parasitoses intestinais de indígenas 13. Moura H, Fernandez O, Viola JPB, Silva SP, Passos RH, Lima DB. Enteric parasites da comunidade Mapuera (Oriximiná, Estado do Pará, Brasil): elevada prevalência de and HIV infection: occurrence in AIDS patients in Rio de Janeiro, Brazil. Mem Inst Blastocystis hominis e encontro de Cryptosporidium sp e Cyclospora cayetanensis. Oswaldo Cruz. 1989;84:527-33. Rev Soc Bras Med Trop. 2009;42:348-50. 14. Nascimento SA, Moitinho ML. Blastocystis hominis and other intestinal parasites in 5. Chandramathi S, Suresh K, Sivanandam S, Kuppusamy UR. Stress exacerbates infectivity a community of Pitanga City, Paraná State, Brazil. Rev Inst Med Trop Sao Paulo. and pathogenicity of Blastocystis hominis: in vitro and in vivo evidences. PLOS One. 2005;47:213-7. 2014;9:e94567. 15. Noureldin MS, Shaltout AA, El Hamshary EM, Ali ME. Opportunistic intestinal protozoal 6. Chen TL, Chan CC, Chen HP, Fung CP, Lin CP, Chan WL, et al. Clinical characteristics infections in immunocompromised children. J Egypt Soc Parasitol. 1999;29:951-61. and endoscopic findings associated with Blastocystis hominis in healthy adults. Am J Trop Med Hyg. 2003;69:213-6. 16. Paboriboune P, Phoumindr N, Borel E, Sourinphoumy K, Phaxayaseng S, Luangkhot E, et al. Intestinal parasitic infections in HIV-infected patients, Lao People’s Democratic 7. Cimerman S, Cimerman B, Lewi DS. Prevalence of intestinal parasitic infections in Republic. PLOS One. 2014;9:e91452. patients with acquired immunodeficiency syndrome in Brazil. Int J Infect Dis. 1999;3:203-6. 17. Rezende CH, Costa-Cruz JM, Gennari-Cardoso ML. Enteroparasitoses in food handlers of the public schools in Uberlândia (Minas Gerais), Brazil. Rev Panam Salud Publica. 8. Hoffman WA, Pons JÁ, Janer JL. Sedimentation concentration method in Schistosomiasis 1997;2:392-7. mansoni. Puerto Rico J Public Health. 1934;9:283-98. 18. Ritchie LS, Lin S, Moon AP, Frick LP, Williams JE, Asakura S, et al. The possible effects 9. Idris NS, Dwipoerwantoro PG, Kurniawan A, Said M. Intestinal parasitic infection of of pH and specific gravity on the ether-sedimentation procedure in concentrating eggs immunocompromised children with diarrhea: clinical profile and therapeutic response. and cysts. Am J Trop Med Hyg. 1960;9:444-9. J Infect Dev Ctries. 2010;4:309-17. 19. Stenzel DJ, Boreham PF. Blastocystis hominis revisited. Clin Microbiol Rev. 1996;9:563- 10. Jantermtor S, Pinlaor P, Sawadpanich K, Pinlaor S, Sangka A, Wilailuckana C, et al. 84. Subtype identification ofBlastocystis spp isolated from patients in major hospital in northeastern Thailand. Parasitol Res. 2013;112:1781-6. 20. Tan KS, Singh M, Yap EH. Recent advances in Blastocystis hominis research: hot spots in terra incognita. Int J Parasitol. 2002;32:789-804. 11. Ludwig KM, Frei F, Álvares Filho F, Ribeiro-Paes JT. Correlação entre condições de saneamento básico e parasitoses intestinais na população de Assis, Estado de São 21. Tan TC, Ong SC, Suresh KG. Genetic variability of Blastocystis sp isolates obtained Paulo. Rev Soc Bras Med Trop. 1999;32:547-55. from cancer and HIV/AIDS patients. Parasitol Res. 2009;105:1283-6.

12. Miné JC, Rosa JA. Frequency of Blastocystis hominis and other intestinal parasites in 22. Velásquez V, Caldera R, Wong W, Cermeño G, Fuentes M, Blanco Y, et al. Elevada stool samples examined at the Parasitology Laboratory of School of Pharmaceutical prevalência de blastocistose em pacientes do Centro de Saúde de Soledad, Estado Sciences at the São Paulo State University, Araraquara. Rev Soc Bras Med Trop. Anzoategui, Venezuela. Rev Soc Bras Med Trop. 2005;38:356-7. 2008;41:565-9. Received: 15 April 2014 Accepted: 26 August 2014

214 Rev. Inst. Med. Trop. Sao Paulo 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 Reports of human infection in Brazil are scarce. The first report of human infection in Brazil was evidenced in Pará, in 197016. In the Saint Louis encephalitis virus (SLEV) is a recognized human 1990’s, detection of anti-SLEV antibodies, including seroconversion, was pathogen classified in the Japanese encephalitis virus complex, Flavivirus reported in residents of an ecological reserve in Vale do Ribeira, SP19. genus, Flaviviridae family, circulating in the Americas. SLEV is an A few human cases have been reported more recently: in a woman from arbovirus maintained by zoonotic cycles involving Culex (Cx.) spp. the city of São Paulo, SP, 200418 and in 20 patients from São José do Rio and other mosquitoes as vectors; birds as amplifiers; humans and other Preto (SP), two years later12,13,23. One case was identified in a suspected animals as accidental final hosts7. Most human infections are subclinical. dengue fever patient from Ribeirão Preto (SP) in 20149. SLEV infection Some are unspecific acute febrile infections rarely accompanied by might not be rare in humans, but instead is often mistaken with dengue meningoencephalitis with increased severity and fatality in the elderly24. virus (DENV) and goes largely undiagnosed in Brazil.

SLEV is widely dispersed throughout the New World, from Canada Serological studies to estimate SLEV prevalence in the population of to Argentina. However, clinical infection has become more frequent in Central Brazil are limited in number. Often, antigenic similarity between the United States of America (USA) and, to a lesser extent, in Central and DENV, SLEV and other flaviviruses compromises seroprevalence studies South America25. The virus was first reported during a human encephalitis due to cross-reactions. A few reports indicate 5% seroprevalence in outbreak in Saint Louis, USA, in 193326. SLEV was first identified in northern and southeastern Brazil20. Seroprevalence studies with SLEV Brazil in the 1960’s in Sabethes belisarioi pools from the state of Pará demonstrate that prevalence ranges from 3-43% within the Brazilian (PA), in the northern region of the country16,24. Later, between 1967 and population9. Acute clinical infections are rarely reported in Brazil, 1969, it was detected in sentinel mice, sylvatic rodents and birds in the possibly because humans are accidental or final hosts, and infections state of São Paulo (SP)8. 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. The minimum infection rate (MIR) was calculated with the formula Despite these difficulties, molecular approaches are important tools for ([number of positive pools / total specimens tested] x 1000), considering population screening and the monitoring of flaviviruses12. the total of Culex quinquefasciatus specimens tested (3,433 mosquitoes). SLEV-positive samples were subjected to inoculation in C6/36 cells16. Among domestic animals, serologic evidence of SLEV circulation in horses has been reported in different states across Brazil, including Nucleotide and amino acid sequence analysis of an envelope gene Minas Gerais (MG), Rio de Janeiro (RJ), Mato Grosso do Sul (MS), region from SLEV: A region of the SLEV envelope gene (477 bp) was Paraíba (PB), São Paulo (SP) and Pará (PA)15,17,20,21. Infected horses amplified in positive samples via semi-nested RT - PCR and sequenced rarely develop clinical symptoms. However, one fatal neurological case for phylogenetic analysis6. A phylogenetic tree was constructed with the of SLEV in a horse was recently described in MG20. Detection of SLEV neighbor-joining method, based on the Tamura-Nei distance model and in birds and serological evidence in horses from the state of Mato Grosso 1,000 bootstrap replicates (Geneious R7 7.1.7, USA) using reference (MT) has also been reported14,18. Concerning vector species, SLEV is SLEV sequences from the GenBank database (PubMed, NCBI, USA). frequently identified in arthropods in the Amazon18 and other regions Deduced amino acid sequences were also analyzed (Geneious R7 version of the country. In 1993, SLEV was identified in Anopheles triannulatus 7.1.7; Molecular Evolutionary Genetics Analysis version 5.05, USA), and Culex spp. in northwestern São Paulo19. Usually, mosquito species including residues present at specific positions characteristic of SLEV serving as SLEV vectors vary according to geographical region. Cx. lineages10. pipiens, Cx. quinquefasciatus and Cx. negripalpus are the most frequently involved in SLEV transmission in the Americas. The virus persists in Nucleotide sequences obtained in this study were deposited Culex spp. and these mosquitoes have been thought of responsible for at GenBank, pubMed (accession numbers: KJ699354; KJ957827; SLEV maintenance between seasons10. KJ847419; KJ801827).

DENV is the most common flavivirus worldwide, representing an RESULTS important health public problem. Reports of dengue fever outbreaks are frequent in Brazil. During epidemics, laboratory diagnosis of non- Clinical and epidemiological findings: During this transversal specific acute febrile illnesses has been directed towards this flavivirus observational study, three patients from the metropolitan area of Cuiabá infection, hindering the detection of other arboviruses. The aim of this who tested positive for DENV without neurological or hemorrhagic study was to investigate, using molecular approaches, other flaviviruses manifestations were also positive for SLEV. The first patient, a 47-year- possibly circulating in MT. old woman working in general services, sought medical care at a local hospital in May, 2012 with hyperthermia and posterior neck pain. She MATERIALS AND METHODS 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. Human and arthropod sampling: After receiving approval from the institutional Ethics Committee (CEP/HUJM/100/2011), serum samples The second patient, a 55-year-old male civil engineer, was treated at and epidemiological data were obtained from 604 patients in 20 cities the same hospital on the same day for hyperthermia, headache, emesis across MT who sought medical care between October, 2011 and July, and epigastric pain. The third patient was a 10-year-old male school pupil 2012 for acute febrile illnesses lasting less than five days. Also, 3,433 who sought medical care in the city of Várzea Grande in March 2012. female Culex spp. were captured with Nasci aspirators from 184 censitary Though clinical information was not available in his case, both patients sectors of Cuiabá between January and May, 2013, identified using GPS were also positive for DENV-4 by RT-PCR. locators. Three places were sampled at each sector. These Culicidae were identified according to dichotomy keys3,4 and a nested-PCR for Culex All patients were urban residents without history of traveling or visits quinquefasciatus22 and allocated to 409 pools of between one and ten to sylvatic or rural areas. None of the patients reported previous cases of mosquitoes; 403 with 3,425 Cx. quinquefasciatus, five with seven Cx. a similar disease. Attempts to recover SLEV from the co-infected serum bidens or Cx. interfor and one with one female of Culex spinosus. were unsuccessful.

Flaviviruses detection: Viral RNA from patient serum and total One pool containing a single Cx. quinquefasciatus female was RNA from mosquito pools were extracted according to manufacturers’ positive for SLEV in the metropolitan area of Cuiabá, with a MIR of instructions (QIAamp Viral RNA Mini Kit, Qiagen and Trizol, Invitrogen, 0.29 per 1000 specimens of this species. respectively). Extracted RNA was subject to a multiplex semi-nested reverse transcription PCR (RT-PCR) for a nucleotide region of flaviviruses The vast majority of the patients included in this study were positive NS5 gene (958 bp), followed by a species-specific secondary reaction for DENV serotypes (331/604 patients, 54.8%) and were admitted during differentiating 11 flaviviruses, as previously described1. Flavivirus- an epidemic coinciding with the introduction of the DENV-4 serotype positive samples were confirmed by at least two independent single in MT. The hyperendemicity of the four serotypes in Cuiabá, MT, nine reactions with the same forward and species-specific reverse primer. PCR co-infections between DENV-1/DENV-4 and one between DENV-2/ products were then submitted to nucleotide sequencing (3500 Genetic DENV-4 are going to be discussed separately. Analyzer, Applied Biossystems, USA). RNA from the SLEV strain genotype V-B BeH 355964 and no template were included as controls Analysis of a partial sequence of the envelope gene: Phylogenetic in all the reactions. Nucleotide sequences obtained from the positive analysis of an envelope glycoprotein gene region with 477 bp showed that control were analyzed to exclude contamination. SLEV identified in a female Cx. quinquefasciatus mosquito (SLEV_BR/

216 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.

MT-CbaAr499/2013) and in human (SLEV_BR/MT-CbaH364/2012) be circulating in MT. Although under-notification is common, data samples in the present study belong to genotype V-A. from the Notifiable Diseases Information System in 2012 included 44,814 notifications of dengue fever cases in MT, 10,742 of them in Envelope amino acid sequences from SLEV genotypes II, V and VIII Cuiabá and 3,133 in Várzea Grande, attributed to DENV-4 (96.2 %) retrieved from GenBank were compared with the human SLEV_BR/ and DENV-1 (3.8 %)2,11. 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).

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 co- circulating 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 DENV- 4 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, Fig 1 - Distribution of patients with acute febrile illness in the state of Mato Grosso between accompanied by hemorrhagic manifestations without increased severity, 2011 and 2012 tested for flaviviruses species by multiplex semi-nested RT-PCR. Cities with was previously reported during a dengue fever epidemic in the city of patients who tested positive for dengue virus (DENV) serotypes 1 and 4 and Saint Louis São José do Rio Preto, SP, southeastern Brazil12. encephalitis virus (SLEV) are identified.

SLEV infection may be underestimated in MT. Only patients with SLEV is currently classified by eight lineages, 15 subtypes, based acute febrile illness for less than five days were included in the present on envelope gene or genome sequences. These lineages correlate with study. Further studies to estimate seroprevalence in the population, as the geographical distribution of the virus. The Brazilian strains reported well as to identify the vector species transmitting the virus, are a matter so far belong to SLEV genotypes II, III, V, and VIII (subtypes A and to be addressed shortly. B), with V and VIII being the most prevalent in the Amazon region18.

One Cx. quinquefasciatus female captured in the Bela Vista Phylogenetic analysis shows that SLEV identified in the present neighborhood of Cuiabá was positive for SLEV (MIR = 0.29). Aedes and study within humans (SLEV_BR/MT-CbaH364/2012) and mosquitoes Culex spp. are involved in DENV and SLEV transmission, respectively24. (SLEV_BR/MT-CbaAr499/2013) belong to genotype V-A, closely related The co-infections described here likely resulted from exposure to both to isolates from animals in the Amazon region, in the state of Pará (Fig. infected mosquito species. However, several other mosquito species have 2). Previously, genotype VIII-B was isolated from birds in the Amazon been described as SLEV vectors. in MT in 197418.

Although SLEV has been detected in both urban and sylvatic The phylogenetic tree, constructed with strains belonging to the environments of Brazil and that Amazônia and Pantanal constitute the eight lineages of SLEV, demonstrates a common ancestry between majority of territory in Mato Grosso, the three human cases reported the human SLEV_BR/MT-CbaH364/2012, mosquito SLEV_BR/MT- here were detected within a large metropolitan area, located in the CbaAr499/2013 and SLEV isolates from PA and Argentina belonging to Cerrado biome (Fig 1). These cases were reported during a large genotype V. The MT SLEV samples clustered with a bootstrap value of DENV-4 outbreak, indicating that flaviviruses besides DENV may 98%, originating a clade in genotype V. They also indicated a homology

217 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.

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 Central and South American strains and some North American isolates be circulating in vectors and hosts. Although in the present study (Fig. from California and West Texas10. Genotype V-A, dispersed throughout 2), the cluster within lineage V is not supported by a high bootstrap value the Americas, was already reported in Brazil, Argentina, Peru and (65.9%), similar results were described by others when analyzing partial Trinidad & Tobago in vertebrate hosts and arthropod vectors. In Brazil, envelope sequences of SLEV strains belonging to the same lineage10,23. this genotype has been detected in different cities of PA and the state The distance between the isolates in the phylogenetic tree belonging of Rondônia. Genotype V-B was only reported in PA18. Genotype VIII to lineage V ranged from 0.008% between the two samples from MT, subtypes A and B are frequent in the Brazilian Amazon and, genotype to 0.060% among the BeAn288398 and BeAn203235 strains, from VIII-B was already isolated from Amazon region birds of MT in 1974 genotypes VA and VB, respectively. and from one horse with a neurological disease from MG in 201318,20.

The SLEV samples circulating in MT demonstrated a greater The analysis of the envelope amino acid sequences revealed a high similarity to the BRA PA BeAn259507 isolate. This isolate belongs to homology between the human SLEV_BR/MT-CbaH364/2012 and genotype V-A, obtained from domestic birds in Altamira, PA. SLEV the mosquito SLEV_BR/MT-CbaAr499/2013. None of the variations strains obtained from animals in Belém (BR PA An BeAn248398, BR described for other lineages in positions that were present in the PA An BeAn246407 and BRA PA An BeAn259507) and from Culex analyzed envelope amino acid partial sequence were observed in the MT spp. in Argentina (ARG Ar 78v6507) are allocated in the same branch samples10. The leucine residue at position 96 in the human SLEV_BR/ as they belong to the same genotype. MT-CbaH364/2012, whereas all the other studied amino acid sequences from genotypes II, V and VII presented a proline residue, has already Although SLEV circulates between arthropod vectors, mammalian been described in the human SLEV sample from São José do Rio and avian hosts, isolates of the virus generally do not exhibit a high Preto, SP23. Additionally, the SLEV_BR/MT-CbaAr499/2013 obtained level of genetic diversity; indeed, the most diverse isolates have a 10.1% from mosquitoes showed an asparagine at position 45, whereas all the nucleotide divergence and, strains within each lineage show less than other SLEV strains analyzed in the present study, including the human 5.5% nucleotide divergence10. In this regard, the sequences included SLEV_BR/MT-CbaH364/2012, have a lysine residue at the same position in the study from genotypes VA and VB indicate nucleotide similarity (Fig. 3). The homology between the human and arthropod SLEV samples, between 96.1 and 99%. identified in MT, indicates that the same virus is perhaps circulating in both vector and vertebrate host populations. The most prevalent genotypes in Brazil are V and VIII, existing throughout the Amazon basin, mainly in PA18. Lineage V is composed of Birds are believed to carry SLEV to different regions, and may be

218 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.

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/MT- CbaH364/2012) 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 estado de Mato Grosso (MT), Centro-Oeste do Brasil, entre 2011- from the Amazon to Pantanal. Culex mosquitoes are abundant in MT, 2012. Concomitantemente, 3.433 fêmeas de Culex spp. capturadas and most likely maintain viral transmission between birds as well as com aspirador de Nasci na cidade de Cuiabá, MT e alocadas em 409 sporadic transmission to horses and humans. pools com 1-10 mosquitos em 2013 foram testadas por multiplex semi- nested RT-PCR para os mesmos flavivírus. O SLEV foi detectado em SLEV infections in humans may occur sporadically in MT and três pacientes co-infectados com o DENV-4 das cidades de Cuiabá e be more frequent than observed in this study, conducted only with Várzea Grande, MT. Um dos pacientes apresentava tripla co-infecção patients who sought medical care during a dengue outbreak. The com DENV-1. Nenhum paciente referiu histórico recente de viagem absence of routine differential diagnosis may contribute to the lack ou acesso a áreas rurais/silvestres. Um pool contendo uma fêmea de of previous reports. Therefore, these findings indicate the necessity Culex quinquefasciatus foi positivo para o SLEV, apresentando taxa de for broad-spectrum clinical-epidemiological investigations during infecção mínima (MIR) de 0,29 por 1000 espécimes desta espécie. A dengue outbreaks. Active surveillance of arboviral circulation should análise filogenética indica que ambas as amostras formam um cluster be routinely performed in MT in the imminence of introduction com isolados do genótipo V-A do SLEV obtidos de animais na região or reintroduction of these viruses. Additional studies involving amazônica do estado do Pará. Este é o primeiro relato de identificação other animal species, birds and vector mosquitoes are necessary molecular do SLEV no MT. to comprehend the epidemiological cycle and magnitude of SLEV circulation in MT. ACKNOWLEDGMENTS

RESUMO The authors thank Ana E. Viniski, Sumako U. Kinoshita (LACEN/ MT, SES, Cuiabá), Liliana V. A. Correa, (FM, UFMT, Cuiabá) for Vírus da encefalite de Saint Louis em Mato Grosso, Centro-Oeste, their assistance. In addition, they thank Fernanda C. Pereira, a medical Brasil graduate student, for her scientific training, Roberta V. M. Bronzoni (UFMT Sinop) for providing RNA of the SLEV positive control and O vírus da dengue (DENV), frequentemente envolvido em Mauricio L. Nogueira (FAMERP) for training and discussion of the epidemias de grande proporção, e o vírus da febre amarela (YFV), results. responsável por surtos silvestres esporádicos, são considerados os flavivírus circulantes mais importantes no Brasil. Por este motivo, o FINANCIAL SUPPORT diagnóstico laboratorial de doença febril aguda indiferenciada durante períodos epidêmicos é frequentemente direcionado para dengue e This study was supported by the National Council for Scientific and febre amarela no país, dificultando a detecção de outros arbovírus Technological Development (CNPq; grant 472890/2011-5). NZ, LBSH, possivelmente circulantes, incluindo o vírus da encefalite de Saint OPS and BFC were recipients of the Coordination for the Improvement Louis (SLEV), que é amplamente disperso nas Américas. O objetivo of Higher Education Personnel (CAPES) scholarships; FCP and BHFG deste estudo foi investigar molecularmente a presença de 11 flavivírus were recipients of the UFMT scientific initiation scholarships. no soro de 604 pacientes durante grande epidemia de dengue no

219 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.

REFERENCES 16. Pinheiro FP, LeDuc JW, Travassos da Rosa AP, Leite OF. Isolation of St. Louis encephalitis virus from a patient in Belém, Brazil. Am J Trop Med Hyg. 1981;30:145- 1. Bronzoni RVDM, Baleotti FG, Nogueira MRR, Nunes M, Figueiredo LTM. 8. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 17. Rodrigues SG, Oliva OP, Araujo FAA, Martins LC, Chiang JD, Henriques DF, et 2005;43:696-702. al. Epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon region and in the State of Mato Grosso do Sul, Brazil: elevated prevalence of antibodies in 2. Capelassi C. Saúde divulga dados da Dengue de 2013 e o fechamento de 2012. horses. Rev Pan-Amazônica Saúde. 2010;1:81-6. [Internet]. Cuiabá (Brazil): Secretaria Estadual de Saúde do Mato Grosso. [cited 2013 Jan 16]. Available from: www.saude.mt.gov.br/noticia/3291 18. Rodrigues SG, Nunes MRT, Casseb SMM, Prazeres ASC, Rodrigues DSG, Silva MO, et al. Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian 3. Forattini OP. Culicidologia médica. São Paulo: EDUSP; 1996. v.1, p. 531. Amazon: genetic divergence and dispersal. J Gen Virol. 2010;91:2420-7.

4. Forattini OP. Culicidologia médica. São Paulo: EDUSP; 2002. v. 2, p.548. 19. Rocco IM, Santos CLS, Bisordi I, Petrella SMCN, Pereira LE, Souza RP, et al. St. Louis encephalitis virus: first isolation from a human in São Paulo State, Brazil. Rev 5. Fulop L, Barrett AD, Phillpotts R, Martin K, Leslie D, Titball RW. Rapid identification Inst Med Trop Sao Paulo. 2005;47:281-5. of flaviviruses based on conserved NS5 gene sequences. J Virol Methods. 1993;44:179-88. 20. Rosa R, Costa EA, Marques RE, Oliveira TS, Furtini R, Bomfim MRQ, et al. Isolation of Saint Louis encephalitis virus from a horse with neurological disease in Brazil. 6. Kramer LD, Chandler LJ. Phylogenetic analysis of the envelope gene of St. Louis PLOS Negl Trop Dis. 2013;7:e2537. encephalitis virus. Arch. Virol. 2001;146:2341-55. 21. Silva JR. Pesquisa de infecções por flavivírus da encefalite de Saint Louis, Rocio e 7. Kuhn R. Flaviviruses. In: Acheson NH, editor. Fundamentals of molecular Virology. Oeste do Nilo em cavalos, por inquérito sorológico e isolamento viral. [Dissertation]. New York: John Wiley & Sons; 2007. p. 181-90. São Paulo: Universidade de São Paulo; 2010.

8. Lopes OS, Sacchetta LA, Coimbra TL, Pereira LE. Isolation of St. Louis encephalitis 22. Smith JL, Fonseca DM. Rapid assays for identification of members of the Culex virus in South Brazil. Am J Trop Med Hyg. 1979;28:583-5. (Culex) pipiens complex, their hybrids and other sibling species (Diptera Culicidae). Am J Trop Med Hyg. 2004;70:339-45. 9. Maia FGM, Chávez JH, Souza WM, Romeiro MF, Castro Jorge LA, Fonseca BAL, et al. Infection with Saint Louis encephalitis virus in the city of Ribeirão Preto, Brazil: 23. Terzian ACB, Mondini A, Bronzoni RVM, Drumond BP, Ferro BP, Cabrera EMS, report of one case. Int J Infect Dis. 2014;269:96-7. et al. Detection of Saint Louis encephalitis virus in dengue-suspected cases during a dengue 3 outbreak. Vector Borne Zoonotic Dis. 2011;11:291-300. 10. May FJ, Li L, Zhang S, Guzman H, Beasley DWC, Tesh RB, et al. Genetic variation of St. Louis encephalitis virus. J Gen Virol. 2008;89:1901-10. 24. Vasconcelos PFC, Travassos da Rosa APA, Pinheiro FP, Shope RE. Arboviruses pathogenic for man in Brazil. In: Travassos da Rosa APA, Vasconcelos PFC, Travassos 11. Ministério da Saúde. Balanço Dengue I Janeiro a abril 2012. [Internet]. Brasilia: da Rosa JFS, editors. An overview of arbovirology in Brazil and neighbouring Ministry Health of Brazil; 2012. Available from: http://www.slideshare.net/MinSaude/ Countries. Belém: Instituto Evandro Chagas; 1998. p. 72-99. balano-dengue-i-jan-a-abr-2012 25. Vasconcelos PFC, Travassos da Rosa JFS, Travassos da Rosa APA, Degallier N. 12. Mondini A, Bronzoni RVDM, Cardeal ILS, Santos TMILS, Lázaro E, Nunes SHP, et Epidemiologia das encefalites por arbovírus na Amazônia Brasileira. Rev Inst Med al. Simultaneous infection by DENV-3 and SLEV in Brazil. J Clin Virol. 2007;40:84-6. Trop Sao Paulo. 1991;33:465-76.

13. Mondini A, Cardeal ILS, Lázaro E, Nunes SH, Moreira CC, Rahal P, et al. Saint 26. Webster LT, Fite GL. A virus encountered in the study of material from cases of encephalitis Louis encephalitis virus, Brazil. Emerg Infect Dis. 2007;13:176-8. in the St. Louis and Kansas city epidemics of 1933. Science. 1933;78(2019):463-5.

14. Ometto T, Durigon EL, de Araujo J, Aprelon R, de Aguiar DM, Cavalcante GT, et Received: 15 June 2014 al. West Nile virus surveillance, Brazil, 2008-2010. Trans R Soc Trop Med Hyg. Accepted: 26 August 2014 2013;107:723-30.

15. Pauvolid-Corrêa A, Tavares FN, Costa EV, Burlandy FM, Murta M, Pellegrin AO, et al. Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil. Mem Inst Oswaldo Cruz. 2010;105:829-33.

220 Rev. Inst. Med. Trop. Sao Paulo 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%) HIV- infected 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 resident bacterial pathogens8,26. The herpesviral-bacterial hypothesis of periodontitis development proposes that active herpesvirus infection Herpesviruses are large DNA-enveloped viruses belonging to initiates periodontal tissue breakdown and that host immune responses the Herpesviridae family. Herpesviruses are highly disseminated in against the herpesvirus infection are important components of the nature. Of more than 200 known, eight are human pathogens: herpes etiopathogeny of the disease28. The herpesvirus infection triggers the simplex virus 1 (HSV-1), herpes simplex 2 (HSV-2), varicella zoster release of proinflammatory cytokines, which have the potential to activate virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), osteoclasts and matrix metalloproteinases and to impair antibacterial and human herpesviruses 6, 7 and 8 (HHV-6, -7, -8)23. Transmission immune mechanisms, causing an upgrowth of periodontopathic bacteria28. occurs by contact, and primary infections generally occur early in life, followed by persistence of the virus in the organism. Herpesvirus diseases High frequencies of EBV and CMV genomes have been noted occur primarily in immunosuppressed individuals; fatal infections in in adults with progressive periodontitis, in localized and generalized immunocompetent hosts are rare23. aggressive (juvenile) periodontitis, HIV-associated periodontitis, acute necrotizing ulcerative gingivitis, periodontal abscesses, and some rare Several studies have implicated herpesviruses in the etiology of types of advanced periodontitis associated with medical disorders26. periodontitis26-29. Apparently, periodontal tissue breakdown occurs Other herpesviruses such as HHV-6, HHV-7, HHV-8, and HSV-1, have more frequently and progresses more rapidly in herpesvirus-infected also been associated with periodontitis4,15,19. In contrast, HSV-2 appears than in herpesvirus-free periodontal sites26-29. Herpesviruses may to be uncommon at periodontal sites7,32. However, the pathogenesis of cause periodontal pathosis as a direct result of virus infection and herpesviruses in periodontitis has not yet been fully elucidated. replication, or as a consequence of virally induced impairment of periodontal immune defenses, resulting in heightened virulence of 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, 21941- 902 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 periodontal probe. Gingivitis was considered to be present when gingival that highly active antiretroviral therapy (HAART) does not significantly bleeding occurred on probing1. The extension of gingivitis was classified reduce the prevalence or the load of HHVs in saliva11,17,22,32. The elevated according to the System for the Classification of Periodontal Diseases frequency of HHVs infections in association with periodontitis in HIV- and Conditions2; patients with gingivitis at <30% of sites surveyed were infected individuals7,8,11,12 suggests that these viruses play a role in the classified as having localized gingivitis, and those with >30% of surveyed disease. In children, the prevalence of some oral manifestations associated sites affected were classified as having generalized gingivitis. with HIV infection was reduced after HAART initiation. However, other lesions emerged24, as these individuals are prone to develop opportunistic Individuals were not allowed to brush their teeth or eat for one h viral infections, especially those caused by Herpesviridae family before providing saliva samples. Five milliliters of paraffin-stimulated members, in the oral mucosa. Little information is available on HHVs saliva were collected in a sterile container. The samples were kept in co-infection in the saliva of HIV-infected children. an ice-filled cooler and submitted for laboratory analysis within two h.

The aims of this study were to detect HHVs in the saliva of HIV- Sample processing: The saliva samples were centrifuged and 1-infected children in comparison with healthy control children, and to pelleted, and DNA was extracted using the Wizard Genomic DNA evaluate possible associations between viral infection and gingivitis and purification kit (Promega, Madison, WI, USA) according to the immunodeficiency stage. manufacturer’s instructions.

MATERIAL AND METHODS Virus detection: All samples were subjected to human β-globin gene amplification to determine the integrity and quality of extracted Samples: The ethics committees of the Hospital Universitário DNA and to avoid false-negative results3. Specimens were analyzed Clementino Fraga Filho and Institute of Pediatrics and Childcare using conventional polymerase chain reaction (PCR) assays, as described Martagão Gesteira, Universidade Federal do Rio de Janeiro (UFRJ), previously, to detect the presence of HSV-1/2, VZV, EBV, and CMV30. Brazil, approved the study protocol. The parents of all children involved PCR products were detected using 1.2% agarose gel electrophoresis and in the study provided written informed consent in accordance with staining with ethidium bromide. Resolution 196/96 of the Brazilian Ministry of Health. First-round PCR reactions consisted of the addition of 5 µL of The study population was composed of patients attending the UFRJ extracted DNA to 20 µL of PCR mix containing 0.5 µM of each of School of Dentistry between August 2009 and July 2010. Participants the primers HHV-F1 and HHV-R1, 0.125 µM of each of the primers were selected by convenience sampling during initial appointments for VZV-F1 and VZV-R1, and 1x PCR buffer; 1.5 mM MgCl2 and 0.2 mM of dental treatment. The HIV-1-infected group was made up of 48 children deoxyribonucleotide triphosphates; and 2.5 U of GoTaq DNA polymerase of both sexes, ranging from six to 12 years old, who were patients at (Promega). First-round PCR was carried out as follows: one cycle at 94 the Institute of Pediatrics and Childcare Martagão Gesteira, UFRJ, with °C for three min, followed by 35 cycles at 94 °C for 45 s, 65.5 °C for one definitive diagnoses of HIV infection. The following medical history data min, 72 °C for one min, and final extension at 72 °C for seven min. For were extracted from their medical records: diagnosis of HIV infection, nested PCR, 0.5 µL of first-round product was transferred to 25 µL PCR results of most recent (closest to the day of saliva sample collection; mix similar to that described above, but containing second-round primers maximum interval, three months) laboratory tests (viral load, CD4 and (HHV-F2, HHV-R2, VZV-F2, and VZV-R2), at the same concentrations CD8 counts, and CD4/CD8 ratio) and use of anti-retroviral agents (at as in the first round. PCR conditions were the same as in the first round, the time of saliva sample collection). The immunodeficiency stages of except that the annealing temperature was changed to 63 °C. Positive and HIV-infected individuals were defined using CD4 counts, according to negative controls were included in each run. Infected cell cultures were the classification of the Centers for Disease Control and Prevention6. 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 The control group consisted of 48 healthy children, ranging from patients with varicella diagnoses confirmed by PCR amplification and seven to 12 years old, who attended the UFRJ Pediatric Dentistry sequencing analysis were used as positive controls. Negative controls Clinic and showed no clinical evidence of systemic or chronic disease. consisted of saliva samples previously demonstrated to be HHVs. The They were considered clinically healthy because they were receiving expected sizes of the PCR products for first-round and nested PCRs, no medical treatment for any disease and showed no clinical sign of respectively, were: HSV-1/2, 742 and 493 pb; VZV, 650 and 356 pb; immunosuppression, systemic disease, and/or had no history of a risk EBV, 748 and 499 pb; and CMV, 817 and 565 pb. factor for HIV infection. These data were collected through medical anamnesis with the patients’ parents and the attending physician. Children Because some PCR products had very similar sizes, sequencing in the control group did not undergo testing to confirm serological HIV analysis was used to confirm their specificity and to differentiate HSV-1 and negativity because there was no reason to justify this procedure, which HSV-2. Amplified DNA from all HSV-positive samples and three CMV- the local ethics committees therefore disallowed. positive samples was purified using the Wizard SV gel and PCR clean-up system kit (Promega), and sequences were determined using the BigDye Prior to saliva sample collection, all children in the HIV-1-infected terminator cycle sequencing kit and the ABI PRISM 3100 automated DNA and control groups underwent oral and oropharyngeal examinations sequencer (Applied Biosystems, Foster City, CA, USA) using the same by a trained and calibrated dentist to identify oral manifestations such PCR primers. DNA sequences were edited using the Chromas software as gum bleeding, mouth ulcers, oral mucosal lesions, and cervical (Technelysium Pty. Ltd., Brisbane, QLD, Australia) and compared with lymphadenopathy. The gingival index was assessed using a sterile the DNA sequences available in GenBank (http://www.ncbi.nlm.nih.gov)

222 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.

using the BLAST tool (http://www.ncbi.nlm.gov/BLAST). 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 Statistical analysis: Using the Epi-Info statistical program (version was more common in immunocompromised HIV-1-infected children. 3.5.1., CDC, Atlanta, GA, USA), data from the two groups were Sequence analysis confirmed the PCR results and allowed differentiation compared using the Mann-Whitney test for means of continuous variables between HSV-1 and HSV-2 strains. (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 Table 2 group, the χ2 and Fischer’s exact tests were used to verify correlations Herpesviruses detected in saliva from HIV-1–infected and healthy control between the presence of HVs in saliva and immunosuppression, HAART, children and the presence of oral manifestations. HIV-1-infected Control subjects RESULTS Virus1 subjects (%) (%) p N = 48 N = 48 The mean age of the 48 HIV-1-infected children was 9.58 years; HSV-1 3 (6.2) 3 (6.2) 1.00 45.8% of these subjects were male, 70.8% were receiving HAART, and 52.1% had no immunosuppression. The mean age of healthy control HSV-2 2 (4.2) 4 (8.3) 0.458 children (47.9% male) was 9.04 years. There groups did no differ in CMV 12 (25.0) 6 (12.5) 0.117 terms of age or sex (p > 0.05). Other clinical and medical data from the study subjects are shown in Table 1. Total 17 (35.4) 13 (27.0) 0.167 1EBV and VZV were not detected. Table 1 Demographic, clinical, and immunological characteristics of children in the HIV-1-infected individuals were classified into three immunologic HIV-1–infected and control groups categories: no evidence of suppression (CD4+ > 500 cells/µL; CD4+ % > 25), moderate suppression (CD4+ = 200-499 cells/µL; CD4+ % [15-24), HIV-1-infected and severe suppression (CD4+ < 200 cells/µL; CD4+ % <15)19 (Table 1). Variable Control subjects subjects Twelve of 25 (48.0%) children with no evidence of immunosuppression, 2/14 (14.3%) children with moderate immunosuppression, and 3/9 Age (years), mean 9.58 (6-12) 9.04 (7-12) (33.3%) children with severe immunosuppression were HHVs positive. (range) No correlation was found between HHV infection and the degree of Sex Male 22 (45.8%) Male 23 (47.9%) immunosuppression (p = 0.447). Female 26 (54.2%) Female 25 (52.1%) Eleven of 34 (32.4%) individuals undergoing HAART and 6/14 HAART Yes, 34 (70.8%) (42.8%) children not receiving HAART were HHVs positive. However, No, 14 (29.2%) no significant correlation between HHVs detection in saliva and receipt of HAART was observed (p = 0.489). <200; 9 (18.7%) CD4+ count (cells/µL) 200-499; 14 (29.2%) Thirty-five of 48 (72.9%) HIV-1-infected children had gingivitis >500; 25 (52.1%) 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 Gingivitis Yes, 35 (72.9%) Yes, 16 (33.3%) were HHVs positive. No significant correlation between the presence of No, 13(27.1%) No, 32 (66.7%) HHVs in saliva and the presence and extension of gingivitis was observed within each group, HIV-1-infected children (p = 0.104) and healthy Oral findings control children (p = 0.251), or when the HIV-1-infected group was Candidosis n = 4 -1 compared with the control group (p = 0.491). However, HIV infection Linear gingival n = 5 - was strongly correlated with gingivitis (p = 0.0001). erythema Four (8.3%) HIV-1-infected children had candidosis, five (10.4%) Angular cheilitis n = 1 - had linear gingival erythema (LGE), one (2.1%) had an oral ulcer, and Oral ulcer n = 1 - one (2.1%) had angular cheilitis (Table 1). One subject with candidosis HAART = highly active antiretroviral therapy; 1 -: absence of symptoms. and LGE and one subject with angular cheilitis were HSV-1 positive; one subject with LGE was CMV positive. HHV detection in saliva was Seventeen (35.4%) of the 48 HIV-1-infected children were positive not correlated with any oral symptom. 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 DISCUSSION (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 Herpesviruses, most commonly CMV, EBV, and HSV-1, have been VZV, EBV or co-infection with those viruses was detected in either group detected in oral samples from immunosuppressed and immunocompetent (Table 2). No significant difference was observed in HHVs detection in individuals with gingivitis7,9,11–13,15,16,18,25,33.

223 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.

Median CMV detection rates in healthy periodontium and in RESUMO individuals with gingivitis are about 8% and 33%, respectively27. CMV has been detected in 25-49% of immunocompetent individuals and Ausência de associação entre a detecção de herpesvírus na saliva e 40-62% of HIV-infected individuals with gingivitis11-13,16,33. Previous gengivite em crianças infectadas pelo HIV studies have not been able to demonstrate a clear association between the presence of CMV and gingivitis because the virus was detected at high Os objetivos deste estudo foram detectar a presença de herpesvírus frequencies in control groups. The present study found CMV in the saliva humanos (HHVs) na saliva de crianças infectadas pelo HIV, em of 25% of HIV-1-infected children and 12.5% of healthy control children. comparação com controles saudáveis e avaliar a associação entre Although CMV was detected more often in immunocompromised infecção viral, gengivite e imunodeficiência. Para este fim, foram colhidas children, CMV could not be clearly associated with gingivitis. amostras de saliva de 48 crianças HIV-positivas e 48 controles saudáveis. O índice gengival e extensão de gengivite foram determinados por um HSV-1 is detected less frequently than CMV and EBV in the saliva of dentista treinado. Informações clínicas e laboratoriais foram obtidas individuals with periodontitis12,13,25, but its detection has been described in durante a consulta odontológica e dos registros médicos. As amostras patients with gingivitis11,16. The present study found HSV-1 in the saliva de saliva foram testadas para detecção de vírus herpes simplex tipos 1 e of 6.2% (3/48) of subjects from both groups. 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 HSV-2 is rarely detected in saliva7,20,27,31,32, but it was detected in crianças HIV-positivas e 16 crianças do grupo controle apresentavam both groups in the present study. Two HIV-1-infected boys aged 10 gengivite. Dezessete (35,4%) crianças HIV-positivas e 13 (27%) crianças and 11 years were HSV-2 positive; one of these subjects, a severely controle testaram positivo para a presença de HHVs. CMV foi o vírus immunosuppressed (CD4+ count = 149 cells/µL [11.27%]) boy who mais comum detectado em ambos os grupos (25% HIV-positivas e 12,5% was not undergoing HAART, had gingivitis. The other HIV-1-infected, de controle), seguido por HSV-1 (6,2% de ambos os grupos) e HSV-2 HSV-2-positive child had no evidence of immunosuppression (CD4+ (4,2% HIV-positivas e 8,3% de controle). Não houve associação entre count = 724 cells/µL [29%]) and was receiving HAART. Four children a detecção de HHVs na saliva e a presença de gengivite em ciranças (aged 7-9 years) in the control group were HSV-2 positive; three of them HIV-positivas (p = 0.104) ou crianças saudáveis (p = 0,251), ou com had gingivitis. imunossupressão em indivíduos HIV-positivos (p = 0,447). Foi observada uma correlação entre a infecção por HIV e a presença de gengivite (p A recent review of HHVs in periodontitis showed that EBV is = 0,0001). Os resultados sugerem que a detecção salivar assintomática detected in association with gingivitis in 20% of cases and with healthy de HHVs é comum entre crianças HIV-positivas e crianças saudáveis, e periodontium in 8% of cases27. Several studies have described EBV não está associada à gengivite. 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 ACKNOWLEDGMENTS present study did not detect EBV in HIV-1-infected or healthy children with or without gingivitis. This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Few studies have reported VZV excretion in saliva. Such excretion Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação is usually observed in stressed individuals20 or those with herpes zoster Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro manifestations10,21. WANG et al.32 detected VZV DNA in the saliva of (FAPERJ), Brazil. 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 The authors thank Soluza dos Santos Gonçalves for technical concluded that such an event is infrequent in the saliva of asymptomatic assistance. HIV-positive persons and that HAART does not reduce the risk of asymptomatic VZV excretion32. Accordingly, VZV has not been CONFLICT OF INTEREST associated with periodontal disease27. Consistent with these findings, the present study did not detect VZV in its study population. The authors declare that they have no conflict of interest.

In this study, HHVs detection in the saliva of HIV-1-infected and AUTHORS’ CONTRIBUTIONS healthy children with and without gingivitis was compared. Although sample size potentially limits the statistical power of the results, the GFC, IPRS, and NS conceived and designed the study and analyzed study’s findings are comparable to those reported in the literature. the data. RAO, FNNN, RCS, and RSL were responsible for data and CMV was the most prevalent virus detected in both groups, followed sample collection and PCR analysis. All authors have read and approved by HSV-1 and HSV-2. EBV and VZV were not detected in either group. the final manuscript. No association was demonstrated between HHV detection in saliva and the presence of gingivitis. No association between the detection of HV REFERENCES DNA in saliva and the level of immunosuppression in HIV-1-infected children was observed. Moreover, HAART did not seem to reduce virus 1. Ainamo J, Bay I. Problems and proposals for recording gingivitis and plaque. Int Dent shedding. However, a strong correlation between HIV infection and J. 1975;25:229-35. gingivitis was confirmed. 2. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol. 1999;4:1-6.

224 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.

3. Bauer HM, Manos MM. PCR detection of genital human papillomavirus. Diagnostic 18. Lucht E, Brytting M, Bjerregaard L, Julander I, Linde A. Shedding of cytomegalovirus and molecular microbiology: principals and applications. Washington: American Society herpesviruses 6, 7, and 8 in saliva of human immunodeficiency virus type 1-infected for Microbiology; 1993. p. 407-13. patients and healthy controls. Clin Infect Dis 1998;27:137-41.

4. Bilichodmath S, Mangalekar SB, Sharma DC, Prabhakar AK, Reddy SB, Kalburgi NB, 19. Mardirossian A, Contreras A, Navazesh M, Nowzari H, Slots J. Herpesviruses 6, 7 and et al. Herpesviruses in chronic and aggressive periodontitis patients in an Indian 8 in HIV- and non-HIV-associated periodontitis. J Periodontal Res. 2000;35278-84. population. J Oral Sci. 2009;51:79-86. 20. Mehta SK, Cohrs RJ, Forghani B, Zerbe G, Gilden DH, Pierson DL. Stress-induced 5. Carvalho KS, Silvestre EA, Maciel SS, Lira HI, Galvão RA, Soares MJ, et al. PCR subclinical reactivation of varicella zoster virus in astronauts. J Med Virol. detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals 2004;72:174-9. in Teresina, State of Piauí, Brazil. Rev Soc Bras Med Trop. 2010;43:620-3. 21. Mehta SK, Tyring SK, Gilden DH, Cohrs RJ, Leal MJ, Castro VA, et al. Varicella-zoster 6. Centers for Disease Control and Prevention (CDC). Revised classification system for virus in the saliva of patients with herpes zoster. J Infect Dis. 2008;197:654-7. human immunodeficiency virus infection in children less than 13 years of age. MMWR Recomm Rep. 1994;43(RR-12):1-19. 22. Miller CS, Berger JR, Mootoor Y, Avdiushko SA, Zhu H, Kryscio RJ. High prevalence of multiple human herpesviruses in saliva from human immunodeficiency virus- 7. Contreras A, Mardirossian A, Slots J. Herpesviruses in HIV-periodontitis. J Clin infected persons in the era of highly active antiretroviral therapy. J Clin Microbiol. Periodontol. 2001;28:96-102. 2006;44:2409-15.

8. Contreras A, Slots J. Herpesviruses in human periodontal disease. J Periodontal Res. 23. Pellett PE, Roizman B. The family Herpesviridae: a brief introduction. In: Fields’virology. 2000;35:3-16. 5th ed. Philadelphia: Lippincott-Raven; 2007. v. 2. p. 2479-99.

9. Di Luca D, Mirandola P, Ravaioli T, Dolcetti R, Frigatti A, Bovenzi P, et al. Human 24. Pinheiro RS, Ferreira DC, Nóbrega F, Santos NSO, Souza IPR, Castro GFBA. Current herpesviruses 6 and 7 in salivary glands and shedding in saliva of healthy and human status of herpes virus identification in the oral cavity of HIV-infected children. Rev immunodeficiency virus positive individuals. J Med Virol. 1995;45:462-8. Soc Bras Med Trop. 2013;46:15-9.

10. Druce J, Catton M, Chibo D, Minerds K, Tyssen D, Kostecki R, et al. Utility of a multiplex 25. Saygun I, Kubar A, Sahin S, Sener K, Slots J. Quantitative analysis of association between PCR assay for detecting herpesvirus DNA in clinical samples. J Clin Microbiol. herpesviruses and bacterial pathogens in periodontitis. J Periodontal Res. 2008;43: 2002;40:1728-32. 352-9.

11. Grande SR, Imbronito AV, Okuda OS, Lotufo RF, Magalhães MH, Nunes FD. Herpes 26. Slots J. Herpesviruses in periodontal diseases. Periodontol 2000. 2005;38:33-62. viruses in periodontal compromised sites: comparison between HIV-positive and -negative patients. J Clin Periodontol. 2008;35:838-45. 27. Slots J. Human viruses in periodontitis. Periodontol 2000. 2010a;53:89-110.

12. Grande SR, Imbronito AV, Okuda OS, Pannuti CM, Nunes FD, Lima LA. Relationship 28. Slots J. Herpesviral-bacterial interactions in periodontal diseases. Periodontol 2000. between herpesviruses and periodontopathogens in patients with HIV and 2010b;52:117-40. periodontitis. J Periodontol. 2011;82:1442-52. 29. Slots J. Oral viral infections of adults. Periodontol 2000. 2009;49:60-86. 13. Grenier G, Gagnon G, Grenier D. Detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment. 30. Tafreshi NK, Sadeghizadeh M, Amini-Bavil-Olyaee S, Ahadi AM, Jahanzad I, Oral Microbiol Immunol. 2009;24:506-9. Roostaee MH. Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections. J Clin Virol. 14. Guilherme BP, Ferreira DC, Rôças IN, Provenzano JC, Santos KR, Siqueira JF Jr. 2005;32:318-24. Herpesvirus carriage in saliva and posttreatment apical periodontitis: searching for association. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112:678-83. 31. Wald A, Ericsson M, Krantz E, Selke S, Corey L. Oral shedding of herpes simplex virus type 2. Sex Transm Infect. 2004;8:272-6. 15. Hernádi K, Csoma E, Adám B, Szalmás A, Gyöngÿosi E, Veress G, et al. Association of human herpesvirus 6 subtypes with symptomatic apical periodontitis. Oral Surg 32. Wang CC, Yepes LC, Danaher RJ, Berger JR, Mootoor Y, Kryscio RJ, et al. Low prevalence Oral Med Oral Pathol Oral Radiol Endod. 2011;112:401-6. of varicella zoster virus and herpes simplex virus type 2 in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral 16. Imbronito AV, Okuda OS, Maria de Freitas N, Moreira Lotufo RF, Nunes FD. Detection therapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;109:232-7. of herpesviruses and periodontal pathogens in subgingival plaque of patients with chronic periodontitis, generalized aggressive periodontitis, or gingivitis. J Periodontol. 33. Wu YM, Yan J, Ojcius DM, Chen LL, Gu ZY, Pan JP. Correlation between infections with 2008;79:2313-21. different genotypes of human cytomegalovirus and Epstein-Barr virus in subgingival samples and periodontal status of patients. J Clin Microbiol. 2007;45:3665-70. 17. Ling PD, Vilchez RA, Keitel WA, Poston DG, Peng RS, White ZS, et al. Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients Received: 30 July 2014 receiving highly active antiretroviral therapy. Clin Infect Dis. 2003;37:1244-9. Accepted: 19 August 2014

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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 in 1962 by MACIEL16. The author compiled his own data with data from literature, as well as from the former Department of Rural Mosquitoes (Diptera: Culicidae) are a group of insects that in their Endemic Diseases. With this, he created a list of the Culicidae in Minas early stages develop in a variety of aquatic habitats, including permanent Gerais and the municipalities where they were found, as well as the (i.e. rivers and lakes) and transient; transient habitats can include any coordinates of the collection sites. Overall, 119 species of Culicidae receptacle that accumulates water, such as hollow trees, bromeliad tanks, were reported as occurring in 168 municipalities. The upper-middle fallen plant material, and even animal tracks21. 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 Studies of Culicidae diversity in Brazil were mainly focused on river, which contains two surveys done in the city of Manga in 1947 and rainforests in the southeastern and southern regions of the country, 1954. Thereafter, the only published work in northern Minas Gerais was which coincide with the location of major national research centers. The by GAMA et al.13, in which the authors present a list of Anophelines Amazon rainforest is another important, well-studied region, primarily collected in the municipality of Varzelândia. because of its significance for the transmission of several diseases, such as malaria and wild-type yellow fever7. However, the authors remain The present study aims to conduct an inventory of the Culicid fauna unsure of the diversity of mosquitoes in Brazilian regions with different in conservation units within a semi-arid region of the state of Minas climatic characteristics and forms of vegetation. Gerais, Brazil, in order to better understand the diversity of Culicidae in this region. Despite the high diversity of plant and animal species in other biomes, such as the Cerrado (Savanna) and Caatinga (Semi-arid forest), MATERIALS AND METHODS there are very few studies of Culicidae diversity in these areas, and in particular, few in the transition zones between these biomes in northern Samples were collected within four conservation units administered Minas Gerais (MG). This region is primarily tropical dry forest (TDF), by the State Forestry Institute (Instituto Estadual de Florestas - IEF). characterized by deciduous forest vegetation and a semi-arid climate, These areas are in the northern region of Minas Gerais, in the mid-São due to low humidity and low rainfall. Francisco Valley, and are as follows: (1) the Mata Seca State Park MSSP (Parque Estadual da Mata Seca - PEMS) (14o48’36’’S - 43o55’12’’), The last major survey of Culicidae in Minas Gerais was conducted 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 trapping utilized two sampling methods, both beginning at dusk: one - 43o56’23’’W) and (3) the Jaíba Biological Reserve - JBR (Reserva Shannon-type light trap exposed for a period of two hours and two CDC Biológica de Jaíba) (15º3’57.81”S - 43º45’45.03”W), both located in light traps exposed for a period of 12 hours per plot. A third sampling the municipality of Matias Cardoso; and (4) the Serra Azul Biological method consisted of “active collections” used to sample mosquitoes Reserve - SABR (Reserva Biológica de Serra Azul) (15º11’32.20”S - with daytime activity, and was performed once at each sample point 43º54’41.1”W), located in the municipality of Jaíba (Fig. 1). for 45 minutes. Briefly, active collections consisted of using a manual vacuum to collect all mosquitoes landing on researcher’s bodies prior As the study areas are located within a Caatinga-Cerrado transition to the attempted blood meal. Transportation and mounting techniques zone, they contain fragments of tropical dry forest (TDF). These for mosquitoes were based on previous reports by FORATTINI10 and formations are broadly defined as having a vegetation type typically CONSOLI & OLIVEIRA6. Specimens were taxonomically identified dominated by deciduous trees (at least 50% deciduousness during the and incorporated into the entomological collection of the Laboratory of dry season), with an average annual temperature ≥ 25 °C, total annual Ecology and Biological Control of Insects (Laboratório de Ecologia e precipitation between 700 and 2,000 mm, and three or more dry months Controle Biológico de Insetos - LECBI) at Montes Claros State University per year (precipitation < 100 mm/month22). According to the Köppen (Universidade Estadual de Montes Claros - Unimontes). Species classification, regions with TDFs have a seasonal tropical climate (Aw) identification was carried out using dichotomous keys by CONSOLI & with an average annual temperature of 24.4 ºC and an average annual OLIVEIRA6, FARAN9, FORATTINI10 and LANE15. precipitation of 871 mm2. 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 During the study period, a total of 11,219 mosquitoes were collected between August 2008 and July 2012, on a total of 18 nights and across (11 genera and 45 species). There were 15 new records for Minas Gerais 504 hours of collections in the dry seasons, with the same sampling overall, and 26 new records for the semi-arid region of Minas Gerais effort taking place in the wet seasons during the study period. Night (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 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.

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

MSSP LCSP JBR SABR SPECIES Total Dry Wet Dry Wet Dry Wet Dry Wet Anophelinae Anopheles (Nys.) albitarsis Lynch Arribalzaga, 1878 19 4 17 9 0 1 0 0 50 An. (Nys.) argyritarsis Robineau-Desvoidy, 1827 23 37 79 13 0 1 0 0 153 An. (Nys.) braziliensis (Chagas, 1907) 0 2 0 1 0 0 0 0 3 An. (Nys.) darlingi Root, 1926 53 76 8 16 0 0 0 0 153 An. (Nys.) deaneorum Rosa-Freitas, 1989 + 0 0 4 2 0 0 0 0 6 An. (Nys.) evansae (Brethes, 1926) + 0 0 0 1 0 0 0 1 2 An. (Nys.) triannulatus triannulatus (Neiva & Pinto, 1922) 33 27 2 3 0 0 0 0 65 An. (Nys.) Albimanus section/Oswaldoi Subgroup 4 5 0 1 2 0 0 0 12 Culicinae Tribe Aedomyiini Aedeomyia (Ady.) squamipennis (Lynch Arribalzaga, 1878)+ 12 31 4 4 0 0 0 0 51 Tribe Aedini Aedes (How.) fulvithorax (Lutz, 1904)+ 0 0 0 1 0 0 0 0 1 Ae. (Och.) fulvus (Wiedemann, 1828) 0 2 0 13 0 0 0 1 16 Ae. (Och.) hastatus Dyar 1922*+ 0 1 0 3 0 0 0 0 4 Ae. (Och.) scapularis (Rondani 1848) 25 393 6 526 1 176 0 802 1,929 Ae. (Och.) serratus (Theobald 1901) 0 0 0 3 0 0 0 1 4 Ae. (Och.) stigmaticus (Edwards 1922)*+ 0 134 0 207 0 1 0 0 342 Ae. (Och.) taeniorhynchus (Wiedemann 1821) 0 0 0 0 0 0 0 1 1 Ae. (Stg.) aegypti (Linnaeus 1762) 0 1 0 0 0 0 0 0 1 Haemagogus (Con.) leucocelaenus (Dyar & Shannon, 1924)+ 0 0 0 2 0 0 0 0 2 Hg. (Hag.) janthinomys Dyar, 1921+ 0 2 0 2 0 0 0 0 4 Hg. (Hag.) spegazzinii Brethés, 1912 0 2 0 0 0 0 0 2 4 Psorophora (Gra.) cingulata Fabricius, 1805 0 0 0 0 0 0 0 2 2 Ps. (Jan.) albigenu (Peryassu, 1908)*+ 0 7 0 30 0 0 0 0 37 Ps. (Jan.) discrucians (Walker, 1856)*+ 0 30 0 557 0 0 0 9 596 Ps. (Jan.) ferox (Von Humboldt, 1819) 0 6 0 63 0 0 0 3 72 Tribe Culicini Culex (Cux.) ameliae Casal, 1967*+ 1 0 0 0 0 0 0 0 1 Cx. (Cux.) bidens Dyar, 1922*+ 0 0 0 1 0 0 0 0 1 Cx. (Cux.) habilitator Dyar & Knab, 1906*+ 0 1 0 0 0 0 0 0 1 Cx. (Cux.) restuans Theobald, 1901*+ 0 1 0 0 0 0 0 1 2 Cx. (Cux.) salinarius Coquillett, 1904*+ 0 0 0 0 0 0 0 1 1 Cx. (Cux.) saltanensis Dyar, 1928*+ 0 0 0 0 0 1 0 0 1 Cx. (Cux.) scimitar Branch & Seabrook, 1959*+ 0 1 0 2 0 0 0 0 3 Cx. (Mel.) complexo Vomerifer 2 0 0 0 0 0 0 0 2 Cx. (Mel.) group Atratus 1 0 0 0 0 0 0 0 1 Cx. (Mel.) section Melanoconion 0 0 0 0 0 0 7 0 7 Tribe Mansoniini Coquillettidia (Rhy.) albicosta (Peryassú, 1908)+ 2 332 0 0 0 0 0 0 334 Cq. (Rhy.) hermanoi (Lane & Coutinho, 1940)*+ 0 10 1 181 0 0 0 0 192 Cq. (Rhy.) juxtamansonia (Chagas, 1907) 0 0 0 1 0 0 0 0 1 Cq. (Rhy.) lynchi Shannon 1931*+ 0 0 0 0 0 2 0 0 2

229 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.

MSSP LCSP JBR SABR SPECIES Total Dry Wet Dry Wet Dry Wet Dry Wet Cq. (Rhy.) nigricans (Coquillett, 1904) 7 810 0 38 0 0 0 0 855 Cq. (Rhy.) venezuelensis (Theobald, 1912) 9 598 0 116 0 0 0 1 724 Mansonia (Man.) humeralis Dyar & Knab 1916+ 65 400 228 510 0 2 0 1 1,206 Ma. (Man.) indubitans Dyar & Shannon 1925+ 3 26 730 113 0 1 0 2 875 Ma. (Man.) pseudotitillans (Theobald, 1901)+ 24 401 0 9 0 0 0 0 434 Ma. (Man.) titillans (Walker, 1848) 161 1,352 508 1,010 3 0 0 6 3,040 Tribe Uranotaeniini Uranotaenia (Ura.) geometrica Theobald, 1901 0 0 0 2 0 0 0 0 2 Ur. (Ura.) lowii Theobald 1901*+ 0 0 1 0 0 0 0 1 2 Ur. (Ura.) pulcherrima Lynch Arribalzaga 1891*+ 0 0 0 2 0 0 1 0 3 Tribe Sabethini Limatus paraensis (Theobald 1903) 0 0 0 17 0 0 0 0 17 Sabethes (Pey.) undosus (Coquillett, 1906)+ 0 1 0 0 0 0 0 0 1 Total 444 4,693 1,589 3,459 6 185 8 835 11,219 * New record to the Minas Gerais State. +new record to the semi-arid region of Minas Gerais.

DISCUSSION these ponds act as major breeding grounds for Mansoniini mosquitoes in the larval and pupal stages as they contain lots of aquatic vegetation, Of all the collected specimens, 8,170 (73%) were characterized the aerenchyma of the roots providing the mosquitoes with oxygen10. by their use of permanent breeding habitats (e.g. ponds, marshes, river Some Mansoniini mosquitoes, such as Coquillettidia venezuelensis, backwaters and puddles) for their larval and pupal (immature) stages; are involved in the transmission of arboviruses, such as Eastern equine these individuals represented Anophelinae subfamily and Mansoniini, encephalitis virus (actual vectors) and Oropouche virus (potential Aedomyiini tribes and, in some cases, Culicini. The remaining 27% vectors)10. In addition, Ma. titillans have been found to be naturally (3,040 specimens) were characterized by their use of temporary infected with the Venezuelan equine encephalitis virus. Thus, the large breeding habitats (e.g., puddles, hollow bamboo, bromeliads and other abundance of mosquitoes of the genus Mansonia in the conservation phytotelmata) in their larval and pupal stages, representing primarily units sampled could potentially impact wild bird conservation, as these Aedini, Uranotaeniini and Sabethini tribes. mosquitoes are ornithophilic and can transmit avian malaria14,26.

Mosquito species belonging to the Mansoniini tribe were the The high abundance of Aedes scapularis was probably related to most abundant (68.3%), of which the species Mansonia titillans alone the vegetational structure of the study area, which is in the process of accounted for 27.10% of all mosquitoes sampled in the study. Mosquitoes natural regeneration from successive anthropogenic pressures, such as of the Aedini tribe were the second most abundant group of all mosquitoes agriculture and livestock farming17. These environments provide ideal collected (26.87%), with Aedes scapularis as the dominant species conditions for the establishment of Ae. scapularis populations, as these within the tribe (17.19%). Among the Anopheles species collected, mosquitoes have a marked tendency to invade artificially modified Anopheles darlingi was the most abundant and amounted to 1.36% of environments8,11,12. Furthermore, the larval and pupal stages of Ae. all mosquitoes sampled. scapularis develop in temporary ground pools formed by rainfall, and are comparable to those known to exist in environments in the initial The large percentage of mosquito species using permanent reservoirs stages of natural regeneration5,10. might be related to the relatively long dry periods, which are characteristic of the study area. Prolonged droughts can have a damaging effect on At least 15 viruses have been isolated from Ae. scapularis, including the viability of Aedini mosquitoes’ eggs24 and can negatively affect the the Rocio virus, Yellow fever virus, and Venezuelan equine encephalitis nutritional quality of the detritus found in temporary breeding habitats4. virus; this species may also be a vector of Bancroftian filariasis18,20. Despite the long dry periods, the community of mosquitoes manages to VASCONCELOS et al.27 isolated a strain of Yellow fever virus from survive, mainly using the vegetation surrounding the ponds located in field-captured Ae. scapularis. Previously, only experimental laboratory PEMS and PELC. infections had been reported in this species. Considering the ecological and epidemiological characteristics reported for this species, these The large abundance of mosquitoes within the Mansoniini tribe can mosquitoes can be a potential bridge between wild arboviruses and be explained by the influence of ponds located on the banks of the São human populations in this region, given the current state of anthropogenic Francisco River, located in the MSSP and LCSP. Even in dry seasons, modifications of the study region.

230 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.

Mosquitoes of the Psorophora genus were the most abundant Aedini (CNPq -563304/2010-3 and 562955/2010-0), Fundação de Amparo à after Ae. scapularis; this might be explained by the fact that these types of Pesquisa de Minas Gerais (FAPEMIG CRA - APQ-00001-11) and the mosquitoes share the same breeding habitats3,19,25. Although Psorophora Inter-American Institute for Global Change Research (IAI-CRN II-021). have been found to carry some types of infection in nature, mosquitoes of this genus are not considered epidemiologically significant vectors. REFERENCES These mosquitoes are, however, treated as potential incidental vectors of disease due to some of their behavioral characteristics, such as eclecticism 1. Andrade RM, Leal JM. Distribuição de anofelinos na bacia hidrográfica do rio São in the choice of blood host and exophilic behavior10. Francisco. Estados de Minas Gerais, Bahia, Goiás, Pernambuco, Alagoas e Sergipe. Rev Bras Malar D Trop. 1960;1:147-63.

The abundance of An. darlingi recorded deserves particular attention, 2. Antunes FZ. Caracterização climática: caatinga do estado de Minas Gerais. Inf as this species is the main vector of malaria parasites in Brazil and is Agropecuário Belo Horizonte. 1994;17:15-9. widely distributed across South America23; additionally, these mosquitoes have an increased capacity for taking blood meals within and around 3. Arnell JH. Mosquito studies (Diptera, Culicidae). XXXIII. A revision of the Scapularis group of Aedes (Ochlerotatus). Contrib Am Entomol Inst (Ann Arbor). 1976;13:1-144. residential regions6. Although Anopheles argyritarsis and Anopheles triannulatus are not the primary vectors of the Plasmodium species 4. Aspbury AS, Juliano SA. Negative effects of habitat drying and prior exploitation on responsible for malaria, these species are of great epidemiological interest the detritus resource in an ephemeral aquatic habitat. Oecologia. 1998;115:137-48. because of their high abundance and anthropophilic nature6. 5. Casanova C, Prado AP. Key-factor analysis of immature stages of Aedes scapularis (Diptera: Culicidae) populations in southeastern Brazil. Bull Entomol Res. The abundance of new Culicid records for Minas Gerais State, and 2002;92:271-7. for the semi-arid region of the state, indicates that studies of mosquito communities in forest remnants are still required, especially with regards 6. Consoli RAGB, Oliveira RL. Principais mosquitos de importância sanitária no Brasil. to the development and maintenance of support programs aimed at the Rio de Janeiro: Ed. Fiocruz; 1994. prevention of disease transmission to humans and other animals. 7. Deane LM. Malaria vectors in Brasil. Mem Inst Oswaldo Cruz. 1986;81(Suppl 2):5- 14. RESUMO 8. Dorvillé LFM. Mosquitoes as bioindicators of forest degradation in southeastern Inventário de mosquitos (Diptera: Culicidae) em unidades de Brazil, a statistical evaluation of published data in the literature. Stud Neotrop Fauna conservação em florestas tropicais secas brasileiras Environm. 1996;31:68-78. 9. Faran ME. Mosquito studies (Diptera, Culicidae) XXXIV. A revision of the Albimanus No Brasil, a maior parte dos estudos relacionados à família Section of the subgenus Nyssorhynchus of Anopheles. Contrib Am Entomol Inst (Ann Culicidae se concentram em regiões de florestas úmidas, existindo uma Arbor). 1980;15:1-214. lacuna no conhecimento da diversidade destes mosquitos em regiões com características climáticas e vegetacionais diferentes. O objetivo 10. Forattini OP. Culicidologia médica. São Paulo: EDUSP; 2002. desse trabalho foi inventariar a fauna de culicídeos em unidades de 11. Forattini OP, Alves AC, Natal D, Santos JLF. Observações sobre atividades de conservação do semi-árido de Minas Gerais, visando assim contribuir mosquitos Culicidae em mata primitiva da encosta no Vale do Ribeira São Paulo, para o conhecimento da diversidade de Culicidae desta região. O Brasil. Rev Saúde Pública. 1986;20:1-20. estudo foi realizado em quatro unidades de conservação localizadas na região norte do estado de Minas Gerais, Brasil, área representada 12. Forattini OP, Kakitani I, Massad E, Marucci D. Studies on mosquitoes (Diptera: Culicidae) and anthropic environment. 9. Synanthropy and epidemiological vector por fragmentos de Floresta Tropical Seca (FTS). Foram utilizados três role of Aedes scapularis in South-Eastern Brazil. Rev Saúde Pública. 1995;29:199- métodos de coleta: armadilha do tipo Shannon, armadilha luminosa do 207. 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, 13. Gama RA, Andrade AJ, Andrade MR, Resende MC, Eiras, AE. Avaliação da armadilha distribuídos em 11 gêneros e 45 espécies. Foram registrados 15 novos HP iscada com diferentes taxas de liberação de octenol na captura de anofelinos (Diptera: Culicidae) em Brejo do Mutambal, Município de Varzelândia, Estado de registros de mosquitos para o estado de Minas Gerais e 26 novos Minas Gerais. Rev Soc Bras Med Trop. 2007;40:408-10. registros para a região do semi-árido de Minas Gerais. O elevado número de novos registros de Culicidae na região demonstra a 14. Lacorte GA, Félix GMF, Pinheiro RRB, Chaves AV, Almeida-Neto G, Neves FS, et importância de estudos de inventário para o aumento do conhecimento al. Exploring the diversity and distribution of neotropical avian malaria parasites: a da biodiversidade de culicídeos em Minas Gerais, e em particular a molecular survey from southeast Brazil. PLOS One. 2013;8:1-9. região do semi-árido do estado. 15. Lane J. Neotropical Culicidae. Tribe Culicini, Deinocerites, Uranotaenia, Mansonia, Orthopodomyia, Aedomyia, Aedes, Psorophora, Haemagogus, tribe Sabethini, ACKNOWLEDGEMENTS Trichoprosopon, Wyeomyia, Phoniomyia, Limatus and Sabethes. São Paulo: Universidade de São Paulo; 1953. v. 2. The authors gratefully acknowledge the staff of the Instituto Estadual 16. Maciel CS. Lista de culicineos do Estado de Minas Gerais, Brasil (Diptera, Culicidae). de Florestas (IEF-MG), for allowing them to work and stay at Mata Rev Bras Malar D Trop. 1962;14:465-94. Seca State Park (MSSP), Lagoa do Cajueiro State Park (LCSP), Jaiba Biological Reserve (JBR) and Serra Azul Biological Reserve (SABR) 17. Madeira GB, Espirito-Santo MM, Neto SA, Nunes YRF, Sánches-Azofeifa GA, for logistical support. This work was carried out with the aid of a grant Fernandes GW, et al. Changes in tree and liana communities along a succecional from Conselho Nacional de Desenvolvimento Científico e Tecnológico gradient in a tropical dry forest in south-eastern Brazil. Plant Ecol. 2009;201:291-304.

231 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.

18. Mitchell CJ, Forattini OP. Experimental transmission of Rocio encephalitis virus 24. Sota T, Mogi M. Interspecific variation in desiccation survival time of Aedes by Aedes scapularis (Diptera: Culicidae) from the epidemic zone in Brazil. J Med (Stegomyia) mosquito eggs is correlated with habitat and egg size. Oecologia. Entomol. 1984;21:34-7. 1992;90:353-8.

19. Piovezan R, Azevedo TS, Von Zuben CJ. Spatial evaluation of larvae of Culicidae 25. Stein M, Ludueña-Almeida F, Willener JA, Almirón WR. Classification of immature (Diptera) from different breeding sites: application of a geospatial method and mosquito species according to characteristics of the larval habitat in the subtropical implications for vector control. Rev Bras Entomol. 2012;56:368-76. province of Chaco, Argentina. Mem Inst Oswaldo Cruz. 2011;106:400-7.

20. Rachou RG, Lima MM, Neto JAF, Martins CM. Inquérito epidemiológico de filariose 26. Valkiūnas G. Avian malaria parasites and other haemosporidia. Boca Raton: CRC bancroftiana em uma localidade de Santa Catarina, como fase preliminar de uma Press; 2005. prova profilática. Constatação de transmissão extradomiciliária por um novo vetor, Aedes scapularis. Rev Bras Malar D Trop. 1955;7:51-70. 27. Vasconcelos PC, Costa ZG, Travassos da Rosa ES, Luna E, Rodrigues SG, Barros VLRS, et al. Epidemic of jungle yellow fever in Brazil, 2000: implications of climatic 21. Rueda LM. Global diversity of mosquitoes (Insecta: Diptera: Culicidae) in freshwater. alterations in disease spread. J Med Virol. 2001;65:598-604. Hydrobiologia. 2008;595:477-87. Received: 20 December 2013 22. Sanches-Azofeifa GA, Quesada M, Rodriguez JP, Nassar JM, Stoner KE, Castillo Accepted: 4 September 2014 A, et al. Research priorities for neotropical dry forests. Biotropica. 2005;37:477-85.

23. Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, et al. A global map dominant malaria vectors. Parasit Vectors. 2012;5:69.

232 Rev. Inst. Med. Trop. Sao Paulo 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 in both rural and urban areas3,11,12,17,24,25.

American visceral leishmaniasis (AVL) has been recorded in The Águas do Miranda district, has fishing tourism as its main increasing numbers of human and canine cases in the state of Mato economic source and presents socio-economic and environmental Grosso do Sul (MS), which is one of the states with the greatest incidences conditions favorable to the transmission of the endemic diseases under in the central-western region of Brazil4,6,26. Meanwhile, American consideration. These facts together with the results of research into the cutaneous leishmaniasis (ACL) has been recorded in the majority of canine population of the district, which have shown 40% out of the 92 municipalities12,17. However, despite the wide distribution and growing animals as seropositive for Leishmania (VLB Nunes, unpublished data), number of human cases, epidemiological studies on leishmaniasis in MS have motivated the present study for the purpose to identify behavioral are few and far between. aspects of the sandfly fauna, including its species abundance, diversity, evenness, monthly distribution and natural infection by flagellates, to In Bonito (MS), which is a municipality classified at a moderate identify potential vectors of Leishmania spp. transmission level of leishmaniasis, studies have indicated the occurrence of both human and canine cases of AVL and ACL, and these diseases MATERIAL AND METHODS are spreading due to the implementation of ecotourism and livestock activities in the area4,16,24. 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 Three species of Leishmania (Ross) have already been reported Campo Grande, the capital of the state of MS. The permanent human in MS: Leishmania (Leishmania) infantum chagasi Cunha & Chagas, population consists of 450 inhabitants, which may rise to as many as Leishmania (Leishmania) amazonensis Lainson & Shaw and Leishmania 10,000 in the fishing season, from March to October. The local economy (Viannia) braziliensis Vianna and their respective vectors, Lutzomyia is based mainly on fishing and the tourist trade29. longipalpis (Lutz & Neiva), Bichromomyia flaviscuttelata (Mangabeira) and Nyssomyia whitmani (Antunes & Coutinho), all of which are found 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. E-mail: [email protected] 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 The flagellates found in the gut of the dissected females of two sand environmental conditions, such as forest patches in areas with more fly species were inoculated intradermally, in the hind legs of hamsters fertile soil and a more plentiful supply of water. Noteworthy is the (Mesocricetus auratus). The animals were observed weekly during vegetation cover of the Bodoquena range, a typical forest associated with 12 months for checking the appearance of lesions. After euthanasia, calcareous rocks known as Dry Forest or Submontane Semideciduous the spleens of these animals were removed and inoculated into NNN Seasonal Tropical Forest. The climate is tropical with an annual average culture medium (blood agar) for isolation of the parasites. Cultures were temperature of 22 ºC29,38. maintained at 25 °C and examined weekly for one month to observe if there was proliferation of flagellates. Capture sites:A total of nine sites were sampled in different environments (Fig. 1): peridomiciliary areas near fruit trees, native The pluviometric data used in the analysis was obtained from the grass and tuberous vegetables (A1), native species of trees, fruit trees Aquidauna meteorological station, the nearest to Águas do Miranda and bamboos (A2) and bordered by a stream, within a bamboo grove, district, at about 30 km away. near fruit and ornamental trees (A3); area of savannah with selective extraction of timber and native species of trees (A4); gallery forests with Data analysis: Species abundance was calculated for all the ecotopes some secondary vegetation which grew after the selective extraction of investigated in accordance with ROBERTS & HSI (1979)31. Initially the timber (A5, A6) and animal shelters such as a pigsty (A7), a henhouse Index of Species Abundance (ISA) was obtained by the application of the (A8) and a perch (A9), near peridomiciliary areas. 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.

The project was submitted to the Ethics Committee on Animal Use in Research (CEUA) Anhanguera-UNIDERP University and approved Fig. 1 - Distribution of capture sites in the district of Águas do Miranda, in the municipality according to opinion No. 63-006/09. of Bonito, Mato Grosso do Sul, from December 2009 to November 2010, Brazil 04/30/2012. Source: Google Earth. RESULTS

Methodology: The phlebotomines were captured on three consecutive A total of 6,699 phlebotomine specimens were captured, Brumptomyia nights, once a month, during the period from December 2009 to November avellari (Costa Lima), Br. brumpti (Larrousse), Brumptomyia sp., 2010 using modified automatic light CDC traps23 from 18h00 to 06h00. Evandromyia sp. (Cortelezzii complex), Ev. lenti (Mangabeira), Ev. One trap was installed per night in each of the nine sites sampled. termitophila (Martins, Falcão & Silva), Lutzomyia longipalpis (Lutz & Neiva), Sciopemyia sordellii (Shannon & Del Ponte), Nyssomyia neivai The insects captured with the CDCs were transferred to nylon cages. (Pinto), Ny. whitmani (Antunes & Coutinho), Psathyromyia aragaoi The females were recaptured with polyethylene tubes, in which they (Costa Lima), Pa. campograndensis Oliveira, Andrade Filho, Falcão were anaesthetized with sulfuric ether. Then, after dissection to expose & Brazil, Pa. hermalenti (Martins, Silva & Falcão), Pa. bigeniculata the gut and spermathecae, under stereomicroscopy, they were examined (Floch & Abonnenc), Pa. punctigeniculata (Floch & Abonnenc) and under an optical microscope (400x) for identification of the phlebotomine Micropygomyia quinquefer (Dyar) (Table 1). species and investigation of flagellates. The male insects were kept in Petri dishes under refrigeration until their clarification in accordance with The greatest phlebotomine species richness (15) and frequency the technique given by FORATTINI (1973)13. Species identification was (95.35%) occurred in the henhouse (A8), representing almost the totality undertaken in accordance with GALATI (2003)18 and the abbreviation of the specimens captured during the period studied. On the other hand, of the species names follows MARCONDES (2007)22. the lowest species richness (3) occurred in the pigsty (A7) and the lowest

234 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.

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 Species ecotopes A1 A2 A3 A4 A5 A6 A7 A8 A9 Brumptomyia sp. 1 - 1 ------2 0.03 0.164 Br. avellari - - - 1 1 - - 1 - 3 0.04 0.107 Br. brumpti - 1 1 3 2 - - 8 - 15 0.22 0.342 Cortelezzii complex 1 2 2 1 24 2 - 95 1 128 2.06 0.678 Ev. lenti - 3 - 23 3 - - 9 - 38 0.57 0.357 Ev. termitophila ------1 - 1 0.01 0.014 Lu. longipalpis 4 5 1 18 5 9 8 885 1 936 13.80 0.853 Mi. quinquefer ------4 - 4 0.07 0.043 Ny. neivai - 1 1 - - 25 - 323 23 373 6.44 0.457 Ny. whitmani 3 - 4 34 9 9 6 3270 7 3342 49.26 0.830 Pa. aragaoi - - - 9 - - - 2 - 11 0.18 0.107 Pa. campograndensis - - 2 - - - - 4 - 6 0.10 0.135 Pa. hermanlenti - - - 3 1 - - 4 - 8 0.12 0.164 Pa. punctigeniculata - - - - 1 - - 8 - 9 0.13 0.114 Pa. bigenicutala - 1 2 9 19 10 2 1773 4 1820 26.90 0.764 Sc. sordellii - - - 1 1 - - 1 - 3 0.06 0.107 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 0.54 100 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 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 Fig. 2 - Number of specimens of both sexes collected per month of the species Lu. Longipalpis, the ecotopes sampled is shown in Figure 2. Lu. longipalpis presented Ny. whitmani and Pa. bigeniculata, in the district of Águas do Miranda, Bonito, Mato Grosso peaks in January and November, Ny. whitmani in July, August, October do Sul, December 2009 to November 2010. and November and Pa. bigeniculatus in November. 0.07% (1/1418) for Ny. whitmani and 0.23% (1/408) for Pa. bigeniculata. The monthly distribution of rainfall, average temperature and relative The flagellates were observed in the hindgut and midgut. humidity is shown in Figure 3. The animals inoculated with the gut Ny. whitmani and Pa. The rates of natural infection detected by optical microscopy were bigeniculata containing flagellates did not develop a lesion during the

235 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.

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.

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 Fig. 3 - Monthly distribution of rainfall (mm), temperature (ºC) and relative humidity (%) in fishing season (March to October), thus indicating the concomitant risk of the municipality of Bonito, from December 2009 to November 2010. the transmission of cutaneous leishmaniasis to both the local population and visiting tourists. observation period and in the cultures in the hamsters’ spleens showed no growth of flagellated form. Lu. longipalpis was found in anthropic environments in which animals are reared, but is also habitual in other environments, both DISCUSSION rural and urban, where AVL and canine visceral leishmaniasis (CVL) occur4,24,25, which suggests that this species may be the vector responsible The greatest frequency and species richness of the phlebotomines for the transmission of Le. i. chagasi to the canine population of the captured occurred in anthropic environments, probably attracted to the district, which, in a research project undertaken in 2009, presented 40% peridomicile due to blood meal sources represented by domestic and seropositive dogs for Le. i. chagasi in a population of 92; the parasite synanthropic animals. The predominance of Ny. whitmani in a henhouse isolated by the Polymerase Chain Reaction (PCR) technique being Le. i. (A8) near native forest suggests that this species has a close relationship chagasi (VLB Nunes, unpublished data). These observations have been with both wild and anthropic environments, in which the forest serves found in other areas of the country and the public health authorities should as a shelter and breeding place for adults, as do the shaded areas in the be alerted, since that CLV cases precede human AVL and the dog has a peridomicile7. Furthermore, the walls and roof of the henhouse, as well as fundamental role in the domestic transmission8, 20. the chickens can serve as substrates for males waiting for the opportunity to mate with the females seeking blood meal sources in this ecotope, Pa. bigeniculata, considered for long time as a junior synonym of Pa. since the males of hematophagous insects, dispersed throughout their shannoni, recently had its status of species resurrected. The difference habitats, may obtain a mating advantage by staying near the host and between these two species is mainly the thoracic coloration, i.e. while Pa. waiting for the females to arrive1. It is noteworthy that one specimen shannoni presents pronotum and paratergite straw, prescutum, scutum, of this species, naturally infected by flagellates, was captured in this and scutellum brown, pleurae off-white, Pa. bigeniculata presents henhouse, suggesting that this ecotope is attractive to the synanthropic pronotum, paratergite, prescutum, scutum, and scutellum brown, upper animals which constitute the Leishmania reservoir or that the infected anepisternum straw and the other pleural off-white sclerites33. sand fly had moved from the forest to the henhouse . Pa. shannoni is considered in the United States an important arbovirus The highest diversity and evenness indices in Águas do Miranda, vector9 and has been captured naturally infected by Leishmania mexicana especially in the peridomiciliary environment, may demonstrate the in peri-urban areas of Mexico34, by Leishmania sp. in Guatemala32 presence of these insects in areas of preserved forests and anthropic and also developed experimental infection with L. i. chagasi when action in these locations. The findings of this study differed from those feeding on infected dog37. Ps. shannoni s. lat has been associated with of NUNES et al. (2008)24 and ANDRADE et al. (2009)4, which found the transmission of Endotrypanum schaudinni, a trypanosomatid of lower values in urban areas of the municipality of Bonito. The highest sloths14, and was found naturally infected by Leishmania sp. in Serra frequency of Ny. whitmani (49.26%) in the henhouse (A8) may explain do Baturité, in the northeastern region of Brazil30. The finding ofPa. the lowest evenness and diversity despite being the ecotope with the bigeniculata naturally infected by flagellates, in a henhouse close to a highest species richness. forest, demonstrates the need to clarify its epidemiological significance in relation to anthropophily and the transmission of the leishmaniasis agent, The most abundant species calculated by SISA were Lu. longipalpis especially because it presents close affinity with Pa. shannoni for which and Ny. whitmani, which showed a wide distribution of these species in the there are records of its infection, being either natural or experimental, ecotopes sampled, this indicates that these species may be participating by Leishmania spp.32,37. 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 Ny. neivai presents no widespread geographical distribution in the Mato Grosso do Sul4,17,24,25. state of Mato Grosso do Sul, being found mainly in the southeastern and eastern mesoregions2,5. It is worth highlighting the considerable The natural infection rate found for Ny. whitmani (0.07%) was abundance of the species found in this study, especially in animal shelters, lower than that recorded by GALATI et al. (1996)17 in the Corguinho because it has been reported as naturally infected by Leishmania and is

236 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.

suspected of involvement in the transmission of cutaneous leishmaniasis support and to Bonito’s municipal government for their logistic support. by Le. braziliensis in such Brazilian states15,22,27,28 and also in neighboring countries10. REFERENCES

The Cortelezzii complex includes the species Ev. cortelezzii, Ev. 1. Alexander B, de Carvalho RL, McCallum H, Pereira MH. Role of the domestic chicken sallesi and Ev. corumbaensis, which are all found in MS2,3,17. The only (Gallus gallus) in the epidemiology of urban visceral leishmaniasis in Brazil. Emerg Infect Dis. 2002;8:1480-5. possible way to distinguish them with confidence is by using males. As only females were identified in this study (data not given), it was 2. Almeida PS, Leite JA, Araújo AD, Batista PM, Touro RB, Araújo VS, et al. Fauna of impossible to identify the species of this complex. Recent studies phlebotomine sand flies (Diptera, Psychodidae) in areas with endemic American have reported the natural infection of Ev. cortelezzii and Ev. sallesi by cutaneous leishmaniasis in the State of Mato Grosso do Sul, Brazil. Rev Bras Entomol. Leishmania3,35, thus calling for studies on their vectorial capacity, since 2013;57:105-12. many of the localities where they have been captured are endemic for 3. Andrade AR, Dorval ME, Andrade SM, Marques A, Lima-Junior MS, Silva BAK, et leishmaniasis, as in the area covered by the present study. al. First report of natural infection of phlebotomines for Leishmania (Leishmania) chagasi captured in Ponta Porã, on the border between Brazil and Paraguay. Asian The District of Águas do Miranda revealed a diverse sandfly fauna, Pac J Trop Dis. 2011;1:253-8. with 16 species, some of them proven vectors of leishmaniasis agents 4. Andrade AR, Nunes VL, Galati EA, de Arruda CC, Santos MF, Rocca ME, et al. in Brazil and others that have been described as naturally infected and Epidemiological study on leishmaniasis in an area of environmental tourism and which may, therefore, act as potential vectors. In light of the above, the ecotourism, state of Mato Grosso do Sul, 2006-2007. Rev Soc Bras Med Trop. local population and visiting tourists should be warned of the threat 2009;42:488-93. posed by leishmaniasis and the health authorities alerted of the need for adopting environmental sanitary measures, especially regarding such 5. Andrade-Filho JD, Galati EA, Falcão AL. Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae: Phlebotominae) geographical animal shelters, as they seem to provide favorable conditions to the distribution and epidemiological importance. Mem Inst Oswaldo Cruz. 2007;102:481- proliferation, maintenance and breeding opportunities of phlebotomines. 7.

RESUMO 6. Brasil. Ministério da Saúde. Sistema de Informação de Notificação de Agravos de Notificação (SINAN). Brasília; Ministério da Saúde, 2010. Available from: http:// portal.saude.gov.br/portal/arquivos/pdf/2_lv_casos_14_10_10.pdf Fauna flebotomínea (Diptera: Psychodidae) em área de turismo pesqueiro no Centro-Oeste do Brasil 7. Casanova C. A soil emergence trap for collection of phlebotomine sand flies. Mem Inst Oswaldo Cruz. 2001;96:273-5. O objetivo deste estudo foi identificar aspectos do comportamento da fauna flebotomínea de área de turismo pesqueiro localizada no município 8. Colla-Jacques FE, Casanova C, Prado AP. Study of sand fly fauna in an endemic area of American cutaneous leishmaniasis and canine visceral leishmaniasis in the de Bonito (MS). Foram realizadas capturas mensais no período de municipality of Espírito Santo do Pinhal, São Paulo, Brazil. Mem Inst Oswaldo Cruz. dezembro de 2009 a novembro de 2010, utilizando armadilhas luminosas 2010;105:208-15. automáticas do tipo CDC das 18:00h às 6:00h, em matas, área de cerrado, peridomicílios e abrigos de animais próximos a áreas peridomiciliares. 9. Comer JA, Tesh RB, Modi GB, Corn JL, Nettles VF. Vesicular stomatitis virus, New De um total de 6.699 espécimes coletados, pertencentes a 16 espécies, Jersey sorotype: replication in and transmission by Lutzomyia shannoni (Diptera: Psychodidae). Am J Trop Med Hyg. 1990;42:483-90. Nyssomyia whitmani foi a mais frequente, seguida de Psathyromyia bigeniculata e Lutzomyia longipalpis, encontradas em todos os tipos 10. Córdoba-Lanús E, De Grosso ML, Piñero JE, Valladares B, Salomón OD. Natural infection de ambientes, porém com maior expressão em abrigos de animais. Ny. of Lutzomyia neivai with Leishmania spp. in northwestwern Argentina. Acta Trop. whitmani apresentou frequências mais elevadas nos meses mais secos, 2006;98:1-5. coincidentes com a estação da pesca, o que eleva o risco de transmissão 11. Dorval ME, Alves TP, Cristaldo G, Rocha HC, Alves MA, Oshiro ET, et al. Sand fly da leishmaniose tegumentar a turistas e moradores da área. Importante captures with Disney traps in area of occurrence of Leishmania (Leishmania) ressaltar o encontro de duas espécies naturalmente infectadas por amazonensis in the State of Mato Grosso do Sul, mid-western Brazil. Rev Soc Bras flagelados:Ny. whitmani e Pa. bigeniculata. A população local e turistas Med Trop. 2010;43:491-5. devem ser advertidos da ameaça que representam as leishmanioses e as autoridades de saúde alertadas para adoção de medidas de saneamento 12. Dorval ME, Oshiro ET, Cupollilo E, Castro AC, Alves TP. Ocorrência de leishmaniose tegumentar americana no Estado do Mato Grosso do Sul associada à infecção por ambiental, principalmente com relação aos abrigos de animais, que Leishmania (Leishmania) amazonensis. Rev Soc Bras Med Trop. 2006;39:43-6. parecem fornecer condições favoráveis para a proliferação, manutenção e reprodução de flebotomíneos. 13. Forattini OP. Entomologia médica. São Paulo; Edgard Blücher; 1973. p. 658.

CONFLICT OF INTEREST 14. Franco AM, Grimaldi G Jr. Characterization of Endotrypanum (Kinetoplastida: Trypanosomatidae), a unique parasite infecting the neotropical tree sloths (Edentata). Mem Inst Oswaldo Cruz. 1999;94:261-8. The authors declare there is no conflict of interests. 15. Galati EA, Fonseca MB, Marassá AM, Bueno EF. Dispersal and survival of Nyssomyia ACKNOWLEDGEMENTS intermedia and Nyssomyia neivai (Diptera: Psychodidae: Phlebotominae) in a cutaneous leishmaniasis endemic area of the speleological province of the Ribeira Valley, state of São Paulo, Brazil. Mem Inst Oswaldo Cruz. 2009;104:1148-58. The authors wish to express their thanks to the Universidade Anhanguera - Uniderp, to the Brazilian agency CAPES for their financial

237 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.

16. Galati EA, Nunes VL, Cristaldo G, Rocha HC. Aspectos do comportamento da fauna 28. Pita-Pereira D, Souza GD, Zwestch A, Alves CR, Britto C, Rangel EF. First report of flebotomínea (Diptera:Psychodidae) em foco de leishmaniose visceral e área Lutzomyia (Nyssomyia) neivai (Diptera: Psychodidae: Phlebotominae) naturally adjacente, estado de Mato Grosso do Sul, Brasil. Rev Patol Trop. 2003;32:235-61. infected by Leishmania (Viannia) braziliensis in a periurban area of south Brazil using a multiplex polymerase chain reaction assay. Am J Trop Med Hyg. 2009;80:593-5. 17. Galati EA, Nunes VL, Dorval ME, Oshiro ET, Cristaldo G, Espíndola MA, et al. Estudo dos flebotomíneos (Diptera: Psychodidae) em área de leishmaniose tegumentar no 29. Prefeitura Municipal de Bonito. Relatório de planejamento da Prefeitura Municipal de estado de Mato Grosso do Sul, Brasil. Rev Saúde Pública. 1996;30:115-28. Bonito. Bonito; Prefeitura Municipal, 2009.

18. Galati EA. Classificação de Phlebotominae e morfologia e taxonomia Phlebotomine. 30. Queiroz RG, Vasconcelos IA, Vasconcelos AW, Pessoa FA, de Sousa RN, David JR. In: Rangel EF, Lainson R, organizadores. Flebotomíneos do Brasil. Rio de Janeiro; Cutaneous leishmaniasis in Ceara State in Northeastern Brazil: incrimination of Fiocruz, 2003. p. 23-51. Lutzomyia whitmani (Diptera: Psychodidae) as a vector of Leishmania braziliensis in Baturité municipality. Am J Trop Med Hyg. 1994;50:693-8. 19. Hayek LA, Buzas MA. Surveying natural populations. New York; Columbia University; 1997. p. 347-89. 31. Roberts DR, Hsi BP. An index of species abundance for use with mosquito surveillance data. Environ Entomol. 1979;7:1007-13. 20. Krauspenhar C, Beck C, Sperotto V, Silva AA, Bastos R, Rodrigues L. Leishmaniose visceral em um canino de Cruz Alta, Rio Grande do Sul, Brasil. Cienc Rural. 32. Rowton E, de Mata M, Rizzo N, Navin T, Porter C. Vectors of Leishmania braziliensis 2007;37:907-10. in the Petén, Guatemala. Parassitologia. 1991;33:501-4.

21. Marcondes CB, Bittencourt IA, Stoco PH, Eger I, Grisard EC, Steindel M. Natural 33. Sábio PB, Andrade AJ, Galati EA. Assessment of the taxonomic status of some species infection of Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae, Phlebotominae) included in the Shannoni Complex, with the description of a new species of by Leishmania (Viannia) spp. in Brazil. Trans R Soc Trop Med Hyg. 2009;103:1093-7. Psathyromyia (Diptera: Psychodidae: Phlebotominae). J Med Entomol. 2014;51:331- 41 22. Marcondes CB. A proposal of generic and subgeneric abbreviations of phlebotomines sandflies (Diptera: Psychodidae: Phlebotominae) of the world. Entomol News. 34. Sánchez-García L, Berzunza-Cruz M, Becker-Fauser I, Rebollar-Téllez EA. Sand flies 2007;118:351-6. naturally infected by Leishmania (L.) mexicana in the peri-urban area of Chetumal city, Quintana Roo, México. Trans R Soc Trop Med Hyg. 2010;104:406-11. 23. Natal D, Marucci D, Reis IM, Galati EA. Modificação da armadilha CDC com testes para coletas de flebotomíneos (Diptera). Rev Bras Entomol. 1991;35:697-700. 35. Saraiva L, Carvalho GM, Gontijo CM, Quaresma PF, Lima AC, Falcão AL, et al. Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by 24. Nunes VL, Galati EA, Cardozo C, Rocca ME, Andrade AR, Santos MF, et al. Estudo Leishmania infantum chagasi in Brazil. J Med Entomol. 2009;46:1159-63. de flebotomíneos (Diptera: Psychodidae) em área urbana do município de Bonito, Mato Grosso do Sul, Brasil. Rev Bras Entomol. 2008;52:446-51. 36. Souza NA, Andrade-Coelho CA, Vilela ML, Peixoto AA, Rangel EF. Seasonality of Lutzomyia intermedia and Lutzomyia whitmani (Diptera: Psychodidae: 25. Oliveira AG, Galati EA, de Oliveira O, de Oliveira GR, Espindola IA, Dorval ME, et al. Phlebotominae), occurring sympatrically in area of cutaneous leishmaniasis in the Abundance of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and State of Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz. 2002;97:759-65. urban transmission of visceral leishmaniasis in Campo Grande, state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz. 2006;101:869-74. 37. Travi BL, Ferro C, Cadena H, Montoya-Lerma J, Adler GH. Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas. Res Vet Sci. 2002;72:83-6. 26. Oliveira AL, Paniago AM, Dorval ME, Oshiro ET, Leal CR, Sanches M, et al. Foco emergente de leishmaniose visceral em Mato Grosso do Sul. Rev Soc Bras Med 38. Veloso HP, Rangel-Filho LR, Lima JC. Classificação da vegetação brasileira, adaptada a Trop. 2006;39:446-50. um sistema universal. Rio de Janeiro; Instituto Brasileiro de Geografia e Estatística; 1991. 27. Oliveira DM, Reinhold-Castro KR, Bernal MV, Legriffon CM, Lonardoni MV, Teodoro U, et al. Natural infection of Nyssomyia neivai by Leishmania (Viannia) spp. in the Received: 16 December 2013 State of Paraná, Southern Brazil, detected by multiplex polymerase chain reaction. Accepted: 26 September 2014 Vector Borne Zoonotic Dis. 2011;11:137-43.

238 Rev. Inst. Med. Trop. Sao Paulo 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 In Brazil, epidemiological studies concerning this ectoparasitosis are concentrated in the southeastern region, while information from Pediculosis, infestation by Pediculus capitis De Geer (head lice), the northern and northeastern regions regarding infestation remains has worldwide distribution, including Brazil11, and is observed in all scarce1,4,13, except for a recent study involving school-age children in age groups, though particularly among children4,23,28. It is considered urban areas of Manaus4 and another one from the state of Acre concerning one of several ectoparasites neglected by the scientific community and infant dermatitis, which included head lice1. healthcare authorities12. Epidemiological features associated with head lice infestation Infestation is characterized by intense itching, secondary infection among young people and adults were studied using analysis of hair cut and anemia in cases of severe infestation and inadequate diet23. in salons and similar establishments, located within the urban area of Severe infestations are associated with low socioeconomic status, hair Manaus1,13. This study provided information concerning the occurrence, characteristics, parasite resistance to insecticides, genetic factors, and its monthly distribution and possible associations of several factors cultural habits6,8,10,24. which, according to the literature3,4, can influence its occurrence, such as hair characteristics, ethnicity, age, socioeconomic status, the location Besides the physical symptoms described above, it can cause of sampling, and seasonal variation. embarrassment among children. Certain population groups, particularly adults, often resist having their heads examined for the diagnosis of the MATERIAL AND METHODS parasite, which is a fairly sensitive diagnostic method3,16. Given this restriction, researchers have sought alternative methods of assessment Manaus, in the Amazonas State, occupies an area of 11,401,092 km² regarding the degree of importance of this disease in certain populations. (4,401,986 m2) and is located at 60°01’30” W and 03°06’07” S. The city Analysis of samples of hair cut in salons and similar establishments is one has 1,982,179 inhabitants and is divided into six administrative zones such technique7,9,16,18. Although it does not present the same sensitivity comprising 63 neighborhoods15. The study was conducted by obtaining as direct scalp examination3, it is an alternative that permits verification and examining hair samples from customers of 18 beauty parlors and of the epidemiological aspects of this parasitosis in population groups barber shops located in five of the six administrative zones of this city. that have such restrictions. 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. E-mail: [email protected] 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 the value of the haircuts charged by the establishments. Thus, customers owners/managers for inclusion in the study. Two to four establishments who sought a haircut in an establishment that charged R$ 5.00 to R$ were sampled in each administrative zone. Each establishment was 10.00 were considered to be of low socioeconomic status, R$ 15.00 of visited around 15 times, with each visit on a different day to obtain hair medium status and $ 20.00 to R$ 30.00 of high status. samples, between August 2010 and July 2013. Customers who attended the establishments and appeared to be between 15 and 65 years of age The monthly distribution of infestation was monitored for three were included in the study. years. School age children seemed to act as reservoirs of this disease4 and studies have demonstrated that significant variation in occurrence The owners or managers of the institutions signed a term of free and appears among children between the academic and vacation periods in informed consent. Details on the ethical procedures adopted are described Minas Gerais3, 20. in the project approved by the Research Ethics Committee of the Federal University of Amazonas, under protocol no. CAAE 0099.0.115.000-09. Thus, this work also investigated whether there was any relation between infestations in children and individuals of other age groups by Hair samples were collected after each hair cut and individually comparing prevalence rates obtained in these two periods of the year, i.e. placed in properly labeled plastic bags. Information concerning their the occurrence determined during months that composed the academic origin and the physical appearance of the customers was recorded periods of kindergarten, elementary and junior education and the months on a form. The samples were sent to the laboratory and analyzed these children were on vacation. with a magnifying glass and microscope to check the condition and characteristics of the hair18 (size, type, color, and thickness). Samples The χ2 statistical test was used to compare two or more proportions. considered positive were those on which any of the developmental stages In cases where significant differences between more than two proportions of lice (eggs, nymphs or adults) were identified, irrespective of whether were observed, the data were submitted to angular transformation (p’ = they were viable at the time of examination. arc. sen √p’), followed by multiple comparisons using the Tukey range test4,29. A 5% level of significance was adopted29. Confidence intervals Hair characteristics were determined according to BORGES & (95%) were also calculated for the occurrence and prevalence ratios MENDES2, following training with their method prior to evaluation. The determined. hair samples were classified according to the following characteristics: length, color and thickness. Hair of up to 3 cm in length was considered RESULTS 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 An occurrence of 2.84% was detected for the 1,860 hair samples four categories: light (blonde and red), dark (brown and black), gray, and obtained from the 18 establishments surveyed. The highest occurrence dyed. Regarding thickness, there were two categories: thin and thick2. rates were found in the south-central (3.5%) and west-central areas (3.47%). However, the differences between these rates and those The ethnicity, sex and age group of the clients whose hair was obtained for other areas, where the rates were lower, were not significant 2 sampled were determined based on observations regarding the physical (χ 0.05,4 = 1.67, p > 0.75) (Table 1). appearance of the individuals during their haircuts. Regarding ethnicity, the individuals were divided into blacks and non-blacks. Age group was Of the 53 positive samples, 37 had non-viable lice eggs, three had established according to WHO30: young people, those who appeared to adult lice and 13 had viable eggs. be 15-29 years old; adults, 30-59 years old; and elderly, those over 60 years old2. In general, the hair characteristics, length, color and thickness, did not significantly influence the prevalence rates obtained (Table 3). The socioeconomic profile of the clients was inferred according to 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

No. of samples No. of samples Occurrence rate (%) Prevalence ratio Establishments Location examined positives 95% confidence interval 95% confidence interval I to IV 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) Total - 1860 53 - - a = occurrence rates with different letters are statistically different from each other by the Tukey test at a 5% level of significance.

240 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.

such that non-black individuals and the elderly had higher occurrence The results gathered here, together with those obtained for school 2 2 4 rates (χ 0.05,1 = 5.05, p = 0.025; χ 0.05,2 = 7.65, p < 0.025) (Table 2). The children in the same city , reflect the degree of importance of this socioeconomic profiles of the clients, inferred from the price of a haircut ectoparasitosis for the population. However, it should be noted that charged by the establishment, did not significantly influence the rate of given the lower sensitivity of this technique in relation to direct scalp 2 head lice infestation either (χ 0.05,2 = 2.16, p > 0.25) (Table 2). examination, the real rate of occurrence of the study population is likely to be greater than that found. The differences in sensitivity are partly Analysis of the monthly distribution of the occurrence rates revealed due to factors like the length of the hair that is cut18 and the number of that the highest rate was obtained in July, a school holiday period (Fig. 1). people aware that they have head lice, who would not choose to cut their The rates obtained for the months that composed the academic periods hair in such establishments. An additional limitation of this study is the and the months that composed the school holiday period of school-age fact that the establishments were not sampled every month of the year. children were grouped and compared; the rate determined for the holiday Another fact that influences the monthly head lice distribution is that 2 period was significantly higher χ( 0.05,1 = 13.28, p < 0.01) (Table 2). part of the inactive infestation results from active infestations acquired in the months prior to those in which the hair samples were obtained. DISCUSSION However, considering that numerous establishments were sampled throughout the study period, the data obtained over the months were The use of hair samples collected from barbershops for estimating grouped into the academic and vacation periods of the year, while the the prevalence of pediculosis capitis was adopted for the first time in number of hair samples obtained were satisfactory from a statistical point Brazil by18. The overall prevalence of head lice infestation in this study of view; possible bias resulting from this sampling procedure does not was relatively low, compared with other studies conducted in the state significantly influence the statistical analysis. of Minas Gerais using this same technique3. However, the data obtained corroborate the results of recent research among school children in It is also worth highlighting, particularly in this study, that the portion Manaus using the head inspection technique4, in which an occurrence of the general population having a less favorable socioeconomic profile rate of 18% was obtained in children, a rate that is also considered low may not be able to easily afford a haircut, and thus this population group for this age group compared with studies conducted in other regions of may not have been adequately sampled. The fact that no significant the country13,23. 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 95% confidence interval 95% confidence interval Sex 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) Ethnicity 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) Age group 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 4.76 (1.89 - 7.63) Aa 2.50 (1.18 - 5.30) Socioeconomic profileb 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) School holidays 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) 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 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.

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

Occurrence rate (%) Prevalence ratio No. of samples examined No. of samples positives 95% confidence interval 95% confidence interval Hair length 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) 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) Hair color Dyed 197 2 1.01 (0 - 2.4) Aa - 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) Hair thickness 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) a = occurrence rates with different letters are statistically different from each other by the Tukey test at a 5% level of significance.

25 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. 20 Analysis of the monthly variations of infestation and comparison with the data obtained for schoolchildren in Manaus and other Brazilian es cities4, 13 indicated that the profile of temporal variation of infestation in 15 adults is different to the temporal variation in children. The profile of

sampl

e infestations in adults appears to be influenced by the difference in time

v 10 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 posi 2,13,20 5 of head lice infestation 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 0 respective families. The greater interaction between children and their jan feb mar apr may jun jul aug sep oct nov dec families during the vacations partially explains the increase in prevalence months in other age groups during this period of the year. *Number of samples examined, N = 1860.

Fig. 1 - Monthly distribution of samples examined and samples positive for head lice, collected Several authors mention the influence of temperature and humidity 13,22 from 18 barber shops and beauty parlors, in Manaus, Amazonas State, Northern Brazil, from on the temporal profile of infestation . The North and Northeastern August 2010 to July 2013. School holiday period: Jan, Jun, Jul and Dec. Academic periods: regions of Brazil are subject to less annual variations in temperature. Feb, Mar, Apr, May, Aug, Sep, Oct and Nov. However, humidity has greater variation and is considered relatively low in some months, in which the lowest occurrence was observed. This rates obtained for the different socioeconomic statuses, according to result is consistent with findings reported by authors who also performed the price of a haircut charged by the establishment, seems to strengthen studies in the northeastern region13. this hypothesis. Thus, procedures must be adopted to obtain more representative samples of this population group. One procedure adopted A higher occurrence rate among the elderly was also observed by BORGES et al.3 to sample this population group was the examination in Minas Gerais3. The data obtained herein, together with those just of samples of free haircuts during events promoted by charities and described, reinforce the hypothesis that the elderly are the second most

242 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.

commonly affected age group in some cities. This is probably due, this work and the National Council of Scientific and Technological at least in part, to the following factors: they spend more time with Development (CNPq) and the Amazon Research Foundation (FAPEAM) children than other family members, and a number of the elderly are for providing study grants. dependent on caregiver and live in or attend nursing homes or similar collective environments, under conditions conducive to the transmission FINANCIAL SUPPORT of ectoparasitic diseases. The National Council of Scientific and Technological Development The higher occurrence among non-blacks verified in this study was (CNPq/MCT-Amazônia 2006/2008) provided support for this study. also observed in American schools that attend a multiracial population of children14. In contrast, several studies have reported higher prevalence CONFLICTS OF INTEREST among black individuals2,4,6. These divergent reports could be related to several factors that have comparative influence on head lice infestation, The authors declare that there are no conflicts of interest. such as different forms of hair among different ethnic groups, variations in cultural habits and different socioeconomic conditions5,17. REFERENCES

It should be emphasized that scalp examination is a more sensitive 1. Bechelli LM, Haddad N, Pimenta WP, Pagnano PMG, Melchior JE, Fregnan RC, et al. technique for diagnosing this parasitosis than the examination of cut hair Epidemiological survey of skin diseases in schoolchildren living in the Purus Valley (Acre State, Amazonia, Brazil). Dermatologica. 1981;163:78-93. samples. Thus, scalp examination should be used whenever possible. Despite their limitations, the diagnostic technique and sampling system 2. Borges R, Mendes J. Epidemiological aspects of head lice in children attending day care adopted allowed for the accomplishment of the main objectives of the centers, urban and rural schools in Uberlândia, Central Brazil. Mem Inst Oswaldo study, i.e. obtain information concerning the degree of importance of Cruz. 2002;97:189-92. this ectoparasitic disease and the factors that most strongly influence 3. Borges R, Silva JJ, Rodrigues RM, Mendes J. Prevalence and monthly distribution of the occurrence of head lice in the study population. However, additional head lice using two diagnostic procedures in several age groups in Uberlândia, State procedures that would make the technique more sensitive while of Minas Gerais, Southeastern Brazil. Rev Soc Bras Med Trop. 2007;40:247-9. maintaining the ease of its application among the target population are required. Measures that improve sampling among the section of 4. Borges-Moroni R, Mendes J, Justiniano SCB, Bindá AGL. Pediculose do couro cabeludo the population that finds it difficult or impossible to pay for a haircut em crianças de creches e escolas de Manaus, Amazonas, Brasil. Rev Patol Trop. 2011;40:263-70. would grant a more accurate view of the degree of importance of this ectoparasitic disease in the population group with low or no purchasing 5. Brenner LR. How to control lice. Nurs Care. 1977;10:20-2. power, which is usually the group most affected by this parasitosis. 6. Carzola D, Ruiz A, Acosta M. Estudio clínico-epidemiológico sobre Pediculosis capitis RESUMO en escolares de Coro, estado Falcón, Venezuela. Invest Clin. 2007;48:445-57. 7. Castro DC, Abrahamovich AH, Cicichino AC, Rigoni AM, Raffaeli C, de Barrio A. Pediculose da cabeça em amostras de cabelos de jovens, adultos e Prevalencia y estacionalidad de la Pediculosis capitis en la población infante-juvenil idosos em Manaus, Estado do Amazonas, Brasil de la región sanitaria, Buenos Aires, Argentina. Rev Saude Publica. 1994;28:295-9.

Estudo sobre a pediculose do couro cabeludo em jovens, adultos 8. Catalá S, Carrizo L, Córdoba M, Khairallah R, Moschella F, Bocca JN, et al. Prevalência e intensidade da infestação por Pediculus humanus capitis em escolares de seis a e idosos foi realizado de agosto de 2010 a julho de 2013 em Manaus onze anos. Rev Soc Bras Med Trop. 2004;37:499-501. - AM, região norte do Brasil. Amostras de cabelos obtidas de 1.860 indivíduos em 18 barbearias e salões de beleza foram examinadas à 9. Catalá S, Junco L, Vaporaky R. Pediculus capitis infestation according to sex and social procura do parasito. Procurou-se verificar a ocorrência da pediculose e factors in Argentina. Rev Saude Publica. 2005;39:438-43. sua associação com fatores tais como: sexo, idade, etnia, características 10. Donaldson RJ. The head louse in England: prevalence amongst schoolchildren. R Soc dos cabelos e perfil socioeconômico dos clientes dos salões, localização Health J. 1976;96:55-7. 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. 11. Falagas ME, Matthaiou DK, Rafailidis PI, Panos G, Pappas G. Worldwide prevalence of Também se observou maior prevalência no período de férias escolares head lice. Emerg Infect Dis. 2008;14:1493-4. do ensino fundamental e médio. Os resultados indicam que a ocorrência 12. Feldmeier H, Heukelbach J. Epidermal parasitic skin diseases: a neglected category of da pediculose em jovens, adultos e idosos em Manaus é relativamente poverty-associated plagues. Bull World Health Organ. 2009;87:152-9. baixa em comparação com as encontradas em crianças e as encontradas em outras regiões do país. Depois das crianças, os idosos seriam os 13. Heukelbach J, Wilcket T, Winter B, Feldmeier H. Epidemiology and morbidity of mais acometidos. O estudo também indica a necessidade de adotar scabies and pediculosis capitis in resource-poor communities in Brazil. Br J Dermatol. procedimentos adicionais para melhor amostrar a faixa da população 2005;153:150-6. com menor ou com nenhum poder aquisitivo, a qual geralmente é a mais 14. Hoffmann G. Epidemiology and control of pediculosis capitis infestation in the acometida por esta ectoparasitose. Federal Republic of Germany. J R Soc Health. 1983;103:88-92.

ACKNOWLEDGMENTS 15. Instituto Brasileiro de Geografia e Estatística (IBGE). Updated 2013 January 2010. Rio de Janeiro: IBGE; 2013. [cited 2013 Oct 29]. Available from: http://www.ibge. gov.br The authors would like to thank the establishments involved in

243 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.

16. Jahnke C, Bauer E, Hengge UR, Feldemeier H. Accuracy of diagnosis of pediculosis 23. Mirza A, Shamsi A. Head lice infestation. Innovait. 2010;3:85-90. capitis: visual inspection vs wet combing. Arch Dermatol. 2009;145:309-13. 24. Moradi AR, Zahirnia AH, Alipour AM, Eskandari Z. The prevalence of pediculosis 17. Linardi PM, Botelho JR, Maria M, Cunha HC. Crendices e falsos conceitos que capitis in primary school students in Bahar, Hamadan Province, Iran. J Res Health dificultam ações profiláticas contra o piolho e a pediculose. J Pediatr. 1988a;64:248- Sci. 2009;9:45-9. 55. 25. Motovali-Emami M, Aflatoonia MR, Fekri A, Yazdi ME. Epidemiological aspects of 18. Linardi PM, Maria M, Botelho JR, Cunha HC, Ferreira JB. Prevalence of nits and pediculosis capitis and treatment evaluation in primary-school children in Iran. Pak lice in samples of cut hair from floors of barbershops and beauty parlors in Belo J Biol Sci. 2008;11:260-4. Horizonte, Minas Gerais State, Brazil. Mem Inst Oswaldo Cruz. 1988b;83:471-4. 26. Oh J, Lee IY, Lee WJ, Seo M, Park SA, Lee SH, et al. Prevalence of pediculosis 19. Linardi PM, De Maria M, Botelho JR, Cunha HC, Ferreira JB. Pediculose capitis: capitis among Korean children. Parasitol Res. 2010;107:1415-9. prevalência em escolares da rede municipal pública de Belo Horizonte, Minas Gerais, Brasil. Mem Inst Oswaldo Cruz. 1989;84:327-31. 27. Toloza A, Vassena C, Gallardo A, González-Audino P, Picollo MI. Epidemiology of Pediculosis capitis in elementary schools of Buenos Aires, Argentina. Parasitol Res. 20. Linardi PM, De Maria M, Botelho JR, Hosken CI, Cunha HC. Alguns fatores 2009;104:1295-8. epidemiológicos relativos à infestação humana por Pediculus capitis (ANOPLURA, PEDICULIDAE) em Belo Horizonte, Minas Gerais, Brasil. Rev Bras Entomol. 28. Vladeni S, Petinaki E, Roussaki-Schultze A. Hair characteristics and lice infestation. 1995;39:921-9. Data from schoolchildren in Greece. J Eur Acad Dermatol Venereol. 2011;25:118-9.

21. Manrique-Saide P, Pavía-Ruz N, Rodriguez-Buenfil JC, Herrera Herrera R, Goméz- 29. Zar JF. Biostatistical analysis. New Jersey: Prentice Hall; 1999. Ruiz P, Pilger D. Prevalence of pediculosis capitis in children from a rural school in Yucatan, Mexico. Rev Inst Med Trop Sao Paulo. 2011;53:325-7. 30. World Health Organization. Young people´s health: a challenge for society. Report of a WHO Study Group on Young People and “Health for All by the Year 2000”. 22. Mimouni D, Ankol OE, Gdalevich M, Groto I, Davidovitch N, Zangvil E. Seasonality Geneva: WHO; 1986. (Technical Report Series 731). trends of Pediculosis capitis and Phthirus pubis in a young adult population: follow- up of 20 years. J Eur Acad Dermatol Venereol. 2002;16:257-9. Received: 15 January 2014 Accepted: 30 July 2014

244 Rev. Inst. Med. Trop. Sao Paulo 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 To combat the formation of free radicals and neutralize them before they cause damage, biological systems use molecules and enzymes such Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi, as reduced glutathione (GSH), superoxide dismutase (SOD), catalase, a flagellate protozoan9 that infects approximately 12–14 million people in glutathione peroxidase (GSH-Px), and vitamin E. Others, such as vitamin Latin America15. After contact with the parasite, patients develop the acute C (ascorbic acid), glutathione reductase (GSH-Rd), and glutathione phase of the illness, which may be asymptomatic in some individuals or peroxidase (GSH-Px) are used to repair damage that has already been may show non-specific symptoms, such as fever, tachycardia, weakness, done42. and lymphadenopathy. This evolves into an indeterminate phase, with no clinical signs, but with subclinical pathologies17. Many years after A study by WEYERS et al.44 showed that appropriate doses of infection, about 30% of the patients develop the chronic phase of the vitamin C can have a preventive effect against the lipid peroxidation disease, which has characteristic signs including megaesophagus, induced by free radicals; both those formed naturally and those caused megacolon and cardiomegaly24. During the chronic phase, the presence by exogenous compounds in mice experimentally dosed with the of microfoci parasites leads to the constant production of interleukin-12, antibacterial drug ciprofloxacin. Vitamin C decreased the oxidative which activates macrophages and generates a Th1 immune response stress on the liver and lipid peroxidation in the mouse kidney caused consistent with delayed hypersensitivity, which leads to tissue damage14,24. by this drug43. According to MAY33, ascorbic acid’s ability to reduce oxidative stress would depend on its concentration and its potential for Much of the damage to the host in Chagas disease is caused by an recycling in blood and endothelium. excess of free radicals, more specifically by the metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These are However researches of CHEN et al.11 showed that pharmacological produced by phagocytic cells stimulated by inflammatory mediators in doses of ascorbic acid favored the cytotoxicity of tumor cells by formation tissues parasitized by T. cruzi. They may harm any cellular component, of hydrogen peroxide (H2O2), leading to the decreased growth of tumors but the components that are affected most strongly are the cellular in mice due to pro-oxidant action. For LEVINE et al.29 ascorbate could be membranes18,23,42,43. 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.

22 pathogens, where the production of H2O2 and ROS could be beneficial day after infection for histopathologic analysis after CO2 euthanasia . in disease progression. The tissues were embedded in paraffin and 5 μm sections were stained with hematoxylin-eosin and examined under a light microscope at A study by MAÇAO et al.30 showed that oxidative stress was a magnification of 400×. Five sequential histological sections were controlled in the myocardium of patients in the chronic phase of Chagas examined for each fragment, and the fragments were analyzed and graded disease when 500 mg of vitamin C and 800 UI of vitamin E were for inflammation and amastigote nests for a total of 10 high magnification administered daily for a six-month period, probably as a consequence of fields for each type of tissue. The investigator examining the sections vitamin E consumption and antioxidant protection. According to KOURY was unaware of the grouping of the mice. & DONANGELO28, vitamins C and E are important “street sweepers” of free radicals. 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. Nonetheless, CHEN10 noted that with a lower concentration of This way, inflammation and/or necrosis were examined separately to the vitamin E, the administration of high doses of vitamin C appears to number of amastigotes for each animal: “+” signified mild inflammation, promote lipid peroxidation and significantly decreases the animals’ necrosis and rare amastigote nests; “++” moderate inflammation, antioxidant potential. necrosis and moderate number of amastigote nests; and “+++”, intense inflammation, necrosis and frequent amastigote nests. Based on these studies, the authors decided to investigate the effectiveness of vitamin C in combating the oxidative insult caused by Biochemistry: Blood samples were collected on the 180th day after progressive T. cruzi inflammation during the evolution of Chagas disease in infection. The mice were exposed to CO2 (100% CO2) for a few seconds its chronic phase, by administering two different daily dosages of vitamin as a form of anesthetizing them, and then the blood was collected by

C to mice experimentally infected with the T. cruzi QM1strain, since the cardiac puncture into heparin. This method of euthanasia was chosen common sense of its role in antioxidant defense makes its use indiscriminate as studies conducted by GODIN & GARNETT22 and BILLERT & among people, including chronic carriers of Chagas disease. DROBNICK5 show that exposure to this gas may not increase oxidative damage to tissues. MATERIALS AND METHODS Blood was centrifuged at 1500 g immediately after collection and the Infection of mice: Thirty 20-day-old male “Swiss” mice, weighing plasma stored at -80 ºC. For the measurement of vitamin C, 200 µL plasma on average 13 grams, were intraperitoneally infected with 5.0 × 104 samples were immediately acidified with 800 µL 5% trichloroacetic acid trypomastigotes of T. cruzi QM1 strain32 via blood from other previously- and stored at -80 ºC for later analysis. One fragment of heart muscle from infected mice, which was obtained by cardiac puncture into heparin. The each mouse was immediately frozen in liquid nitrogen and subsequently trypomastigotes were counted following the BRENER6 method and the stored at -80 °C. Oxidative stress markers were determined around 3 - 5 intraperitoneal infection was performed by inoculating 0.1 mL of blood weeks after sampling. All samples were stored at -80 ºC before analysis. 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 Thiobarbituric acid reactive substances (TBARS) were used as that received the dose of vitamin C (D60); see next section for the doses biomarkers of lipid peroxidation in plasma and heart muscle and given), and Vit C 500 (mice that received a dosage of vitamin C (D500)). measured using a method adapted from COSTA et al.13. The total The animals were kept in individual cages to facilitate handling and fed concentration of TBARS was determined by the difference in absorbance with the standard Nuvilab CR-1® diet, which is composed of: ground between samples and in a standard solution of malondialdehyde. The whole corn, soybean meal, wheat bran, calcium carbonate, calcium total peroxide in the cardiac muscle and plasma was determined using phosphate, sodium chloride, vegetable fat, mineral vitamin premix, amino the FOX method, as described by SÖDERGREN et al.38. This method acid, and water ad libitum. uses a comparison with the standard curve of H2O2. Quantification of plasma glutathione was performed by adapting the method described by Calculation of vitamin C dosage and treatment: Vit C 60 group COSTA et al.13 using the standard curve of GSH. (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 The vitamin C concentration in plasma and heart muscle was in 10 μL of human mineral water (Soft®). Vit C 500 group (a D500 dose) determined according to BESSEY’s4 method, 100 µL of a solution was given a daily dose of 500 mg of vitamin C, equal to the average containing 2,4-dinitrophenylhydrazine (2%), thiourea (5%), and copper weight of the mice × (7.14 × 10–3 mg per gram of weight) diluted in 10 sulfate (0.6%) in sulfuric acid (25%) added to 300 µL of acidified μL of human mineral water (Soft®). The P group was a placebo group: plasma. After a 4-hour incubation in a 37 °C water bath, 200 µL sulfuric each animal received 10 μL of human mineral water (Soft®) daily. Every acid (65%) was added and the solution was incubated for 20 min at morning, all mice were treated orally with 10 μL of vitamin C (D60 or room temperature. Reading was performed on a spectrophotometer D500) in mineral water or mineral water (Soft®) alone, using a Gilson (Spectramax M5, Molecular Devices) at 520 nm and compared with the automatic pipette. The three groups were treated for 180 days, starting standard curve of vitamin C. from the infection date. Vitamin C used in this research was Cewin® (in drops doses) from Sanifi-Aventis Laboratory. Statistics: The results were analyzed by Normality Test (Shapiro- Wilks) to verify that the data followed a normal distribution and Histopathologic analysis: A fragment of heart, colon and skeletal homogeneity of variances in the groups of Levene’s test. Statistical thigh muscle from all mice that survived was collected on the 180th inference was performed using ANOVA one-way and Post-hoc analyses

246 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.

were performed with the Tukey (HSD) test1. The significance level used (lipid oxidation levels) in the myocardium showed no statistically was 5%. significant differences between the dosages.

This study was approved by the Ethics Committee of the Faculty The vitamin C results showed no statistical differences in plasma and of Medicine of Marília (FAMEMA) under number 133/10. The manner myocardium between the three groups with p > 0.05 for all comparisons. of treatment, care, and euthanasia of mice followed the standards set by Colégio Brasileiro de Experimentação Animal/Brazilian College of DISCUSSION Animal Experimentation (Cobea)39. To date, no research has been done to test the effectiveness of vitamin RESULTS C in combating ROS and RNS produced during the evolution of Chagas disease. This study has shown that oral administration of 500 mg of After 180 days of treatment two animals had died in the placebo vitamin C, per 180 days, in mice infected with T. cruzi can be harmful group, three had died in group Vit C 60, and four had died in group Vit to the host, leading to increased total peroxide and TBARS, which may C 500. The study was therefore performed on eight animals in group P, increase the severity of lesions, characteristic of chronic phase of this seven in group Vit C 60, and six in group Vit C 500. Group Vit C 500 disease. suffered greater damage to the skeletal muscle, showing a greater degree of inflammation than group Vit C 60 and a greater degree of necrosis In this study, the vitamin C doses of 60 mg and 500 mg provided than group P (Table 1). With regards to cardiac muscle and the colon, all to groups Vit C 60 and Vit C 500 resulted in no statistically significant animals in group Vit C 500 had inflammation, with mice of this group differences in myocardial and plasma vitamin C concentrations relative showing a greater level of damage. 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 An analysis of TBARS, total peroxide, vitamin C, and plasma GSH is tissue collection could be explained by the physiological mechanism of shown in Table 2 and an analysis of TBARS, total peroxide and vitamin C control of concentrations of vitamin C, where ascorbate concentrations in the myocardium is shown in Table 3. Group P had lower total peroxide in plasma and tissue are tightly controlled. According to LEVINE et al.29, concentrations in plasma than group Vit C 500 (p = 0.021, Table 2). The the vitamin C concentration was tightly controlled by absorption, tissue TBARS analysis in plasma showed a statistically significant difference transport and renal reabsorption and excretion. 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 Surprisingly, the dosage of 500 mg caused detrimental repercussions statistically significant difference between the groups in the plasma GSH to the host. This is confirmed by the study’s results regarding the number (p > 0.05, Table 2). of mice that survived the treatment and the histopathologic findings, in which group Vit C 500 was shown to have suffered greater skeletal, There was a higher total production of peroxides in the myocardium cardiac muscle and colonic tissue damage, causing the death of a large of group Vit C 500 than in group P (p = 0.050, Table 3). TBARS analysis 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 VitC60 VitC500

Amastigote Amastigote Amastigote Inflammation Necrosis Inflammation Necrosis Inflammation Necrosis nests nests nests % (8) % (8) % (8)* % (7) % (7) % (7) % (6) % (6) % (6) + 12.5% (1) — 50.0% (4) — — 42.6% (3) 50.0% (3) — 100% (6) Skeletal ++ — — — — — 14.3% (1) — — — muscle +++ — — — — — — — — — + 50.0% (4) 12.5% (1) — 85.7% (6) — — 50.0% (3) — — Cardiac ++ 25.0% (2) — — — — — 50.0% (3) — — muscle +++ — — — — — — — — — + 62.5% (5) — — 42.9% (3) 28.6% (2) — 50.0% (3) — — Colon ++ 25.0% (2) — — 28.6% (2) — — 50.0% (3) — — +++ — — — — — — — — —

247 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.

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 P & VitC60 TBARS (µmol/L) 34.8 (SD = 2.5) 34.8 (SD = 2.8) 41.0 (SD = 6.1) P & VitC500* VitC60 &VitC500* P & VitC60 Total peroxide (µmol H O 2 2 88.4 (SD = 35.2) 134.3 (SD = 38.1) 148.6 (SD = 40.0) P & VitC500* equivalent/L) VitC60 & VitC500 P & VitC60 Vitamin C (mg/dL) 0.28 (SD = 0.07) 0.29 (SD = 0.07) 0.33 (SD = 0.04) P & VitC500 VitC60 & VitC500 P & VitC60 GSH (mmol/L) 237.13 (SD = 29.45) 203.30 (SD = 46.77) 183.32 (SD = 25.59) P & VitC500 VitC60 & VitC500 * 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 P & VitC60 TBARS (µmol/g protein) 82.4 (SD = 20.3) 73.6 (SD = 14.3) 100.4 (SD = 27.1) P & VitC500 VitC60 &VitC500 P & VitC60 Total peroxide (µmol H O 2 2 161.4 (SD = 82.3) 304.1 (SD = 172.7) 368.4 (SD = 188.6) P & VitC500* equivalent/g protein) VitC60 &VitC500 P & VitC60 Vitamin C (μg/g prot) 6.61 (SD = 1.13) 6.76 (SD = 0.74) 6.97 (SD = 0.76) P & VitC500 VitC60 &VitC500 * Groups differ by Tukey test at 5% probability.

Corroborating the histopathologic findings, a greater total peroxide Nevertheless, the aim of this study was to evaluate the effects production in group Vit C 500 than in group P was observed, both in of the isolated uses of ascorbic acid. These results show that daily plasma (p = 0.021) and in the myocardium (p = 0.050), as well as an supplementation, only with the equivalent of 500 mg of vitamin C, is increase in plasma TBARS concentrations in group Vit C 500 (p = 0.021). coupled with a statistically significant increase in production of free Although a tendency of decrease in GSH concentration of group Vit C radicals and lipid peroxidation between Vit C 60 Vit C 500 (p = 0.022), 500 was observed, suggesting that antioxidant response was activated to as shown by the result. neutralize ROS and RNS during disease progression, these results were not statistically significant. Although the antioxidant properties of vitamin C are well established2,20,26, it is still debatable that it beholds a possible pro- Researches of MAÇAO et al.30 demonstrated a decrease in TBARS and oxidant8,16,37 and an anti / pro-inflammatory effect27,34. increase in GSH when chronic chagasic patients were supplemented daily with 500 mg vitamin C and 800 IU vitamin E. The effectiveness of these In vitro studies performed by CLEMENT et al.12, showed that, in antioxidants has been studied by various authors3,46, who indicated that both high concentrations, vitamin C can exert a pro-oxidant effect before vitamins C and E operate in the context of an integrated system in which the antioxidant, but in vivo the biological relevance of these events remains overall antioxidant status is under homeostatic control, so that the change controversial8. 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. However, in case of an injury inflicted by these tissues, the possibility

248 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.

of free transition metal Fe3+ and Cu2+ increases, which may be reduced esquelético. Resultados: Animais que receberam diariamente uma by ascorbate7. In vitro, the interaction of these reduced metals with dosagem equivalente a 500 mg de vitamina C apresentaram aumento . H2O2 produce hydroxyl radicals and lipid alkoxyl radicals (LO ), causing na produção de ROS e RNS no plasma e no miocárdio e maior grau de oxidative damage, and the ascorbate would then act as pro oxidant. inflamação e necrose em músculo esquelético em comparação àqueles que receberam doses menores ou nenhuma vitamina C. Conclusão: Embora Therefore, it is possible that a high intake of vitamin C could muitas pesquisas tenham mostrado o efeito antioxidante da vitamina C, be harmful to persons who have chronic inflammatory conditions19, nossos resultados mostraram que os animais que foram expostos a 500 mg because it would cause the interaction of these catalytically active de vitamina C apresentaram maior dano tecidual na fase crônica da doença metals with ascorbate, as in the chronic phase of Chagas disease. This de Chagas, provavelmente devido a ações paradoxais desta substância, would also cause damage to surrounding tissues, which contribute to the onde nesta patologia, poderá agir como pró-oxidante ou pró-inflamatória. inflammatory process, besides the fact that their own ROS amplify the release of pro-inflammatory cytokines. ACKNOWLEDGEMENTS

Although several studies in humans and other species delineate the Statistical assistance made through the Extension Project “Statistical functional significance of vitamin C in resistance to infection40,41, the advice to students and teachers of undergraduate and graduate programs effects of a high dose on the activity of the immune system is still scarce. of FFC-CM, UNESP and external researchers,” coordinated by Prof. Dr. Sebastião Marcos Ribeiro de Carvalho, Department of Educational Studies of HORNIG et al.25 reported the increased bioavailability of Psychology, the FFC - Campus Marília, UNESP. nitric oxide in patients with chronic heart failure after arterial and oral administration of vitamin C and NOH et al.36, discovered that megadoses CONFLICT OF INTEREST 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 The authors of “Effects of vitamin C supplementation on the chronic inflammatory cytokines. Moreover, MIKIROVA et al.34 discovered that phase of Chagas disease” declare no conflicts of interest in developing high doses of vitamin C reduces inflammation in patients with cancer. this study.

These results showed an increase in the inflammatory process with FINANCIAL SUPPORT 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 authors thank the São Paulo State Foundation for Research the administration of 500 mg of vitamin C provides a pro-oxidant and Support (FAPESP) for financial support for this project. pro-inflammatory medium to these animals. Since the effects were not observed in shorter periods of supplementation31, these results may indicate REFERENCES 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 1. Armitage P, Berry G. Estadística para la investigación biomédica. 3ª ed. Madrid: results will be elucidated through future research on the technique of Harcourt Brace; 1997. immunohistochemistry and may highlight whether the exacerbation of the 2. Barreiros ALBS, David JM, David PJ. Estresse oxidativo: relação entre geração de inflammatory process is associated with the presence of the parasite or its espécies reativas e defesa do organismo. Quim Nova. 2006;29:113-23. cell debris, or if it occurred due to the actions of vitamin C. 3. Benzie IF, Strain JJ. Effect of vitamin C supplementation on concentrations of vitamins Therefore, these results indicate the need for further investigation C and E in fasting plasma. Asia Pac J Clin Nutr. 1999;8:207-10. to elucidate the mechanisms whereby the high dosage of vitamin C 4. Bessey OA. Ascorbic acid. Microchemical methods. In: Vitamin methods. New York: 35,45 may operate in the parasite or in the harmful events identified by Academic Press; 1960. this research. 5. Billert H, Drobnick L. Carbon dioxide and oxidative stress. Nowiny Lekarskie. RESUMO 2001,70:753-62. 6. Brener Z. Contribuição ao estudo da terapeutica experimental da doenca de Chagas. Efeitos da suplementação de vitamina C na fase crônica da doença [Dissertação]. Belo Horizonte: Universidade Federal de Minas Gerais; 1961. de Chagas 7. Buettner GR, Anne Jurkiewicz BA. Catalytic metals, ascorbate and free radicals: Introdução: Para verificar a eficácia da vitamina C em combater o combinations to avoid. Radiat Res. 1996;145:532-41. insulto oxidativo causado pelo Trypanosoma cruzi durante a evolução 8. Carr A, Frei B. Does vitamin C act as a pro-oxidant under physiological conditions? da fase crônica da doença de Chagas, camundongos Swiss foram FASEB J. 1999;13:1007-24. previamente infectados via intraperitoneal com 5.0 × 104 tripomastigotas da cepa QM1 de T. cruzi. Métodos: Camundongos foram suplementados 9. Catalán LN. Enfermedad de Chagas. Acta Gastroenterol Latinoam. 2010;21:292-7. com duas diferentes doses de vitamina C por 180 dias. Foram mensurados 10. Chen LH. Interaction of vitamin E and ascorbic acid. In vivo. 1989;3:199-209. os níveis de peroxidação lipídica (indicado por substâncias reativas ao ácido tiobarbitúrico-TBARS), peróxido total, vitamina C, e glutationa 11. Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, et al. Pharmacologic reduzida no plasma e TBARS, peróxido total e vitamina C no miocárdio, doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor e foi realizado o estudo histopatológico em coração, cólon e músculo xenografts in mice. Proc Natl Acad Sci USA. 2008;105:11105-9.

249 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.

12. Clément MV, Ramalingam J, Long LH, Halliwell B. The in vitro cytotoxicity of 31. Marim RG, Gusmão AS, Castanho REP, Demenici R, Therezo ALS, Jordão Júnior ascorbate depends on the culture medium used to perform the assay and involves AA, et al. Effects of vitamin C supplementation in Chagas disease acute phase on hydrogen peroxide. Antioxid Redox Signal. 2001;3:157-63. experimentally infected mice with Trypanosoma cruzi QM1 strain. Rev Inst Med Trop Sao Paulo. 2012;54:319-23. 13. Costa CM, Santos RCC, Lima EA. A simple automated procedure for thiol measurement human and serum samples. J Bras Patol Med Lab. 2006;42:345-50. 32. Martins LPA, Marcili A, Castanho REP, Therezo ALS, Oliveira JCP, Suzuki RB, et al. Rural Triatoma rubrovaria from southern Brazil harbors Trypanosoma cruzi of 14. Cunha-Neto E, Rizzo LV, A lbuquerque F, Abel L, Guilherme L, Bocchi E, et lineage IIc. Am J Trop Med Hyg. 2008;79:427-34. al. Cytokine production profile of heart-infiltrating T cells in Chagas’disease cardiomyopathy. Braz J Med Biol Res. 1998;31:133-7. 33. May JM. How does ascorbic acid prevent endothelial dysfunction? Free Radic Biol Med. 2000;28:1421-9. 15. Dias JCP. Globalização, iniquidade e doença de Chagas. Cad Saúde Pública. 2007;23(Suppl 1):S13-S22. 34. Mikirova N, Casciari J, Rogers A, Taylor P. Effect of high-dose intravenous vitamin C on inflammation in cancer patients. J Transl Med. 2012;10:189. 16. Duarte TL, Lunec J. Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Radic Res. 2005;39:671-86. 35. Monteiro G, Horta BB, Pimenta DC, Augusto O, Netto LE. Reduction of 1-Cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing 17. Dutra WO, Menezes CAS, Villani FNA, Costa GC, Silveira ABM, Reis DA, et another function of vitamin C. Proc Natl Acad Sci USA. 2007;104:4886-91. al. Cellular and genetic mechanisms involved in generation of protective and pathogenic immune responses in human Chagas disease. Mem Inst Oswaldo Cruz. 36. Noh K, Lim H, Moon S, Kang JA, Wang JL, Lee D, et al. Mega-dose vitamin C 2009;104(Suppl 1):208-18. modulates T cell functions in Balb/c mice only when administered during T cell activation. Immunol Lett. 2005;98;63-72. 18. Ferreira ALA, Matsubara LS. Radicais livres: conceitos, doenças relacionadas, sistema de defesa e estresse oxidativo. Rev Assoc Med Bras. 1997;43:61-8. 37. Podmore ID, Griffiths HR, Herbert KE, Mistry P, Lunec J. Vitamin C exhibits pro- oxidant properties. Nature. 1998;392:559. 19. Fisher AEO, Naughton DP. Vitamin C contributes to inflammation via radical generating mechanisms: a cautionary note. Medical Hypotheses. 2003;61(5-6):657- 38. Södergren E, Nouroz-Zadeh J, Berglund L, Vessby B. Re-evaluation of the 60. ferrous oxidation in xylenol orange assay for the measurement of plasma lipide hydroperoxides. J Biochem Biophys Methods. 1998;37:137-46. 20. Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci USA. 1989;86:6377-81. 39. Sogayar R. Ética na experimentação animal: consciência & ação. Botucatu: Fundação de Estudos e Pesquisas Agrícolas e Florestais; 2006. 21. Gey KF. Vitamins E plus C and interacting constituents required for optimal health. A critical and constructive review of epidemiology and supplementation data regarding 40. Ströhle A, Wolters M, Hahn A. Micronutrients at the interface between inflammation cardiovascular disease and cancer. Biofactors. 1998;7:113-74. and infection - ascorbic acid and calciferol. Part 1: general overview with a focus on ascorbic acid. Inflamm Allergy Drug Targets. 2011;10:54-63. 22. Godin DV, Garnett ME. Effects of various anesthetic regimens on tissue antioxidant enzyme activities. Res Commun Chem Pathol Pharmacol. 1994;83:93-101. 41. Thurnham DI. Micronutrients and immune function: some recent developments. J Clin Pathol. 1997;50:887-91. 23. Gupta S, Wen JJ, Garg NJ. Oxidative stress in Chagas disease. Interdiscip Perspect Infect Dis. 2009;190354. 42. Wen JJ, Garg N. Oxidative modification of mitochondrial respiratory complexes in response to the stress of Trypanosoma cruzi infection. Free Radic Biol Med. 24. Higuchi ML, Benvenuti LA, Martins Reis M, Metzger M. Pathophysiology of the 2004;37:2072-81. heart in Chagas disease: current status and new developments. Cardiovasc Res. 2003;60:96-107. 43. Wen JJ, Yachelini PC, Sembaj A, Manzur RE, Garg NJ. Increased oxidative stress is correlated with mitochondrial dysfunction in chagasic patients. Free Radic Biol 25. Hornig B, Arakawa N, Kohler C, Drexler H. Vitamin C improves endothelial function Med. 2006;41:270-6. of conduit arteries in patients with chronic heart failure. Circulation. 1997;97:363-8. 44. Weyers A, Ugnia LI, Ovando HG, Gorla NB. Antioxidant capacity of vitamin C in 26. Iqbal K, Khan A, Khattak MMAK. Biological significance of ascorbic acid (vitamin mouse liver and kidney tissues. Biocell. 2008;32:27-31. C) in human health: a review. Pakistan J Nutr. 2004;3:5-13. 45. Wilkinson SR, Prathalingam R, Taylor MC, Horn D, Kelly JM. Vitamin C biosynthesis 27. Jialal I, Singh U. Is vitamin C an antiinflammatory agent? Am J Clin Nutr. in trypanosomes: a role for the glycosome. Proc Natl Acad Sci USA. 2005;102:11645- 2006;83:525-6. 50.

28. Koury JC, Donangelo CM. Zinco, estresse oxidativo e atividade física. Rev Nutr. 46. Witting LA. Vitamin E: do we need it? In: Wolinsky I, Hickson JF, editors. Nutrition 2003;16:433-41. and health. Topics and controversies. Boca Raton: CRC Press; 1995. p. 157-79.

29. Levine M, Padayatty SJ, Espey MG. Vitamin C: a concentration-function approach Received: 9 January 2014 yields pharmacology and therapeutic discoveries. Am Soc Nutr. 2011;2:78-88. Accepted: 10 September 2014

30. Maçao LB, Wilhelm Filho D, Pedrosa RC, Pereira A, Backes P, Torres MA, et al. Antioxidant therapy attenuates oxidative stress in chronic cardiopathy associated with Chagas disease. Int J Cardiol. 2007;123:43-9.

250 Rev. Inst. Med. Trop. Sao Paulo 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 Therapy consists of a prolonged course, its success depending on the species of bacteria, the virulence of the strain, the organs Nocardiosis is a chronic and severe pyogranulomatous disease affected, the time of evolution and the health status of the susceptible caused by the environmentally ubiquitous actinomycete of the Nocardia individual(s)1. Nocardia spp. is refractory to conventional antimicrobial genus1. Nocardiosis is an emerging disease in humans and animals therapy. Sulfonamides potentiated by trimethoprim, minocycline, worldwide2,13,26,32,34. According to BAIO et al. (2013) nocardiosis aminoglycosides (amikacin, gentamicin) and cephalosporins (ceftiofur, is a neglected disease, particularly in patients with some degree ceftriaxone, cephalexin) alone or in combination, based on in vitro tests3, of immunosuppression1. For many years, since Edmond Nocard’s are the choices of treatment for human and animal nocardiosis26,28,31. first description of the pathogen in 1888, its diagnosis was based on phenotypic methods17. To date, 102 species of Nocardia have been The purpose of this case report is to present the species of Nocardia discovered using molecular methods, of which seven have been involved in human nocardiosis in Brazil, and their respective drug reclassified and 90 currently stand on the list of prokaryotic names with susceptibility pattern. standing nomenclature24. Among these species, at least 25 are pathogenic to humans and animals8,13,17,29. N. asteroides, N. brasiliensis, N. farcinica, MATERIAL AND METHODS N. nova, N. cyriacigeorgica and N. veterana are the main species related to nocardiosis in humans1,11,14,17. 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 Either tegumentary injury or the inhalation of bacteria is considered and five in Paraná), were isolated from clinical cases of nocardiosis. The the most common route of transmission of Nocardia spp. in humans7. strains were isolated from different specimens (bronchial wash, cutaneous Clinically, the main manifestations of human nocardiosis are pneumonia, and organ fragments) in defibrinated sheep blood agar (5%), Sabouraud encephalitis, lymphadenitis, lymphangitis and cutaneous tissue lesions33. 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, 18618- 970 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 results were recorded after 48 - 72 hours because of the organisms’ dry, convex, adherent, and white to orange color aspects. After 48 to 72 fastidious growth3,20. The following antibiotics were used: amikacin hours post-inoculation, colonies were submitted to Gram and Kinyoun (0.016 - 256 µg/mL), ampicillin (0.016 - 256 µg/mL), amoxicillin/ stain8,27. Gram-positive, filamentous to cocobacillary, partially acid-fast clavulanate (0.016 - 256 µg/mL), ceftriaxone (0.016 - 256 µg/mL), organisms were identified as Nocardia. gentamicin, sulfamethoxazole-trimethoprim (0.002 - 32 µg/mL) and imipenem (0.002 - 32 µg/mL). Molecular identification. Molecular analysis was carried out in the Medical Mycology Research Center, Chiba, Japan. Genomic DNA The similarities between the results from the disk-diffusion test and for sequencing was performed according to KAGEYAMA et al., 2004. E-test were analyzed using the Kappa agreement index. According to The 16S rRNA gene was amplified by PCR using a DNA thermal cycler the values obtained, the agreement analysis between the tests followed (TaKaRa Bio Inc., Chiba, Japan) under the following conditions: 35 the criteria established by THRUSFIELD (1995). All statistical analysis cycles at 94 ºC for 60 seconds for denaturation, 60 ºC for 60 seconds was done using Bioestat v.5.0 and SPSS v.14 packages. The Chi-squared for primer annealing, and 72 ºC for 120 seconds for primer extension. and Fisher’s exact tests were used to analyze whether the resistance PCR primers were the prokaryotic 16S rRNA universal primer pairs, percentages of Nocardia spp. were normally distributed between strains 8F and 691R, 520F and 1100R, and 926F and 1542R. DNA sequences for each antibiotic tested with the disk-diffusion method. The level of were determined with an automatic sequence analyzer (ABI PRISMTM significance for this test was p < 0.0533. 3130; Applied Biosystems, Japan), using the same primers and a dye terminator cycle sequencing kit (Applied Biosystems). Near-complete RESULTS 16S rRNA gene sequences consisting of approximately 1400 bases pairs were obtained. The sequence of the 16S rRNA gene was compared to the All strains were taken from young adult males between 28 and GenBank database using BLAST, and 16S rRNA sequences of related 35 years of age. Three patients displayed cutaneous manifestations Nocardia-type strains were retrieved from the database. These sequences (exhibiting fistulous mycetomas due to traumatic inoculation), while two were submitted to GenBank/JJBJ/EMBL. showed pulmonary, one neurological and one systemic. Two individuals with pulmonary symptoms, one with neurological, and one with In vitro drug susceptibility tests. All strains were examined using systemic were co-infected with immunosuppressive diseases, or were the disk-diffusion test and minimum inhibitory concentration test (MIC undergoing/had undergone prolonged treatments with chemotherapy or test) based on E-test™ (E-test™, AB biodisk, BioMérieux, Dalvägen, corticotherapy. Of all the patients, four died due to the severity of their Sweden), according to the procedures described by GLUPCKZYNSKI disease (these were pulmonary, neurological and systemic cases), two et al. 2006 and NCCLS 2006. Isolates resistant to three or more recovered (cutaneous manifestations), and one case had no documented antimicrobials were considered multi-resistant23. outcome (cutaneous manifestation) [Table 1].

The study of drug susceptibility in Actinomycetes is more laborious The microbiological culture of samples showed dry, convex, strongly because this group of bacteria usually grows in clumps. Due to this adherent, whitish to orange-brown colonies with a powdery surface adherent characteristic during bacterial growth, the observed irregular after 48-96 hours of incubation at 37 ºC. Gram and modified Ziehl- broth turbidity makes it difficult to measure the optimum inoculum Neelsen stains showed thin, branched filaments sometimes fragmented concentration. Naturally, the precise concentration of unit colony in cocobacillary forms, suggestive of the Nocardia genus. formation is essential for the correct interpretation of both antimicrobial tests11,20. The sequence of the 16S rDNA gene enabled the identification of four strains - N. farcinica, two N. nova, and one N. asiatica - based on The isolates were briefly subcultured twice in blood agar (5%) to its 99.6% or higher sequence similarity to the reference sequence in ensure their purity. After 48 hours, they were inoculated in brain-heart- GenBank (DDBJ/GenBank/EMBL), using BLAST as recommended infusion at 37 ºC for another 48 hours. Sterile glass beads were added at by CLSI, 2008. The access number for the isolates in GenBank is as the time the strains were vortexed to decrease the formation of clumps follows: IFM 11128/ AB 633331 - N. nova; IFM 11096/ AB 630965 - and subsequently obtain a more accurate optical density (OD) equivalent N. farcinica; IFM 11231/ AB 636474 - N. farcinica; IFM 11232/ AB to a 0.5 McFarland standard to disk-diffusion test and 1.0 McFarland 636475 - N. farcinica; IFM 11099/ AB 630966 - N. nova; IFM 11100/ standard for E-test. AB 630967 - N. asiatica; IFM 11285/ AB 638765 - N. farcinica.

All of the strains were submitted to a disk-diffusion test and the In this study, N. farcinica was observed in two cutaneous, one inhibition zones interpreted following standards according to BAUER systemic and other pulmonary cases; N. nova was present in one et al., 1966 and AMBAYE et al. 1997. The antibiotics selected were neurological manifestation and one pulmonary; and Nocardia asiatica amikacin (30 μg), ampicillin (10 μg), amoxicillin/clavulanate (20 in one cutaneous case (Table 1). μg/10 μg), ceftiofur (30 μg), cefoperazone (75 μg), ceftriaxone (30 μg), cephalexin (30 μg), cefuroxime (30 μg), cefalonium (30 μg), imipenem The in vitro drug susceptibility test, based on disk-diffusion, is (10 μg), gentamicin (10 μg), mynocicline (30 μg) and sulfamethoxazole/ presented in Table 2. Based on the disk-diffusion test, Nocardia spp. trimethoprim (25 μg). isolates were sensitive to amikacin, amoxicillin/clavulanate, cephalexin, cefalonium, ceftiofur, ceftriaxone and minocycline. However, around In the MIC test, a maximum of five E-test strips were attached 50% of the Nocardia spp. isolates were resistant to ampicillin, gentamicin to Mueller Hinton agar and were then incubated at 37 ºC. The and sulfamethoxazole/trimethoprim. Of the seven isolates, two were

252 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.

Table 1 Summary of clinical presentation and outcomes of seven patients with nocardiosis. Santa Maria, RS; Curitiba, PR and Botucatu, SP, Brazil

Clinical Underlying Nocardia species Site of isolate Age/sex Treatment Outcome presentation condition Neurologic Liquor 32/male Chemotherapy NA* Death N. nova 1 Pulmonary Sputum 33/male HIV 4 drugs*2 Death Dermatologic Biopsy 30/male None NA Recovery Dermatologic Biopsy 28/male None NA Recovery N. farcinica Pulmonary Broncoalveolar wash 35/male HIV NA Death Systemic Liquor 34/male Chemoterapy NA Death N. asiatica Dermatologic Biopsy 35/male 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 the tested antimicrobials (Table 3) except for one isolate of Nocardia was resistant to ampicillin, cefoperazone and gentamicin; and the other farcinica, which was resistant to sulfamethoxazole/trimethoprim. isolate of N. farcinica was resistant to ampicillin, sulfamethoxazole/ trimethoprim, gentamicin and cefuroxime. Table 3 Minimum inhibitory concentrations (µg/mL) and susceptibility proportion The minimum inhibitory concentration of tested antimicrobials estimates of Nocardia spp. isolated from seven case reports. UNESP, Botucatu, showed suitable breakpoints, with all the isolates being susceptible to SP, Brazil

Table 2 MIC a MIC a %Susceptible Antimicrobials 50 90 Standard inhibition zone diameter and percentage of susceptibility of 7 (µg/mL) (µg/mL) (Overall n=7) Nocardia spp. isolates to selected antimicrobials in disk-diffusion test. UNESP, Botucatu, SP, Brazil Amikacin 0.20 0.40 100 Amoxicillin/ clavulanate 0.5 3 100 Zone diameter (mm)a % Suscep- Ampicillin 0.25 1 100 Antimicrobial R I S tible Cephalexin 1 2 100 Amikacin ≤14 15-16 ≥17 100 Ceftriaxone 1.5 2 100 Amoxicillin/ ≤13 14-17 ≤13 100 Gentamicin 1 2 100 clavulanate Imipenem 0.75 1,5 100 Ampicillin ≤13 14-16 ≥17 29 Sulfamethoxazole/ 1.5 3 86 Cephalexin ≤14 15-17 ≥18 100 trimethoprim a MIC and MIC : values are concentrations at which ≥ 50% and ≥90% of isolates Cephalonium ≤14 15-17 ≥18 100 50 90 are inhibited by antimicrobials. Cefoperazone ≤15 16-20 ≥21 43 The agreement between the tests was considered low by the statistical Ceftiofur ≤17 18-20 ≥21 100 analysis. Ceftriaxone ≤13 14-20 ≥21 86 DISCUSSION Cefuroxime ≤14 15-17 ≥18 72 Imipenem ≤13 14-15 ≥16 72 These findings reinforce that molecular techniques are a reliable, suitable and quick method for the diagnosis of species of Nocardia genus Gentamicin ≤10 11-14 ≥15 57 taken from a human origin. Phenotypic evaluations could be performed Mynocicline ≤14 15-19 ≥19 100 to identify the Nocardia species, combining tests such as the hydrolysis of organic compound (adenine, xanthine, hypoxanthine, casein, esculin Sulfamethoxazole/ ≤10 11-15 ≥16 43 and tyrosine), carbohydrate assimilation, antimicrobial susceptibility trimethoprim pattern, citrate utilization and acetamid and arylsulfatase utilization, a Zone of inhibition diameter (mm) by disk diffusion method susceptibility inter- among others8,27,36. However, they are usually laborious, time-consuming, pretative guidelines based on NCCLS (2006), AMBAYE et al. (1997), and BAUER and require experience in evaluating the results. For this reason, molecular et al. (1966). R = resistant, I = intermediate, S = susceptible. methods emerged as an alternative that can be used on several different

253 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.

body fluids and tissue samples, and can also be used for identifying strains as therapy20,31. However, the high efficacy of cephalosporins against N. that are difficult to grow in a conventional medium8,29. farcinica isolates and the high success rate of amoxicillin/clavulanate against N. nova could be justified by the variability inβ -lactamase The 16S rRNA gene is highly conserved with constant regions and, to activity present in the bacteria’s cell wall3,20. In agreement with previous date, its complete sequence of approximately 1400 bp has a considerably results, minocycline appeared to be highly effective as in vitro against large database on GenBank, which allows for the identification of the Nocardia species isolated in these reports3,31, however MUÑOZ et al. most Nocardia species29. However, because 16S rRNA shows minimal (2007) observed resistance to this drug in N. nova and N. cyriacigeorgica. 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 Sulfamethoxazole/trimethoprim presented a lower efficacy in of CLSI, which recommends a similarity higher than 99.6%. Nowadays, disk-diffusion tests against the isolates and are perhaps, therefore, not other researchers are reporting on the sequencing of housekeeping genes appropriate for therapy. Similar observations with a larger number of with a better discriminatory power, even with partial gene sequencing16,29. clinical isolates were made by TREMBLAY et al. (2011), informing of the importance of establishing treatment protocols for in vitro tests. The incidence of human nocardiosis has increased in the last two However, despite the worldwide resistance pattern to sulfonamides decades across several countries, particularly in patients affected by recorded in clinical isolates, few patients failed therapy with these drugs, immunosuppressive therapies or diseases1,8. In Brazil, the 22 cases of suggesting that the inhibition breakpoint for sulfonamides still poses a human nocardiosis, predominantly displaying pulmonary symptoms, challenge and varies among different laboratories9. This can result from were most prevalent in adult males (59.2%) between the ages of 21 and 84. the methodology suggested by CLSI in which sulfonamide breakpoints Most cases were related to immunosuppressed conditions (69.9%), such are based on 80% of inhibition of growth endpoint compared to the as transplants, corticotherapy and being HIV-positive30. These findings 100% inhibition of growth by other drugs. In future research, more agree with similar observations that described immunocompromised isolates should have the antimicrobial profile analyzed using the same conditions in humans affected by nocardiosis10,19,21,30, reinforcing the CLSI specifications, as performed in this study, and new breakpoints opportunistic behavior of the Nocardia genus7. However, it is important to for Nocardia species should perhaps be considered. Moreover, the stress that cutaneous presentations are not always associated to a previous indiscriminate use of the drug for comorbidities can induce resistance health condition, indicating possible transmission through cutaneous of Nocardia to sulfonamides and molecular mechanisms, thus it should trauma, as suggested by other studies4. In this study, nocardiosis only be analyzed8,9,26. affected men between the ages of 28 and 35. This is consistent with the results of other studies, which have also identified similar occurrences Some studies showed the applicability of the E-test for antimicrobial of nocardiosis in young adult males, indicating the occupational risk of susceptibility testing for the analysis of actinomycete resistance20. human infection by Nocardia species in mainly immunocompromised However, disagreement between MIC and disk diffusion tests has been patients exposed to the agent within their environment10,21,34. indicated in other papers3,25. These differences probably occur due to colony lumps formed during culture growth, which makes it difficult to The clinical picture of human nocardiosis is diverse, though obtain a precise McFarland scale, or an ideal count of colony-forming cutaneous and pulmonary manifestations are present in the majority of units25. So far, for Nocardia the broth dilution method is still favorable, cases4,5. In the last few years, N. farcinica has been the most isolated in comparison to the E-test, in order to assure the significance of the species in reviews of different kinds of patient infection, whether with or minimum inhibitory concentration method. without immunosuppression, representing around 22% and 45% of cases respectively19,26,32,34. Evidence shows that N. farcinica is widely distributed Despite the small number of isolates, it was possible to notice in the environment, has a high potential of virulence and is closely that nocardiosis in Brazil mainly affects men and immunosuppressed associated to a great number of fatal nocardiosis cases, including those patients with localized or disseminated infection. Clinical manifestations with systemic dissemination8. Cases related to N. farcinica confirmed could vary depending on the species, virulence of isolates, and the severity of the disease in immunosuppressed patients. immunocompromised factors of the patients. The cases reported in this study were seen in patients with a higher comorbidity, predominantly In other reports, N. nova has been associated to approximately 20% associated with pulmonary or disseminated forms. Antimicrobial of isolates in the United States and was the most pathogenic species in resistances of isolates reinforce the importance of prior in vitro tests Canada until 200834,35. N. nova has been attributed to different clinical before initiating therapy. Further investigation with a larger number of presentations in humans, mainly related to immunosuppressive conditions cases and isolates is necessary. or trauma4,35. These findings reinforce this species’ pathogenic potential for humans, and the risks of developing systemic nocardiosis, resulting RESUMO in death. Identificação molecular e perfil de sensibilidade a antimicrobianos N. asiatica was recently identified in human nocardiosis in Brazil, and de sete isolados clínicos de Nocardia spp. no Brasil this is one of few reports worldwide5,21,22 which reinforces the necessity of identifying more isolates and understanding their epidemiology. Nocardia é um microorganismo ubiquitário relacionado a infecções piogranulomatosas, com difícil resolução tecidual em humanos e animais. The lower efficacy of human strains to gentamicin and ampicillin in the A doença é mundialmente emergente devido ao aumento de doenças disk- diffusion test is probably related to the pattern of low susceptibility e tratamentos imunossupressores. Este relato de casos ocorridos no of N. farcinica resistance, indicating that these are not recommended Brasil visa apresentar a identificação molecular dos isolados e o padrão

254 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.

de sensibilidade a antimicrobianos por disco-difusão e concentração 11. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial inibitória mínima (CIM) através de fitas E-test®. Os casos ocorreram disk susceptibility tests; approved standard. NCCLS. 2006. Susceptibility testing of Mycobacteria, Nocardiae, and other aerobic Actinomycetes. 8th ed. Approved standard em homens, em idade adulta. Três quadros foram cutâneos, dois CLSI document M2-A9, Wayne, PA; 2006. pulmonares, um neurológico e um sistêmico. O quadro respiratório, o neurológico e um sistêmico estavam associados à doença ou terapia 12. Clinical and Laboratory Standards Institute. Interpretive criteria for identification of imunossupressoras. O sequenciamento do gene 16S rRNA (1491pb) bacteria and fungi by DNA target sequencing; approved guideline. CLSI document possibilitou a identificação de quatro isolados de Nocardia farcinica, dois MM18-A.Wayne, PA; 2008. de Nocardia nova e um de Nocardia asiatica. N. farcinica foi observada 13. Condas LAZ, Ribeiro MG, Yazawa K, Vargas APC, Salerno T, Giuffrida R, et al. em dois casos dermatológicos, um pulmonar e um quadro sistêmico, N. Molecular identification and antimicrobial susceptibility ofNocardia spp. isolates nova foi isolada de um caso neurológico e outro pulmonar; e N. asiatica from bovine mastitis in Brazil. Vet Microbiol. 2013;167:708-12. em um caso dermatológico. O teste de disco-difusão mostrou que amicacina (100%), amoxicilina/clavulanato (100%), cefalexina (100%) 14. Conville PS, Brown JM, Steigerwalt AG, Lee JW, Byrer DE, Anderson VL, et al. Nocardia veterana as a pathogen in North American Patients. J Clin Microbiol. e ceftiofur (100%) foram mais efetivos; enquanto gentamicina (43%), 2003;41:2560-8. sulfametoxazol/trimetoprim (43%) e ampicilina (29%) foram menos efetivos. No entanto, no teste de concentração inibitória mínima (CIM), 15. Conville PS, Brown-Elliott BA, Wallace Jr RJ, Witebsky FG, Koziol D, Geraldine S, apenas um dos quatro isolados da espécie Nocardia farcinica mostrou-se et al. Multisite reproducibility of the Broth microdilution method for susceptibility resistente a sulfametoxazole-trimetropina. testing of Nocardia species. J Clin Microbiol. 2012;50:1270-80.

16. Conville PS, Witebsky FG. Multiple Copies of the 16S rRNA gene in Nocardia ACKNOWLEDGMENTS nova isolates and implications for sequence-based identification procedures. J Clin Microbiol. 2005;2881-5. To FAPESP Funding (Protocols 2009/56037-1 and 2010/53494-5) and Mycology Research Center of University of Chiba - Japan. 17. Conville PS, Witebsky F. Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces, and other aerobic actinomycetes. In: Vervalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, Warnock DW. Manual of Clinical Microbiology. 10th REFERENCES ed. Washington: AMS Press; 2011.

1. Acha, PN, Szyfres B. Nocardiosis. In: Acha, PN, Szyfres B. Zoonosis y enfermedades 18. Corti ME, Villafañe-Fioti MF. Nocardiosis: a review. Int J Infect Dis. 2003;7:243-50. transmisibles comunes al hombre y a los animales, Vol.I. Washington: Organización Panamericana de la Salud; 2003. p. 212-6. 19. Farina C, Boiron P, Ferrai I, Provost F, Goglio A. Report of human nocardiosis in Italy between 1993 and 1997. Eur J Epidemiol. 2001;17:1019-22. 2. Agterof MJ, Van Der Bruggen T, Tersmette M, Ter Borg EJ, Van Den Bosch JMM, Biesma DH. Nocardiosis: a case series and a mini review of clinical and 20. Glupczynski Y, Berhin C, Janssens M, Wauters G. Determination of antimicrobial microbiological features. Neth J Med. 2007;65:199-202. susceptibility patterns of Nocardia spp. from clinical specimens by Etest. Clin Microbiol Infect. 2006;12:905-12. 3. Ambaye A, Kohner PC, Wollan PC, Roberts KL, Roberts GD, Cockerill FR. Comparison of agar dilution, broth microdilution, disk diffusion, E-Test, and BACTEC 21. Iona E, Giannoni F, Brunori L, Gennaro M, Mattei R, Fattorini L. Isolation of Nocardia radiometric methods for antimicrobial susceptibility testing of clinical isolates of the asiatica from cutaneous ulcers of a human immunodeficiency virus-infected patient Nocardia asteroides complex. J Clin Microbiol. 1997;35:847-52. in Italy. J Clin Microbiol. 2007;45:2088-9.

4. Anzalone CL, Cohen PR, Tarrand JJ, Diwan AH, Prieto VG. Nocardia yamanashiensis 22. Kageyama A, Poonwan N, Yazawa K, Mikami Y, Nishimura K. Nocardia asiatica sp. in an immunocompromised patient presenting as an indurated nodule on the dorsal nov., isolated from patients with nocardiosis in Japan and clinical specimens from hand. Tumori. 2013;99:156e-8e. Thailand. Int J Syst Evol Microbiol. 2004;54:125-30.

5. Baio PV, Ramos JN, Dos Santos LS, Soriano MF, Ladeira EM, Souza MC, et al. 23. Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to Molecular identification of Nocardia isolates from clinical samples and an overview identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. of human nocardiosis in Brazil. PlOs Negl Trop Dis. 2013;7(12):e2573. 1983;46:165-70.

6. Bauer Aw, Kirby W, Sherris JC, Turck M. Antibiotic susceptibility testing by a 24. List of prokaryotic names with standing in nomenclature. [Internet] 2012. Available standardized single disk method. Am J Clin Pathol. 1966;45:493-6. from: www.baterio.net/nocardia.html

7. Beaman BL & Beaman L. Nocardia species: host-parasite relationships. Clin 25. Lowman W, Aithma N. Antimicrobial susceptibility testing and profiling of Nocardia Microbiol Rev. 1994;7:213-64. species and other aerobic actinomycetes from South Africa: comparative evaluation of broth microdilution versus the Etest. J Clin Microbiol. 2010; 8:4534-40. 8. Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol 26. Muñoz J, Mirelis B, Aragón LM, Gutiérrez N, Sánchez F, Español M, et al. Clinical and Rev. 2006;19:259-82. microbiological features of nocardiosis 1997-2003. J Med Microbiol. 2007;56:545-50.

9. Brown-Elliott BA, Biehle J, Conville PS, Cohen S, Saboulle M, Sussland D, et al. 27. Quinn PJ, Carter ME, Markey BK, Carter GR. The Actinomycetes. In: ___ Clinical Sulfonamide resistance in isolates of Nocardia spp. from a U.S. Multicenter Survey. Veterinary Microbiology. London: Wolfe; 2004. J Clin Microbiol. 2012;50:670-2 28. Ribeiro MG, Salerno T, Mattos-Guaraldi AL, Camello TCF, Langoni H, Siqueira 10. Chedid MB, Chedid MF, Porto NS, Severo CB, Severo LC. Nocardial infections: AK, et al. Nocardiosis: an overview and additional report of 28 cases in cattle and report of 22 cases. Rev Inst Med Trop Sao Paulo. 2007;49:239-46. dogs. Rev Inst Med Trop Sao Paulo. 2008;50:177-85.

255 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.

29. Roth A, Andress S, Kroppenstedt RM, Harmsen D, Mauch H. Phylogeny of the genus 33. Thrusfield M. Veterinary epidemiology. 2. ed. Cambridge: Blackwell Science; 1995. Nocardia based on reassessed 16S rDNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species 34. Tremblay J, Thibert L, Alarie I, Valiquette L, Pépin J. Nocardiosis in Quebec, Canada, and to unnamed taxons. J Clin Microbiol 2003;41:851-6. 1988-2008. Clin Microbiol Infect. 2011;17:690-6.

30. Santos IS. Actinomicetoses no Rio Grande do Sul: a propósito de 59 casos, atualizando 35. Wallace JR, Brown, BA, Tsukamura M, Brown JM, Grace O. Clinical and laboratory Actinomicose, Nocardiose e Rodococose. [Dissertação]. Porto Alegre: Universidade features of Nocardia nova. J Clin Microbiol. 1991;29:2407-11. Federal do Rio Grande do Sul, Faculdade de Medicina; 2010. 36. Wallace JR, Steele LC, Sumter G, Smith JM. Antimicrobial susceptibility patterns 31. Saubolle MA, Sussland D. Nocardiosis: review of clinical and laboratory experience. of Nocardia asteroides. Antimicrob Agents Chemother. 1988;2:1776-9. J Clin Microbiol 2003;41:4497-4501. Received: 12 January 2014 32. Tan CK, Lai CC, Lin SH, Liao CH, Chou CH, Hsu HL, et al. Clinical and Accepted: 23 Septembeer 2014 microbiological characteristics of Nocardiosis including those caused by emerging Nocardia species in Taiwan, 1998-2008. Clin Microbiol Infect. 2010;16:966-72.

256 Rev. Inst. Med. Trop. Sao Paulo 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 wide geographic distribution1,7,16,18,28.

The Leishmania genus causes leishmaniasis, which constitutes a ATL is widely distributed across the Americas. Between 2001 and variety of chronic diseases. There is a wide spectrum of clinical forms, 2011, around 270,500 cases were reported, with an average of 27,500 including those affecting the skin, mucosa, or internal organs16,18. new cases/year. Around 3 - 5% of patients who develop cutaneous lesions are also susceptible to mucosal leishmaniasis23,30. In the state The subgenera Leishmania Viannia is the causative agent of new- of São Paulo there are approximately 400 new cases per year. Another world cutaneous leishmaniasis, comprising the species L. (V.) braziliensis, substantial problem is the urbanization of the infection. Autochthonous L. (V.) panamensis and L. (V.) guyanesis, among others18,26. Infections cases have been reported in urban areas. The incidence of peri-urban and by these species cause three clinical types of American tegumentary urban cases has been increasing. Approximately 10% of the population leishmaniasis (ATL): localized cutaneous, mucosal, and disseminated living in endemic areas is at risk of acquiring the infection29. ATL is also leishmaniasis. Cutaneous lesions are restricted to the entry site of the considered one of the most common dermatological syndromes diagnosed parasites, whereas the mucosal strain is defined by its spreading to the in travelers (or tourists) who have visited endemic areas15. mucosal surfaces of the upper digestive and airway tracts. Disseminated leishmaniasis is characterized by large-scale spreading to distant The life cycle of L. (V.) braziliensis includes different reservoirs, such cutaneous sites2,14,15,24. as humans and wild and domestic mammals, as well as various vector species. Therefore, Leishmania strains can be maintained in both rural and Despite the fact that cutaneous leishmaniasis is caused by at least urban settings, thereby affecting the epidemiology of the infection. Due seven different Leishmania species in Brazil, the vast majority of cases to the proximity of dogs and humans, studies have shown the important are caused by the L. (V.) braziliensis sub-genera, which can be transmitted role of domestic dogs in ATL19,21. Studies using molecular techniques to by different phlebotomine sandfly vectors via animal reservoirs across a 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 twice in phosphate-buffered saline (pH 7.2) at 1,000g for 10 min. The adapting to changes in their original forest habitats, and the consequent parasite pellets were used for DNA extraction. L. (V.) braziliensis strain public health implications13. DNA also was used in reactions as a positive control.

Despite the significance of ATL to the Brazilian public health DNA purification: Before performing DNA extraction, clinical system, the genetic diversity of L. (V.) braziliensis in some endemic samples and WHO Leishmania reference strains were crushed and areas of Brazil has been poorly researched, as in the state of São Paulo. digested in a lysis buffer until tissue lysis was complete, (This-HCl, Therefore, this study aims to analyze the genetic diversity of a L. (V.) 10 mM, pH 8.0; EDTA 10mM; SDS, 0,5%; N-laurilsarcozil, 0.01%; braziliensis population collected from patients and dogs in the state of proteinase K, 100 µg/mL) by incubation in water bath at 56 °C. Then, São Paulo with cutaneous lesions, avoiding in vitro cultivation. The DNA molecules were extracted by a QIAamp DNA Mini Kit (Qiagen), reason for evaluating polymorphism in humans and dogs was due to the according to the manufacturer’s instructions. DNA concentration and importance of both species within the parasite's life cycle. The results purity was determined by the ratio of O. D. at 260 and 280 nm in a indicate a high variability in isolates collected in patients and dogs from NanoDrop ND1000 (Thermo Scientific). the state of São Paulo. Additionally, this study has shown the possibility of performing genotyping directly on clinical samples without having Routine Leishmania diagnosis to isolate the parasite. Parasitological diagnosis: Skin biopsy imprints were plated onto a MATERIAL AND METHODS glass slide, fixed with methanol and stained with Giemsa11. The presence of amastigotes was observed microscopically with an immersion objective Human and dog samples: The selection of positive samples was (×1,000). made in biopsies received by an in-house Laboratory over a period of nine years (2003 - 2012). The biopsies were collected by medical or PCR targets for Leishmania and internal controls: The Leishmania veterinary health services. The human or canine lesions were cleansed genus was identified by a 120-bp PCR product, amplified from a with antiseptics after the administration of a local anesthetic. The borders conserved region of kDNA minicircles of Leishmania spp., using the of the lesions were scraped or smears of material were obtained by a primer set 150/15223. L. (V.) braziliensis was identified by an amplified punch biopsy of the lesions and immediately added to tubes containing fragment of 146-149 bp from the multicopy spliced leader (SL) RNA 1-2 mL of a sterile 0.85% NaCl and 200 µg/mL gentamicin solution, gene using the primer set LU-5A/LB-3C, which amplifies a 146-149 bp sent to the laboratory within 48 hours and promptly processed to sequence from the SL12,17. These tests were carried out under the same confirm clinical diagnosis. All biopsies recorded were from patients aforementioned conditions11,12. To check PCR inhibitors, canine and human with the cutaneous clinical form. Samples were tested by routine samples were assayed using a reference gene, whose primer sets were diagnosis, which included molecular and parasitological methods. The GAPDH4F/GAPDH4R and β1-β2, respectively, in the same conditions methodologies applied were PCR, using two different sets of primers, and as previously described3,11. After the thermal cycles, PCR products were a parasitological method (microscopic observation). These DNA samples electrophoresed in 2% agarose gel and stained with ethidium bromide. were from patients and dogs living in 36 different municipalities and DNA fragments were made visible under UV illumination. endemic areas for ATL in the state of São Paulo, Brazil (Alumínio, Aruja, Avaré, Bauru, Bragança Paulista, Cajamar, Campinas, Caraguatatuba, L. (V.) braziliensis genotyping by RFLP-PCR (restriction Cerquilho, Conhal, Cubatão, Guapiara, Guarulhos, Ibirá, Ilha Bela, fragment length polymorphism-PCR): Originally, 132 DNA extracts Indaiatuba, Iperó, Iporanga, Itapera, Itupeva, Jaboticabal, Jundiaí, from biopsies, positive for L. (V.) braziliensis, were analyzed for Mairiporã, Marília, Miracatu, Mirandópolis, Mogi Guaçu, Monte Mor, genotype determination. PCR was used for diagnosis and genotyping Pilar do Sul, Ribeira, Salto, São Paulo, Sorocaba, Suzano, Tatuí, Tietê). directly from clinical DNA samples. Each test was performed by Epidemiological registers of the different Public Dermatology Clinics adding 5 µL from each DNA template and 25 pmol from each primer or Centers for Zoonosis Control were analyzed to determine the locality for a final volume of 25µ L. The amplifications were carried out with of the Leishmania infection of each patient (or dog). 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,

Ethical considerations: This study was performed according to the 10 mM Tris-HCl, pH 8.5; 50 mM KCl; 1.5 mM MgCl2; and 200 mM recommendations of the Human Ethics Committee (CONEP-IAL) and of each dNTP. In genotype reactions the primer set used was IR1/ “Sociedade Brasileira de Ciência em Animais de Laboratório/Colégio IR2 (5’-GCTGTAGGTGAACCTGCAGCAGCTGGATCATT-3’ and Brasileiro de Experimentação Animal” (SBCAL/COBEA). Both Ethic 5’-GCGGGTAGTCCTGCCAAACACTCAGGTCTG-3’), which Committees of Instituto Adolfo Lutz have approved of this study. 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. Leishmania strains: For genotype standardization, the following PCR-amplified products were digested with a HhaI restriction enzyme, WHO standard Leishmania strains were used: L. (V.) guyanensis which were separated by electrophoresis in an 8% polyacrylamide gel (MHOM/BR/1975/M4147), L. (L.) amazonensis (IFLA/BR/1967/PH8), and stained with ethidium bromide. DNA fragments were made visible L. (L.) major (MHOM/SU/1973/5-ASKH), L. (L.) infantum (MHOM/ under UV illumination. The images from reactions for diagnosis and BR/1974/PP75), and L. (V.) braziliensis (MHOM/BR/1975/M2903). The genotyping were analyzed by a MiniBIS Gel Imager and Documentation Leishmania strains were maintained by serial passages and grown at 24 system (BioSystematica). The size of the fragments was based on a ºC in M-199 medium, supplemented with 10% calf serum and 0.25% comparison with molecular-weight size markers. In genotyping reactions, hemin25. In the log phase, 1 x 108 parasites were harvested and washed the banding patterns were used to group the isolates into genotypes with

258 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.

the same banding pattern for the restriction enzyme. Next, genotype experiments were conducted on the 132 DNA samples taken from biopsies with a positive parasitological and molecular Quality assurance: Each DNA extraction batch included a DNA diagnosis. All samples also tested positive for L. (V.) braziliensis (in extraction from Leishmania-free eukaryotic samples as a negative PCR), which was previously determined by the LU-5A/LB-3C primer control. In each reaction, a tube containing nuclease-free water and PCR set, whose products range in size from 146 to 149 bp9,12,17. According the mix was used as a blank control. Separate rooms were used for i. DNA epidemiological registers of the Public Dermatology Clinics and Centers extraction, ii. PCR mix and primer preparation, iii. the adding of DNA for Zoonosis Control, all samples analyzed were from patients or dogs from clinical samples and positive control; and iv. post-PCR agarose-gel with an autochthonous Leishmania infection (in the same locality as the electrophoresis analysis. DNA samples were assayed in duplicate and biopsy collection). at least twice. Of the 132 DNA samples, only 52 (40%) were successfully RESULTS 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 The first experiments were conducted using the DNA extracted from amplified by this primer set. As expected, no amplification was detected WHO reference strains to establish the genotype by RFLP-PCR, using in DNA extracted from DNA as a negative control and PCR products the primer set IR1/IR2 and additional treatment with HhaI enzymes. were obtained for all positive controls. Figure 1 shows the restriction patterns of the six WHO reference strains. Table 1 shows the specification of the 52 genotyped samples in L. (V.) guyanensis and L. (V.) braziliensis showed the same restriction detail, which included the collection date of the biopsies (2003 - 2012), profile. On the other hand, L. (L.) amazonensis, L. (L.) major and L. (L.) as well as the host (human or canine) and locality within the state of infantum had specific restriction profiles. 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. 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/ DISCUSSION M4147) (1), L. (L.) amazonensis (IFLA/BR/1967/PH8) (2), L. (L.) major (MHOM/SU/1973/5- ASKH) (3), L. (L.) infantum (MHOM/BR/1974/PP75) (4), and L. (V.) braziliensis (MHOM/ ATL has been growing worldwide in both incidence and range, BR/1975/M2903) (5). Digested products were resolved in 2% agarose gel stained with ethidium bromide. MM, 50-bp ladder. 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 fromL. (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 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.

Table 1 Clinical samples genotyped by RFLP-PCR in this study

Sample code-month/year Host Municipality Sample code-month/year Host Municipality 07-09/2003 Human Sorocaba 1063-07/2008 Human Sorocaba 18-09/2003 Human Sorocaba 1153-11/2008 Human Campinas 20-09/2003 Canine Sorocaba 1324-02/2009 Human Sorocaba 26-10/2003 Canine Marilia 1622-08/2009 Human Sorocaba 64-09/2004 Human Sorocaba 1758-02/2010 Human Jundiai 65-09/2004 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. The idea of conducting this study in the state of São Paulo was To prevent new cases in these areas, epidemiological strategies must be motivated by the state's increase of ATL incidences for the last 20 years. implemented, such as rapid diagnosis, treatment and vector control. The Currently, 147 municipalities have already recorded transmission. Thus, importance of the study of genetic variability of Leishmania is mainly in this study, biopsies from 24.5% of these municipalities (36) were due to its correlation with the epidemiological aspects of the disease, investigated. However, due to the low sensitivity of the IR1/IR2 primer such as geographic location, clinical forms, virulence, pathogenicity, set, samples from only 15 municipalities were genotyped. drug resistance and antigenic variation, among others6,13. One of the methods used to evaluate the genetic polymorphism of Species belonging to the L. (V.) braziliensis sub-genera are highly L. (V.) braziliensis isolates in different Brazilian regions is the analysis prevalent in patients with ATL in Brazil. Other Brazilian studies have of RFLP in the internal transcribed spacers (ITS) of the ribosomal DNA shown the genetic variability of these parasites, which would explain their (rDNA) locus. These studies have shown that molecular markers are adaptation to changes in diverse environmental conditions4,6. Thus, with suitable for population genetics and epidemiological studies4,5,6. such resilience, these parasites are more likely to infect multiple hosts. Although different genetic studies have analyzed L. (V.) braziliensis Despite the low sensitivity of the IR1/IR2 primer set and that the isolates from other Brazilian regions4,6,10,11,21,27, none have been conducted clinical samples presented a low quantity of parasites in comparison in the state of São Paulo. with culture isolates, 40% (52/132) of them were genotyped. Similar data

260 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.

Table 2 has previously shown20 the possibility of performing RFLP-PCR using Distribution of the nine L. (V.) braziliensis profiles isolated from human and small amounts of Leishmania DNA from host tissues. Consequently, it is canine clinical samples in 15 municipalities of the state of São Paulo possible to genotype Leishmania populations with the analysis of DNA extracted directly from clinical samples. This information is important, L. (V.) braziliensis Municipality because in many laboratories there are no conditions in which to isolate Human Canine genotyping (n) (n=15) and culture parasites from clinical samples20. Another interesting finding Bauru 1 - 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, Cajamar 1 - as shown in Table 1. Caraguatatuba - 1 Iperó 2 - Results showed that L (V.) braziliensis seems to be a species with Iporanga - 2 great genetic diversity, as nine different patterns were observed in 52 Pattern 1 (29 samples) Itapera 1 - different DNA samples from 15 municipalities using PCR-RFLP. As shown in Figure 3, the different L (V.) braziliensis patterns were spread Itupeva 2 - throughout the regions. This genetic variability has already been shown in Jundiai 2 - other Brazilian studies4,10,11,21,27. Additionally, the parasite polymorphism Mairiporã 1 - was correlated with different clinical forms of the disease, effectiveness 10,21,27 Ribeira 2 - of treatment and cytokines expression . Sorocaba 13 1 According to other studies4,6,23,29, genotypic variations exhibited by Jundiai 1 - L (V.) braziliensis could be explained by the adaption of parasites to Pattern 2 (9 samples) Ribeira 1 - changes in the transmission process, as originally, the biological cycle was Sorocaba 7 - restricted to forest environments. Similar to in other Brazilian regions, Avaré - 1 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 Pattern 3 (3 samples) Marilia - 1 diversity of sand flies and reservoirs. Sorocaba 1 - Campinas 1 - RESUMO Pattern 4 (2 samples) Sorocaba 1 - Caracterização genotípica de isolados de Leishmania (Viannia) Pattern 5 (1 sample) Sorocaba 1 - braziliensis provenientes de biopsias de humanos e cães com Pattern 6 (2 samples) Sorocaba 2 - leishmaniose tegumentar americana Pattern 7 (2 samples) Sorocaba 2 - Iporanga - 1 Introdução: A leishmaniose tegumentar americana (LTA) é causada Pattern 8 (2 samples) Sorocaba 1 - pelo sub-gênero Leishmania (Viannia) braziliensis. A evolução da LTA resulta com a evolução das lesões iniciais. A diversidade genética de Ilha Bela - 1 Pattern 9 (2 samples) L. (V.) braziliensis em algumas áreas endêmicas brasileiras, como no Sorocaba 1 - estado de São Paulo, é pouco conhecida. Assim, este estudo teve como Total of samples 44 8 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 Fig. 3 - Map of South America and Brazil (A) indicating the location of the state of São Paulo circulam no estado de São Paulo. Estes dados são concordantes com (A). Map of the state of São Paulo (B) indicating the municipalities studied and geographical estudos em outras regiões do Brasil, mostrando grande variabilidade distribution of the L. (V.) braziliensis patterns: 1 (red), 2 (yellow), 3 (green), 4 (blue), 5 destas populações naturais de focos endêmicos. (purple), 6 (orange), 7 (pink), 8 (light blue) and 9 (gray).

261 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.

ACKNOWLEDGMENTS 15. Goto H, Lindoso JA. Cutaneous and mucocutaneous leishmaniasis. Infect Dis Clin North Am. 2012;26:293-307.

This study was supported by grants from the FAPESP (Fundação 16. Grimaldi G Jr, Tesh RB. Leishmaniasis of the New Word: current concepts and de Amparo à Pesquisa do Estado de São Paulo, Brazil). Proc- implications for the future research. Clin Microbiol Rev. 1993;6:230-50. 2011/13939-8. L.T.F. was supported by a fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, 17. Harris E, Kropp G, Belli A, Rodriguez B, Agabian N. Single-step multiplex PCR Brazil). V.L.P.C. was supported by a fellowship from CNPq (Conselho assay for characterization of New World Leishmania complexes. J Clin Microbiol. 1998;36:1989-95. Nacional de Desenvolvimento Científico e Tecnológico, Brazil) Produtividade em Pesquisa, Proc. 303489/2012-0. Jim Hesson of 18. Lainson R, Shaw JJ. New world leishmaniasis: the neotropical Leishmania species. In: AcademicEnglishSolutions.com proofread the English. Collier L, Balows A, Sussman M, editors. Topley & Wilson’s microbiology and microbial infections. 9th ed. London: Arnold, 1998. p. 241-66. REFERENCES 19. Madeira MF, Uchôa CM, Leal CA, Silva RM, Duarte R, Magalhães CM, et al. Leishmania (Viannia) braziliensis em cães naturalmente infectados. Rev Soc Bras Med Trop. 1. Azulay RD, Azulay Junior DR. Immune-clinical-pathologic spectrum of leishmaniasis. 2003;36:551-5. Int J Dermatol. 1995;34:303-7. 20. Motoie G, Ferreira GE, Cupolillo E, Canavez F, Pereira-Chioccola VL. Spatial distribution 2. Carvalho EM, Barral A, Costa JM, Bittencourt A, Marsden P. Clinical and and population genetics of Leishmania infantum genotypes in São Paulo State, Brazil, immunopathological aspects of disseminated cutaneous leishmaniasis. Acta Trop. employing multilocus microsatellite typing directly in dog infected tissues. Infect 1994;56:315-25. Genet Evol. 2013;18:48-59.

3. Colombo FA, Odorizzi RM, Laurenti MD, Galati EA, Canavez F, Pereira-Chioccola VL. 21. Oliveira GM, Madeira MF, Oliveira FS, Pires MQ, Pacheco RS. Canine cutaneous Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected leishmaniasis: dissemination and tissue tropism of genetically distinct Leishmania from naturally infected dogs. Parasitol Res. 2011;109:267-74. (Viannia) braziliensis populations. Vet Med Int. 2013;2013:982183.

4. Cupolillo E, Brahim LR, Toaldo CB, Oliveira-Neto MP, Brito ME, Falqueto A, et 22. Pan American Health Organization. Leishmaniases: epidemiological report of the al. Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) Americas. PAHO; 2013. [Cited Jan 2014]. Available at: http://www.paho.org/hq/ braziliensis from different hosts and geographic areas in Brazil. J Clin Microbiol. index.php?option=com_docman&task=doc_view&gid=21608&Itemid=%20 2003;41:3126-32. 23. Passos VM, Fernandes O, Lacerda PA, Volpini AC, Gontijo CM, Degrave W, et al. 5. Cupolillo E, Grimaldi Júnior G, Momen H, Beverley SM. Intergenic region typing (IRT): Leishmania (Viannia) braziliensis is the predominant species infecting patients with a rapid molecular approach to the characterization and evolution of Leishmania. Mol American cutaneous leishmaniasis in the state of Minas Gerais, Southeast Brazil. Biochem Parasitol. 1995;73:145-55. Acta Trop. 1999;72:251-8.

6. Cupolillo E, Momen H, Grimaldi G Jr. Genetic diversity in natural populations of New 24. Queiroz A, Sousa R, Heine C, Cardoso M, Guimarães LH, Machado PR, et al. Association World Leishmania. Mem Inst Oswaldo Cruz. 1998;93:663-8. between an emerging disseminated form of leishmaniasis and Leishmania (Viannia) braziliensis strain polymorphisms. J Clin Microbiol. 2012;50:4028-34. 7. Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 2001;95:239-43. 25. Reimão JQ, Colombo FA, Pereira-Chioccola VL, Tempone AG. In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: detection of viable 8. Fernandes O, Bozza M, Pascale JM, Miranda AB, Lopes UG, Degrave WM. An Leishmania (L.) chagasi by real-time PCR. Exp Parasitol. 2011;128:111-5. oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia). Mem Inst Oswaldo Cruz. 1996;91:279-84. 26. Rioux JA, Lanotte G, Serres E, Pratlong F, Bastien P, Perieres J. Taxonomy of Leishmania. Use of isozymes. Suggestions for a new classification. Ann Parasitol Hum Comp. 9. Garcia AL, Parrado R, De Doncker S, Bermudez H, Dujardin JC. American tegumentary 1990;65:111-25. leishmaniasis: direct species identification of Leishmania in non-invasive clinical samples. Trans R Soc Trop Med Hyg. 2007;101:368-71. 27. Schriefer A, Schriefer AL, Góes-Neto A, Guimarães LH, Carvalho LP, Almeida RP, et al. Multiclonal Leishmania braziliensis population structure and its clinical implication 10. Garcia L, Kindt A, Bermudez H, Lianos-Cuentas A, De Doncker S, Arevalo J, et al. in a region of endemicity for American tegumentary leishmaniasis. Infect Immun. Culture-independent species typing of neotropical Leishmania for clinical validation 2004;72:508-14. of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol. 2004;42:2294-7. 28. Shaw JJ. Taxonomy of the genus Leishmania: present and future trends and their implications. Mem Inst Oswaldo Cruz. 1994;89:471-8. 11. Gomes AH, Armelin IM, Menon SZ, Pereira-Chioccola VL. Leishmania (V.) braziliensis: detection by PCR in biopsies from patients with cutaneous leishmaniasis. Exp 29. Silva RA, Mercado VT, Henriques LF, Ciaravolo RM, Wanderley DM. Magnitude and Parasitol. 2008;119:319-24. trend of American tegumentary leishmaniasis in the State of São Paulo, Brazil, 1975 to 2008. Rev Bras Epidemiol. 2012;15:617-26. 12. Gomes AH, Ferreira IM, Lima ML, Cunha EA, Garcia AS, Araujo MF, et al. PCR identification of Leishmania in diagnosis and control of canine leishmaniasis. Vet 30. World Health Organization. Leishmaniasis: Fact sheet N° 375, updated January 2014. Parasitol. 2007;144:234-41. WHO; 2014. [Cited Feb 2014]. Available from: http://www.who.int/mediacentre/ factsheets/fs375/en/index.html 13. Gontijo B, de Carvalho M de L. Leishmaniose tegumentar Americana. Rev Soc Bras Med Trop. 2003;36:71-80. Received: 23 June 2014 Accepted: 24 September 2014 14. Goto H, Lindoso JA. Current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. Expert Rev Anti Infect Ther. 2010;8:419-33.

262 Rev. Inst. Med. Trop. Sao Paulo 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 thereby increasing malaria cases in the region. Thus, the assessment of mosquito population density and species occurrence in areas where these Although cases of malaria have decreased in Brazil since 2005, the activities take place or will take place is essential for evaluating the risk disease remains an important public health problem in the country. In of malaria transmission and for designing and implementing effective 2012 alone, approximately 240,000 cases were recorded here, of which measures of control and/or prevention. more than 90% occurred in the Amazon region3. Therefore, our study aimed to assess the changes in the number and Anopheline mosquitoes are the only vectors of Plasmodium spp. composition of anopheline species along the construction corridor of a parasites to humans. These mosquitoes are found in tropical and power transmission line. neotropical regions. As of 2004, 476 species had been recorded26, of which approximately 100 are considered vectors or potential vectors MATERIALS AND METHODS of Plasmodium spp. to humans22,38. In the Amazon region, the main disease vector is Anopheles darlingi, although other species such Mosquito capture was conducted along a 380 km stretch of the as An. deaneorum, An. triannulatus, and An. nuneztovari may play BR 364 highway, between Porto Velho (RO) and Rio Branco (AC). important roles in the epidemiology of the disease35,36,43. The occurrence Six distinct localities with the highest population densities along the of an anopheline species in an endemic area is associated with the stretch were selected. These localities were Extrema (09°45'29.1"S environmental characteristics of the region. 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 South America is currently among the regions most affected by 64°56'24.4"W), Jaci Paraná (09°15'37.2"S 64°23'44.1"W), and Porto environmental changes, mainly because of the great pressure caused Velho (08°48'35.5"S 63°56'30.3"W) (Fig. 1). Vegetation in these areas by humans in the area. The construction of hydroelectric power plants, predominantly consists of open rain forest with a high degree of human family farming, and selective logging, among many other activities, has disturbance, as evidenced by the removal of native vegetation and its become common in the region44. The changes caused by these activities replacement by pasture. can drastically influence the population dynamics of malaria vectors,

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 Total Frequency Area Anopheles albitarsis l.s. 2 0.09 AB; MP Anopheles benarrochi 2 0.09 JP Anopheles braziliensis 8 0.37 MP; PVH Anopheles darlingi 2,078 95.10 PVH; EX; VA; AB; MP; JP Anopheles deaneorum 38 1.74 EX; VA Anopheles konderi l.s. 17 0.78 JP; PVH Fig. 1 - Locations of capture sites. (A) State of Rondônia location in Brazil. (B) Magnified Anopheles triannulatus l.s. 40 1.83 PVH; JP; MP view of Porto Velho municipality and localities of anopheline mosquito capture sites (circles) and hydroelectric power plants (triangle) location. AB = Abunã; EX = Extrema; JP = Jaci Paraná; MP = Mutum Paraná; PVH = Porto Velho; VA = Vista Alegre Abunã. 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 Fig. 2 - Scoring of the areas according to the composition of anopheline species. 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%. Table 2 Number and distribution of Anopheles darlingi mosquitoes RESULTS collected by locality

We collected 2,185 anopheline mosquitoes belonging to seven Contrast Locality Mean ± SD p value species in the six localities. Of the mosquitoes collected, 95.1% were analysis identified as An. darlingi, 1.8% as An. triannulatus l.s., 1.7% as An. Porto Velho 144.55 ± 59.81 a < 0.001 deaneorum, 0.8% as An. konderi l.s., 0.4% as An. braziliensis, 0.1% as Jaci Paraná 37.22 ± 10.40 b An. albitarsis l.s., and 0.1% as An. benarrochi l.s. Anopheles darlingi Extrema 26.55 ± 12.27 b 0.30 was collected in all localities. The remaining species were found in sites with unique characteristics (e.g., An. braziliensis was predominantly Mutum Paraná 23.77 ± 2.78 b Vila Abunã 7.11 ± 1.04 c found in areas of secondary growth, such as forest clearings and 0.63 pastures) (Table 1). Mutum Paraná, Porto Velho, and Jaci Paraná Vista Alegre do Abunã 5.66 ± 2.71 c exhibited four of the seven collected species, whereas only two were SD: standard deviance; columns followed by different letters indicate statistical found in Extrema, Vista Alegre do Abunã, and Vila Abunã. Moreover, difference. 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 (ANOVAχ 2 = 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 Fig. 3 - Mean of Anopheles darlingi collected per night in and around dwellings of various 2 in outdoor than indoor dwellings (ANOVA χ = 27.79, p < 0.001; localities under study. AB = Abunã; EX = Extrema; JP = Jaci Paraná; MP = Mutum Paraná; Fig. 3). PVH = Porto Velho; VA = Vista Alegre Abunã.

264 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.

DISCUSSION Because anopheline mosquitoes are key determinants in the transmission of Plasmodium, which can infect and cause malaria in In Brazil, six species of anopheline mosquitoes play significant humans, the regular monitoring of the disease transmission of these roles in the transmission of Plasmodium spp. parasites to humans. vectors in relation to regional climate cycles is of paramount importance. Three of these species were collected in our study: An. darlingi, An. Moreover, scientifically-based joint and coordinated action between triannulatus, and An. braziliensis. Other species such as An. albitarsis public authorities and construction entrepreneurs should be conducted l.s. may be secondary or specific vectors in particular areas29,35,36,43. The to control malaria transmission in the Brazilian Amazon. prevalence of An. darlingi has remained consistently higher than that of other species since the first studies of malaria vectors in Rondônia, RESUMO 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 Distribuição sazonal de vetores da malária (Diptera: Culicidae) darlingi in this study was expected, as this species presents the most em localidades rurais de Porto Velho, Rondônia, Amazônia anthropophilic behavior among the sample collected. Furthermore, many brasileira 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. Foi realizado levantamento de vetores de malária na área que compreende a construção da linha de transmissão entre os municípios de The peak number of species occurred in July. Anopheles darlingi Porto Velho e Rio Branco, estados de Rondônia e Acre, respectivamente. showed two distinct population peaks, the first in March/May (during Os dados aqui apresentados mostram os resultados do levantamento the rainy season) and the second in August/September (end of the dry da fauna dos Anopheles realizado em Rondônia. As capturas foram season)18. The peak in July may be explained by the environmental realizadas no município de Porto Velho em seis aglomerados changes in the area due to the implementation of two hydroelectric power populacionais ao longo da rodovia federal BR 364, denominados Porto plants, or a delay in the response of An. darlingi to the rainy season. Velho, Jaci Paraná, Mutum Paraná, Vila Abunã, Vista Alegre do Abunã e Extrema. As capturas ocorreram em três diferentes pontos de cada The susceptibility of anopheline mosquitoes to infection by the uma das localidades nos meses de fevereiro, julho e outubro de 2011, predominant Plasmodium species recorded in the region, P. falciparum seguindo a metodologia de coleta por atração humana protegida em dois and P. vivax, has been previously reported9,27,28,31. Although the highest ambientes, sendo no intradomicílio e no peridomicílio simultaneamente number of mosquitoes occurred around dwellings, the results highlight com duração de seis horas. Nas áreas amostradas foram capturados 2.185 that An. darlingi exhibits highly anthropophilic behavior. Moreover, anofelinos pertencentes a sete espécies de Anopheles sp. dos quais 95,1% although we did not find a higher number of mosquitoes inside dwellings, foram identificados como Anophels darlingi, 1,8% An. triannulatus l.s., these species are highly endophilic15,21,30, making the study region a high- 1,7% An. deaneorum, 0,8% An. konderi l.s., 0,4 An. braziliensis, 0,1% risk area for the transmission and prevalence of malaria. An. albitarsis l.s., e 0,1% An. benarrochi. Anopheles darlingi foi a única espécie amostrada em todas as localidades enquanto as demais espécies, New farming frontiers in areas where malaria is endemic require more ocorreram em locais com características singulares. public policies for mosquito control8,34,41. Climate change, urbanization, and new settlements for agriculture and the rearing of livestock are among FINANCIAL SUPPORT the factors that can lead to epidemics of malaria and other vector-borne diseases41. This study received financial and logistical support from Cepemar Serviços de Consultoria em Meio Ambiente Ltda. As the localities of the present study are situated within the area of influence of two hydroelectric power plants in the Madeira River, REFERENCES with intense anthropization and increased water surface, and the results obtained indicate high vector densities in urbanized areas, the current 1. Alves FP, Durlacher RR, Menezes MJ, Krieger H, Silva LH, Camargo EP. High prevalence malaria situation requires attention. The Madeira River carries a large of asymptomatic Plasmodium vivax and Plasmodium falciparum infections in native Amazonian populations. Am J Trop Med Hyg. 2002;66:641-8. quantity of suspended sediment, which favors the predominance of mosquitoes of the Mansonia genus. The river water does not exhibit 2. Alves FP, Gil LH, Marrelli MT, Ribolla PE, Camargo EP, da Silva LH. Asymptomatic this characteristic, however, the conditions in this water promote the carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the proliferation of An. darlingi39. Therefore, despite the decreased number Brazilian Amazon. J Med Entomol. 2005;42:777-9. of malaria cases over the last few years in Brazil3, urban expansion in 3. Brasil. Ministério da Saúde. Secretaria de Vigilância Epidemiológica. Sistema de the proximity of these new water reservoirs increases the risk of malaria Informação de Vigilância Epidemiológica-SIVEP-Malária. [cited 15 Sep 2013]. 14,40 transmission . Available from: http://www.saude.gov.br/sivep_malaria

Factors such as the creation of water reservoirs and deforestation 4. Camargo EP, Alves F, Pereira da Silva LH. Symptomless Plasmodium vivax infections caused by the construction of hydroelectric power plants and power in native Amazonians. Lancet. 1999;353:1415-6. 18,42 lines, as well as peri-urban transmission of disease , have increased 5. Camargo LM, Noronha E, Salcedo JM, Dutra AP, Krieger H, Pereira da Silva LH, et al. 1,2,4,5,23,24,25 the density of malaria vectors , thereby elevating the risk of The epidemiology of malaria in Rondonia (Western Amazon region, Brazil): study epidemics within the next several decades19,20. of a riverine population. Acta Trop. 1999;72:1-11.

265 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.

6. Consoli RA, Oliveira RL. Principais mosquitos de importância sanitária no Brasil. Rio 24. Katsuragawa TH, Gil LH, Tada MS, Pereira da Silva LH. Endemias e epidemias na de Janeiro; Fiocruz; 1994. Amazônia. Malária e doenças emergentes em áreas ribeirinhas do Rio Madeira. Um caso de escola. Estudos Av. 2008;22:111-41. 7. Cruz RM, Gil LH, de Almeida e Silva A, da Silva Araújo M, Katsuragawa TH. Mosquito abundance and behavior in the influence area of the hydroelectric complex on the 25. Katsuragawa TH, Gil LH, Tada MS, de Almeida e Silva A, Costa JDN, Araújo MS, et al. Madeira River, Western Amazon, Brazil. Trans R Soc Trop Med Hyg. 2009;103:1174- The dynamics of transmission and spatial distribution of malaria in riverside areas 6. of Porto Velho, Rondônia, in Amazon Region of Brazil. PLOS One. 2010;5:e9245.

8. de Castro MC, Monte-Mór RL, Sawyer DO, Singer BH. Malaria risk on the Amazon 26. Kiszewski A, Mellinger A, Spielman A, Malaney P, Sachs SE, Sachs J. A global index frontier. Proc Natl Acad Sci. 2006;103:2452-7. representing the stability of malaria transmission. Am J Trop Med Hyg. 2004;70:486- 98. 9. de Oliveira-Ferreira J, Lourenço-de-Oliveira R, Teva A, Deane LM, Daniel-Ribeiro CT. Natural malaria infections in anophelines in Rondonia State, Brazilian Amazon. Am 27. Klein TA, Lima JB, Tada MS, Miller R. Comparative susceptibility of anopheline J Trop Med Hyg. 1990;43:6-10. mosquitoes in Rondonia, Brazil to infection by Plasmodium vivax. Am J Trop Med Hyg. 1991;45:463-70. 10. Deane LM. Malaria vectors in Brazil. Mem Inst Oswaldo Cruz. 1986;81(Suppl 2):5-14. 28. Klein TA, Lima JB, Tada MS. Comparative susceptibility of anopheline mosquitoes to 11. Deane LM, Causey CR, Deane MP. Notas sobre a distribuição e a biologia dos anofelinos Plasmodium falciparum in Rondonia, Brazil. Am J Trop Med Hyg. 1991;44:598-603. das regiões nordestina e amazônica do Brasil. Rev Serv Espec Saúde Pública. 1948;1:827-965. 29. Laporta GZ, Ramos DG, Ribeiro MC, Sallum MAM. Habitat suitability of Anopheles vector species and association with human malaria in the Atlantic Forest in south- 12. Deane LM, Daniel Ribeiro C, Lourenço de Oliveira R, Oliveira-Ferreira J, Guimarães eastern Brazil. Mem Inst Oswaldo Cruz. 2011;106(Suppl 1):239-45. AE. Study on the natural history of malaria in areas of the Rondonia State-Brazil and problems related to its control. Rev Inst Med Trop Sao Paulo. 1988;30:153-6. 30. León W, Valle J, Naupay R, Tineo E, Rosas A, Palomino M. Comportamiento estacional del Anopheles (Nyssorhynchus) darlingi Root 1962 en localidades de Loreto y Madre 13. Forattini OP. Culicidologia médica: identificação, biologia, epidemiologia. São Paulo; de Dios, Peru 1999-2000. Rev Peru Med Exp Salud Publica. 2003;20:22-7. EDUSP; 2002. 31. Marrelli MT, Honório NA, Flores-Mendoza C, Lourenço-de-Oliveira R, Marinotti O, 14. Freitas FT, Costa CM, Nóbrega AA, Araújo WN. Surto de malária no distrito de Jaci- Kloetzel JK. Comparative susceptibility of two members of the Anopheles oswaldoi Paraná, município de Porto Velho-RO, em 2009. Bol Epidemiol Eletron. 2010;10:1-4. complex, An. oswaldoi and An. konderi, to infection by Plasmodium vivax. Trans R Soc Trop Med Hyg. 1999;93:381-4. 15. Gama RA. Periodicidade de hematofagia de Anopheles darlingi, em Porto Velho (RO), e modificação da armadilha BG-Sentinel® para a captura de anofelinos, visando à 32. Morais SA, Urbinatti PR, Sallum MA, Kuniy AA, Moresco GG, Fernandes A, et al. substituição da atração humana. [PhD thesis]. Belo Horizonte: Universidade Federal Brazilian mosquito (Diptera: Culicidae) fauna. I. Anopheles species from Porto de Minas Gerais, Instituto de Ciências Biológicas; 2009. Velho, Rondônia State, western Amazon, Brazil. Rev Inst Med Trop Sao Paulo. 2012;54:331-5. 16. Gama RA, Silva IM, Monteiro HA, Eiras AE. Fauna of Culicidae in rural areas of Porto Velho and the first record of Mansonia (Mansonia) flaveola (Coquillet, 1906), for 33. Moutinho PR, Gil LH, Cruz RB, Ribolla PE. Population dynamics, structure and behavior the state of Rondônia, Brazil. Rev Soc Bras Med Trop. 2012;45:125-7. of Anopheles darlingi in a rural settlement in the Amazon rainforest of Acre, Brazil. Malar J. 2011;10:174. 17. Gil LH, Alves FP, Zieler H, Salcedo JM, Durlacher RR, Cunha RP, et al. Seasonal malaria transmission and variation of anopheline density in two distinct endemic areas in 34. Natal D, Barata JM, Lagos CB, Rocha RM. Nota sobre culicídeos (Diptera: Culicidae) Brazilian Amazonia. J Med Entomol. 2003;40:636-41. da bacia do rio Purus, Acre, Amazônia (Brasil). Rev Saúde Pública. 1992;26:129-31.

18. Gil LH, Tada MS, Katsuragawa TH, Ribolla PEM, Pereira da Silva LH. Urban 35. Póvoa MM, Conn JE, Schlichting CD, Amaral JC, Segura MN, Da Silva AN, et al. Malaria and suburban malaria in Rondônia (Brazilian Western Amazon). II. Perennial vectors, epidemiology, and the re-emergence of Anopheles darlingi in Belém, Pará, transmissions with high anopheline densities are associated with human environmental Brazil. J Med Entomol. 2003;40:379-86. changes. Mem Inst Oswaldo Cruz. 2007;102:271-6. 36. Póvoa MM, de Souza RT, Lacerda RN, Rosa ES, Galiza D, de Souza JR, et al. The 19. Gomes AC, Paula MB, Duarte AM, Lima MA, Malafronte RS, Mucci LF, et al. importance of Anopheles albitarsis E and An. darlingi in human malaria transmission Epidemiological and ecological aspects related to malaria in the area of influence of in Boa Vista, state of Roraima, Brazil. Mem Inst Oswaldo Cruz. 2006;101:163-8. the lake at Porto Primavera dam, in western São Paulo State, Brazil. Rev Inst Med Trop São Paulo. 2008;50:287-95. 37. R Development Core Team. R: a Language and Environment for Statistical Computing. Vienna: R Development Core Team; 2014. Available from: http://www.r-project.org 20. Gomes AC, Paula MB, Natal D, Gotlieb SL. Ecologia de Anopheles (Nyssorhynchus) darlingi Root em área de implantação de empreendimento hidrelétrico, na divisa dos 38. Raghavendra K, Barik TK, Reddy BP, Sharma P, Dash AP. Malaria vector control: from estados de Mato Grosso do Sul e São Paulo. Rev Soc Bras Med Trop. 2010;43:272-6. past to future. Parasitol Res. 2011;108:757-79.

21. Guimarães AE, Gentile C, Lopes CM, de Mello RP. Ecology of mosquitoes (Diptera: 39. Rodrigues IB, Tadei WP, Dias JM, Lima CA. Atividade larvicida de Bacillus sphaericus Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. III. Daily 2362 contra Anopheles sp. (Diptera, Culicidae) em rios do Amazonas, Brasil. biting rhythms and lunar cycle influence. Mem Inst Oswaldo Cruz. 2000;95:753-60. BioAssay. 2013;8:2. [cited 15 Jan 2014]. Available from: http://www.bioassay.org. br/bioassay/article/download/95/178 22. Harbach RE. The classification of genus Anopheles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships. Bull Entomol Res. 2004;94:537-53. 40. Saraiva MD, Amorim RD, Moura MA, Martinez-Espinosa FE, Barbosa MD. Expansão urbana e distribuição espacial da malária no município de Manaus, Estado do 23. Katsuragawa TH, Cunha RP, de Souza DC, Gil LH, Cruz RB, Silva AA, et al. Malária e Amazonas. Rev Soc Bras Med Trop. 2009;42:515-22. aspectos hematológicos em moradores da área de influência dos futuros reservatórios das hidrelétricas de Santo Antônio e Jirau, Rondônia, Brasil. Cad Saúde Pública. 41. Sutherst RW. Global change and human vulnerability to vector-borne diseases. Clin 2009;25:1486-92. Microbiol Rev. 2004;17:136-73.

266 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.

42. Tada MS, Marques PR, Mesquita E, Dalla-Martha RC, Rodrigues AJ, Costa JDN, et 44. Tadei WP, Thatcher BD, Santos JM, Scarpassa VM, Rodrigues IB, Rafael MS. Ecologic al. Urban malaria in the Brazilian Western Amazon Region. I. High prevalence of observations on anopheline vectors of malaria in the Brazilian Amazon. Am J Trop asymptomatic carriers in an urban riverside district is associated with a high level of Med Hyg. 1998;59:325-35. clinical malaria. Mem Inst Oswaldo Cruz. 2007;102:263-9. Received: 26 March 2014 43. Tadei WP, Dutary Thatcher B. Malaria vectors in the Brazilian Amazon: Anopheles of Accepted: 30 September 2014 the subgenus Nyssorhynchus. Rev Inst Med Trop Sao Paulo. 2000;42:87-94.

267 LIBRARY OF THE SÃO PAULO INSTITUTE OF TROPICAL MEDICINE

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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 findingT. 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 in a house and keeping chickens in a corral were strong determinants zoonosis transmitted domestically by Triatoma dimidiata Latreille 1811 for house infestation in rural communities. Such findings from Yucatan (Hemiptera: Reduviidae), the only locally proven vector. T. dimidiata can agree with reports of house infestation and colonization by T. dimidiata be collected in domestic, peridomestic and sylvatic habitats of Yucatan. in Guatemala11. This study confirms the significance of the peridomicile House infestation is described as seasonal, occurring mainly due to the environment, and particularly of chicken coops, as a source of T. dimidiata dispersal of adult insects from peridomestic and sylvatic habitats during populations and a potential risk factor for T. cruzi transmission in a rural the late dry season with non-apparent or limited colonization2,5. village in Yucatan, Mexico.

Few studies in Yucatan have examined the importance of household Fieldwork was carried out between November 2009 and May 2010 in and backyard characteristics in the prevalence of triatomine infestations a sample of 101 homesteads (each homestead including the house and all and their infection rates with T. cruzi in and around houses. GUZMÁN- peridomestic structures found in the front and backyard) from Molas, a MARÍN et al.8 reported that household triatomine infestation in rural village located in the Southeast of Mexico (20° 48’58’’ N and 089° rural communities was associated with the type/quality of housing, 37’54’ W). The community has a population of 2,014 inhabitants, living e.g. construction with natural materials, thatched roofs, unplastered in 553 houses and surrounded by a subtropical deciduous forest within the walls or walls with adobe plastering and the lack of cemented floors. Cuxtal ecological reserve. Altitude is 10 m. a. s. l. Climate is characterized However, other studies have reported that the location of a house within by an average annual temperature of 25.9 °C, with an annual rainfall of 800- a community (especially if located on the periphery) is a significant risk 1000 mm, occurring mainly between June and November. Molas is located factor for infestation and the invasion of dispersing adult insects2,5,13. within the highest risk area for Chagas disease in the state of Yucatan2,3. REYES-NOVELO et al.14 showed that T. dimidiata did colonize animal shelters (e. g. chicken and dove coops, dog houses and opossum nests). Homestead infestation with triatomines was evaluated through: More recently, DUMONTEIL et al.5 quantified that the number of dogs 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 Using a Fisher’s exact test, statistical analyses compared the sex manual active searches for triatomines (described by Gürtler et al. 1999) and stage of development of T. dimidiata between locations (intra- and were performed, one during the 2009 rainy season (November) and the peridomicile). Comparisons of infection by sex between seasons were other one during the 2010 dry season (May). not performed because of the low number of insects collected. Tests were carried out to study the association between triatomine infestation Collections were performed inside houses (interdomiciliary) and (positive homesteads) and T. cruzi infection with household-level in front/backyards (peridomiciliary) by teams of two trained research potential risk factors. Variables of interest were analyzed using c2 to personnel (30 min in each ecotope to complete one man/hour/homestead reduce the model, by comparing percentages in contingency tables. Those between 8:00 a.m. and 1:00 p.m.). Intradomiciliary searches included with p < 0.25 were included in a logistic regression analysis. Adjusted inside walls, the base of the roof and furniture. Peridomiciliary searches Odds Ratio and Confidence Intervals α( = 0.05) were calculated with focused on animal housing, rock and woodpiles, tree trunks, and any SPSS® (v17.0). other potential triatomine refuges. In addition, householders were invited to take part in a six-month participatory vector surveillance strategy4 A total of 200 T. dimidiata specimens were collected from 35.6% between December and March, 2010. (36/101) homesteads throughout the study period (Table 1). Overall, a greater number of adults were collected than nymphs (p < 0.05), with a Trypanosoma cruzi DNA extraction from individual triatomines higher male:female abundance ratio (p < 0.05) between ecotopes (Table and PCR amplification of T. cruzi kinetoplast DNA were performed 1). The majority of specimens (73%) - both adults and nymphs - were as described by REYES-NOVELO et al.14 based on the EDWARDS collected in the peridomicile environment; nevertheless, 22.5% of the et al.6 and MOSER et al.12 protocols. The primers used were adults and 4.5% of the nymphs collected were reported to have been TcZ1: 5’-CGAGCTCTTGCCCACACGGGTGCT-3’- and TcZ2 found intradomiciliary. 5’-CCTCCAAGCAGCGGATAGTTCAGG-3’. Amplification was performed in a Techne TC132 (Barloworld Scientific LTD, Collection methods were complementary. Active collections yielded Staffordshire, UK) thermal cycler. A 188bp fragment identified the more specimens (65%) than participatory collections. 130 T. dimidiata presence of T. cruzi DNA following the electrophoresis of a percentage specimens were captured by active collection, mostly peridomiciliary of PCR product in a 2% agarose-TBE stained with ethidium bromide (10 (97.7%), with a sample composed by adults and nymphs in a similar µg/mL) and further documentation in an EDAS 290 gel documentation ratio. 70 specimens of T. dimidiata were captured through householders’ system (Kodak, Rochester, USA). Local strains of T. cruzi were used collections, mostly reported as intradomiciliary (74.3%) and consisting as positive controls, whereas the whole mixture minus DNA was used mostly of adult triatomines (77%). as a negative control. Overall, 48% (96/200) of the T. dimidiata specimens collected A household survey was performed to investigate a range of tested positive for T. cruzi (Table 1) and were found in 31.6% (32/101) household/backyard characteristics previously reported as significant in of homesteads. The infected specimens were mostly adults (p < 0.05); the infestation of T. dimidiata and other triatomines1,10,15 - type/material but the proportion of nymphs infected was high (37.5%). Slightly more of the house (roof, walls, floor); use of window screening; presence of infected T. dimidiata were found intradomiciliary (54.1%) than in the rubbish, rock/wood piles, stone walls, abandoned lots on the sides; the peridomicile environment (45.9%). Infection prevalence detection was presence of domestic animals e. g. dogs, cats, poultry, horses, sheep, higher in participatory collections (59/70) than in active collections cattle, and the presence of animal housing structures (organized by (37/130) (p < 0.05). species) e. g. chicken coops, pig corrals, house stables and kennels.

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 (%) ♂ (%) ♀ (%) Infestation 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)* Infection 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) * 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 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.

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 (%) ♂ (%) ♀ (%) 1st active collection Rainy season Infestation 41 23 (56.1) 18 (43.9) 15 (36.6)* 3 (7.3) Infection 20 19 (95)* 1 (5) 0 (0) 1 (5) 2nd active collection Dry season Infestation 26 13 (50.0) 13 (50.0) 5 (19.2) 8 (30.8) Infection 10 4 (40) 6 (60) 3 (30) 3 (30) *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 rabbit hutch had a large population of triatomines, this type of refuge presence of a chicken coop in the backyard, the type of walls in the was not as commonly found in the peridomiciles as chicken coops. Piles house and wood storage; but only the presence of a chicken coop was of rocks and wood were quite common, but only one was found to be significantly and positively associated with the presence of T. dimidiata infested with triatomines. (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. While the debate concerning whether house infestation by dimidiata infected with T. cruzi (OR = 3.37, p = 0.006; 95% CI = 1.36- triatomines is influenced by the peridomicile and/or the sylvatic habitats 8.33). 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 Fifty-four homesteads were found in the area with at least one populations. Preceding studies in Yucatan report that T. dimidiata chicken coop. The general structure of local chicken coops consists of infestations occur seasonally but transiently, i.e. with a limited capacity cages of 1.30 - 1.50m in height, square in shape, and with sides of 2 - 3m for colonizing households in Yucatan2,7. These findings indicate the in length. Coops are built on a 4-log base, one on each corner, holding existence of peri- and intradomiciliary infestation and the high prevalence a roof made of either zinc or cardboard and surrounded by a chicken of infected triatomines not only during the dry season, but also in the wire fence. The ground is commonly covered with compacted dirt and rainy season. Colonization in houses (based on the collection of nymphs) small stones. Approximately 26% (14/54) of the chicken coops had is indeed uncommon during the rainy season, but increases during the T. dimidiata, and 64.3% (9/14) had triatomines infected with T. cruzi. dry season. Of all peridomestic triatomines, 45.9% (67/146) were collected from chicken coops, with nymphs and adults found in a similar ratio (36:31, This study shows that chicken coops are a risk factor for insect respectively. 44.8% of all specimens collected from chicken coops were infestation and parasite infection. Nonetheless these findings should be infected (30/67), with a higher percentage of infected nymphs (76.7% re-evaluated in other communities infested by T. dimidiata. compared to 23.3% in adults). RESUMEN Triatomine specimens collected from chicken coops were obtained exclusively by active collection and were found on the floor and under Gallineros, la infestación por Triatoma dimidiata y su infección con stones. During the sectional-active collection in the rainy season, 9.3% Trypanosoma cruzi en una localidad rural de Yucatán, México (5/54) of homesteads with chicken coops were positive for triatomines. During the second active collection in the following dry season, 16.7% Investigamos longitudinalmente la asociación entre la infestación (9/54) of homesteads with chicken coops were positive for triatomines por Triatoma dimidiata, su infección con Trypanosoma cruzi y las (the majority identified positive for the first time and only two were características ambientales de los domicilios/peridomicilios en 101 consistent from the collection four months earlier (Table 2). viviendas de Molas, Yucatán, México entre Noviembre de 2009 (temporada lluviosa) y Mayo de 2010 (temporada seca). Mediante Chicken coops are known to play an important role in the maintenance modelos de regresión logística se probaron asociaciones entre la of T. dimidiata populations, both as a refuge for invading insects11,16 and as infestación/infección de T. dimidiata y factores de riesgo potenciales a primary source of blood for triatomines9. All coops that tested positive a nivel de las viviendas. Se colectó un total de 200 individuos de T. in Molas had chickens, except for one, where chickens were removed dimidiata en el 35.6% de las viviendas, mayormente del peridomicilio two weeks before the survey. Among the total homesteads sampled in (73%). De todos los triatominos colectados el 48% se encontraron the locality, only two other sites were found to be used by triatomines infectados con T. cruzi. Los triatomas infectados fueron colectados as refuges: a rabbit hutch and a pile of rocks and wood. Although the en el 31.6% de las viviendas (54.1% y 45.9% en intra y peridomicilio

271 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. respectivamente). Aproximadamente el 30% de todos los triatominos 6. Edwards K, Johnstone C, Thompson C. A simple and rapid method for the preparation colectados, fueron encontrados en gallineros. La presencia de un gallinero of plant genomic DNA for PCR analysis. Nucleic Acid Res. 1991;19:1349. en el peridomicilio de una vivienda se asoció significativamante tanto 7. Gourbiere S, Dumonteil E, Rabinovich JE, Minkoue R, Menu F. Demographic and con las posibilidades de encontrar T. dimidiata (OR = 4.10, CI 95% = dispersal constraints for domestic infestation by non-domicilated Chagas disease 1.61-10.43, p = 0.003) como con la presencia de triatominos infectados vectors in the Yucatan Peninsula, Mexico. Am J Trop Med Hyg. 2008;78:133-9. 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 8. Guzmán-Marín E, Barrera-Perez M, Rodríguez-Felix ME, Escobedo-Ortegon F, Zavala- Velázquez J. Índices entomológicos de Triatoma dimidiata en el estado de Yucatán. putativa de poblaciones de T. dimidiata y como una fuente potencial de Rev Biomed. 1991;2:20-9. riesgo de transmisión de T. cruzi. 9. Guzmán-Marín E, Barrera-Perez M, Rodríguez-Félix ME, Zavala-Velázquez J. Hábitos ACKNOWLEDGEMENTS 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 This study was funded by the project “Estudio multidisciplinario para dimidiata in the city of Mérida, Yucatán, México. Vector Borne Zoonotic Dis. la identificación de variables asociadas a la transmisión de enfermedades 2007;7:597-606. zoonóticas y enfermedades transmitidas por vector en Yucatán” of the Red epidemiológica de Enfermedades Zoonóticas y Transmitidas por 11. Monroy CM, Bustamante DM, Rodas A, Enriquez ME, Rosales R. Habitats, dispersion Vector (ETV’s) de Importancia en Salud Pública (PROMEP 2008- and invasion of sylvatic Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) in Peten, Guatemala. J Med Entomol. 2003;40:800-6. 103.5/09/12.58. SISTPROY CIRB-2009-0006). 12. Moser D, Kirchoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA REFERENCES 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 13. Ramírez-Sierra MJ, Herrera-Aguilar M, Gourbiere S, Dumonteil E. Patterns of house pallidipennis (Hemiptera: Reduviidae: Triatominae) is associated with housing infestation dynamics by non-domiciliated Triatoma dimidiata reveal a spatial gradient characteristics in rural Mexico. J Med Entomol. 2006;43:1252-60. 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, Baños- López O, et al. Geographic distribution of Triatoma dimidiata and transmission 14. Reyes-Novelo E, Ruiz-Piña HA, Escobedo-Ortegon J, Barrera-Perez M, Manrique-Saide dynamics of Trypanosoma cruzi in the Yucatan peninsula of Mexico. Am J Trop Med P, Rodríguez-Vivas RI. Triatoma dimidiata (Latreille) abundance and infection with Hyg. 2002;67:176-83. Trypanosoma cruzi in a rural community of Yucatan, Mexico. Neotrop Entomol. 2013;42:317-24. 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 15. Starr MD, Rojas JC, Zeledón R, Hird DW, Carpenter TE. Chagas’ disease: risk factors Mexico. Am J Trop Med Hyg. 2004;70:514-9. for house infestation by Triatoma dimidiata, the major vector of Trypanosoma cruzi in Costa Rica. Am J Epidemiol. 1991;133:740-7. 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 16. Zeledón R, Montenegro VM, Zeledón O. Evidence of colonization of man-made ecotopes non-domiciliated triatomines. J Parasitol. 2009;95:469-71. by Triatoma dimidiata (Latreille, 1811) in Costa Rica. Mem Inst Oswaldo Cruz. 2001;96:659-60. 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 non- Received: 23 April 2014 domiciliated Triatoma dimidiata in the Yucatan peninsula, Mexico. PLOS Negl Trop Accepted: 16 September 2014 Dis. 2013;7:e2466.

272 Rev. Inst. Med. Trop. Sao Paulo 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 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. Invasive candidiasis is a clinical condition that generally occurs in The remainder of the systemic examination was unremarkable. immunosuppressive patients and those with general defects in the immune system. Candida albicans is the most common fungal pathogen capable Laboratory results included a leukocyte count of 14800×103/mm3, of causing infections, ranging from slight mucocutaneous disorders to serum glucose level of 143 mg/dL, blood urea nitrogen of 40 mg/ invasive diseases which affect multiple organs3. Deep Candida infections dL and creatinine level of 1.7 mg/dL. Inflammatory markers were rarely occur in healthy hosts, but the incidence is greatly increased elevated with an erythrocyte sedimentation rate (ESR) of 120 mm/h in immunosuppressive patients. Tuberculosis remains a major global and C-reactive protein (CRP) level of 11.1 mg/L. Both a tuberculin health problem and has become significantly more prevalent in the past skin test and human immunodeficiency virus (HIV) antibody exam decade. Moreover, spinal tuberculosis may not display typical symptoms were negative. Comprehensive immunological studies, including and, sometimes, show predominant extrapulmonary manifestations that serum immunoglobulins and complement levels, tests for cell-mediated result in delayed or missed diagnosis. Furthermore, multiple deep organ immunity (NK, CD3, CD4, CD8, CD4/CD8 and CD19) and autoantibody infections caused by Candida albicans and spinal tuberculosis occurring tests, were normal. 1, 3-β-D-glucan assay levels and the galactomannan simultaneously in the same patient are very rare. In view of this, the test were normal. Blood cultures were also negative. Whole-body positron following report presents the rare case of multiple deep abscesses caused emission tomography/computed tomography (PET/CT) revealed multiple by Candida albicans with simultaneously occurring spinal tuberculosis abscesses in the right of the patient’s neck, liver and right psoas major in an immunocompetent patient. area respectively (Fig.1A); and there was a raised uptake of [18F] FDG in vertebral bodies of T11, T12 and L1. Consequently, percutaneous CASE REPORT abscess drainage was conducted on the upper body using B-mode ultrasonography and drained brown fluids (30 mL, 280 mL and 130 mL, A 19-year-old man complained of recurrent fever and lower back pain respectively, Fig.1B) were sent to the microbiology lab. Also, drainage for a month. He had a history of scalded mouth syndrome with no regular tubes were inserted into abscesses in the liver and psoas major, but were antibiotic or antifungal drug treatment six months prior to hospitalization. removed after no drainage took place. Results of the Chromagar Candida He had neither history of taking immunosuppressors, nor of any disease Medium (Chromagar, France) cultures were positive for Candida albicans indicative of immunodeficiency. He had, however, received the Bacillus (Fig. 1C), but no acid-fast bacilli were detected. The germ tube test was Calmette-Guérin (BCG) vaccine during childhood. On admission, his positive and the documented diagnosis using API 20C Aux systems vital signs included an oral temperature of 39 ºC, heart rate of 92 beats (BioMeriux, France) was Candida albicans, which was sensitive to per minute, respiratory rate of 20 breaths per minute and blood pressure 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. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 273-5, 2015.

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.

term therapy with broad-spectrum antibiotics or corticosteroids11. The incidence of IC in the US between 1996 and 2003 was 19-24 infections Fig. 1 - A. Whole-Body positron emission tomography/computed tomography (PET/CT) per 10,000 annual hospital discharges12. 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 As one knows, microscopic examination and the cultivation of ultrasonography inducted. C. Microphotography of Candida albicans Gram staining 1000×. clinical samples for the diagnosis of fungal infection are gold-standard methods. Diagnosis of invasive candidiasis remains difficult and is However, there was no improvement in the patient’s lower back generally confirmed by the direct microscopic examination of the fluid pain. Spinal magnetic resonance imaging (MRI) showed altered signal drained during percutaneous or surgical abscess drainage. Candida intensity in T11, T12, L1 and unique osteolytic lesions in upper vertebral infection in patients is clinically very difficult to recognize and its bodies (Fig. 2A). Subsequently, computed tomography (CT) guided diagnosis is frequently missed because of non-specific clinical features, percutaneous vertebral biopsy specimens from the T12 vertebrae revealed poor diagnostic yield of traditional microbiological techniques and the caseous material and the acid fast bacilli were identified (Fig.2B, C). non-specificity of radiological imaging9. The results of blood cultures Also, the aspirate culture showed growth of the M. tuberculosis. Chest are frequently negative, as was the case for this patient. In addition, 1, CT, urine and sputum examinations were normal. Taking histological and 3-β-D-glucan assay levels and galactomannan tests were normal in this microbiological findings into consideration, the patient was treated with case. Recently, some studies testified the importance of 18[ F] FDG-PET/ antitubercular therapy (rifampicin, isoniazid, pyrazinamide, ethambutol) CT in detecting non-central nervous system invasive fungal infection for 12 months. In addition, he was treated with a total dose of 1500 at an early stage and assessing the efficiency of antifungal therapy5. mg of amphotericin B, while oral fluconazole and 5-flucytosine were With the help of whole-body PET/CT scans, the patient’s multiple subsequently prescribed for six months. By his 2-year follow up, there deep abscesses were found in time. Although the whole-body PET/CT were complete resolutions of the lesions in the upper vertebral bodies provides a new, more sensitive way of observing deep organ infections, and no evidence of new abscesses. its high costs mean that it cannot be widely used in clinical examinations. According to the clinical practice guidelines for the management of DISCUSSION candidiasis by the Infectious Diseases Society of America and antifungal susceptibility testing, amphotericin B is the current antifungal treatment Candida is an opportunistic fungal pathogen which generally of choice10. The patient responded to antifungal therapy and the number forms part of the normal flora of the oropharynx, gastrointestinal tract, of abscesses was greatly decreased. In spite of the high death rates of and urinary tract in the healthy human. Candida albicans is the most invasive candidiasis, the patient survived due to the combination of early common pathogenic cause of fungal infections in humans, which can diagnosis, percutaneous drainage and antifungal therapy. involve multiple organs, including the brain, mediastinum, kidney, heart, lung, pancreas, liver and peritoneum. Invasive candidiasis (IC) involves Tuberculosis (TB), a disease more common in immunocompromised the infection and spread of Candida via the bloodstream and normally persons, is a major global problem still prevalent in developing countries, affects the immunocompromised or immunodeficient, such as those despite the availability of highly effective treatment for decades. China with diabetes, neutropenia or burns; people undergoing hemodialysis, has the second highest level of tuberculosis in the world, registering abdominal surgery or total parenteral nutrition; and those undergoing long 12% of global cases (0.9 million - 1.1 million) in 201214. Tuberculosis’

274 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. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 273-5, 2015.

insidious nature often leads to delayed or missed diagnosis, sometimes em múltiplos órgãos por Candida albicans e neuro tuberculose em with devastating consequences for the patient. In this patient, Candida homem jovem saudável. Um jovem de 19 anos de idade queixou-se spondylitis was initially thought of as the cause of his back pain, yet it de febre e lombalgia há um mês. Relatava ainda histórico de síndrome showed no improvement following antifungal treatment. However, the da boca escaldada. Foi diagnosticada co-infecção por Mycobacterium patient’s history of BCG vaccination, negative skin tests and normal chest tuberculosis e Candida albicans em cultura do aspirado de diferentes scans were not disregarded when considering the diagnosis of tuberculosis. regiões do organismo. Os sintomas melhoraram significativamente após Percutaneous vertebral biopsy was very useful in confirming diagnosis and a terapia antifúngica e antituberculosa. Este caso é apresentado para could help avoid open surgical biopsy in future patients. The mainstay in mostrar que a tuberculose pode prejudicar o sistema imune do hospedeiro the treatment of spinal tuberculosis is the conservative management of e aumentar o risco de candidíase invasiva em paciente imunocompetente. antituberculous drugs. Surgery is needed only if there is neurological deficit or spinal instability. In the management of osteoarticular tuberculosis, REFERENCES chemotherapy including isoniazid, rifampin, ethambutol, and pyrazinamide is recommended for approximately 12 - 18 months8. The patient was 1. Almeida AS, Lago PM, Boechat N, Huard RC, Lazzarini LC, Santos AR, et al. administered antituberculous treatment with the above four drugs for a Tuberculosis is associated with a down-modulatory lung immune response that impairs Th1-type immunity. J Immunol. 2009;183:718-31. 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 2. Bonecini-Almeida MG, Ho JL, Boéchat N, Huard RC, Chitale S, Doo H, et al. for increased sclerosis at the margin of the lesion. Down-modulation of lung immune responses by interleukin-10 and transforming growth factor beta (TGF-beta) and analysis of TGF-beta receptors I and II in active Deep Candida infections generally occur in immunosuppressive tuberculosis. Infect Immun. 2004;72:2628-34. patients, especially those who have been treated with immunosuppressors. 3. Fridkin SK. The changing face of fungal infections in health care settings. Clin Infect Recent studies showed that M. tuberculosis promoted down-modulatory Dis. 2005;41:1455-60. immune mediators to counteract Th1-type cells and patients’ innate immunity, and might have suppressive effects on the host’s immune 4. Grubb SE, Murdoch C, Sudbery PE, Saville SP, Lopez-Ribot JL, Thornhill MH. system1,2. Also, levels of FoxP3 gene expression and IL-10 secretion Candida albicans-endothelial cell interactions: a key step in the pathogenesis of systemic candidiasis. Infect Immun. 2008;76:4370-7. were raised in both pulmonary TB and extra-pulmonary TB7. The secretion of IL-10 by regulatory T cells can account for the inhibition 5. Hot A, Maunoury C, Poiree S, Lanternier F, Viard JP, Loulergue P, et al. Diagnostic of T cell responses and increase the risk of tuberculosis reactivation6. contribution of positron emission tomography with [18F] fluorodeoxyglucose for A case of coinfection with M. tuberculosis and Cryptococcus gattii in invasive fungal infections. Clin Microbiol Infect. 2011;17:409-17. a healthy young woman indicated that infection with TB predisposed 6. Kursar M, Koch M, Mittrücker HW, Nouailles G, Bonhagen K, Kamradt T, et al. to infection with Cryptococcus by down-regulate immune system and Cutting Edge: regulatory T cells prevent efficient clearance of Mycobacterium 13 altered host defenses . In the case featured in this report, the patient was tuberculosis. J Immunol. 2007;178:2661-5. immunocompetent and had no history of using immunosuppressive drugs or having any recent major surgery. It is likely that Candida albicans 7. Masood KI, Rottenberg ME, Salahuddin N, Irfan M, Rao N, Carow B, et al. Expression invaded his bloodstream through impaired oral mucosa membranes and of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis. BMC stuck to the endothelial cells of his blood vessels, in turn transmigrating Infect Dis. 2013;13:13. into the tissue4. This indicated that TB infection may impair the host’s immune system and that the spread of Candida albicans maybe ultimately 8. Moon MS, Moon YW, Moon JL, Kim SS, Sun DH. Conservative treatment lead to the occurrence of multiple deep organ infections in the healthy of tuberculosis of the lumbar and lumbosacral spine. Clin Orthop Relat Res. young man. 2002;398:40-9. 9. Oz Y, Kiraz N. Diagnostic methods for fungal infections in pediatric patients: In conclusion, multiple deep organ infections with Candida albicans microbiological, serological and molecular methods. Expert Rev Anti Infect Ther. and spinal tuberculosis are rare in healthy young men. Moreover, as it 2011;9:289-98 is always difficult to identifyM. tuberculosis, early diagnosis should be considered in patients with clinical and radiological findings suggesting 10. Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr, et al. Clinical practice guidelines for the management of candidiasis: 2009 update tuberculosis to avoid missing diagnosis, particularly in developing by the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:503-35. countries where tuberculosis is endemic. Furthermore, the present case report suggests that tuberculosis infection might increase the risk of 11. Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs, and modes of invasive candidiasis in patients without significant immunodeficiency. transmission. Clin Infect Dis. 1996; 22(Suppl 2):S89-94. This hypothesis needs to be confirmed by prospective cohorts with 12. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent problem. sufficiently large sample sizes. Clin Microbiol Rev. 2007;20:133-63.

RESUMO 13. Van Tongeren L, Shaipanich T, Fleetham JA. Coinfection with Cryptococcus gattii and Mycobacterium tuberculosis in an otherwise healthy 18-year-old woman. Can Tuberculose pode aumentar o risco de candidíase invasiva em Respir J. 2011;18:e62-3. paciente imunocompetente 14. World Health Organization. Global tuberculosis report. Geneva: WHO; 2013.

As infecções profundas por Candida ocorrem geralmente em Received: 23 June 2014 pacientes imunossuprimidos. Relatamos caso raro de infecções profundas Accepted: 30 September 2014

275 Rev. Inst. Med. Trop. Sao Paulo 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 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 During the initial reports in some countries of Africa and Asia and until the 1990s, clinical, cerebrospinal fluid data, radiological and electroencephalographic “patterns” of West Nile fever (WNF) was only considered a “minor” public health problem. The disease WNV encephalitis hinders its recognition. Thus, the reporting of suspected cases, which is gained awareness after outbreaks occurred in Israel, Australia and European countries and, an essential step for the national reference laboratories to perform specific diagnostic tests, is in particular, due to the large number of people and animals affected in the United States of fairly limited. The classical assumption of the herpetic nature of viral encephalitis, the lack of America between the end of the 1990s and the beginning of the new century. Thereafter, signs specific therapies against most viruses and the lack of diagnostic methods in most Brazilian of West Nile virus (WNV) circulation were detected in the Cayman Islands, El Salvador, hospitals are factors that combined lead to the non-recognition of the etiologic agents involved Guatemala, Belize, Colombia, Venezuela, and Argentina. However, viral isolation was rarely in central nervous system infections. These assertions may indicate that other cases of WNV achieved, and records of human, equine and avian morbidity in Latin America are lacking. encephalitis may have occurred without clinical recognition (which is admittedly difficult) of Moreover, for unclear reasons, there was no correspondence between the expansion of the the disease and without performing the tests necessary for an etiological diagnosis. Brazilian geographic range of viral circulation and the occurrence of significant animal or human clinicians, researchers and epidemiologists have a challenge ahead, given that the clarification morbidity by WNF in these regions7. Given the evidence of WNV circulation in South of the current status of WNV circulation does have an epidemiological relevance to Teresina American countries beginning in 2003, the Brazilian Ministry of Health adopted the reporting municipality, for Piauí State and to Brazil. 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 Marcelo A. Cunha e Silva VIEIRA(1), Aline de Almeida Xavier AGUIAR(2), Amaríles posed the question: “West Nile Encephalitis: our next epidemic?”3. Between 2002 and 2013, de Souza BORBA(3), Herlon Clístenes Lima GUIMARÃES(4), Kelsen Dantas serological evidence of viral circulation was found in horses and birds in the Amazon and EULÁLIO(5), Linduarte Leitão de ALBUQUERQUE-NETO(5), Maria do Amparo Pantanal regions4,6,8. Serological surveys conducted in the states of Rio Grande do Sul (2002) SALMITO(3) & Oriana Bezerra LIMA(3) and Rio Grande do Norte (2003), which included a significant number of birds of various (1) Natan Portella Institute of Tropical Medicine, Dept. Neurology, species, found no evidence of WNV circulation in the country1. R. Governador Artur de Vasconcelos 151, 64001-450 Teresina, PI, Brazil. In 2010, despite the negative results of the study “Is West Nile virus a potential cause of Telephone: +55 86 3221 3413. E-mail: [email protected]. central nervous system infection in Brazil?”, SOARES et al.10 concluded that “With the recent (2) Municipal Health Dept., Zenon Rocha Emergency Hospital, Teresina, PI, Brazil. activity in Argentina, it is fundamental to continue to monitor for this virus as an emerging (3) Municipal Health Dept., Surveillance Secretariat, Teresina, PI, Brazil. cause of neurological disease in South America”. Similarly, in early 2014 FIGUEIREDO & (4) State Health Dept., Surveillance Secretariat, PI, Brazil. FIGUEIREDO2 advised: “It is necessary to improve the surveillance of SLEV, ROCV, and (5) Natan Portella Inst. of Tropical Medicine, Dept. Infectious Diseases, Teresina, PI, Brazil. WNV in Brazil. Therefore, doctors must include flaviviruses (not only dengue) and other Correspondence to: Marcelo A.C.S. VIEIRA arboviruses in their differential diagnosis of acute febrile disease and of meningoencephalitis. Funding: This research received no grant from any funding agency in the public, In fact, if the doctors do not think on these pathogens, it will perpetuate the mistaken idea commercial or not-for-profit sectors. 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, REFERENCES 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 1. Araújo FAA, Vianna RST, Wada MY, Silva EV, Doretto L, Cavalcante GC, et al. Department of Teresina. A research protocol established in partnership with the Evandro Inquérito sorológico em aves migratórias e residentes de Galinhos/RN para detecção Chagas Institute (Ananindeua, Pará State, Brazil) enabled the shipment of blood, cerebrospinal do vírus do Nilo Ocidental e outros vírus. Bol Eletrôn Epidemiol. 2004;4(2):1-12. fluid and fecal samples in an attempt to isolate and molecularly and serologically detect herpes 2. de Figueiredo ML, Figueiredo LT. Review on infections of the central nervous system viruses, enteroviruses and arboviruses. From the start of the program until the admission of by St. Louis encephalitis, Rocio and West Nile flaviviruses in Brazil, 2004-2014. Adv this patient, samples from 36 patients had been examined. In the second half of November Microbiol. 2014;955-61. 2014, the Evandro Chagas Institute released the results of the examinations that undoubtedly 3. Luna EJA, Pereira LE, Souza RP. Encefalite do Nilo Ocidental, nossa próxima confirmed that Piauí had recorded the first human case of WNF in Brazil12. epidemia? Epidemiol Serv Saúde. 2003;12(1):7-19. At the announcement of the first confirmed case of WNF in Brazil, the Ministry of 4. Melandri V, Guimarães AÉ, Komar N, Nogueira ML, Mondini A, Fernandez-Sesma Health released the following statement: “It is noteworthy that this case was an isolated A, et al. Serological detection of West Nile virus in horses and chicken from Pantanal, event; the chain of transmission was not identified, and an in-depth investigation is being Brazil. Mem Inst Oswaldo Cruz. 2012;107:1073-5. performed to clarify the mode of transmission. It does not have epidemiological significance 5. Ministério da Saúde, Brasil, 2014. Saúde confirma primeiro caso de Febre do Nilo. to Brazil nor does it represent a risk to the public health of Piauí or Brazil”5. Thereafter, (cited 2015 Mar 30). Available from: http://portalsaude.saude.gov.br/index.php/ the Brazilian Society of Tropical Medicine expressed concern about the circumstances in cidadao/principal/agencia-saude/15962-saude-confirma-primeiro-caso-de-febre-do- which the diagnosis of the first case of WNF in Brazil was conducted: “Even though this nilo was an isolated case, the situation is worrisome. Its absence in Brazil, until this case, was 6. Ometto T, Durigon EL, de Araujo J, Aprelon R, de Aguiar DM, Cavalcante GT, et enigmatic. After all, why would a virus disseminated throughout North America and the al. West Nile virus surveillance, Brazil, 2008-2010. Trans R Soc Trop Med Hyg. Caribbean not enter into Brazil where its insect vectors and animal reservoirs are present? 2013;107:723-30. Then, suddenly it appears in the State of Piauí? (...) This question is even more troublesome 7. Pauvolid-Corrêa A, Varella RB. Aspectos epidemiológicos da febre do oeste do Nilo. because the virus was identified just after routine surveillance was instituted in a referral Rev Bras Epidemiol. 2008;11:463-72. hospital. The conclusion is obvious: the virus has already been circulating undetected in 8. Pauvolid-Corrêa A, Campos Z, Juliano R, Velez J, Nogueira RM, Komar N. Serological Brazil for some time”11. evidence of widespread circulation of West Nile virus and other flaviviruses in equines The epidemiology news portal from the International Society for Infectious Diseases, of the Pantanal, Brazil. PLOS Negl Trop Dis. 2014;8:e2706. ProMED-mail, also expressed moderate concern following the disclosure of the diagnosis: 9. ProMED-mail. Febre do Nilo Ocidental: Brazil (PI), humanos, casos suspeitos, “Vector? Isn’t missing ... There: all the elements of a transmission cycle are present. To deny surto? International Society for Infectious Diseases. 2014. Archive number that the risks of dissemination and, eventually, that outbreaks are a tangible reality would 20141224.3052950. (cited 2015 Mar 30). Available from: http://promedmail.org/ be naive, reckless and, to some degree, irresponsible”9. direct.php?id=3052950 The detection of the first case of WNF in Brazil may have distinct epidemiological 10. Soares CN, Castro MJ, Peralta JM, Freitas MR, Puccioni-Sohler M. Is West Nile virus meaning expressed by the assumptions that the encephalitis surveillance system was able to a potential cause of central nervous system infection in Brazil? Arq Neuropsiquiatr. detect: (1) the initial phase of an outbreak or an epidemic of WNF in the State of Piauí; (2) 2010;68:761-3. a case of an already endemic disease at low levels that so far had an unknown occurrence 11. Sociedade Brasileira de Medicina Tropical. Caso de febre do Nilo Ocidental no Piauí or was undetected in the region or in the country or (3) a sporadic case that emerged under é preocupante, alertam especialistas. Brasília: Newsletter SMBT; 2014. Edição 41. exceptional circumstances that is so far unexplained. 15 de dezembro de 2014. (cited 2015 Mar 30). Available from: http://sbmt.org.br/ The capacity of Brazilian biomes to provide an ecological niche conducive to the spread portal/caso-de-febre-do-nilo-ocidental-no-piaui-e-preocupante-alertam-especialistas of WNV remains enigmatic. To date, there have been no other cases with a confirmed diagnosis 12. Vieira MA, Romano AP, Borba AS, Silva EV, Chiang JO, Eulalio KD, et al. West of WNF in the country. The team involved in the surveillance of viral encephalitis in Teresina Nile virus encephalitis: the first human case recorded in Brazil. Am J Trop Med Hyg. is advocating the hypothesis of a silent endemic (at low levels) of WNF in Piauí and, perhaps, 2015;pii:15-0170. (Epub ahead of print).