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I ISSN 0036-4665 ISSN 1678-9946 on line Established: 1959. The year 2012 is the 53 rd anniversary of continuous publication UNIVERSIDADE DE SÃO PAULO - BRAZIL FACULDADE DE MEDICINA Instituto de Medicina Tropical de São Paulo Director: Prof. Dr. Paulo C. Cotrim EDITOR-IN-CHIEF EMERITUS EDITORS Prof. Dr. Thales F. de Brito Prof. Dr. Luis Rey (Founding Editor) Associate Editors: Prof. Dr. Marcello Fabiano de Franco Prof. Dr. Carlos da Silva Lacaz Prof. Dr. Pedro Paulo Chieffi EDITORIAL BOARD Alan L. de Melo (Belo Horizonte, MG) Alberto Duarte (S. Paulo, SP) Angela Restrepo M. (Medellin, Colombia) Anna Sara S. Levin (S. Paulo, SP) Antonio A. Barone (S. Paulo, SP) Antonio Carlos Nicodemo (S. Paulo, SP) Antonio Sesso (S. Paulo, SP) Antonio W. Ferreira (S. Paulo, SP) Barnett L. Cline (New Orleans, USA) Carlos F. S. Amaral (Belo Horizonte, MG) Celso Granato (S. Paulo, SP) Cesar A. Cuba Cuba (Brasília, DF) César Naquira V. (Lima, Peru) Clarisse M. Machado (S. Paulo, SP) Claudio S. Pannuti (S. Paulo, SP) Cláudio Santos Ferreira (S. Paulo, SP) Dalton L. F. Alves (Belo Horizonte, MG) Eridan Coutinho (Recife, PE) Ernesto Hofer (Rio de Janeiro, RJ) Euclides A. Castilho (S. Paulo, SP) Eufrosina S. Umezawa (S. Paulo, SP) Fan Hui Wen (S. Paulo, SP) Fernando A. Corrêa (S. Paulo, SP) Fernando Montero-Gei (San José, Costa Rica) Flair J. Carrilho (S. Paulo, SP) Gil Benard (S. Paulo, SP) Gioconda San-Blas (Caracas, Venezuela) Govinda Visvesvara (Atlanta, USA) Heitor F. Andrade Jr. (S. Paulo, SP) Henrique L. Lenzi (Rio de Janeiro, RJ) Hiro Goto (S. Paulo, SP) Ises A. Abrahamsohn (S. Paulo, SP) João Carlos Pinto Dias (Belo Horizonte, MG) João Renato Rebello Pinho (Sao Paulo, SP) José Eduardo Levi (S. Paulo, SP) José M. R. Zeitune (Campinas, SP) Julia Maria Costa-Cruz (Uberlândia, MG) Julio Litvoc (S. Paulo, SP) Luiz Caetano da Silva (S. Paulo, SP) Luiz Carlos Severo (P. Alegre, RS) Luiz Jacintho da Silva (Campinas, SP) Luiz T. M. Figueiredo (Rib. Preto, SP) Lygia B. Iversson (S. Paulo, SP) Marcos A. Rossi (Ribeirão Preto, SP) Marcos Boulos (S. Paulo, SP) M. A. Shikanai-Yasuda (S. Paulo, SP) Maria I. S. Duarte (S. Paulo, SP) Maria L. Higuchi (S. Paulo, SP) Mario Mariano (S. Paulo, SP) Mirian N. Sotto (S. Paulo, SP) Moisés Goldbaum (S. Paulo, SP) Moysés Mincis (S. Paulo, SP) Moysés Sadigursky (Salvador, BA) Myrthes T. Barros (S. Paulo, SP) Nilma Cintra Leal (Recife, PE) Paulo C. Cotrim (São Paulo, SP) Paulo M. Z. Coelho (Belo Horizonte, MG) Pedro Morera (San José, Costa Rica) Regina Abdulkader (S. Paulo, SP) Ricardo Negroni (B. Aires, Argentina) Robert H. Gilman (Baltimore, USA) Roberto Martinez (Rib. Preto, SP) Semíramis Guimarães F. Viana (Botucatu, SP) Silvino A. Carvalho (S. Paulo, SP) Silvio Alencar Marques (Botucatu, SP) Sumie Hoshino-Shimizu (S. Paulo, SP) Thelma S. Okay (S. Paulo, SP) Tsutomu Takeuchi (Tokyo, Japan) Venâncio A. F. Alves (S. Paulo, SP) Vicente Amato Neto (S. Paulo, SP) Zilton A. Andrade (Salvador, BA) Executive Board - Librarians: Maria do Carmo Berthe Rosa; Sonia Pedrozo Gomes; Maria Ângela de Castro Fígaro Pinca; Carlos José Quinteiro The Revista do Instituto de Medicina Tropical de São Paulo is abstracted and/or indexed in: Index Medicus, Biological Abstracts, EMBASE/Excerpta Medica, Hepatology/Rapid Literature Review, Tropical Diseases Bulletin, Referativnyi Zhurnal: All-Russian Institute of Scientific and Technical Information (VINITI), Periódica - Índice de Revistas Latinoamericanas en Ciencias, Helminthological Abstracts, Protozoological Abstracts, Review of Medical and Veterinary Mycology, PubMed, UnCover, HealthGate, OVID, LILACS, MEDLINE, New Jour, ExtraMED, Free Medical Journals, ISI (Institute for Scientific Information), BIOSIS Previews, Scopus, Science Citation Index Expanded (SciSearch), Journal Citation Reports/Science Edition, Current Contents®/Clinical Medicine and Index Copernicus. ON LINE ACCESS - http://www.imt.usp.br/portal/ - FREE PDF ACCESS TO ALL PAST ISSUES, 1959-1989 (Financial support by “Alves de Queiroz Family Fund for Research). http://www.scielo.br/rimtsp - FULL TEXT, SINCE 1987. E-mail: [email protected] Reprints may be obtained from Pro Quest Inf. and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106-1346 - USA. The Revista do Instituto de Medicina Tropical de São Paulo is supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Universidade de São Paulo and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This issue was financed by: CNPq Proc. 402279/2011-5 and Programa de Apoio às Publicações Científicas Periódicas da Universidade de São Paulo. Desktop Publishing by: Hermano - e-mail: [email protected]. Phone: 55.11.5571-8937. - Printed by: Global Print Editora Gráfica, Phone: 55.31.3198-1100. English Revision: [email protected]

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I

ISSN 0036-4665ISSN 1678-9946 on line

Established: 1959.

The year 2012 is the 53rd anniversary

of continuous publication

UNIVERSIDADE DE SÃO PAULO - BRAZILFACULDADE DE MEDICINA

Instituto de Medicina Tropical de São PauloDirector: Prof. Dr. Paulo C. Cotrim

EDITOR-IN-CHIEF EMERITUS EDITORS Prof. Dr. Thales F. de Brito Prof. Dr. Luis Rey (Founding Editor)Associate Editors: Prof. Dr. Marcello Fabiano de Franco Prof. Dr. Carlos da Silva Lacaz Prof. Dr. Pedro Paulo Chieffi

EDITORIAL BOARDAlan L. de Melo (Belo Horizonte, MG) Alberto Duarte (S. Paulo, SP) Angela Restrepo M. (Medellin, Colombia) Anna Sara S. Levin (S. Paulo, SP)Antonio A. Barone (S. Paulo, SP)Antonio Carlos Nicodemo (S. Paulo, SP) Antonio Sesso (S. Paulo, SP) Antonio W. Ferreira (S. Paulo, SP) Barnett L. Cline (New Orleans, USA) Carlos F. S. Amaral (Belo Horizonte, MG) Celso Granato (S. Paulo, SP) Cesar A. Cuba Cuba (Brasília, DF) César Naquira V. (Lima, Peru) Clarisse M. Machado (S. Paulo, SP) Claudio S. Pannuti (S. Paulo, SP) Cláudio Santos Ferreira (S. Paulo, SP) Dalton L. F. Alves (Belo Horizonte, MG) Eridan Coutinho (Recife, PE) Ernesto Hofer (Rio de Janeiro, RJ) Euclides A. Castilho (S. Paulo, SP)Eufrosina S. Umezawa (S. Paulo, SP) Fan Hui Wen (S. Paulo, SP) Fernando A. Corrêa (S. Paulo, SP) Fernando Montero-Gei (San José, Costa Rica)

Flair J. Carrilho (S. Paulo, SP)Gil Benard (S. Paulo, SP)Gioconda San-Blas (Caracas, Venezuela)Govinda Visvesvara (Atlanta, USA) Heitor F. Andrade Jr. (S. Paulo, SP) Henrique L. Lenzi (Rio de Janeiro, RJ) Hiro Goto (S. Paulo, SP)Ises A. Abrahamsohn (S. Paulo, SP) João Carlos Pinto Dias (Belo Horizonte, MG) João Renato Rebello Pinho (Sao Paulo, SP) José Eduardo Levi (S. Paulo, SP)José M. R. Zeitune (Campinas, SP) Julia Maria Costa-Cruz (Uberlândia, MG)Julio Litvoc (S. Paulo, SP) Luiz Caetano da Silva (S. Paulo, SP) Luiz Carlos Severo (P. Alegre, RS) Luiz Jacintho da Silva (Campinas, SP) Luiz T. M. Figueiredo (Rib. Preto, SP) Lygia B. Iversson (S. Paulo, SP) Marcos A. Rossi (Ribeirão Preto, SP)Marcos Boulos (S. Paulo, SP)M. A. Shikanai-Yasuda (S. Paulo, SP)Maria I. S. Duarte (S. Paulo, SP)Maria L. Higuchi (S. Paulo, SP)

Mario Mariano (S. Paulo, SP)Mirian N. Sotto (S. Paulo, SP)Moisés Goldbaum (S. Paulo, SP)Moysés Mincis (S. Paulo, SP)Moysés Sadigursky (Salvador, BA)Myrthes T. Barros (S. Paulo, SP)Nilma Cintra Leal (Recife, PE)Paulo C. Cotrim (São Paulo, SP)Paulo M. Z. Coelho (Belo Horizonte, MG)Pedro Morera (San José, Costa Rica)Regina Abdulkader (S. Paulo, SP)Ricardo Negroni (B. Aires, Argentina)Robert H. Gilman (Baltimore, USA)Roberto Martinez (Rib. Preto, SP)Semíramis Guimarães F. Viana (Botucatu, SP)Silvino A. Carvalho (S. Paulo, SP)Silvio Alencar Marques (Botucatu, SP)Sumie Hoshino-Shimizu (S. Paulo, SP)Thelma S. Okay (S. Paulo, SP)Tsutomu Takeuchi (Tokyo, Japan)Venâncio A. F. Alves (S. Paulo, SP)Vicente Amato Neto (S. Paulo, SP)Zilton A. Andrade (Salvador, BA)

Executive Board - Librarians: Maria do Carmo Berthe Rosa; Sonia Pedrozo Gomes; Maria Ângela de Castro Fígaro Pinca; Carlos José Quinteiro

The Revista do Instituto de Medicina Tropical de São Paulo is abstracted and/or indexed in: Index Medicus, Biological Abstracts, EMBASE/Excerpta Medica, Hepatology/Rapid Literature Review, Tropical Diseases Bulletin, Referativnyi Zhurnal: All-Russian Institute of Scientific and Technical Information (VINITI), Periódica - Índice de Revistas Latinoamericanas en Ciencias, Helminthological Abstracts, Protozoological Abstracts, Review of Medical and Veterinary Mycology, PubMed, UnCover, HealthGate, OVID, LILACS, MEDLINE, New Jour, ExtraMED, Free Medical Journals, ISI (Institute for Scientific Information), BIOSIS Previews, Scopus, Science Citation Index Expanded (SciSearch), Journal Citation Reports/Science Edition, Current Contents®/Clinical Medicine and Index Copernicus.

ON LINE ACCESS - http://www.imt.usp.br/portal/ - FREE PDF ACCESS TO ALL PAST ISSUES, 1959-1989 (Financial support by “Alves de Queiroz Family Fund for Research).

http://www.scielo.br/rimtsp - FULL TExT, SINCE 1987. E-mail: [email protected]

Reprints may be obtained from Pro Quest Inf. and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106-1346 - USA.

The Revista do Instituto de Medicina Tropical de São Paulo is supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Universidade de São Paulo and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

This issue was financed by: CNPq Proc. 402279/2011-5 and Programa de Apoio às Publicações Científicas Periódicas da Universidade de São Paulo.

Desktop Publishing by: Hermano - e-mail: [email protected]. Phone: 55.11.5571-8937. - Printed by: Global Print Editora Gráfica, Phone: 55.31.3198-1100. English Revision: [email protected]

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The purpose of the “Revista do Instituto de Medicina Tropical de São Paulo” (Journal of the São Paulo Institute of Tropical Medicine) is to publish the results of researches which contri-bute significantly to knowledge of all transmissible diseases.

REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO(JOURNAL OF THE S. PAULO INSTITUTE OF TROPICAL MEDICINE).

São Paulo, SP-Brasil, 1959 -v. ilust. 28 cm

1959-2011, 1-531973-2002 (supl. 1-12)2003 (supl. 13 - on-line only)2005-2012 (supl. 14-18)2012, 54 (1-5)

ISSN 0036-4665ISSN 1678-9946 on line

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Rev. Inst. Med. Trop. Sao Paulo Vol. 54 No. 5 P. 239-298 September-October, 2012

ISSN 0036-4665ISSN 1678-9946 on line

ADDRESSINSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO

Av. Dr. Enéas de Carvalho Aguiar, 47005403-000 São Paulo, SP - Brazil

Phone/Fax: 55.11.3062.2174; 55.11.3061-7005e-mail: [email protected]

SUBSCRIPTIONSFOREIGN COUNTRIESOne year (six issues) ........ U$ 200.00Single issue ...................... U$ 50.00

CONTENTS

TOxOPLASMOSISCase-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil - C.C.J. EKMAN, M.F.V. CHIOSSI, L.R. MEIRELES, H.F. ANDRADE JÚNIOR, W.M. FIGUEIREDO, M.A.M. MARCIANO & E.J.A. LUNA ...............................239

SCHISTOSOMIASISTwo sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load - M.C.C. ESPÍRITO-SANTO, M.V. ALVARADO-MORA, P.L.S. PINTO, F.J. CARRILHO, J.R.R. PINHO & R.C.B. GRYSCHEK .........................................................................245

VIROLOGYAcute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil - M.C.M. ALBUQUERQUE, R.B. VARELLA & N. SANTOS ....................................................................................................................................................................................................................249

HIVTuberculosis among HIV-1-infected subjects in a tertiary out-patient service in São Paulo city, Brazil - J. CASSEB, L.A.M. FONSECA, L.A. MEDEIROS, C.R. GONSALEZ, E.R. LAGONEGRO, A.P.R. VEIGA, D.C. DA SILVA, M. MENDONçA & A.J.S. DUARTE .......................257

DENGUESpatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools - R.M. CARVALHO & L.F.C. NASCIMENTO ....................................................................................................................................................................................................261

PARASITOLOGYContamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by Toxocara spp.and Ancylostoma spp. - J.P. MARQUES, C.R. GUIMARÃES, A. VILAS BOAS, P.U. CARNAÚBA & J. MORAES ......................................................................................267

MEDICINAL PLANTSPharmacological effects of Sapindus mukorossi - A. UPADHYAY & D.K. SINGH ......................................................................................................273

VECTOR CONTROLSMolluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. - M.M. SALAMA, E.E. TAHER & M.M. EL-BAHY .......................................................................................................................................................281

Genetic control of mosquitoes: population suppression strategies - A.B.B.WILKE & M.T. MARRELLI ....................................................................287

CASE REPORTWhipple’s disease: rare disorder and late diagnosis - V.P. RENON, M.C.APPEL-DA-SILVA, R.B. D’INCAO, R.M. LUL, L.S. KIRSCHNICK & B. GALPERIM ..........................................................................................................................................................................293

LETTER TO THE EDITORAnalogies in medicine: picture frame and tapir’s nose - J.S. ANDRADE FILHO .........................................................................................................298

Impact Factor: 1.000

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ENDEREçOINSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO

Av. Dr. Enéas de Carvalho Aguiar, 47005403-000 São Paulo, SP - Brasil

Fone/Fax: 55.11.3062.2174; 55.11.3061-7005e-mail: [email protected]

Rev. Inst. Med. Trop. Sao Paulo Vol. 54 No. 5 P. 239-298 Setembro-Outubro, 2012

CONTEÚDO

ISSN 0036-4665ISSN 1678-9946 on line

TOxOPLASMOSEEstudo caso-controle de surto de toxoplasmose aguda em indústria no estado de São Paulo, Brasil - C.C.J. EKMAN, M.F.V. CHIOSSI, L.R. MEIRELES, H.F. ANDRADE JÚNIOR, W.M. FIGUEIREDO, M.A.M. MARCIANO & E.J.A. LUNA ............................................................239

ESQUISTOSSOMOSEDuas amplificações sequenciais por PCR para detecção de Schistosoma mansoni em amostras de fezes com baixa carga parasitária - M.C.C. ESPÍRITO-SANTO, M.V. ALVARADO-MORA, P.L.S. PINTO, F.J. CARRILHO, J.R.R. PINHO & R.C.B. GRYSCHEK ..........................245

VIROLOGIAInfecções respiratórias agudas causadas por vírus em crianças do Rio de Janeiro e de Teresópolis, Brasil - M.C.M. ALBUQUERQUE, R.B. VARELLA & N. SANTOS .....................................................................................................................................................................................249

HIVTuberculose entre pacientes infectados pelo HIV-1 em ambulatório terciário de São Paulo, Brasil - J. CASSEB, L.A.M. FONSECA, L.A. MEDEIROS, C.R. GONSALEZ, E.R. LAGONEGRO, A.P.R. VEIGA, D.C. DA SILVA, M. MENDONçA & A.J.S. DUARTE .......................257

DENGUEDistribuição espacial da dengue no município de Cruzeiro, Estado de São Paulo, Brasil: o uso do geoprocessamento - R.M. CARVALHO & L.F.C. NASCIMENTO ................................................................................................................................................................261

PARASITOLOGIAContaminação de parques e praças públicas por Toxocara spp. e Ancylostoma spp., no município de Guarulhos, São Paulo, Brasil - J.P. MARQUES, C.R. GUIMARÃES, A. VILAS BOAS, P.U. CARNAÚBA & J. MORAES ......................................................................................267

PLANTAS MEDICINASEfeitos farmacológicos do Sapindus mukorossi - A. UPADHYAY & D.K. SINGH .......................................................................................................273

CONTROLE DE VETORESAtividades moluscicida e mosquitocida de óleos essenciais de Thymus capitatus Hoff. et Link. e de Marrubium vulgare L. - M.M. SALAMA, E.E. TAHER & M.M. EL-BAHY .......................................................................................................................................................281

Controle genético de mosquitos: estratégias de supressão de populações - A.B.B WILKE & M.T. MARRELLI .........................................................287

RELATO DE CASODoença de Whipple: patologia rara e de diagnóstico tardio - V.P. RENON, M.C.APPEL-DA-SILVA, R.B. D’INCAO, R.M. LUL, L.S. KIRSCHNICK & B. GALPERIM ..........................................................................................................................................................................293

CARTA AO EDITORAnalogies in medicine: picture frame and tapir’s nose - J.S. ANDRADE FILHO .........................................................................................................298

Impact Factor: 1.000

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Rev. Inst. Med. Trop. Sao Paulo54(5):239-244, September-October, 2012doi: 10.1590/S0036-46652012000500001

(1) Instituto de Medicina Tropical, Universidade de São Paulo, São Paulo, SP, Brasil.(2) Serviço Especial de Saúde de Araraquara, Faculdade de Saúde Publica, Universidade de São Paulo, Araraquara, SP, Brasil.(3) Instituto Adolfo Lutz, Secretaria de Estado da Saúde, São Paulo, SP, Brasil.Correspondence to: Luciana Regina Meireles, D.V.M., M.Sc., Ph.D., Laboratório de Protozoologia, IMTSP, Av. Dr. Enéas de Carvalho Aguiar 470, 05403-000 São Paulo, SP, Brasil. Phone

+55.11.3061-7010; Fax +55.11.3088-5237. E-mail: [email protected]

CASE-CONTROL STUDY OF AN OUTBREAK OF ACUTE TOXOPLASMOSIS IN AN INDUSTRIAL PLANT IN THE STATE OF SÃO PAULO, BRAZIL

Claudio Cesar Jaguaribe EKMAN(1), Maria Fernanda do Valle CHIOSSI(2), Luciana Regina MEIRELES(1), Heitor Franco de ANDRADE JÚNIOR(1), Walter Manso FIGUEIREDO(2), Maria Aparecida Moraes MARCIANO(1,3) & Expedito José de Albuquerque LUNA(1)

SUMMARY

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

KEYWORDS: Toxoplasmosis; Outbreak; Foodborne disease; Green vegetables; Case-control study; ELISA; IgG avidity.

INTRODUCTION

Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and animals worldwide5,18. Toxoplasmosis is generally asymptomatic, except in immunocompromised adults and congenitally infected children18.

Humans acquire infection postnatally mainly by ingesting food and water contaminated with oocysts shed in the feces of infected cats or by ingesting viable tissue cysts in raw or undercooked meat. The major routes of transmission vary between different human populations and depend on social culture, eating habits and environmental factors20,21. Furthermore, estimates of the rate of infection of meat vary widely depending on the animal species12.

Several reports concerning toxoplasmosis outbreaks have been published in recent decades, mainly regarding outbreaks associated with the consumption of undercooked meat7,10. These outbreaks usually affect a small group of individuals and are associated with exposure to a common source, such as that described by a group of American medical students who had eaten undercooked hamburgers11 and that described in the same year by a community of students from a Brazilian university who ate their meals together at the cafeteria17. Another well-documented

Brazilian outbreak occurred in1993, with 17 cases of acute toxoplasmosis transmitted by raw lamb meat2.

Until recently, toxoplasmosis was not often considered a waterborne zoonosis, and the occurrence of outbreaks of Toxoplasma infection involving more than a single family or small group were rarely reported. However, a major outbreak of acute toxoplasmosis in humans occurred in Brazil in 20014, which was associated with the T. gondii contamination of a town’s water supply. A similar waterborne outbreak was reported in Canada but with a smaller number of cases3. Oocyst-related Brazilian outbreaks of toxoplasmosis had previously been reported in a specific region of the State of São Paulo, the mesoregion of Araraquara, which featured excellent sanitary conditions. One such incident was a large outbreak that affected 113 students from the one large public university campus at São Carlos near our study area, with numerous cats on campus including inside the cafeteria8, which were linked to the contamination of a water reservoir by cat feces. Here, we describe an outbreak of 11 cases of acute toxoplasmosis in the same mesoregion at an industrial plant with approximately 2300 employees.

MATERIAL AND METHODS

Epidemiologic investigation: In April 2009, the Institute of Tropical

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EKMAN, C.C.J.; CHIOSSI, M.F.V.; MEIRELES, L.R.; ANDRADE JÚNIOR, H.F.; FIGUEIREDO, W.M.; MARCIANO, M.A.M. & LUNA, E.J.A. - Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 239-44, 2012.

240

Medicine of the University of Sao Paulo (IMTSP - USP) became aware of a cluster of acute toxoplasmosis cases in an industrial plant located in the mesoregion of Araraquara, Sao Paulo State, Brazil, through the Adolfo Lutz Institute, the State of São Paulo’s public health laboratory (IAL). In a collaborative research effort with the State’s Epidemiologic Surveillance Center (CVE), we developed a case-control study to investigate the outbreak. The investigation was conducted by professionals of the IMTSP - USP, IAL and the Special Health Service of Araraquara, School of Public Health of University of São Paulo (SESA - FSP- USP). Upon the manifestation of symptoms, the patients sought the company’s outpatient services, which then referred them to SESA - FSP - USP to confirm the diagnosis. The initial information provided indicated the existence of eight laboratory-confirmed cases of acute toxoplasmosis and one suspected case diagnosed between March and April 2009. The case subjects shared the common characteristic of working in a large industrial plant located in the region, and the temporal distribution of the dates of the onset of symptoms suggested a common source of exposure.

Initially, we conducted a descriptive study aimed at addressing active cases. The study began in July 2009 with an active search of suspected cases through the medical records of the company’s outpatient services and the Special Health Service of Araraquara (SESA). A suspected case was defined as “any person assisted in the services mentioned above who had at least two of the following symptoms: lymphadenopathy, fever, headache and fatigue, or who had laboratory results with serologic evidence of toxoplasmosis in the period from March 15 to March 31, 2009.” Moreover, an investigation of the risk factors for toxoplasmosis was conducted through an interview with the nutritionist responsible for the company’s industrial kitchen, who provided information regarding the origin and suppliers of the ingredients used in preparing meals, as well as the location and storage temperature of meat products. On this occasion, we requested menus with a list of all of the food items used in each meal for the period from February 2 to March 15, 2009, and search for T.gondii cysts in meat samples (50 g) from the alleged suppliers used during the period of employee infection.

To identify risk factors for toxoplasmosis and possible sources of infection, such as food and water, we performed a case-control study. Initially, the study included the eight laboratory-confirmed cases and one suspected case that did not have laboratory confirmation. As controls, we selected 36 asymptomatic employees who worked in the same teams and during the same work shifts as the laboratory-confirmed cases. All symptomatic individuals (cases) were seropositive for IgM and IgG anti-T. gondii, and only one case with clinical suspicion had not been evaluated by laboratory testing.

Because we selected controls based on clinical symptoms, the control group may have included asymptomatic chronic toxoplasmosis individuals who were protected and did not become cases in the investigated outbreak. We carried out the serological testing of all asymptomatic toxoplasmosis controls and all 45 individuals selected for the study underwent laboratory tests for antibodies IgG and IgM anti-T. gondii IgG avidity to confirm whether reagent individuals had acute or chronic toxoplasmosis.

Blood samples used for laboratory tests were obtained from May 19 to May 21, 2009, from the staff of the company’s outpatient clinic with the written consent of each participant. All samples were processed at

the Laboratory of Protozoology of IMTSP - USP. At the time of blood collection, subjects were interviewed by the IMTSP / USP and SESA team through a standardized questionnaire featuring questions regarding culture, hygiene, food habits and the menu of meals offered by the company during the period from February 2 to March 15, 2009. Only questionnaires from patients with acute toxoplasmosis and susceptible, laboratory-confirmed cases were analyzed, excluding those with chronic toxoplasmosis, for whom serological evidence of previous exposure had already been established.

Serological detection: Sera were assayed by in-house standard ELISA with determination of IgG avidity. Sera were screened at a dilution of 1/100, with a cut-off (0.417), which was previously determined and confirmed by a test set of 16 negative sera. The reaction was performed as described elsewhere22 using T. gondii whole-saline antigen and conjugated anti-human IgG, with subsequent titration of positive samples.

IgG avidity was determined by measuring antibody resistance to washing with 6 M urea chaotropic solution and expressed as the percentage of antibody-resistant urea. IgG avidity was considered low when the percentage of resistant antibodies was less than 30%, it was considered undetermined when this percentage was between 30 and 50% and high (chronic infection of > six months duration) when this percentage was greater than 50%.

IgM antibodies were determined by classic ELISA, with a cut-off of (0.331). Serum samples with high titers of IgG were previously treated for the removal of rheumatoid factor.

Detection of T. gondii cysts in meat samples: T. gondii tissue cysts were detected by molecular biology techniques (PCR) and bioassay in Swiss mice. Briefly, 50 g of meat from each sample was subjected to peptic digestion using commercial pepsin (1:10,000) according to a protocol described elsewhere6. The sediment from the digestion by pepsin containing T. gondii cysts was used to bioassay the samples and was also used for DNA extraction in PCR. The DNA extraction of samples was performed using a commercial extraction kit (Brasílica, LCG®) according to the manufacturer’s instructions.

PCR was performed to detect a 115-bp fragment from the T. gondii B1 gene. In each reaction, we included DNA samples extracted from the T. gondii RH strain (positive control) and a sample of sterile ultra-pure water as a control negative. The PCR products were observed by electrophoresis on 2% agarose gel stained with ethidium bromide solution. The bands were observed by transillumination with ultraviolet light and recorded using a digital imaging system (AlphaImager ® EC, Alpha Innotech Corporation).

The bioassay was performed by the subcutaneous inoculation of one mL of digested sample in Swiss mice. The animals were kept in the Laboratory of Protozoology IMTSP. After a period of 30 days, the animals were euthanized for the microscopic study of brain cysts and T. gondii antibodies by ELISA.

Statistical analyses: Risk factors assessed by the questionnaire were analyzed by 2x2 tables comparing the infected and control groups using Fisher’s exact test or a χ2 test at a significance level of p < 0.05.

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RESULTS

Screening for T. gondii IgG antibodies by ELISA in individuals selected for the case-control study revealed the presence of 25 anti-T. gondii IgG-positive sera, including the nine indexed cases, and 20 IgG-negative samples. All 25 reactive sera were tested for IgG avidity by ELISA via serial dilution using 6 M urea as a chaotrope, 14 samples exhibited high avidity (> 50%), indicating chronic infection from candidate controls and one indexed case, and 11 samples exhibited low avidity (< 30%), including eight indexed cases and three asymptomatic controls, indicating recent infection (Fig. 1). All eleven samples of low avidity were positive for T. gondii IgM antibodies, as determined by ELISA.

Table 1 shows the sorting of the tested population according to the serological classification. Using the clinical classifications of the case-control population, nine cases from among 45 individuals were selected to participate in the study, giving a 20% clinical infection rate. Additionally, three candidates from the control group presented serological evidence

of acute asymptomatic toxoplasmosis. The selection of the patients according to the incidence of susceptible and acute infection resulted in an unprotected population of 31 participants. The final classification revealed 11 cases and 20 controls, with an infection rate of 35.5%, excluding chronically infected subjects. In the group of cases with compatible clinical symptoms, 8/9 individuals showed evidence of recent infection, but one case showed evidence of chronic infection. This patient visited the infirmary more than one week after the other acute cases had visited the infirmary and presented chronic toxoplasmosis. The incidence of asymptomatic infections was therefore 9.67% (3/31). According to laboratory results, we reclassified the case and control groups, resulting in 11 cases of acute toxoplasmosis and 20 susceptible controls. These results clearly show that there was a recent outbreak of toxoplasmosis in the population but that the clinical symptoms did not occur in all of the infected patients, with a percentage of at least 27% (3/11) of infected patients without clinical symptoms.

According to the dates during which the study was conducted, the first index case developed symptoms on March 15, 2009. The main reported symptoms were fever (9/11 = 81.81%), night sweats (9/11 = 81.81%), asthenia (9/11 = 81.81%), malaise (9/11 = 81.81%), lymphadenopathy (7/11 = 63.64%), headache (7/11 = 63.64%), appetite loss (6/11 = 54.55%), myalgia (6/11 = 54.55%), arthralgia (4/11 = 36.36%), nausea (3/11 = 27.27%), sore throat (3/11 = 27.27%), diarrhea (2/11 = 18.18%), vision problems (2/11 = 18.18%), dysuria (2/11 = 18.18%), abdominal pain (1/11 = 9.09%), neurological symptoms (1/11 = 9.09%), dizziness (1/11 = 9.09%) and irritability (1/11 = 9.09%). The outbreak peaked between March 17 and March 22, 2009. Some symptoms were reported in interview by alleged previously asymptomatic patients.

The analysis of possible associations between the exposure to risk factors and disease is described in Table 2. Significant events were analyzed in connection with the risk association of habits, environments and food consumption in case-control analysis using the serologically defined acute cases (11 individuals) and controls (20 individuals). As shown, the individual consumption of a vegetable item (escarole) on March 7, 2009, was the single variable significantly associated with the incidence of disease (p < 0.05). The potential impact of this variable is limited as only two infected cases had consumed this recipe, despite the significant association. None of the other variables investigated were

Table 1Classification of participants in case-control analysis using both clinical presentation and serological definition of recent,

chronic or absence of Toxoplasma gondii infection

Clinical picture n Serology nCases

nControls

nChronic infection

(excluded)

Acute (indexes)

Recent 8 8

9 Chronic 1 1

Negative 0

Asymptomatic Controls

Recent 3 3

36 Chronic 13 13

Negative 20 20

Total 45 11 20 14

Fig. 1 - IgG and IgG Avidity ELISA results for both clinical cases and controls. The circle in the

case group represents an individual with chronic infection. The arrows indicate all individuals

of a control group, sorted as non-infected, without antibodies or IgG positive with IgG

avidity. The circle in control group represents asymptomatic individuals with acute infection.

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significantly associated with the illness. When looking for an association with meals, green vegetable ingestion was strongly associated with infection (p < 0.005). Protection factors were associated with individual habits such as the consumption of filtered water, ingestion of well-cooked meat and good hygiene practices. The DNA extracted by PCR and the mice bioassay were negative in the frozen meat obtained from restaurants two months after the outbreak.

DISCUSSION

This epidemic outbreak of toxoplasmosis revealed an unspecific clinical picture of acute toxoplasmosis in most infected patients; similar to what has been reported in other studies19. This clinical picture overlaps with that of several other acute infections, and we found a reported case that lacked serological evidence of acute toxoplasmosis and so was excluded from our group of cases. Among our candidate controls, we also found three patients who presented asymptomatic disease and were included in our infected group. These data show the importance of accurate serology for diagnosis of acute toxoplasmosis.

Our study did not identify other factors common to the cases outside the workplace or individual behavioral risks. The epidemic curve showed common and isolated exposure because the onset of symptoms in cases occurred over a period of 14 days and peaked within a eight days interval. These results are consistent with the incubation period of 5-23 days described in the literature18.

We searched and detected the absence of cats in the surroundings of the plant, without access to water reservoirs, which could have been an alternative source of oocysts, as elsewhere described4. The clustered and

small number of symptomatic cases does not suggest water as a factor of exposure to Toxoplasma gondii because water contaminated with oocysts spreads infection to a large number of people and the number of cases increases over time, as illustrated by the outbreaks in Canada3 and Brazil4, which was not observed for this outbreak. Reported outbreaks featuring water as a mode of transmission have led to high attack rates, due to a high dosage of oocysts10 or a particularly virulent strain1, distinct from our typical self-limited benign disease infected group, with three asymptomatic infections. Our data, as well as those previously described in the literature8, suggest that acute cases of human toxoplasmosis in the Araraquara mesoregion are associated with transmission by oocysts, reinforcing the need for preventive measures in this region, with a focus on maintaining the quality of water and processing of raw foods.

The other exposure factor that could be implicated in the outbreak of toxoplasmosis is food, such as fresh vegetables and raw or undercooked meat20. We investigated the list of all food items provided for each meal in the company’s menu over the three-week period corresponding to the exposure period. Except for escarole, no other individual food item was implicated in the outbreak, but ingestion of a meal with green vegetables was intensely associated with the infection. Those vegetables are composed of hard green leaves, which are considered to be difficult to clean, as evidenced by Salmonella assays14. The food supplier of the plant also informed us that aside from mechanical washing, the main method used to wash fresh vegetables is low-concentration chlorine immersion, which does not affect the viability of T. gondii oocysts23. Another problem with cleaning escarole leaves is the fact that this vegetable possesses isolated leaves without an unrolling leaf pattern, such as that of cabbage. In fact, it is more similar to arugula, which has also been reported to be easily contaminated by Salmonella9. This leaf organization allows

Table 2Distribution of demographic data, habits and food ingestion in serologically defined cases and control groups

Events Cases (11) Controls (20)

Gender (% males) 91% (10/11) 85% (17/20) NS

Mean Age Year ± SD 28.3 ± 6.7 27.5 ± 7.3 NS*

Restaurant Breakfast 37% (4/11) 25% (5/20) NS

Lunch 100% (11/11) 85% (17/20) NS

Dinner 0 (0/11) 20% (4/20) NS

Meat Barbecue 73% (8/11) 90% (18/20) NS

Soil contact Gardening 0 (0/11) 30% (6/20) NS

Washing hands 89% (8/9) 100% (13/13) NS

Green vegetables Lettuce 100% (11/11) 83% (15/18) NS

Arugula 100% (5/5) 60% (6/10) NS

Escarole 100% (2/2) 0 (0/5) p < 0.05

Chicory 45% (5/11) 26% (5/19) NS

Water cress 55%(6/11) 25%(5/20) NS

Green vegetables referred meals (29/40) (31/72) p < 0.005

Arabian crude recipes Tabule 55% (5/9) 28% (5/18) NS

Kafta 100% (11/11) 83% (15/18) NS

Water Filtered water 90% (9/10) 55% (11/20) NS

Bottled water 82% (9/11) 100% (20/20) NS(*) Comparison performed by Student T test. Categorical data analyzed by Fisher´s Exact test.

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contaminants to reach all leaves, especially if contaminated fertilizer is dispersed over the vegetables. This relationship between the consumption of raw green vegetables and toxoplasmosis has also been observed in other areas of Brazil15.

The absence of the implication of other possible food items as sources of the outbreak may be due to the small sample size, because the small number of cases and controls reduces the power of the study16. Another factor was the long interval between exposure and the investigation period, which lasted approximately three months. This interval means that participants may have difficulty in accurately reporting the foods they consumed at the time. Difficulty in remembering facts in epidemiologic studies is known as bias, which sometimes leads to mistakes in the classification of exposure to the factors investigated; in this case, the factor is the consumption of food13.

Our study exhibited the same difficulties associated with other Toxoplasma outbreaks. Private organizations usually allow only small studies to be performed, without testing the whole group of employees, which precludes the exact determination of the extent of the outbreak. Food suppliers usually offer biased or outdated samples. Serology without the definition of acute infection leads to an inaccurate estimate of incidence due to imprecise serology and a large proportion of Toxoplasma chronic infection in the population. We performed IgG avidity assays to confirm the incidence of acute infections; this approach must be used together with IgM determination. We also suggest that food restaurant suppliers and nutritionists avoid the use of raw green vegetables with open leafs, such as Brazilian lettuce, escarole, chicory, broccoli and arugula in their salads to prevent exposure to toxoplasmosis caused by viable oocysts after chlorine treatment.

Outbreaks of infectious diseases due to food consumption are difficult to study, and our report dealt with most of these difficulties. We highlight the importance of the accurate diagnosis of indexed cases and also the importance of detecting asymptomatic infections for determining the accurate extent of outbreaks. Outbreaks of toxoplasmosis present a very good opportunity to test this rationale and all of the operative procedures used in outbreak studies. All possible efforts must be made to generate collaborative discussions for the implementation of adequate preventive measures.

RESUMO

Estudo caso-controle de surto de toxoplasmose aguda em indústria no estado de São Paulo, Brasil

Doenças transmitidas por alimentos representam riscos operacionais em restaurantes industriais. Descrevemos surto de nove casos agrupados de doença aguda semelhante à toxoplasmose em indústria de 2300 funcionários. Estes pacientes e outros 36 funcionários assintomáticos foram diagnosticados por ELISA para o título e avidez de IgG anti-T. gondii. Foram excluídos 14 pacientes com toxoplasmose crônica e alta avidez: 13 de controles e um de doença aguda não relacionada à infecção por T. gondii. Também identificamos três empregados assintomáticos com infecção aguda por T.gondii, que como os restantes agudos apresentavam anti-T.gondii IgM ELISA positivo. Conduzimos estudo caso controle por entrevista em 11 infecções agudas e 20 controles negativos. A ingestão de vegetais, mas não de carne ou água, foi associada com a

incidência da doença aguda. Esses dados reforçam a importância do controle sanitário em restaurantes industriais e também demonstram a necessidade de melhoria no controle de qualidade sobre vegetais em risco de contaminação por oocistos de T. gondii. Enfatizamos o diagnóstico preciso de casos e a detecção de infecções assintomáticas para determinar a extensão do surto de toxoplasmose.

REFERENCES

1. Benenson MW, Takafuji ET, Lemon SM, Greenup RL, Sulzer AJ. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N Engl J Med. 1982;307:666-9.

2. Bonametti AM, Passos JN, da Silva EM, Bortoliero AL. Surto de toxoplasmose aguda transmitido através da ingestão de carne crua de gado ovino. Rev Soc Bras Med Trop. 1997;30:21-5.

3. Bowie WR, King AS, Werker DH, Isaac-Renton JL, Bell A, Eng SB, et al. Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team. Lancet. 1997;350:173-7.

4. De Moura L, Bahia-Oliveira LMG, Wada MY, Jones JL, Tuboi SH, Carmo EH, et al. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg Infect Dis. 2006;12:326-9.

5. Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton: CRC Press; 1988. p. 220.

6. Dubey JP. Refinement of pepsin digestion method for isolation of Toxoplasma gondii from infected tissues. Vet Parasitol. 1998;74:75-7.

7. Dubey JP. The history of Toxoplasma gondii--the first 100 years. J Eukaryot Microbiol. 2008;55:467-75.

8. Gattás VL, Nunes EM, Soares ALB, Pires MA, Pinto PLS, Andrade Jr HF. Acute toxoplasmose outbreak at campus of the University of Sao Paulo related to food or water oocyst contamination. In: Annals of the International Conference on Emerging Infectious Diseases; 2000; Atlanta, Georgia. p. 135.

9. Golberg D, Kroupitski Y, Belausov E, Pinto R, Sela S. Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs. Int J Food Microbiol. 2011;145:250-7.

10. Jones JL, Dubey JP. Waterborne toxoplasmosis-recent developments. Exp Parasitol. 2010;124:10-25.

11. Kean BH, Kimball AC, Christenson WN. An epidemic of acute toxoplasmosis. JAMA. 1969;208:1002-4.

12. Kijlstra A, Jongert E. Control of the risk of human toxoplasmosis transmitted by meat. Int J Parasitol. 2008;38:1359-70.

13. Kopec JA, Esdaile JM. Bias in case-control studies. A review. J Epidemiol Community Health. 1990;44:179-86.

14. Kroupitski Y, Pinto R, Belausov E, Sela S. Distribution of Salmonella typhimurium in romaine lettuce leaves. Food Microbiol. 2011;28:990-7.

15. Lopes FM, Mitsuka-Breganó R, Gonçalves DD, Freire RL, Karigyo CJ, Wedy GF, et al. Factors associated with seropositivity for anti-Toxoplasma gondii antibodies in pregnant women of Londrina, Paraná, Brazil. Mem Inst Oswaldo Cruz. 2009;104:378-82.

16. Lwanga SK, Lemeshow S. Sample size determination in health studies: a practical manual. Geneva: World Health Organization; 1991.

17. Magaldi C, Elkis H, Pattoli D, Coscina AL. Epidemic of toxoplasmosis at a university in São-José-dos Campos, S.P. Brazil. Clinical and serologic data. Rev Latinoam Microbiol Parasitol (Mex). 1969;11:5-13.

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18. Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmosis. In: Remington JS, Klein JO, Wilson CB, Baker CJ, editors. Infectious diseases of the fetus and newborn infant. 6th ed. Philadelphia: Elsevier Saunders; 2006. p. 947-1091.

19. Renoiner EIM, Siqueira AA, Garcia MH, Alves RM, Cardoso ME, Ferreira ABPL, et al. Surto de toxoplasmose adquirida, Anápolis-GO, fevereiro de 2006. Bol Eletrôn Epidemiol. 2007;7(8):1-6. [cited: 2011 Oct 2]. Available from: http://portal.saude.gov.br/portal/arquivos/pdf/ano07_n08_toxopl_adquirida_go.pdf

20. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000;30:1217-58.

21. Tenter AM. Toxoplasma gondii in animals used for human consumption. Mem Inst Oswaldo Cruz. 2009;104:364-9.

22. Venkatesan P, Wakelin D. ELISAs for parasitologists: or lies, damned lies and ELISAs. Parasitol Today. 1993;9:228-32.

23. Wainwright KE, Miller MA, Barr BC, Gardner IA, Melli AC, Essert T, et al. Chemical inactivation of Toxoplasma gondii oocysts in water. J Parasitol. 2007;93:925-31.

Received: 24 February 2012Accepted: 11 June 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):245-248, September-October, 2012doi: 10.1590/S0036-46652012000500002

(1) Department of Infectious and Parasitic Diseases, School of Medicine, University of São Paulo, São Paulo, SP, Brazil (LIM-06). E-mails: [email protected]; [email protected] (2) Laboratory of Gastroenterology and Hepatology, Institute of Tropical Medicine, Department of Gastroenterology, School of Medicine, University of São Paulo, São Paulo, SP, Brazil.

E-mails: [email protected]; [email protected] (3) Enteroparasitosis Section of the Parasitology and Mycology Division, Adolfo Lutz Institute, São Paulo, SP, Brazil. E-mail: [email protected] (4) Department of Gastroenterology, School of Medicine, University of São Paulo, São Paulo, SP, Brazil. E-mail: [email protected] Correspondence to: Maria Cristina Espírito-Santo. E-mail: [email protected]

TWO SEQUENTIAL PCR AMPLIFICATIONS FOR DETECTION OF Schistosoma mansoni IN STOOL SAMPLES WITH LOW PARASITE LOAD

Maria Cristina Carvalho do ESPÍRITO-SANTO(1), Mónica Viviana ALVARADO-MORA(2), Pedro Luiz Silva PINTO(3), Flair José CARRILHO(4), João Renato Rebello PINHO(2) & Ronaldo Cesar Borges GRYSCHEK(1)

SUMMARY

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

KEYWORDS: S. mansoni; PCR reamplification; Eggs; Human feces; Diagnosis.

INTRODUCTION

Schistosomiasis is a major public health problem, with an estimated 200 million individuals infected worldwide, of whom 120 million are symptomatic and 20 million have severe forms of the disease. Seven hundred million people are at risk of infection by six species that can infect humans2.

In Brazil, the only species of medical and sanitary interest is Schistosoma mansoni16. Schistosomiasis mansoni is considered an endemic disease in 19 Federative Units. For this reason, the Brazilian Ministry of Health aims to reduce the occurrence of the severe forms of the disease and the number of deaths caused by it, as well as to reduce the prevalence of infection and the risk of geographic expansion of the disease1.

Parasitological methods continue to be the gold standard for laboratory diagnosis of this parasitosis. However, despite being less costly and technically simple, these diagnostic techniques lack sensitivity in areas of low prevalence (prevalence below 10%, with most infected asymptomatic subjects, eliminating less than 96 eggs/gram of feces) of schistosomiasis6,17.

Thus, diagnosis of schistosomiasis requires the use of more sensitive

techniques than the search for eggs in feces, both in areas of low endemicity and for the evaluation of parasitologic cure after treatment. This is important from the epidemiologic view, because the permanence of infected individuals with false-negative results in coproscopic inquiries is enough to continue the risk of transmission, even after suitable interventions are carried out by means of sanitary measures18.

Extraction of the sample DNA is an important stage when carrying out molecular techniques. There are several different kits for extracting DNA from blood, urine, solid tissues and fecal samples. Among biological samples, feces pose the greatest problems for DNA extraction and require the development of new strategies, especially when searching for S. mansoni eggs because they have complex shells that are not easily removed by chemical digestion5. Hence, the aim of this study is to present the PCR reamplification protocol for the detection of S. mansoni in samples with low parasite load (with few numbers of eggs per gram (epg) of feces).

MATERIAL AND METHODS

Laboratory cycle of S. mansoni: The biologic cycle of S. mansoni is maintained in the Laboratory of Immunopathology of Schistosomiasis (LIM-06) of the Institute of Tropical Medicine of the University of

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São Paulo, Brazil, by infecting hamsters (Mesocricetus auratus) and Biomphalaria glabrata snails (BH strain) periodically. Animals that had been subcutaneously infected with 200-300 cercariae of S. mansoni were sacrificed six to seven weeks after the infection in order to collect the parasite eggs from their livers and intestines. The methodology implemented was in compliance with the National Animal Ethics Guidelines, COBEA (Comitê Brasileiro de Experimentação Animal).

Extraction and cleaning of S. mansoni eggs from liver and intestine of hamsters: The extraction and cleaning of S. mansoni eggs was performed according to DESDREN & PAYNE7 and PINTO et al.14. The livers and intestines of three hamsters were minced into small pieces and ground in a blender with 1.4% saline solution. Thereafter, the sediment was passed sequentially through metal mesh (Granust) sieves: Nº 100 (0.150 mm), Nº 200 (0.75 mm) and Nº 400 (0.038 mm) for the complete removal of liver and intestine tissues. The eggs retained in the latter sieve were then removed by successive rinsing with 1.4% saline solution, and observed and counted by light microscopy. A solution containing 150,000 eggs/mL was obtained for the standardization of the DNA extraction method, and another solution containing 1,380 eggs/mL was obtained for the standardization of PCR reamplification.

DNA extraction methods: In order to obtain the highest amount of DNA from the S. mansoni eggs, three different physical methods and two techniques of DNA extraction were tested.

DNA extraction of S. mansoni eggs - DNA extraction was carried out using 1,200 µL of PBS solution (0.01 M) containing 150,000 eggs/mL. Six aliquots of 200 µL were prepared from the PBS solution: 1) two aliquots were placed in liquid nitrogen for one minute, incubated at 95 ºC for one minute, placed in liquid nitrogen for one minute and incubated at 95 ºC for five minutes; 2) two aliquots were put into a whirlpool with five glass beads for five minutes; 3) two aliquots were put into a whirlpool with 0.1 mg of glass fiber for five minutes. DNA extraction was then carried out with GT3 in one of the two aliquots from each test, whereas QIAamp® DNA Stool Mini Kit was used in the other aliquot. After extraction the DNA was quantified in duplicate by NanoDrop (ND-1000 Spectrophotometer V3.5).

DNA extraction of S. mansoni eggs in feces and detection of two sequential PCR amplifications

Preparation of fecal samples artificially marked with S. mansoni eggs - Fresh human fecal samples negative for S. mansoni, using the Kato-Katz and Hoffman methods (two slides for each technique), were obtained from the Central Laboratory of Hospital das Clínicas (Medical School of the University of São Paulo). Seven samples were prepared in 2 mL tubes, each containing 500 mg of feces. A measuring plate of stainless steel was used for determining the weight of the fecal samples, which was 40 x 37 mm thick and had a central orifice of 14.1 mm in diameter developed to ensure the approximate weight of 500 mg of feces.

The solution containing 1,380 eggs/mL was used in this test. Through direct observation by light microscopy and using an Olympus-CX41 microscope, the eggs were counted and five volumes of the solution containing 5, 10, 20, 30 and 50 eggs/mL were added into five of the seven 2 mL tubes. Similarly, a positive control containing 1,000 eggs/mL was used, in addition to a negative control.

DNA extraction from feces artificially marked with S. mansoni eggs - The total DNA of 500 mg of feces artificially marked with S. mansoni eggs was extracted using the GT3 extraction technique, and modified by adding five glass beads in each 2 mL plastic tube.

The extraction was carried out in two stages: 1) each sample was resuspended in 500 µL of ASL Buffer from the QIAamp® DNA Stool Mini Kit. Five glass beads were added and homogenized for five minutes. Subsequently, 400 µL of the supernatant was removed and added to another tube containing 200 µL of AL Buffer and 30 µL of proteinase K (QIAamp® DNA Stool Mini Kit). The samples, prepared as described, were incubated at 37 ºC for 12 hours. 2) 300 µL of GT and 50 µL of chloroform at -20 ºC were added to each sample, followed by homogenization and incubation for 10 minutes and centrifugation at 13,200 rpm. The supernatant was transferred to another tube and precipitated with 300 µL of isopropanol at -20 ºC. Once the DNA was precipitated, the isopropanol was discarded and 300 µL of ethanol absolute was added, followed by centrifugation at 13,200 rpm. Finally, the ethanol was discarded and the samples were incubated at 95 ºC for one minute and resuspended in 100 µL of DEPC-treated Milli-Q water. The extracted DNA was stored at -20 ºC.

Amplification of the S. mansoni DNA - Once the DNA extraction was carried out the samples were submitted to two sequential amplifications by PCR.

Primers that amplify a 121 bp tandem repeat DNA fragment of the S. mansoni genome were used for the amplification of this region previously cited by PONTES et al.15. Bovine Serum Albumin (BSA) (SIGMA, USA) was also added in order to neutralize the action of any inhibitors that could be present at the fecal samples11.

The first PCR reactions were performed in a final volume of 40 µL under the following conditions: 1X Buffer (10X), 2.5 mM MgCl2, 0.4 mM of each dNTP (10 mM), 0.6 pmol/µL of each primer, 0.1 µg/µL BSA, 2.0 U of Platinum Taq DNA polymerase (Invitrogen TM Life Technologies, Carlsbad, CA, USA) and 10 µL of DNA. The second PCR was performed under the same conditions plus 7 µL of amplified DNA from the first reaction. The PCR program used had been described by GOMES et al., 200911. The detection of the amplified DNA was performed by electrophoresis in 2.5% agarose gel, which was stained with ethidium bromide and Low Mass Ladder in order to check the size of the amplicon generated.

A 120 bp fragment of the gene encoding human beta actin (ACTB) was amplified, as an internal control of the DNA extraction with the previously described primers Aco1 and Aco212. The amplification conditions were performed according to GOMES et al., 201010.

Purification and sequencing of the positive control - The amplified DNA from the positive control (sample with 1,000 eggs/mL) was purified using the ChargeSwitch® PCR Clean-Up Kit. The sequencing was then carried out in the ABI Prism® 3100 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA) using deoxynucleotide triphosphates (dNTPs) with fluorescent markers (Big Dye® Terminator v3.1 Cycle Sequencing Ready Reaction kit - Applied Biosystems, Foster City, CA, USA). In order to obtain the consensus sequence, the forward and reverse sequences obtained were initially analyzed using the Phred-Phrap-Consed

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programs8,9. The consensus sequence was observed, analyzed and edited using the SE-AL program (available at: http://tree.bio.ed.ac.uk/software/seal/). Finally, a BLAST was performed to confirm that the obtained sequence belonged to the expected fragment of the S. mansoni genome.

RESULTS

DNA extraction method: The sample initially containing 150,000 eggs/mL was homogenized and six samples with approximately 30,000 eggs/mL were taken from it and prepared. After DNA extraction, the DNA obtained was quantified using the ND-1000 Spectrophotometer V3.5. The quantification results were: liquid nitrogen/GT = 181 ng/µL; liquid nitrogen/extraction kit = 9 ng/µL; milled glass/GT = 313.2 ng/µL; milled glass/extraction kit = 60.9 ng/µL; glass beads/GT = 437.9 ng/µL; glass beads/extraction kit = 106.6 ng/µL.

Detection of S. mansoni DNA through PCR reamplification: Once the standardization of the DNA extraction technique was performed, the method for the detection of S. mansoni DNA was developed for samples with low parasite load. All the dilutions tested (5, 10, 20, 30 and 50 eggs/mL) were amplified by two sequential reactions of the same DNA fragment (Fig. 1). Similarly, the 120 bp fragment of the internal control (ACTB) was amplified in all the samples, including the negative control, and finally the sequence obtained was considered to be the one from the positive control.

DISCUSSION

The development of laboratory tools for the diagnosis of infections with low parasite load is one of the priorities in the schistosomiasis research agenda of the World Health Organization’s control programs. Among these tools, the research agenda points to the development and standardization of molecular methods for the surveillance of infections in humans and snails which can be applied to individual and public health studies4.

In the present study, a new DNA extraction technique was implemented, followed by PCR reamplification (two sequential amplifications of the same PCR fragment), with the detection of the amplified fragment in 2.5% agarose gel. This is a sensitive method, its detection limit was 5 eggs/500 mg of artificially marked feces, which

represents the equivalent of 0.4 eggs/40 mg of feces, according to the Kato-Katz method or an estimate of 9.6 eggs/gram of feces.

The PCR analytical sensitivity was evaluated by 5-fold dilutions extracted from 500 mg stool samples each labeled as having artificially added 5, 10, 20, 30 and 50 eggs/mL of S. mansoni.

The detection limit was 5 eggs/500 mg, approximately 43 fg of genomic DNA extracted from the eggs of S. mansoni.

The first study conducted with fecal samples and serum revealed that the conventional PCR can detect 2.4 fg of DNA of S. mansoni per gram of feces, which makes this method more sensitive than the Kato-Katz method15.

More recently, other studies have been trying to improve the sensitivity of molecular methods. GOMES et al., 200911, achieved a detection limit of 3 fg of DNA from S. mansoni in feces. OLIVEIRA et al.13 obtained sensitivity using the conventional PCR of an egg in a suspension of 300 µL of feces. Eventually, GOMES et al., 201010, obtained a sensitivity of 0.15 eggs/gram of feces (1.3 fg of DNA), using the PCR-ELISA detection system. Although these studies may report DNA detection values in lower concentrations, it is important to consider that the observation of the bands of DNA concentration may not be suitable for use in routine diagnosis of this infection.

It is important to highlight that the PCR reamplification may show a high level of amplicon contamination when it is not performed under suitable laboratory conditions. Similarly, one must bear in mind that for the diagnosis of the infection the amplified DNA band in the gel, as a final product of the test, should be sufficiently intense in order to ensure the presence of the pathogen in the sample. Thus the DNA bands obtained in this study showed increased DNA concentration, which can justify its use in routine assessments.

Finally, since other factors may compromise results (e.i. stool consistency, medication use, stool conservation, the presence of other parasites in the samples, etc.), further studies are necessary to evaluate our protocol in the future.

CONCLUSIONS

In conclusion, our results have indicated that the extraction technique, which mixed glass beads with the GT, produced good results in the DNA extraction of S. mansoni eggs. With regard to PCR reamplification, despite its limitations, the use of two sequential amplifications per PCR to detect S. mansoni in feces may be a promising new tool.

RESUMO

Duas amplificações sequenciais por PCR para detecção de Schistosoma mansoni em amostras de fezes com baixa carga

parasitária

A esquistossomose constitui grande problema de saúde pública, sendo que estimativas apontam para 200 milhões de pessoas infectadas no mundo e 700 milhões de pessoas em áreas de risco. No Brasil, existem áreas de alta, média e baixa endemicidade. Estudos demonstram

Fig. 1 - Detection of S. mansoni DNA by two sequential PCR amplifications: (1) Low DNA

Mass Ladder; (2) Positive Control (1,000 eggs/mL); (3) Negative Control; (4) Sample 1 (5

eggs/mL); (5) Sample 2 (10 eggs/mL); (6) Sample 3 (20 eggs/mL); (7) Sample 4 (30 eggs/

mL); (8) Sample 5 (50 eggs/mL).

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que nas áreas endêmicas de baixa prevalência da infecção, a reduzida sensibilidade dos métodos parasitológicos torna-se evidente. Isto dificulta o diagnóstico, pela presença de resultados falso-negativos. O objetivo deste estudo foi a padronização de um protocolo de reamplificação da PCR (Re-PCR) para a detecção de Schistosoma mansoni em amostras com menos de 100 ovos por grama (opg) de fezes. Foram utilizados três métodos para ruptura dos envoltórios dos ovos de S. mansoni e duas técnicas de extração de DNA foram aplicadas. O DNA extraído foi quantificado e os resultados sugerem que a técnica de extração de melhor produtividade foi a que associa esferas de vidro a uma solução de isotiocianato de guanidina/fenol/clorofórmio (GT). Aplicou-se a Re-PCR, que demonstrou sensibilidade para a detecção de cinco ovos/500 mg de fezes artificialmente marcadas. Assim, essas novas ferramentas são potencialmente aplicáveis nas infecções por S. mansoni com baixa carga parasitária.

ACKNOWLEDGMENTS

This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo, processes 07/53457-7, 08/50461-6 and 10/52615-0. We thank Dr. Luciana Inacia Gomes, from Laboratório de Pesquisas Clínicas (Laboratory of Clinical Research), Instituto de Pesquisas René Rachou-Fiocruz - Belo Horizonte, Brazil, for her advice on the standardization process. Finally, we thank Miss Maria Cristina Conceição de Mello from LIM-06 FMUSP for her technical support in the biological cycle of S. mansoni.

AUTHOR CONTRIBUTIONS

MCCES participated in the design of the study, in the extraction and cleaning of S. mansoni eggs, standardization of the DNA extraction method and in the two sequential PCR amplifications. MVAM participated in the design of the study, the standardization of the DNA extraction method and in the two sequential PCR amplifications. PLSP participated in the design of the study and in the extraction and cleaning of S. mansoni eggs. FJC participated in the design of the study. MCCES, MVAM, JRRP and RCBG participated in the elaboration of the manuscript. All authors read and approved the final manuscript.

REFERENCES

1. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Esquistossomose Mansoni. In: Doenças infecciosas e parasitárias: guia de bolso. 8ª ed. rev. Brasília: Ministério da Saúde; 2010. p. 169-73.

2. Chitsulo L, Engels D, Montresor A, Savioli L. The global status of schistosomiasis and its control. Acta Trop. 2000;77:41-51.

3. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-9.

4. Colley DG, Secor WE. A schistosomiasis research agenda. PLoS Negl Trop Dis. 2007;1:e32.

5. Da Silva AJ, Bornay-Llinares FJ, Moura IN, Slemenda SB, Tuttle JL, Pieniazek NJ. Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Mol Diagn. 1999;4:57-64.

6. Doenhoff MJ. A vaccine for schistosomiasis: alternative approaches. Parasitol Today. 1998;14:105-9.

7. Dresden MH, Payne DC. A sieving method for the collection of schistosome eggs from mouse intestines. J Parasitol. 1981; 67:450-2.

8. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186-94.

9. Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175-85.

10. Gomes LI, Dos Santos Marques LH, Enk MJ, de Oliveira MC, Coelho PM, Rabello A. Development and evaluation of a sensitive PCR-ELISA system for detection of schistosoma infection in feces. PLoS Negl Trop Dis. 2010;4:e664.

11. Gomes LI, Marques LH, Enk MJ, Coelho PM, Rabello A. Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples. Mem Inst Oswaldo Cruz. 2009;104:1194-6.

12. Musso O, Sommer P, Drouet E, Cotte L, Neyra M, Grimaud JA, et al. In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods. J Virol Methods. 1996;56:125.

13. Oliveira LM, Santos HL, Gonçalves MM, Barreto MG, Peralta JM. Evaluation of polymerase chain reaction as an additional tool for the diagnosis of low-intensity Schistosoma mansoni infection. Diagn Microbiol Infect Dis. 2010;68:416-21.

14. Pinto PLS, Floriano LD, Ferreira SC, Suto LM, Vellosa SAG. Purificação de ovos de Schistosoma mansoni a partir de vísceras de hamsters (Crycetus auratus) experimentalmente infectados. In: XIV Congresso da Sociedade Brasileira de Parasitologia, Goiânia, Brasil; 1995. p. 264. (Revista de Patologia Tropical; vol. 23, n. 2).

15. Pontes LA, Dias-Neto E, Rabello A. Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces. Am J Trop Med Hyg. 2002;66:157-62.

16. Rey L. Esquistossomíase mansônica: o parasito. In: Rey L. Bases da Parasitologia médica. 2ª ed. Rio de Janeiro: Guanabara-Koogan; 2002. p. 153-82.

17. Ruiz-Tiben E, Hillyer GV, Knight WB, Gómez de Rios I, Woodall JP. Intensity of infection with Schistosoma mansoni: its relationship to the sensitivity and specificity of serologic tests. Am J Trop Med Hyg. 1979;28:230-6.

18. Teles HMS. Estudo parasitológico, da transmissão e dos impactos da profilaxia da esquistossomose mansônica no Município de Bananal, Estado de São Paulo, Brasil. [Tese]. Campinas: Instituto de Biologia, Unicamp; 2001.

Received: 12 November 2011Accepted: 22 June 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):249-255, September-October, 2012doi: 10.1590/S0036-46652012000500003

(1) Doctors in Science (Microbiology), Departament of Virology, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-972 Rio de Janeiro, RJ, Brazil. E-mails: [email protected]; [email protected]

(2) Doctor in Medicine (Infectious and Parasitary Diseases), Departament of Microbiology and Parasitology, Instituto Biomédico, Universidade Federal Fluminense, 24210-130 Rio de Janeiro, RJ, Brazil. E-mail: [email protected]

Correspondence to: Norma Santos, Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS-Bl. I, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil. Phone: 55 21 2560-8344 ext. 165, Fax: 55 21 2560-8028. E-mail: [email protected]

ACUTE RESPIRATORY VIRAL INFECTIONS IN CHILDREN IN RIO DE JANEIRO AND TERESÓPOLIS, BRAZIL

Maria Carolina M. ALBUQUERQUE(1), Rafael B. VARELLA(2) & Norma SANTOS(1)

SUMMARY

The frequency of viral pathogens causing respiratory infections in children in the cities of Rio de Janeiro and Teresópolis was investigated. Nasal swabs from children with acute respiratory illnesses were collected between March 2006 and October 2007. Specimens were tested for viral detection by conventional (RT)-PCR and/or real time PCR. Of the 205 nasal swabs tested, 64 (31.2%) were positive for at least one of the viral pathogens. Single infections were detected in 56 samples, 50 of those were caused by RNA viruses: 33 samples tested positive for rhinovirus, five for influenza A, five for metapneumovirus, four for coronavirus and, three for respiratory syncytial virus. For the DNA viruses, five samples were positive for bocavirus and one for adenovirus. Co-infections with these viruses were detected in eight samples. Our data demonstrate a high frequency of viral respiratory infections, emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses. The fact that the emerging respiratory viruses were present in 9.2% of the tested samples suggests that these viruses could be important respiratory pathogens in the country.

KEYWORDS: Respiratory infection; Viral infection; Viral diagnosis.

INTRODUCTION

Acute respiratory infections represent a major cause of human disease and one of the most important causes of child mortality throughout the world30,46. Although there are considerable variations in infantile mortality, the World Health Organization has classified the infections of the lower respiratory tract as the second most important cause of death among children under five years of age.

Viral respiratory diseases are among the most common illnesses affecting children and adults52. The respiratory tract can be infected by RNA and DNA viruses, which induces several clinical syndromes. The human respiratory syncytial virus (HRSV), human parainfluenza virus (HPIV), influenza A virus (FLUAV) and influenza B virus (FLUBV), human adenovirus (HAdV), human coronavirus (HCoV), and human rhinovirus (HRV) are recognized as agents of human respiratory disease. Several other viruses, such as human metapneumovirus (HMPV), human bocavirus (HBoV), and the newly discovered human coronaviruses HCoV-NL63 and HCoV-HKU1, have been identified as potential respiratory pathogens30,46. In addition, two new human polyomaviruses (HPyV), KIPyV and WUPyV, have been detected in patients with respiratory infections.

The main goal of this study was to identify the frequency of viral pathogens causing respiratory infections in children with respiratory

illness in the cities of Rio de Janeiro and Teresópolis.

MATERIAL AND METHODS

Sample Collection: Nasal swabs from 205 children (median age 3.3 years; range one month to 15 years) with both upper and lower acute respiratory illnesses were collected between March 2006 and October 2007 and tested for viral pathogens. The nasal swabs were immersed in 1 mL of virus transport media and kept at -70 oC until processing. Acute respiratory illness was defined by the presence of rhinorrhea and/or cough and/or respiratory distress and/or sore throat, associated or not to fever, with maximal duration of seven days. The specimens were collected from hospitalized patients, emergency departments and walk-in clinics at two university hospitals in the cities of Rio de Janeiro (137 samples) and Teresópolis (68 samples). Relevant clinical information, including hospitalization status, age, sex, and clinical symptoms, was collected during the first medical visit by means of a standard questionnaire.

The study protocol was reviewed and approved by the Ethics Committees in Research of the Instituto de Puericultura e Pediatria Martagão Gesteira of the Universidade Federal do Rio de Janeiro and Fundação Educacional Serra dos Órgãos, of the Centro Universitário Serra dos Órgãos. The parents of all children involved in the study had given informed consent, in accordance with Resolution 196/96, of the Brazilian Ministry of Health.

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Nucleic acid extraction: Nucleic acid was extracted from 200 µL of the sample by using Wizard® Genomic DNA Purification KIT (Promega, Madison, WI, USA) or RNAgents® kit (Promega) according to the manufacturer’s instructions.

Viral detection: Specimens were tested for presence of FLUAV and FLUBV48, HRSV48, HPIV1, HAdV4, HRV34, HMPV32, HBoV45, WUPyV28, and KIPyV3 by conventional PCR and (RT)-PCR assays as previously described. A real time PCR protocol was used for detection of HCoVs (229E, OC43, NL63 and HKU1)18,57. All assays were included positive and negative external controls. Conventional PCR and RT-PCR were performed in a Veriti 96 well (Applied Biosystems, Foster City, CA, USA) thermocycler and the amplified products were detected by agarose gel electrophoresis and staining with ethidium bromide. Real-time RT-PCR was performed in an ABI StepOne Real-time PCR System (Applied Biosystems).

RESULTS

Patient demographics: Among the 205 samples, 73 (35.6%) were collected from hospitalized patients (inpatients) and 132 from outpatients children (59 [28.8%] from the emergence department and 73 [35.6%] from the walk-in clinics). Only one specimen was obtained per patient. The age distribution of the subjects was as follow: 95 (46.3%) were < one year of age; 61 (29.8%) were between 1.1 - 5 years; 30 (14.6%) were between 5.1 - 10 years; 16 (7.8%) between 10.1 - 15 years; and for three patients the age was not informed. The most common symptoms observed among the patients were a cough (54%; 111/205), fever (53.6%; 110/205), rhinorrhea (47.8%; 98/205), nasal congestion (27.3%; 56/205), pneumonia (24.4%; 50/205), wheezing (23.4%; 48/205), bronchitis/bronchiolitis (18%; 37/205), broncospasm (8.3%; 17/205), asthma (5.9%; 12/205) and diarrhea (4.4%; 9/205). Few patients presented headache, sore throat, tonsillitis, exanthema, laryngomalacia, vomit or otitis.

Virus detected: A total of 64 (31.2%; n = 205) samples were positive for at least one of the viral pathogens specified above: 42 positive samples from the city of Rio de Janeiro (30.7%; 42/137) and 22 from Teresópolis (32.4%; 22/68). RNA viruses were detected in higher frequency than DNA virus. Thirty-three single infections were detected with HRV, five with FLUAV, five with HMPV, five with HBoV, three with HRSV, three with HCoV-HKU1 or -NL63 one with HCoV-OC43, and one with HAdV. Co-infections with these viruses were observed in eight samples including two samples positive for KIPyV and WUPyV and HPIV was not detected (Table 1).

Seasonality: The majority of viral infections (79.7%; 51/64) was detected between June and November (late autumn through spring months) (Fig. 1). Yet, positive samples were detected all year round except for the months of February and May.

Virus detection x patient status: Among patients positive for viral pathogen 14 (22%; 14/64) were inpatients (hospitalized) at the time of samples collection; while 18 (28%; 18/64) patients attended the emergency department and 32 (50%; 32/64) the walk-in clinics.

The age of the patients infected with viruses ranged from three months to 15 years (median 3.3 years); 50% (32/64) of the patients positive for viral infections were under one year of age; 25% (16/64)

were between 1.1 - 5 years of age; 17.2% (11/64) were between 5.1 - 10 years; three patients were over 10.1 years of age. For two patients the ages were not informed.

Virus detected x patient symptoms: The symptoms most frequent observed among patients with HRV single infections were cough (67%), rhinorrhea (67%) and fever (52%). The age ranged from one month

Table 1Respiratory viruses detected in the study†

Virus detectedNumber of positive

samples

FLUAV 5

HRSV 3

HMPV 5

HRV 33

HCoV-NL63 2

HCoV-HKU1 1

HCoV-OC43 1

HAdV 1

HBoV 5

HCoV-NL63 + HMPV 1

HCoV-NL63 + HRV 1

HMPV + HRV + HCoV-OC43 + HAdV 1

HMPV + KIPyV 1

HBoV + HRV 1

HBoV + WUPyV 1

FLUAV + HRV + HCoV-OC43 1

HRSV+HCoV-OC43 1

Total 64†FLUBV and HPIV were not detected.

Fig. 1 - Monthly distribution of acute respiratory viral infections in children in Rio de Janeiro

and Teresópolis between March 2006 and October 2007.

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ALBUQUERQUE, M.C.M.; VARELLA, R.B. & SANTOS, N. - Acute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 249-55, 2012.

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to 12 years. Most patients were treated at the walk-in clinics. Of the four patients with co-infections involving HRV (HRV+HMPV+HCoV-OC43+HAdV; HRV+HCoV-NL63; HRV+HBoV; HRV+FLUAV) three were also treated at the walk-in clinics and one attended the emergency department.

Patients only infected with FLUAV were older (between 1 - 15 years of age - three of them were between nine and 15 years of age). Two out of five influenza-positive patients were hospitalized; the most frequent symptoms presented were rhinorrhea (80%), nasal congestion (60%), cough and fever (40% each). One patient was co-infected with FLUAV + HRV.

Patients with HMPV infections had a myriad of symptoms, including fever, cough, rhinorrhea, wheezing, and sore throat. Of the five patients with single infections, four were treated at walk-in clinics and one was treated at an emergency department. A patient co-infected with HMPV and HCoV-NL63 was treated at a walk-in clinic, and a patient co-infected with HMPV and KIPyV and one co-infected with HMPV, HCoV-OC43, HAdV, and HRV were treated at an emergency department. The patient co-infected with KIPyV was a 4-year-old boy with cough, fever, rhinorrhea, and wheezing.

HBoV was detected in samples from five patients as a single infection and in samples from two patients as a co-infection with HRV or WUPyV. Three patients had pneumonia (two single infections and one co-infection with HRV). Two of the three were treated at walk-in clinics; one of the patients with only HBoV infection was hospitalized. The patient co-infected with WUPyV was a 10-month-old boy who had been treated at an emergency department after exhibiting cough, rhinorrhea, and laryngomalacia.

HRSV was only detected among children younger than one year of age treated at an emergency department. Patients presented rhinorrea, fever, cough and nasal congestion. One patient was co-infected with HCoV-OC43.

The diagnosis for most patients infected with HCoV-NL63 or HCoV-HKU1 was pneumonia. Of the three patients with single infections, two were hospitalized and one was treated at the emergency department; the two patients with mixed infection of HCoV-NL63 and HMPV or HRV were treated at walk-in clinics. The patient positive for HCoV-OC43 was a three-month-old child who was hospitalized with bronchiolitis.

A five-year-old child positive for HAdV presented fever, wheezing and nasal congestion and was treated at the walk-in clinic.

DISCUSSION

Viral infection is a major cause of respiratory illness among children. The majority of these infections are caused by HRSV, FLUAV and FLUBV, HPIV and HAdV45. Newly discovered viruses such as HMPV, HBoV, HCoV-NL63 and HCoV-HKU1, have also been demonstrated to be important respiratory pathogens26,39. Moreover, two new human polyomaviruses (KIPyV and WUPyV) are been described among patients with respiratory infections26,39.

In Brazil , epidemiological studies have demonstrated

the importance of viruses as etiological agents of respiratory disease5,7,8,10,14,16,17,19,25,29,33,35,38,40,43,51,53,59,60. However, because such studies are mostly focused on the most common respiratory pathogens (FLUAV, HRSV, HPIV, HRV and HAdV), the role of emerging viruses as etiological agents of respiratory disease among Brazilian children remains unknown.

Generally, RNA viruses, particularly HRSV, FLUAV and HRV, are more often associated to respiratory infections than DNA viruses30,46. In the present study infections caused by RNA viruses represented 89% (57/64) of the positive samples while DNA viruses (HAdV, HBoV and HPyV) were detected in 15.6% (10/64) of the samples, including mixed infections.

Among the RNA viruses, HRSV is usually the pathogen more often detected in children with acute respiratory disease hospitalized or not7,11,13,14,33,37,51,53. Yet, HRV was the most common virus detected in our study (57.8%; 37/64, including co-infections). One possible explanation for these findings could be the fact that HRV is predominantly found in upper respiratory tract infection, with high levels of viral concentration in nasal secretions55 and, material of choice in our study as nasal swabs. On the other hand, HRSV is detected in higher concentration in secretion from the lower respiratory tract. Still, other studies using nasal swabs or nasopharyngeal aspirate from hospitalized and non-hospitalized patients also showed a predominance of HRV8,12,44,60. In the city of Fortaleza, Brazil, an epidemiological surveillance carried out during 29 months showed a high incidence of respiratory illness and a predominance of infections caused by HRV (45.6%)5. Other studies in the country also demonstrated a predominance of HRV. In a longitudinal study among children in daycare centers in Salvador, Bahia, SOUZA et al. showed a high prevalence (48.3%) of HRV infections47. In another study performed in Salvador by NASCIMENTO-CARVALHO et al. HRV was the most common viral pathogen (21%), detected in the same proportion as Streptococcus pneumonia36. BELLEI et al. evaluated the frequency of viral infections among healthcare workers in São Paulo and showed a predominance of HRV (37.7%)6. BONFIM et al. studies on the frequency of respiratory pathogens in children attending daycare centers in São José do Rio Preto, São Paulo, between 2003 and 2005, also showed the predominance of HRV (37.7%)8.

Similar to other respiratory pathogens, HRV seasonality varies geographically due to temperature variations. In the temperate areas of the north hemisphere one peak of infections occurs in September (autumn) and a second peak occurs in April/May (spring). In the temperate areas of the south hemisphere, the seasonal incidence of these infections is similar to that seen at the north hemisphere55. In Brazil, a study in Salvador showed a peak of infection by HRV during the autumn47. Another study in São José do Rio Preto found that HRV was detected more frequently in autumn, but the virus occurred throughout the study, with low frequency in the summer months (December, January and, February)8. In our study, carried out at Rio de Janeiro and Teresópolis, we detected a peak of HRV infection during the late autumn/winter and early of spring as 30 out of 33 HRV-positive single infections were detected between June and September with the highest peak in August (45.5%; 15/33).

Many studies have suggested an association between HRV infections and exacerbation of asthma31,55. Twelve patients participating in our study presented symptoms of asthma at the time of sample collection. Four of

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those individuals were positive for HRV infection. However, we could not draw a conclusion because the number of patients with asthma was small.

FLUAV was only detected in 9.4% (6/64) of the samples. During a surveillance done in São Paulo, in 2007, THOMAZELLI et al. detected FLUAV in 5% of the samples51. Another study in the same state performed between 2003 and 2005 detected FLUAV and FLUBV in 23.8% of the samples8. In Curitiba, this virus was responsible for 8.3% of infections among pediatric and transplant patients51. In Minas Gerais FLUAV and FLUBV were detected in 9.5% of samples collected from children younger than five years of age15. In Brazil, the flu vaccine program began in 1999 and since then the vaccine has been distributed off charge for children and elderly over 60 years of age, healthcare workers and incarcerate population. Surveillance studies carried out in São Paulo and Minas Gerais indicate that the vaccine coverage reached between 63% and 66% of the target population15,22,26. This fact could explain the low frequency of FLUV infections found in the country recently.

Generally the epidemiological studies performed in Brazil show a high frequency of infection by HRSV8,14,33,37,42,49-51. Differently, we only detect four (6.3%; 4/64) HRSV infections. Nevertheless, the above mentioned studies were done in subtropical areas of the country such as São Paulo33,37,51, Porto Alegre49,50 and Minas Gerais14 where the average temperature is below 18 °C, with a thermal amplitude between 9 °C and 13 °C, whereas the state of Rio de Janeiro is a more tropical area with the average temperature between 18 oC and 22 °C and a thermal amplitude between 7 °C and 9 °C. Perhaps this thermal variation could explain the low frequency of HRSV found in our study, since other surveillance done in northern Brazil, a tropical area where the average temperature exceed 20 °C, the HRV is more prevalent than HRSV5,36,47. Corroborating this theory, a study in Wales between 1991 and 2004, demonstrated that a rise of 1 °C in the annual temperature average during this period shortened the HRSV season in 3.1 weeks21. On the other hand, a recent study performed in the city of Recife, northeast Brazil, found an incidence of HRSV infection of 37% testing nasopharyngeal aspirates of children less than five years of age7.

In Brazil, the frequency of HAdV infections usually varies between 2.3% and 9.9%2,5,14,33,49-51. We found a frequency of 3.1% (2/64) of HAdV.

The HPIV presents a distinct seasonal pattern, with a biannual epidemic pattern, particularly for HPIV-1, 2 and, 361. We did not detect any sample positive for HIPV. It seems that, depending on the region studied, season and size of the study population, this virus may not present a significant prevalence. In Fortaleza, Ceará, a study carried out between 2001 and 2006, 3070 nasopharyngeal aspirates were analyzed and 976 (31.8%) were positive for viral infection. Nevertheless, only 3.8% tested positive for HPIV25. In São Paulo, THOMAZELLI et al. analyzed 336 respiratory samples and found 28 (8.3%) to be positive for HPIV-3 and two (0.6%) for HPIV-151. In Uberlândia, Minas Gerais, HIPV was detected in 6.3% of the 379 samples tested14. In Porto Alegre, Rio Grande do Sul, STRALIOTTO et al. found only 2.3% positivity for HPIV in samples collected in 1996 and 1.5% in samples from 1990-199249,50. In São José do Rio Preto, BONFIM et al. found HIPV in 2.4% (19/782) samples collected between 2003 and 20058.

The literature indicates that 20% to 60% of confirmed cases of pneumonia the pathogen cannot be identified. The published data have

also showed that viral pneumonia are more frequent than bacterial pneumonia, especially among < 2-year-old children24. Yet, only in 40% of those cases a viral agent is identified, even when highly sensitive molecular tests are applied30,46. It suggests that unknown viruses could be circulating among the population. Since 2001, new viruses are being identified and the epidemiology has demonstrated that some of those could be associated to respiratory illnesses30,46.

The HMPV was first described in 2001 and since then has been frequently detected in association to respiratory infections30,46. We found that 12.5% (8/64) of our samples were positive for HMPV: five patients had single infections (mean age 2.2 years old, range six months-five years old) and three patients presented mixed infections (Table 1); none of them were hospitalized but one patient with single infection attended the emergency department presenting lower respiratory tract symptoms. DEBURet al. performed a retrospective study in Curitiba, Paraná, and analyzed nasopharyngeal aspirates from 1,572 individuals collected between 2006 and 2008. They found HMPV in 3.9% of the samples. The incidence was higher in outpatients (5.9%; n = 493), whose mean age was 19.7 years (range six months-75 years old), than in inpatients (3%; n = 1079), whose mean age was 7.6 months (range one month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections19. Other studies have shown a significant positivity for HMPV in Brazil as well7,8,16,20,38,40,51,59,60 .

HBoV is another emergent virus associated to illness of the respiratory tract. It was first described in 2005 and has been detected in 1.5% to 19% of patients with acute respiratory infection30,46. In this study we detected HBoV in 10.5% (7/64) of the samples. Other studies have found similar rates in Brazil7,27,40.

Prior to 2003 there were only two coronavirus species known to infected human, the HCoV-OC43 and the HCoV-229E. In 2005 three new HCoV species were recognized as human pathogens: SARS-CoV, HCoV-NL63 and HCoV-HKU130. The SARS-CoV caused a pandemic in 2002 but has not been detected in the human population since 200430. The HCoV-NL63 has been detected in 1% - 10% of children with respiratory infection and has been associated to pneumonia41. The HCoV-HKU1 has been detected in 0.3% to 2.4% of individuals with respiratory disease23,62. We found eight samples positive for HCoV being one sample positive for HCoV-HKU1, four positive for HCoV-NL63 and three for HCoV-OC43. Three HCoV-NL63-positive patients presented pneumonia. Patients infected with HCoV-HKU1 usually present rhinorrhea, cough, wheezing, fever, bronchiolitis or pneumonia46. The patient positive for HCoV-HKU1 was a 4-month-old child who presented fever, bronchiolitis, nasal congestion and wheezing. Generally, the HCoV-OC43 causes common cold, although it has already been described in cases of severe infection of the lower respiratory tract. Rhinorrea and bronchiolitis were the most frequent symptoms associated to HCoV-OC43 in our study; two patients were under six months of age. Few cases of the emerging HCoV in Brazil were described. GÓES et al. did a retrospective analysis of nasopharyngeal aspirate samples collected from children in 1995 and detected six (3.6%; n = 169) cases of HCoV-HKU1 infections. The positive samples were associated with pertussis, pneumonia, bronchiolitis, and diarrhea29. More recently, CABEçA & BELLEI described a case of HCov-NL63 infection in a hospitalized patient suspected of influenza virus H1N1 2009 infection. The patient was

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a 46-year-old female with diabetes presenting influenza-like symptoms that progressed to hemorrhagic pneumonia, respiratory and renal failure, brain edema and death10.

Recently two new polyomaviruses, KIPyV and WUPyV, were detected in respiratory samples3,28. Currently these agents have been described in different countries throughout the world9,56 suggesting a global distribution. In our study, these viruses were only detected in co-infections (WUPyV+HBoV and KIPyV+HMPV). Interestingly, a significant high rate of co-infection with other respiratory viruses (up to 80%) is observed for both viruses9. Therefore, the role of the polyomaviruses in respiratory illness is yet to be determined.

The data presented in this study contribute to a better understanding of the impact of viruses in respiratory infections in the infancy emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses.

RESUMO

Infecções respiratórias agudas causadas por vírus em crianças do Rio de Janeiro e de Teresópolis, Brasil

Neste estudo foi investigada a frequência de patógenos virais causando infecção em crianças nas cidades do Rio de Janeiro e Teresópolis. Foram coletados 205 swabs nasais de crianças com infecção aguda do trato respiratório no período de março de 2006 a outubro de 2007. Os espécimes foram testados para detecção de vírus através de (RT)-PCR e/ou PCR em tempo real. Dentre as 205 amostras testadas, 64 (31,2%) foram positivas para pelo menos um vírus. Infecções causadas por um único agente viral foram detectadas em 56 amostras, 50 das quais eram causadas por vírus de RNA: 33 amostras foram positivas para rinovírus, cinco amostras foram positivas para influenza A, cinco amostras foram positivas para metapneumovírus, quatro amostras foram positivas para coronavírus e três amostras foram positivas para vírus respiratório sincicial. Para os vírus de DNA foram detectadas cinco amostras positivas para bocavírus humano e uma amostra positiva para adenovírus. Foram identificados oito casos de co-infecção. Nossos dados demonstram frequência elevada de infecções respiratórias virais, enfatizando a necessidade de um diagnóstico mais acurado destes patógenos, principalmente os vírus considerados emergentes. O fato de alguns vírus respiratórios emergentes terem sido detectados em 9,2% das amostras testadas sugere que estes vírus podem ser patógenos respiratórios importantes no país.

ACKNOWLEDGMENTS

We thank Soluza dos Santos Gonçalves for the technical assistance.

This manuscript represents part portion of a thesis submitted by M. C. M. A. to the Universidade Federal do Rio de Janeiro, Brazil, as a partial fulfillment of the requirements for a Doctor of Science degree.

This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tenológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by Fundação Carlos Chagas de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

CONFLICT OF INTEREST

The authors of this work have no financial or personal relationship with other people or organization that could inappropriately influence their work during the submission process.

STATEMENTS OF THE AUTHORS’ CONTRIBUTION

MCMA and NS conceived and designed the study and analyzed the data. MCMA was responsible for data and sample collection, PCR analyzed and wrote the paper; NS and RBV revised the manuscript. The final manuscript has been seen and approved by all the authors.

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Received: 27 February 2012Accepted: 31 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):257-259, September-October, 2012doi: 10.1590/S0036-46652012000500004

(1) Secondary Immunodeficiencies outpatient clinic, Dermatology Department, São Paulo University Medical School. São Paulo, SP, Brazil. (2) Preventive Medicine Department, São Paulo University Medical School. São Paulo, SP, Brazil.(3) Institute of Tropical Medicine of São Paulo, Laboratory of Dermatology and Immunodeficiencies/LIM56, University of São Paulo. São Paulo, SP, Brazil.Correspondence to: Jorge Casseb. Instituto de Medicina Tropical de São Paulo, Av. Dr. Enéas de Carvalho Aguiar 470, 05403-000 São Paulo, SP, Brasil. E-mail: [email protected]

TUBERCULOSIS AMONg HIV-1-INFECTED SUBJECTS IN A TERTIARY OUT-PATIENT SERVICE IN SÃO PAULO CITY, BRAZIL

Jorge CASSEB(1,3), Luiz Augusto M. FONSECA(2), Lucas A. MEDEIROS(1), Claudio R. GONSALEz(1), Eduardo R. LAGONEGRO(1), Ana Paula Rocha VEIGA(1), Daniela C. DA SILVA(1) Marcelo MENDONçA(1) & Alberto J. S. DUARTE(1)

SUMMARY

TB is currently considered to be the most important infectious disease among HIV-1-infected subjects in developing countries, such as Brazil. A retrospective analysis of TB cases was performed, occurring from January 1995 to December 2010 in our cohort of 599 HIV positive patients. The primary outcome was the occurrence of active TB. Forty-one TB cases were diagnosed over this period of 16 years, among 599 HIV positive patients in an open cohort setting in the city of Sao Paulo, Brazil. All-time lowest mean CD4 T cell count at the time of TB diagnosis was 146 and 186 cells/mm3, respectively. The mean HIV viral load was 5.19 log

10

copies/mL, and 59% of the patients were on HAART. TB incidence was 1.47 per 100 person-years, for a total follow-up time of 2775 person-years. The probability of surviving up to 10 years after diagnosis was 75% for TB patients as opposed to 96% for patients with other, non-TB opportunistic diseases (p = 0.03). TB can be considered a public health problem among people living with HIV in Brazil despite of the widespread use of antiretrovirals for the treatment of HIV infection/AIDS.

KEYWORDS: AIDS; Brazil; Co-infection; Incidence; HIV; Mortality; TB.

INTRODUCTION

In 2008, there were 1.37 million new clinical cases of tuberculosis (TB) and an estimated half-million deaths from TB occurred in HIV-infected persons worldwide3. TB is currently considered to be the most important opportunistic infection among HIV-1-infected subjects in Brazil1 and is responsible for a substantial proportion of morbidity and mortality among HIV-infected patients, with approximately 70,000 new cases reported annually3,4,5,13. São Paulo State reported 1/3 of Brazilian cases of TB/HIV infection in 20111. In fact, 14% of TB cases in the country are associated with HIV infection and TB is the major cause of death among HIV-infected patients3. Although TB/HIV co-infection is a major public health problem in Brazil, few studies have reported its occurrence and clinical outcomes during the HAART era6. Thus, the goal of this brief report is to describe the prevalence, incidence and survival of TB among HIV/AIDS patients from an HIV outpatient service located in the City of São Paulo, one of the areas most affected by HIV in Brazil.

MATERIAL AND METHODS

We retrospectively analyzed cases of active TB diagnosed between January 1995 and December 2010 in our cohort of HIV-infected patients. This open cohort was made up of HIV-1-infected subjects who had been followed for up to 16 years. We classified individuals as having active TB

if they had at least one positive culture for M. tuberculosis, a positive smear for alcohol-acid resistant bacilli, or if they started a TB treatment regimen. We defined the date of onset of TB as the date of the first positive smear or culture. If neither a smear nor culture result were available before a TB treatment regimen was initiated, the starting date of the treatment was considered to be the date of onset. All physicians providing care to patients were infectious disease specialists, and patients were seen at 4-6 month intervals. TB was treated in accordance with Brazilian guidelines for TB control2,7. We defined patients as ‘lost to follow up’ if they did not visit the clinic within a 6-month period and did not respond to three attempts of us contacting them by telephone and/or telegrams. PPD was performed as a routine screening at the beginning of the follow up; however, prophylaxis was not reported for most patients from this cohort. The study was approved by the Ethical Board (Cappesq number 0211/2010).

Statistical analysis: We analyzed data using Epidata software (Centers for Disease Control and Prevention, Atlanta, GA) and compared predictor variables among TB/HIV patients and those HIV patients without TB. For continuous variables, we used nonparametric Kruskal-Wallis tests, and for discrete variables we used the Fisher exact test. We calculated survival estimates by the Kaplan-Meier method and compared survival curves by the log-rank test using Stata 11.0 software (Stata Corporation, College Station, TX). A pH value lower than 0.05 was considered significant.

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CASSEB, J.; FONSECA, L.A.M.; MEDEIROS, L.A.; GONSALEZ, C.R.; LAGONEGRO, E.R.; VEIGA, A.P.R.; DA SILVA, D.C.; MENDONÇA, M. & DUARTE, A.J.S. - Tuberculosis among HIV-1-infected subjects in a tertiary out-patient service in São Paulo city, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 257-9, 2012.

258

RESULTS

Five hundred and ninety-nine patients were enrolled in the open cohort, and 41 (6.8%) met our case definition for TB. Twenty-nine (71%) were newly diagnosed with TB, twelve (29%) had known TB at the time of cohort entry, and most were on HAART. Thirty patients had pulmonary tuberculosis and eleven had extra-pulmonary disease (three patients had lymph node tuberculosis, two had intra-abdominal disease, two had disseminated tuberculosis, two neuro-tuberculosis, one had pericardial and another one had pleural disease). In general, patients with TB were younger, more likely to be male, and had higher viral loads (5.76 log

10

copies/mL vs. 4.65 log10

copies/mL) at the time of TB diagnosis than those without TB, although none of those characteristics has reached statistical significance (Table 1). Twenty-one TB patients were on HAART, but only three had undetectable viral loads. The remaining 20 patients were not on antiretrovirals. Among patients with other, non-TB opportunistic infections 59 had detectable viral loads, while 19 had undetectable levels.

Tuberculosis incidence was 1.47 cases per 100 person-years over a total cohort follow-up time of 2775 person-years. Comparing patients diagnosed with TB to those diagnosed with other AIDS-defining opportunistic infections, eight (19.5%) of 41 patients with TB were lost to follow up and six (14.6%) died, in comparison with eight deaths (4.8%) among 168 non-TB AIDS patients. All TB patients who died were on HAART, five of them with rescue regimens. Death rates were 2.17 per 100 person-year among TB patients and 0.9 per 100 person-years among non-TB AIDS patients. The probability of surviving up to 10 years after diagnosis was 75% for TB patients as opposed to 96% for patients with other, non-TB opportunistic infections (p = 0.03) (Fig. 1).

DISCUSSION

We found that 6.8% of HIV-infected patients in our cohort had TB on cohort entry or developed it during follow up, corresponding to an incidence of 1.47 cases per 100 person-year. TB is endemic in Brazil, especially in metropolitan areas where the living conditions and poverty of parts of the population facilitate the spread of airborne diseases. The relatively high incidence of TB in our study population of HIV-infected patients is similar to that found by other investigators studying poor, urban populations in Latin America2,6.

We also found that TB was an important cause of mortality in our study, with six confirmed deaths and eight lost to follow up, some or all of whom may have died from TB, although we were unable to ascertain this fact. This number, although substantially lower than the numbers found in several African countries where TB reaches up to 90% of prevalence among AIDS patients8, is nonetheless a cause for concern since the risk of dying from TB is more than twice that of dying from any other opportunistic disease9. Even so, confirmed mortality from TB was not as high in this cohort (1%) as it is in Africa and Asia, possibly due to the more widespread use of ART10, as well as to an earlier diagnosis of HIV infection11,12 . In this respect, we found that only slightly more than half of our TB/HIV patients were on HAART, and just three of them had undetectable viral loads, suggesting an unsatisfactory adherence to treatment.

Our findings provide evidence in support of the current Brazilian national tuberculosis treatment guidelines, which recommend an

Table 1Demographic, clinical and laboratory characteristics of TB patients compared to non-TB patients*. São Paulo, Brazil, 1995-2010

VariableTB/HIV cases

(n=41)Non-TB AIDS cases

(n=168)p value

Age (mean ±sd) years 41.2 (9.4) 43.4 (10.4) 0.5

Gender (% male) 78.0 72.6 0.6

Mean time of HIV infection to disease diagnosis (years) 5.96 2.44 0.01

Nadir of T CD4 cells/mm3 (Median, 25%-75%) 173 (73-249) 150 (58-239) 0.7

T CD4 cells/mm3 at disease diagnosis (Median, 25%-75%) 193 (66-404) 167 (178-344) 0.6

Proportion on HAART(%) 59 48 0.8

RNA HIV viral load log10

/mL 5.19 (4.03-5.7) 4.65 (4.1-5.3) 0.7

Deaths, n (%) 6 (15) 8 (5) 0.03

ns: Not statistically significant; * All comparisons between TB/HIV cases vs Non-TB AIDS cases.

Fig. 1 - Survival estimates of AIDS patients with tuberculosis and of AIDS patients with

other opportunistic diseases. São Paulo, Brazil, 1995-2010. Notes: Analysis time in days;

D = Patients with other opportunistic non-TB diseases; TB = Patients with tuberculosis; *

Log-rank test.

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CASSEB, J.; FONSECA, L.A.M.; MEDEIROS, L.A.; GONSALEZ, C.R.; LAGONEGRO, E.R.; VEIGA, A.P.R.; DA SILVA, D.C.; MENDONÇA, M. & DUARTE, A.J.S. - Tuberculosis among HIV-1-infected subjects in a tertiary out-patient service in São Paulo city, Brazil. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 257-9, 2012.

259

evaluation of HIV-infected patients for TB on entry into care and to treat those found to be infected regardless of clinical stage. In our cohort, all patients with clinical TB received a TB treatment regimen based on the then-current guidelines, which consisted of triple anti-TB therapy for six months (rifampin, isoniazid and pyrazinamide). More recently, the national treatment guidelines have been reviewed to include ethambutol in the treatment regimen and to provide drugs in a fixed-dose combination once daily for six months, free of charge2.

There are several limitations for this study. Firstly, this study was conducted in a tertiary care hospital, and patient care provided in our institution may not be representative of care in other types of clinical facilities in Brazil. Secondly, as this was an observational study, we were unable to control adherence, which could have led to an underestimation of the effectiveness of TB treatment in this population. Finally, much of the data regarding isoniazid prophylaxis was not systematically recorded, and so we were unable to determine the impact of this important intervention on TB incidence.

However, despite these limitations, we believe that our finding that TB is a major cause of morbidity and mortality is valid, despite the widespread use of ART in Brazil. This in turn suggests the need for additional interventions, such as the use of new diagnostic tools, INH prophylaxis and directly observed therapy to minimize the burden of HIV-related TB in Brazil. Thus the early diagnosis and treatment, either of TB and HIV infection, the identification of risk factors and the use of INH, should be improved in this population.

RESUMO

Tuberculose entre pacientes infectados pelo HIV-1 em ambulatório terciário de São Paulo, Brasil

Atualmente, a tuberculose (TB) é considerada a doença infecciosa mais importante entre os pacientes infectados pelo HIV-1 nos países em desenvolvimento, como o Brasil. Análise retrospectiva dos casos de tuberculose ocorridos a partir de janeiro 1995 até dezembro de 2010 foi realizada em nossa coorte de 599 pacientes HIV positivos. O desfecho primário foi a ocorrência de TB ativa, e 41 casos da doença foram diagnosticados durante este período de 16 anos. As contagens médias do nadir de células T CD4 e ao momento do diagnóstico de TB foram de 146 e 217 células/mm3, respectivamente. A carga viral média de HIV foi de 5,19 log

10 cópias/mL, e 59% dos pacientes estavam em tratamento

com ART. A incidência de TB foi de 1,47 casos por 100 pessoas-ano, para um tempo total de seguimento da coorte de 2775 pessoas-ano. A probabilidade de sobreviver até 10 anos após o diagnóstico foi de 75% para pacientes com TB, em oposição a 96% para pacientes com outras doenças oportunistas não-TB (p = 0,03). A tuberculose pode ser considerada problema de saúde pública entre as pessoas que vivem com HIV no Brasil, apesar da ampla utilização de anti-retrovirais para o tratamento da infecção pelo HIV / AIDS

REFERENCES

1. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Tuberculose no Brasil: realidade e perspectivas. Boletim 1/2012 Tuberculose no Brasil. Available from: http://portalsaude.saude.gov.br/portalsaude/index.cfm?portal=pagina.visualizarTexto&codConteudo=6406&codModuloArea=783&chamada=boletim-1/2012-_-tuberculose-no-brasil

2. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Programa Nacional de Controle da Tuberculose. Manual de recomendações para o controle da tuberculose no Brasil. 2010. Available from: http://portal.saude.gov.br/portal/arquivos/pdf/manual_de_recomendacoes_controle_tb_novo.pdf

3. Casseb J, Fonseca LA, Veiga AP, de Almeida A, Bueno A, Ferez AC, et al. AIDS incidence and mortality in a hospital-based cohort of HIV-1-seropositive patients receiving highly active antiretroviral therapy in São Paulo, Brazil. AIDS Patient Care STDS. 2003;17:447-52.

4. Corbett EL, Marston B, Churchyard GJ, De Cock KM. Tuberculosis in sub-Saharan Africa: opportunities, challenges, and change in the era of antiretroviral treatment. Lancet. 2006;367:926-37.

5. Golub JE, Saraceni V, Cavalcante SC, Pacheco AG, Moulton LH, King BS, et al. The impact of antiretroviral therapy and isoniazid preventive therapy on tuberculosis incidence in HIV-infected patients in Rio de Janeiro, Brazil. AIDS. 2007;21:1441-8.

6. Granich R, Akolo C, Gunneberg C, Getahun H, Williams P, Williams B. Prevention of tuberculosis in people living with HIV. Clin Infect Dis. 2010;50(Suppl 3):S215-22.

7. Gutierrez EB, Gomes V, Picone CM, Suga H, Atomiya AN. Active tuberculosis and Mycobacterium tuberculosis latent infection in patients with HIV/AIDS. HIV Med. 2009;10:564-72.

8. Pacheco AG, Saraceni V, Tuboi SH, Lauria LM, Moulton LH, Faulhaber JC, et al. Estimating the extent of underreporting of mortality among HIV-infected individuals in Rio de Janeiro, Brazil. AIDS Res Hum Retroviruses. 2011;27:25-8.

9. Pettit AC, Barkanic G, Stinnette S, Rebeiro P, Blackwell R, Raffanti SP, et al. Potentially preventable tuberculosis among HIV-infected persons in the era of highly active antiretroviral treatment. Int J Tuberc Lung Dis. 2009;13:355-9.

10. Santoro-Lopes G, de Pinho AM, Harrison LH, Schechter M. Reduced risk of tuberculosis among Brazilian patients with advanced human immunodeficiency virus infection treated with highly active antiretroviral therapy. Clin Infect Dis. 2002;34:543-6.

11. Saraceni V, King BS, Cavalcante SC, Golub JE, Lauria LM, Moulton LH, et al. Tuberculosis as primary cause of death among AIDS cases in Rio de Janeiro, Brazil. Int J Tuberc Lung Dis. 2008;12:769-72.

12. Whalen C, Horsburgh CR Jr, Hom D, Lahart C, Simberkoff M, Ellner J. Site of disease and opportunistic infection predict survival in HIV-associated tuberculosis. AIDS 1997;11:455-60.

13. World Health Organization. Global tuberculosis control: a short update to the 2009 report. [Cited: 2010 Nov 3]. Available from: http://www.who.int/tb/publications/global_report/2009/update/en/index.html

Received: 7 November 2011Accepted: 3 July 2012

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LIBRARY OF THE

SÃO PAULO INSTITUTE OF TROPICAL MEDICINE

Website: www.imt.usp.br/portal Address: Biblioteca do Instituto de Medicina Tropical de São Paulo da Universidade de São Paulo

Av. Dr. Enéas de Carvalho Aguiar, 470. Prédio 1 – Andar térreo.05403-000 São Paulo, SP, Brazil.

Telephone: 5511 3061-7003 - Fax: 5511 3062-2174

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. To reach this objective, we select and acquire by donation and / or exchange appropriate material to be used by researchers and we maintain interchange between Institutions thorough the Journal of the São Paulo Institute of Tropical Medicine, since the Library has no funds to build its own patrimony.

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

The collection fo journals existing in the Library can be verified through the USP – Bibliographic Database – OPAC – DEDALUS http://dedalus.usp.br:4500/ALEPH/eng/USP/USP/DEDALUS/start of the USP network.

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Rev. Inst. Med. Trop. Sao Paulo54(5):261-266, September-October, 2012doi: 10.1590/S0036-46652012000500005

(1) Departamento de Engenharia Mecânica. Faculdade de Engenharia de Guaratinguetá, UNESP, Guaratinguetá, SP, Brazil. (2) Departamento de Medicina, Universidade de Taubaté. Taubaté, SP, Brazil.(3) Programa de Pós-Graduação em Ciências Ambientais. Universidade de Taubaté. Correspondence to: Luiz Fernando Costa Nascimento, Rua Durval Rocha 500, Vila Paraíba, 12515-710 Guaratinguetá, SP, Brasil. Tel.: +55.12.3125-1103. E-mail: [email protected]

SPATIAL DISTRIBUTION OF DENgUE IN THE CITY OF CRUZEIRO, SÃO PAULO STATE, BRAZIL: USE OF gEOPROCESSINg TOOLS

Renata Marzzano de CARVALHO(1) & Luiz Fernando Costa NASCIMENTO(2,3)

SUMMARY

The aim of this article is to identify patterns in spatial distribution of cases of dengue fever that occurred in the municipality of Cruzeiro, State of São Paulo, in 2006. This is an ecological and exploratory study using the tools of spatial analysis in the preparation of thematic maps with data from Sinan-Net. An analysis was made by area, taking as unit the IBGE census, the analysis included four months in 2006 which show the occurrence of the disease in the city. The thematic maps were constructed by TerraView 3.3.1 software, the same software provided the values of the indicators of Global Moran (I

M) every month and the Kernel estimation. In

the year 2006, 691 cases of dengue were georeferenced (with a rate of 864.2 cases/100,000 inhabitants); the indicators of Moran and p-values obtained were I

M = 0.080 (March) p = 0.11; I

M = 0.285 (April) p = 0.01; I

M = 0.201 (May) p = 0.01 and I

M = 0.002 (June) p

= 0.57. The first cases were identified in the Northeast and Central areas of Cruzeiro and the recent cases, in the North, Northeast and Central. It was possible to identify census tracts where the epidemic began and how it occurred temporally and spatially in the city.

KEYWORDS: Dengue; Geographic Information Systems; Epidemiologic surveillance; Ecological studies; Spatial distribution of population.

INTRODUCTION

The National Department of Health warns: “Dengue is everyone’s problem. Combating it is our social obligation, and everyone should do it every day. Embrace this commitment and do your part”.

In the 1980s, new cases of dengue were reported in Brazil16, and in the São Paulo State, dengue was included in the list of diseases of compulsory notification in 19865. In 2000, the infestation of Ae. aegypti was detected in the Paraiba Valley, SP, where dengue cases began to be recorded in 2002 in the municipality of Potim, SP20, and where, also, overlap Ae. Albopictus was observed. The same occurred in São Sebastião, in the Paraíba Valley, north coast of São Paulo; this area was affected by a dengue epidemic that began in 2002. The number of cases in the city reached 972 autochthonous contamination18.

The introduction of DEN-1 serotype in São Paulo State occurred in 1987; the DEN-2 and DEN-3 serotypes occurred in 1997 and 2003 respectively; more recently serotype DEN-417 has been diagnosed. Fifty thousand and twenty-two cases were confirmed in 2006 in the São Paulo State (incidence close to 125 cases/100,000 inhabitants)19 and all the cases confirmed in Paraíba Valley were the cases occurred in Cruzeiro.

According to SOUZA-SANTOS & CARVALHO, georeferencing and spatial analysis techniques have been increasingly used to evaluate

the distribution of vectors and the diseases they transmit, contributing to the improvement of surveillance and vector controlling actions. Thus, the use of these tools enables the identification of risk areas that require intensification and/or prioritization of control measures21.

Its greatest advantage lies in treating the city as a place composed of various realities, deserving distinct approaches, contrary to what has been seen, in which the proposed action may differ according to the municipalities, but are always uniform everywhere21.

The use of spatial analysis techniques in a study performed in a city of the central region of São Paulo enabled the visualization of areas of clusters of containers with larvae, indicating that this method can be used to improve entomological surveillance activities2. In Niteroi, RJ, a similar strategy was used to identify geographic areas with the highest number of cases8.

The aim of this study is to identify patterns in the spatial and temporal distribution of dengue cases in the city of Cruzeiro in March, April, May and June of 2006 using geoprocessing tools.

METHODS

We outlined an epidemiological study of the ecological and exploratory type. Dengue cases were confirmed by laboratory or

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CARVALHO, R.M. & NASCIMENTO, L.F.C. - Spatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 261-6, 2012.

262

clinical-epidemiological criteria, according to guidelines of the National Department of Health13. Information about dengue cases was provided by the technical staff of the Center for Vector Control of the municipality, grouped by month and year of occurrence.

There are 80,000 inhabitants in Cruzeiro and the proportion between genders9 is roughly even. Cruzeiro is situated at the eastern end of São Paulo, in the Paraíba Valley, latitude 22° 33’ South and longitude 44º 60’ West, at an altitude of 514 meters and covers an area of 331 km², of which 36 km² are of urban area7. The most relevant economical activities are the industry sector, services and commerce; industry is the sector that employs the most (48.8%), especially the metallurgical industries7.

Ninety seven percent of households in Cruzeiro have running water, 94% of households are connected to the sewage system and 97% of the households have regular garbage collection. The local human development index was 0.81 for the year 2000.

Its territorial divisions are: to the north with the municipalities of Passa Quatro and Marmelópolis, Minas Gerais State; to the south with the municipality of Silveiras; to the East with the municipality of Lavrinhas and to the West with the municipalities of Piquete and Cachoeira Paulista, São Paulo State (Fig. 1). The weather is tropical (hot and humid), with temperatures ranging between 20 and 27 Celsius degrees. The hottest months are between November and April and the coldest, between May and October. The rains are more frequent from December to March, with January being the rainiest month. The driest period lasts from June to September. The municipality was built on the margins of the Paraíba do Sul River, which, cuts the city in half, and into which the sanitary sewage is dumped with no treatment.

The cases were investigated in Cruzeiro, according to the date of appearance of first symptoms, determining address and occurrence in the city map according to the 96 urban census tracts existing in 2000, supplied by IBGE, in order to determine the location and date of the start of the epidemic.

From a cartographic database in digital format, obtained from IBGE, georeferenced in the Cartesian system, containing designs of the city’s census tracts, we inserted the confirmed cases of dengue fever which occurred in the municipality and mapped the incidence of dengue in 2006.

To identify the presence of spatial autocorrelation we used indicators of global Moran (I

M)3 which analyze if there are clusters of sectors with

higher rates next to clusters of low rates. The IM

varies between -1 and +1 and to it can be assigned a level of significance (p-value). The closer to 1 (one), the most similarity between neighbors. The value 0 (zero) indicates no correlation, and negative values indicate dissimilarity14. We used the public domain program TerraView (available at: http:// www.inpe.gov.br).

The expression of Moran’s coefficient is:

In this equation we have n corresponding to the number of areas (sectors), w

(i,j) is equal to the relevance of the neighborhood, X

(i) represents

the square root of the average incidence rate for municipality i in the studied periods and X refers to the mean of X

(i) for the entire studied region.

Fig. 1 - Municipalities of Paraíba Valley, 2006.

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CARVALHO, R.M. & NASCIMENTO, L.F.C. - Spatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 261-6, 2012.

263

Among the procedures for estimating density of events, the kernel estimator is the most widely used of them and plays an important role in the epidemiological context to identify the concentration of cases and identify the hot spots4. The kernel estimator is an exploratory interpolation technique that generates a surface density for visual identification of hot spots, and is best used in the analysis by points, but also used in the analysis by area, when it assigns the center of the study area, the location of all events. The kernel estimator produces a continuous density surface calculated in all locations, the researcher does not need to define where to arbitrarily “cut” a “hot area” 12.

Thematic maps were created in this study with the kernel estimator, with dengue cases referring to the months of March, April, May and June of 2006.

RESULTS

Seven hundred and two cases of dengue fever were reported and confirmed in 2006. Six hundred ninety-one cases (98.4%) were georeferenced (with a rate of 864.2 cases/100,000 inhabitants).

Thematic maps with the distribution of cases (Fig. 2), by the months of 2006, show that the first cases occurred in March, in the North, Northeast and central region, on the banks of Paraíba do Sul River and its tributary, Barrinha stream, also following the path of the railroad that crosses the central region of the city and the road linking the city of Cruzeiro to Lavrinhas. In April, there is the emergence of cases in almost all the census tracts and moving to the south, southeast, central region and northeast. In May, 76 (79%) census tracts had at least one case of dengue, with the largest number of cases in the central, south, southeast and east regions, following the course of the Paraíba do Sul River and Barrinha stream. In June, only two sectors showed three cases, located in the central and south regions.

Moran Indicators and obtained p-values are in Table 1; we can see positive spatial autocorrelation in all the months, but with statistical significance in the months of April and May. The number of cases per month is in Table 1, and the increase in occurrences can be seen in the months of April and May (p < 0.05).

The kernel estimator showed the highest densities of cases in the

Fig. 2 - Dengue cases in according to months; (a) March, (b) April, (c) May and (d) June, Cruzeiro, SP, 2006.

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CARVALHO, R.M. & NASCIMENTO, L.F.C. - Spatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 261-6, 2012.

264

month of March in central and north regions; in the months of April and May, the highest densities occurred in central and south regions; and in June, the highest densities occurred in central and west (Fig. 3).

DISCUSSION

This is the first study carried out in a municipality of the Paraíba Valley, using the techniques of geoprocessing to study the spatial-temporal distribution of the dengue epidemic, making it possible to identify a pattern of the epidemic. The infestation of Aedes aegypti in the Paraiba Valley began in the city of Potim, around the year 2000. Since 1996, the

municipalities already had the plans for the Eradication of Aedes aegypti (PEAa) instituted by the Ministry of Health, performing controlling actions of mosquito infestations, disease prevention, which included the monitoring of traps and strategic places, home visits, and collection of scrap materials. Throughout the process of implementation of this program the mosquito eradication technique was not feasible in the short to medium term. It was necessary to intensify actions to better address the problem and reduce the impact of dengue in Brazil. This plan was abandoned and in 2002 the Plano Nacional de Controle da Dengue-PNCD came into effect.

The epidemic started in the central region, spread to almost all regions of the urban area, following the margins of the Paraíba do Sul river, railroads and roads which cross the municipality and are responsible for the vast movement of people, products and vehicles.

From June there was a decrease in new cases, possibly due to the decrease in the number of susceptible individuals in the region. The municipality intensified vector controlling activities during this period which possibly led to a decrease in the number of breeding. In June, the registered temperatures were milder and humidity is the lowest. This fact strongly suggests that the adverse weather conditions for the proliferation of the vector play an important role in ending the transmission of the disease in the city2.

Table 1 Moran index (I

M) and p-values of spatial distribution of dengue cases,

Cruzeiro, 2006

Months IM

p-value Cases

March 0.080 0.11 35

April 0.285 0.01 258

May 0.201 0.01 271

June 0.002 0.57 15

Fig. 3 - Kernel estimators to dengue cases in according to months; (a) March, (b) April, (c) May and (d) June, Cruzeiro, SP, 2006.

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CARVALHO, R.M. & NASCIMENTO, L.F.C. - Spatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 261-6, 2012.

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Just one type of virus circulated in the city (the DEN-3 serotype), which was confirmed by viral isolation performed during the occurrence of the epidemic and, therefore, people became immune after exposure to the virus.

Three factors must be considered in the decrease of the number of cases of an epidemic: the decrease of susceptible individuals; vector controlling interventions and unfavorable natural conditions, such as the reduction of temperature and humidity, reducing the vector population.

The occurrence of dengue was also identified in areas of high concentration of people, great economic diversity, high population density, high residential density. These findings were similar to those found in Niteroi8.

The accelerated growth of the urban population provides a great source of infected and susceptible individuals, concentrated in restricted areas. This fact, associated with poor sanitation conditions, housing and specific cultural factors of large urban centers, provides ecological conditions favor to the transmission of dengue virus11.

The Moran Indicators obtained during March, April and May showed positive values, indicative of positive spatial autocorrelation. The values tend to be similar to neighboring sectors, indicating that the occurrence of dengue cases in the city is connected, there being a strong relationship between the sectors, and is even stronger among the sectors, closest neighbors. The index showed significant positive spatial correlation for the months of April and May 2006.

The kernel estimator presented a hot spot in the central area in March, while in the following months the highest concentration of cases spread to other non-central areas of the city, probably due to decrease of the number of susceptible individuals in these sectors, and also due to late diagnosis and the natural development of the disease. The central area should be prioritized in the planning of actions in order to control Aedes aegypti breeding sites and disease prevention.

The measures of combating dengue have been based only in the chemical vector control at the expense of important factors that modulate the dynamics of disease transmission, especially those related to social determinants, such as spatial occupation and living conditions. Thus, for each introduction of a new serotype a new epidemic occurs8.

Therefore, it is understandable that cities with larger proportion of urban population can present higher incidence of dengue, because urban growth provides an increase in the number of infected and susceptible individuals concentrated in restricted areas, as shown in a study conducted in São José do Rio Preto6.

It is important that the spatial relationship between dengue transmission and other variables is checked, as the degree of immunity of population, effectiveness of controlling measures, the degree of infestation by the vector, the habits and attitudes of the population, among others. Note that studies with approaches of spatial locations and use of SIG, although recent in the health area, gradually become indispensable for the analysis of the determination of diseases8.

Some limitations of this ecological study can be attributed to working

with secondary data developed for the interest of surveillance and not for research, hindering the recovery of some lost information2. In a study conducted in São José do Rio Preto, out of 14,431 autochthonous cases of dengue occurred in the urban area between September 1994 and August 2002, 433 cases (3%) were not goaded due to lack of or inadequate address of the address with the cartographic basis15.

During the epidemical periods there were changes in staff, as well as in the coordination of activities, causing disruption of routine actions.

Therefore, analysis considering patient’s address as the site of exposure to the risk factors assume an immobility of individuals and ignore any possibility of exposure in other places, such as working or leisure environment. But we cannot say where these people contracted the disease, because it may have been in their own homes, but also may have been at work, in transit or elsewhere in the city.

As ALMEIDA et al. note1, despite the guidelines of the epidemiological surveillance of dengue in investigating the probable sites of infection considering residence, work and in transit in the investigation form standardized by the National Department of Health, only data concerning the residence address are structured to allow the precise location of a case.

As possible sources of error we can consider the clinical-epidemiological character of the criteria for completion of the cases in the municipality with fewer than 100,000 inhabitants, after exceeding the limit of 300 confirmed cases the confirmation of the same is made by clinical and epidemiological criterion rather than through a blood test. This condition is used by the Secretary of Health of the State of São Paulo to stop serological tests (Center for Epidemiological Surveillance: unpublished data), the possible underreporting of people who are affected by the milder form of the disease and who may not seek a health unit, and therefore are not reported; other limitations are indeterminate diagnoses and addresses that were not found. Difficulties found in the georeferencing of the reported cases of disease, due to address problems also occurred in other studies1,15 and prevented the location of cases in census tracts.

Although there are the limitations outlined above, this study indicates that a disease like dengue, with vector transmission, whose mosquito usually has a small displacement during its life cycle, the punctual location of the cases or the aggregation of information in small areas such as the census tracts, can be quite enlightening10.

Thus, it was possible, using the technique of geoprocessing, to map the location of housing for those people affected by dengue epidemic that ravaged the city of Cruzeiro in 2006 and show how the disease spread through the city.

The present study responded to the proposed objective, identifying patterns in the spatial and temporal distribution of dengue cases in the city of Cruzeiro during the months of March, April, May and June of 2006, analyzing the information obtained via the Dengue Control Program of the São Paulo State using geoprocessing tools. This information may support health authorities to propose strategies for prevention and control of dengue cases in the city and prevent the outbreak of another epidemic in the coming years.

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CARVALHO, R.M. & NASCIMENTO, L.F.C. - Spatial distribution of dengue in the city of Cruzeiro, São Paulo State, Brazil: use of geoprocessing tools. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 261-6, 2012.

266

RESUMO

Distribuição espacial da dengue no município de Cruzeiro, Estado de São Paulo, Brasil: o uso do geoprocessamento

Este artigo tem por objetivo identificar padrões na distribuição espacial dos casos de dengue ocorridos no município de Cruzeiro/SP, no ano de 2006. Trata-se de um estudo ecológico e exploratório que utiliza as ferramentas de análise espacial na elaboração de mapas temáticos, com dados obtidos do SinanNet. Foi feita uma análise por área, tomando-se como unidade o setor censitário do IBGE; a análise considerou quatro meses do ano de 2006 que mostra a ocorrência da doença no município. Os mapas temáticos foram construídos pelo programa computacional TerraView 3.3.1; assim como os valores dos índices de Moran Global (I

M) mês a mês e o estimador de Kernel. Foram georreferenciados 691

casos de dengue (taxa de 864,2 casos/100.000 habitantes); os Índices de Moran e p-valores obtidos foram I

M = 0,080 (março) p = 0,11; I

M =

0,285 (abril) p = 0,01; IM

= 0,201 (maio) p = 0,01 e IM

= 0,002 (junho) p = 0,57. Os primeiros casos foram identificados na região nordeste e central e os últimos casos, na região norte, nordeste e central. Foi possível identificar os setores censitários onde a epidemia teve início e como ocorreu têmporo-espacialmente no município.

REFERENCES

1. Almeida MC, Assunção RM, Proietti FA, Caiaffa WT. Dinâmica intra-urbana das epidemias de dengue em Belo Horizonte, Minas Gerais, Brasil, 1996-2002. Cad Saúde Pública. 2008;24:2385-95.

2. Barbosa GL, Lourenço RW. Análise da distribuição espaço-temporal de dengue e da infestação larvária no município de Tupã, Estado de São Paulo. Rev Soc Bras Med Trop. 2010;43:145-51.

3. Barcellos C, Santos SM. Colocando dados no mapa: a escolha da unidade de agregação e integração de bases de dados em saúde e ambiente através do geoprocessamento. Inf Epidemiol SUS. 1997;6:21-9.

4. Carvalho MS, Pina MF, Santos S. Conceitos básicos de sistemas de informação geográfica e cartografia aplicados à saúde. Brasília: Organização Pan-Americana da Saúde/Ministério da Saúde; 2000.

5. Costa AIP. Identificação de unidades ambientais urbanas como condicionantes da ocorrência de Aedes aegypti (Diptera Culicidae) e de dengue na cidade de São José do Rio Preto, S.P., em 1995. [Dissertação]. São Paulo: Universidade de São Paulo, Faculdade de Saúde Pública; 1996.

6. Costa AIP, Natal D. Distribuição espacial da dengue e determinantes socioeconômicos em localidade urbana no Sudeste do Brasil. Rev Saúde Pública. 1998;32:232-6.

7. Cruzeiro. Prefeitura Municipal. Plano Municipal de Saúde 2010-2013. 49p.

8. Flauzino RF, Souza-Santos R, Oliveira RM. Dengue, geoprocessamento e indicadores socioeconômicos e ambientais: um estudo de revisão. Rev Panam Salud Publica. 2009;25:456-61.

9. Fundação Instituto Brasileiro de Geografia e Estatística. IBGE. Planejamento. Ministério do Planejamento, Orçamento e Gestão. Cidades. Rio de Janeiro, FIBGE, 2000. Available from: http://www.ibge.gov.br/cidadesat/topwindow.htm?1

10. Galli B, Chiaravalloti Neto F. Modelo de risco tempo-espacial para identificação de áreas de risco para ocorrência de dengue. Rev Saúde Pública. 2008;42:656-63.

11. Gubler DJ, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis. 1995;1:55-7.

12. Levine, N. CrimeStat. II: a spatial statistics program for the analysis of crime incident locations. Ned Levine & Associates, Houston, TX, and the National Institute of Justice. Washington, DC, 2002. Available from: http://www.spatial.cs.umn.edu/Courses/Fall07/8715/papers/IM10_levine.pdf

13. Ministério da Saúde. Fundação Nacional de Saúde. Manual de dengue: vigilância epidemiológica e atenção ao doente. Brasília: FNS; 1995.

14. Ministério da Saúde. Secretaria de Vigilância em Saúde. Fundação Oswaldo Cruz. Introdução à estatística espacial para a saúde pública. Brasília; 2007. (Série Capacitação e Atualização em Geoprocessamento em Saúde, v. 3).

15. Mondini A, Chiaravalloti Neto F, Gallo Y, Sanches M, Lopes JCC. Análise espacial da transmissão de dengue em cidade de porte médio do interior paulista. Rev Saúde Pública. 2005;39:444-51.

16. Pontes RJS, Ruffino-Netto A. Dengue em localidade urbana da região sudeste do Brasil: aspectos epidemiológicos. Rev Saúde Pública. 1994;28:218-27.

17. Secretaria da Saúde. Centro de Vigilância Epidemiológica. Dengue. São Paulo; 2011. Available from: http://www.cve.saude.sp.gov.br/htm/zoo/if_sem_den02.htm

18. Secretaria de Saúde. Centro de Vigilância Epidemiológica. Dengue. São Paulo, 2011. Available from: http://www.saude.cve.sp.gov.br/htm/cve_dengue.html

19. Secretaria de Saúde. Centro de Vigilância Epidemiológica. Dengue. São Paulo, 2011. Available from: http://www.saude.cve.sp.gov.br/htm/zoo/Den_dir06.htm

20. Serpa LLN, Costa KVRM, Voltolini JC, Kakitani I. Variação sazonal de Aedes aegypti e Aedes albopictus no município de Potim, São Paulo. Rev Saúde Pública. 2006;40:1101-5.

21. Souza-Santos R, Carvalho MS. Análise da distribuição espacial de larvas de Aedes aegypti na Ilha do Governador, Rio de Janeiro, Brasil. Cad Saúde Pública. 2000;16:31-42.

Received: 15 September 2011Accepted: 11 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):267-271, September-October, 2012doi: 10.1590/S0036-46652012000500006

Supervisão de Vigilância em Saúde, SUVIS Casa Verde, Secretaria de Saúde da Cidade de São Paulo, SP, Brazil.Correspondence to: Josué de Moraes, Supervisão de Vigilância em Saúde, SUVIS Casa Verde, Rua Ferreira de Almeida 73, 02517-150 São Paulo, SP, Brasil; Fax: +55 11 39317092; Phone:

+55 11 39317092 R. 25. E-mail: [email protected], [email protected].

CONTAMINATION OF PUBLIC PARKS AND SQUARES FROM gUARULHOS (SÃO PAULO STATE, BRAZIL) BY Toxocara spp. AND Ancylostoma spp.

Jacó Pereira MARQUES, Catarina de Rezende GUIMARÃES, Ailton VILAS BOAS, Paulo Usignolo CARNAÚBA & Josué de MORAES

SUMMARY

The contaminated soil with mammal feces is an important factor of risk to infection with zoonotic diseases. Amongst these zoonoses are visceral larva migrans and cutaneous larva migrans caused by Toxocara spp. and Ancylostoma spp., respectively. The aim of this study was to assess the environmental contamination by Toxocara spp. eggs and hookworms (Ancylostoma spp.) in public parks and squares in the city of Guarulhos, a metropolitan area of São Paulo, São Paulo State, Brazil. Soil samples were collected, between September and December 2010, and examined using the centrifugal flotation technique with sodium dichromate and zinc sulphate as well as the modified Baermann method. Notably, 35 (74.5%) of the 47 districts surveyed in Guarulhos possessed samples contaminated with Toxocara spp. and/or eggs or larvae of Ancylostoma spp. The frequency of Toxocara spp. and Ancylostoma spp. in the samples from public areas was 68.1% and 46.8%, respectively. Overall, the eastern side of Guarulhos is the region with the highest occurrence of causative agents of larva migrans. In all collection sites, the presence of feces from dogs and cats accompanied by their owners and stray animals were observed. Notably, it is important to adopt measures to control dog and cat breeding, to treat infected animals, and provide health education to the population.

KEYWORDS: Larva migrans; Toxocara spp.; Ancylostoma spp.; Soil contamination; Guarulhos.

INTRODUCTION

The presence of mammal feces in the soil of urban areas is an important public health problem in several countries, mainly due to the presence of parasites that can cause larva migrans syndrome in humans. There are traditionally acknowledged zoonotic diseases, such as visceral larva migrans caused by Toxocara spp. and cutaneous larva migrans caused by Ancylostoma spp. These parasitic zoonoses are associated with the presence of animals, mainly dogs and cats in places where humans can become infected, such as squares and public parks12,17,18,37 .

The growing number of domestic animals, especially in large urban centers, has increased the contact between animals and humans and heightened exposure risk to parasites responsible for zoonosis. The invasion of the human body by nematode parasites of other animals is an atypical development of the parasitic species involved, which are unable to complete its life cycle. Under these conditions, it is observed that the infective larval form cannot progress to the adult worm, and its abnormal migration in the host stops in different tissues such as skin, eye, liver, lungs or other organs12,17,18. Nematodes that penetrate through the skin but still wander between the epidermis and dermis, resulting in clinical cutaneous larva migrans12,18, whereas nematodes that enter the body orally and should experience the typical pulmonary

cycle after reaching the digestive tract are likely to finally reside in the liver or lungs or other organs, causing the clinical syndrome visceral larva migrans17.

The parasites causing larva migrans syndrome persist everywhere there are dogs and cats infected with nematodes, especially Ancylostoma spp. and Toxocara spp. Given the ubiquity of dogs and cats, which is exacerbated in cities by human population density, pets and stray animals, the control of parasitism is very difficult17,26,37. In fact, in many cities, large population of animals can circulate freely through the streets and public squares that may be taken by the owners. In these places, the animals defecate, contaminating the environment with eggs or larvae of parasites, which favors zoonotic transmission.

Larva migrans syndromes, especially toxocariasis and hookworm infections, are amongst the most prevalent worldwide, including industrialized countries, but these diseases remain unknown by the population18,44. Environmental contamination with helminth eggs is common in urban public places in most countries. For example, the percentage of soil contaminated by Toxocara spp. eggs has ranged between 12% and 60.3% in Brazil4,21,37,43, 14.4% and 20.6% in the United States of America, 13.0% and 87.1% in Europe, 6.6% and 63.3% in Asia and 30.3% and 54.5% in Africa37.

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MARQUES, J.P.; GUIMARÃES, C.R.; VILAS BOAS, A.; CARNAÚBA, P.U. & MORAES, J. - Contamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by Toxocara spp. and Ancylostoma spp. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 267-71, 2012.

268

Despite the high incidence of larva migrans syndromes in various regions, few studies have assessed soil contamination in large urban centers3,10,14,15,18,37,39. For example, in Guarulhos, a metropolitan area of São Paulo (São Paulo State, Brazil), there is little analysis of soil contamination by helminths that cause zoonoses. Thus, the objective of this study was to analyze soil samples collected from public parks and squares of the city of Guarulhos. Guarulhos is the second largest city in São Paulo, with a population of approximately 1.3 million people distributed in its 47 districts. Currently, Guarulhos is the 8th richest city in Brazil with a production that represents more than 1% of Gross Domestic Product (GDP)32. Over the last decades, several population groups have moved to Guarulhos in search of employment opportunities and housing. Influx of new residents led to erratic construction of an environment marked by strong demand, particularly in the health sector. The diversity of squares and parks provide leisure options for the population.

MATERIAL AND METHODS

Guarulhos is one of 39 municipalities that is in the metropolitan area of São Paulo (São Paulo State, Brazil) and is located 17 km from the capital in the northeast. The city is strategically located between two major national highways, Presidente Dutra Highway and Fernão Dias, the main axis of development of the country (São Paulo/Rio de Janeiro and São Paulo/Minas Gerais). Guarulhos has favorable environmental conditions, such as humid subtropical climate, mean annual temperature of 19 °C, relative humidity average of 81.1% and average annual rainfall of 1470 mm to support the physical expansion of parasitic diseases27,32.

From September to December 2010, soil samples from 120 public parks and squares distributed within 47 districts of Guarulhos were examined. Each sample consisted of approximately 250 grams of soil collected by scraping with a metal spoon the soil surface to a 5 to 10 cm depth at five different points per site5,8,24. After mixing the five collected samples, the pooled samples analyzed on the same day as collected by the centrifugal flotation technique with sodium dichromate (d = 1.35) and zinc sulphate (d = 1.20) and the modified Baermann method9,38. The identification of larvae and eggs of parasites causing larva migrans, Toxocara spp. and Ancylostoma spp., was performed based on morphology using a light microscope.

RESULTS

Notably, 35 (74.5%) of the 47 districts surveyed in Guarulhos possessed samples contaminated with Toxocara spp. and/or eggs or larvae of Ancylostoma spp. (Fig. 1).

The frequency of Toxocara spp. and Ancylostoma spp. in soil samples from public parks and squares in Guarulhos was 68.1% and 46.8%, respectively, whereas concomitant occurrence of these two nematodes was recorded in 19 districts. Moreover, in general, districts in the eastern part of the city were those with the highest prevalence of Toxocara spp. and Ancylostoma spp., whereas the contamination was lower in the central region (Fig. 1). Overall, regarding the Toxocara eggs in soil samples, the light microscopic investigations showed the presence of undeveloped and embryonated eggs.

In the public areas of all districts, the presence of stray dogs and cats and often-domiciled animals, which were accompanied by their owners,

was noted. Additionally, we detected the presence of dog and cat feces scattered across several streets, squares and parks of the city.

DISCUSSION

The soils of squares and public parks are the main transmission route of parasitic zoonoses to the human population. Amongst these zoonoses are visceral larva migrans and cutaneous larva migrans caused by Toxocara spp. and Ancylostoma spp., respectively2,12,17,18. Studies on the prevalence of parasites causing larva migrans have been conducted in various regions of the world, especially by either stool examinations of dogs and cats, or soil contamination assessments for helminth eggs and larvae2,4,20,26. In general, many authors have sought to assess the degree of soil contamination in public places by measuring Toxocara spp. levels and are less interested in environmental contamination by eggs or larvae of Ancylostoma spp.11. This is mainly because human toxocariasis ranks among the most common zoonotic infections worldwide, even found in industrialized and developing countries, with an important clinical expression18,37.

Eggs and larvae of Toxocara spp. and Ancylostoma spp. have been found in soil samples worldwide. They are present in public and private

Fig. 1 - Contaminated regions of Guarulhos, São Paulo, Brazil with egg and/or larvae of

Toxocara spp. and Ancylostoma spp. (September to December, 2010).

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MARQUES, J.P.; GUIMARÃES, C.R.; VILAS BOAS, A.; CARNAÚBA, P.U. & MORAES, J. - Contamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by Toxocara spp. and Ancylostoma spp. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 267-71, 2012.

269

places such as playgrounds, parks, sandpits, pavements, streets, gardens and fields. In the present study there was a high frequency (74.5%) of Ancylostoma spp. and Toxocara spp. in the 47 districts in Guarulhos. This high rate is further justified by the recovery of Toxocara spp. eggs (68.1%) than hookworms (46.8%). This percentage rate is markedly higher than the figures published by other researchers in different regions of the world1,10,13,14,15,22,24,25. For example, the frequency of Toxocara ssp. observed in Guarulhos was higher than that found in Bolivar, Venezuela (55%)13, Kaduna, Nigeria (50.4%)22, Prague, Czech Republic (20.4%)14, Sapporo, Japan (7.47%)25 and London, United Kingdom (6.3%)15 as well as in different regions of Poland (2-37%)26 and Spain (3.8-52%)10,24. However, the level of soil contamination in Guarulhos was similar to the 67% recorded in Murcia (Spain) by RUIZ DE YBÁÑEZ et al. (2001)39 and the 62.5% in Kirikkale (Turkey) by AYDENIZÖZ OZKAYHAN (2006)1. Concerning the Brazilian cities, in general, the degree of contaminated places in the present study by Toxocara spp. is higher than many cities, where studies found that soil contamination by Toxocara spp. varied from 12 to 60.3%4,21,37,43. Indeed, for example, in Moreno (Pernambuco State), LIMA et al. (2005)21 found contamination with Toxocara spp. eggs in 12% of the soil samples, whereas TIYO et al. (2008)43 reported the occurrence of Toxocara spp. eggs in 60.3% of the soil samples from public lawns of the town of Maringá, Paraná State. Furthermore, in the State of São Paulo, the frequency of Toxocara spp. observed in Guarulhos was higher than that found in Sorocaba (53.5%)7, Fernandópolis (20.89%)5, Ribeirão Preto (20.5%)3 and Botucatu (17.5%)40.

With regards to soil samples with eggs and larvae of Ancylostoma spp., the frequency in this study (46.8%) was higher than the cities of some countries, such as Bogotá, Colombia (10.7%)31, Kaduna, Nigeria (9%)22, Wrocław, Poland (4.9%)30, Madrid, Spain (3%)10 and different regions of Costa Rica (2%)29. Concerning the Brazilian cities, our results with hookworms in Guarulhos were similar to the recorded in Araçatuba (São Paulo State) by NUNES et al. (2000)28, who found 46% of the sand samples contaminated with Ancylostoma spp. larvae. On the other hand, the degree of contamination by hookworm was higher than recovered in some cities, such as Fernandópolis, São Paulo (1.8%)5, Santos, São Paulo (15%)35, Duque de Caxias, Rio de Janeiro (33.5%)42 and Araçatuba, São Paulo (37.5%)28. In contrast, contamination rates higher than our results were noted in Santa Maria (Rio Grande do Sul State) by CORRÊA & MOREIRA (1996)8, who found 93.3% of the public squares’ land contaminated with eggs of Ancylostoma spp. In addition, GUIMARÃES et al. (2005)16 reported the occurrence of eggs and larvae of hookworms in 69.6% of the soil samples from public squares of the town of Lavras (Minas Gerais State).

As shown in Figure 1, the central region of Guarulhos had the lowest rate of contamination by nematode Toxocara spp. and Ancylostoma spp. In other areas, soil contamination was higher with Toxocara spp. These data agree with the observations of CAPUANO & ROCHA (2005)3 who examined soil samples from public squares in Ribeirão Preto (São Paulo State) and found environmental contamination by Toxocara spp., whose frequency was lower in the central region. Moreover, the results of this study also agree with CORRÊA & MOREIRA (1996)8; in this case, the authors showed that in Santa Maria (Rio Grande do Sul State), the prevalence of Ancylostoma spp. was lower in the soil of the central public squares. The difference in the centre compared with other regions is most likely due to a more targeted public intervention to those regions, such as cleaning, maintenance, presence of refuse dumps and controlled access by

the use of fences, while those regions still contained a high level of soil contamination. In fact, in public spaces where animals are allowed access, they can liberate eggs and or larvae of parasites into the environment through their feces and thus generate a risk for the population36,41. The high prevalence in other areas indicates that socioeconomic indicators, such as low income and education level, are related to the spread of parasites with zoonotic potential in the environment17,41. Additionally, the canine population density varies from region to region, especially stray dogs. This one factor is related to socio-economic aspects of each population group in the same city41,42; in this context, the peripheral areas of large cities offer better conditions for the high prevalence of helminth parasites1,10,13,14,15,22,24,25. The study results obtained in Guarulhos, where approximately 75% of districts had contaminated the soil with parasites responsible for zoonoses, indicate that the majority of the municipal population is at zoonotic risk.

Human nematode diseases are more prevalent in tropical settings than in temperate regions, and are more prevalent in rural populations than in urban populations in the same region37. Egg resistance, to both chemical and climatic factors, influences their viability in the environment over long periods of time41. Type of soil, ambient temperature and humidity are the main factors that determine the time it takes to evolve from egg to larvae26. In Guarulhos, between September and December 2010, the spring season, the average temperature was 22.5 oC (16.4 - 25.8 oC)6 and thus it has the correct temperature to favor maintenance and development to nematode eggs or larvae.

Analyses of fecal samples found in public places can predict levels of soil contamination. However, comparison between different reports should consider several methodological biases. MIZGAJSKA (2001)26 related that during the course of sampling and laboratory analyses many factors influence the results of soil examinations and these include: sample site selection, number and volume of samples, depth of sampling, season of examination, method of egg recovery, type of soil examined, preservation of samples and laboratory skills. In this context, many techniques have been described, and they vary in the percentage of parasites recovered23. In this study, soil samples were analyzed using the flotation technique according to DADA (1979)9 with sodium dichromate (d = 1.35) and zinc sulphate (d = 1.20) for helminth eggs and the modified Baermann method for larvae recovered according to RUGAI et al. (1954)38. Although the methods used to recover zoonotic parasites in soil are not standardized and are not always comparable23,26, the techniques used in our study are in agreement with others studies and they are considered effective16,21,23,28. Another important aspect in parasite detection is the type of soil21,22,23,26,41. In this study, we observed that soil from all studied place and squares were primarily composed of sand, and more rarely clay soil. It has been described that sandy soils represent an important source of human infection by parasites35.

Approximately one decade ago, RAGOZO et al. (2002)33 analyzed stool samples from 31 cats that were captured in the streets of Guarulhos, and amongst helminths, Toxocara cati and Ancylostoma spp. were the most prevalent. Additionally, the authors have emphasized the importance of stray cats in the epidemiology of zoonoses in the city. In our study, it was noted that many animals (resident, domiciled or semi-errant) defecated in the soil of most parks and public squares of Guarulhos. These dogs and cats are on public roads and thus represent an important aspect in the transmission of zoonoses in urban areas. Moreover, from

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MARQUES, J.P.; GUIMARÃES, C.R.; VILAS BOAS, A.; CARNAÚBA, P.U. & MORAES, J. - Contamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by Toxocara spp. and Ancylostoma spp. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 267-71, 2012.

270

an epidemiological point of view, the World Health Organization (WHO) recommends that the dog population in each location should not exceed 10% of the human population45. In Guarulhos, the estimate is seven dogs per person19, which corresponds to approximately 15% of the human population. Thus, considering that Guarulhos is one of the most populous cities in Brazil, with approximately 1.3 million inhabitants, it is estimated that the dog population is almost 200,000.

Finally, larva migrans syndrome represents a global public health problem and remains neglected in many countries17,18,20,37,44,45. The intervention of the public service to preserve the population’s health and animal welfare is a great challenge12,18,34,44. Considering the high incidence of parasites with zoonotic potential in the soil of the public areas of Guarulhos, it is necessary to highlight the importance of adopting educational and dog and cat breeding control measures to reduce the risk of exposure to these parasites that cause zoonoses in adults and especially children.

CONFLICT OF INTEREST

The authors have declared that no competing interests exist.

RESUMO

Contaminação de parques e praças públicas por Toxocara spp. e Ancylostoma spp., no município de Guarulhos, São Paulo, Brasil

A contaminação do solo com fezes de animais mamíferos representa importante fator de risco para a ocorrência de zoonoses, sobretudo a larva migrans visceral e a larva migrans cutânea, causadas por Toxocara spp. e Ancylostoma spp., respectivamente. O presente estudo teve como objetivo verificar a contaminação ambiental por Toxocara spp. e Ancylostoma spp. em amostras de solos coletadas de praças e parques públicos do município de Guarulhos, região metropolitana de São Paulo (São Paulo, Brasil). No período de setembro a dezembro de 2010 as amostras do solo foram coletadas e examinadas pela técnica de centrífugo-flutuação, com dicromato de sódio e sulfato de zinco, e pelo método de Baermann modificado. Dos 47 distritos analisados, 35 (74,5%) estavam contaminadas com ovos de Toxocara spp. e/ou ovos ou larvas de Ancylostoma spp., sendo que a frequencia de Toxocara spp. e Ancylostoma spp. foi de 68,1% e 46,8%, respectivamente. No geral, a Zona Leste de Guarulhos foi a região com maior ocorrência desses parasitas com potencial zoonótico. Em todas as áreas públicas, dos 47 distritos, notou-se a presença de fezes de cães e gatos, assim como animais errantes ou acompanhados de seus proprietários. Faz-se necessário salientar a importância da adoção de medidas de controle reprodutivo de cães e gatos, o tratamento dos animais infectados, assim como medidas educativas para reduzir os riscos de crianças e adultos à exposição desses parasitas causadores de zoonoses.

ACKNOWLEDGMENTS

The authors would like to thank Jairo de Moraes and Aline A. L. Carvalho for their contribution to the physical and geographical aspects of Guarulhos and Etielle B. Andrade for her artwork. We are indebted to Fabiana Barros, Andréia Assumpção, Sandra E. Oshiro, Roseli S. Lima and many colleagues from Faculdades Integradas Torricelli (Guarulhos, São Paulo) for their help in sample collection from the very beginning of

this project. The authors are also grateful to Dr. Kendi Okuda (Department of Medicine, University of Massachusetts Medical School, USA) for criticism and revision of the manuscript. This work received financial support from Clínica Veterinária Fullpet (Guarulhos, São Paulo).

AUTHORS CONTRIBUTIONS

Conceived and designed the experiments: JP Marques, J Moraes. Performed the experiments: JP Marques, CR Guimarães, A Vilas Boas. Analyzed the data: JP Marques, J Moraes. Contributed reagents/materials/analysis tools: PU Carnaúba, JP Marques, CR Guimarães, A Vilas Boas, J Moraes. Wrote the paper: J Moraes.

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MARQUES, J.P.; GUIMARÃES, C.R.; VILAS BOAS, A.; CARNAÚBA, P.U. & MORAES, J. - Contamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by Toxocara spp. and Ancylostoma spp. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 267-71, 2012.

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42. Thomé SM, Lafayette EP, Pessoa Neto GR. Contaminação ambiental por ovos de Toxocara spp. e Ancylostoma spp. em praças públicas da cidade de Duque de Caxias, estado do Rio de Janeiro. Saude & Amb Rev. 2008;3:30-32.

43. Tiyo R, Guedes TA, Falavigna DL, Falavigna-Guilherme AL. Seasonal contamination of public squares and lawns by parasites with zoonotic potential in southern Brazil. J Helminthol. 2008;82:1-6.

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45. World Health Organization. Guidelines for dog population management. Geneve: WHO; 1990.

Received: 14 March 2012Accepted: 10 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):273-280, September-October, 2012doi: 10.1590/S0036-46652012000500007

Malacology Laboratory, Department of Zoology, DDU Gorakhpur University, Gorakhpur, 273009, U.P. India.Correspondence to: Prof. D.K. Singh, Malacology Laboratory, Department of Zoology, DDU Gorakhpur University, Gorakhpur, 273009, UP, India. Tel: +91 551 2202187(O)/0509(R).

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PHARMACOLOgICAL EFFECTS OF Sapindus mukorossi

Aparna UPADHYAY & D.K. SINGH

SUMMARY

Sapindus mukorossi is an extremely valuable medicinal plant, distributed in tropical and sub-tropical regions of Asia. The aim of present review is to form a short compilation of the phytochemical composition and pharmacological properties of this multipurpose tree. The main phytoconstituents isolated and identified from different parts of this plant are triterpenoidal saponins of oleanane, dammarane and tirucullane type. The structure and chemical names of all the types of triterpenoidal saponins reported in Sapindus mukorossi are included in this review. Many research studies have been conducted to prove the plant’s potential as being spermicidal, contraceptive, hepatoprotective, emetic, anti-inflammatory and anti-protozoal. The present review highlights some of the salient pharmacological uses of Sapindus mukorossi.

KEYWORDS: Sapindus mukorossi; Pharmacology; Saponins.

INTRODUCTION

Sapindus mukorossi Gaertn., a member of the family Sapindaceae, is commonly known by several names such as soapnut, soapberry, washnut, reetha, aritha, dodan and doadni. It is a deciduous tree widely grown in upper reaches of Indo-Gangetic plains, Shivaliks and sub Himalayan tracts at altitudes from 200 m to 1500 m. The Sapindus mukorossi is a fairly large, deciduous tree with a straight trunk up to 12 meters in height, sometimes attaining a height of 20 m and a girth of 1.8 m, with a globose crown and rather fine leathery foliage. Bark is dark to pale yellow, fairly smooth, with many vertical lines of lenticels and fine fissures exfoliating in irregular wood scales. The blaze is 0.8-1.3 cm, hard, not fibrous, pale orange brown, brittle and granular. Leaves are 30-50 cm long, alternate, paripinnate; common petiole very narrowly bordered, glabrous; leaflets 5-10 pairs, opposite or alternate, 5-18 by 2.5-5 cm, lanceolate, acuminate, entire, glabrous, often slightly falcate or oblique; petioles 2-5 m long. Inflorescence is a compound terminal panicle, 30 cm or more in length, with pubescent branches. Flowers are about 5 mm across, small, terminal, polygamous, greenish white, subsessile, numerous, mostly bisexual. Sepals 5, each with a woolly scale on either side above the claw. Fruits are globose, fleshy, 1-seeded drupe, sometimes two drupels together, about 1.8-2.5 cm across. Seeds are 0.8-1.3 cm in diameter, globose, smooth, black and loosely placed in dry fruit7.

The fruit is valued for the saponins (10.1%) present in the pericarp and constitutes up to 56.5% of the drupe known for inhibiting tumor cell growth37. In China and Japan it has been used as a remedy for centuries. In Japan its pericarp is called “enmei-hi”, which means “life prolonging

pericarp” and in China “wu-huan-zi”, the “non-illness fruit”37. The major compounds isolated from Sapindus mukorossi are triterpenoidal saponins of mainly three oleanane, dammarane and tirucullane types. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial19, cytotoxic29, molluscicidal16,42, insecticidal12,26, piscicidal44 and fungicidal37,41 activities. One of the most talked about activities of this plant is the contraceptive activity of the saponins extracted from the pericarp of the fruit11,27.

Sapindus mukorossi is well known for its folk medicinal values30. Pericarps of Sapindus mukorossi have been traditionally used as an expectorant as well as a source of natural surfactant20. Due to the presence of saponins, soapnut is well known for its detergent and insecticidal properties and it is traditionally used for removing lice from the scalp. The fruits are of considerable importance for their medicinal value for treating a number of diseases like excessive salivation, pimples, epilepsy, chlorosis, migranes, eczema and psoriasis21. The powdered seeds are employed in the treatment of dental caries, arthritis, common colds, constipation and nausea8. The seeds of Sapindus mukorossi are used in Ayurvedic medicine to remove tan and freckles from the skin. It cleanses the skin of oily secretion and is even used as a cleanser for washing hair as it forms a rich, natural lather. The leaves are used in baths to relieve joint pain and the roots are used in the treatment of gout and rheumatism. Since ancient times Sapindus mukorossi has been used as a detergent for shawls and silks. The fruit of Sapindus mukorossi was utilized by Indian jewelers for restoring the brightness of tarnished ornaments made of gold, silver and other precious metals33.

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UPADHYAY, A. & SINGH, D.K. - Pharmacological effects of Sapindus mukorossi. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 273-80, 2012.

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PHYTOCHEMICAL CONSTITUENTS

The major constituents of Sapindus mukorossi fruit are saponins (10%-11.5%), sugars (10%) and mucilage10. Saponins are secondary plant metabolites with divergent biological activities10. Sapindus saponins are a mixture of six sapindosides (sapindosides A, B, C, D and mukorozi saponins (E1 and Y1), with sapindoside B as one of the major constituents, isolated by n-butanol extraction of the ethanolic extract of fruit pericarp of Sapindus mukorossi and identified by liquid chromatography and mass spectroscopy28. Saponins are a large family of structurally-related compounds of steroid or triterpenoid aglycone (sapogenin) linked to one or more oligosaccharide moieties by glycosidic linkage. The aglycone, or sapogenin, may contain one or more unsaturated C-C bonds. The oligosaccharide chain is normally attached at the C3 position (monodesmosidic), but many saponins have an additional sugar moiety at the C2,6 or C2,8 position (bidesmosidic)10. The great complexity of the saponin structure arises from the variability of the aglycone structure, the nature of the side chains and the position of attachment of these moieties on the aglycone10. The carbohydrate moiety consists of pentoses, hexoses or uronic acids. Due to this complexity, saponins are difficult to classify. Because it is no longer customary to classify compounds based on their physicochemical or biological properties, a state of the art classification based on the biosynthesis of the saponin carbon skeletons was proposed by VINCKEN et al.43.

Different types of triterpene, saponins of oleanane, dammarane and tirucullane type were isolated from the galls, fruits and roots of Sapindus mukorossi. Oleanane type triterpenoid saponins named Sapindoside A&B (Fig. 34 & 35) were reported from the fruits of Sapindus mukorossi6. Sapindoside C (Fig. 36)4, Sapindoside D (Fig. 37)5, which is a hexaoside of hederagenin, and Sapindoside E (Fig. 38)3, a nonaoside of hederagenin, was isolated and identified from the methanolic extract of the fruits of Sapindus mukorossi.

Dammarane-type saponins, named Sapinmusaponins A & B (Fig. 11 & 12), C-E (Fig. 15, 16, 17), together with three known phenylpropanoid glycosides, were isolated from the galls of Sapindus mukorossi45. Tirucallane-type saponins, sapinmusaponins F-J (Fig. 18-22), were isolated from the galls of Sapindus mukorossi as reported by HUANG et al.17. The structures of these saponins were elucidated on the basis of spectroscopic analysis including 1D and 2D NMR techniques.

Triterpene saponins of oleanane type like, Sapinmusaponin K-N (Fig. 25-28), Mukorozisaponin G & E1 (Fig. 29-30), Sapindoside A & B along with dammarane types like Sapinmusaponin O and P (Fig. 13 & 14) were isolated from fruits and the galls of Sapindus mukorossi as per HUANG et al.15. In another study by NAKAYAMA et al.23, Mukorozisaponin Y1 (Fig. 31), Y2 (Fig. 32), X (Fig. 33) were isolated from the pericarp of Sapindus mukorossi.

Fractionation of an ethanolic extract of the galls of Sapindus mukorossi has resulted in the isolation of two tirucallane type triterpenoid saponins, sapinmusaponin Q and R (Fig. 23-24), along with three known oleanane type triterpenoid saponins: sapindoside A, sapindoside B, and hederagenin-3-O-[β-D-xylopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)+-α-L arabinopyranoside14. The roots of Sapindus mukorossi contain tirucallane-type triterpenoid saponins like Sapimukoside A & B38, Sapimukoside C &D25. Further investigation of the roots of Sapindus mukorossi by NI

et al.24 reported the presence of Sapimukosides E-J24. The structures of Sapimukosides A-J are shown in Fig. 1 to Fig. 10 respectively. Table 1 shows whole view of all the saponins isolated from Sapindus mukorossi.

BIOLOGICAL EFFECTS

1. Anti-bacterial activity: IBRAHIM et al.19 evaluated that ethanolic and chloroform extracts of Sapindus mukorossi inhibited the growth of Helicobacter pylori (both sensitive and resistant), at very low concentrations, when given orally for seven days to male wister rats. In the in vitro study, the isolates show a considerable zone of inhibition at very low concentration (10 µg/mL) and in the in vivo

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6 3-AraGlc 2-Rha3-Xyl

Et

7 3-AraGlc 2-Rha3-Xyl

Me

8 3-AraGlc 2-Rha3-Ara

Et

9 3-RhaGlc2-Rha3-Ara

Me

10 Glc6-Rha Et

AbbreviationsGlc:β-D-GlucopyranosylRha:α-L-hamnopyranosyl Ara:α-L-rabinopyranosyl Xyl:β-D-Xylopyranosyl

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Table 1List of Saponins isolated from Sapindus mukorossi

Saponins Chemical name Tirucullane/oleanane/dammarane type

Structure Reference

Sapindoside A Hederagenin-3-O-α-L-arabinosyl-(2→1)-α-L-

rhamnopyranoside Oleanane 34 Chirva et al., 1970 a

B Hederagenin-3-O-α-L-arabinosyl-(2→1)-O-α-L-rhamnopyranosyl-(3→1)-β-D-xylanopyranoside

Oleanane 35 Chirva et al., 1970 a

C Hederagenin-3-O-β-D-glucosyl(1→4)-β-D-xylosyl (1→3)-α-L-rhamnosyl(1→2)- α-L-arabinoside

Oleanane 36 Chirva et al., 1970 b

Sapinmusaponin A 3,7,20(S),22-tetrahydroxydammar-24-ene-3-O-α-L-

rhamnopyranosyl-(1→2)-D-glucopyranoside Dammarane 11 Yao et al., 2005

B 3,7,20(S),22,23-pentahydroxydammar-24-ene-3-O-α-L-rhamnopyranosyl-(1→2)-D-glucopyranoside

Dammarane 12 Yao et al., 2005

C 3,7,20(S),22,25-pentahydroxydammar-23-ene-3-O-α-L-rhamnopyranosyl-(1→2)-D-glucopyranoside

Dammarane 15 Yao et al., 2005

D 25-methoxy-3,7,20(S),22-tetrahydroxydammar-23-ene-3-O-α-L-rhamnopyranosyl-(1→2)-D-glucopyranoside,

Dammarane 16 Yao et al., 2005

E 25-methoxy-3,7,20(R)-trihydroxydammar-23-ene-3-O-α-L-rhamnopyranosyl-(1→2)-D-glucopyranoside

Dammarane 17 Yao et al., 2005

F 21 β-methoxy-3-β-21(S), 23I-epoxy tirucall-7,24-diene-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl

Tirucullane 18 Huang et al., 2006

G 21α-methoxy-3-β-21(S), 23I-epoxy tirucall-7,24-diene-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl

Tirucullane 19 Huang et al., 2006

H 21α-methoxy-3-β-21(S), 23I-epoxy tirucall-7,24-diene-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl

Tirucullane 20 Huang et al., 2006

I 21β-methoxy-3-β-21(S), 23I-epoxy tirucall-7,24-diene-3-O-α-L-dirhamnopyranosyl-(1→2,6)-β-D-glucopyranosyl

Tirucullane 21 Huang et al., 2006

J 21α-methoxy-3-β-21(S), 23I-epoxy tirucall-7,24-diene-3-O-α-L-dirhamnopyranosyl-(1→2,6)-β-D-glucopyranosyl

Tirucullane 22 Huang et al., 2006

K hederagenin-3-O-(3-O-acetyl-alpha-L-arabinopyranosyl)-(1→3)-alpha-L-rhamnopyranosyl-(1→2)-alpha-L-arabinopyranoside

Oleanane 25 Huang et al., 2008

L hederagenin-3-O-(4-O-acetyl-alpha-L-arabinopyranosyl)-(1→3)-alpha-L-rhamnopyranosyl-(1→2)-alpha-L-arabino-pyranoside,

Oleanane 26 Huang et al., 2008

M hederagenin-3-O-(2,3-O-diacetyl-beta-D-xylopyranosyl)-(1→3)-alpha-L-rhamnopyranosyl-(1→2)-alpha-L-arabinopyranoside

Oleanane 27 Huang et al., 2008

N hederagenin-3-O-(2,4-O-diacetyl-beta-D-xylopyranosyl)-(1→3)-alpha-L-rhamnopyranosyl-(1→2)-alpha-L-arabinopyranoside

Oleanane 28 Huang et al., 2008

O 3,7,20(S)-trihydroxydammar-24-ene-3-O-alpha-L-rhamnopyranosyl-(1→2)-beta-D-glucopyranoside

Dammarane 13 Huang et al., 2008

P 3,7,20(R)-trihydroxydammar-24-ene-3-O-alpha-L-rhamnopyranosyl-(1→2)-beta-d-glucopyranoside

Dammarane 14 Huang et al., 2007

Q 21α-methoxy-3β, 21I, 23(S)-epoxytirucall-7,24-diene-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside

Tirucullane 23 Huang et al., 2007

R 21α-methoxy-3β, 21I, 23(S)-epoxytirucall-7,24-diene-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside

Tirucullane 24 Huang et al., 2007

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Sapinmukoside A 3-O-α-L-rhamnopyranosyl-(1→2)-*α-L-

arabinopyranosyl-(1→3)+ – β-D-glucopyranosyl-21, 23R-epoxyl tirucall-7, 24R-diene-3 β, 2-diol

Tirucullane 1 Teng et al., 2003

B 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl-21, 23R-epoxyl tirucall-7, 24R-diene-3 β, 21-diol

Tirucullane 2 Teng et al., 2003

C 3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl (21,23R)-epoxyl tirucalla-7,24-diene-(21S)-ethoxyl-3β-ol

Tirucullane 3 Teng et al., 2004

D 3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl (21,23R)-epoxyl tirucall-7, 24-diene-(21S)-methoxyl-3β-ol .

Tirucullane 4 Teng et al., 2004

E 3-O-α-L-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl (21,23R)-epoxyl tirucalla-7,24-diene-21β-ethoxyl-3β-ol}

Tirucullane 5 Ni et al., 2006

F {3-O-β-D-xylanopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl 21,23R-epoxyl tirucalla-7,24-diene-21β-ethoxyl-3β-ol}

Tirucullane 6 Ni et al., 2006

G {3-O-β-D-xylanopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-glucopyranosyl (21,23R)-epoxyl tirucalla-7,24-diene-21β-methoxy-3β-ol}

Tirucullane 7 Ni et al., 2006

H {3-O-α-L-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranosyl 21,23R-epoxyl tirucalla-7,24-diene-21β-ethoxy-3β-ol}

Tirucullane 8 Ni et al., 2006

I {3-O-α-L-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranosyl 21,23R-epoxyl tirucalla-7,24-diene-21β-methoxy-3β-ol}

Tirucullane 9 Ni et al., 2006

J {3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl 21,23R-epoxyl tirucalla-7,24-diene-21β-ethoxyl-3β-ol}

Tirucullane 10 Ni et al., 2006

Mukorozi-saponin G Hederagenin-3-O-(2-O-acetyl-β-D-xylanopyranosyl)-(1→3)-

α-L-rhamnopyranosyl-(1→2)-α-L-arabinoside. Oleanane 29 Huang et al., 2008

E1 Hederagenin-3-O-α-L-arabinosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinoside.

Oleanane 30 Huang et al., 2008

Table 1List of Saponins isolated from Sapindus mukorossi (cont.)

study the Helicobacter pylori infection was cleared with minimal dose extracts of 2.5 mg/mL.

2. Insecticidal activity: Saponins possess insecticidal activity, causing mortality and/or growth inhibition in the insects tested, the cotton leafworm Spodoptera littoralis caterpillars and the pea aphid Acyrthosiphon pisum. In the experiments with Acyrthosiphon pisum, 0.1% saponin killed all aphids, whereas with Spodoptera some caterpillars were still able to develop into apparently normal adults on food containing 7% saponin12. Saponins can be employed as novel natural tactics in integrated pest management (IPM) to control pest insects, which fit in modern agriculture and horticulture13. Ethanolic extract of Sapindus mukorossi was investigated for repellency and insecticidal activity against Sitophilus oryzae and Pediculus humanus. Average mortality percentage indicated that the extracts caused significant mortality and repellency on the target insects and bioassays indicated that toxic and repellent effect was proportional to the concentration26.

3. Spermicidal activity: Saponins from Sapindus mukorossi are known to be spermicidal11,27. Morphological changes in human ejaculated spermatozoa after exposure to this saponin were evaluated under scanning electron microscopy. The minimum effective concentration (0.05% in spot test) did not affect the surface topography after exposure for one minute. However, incubation of spermatozoa for 10 minutes resulted in extensive vesiculation and a disruption of the plasma membrane in the head region. Higher concentrations (0.1%, 1.25%, 2.5% and 5.0%) caused more or less similar changes which included vesiculation, vacuolation, disruption or erosion of membranes in the head region. These findings suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of the plasma membrane of spermatozoa8. This spermicidal property has been used in contraceptive cream9.

4. Anti-Trichomonas activity: TIWARI et al.39 demonstrated that the Sapindus saponin mixture shows anti-Trichomonas activity at a 10-fold lower concentration (0.005%) than its minimal effective

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spermicidal concentration against human spermatozoa (0.05%)11. Saponin concentration dependently inhibited the ability of parasites to adhere to HeLa cells and decreased the proteolytic activity of the parasite’s cysteine proteinases. This was associated with the decreased expression of adhesin AP65 and membrane-expressed cysteine proteinase TvCP2 genes. Saponins produced no adverse effect on host cells in the mitochondrial reduction potential measurement assay. Saponin disrupts the actin cytoskeleton network beneath the cell membrane and affects membrane-mediated adherence of Trichomonas to the host cells.

5. Anti-cancer activity: Due to the great variability in saponin structure, saponins always display anti-tumorigenic effect through varieties of anti-tumor pathways. There are more than 11 distinguished classes of saponins including dammaranes, tirucallanes, lupanes, hopanes, oleananes, taraxasteranes, ursanes, cycloartanes, lanostanes, cucurbitanes and steroids. Ginsenosides, belonging to dammaranes, have been found beneficial in the inhibition of tumor angiogenesis by suppressing its inducer in the endothelial cells of blood vessels, and then in the prevention of adhering, invasion and metastasis of tumor cells22.

Dioscin, one of the steroidal saponins, and its aglycone diosgenin also has an extensive anti-tumor effect by cell cycle arrest and apoptosis22. The preliminary bioassay data revealed that saponins [39,4]-43] showed moderate cytotoxic activity (ED50~9-18µg/mL) against human tumor cell lines (Hepa59T/VGH, NCL, HeLa and Med)29. Strychnopentamine was the reference compound used in the study. All saponins were reported to be at least five times less active than the reference compound36.

6. Hepatoprotective activity: IBRAHIM et al.18 reported that the extracts of Sapindus mukorossi (2.5 mg/L) and Rheum emodi (3.0 mg/L) have a protective capacity both in vitro on primary hepatocytes cultures and in vivo in a rat model of tetrachloride carbon (CCl4) mediated liver injury as judged from serum marker enzyme activities. These cultures were treated with CCl4 and extracts of Sapindus mukorossi & Rheum emodi. A protective activity could be demonstrated in the CCl4 damaged primary monolayer culture. For the in vivo study, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of Rheum emodi was analyzed in liver injured CCl4- treated male rats. Extracts of the fruit pericarp of Sapindus mukorossi (2.5 mg/mL) and rhizomes of Rheum emodi (3.0 mg/mL) were found to have protective properties in rats with CCl4 induced liver damage as judged from serum marker enzyme activities. Thus, it was concluded that the extracts of Sapindus mukorossi and Rheum emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl4 mediated liver injury.

7. Anxiolytic activity: Methanolic extracts of Sapindus mukorossi (200 and 40 mg/L) show significant anxiolytic activity as compared to standard anxiolytics Diazepam (2 mg/Kg) and Fluoxetine (10 mg/Kg)1.

Fig R1 R2 R3 R4 R5

11 Glc2-Rha H OH OH H

12 Glc2-Rha H OH OH OH

13 Glc2-Rha OH CH3 H H

14 Glc2-Rha CH3 OH H H

Fig R1 R2 R3

15 Glc2-Rha OH OH

16 Glc2-Rha OH OCH3

17 Glc2-Rha H OCH3

Fig R1 R2

18 Glc6-Rha β-OCH3

19 Glc6-Rha α-OCH3

20 Glc2-Rha α-OCH3

2-Rha

21 Glc β-OCH3

6-Rha

2-Rha

22 Glc α-OCH3

6-Rha

23 Glc2-Glc α-OCH3

2-Glc

24 Glc α-OCH3

6-Rha

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8. Molluscicidal activity: Extracts of Sapindus mukorossi showed molluscicidal effect against the golden apple snail, Pomacea canaliculata Lamarck. (Ampullariidae) with LC50 values of 85, 22 and 17 ppm at 24, 48 and 72h exposure period, respectively16. Bioassay-directed fractionation of Sapindus mukorossi resulted in the isolation of one new hederagenin-based acetylated saponin, hederagenin 3-O-(2,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [1], along with six known hederagenin saponins, hederagenin 3-O-(3,4-O-di-acetyl-α-L-arabinopyranoside)-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [2], hederagenin 3-O-(3-O-acetyl-β-D-xylopyranosyl)-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [3], hederagenin 3-O-(4-O-acetyl-β-D-xylopyranosyl)-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [4],

hederagenin 3-O-(3,4-O-di-acetyl-β-D-xylopyranosyl)-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [5], hederagenin 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside [6], and hederagenin 3-O-α-L-arabinopyranoside [7]. The bioassay data revealed that 1-7 were molluscicidal, causing 70-100% mortality at 10 ppm against the golden apple snail16.

UPADHYAY & SINGH42 reported that Sapindus mukorossi fruit pericarp is a potential source of botanical molluscicides against Lymnaea acuminata. These snails are the intermediate host of liver fluke Fasciola gigantica, which causes 94% fascioliasis in the buffalo population of northern India32. The active molluscicidal component of Sapindus mukorossi fruit is soluble in chloroform, ether, acetone and ethanol. The toxicity of ethanolic extract of Sapindus mukorossi fruit powder is higher than other extracts which indicates that the molluscicidal component present is more soluble in ethanol than other organic solvents. UPADHYAY & SINGH42 characterized that saponin is the active component present in Sapindus mukorossi fruit by High Performance Liquid Chromatography. A comparison of the molluscicidal activity of the column-purified fraction of Sapindus mukorossi fruit powder with synthetic molluscicides clearly demonstrates that the purified fraction of Sapindus mukorossi is more potent. The LC50 at 96 h of the column–purified fraction of Sapindus mukorossi fruit powder (5.43 mg/L) against Lymnaea acuminata is lower than those of synthetic molluscicides-carbaryl (14.40 mg/L), phorate (15.0 mg/L), formothion (8.56 mg/L) and niclosamide (11.8 mg/L)32. LC50 at 96 h of crude powder of Sapindus mukorossi (119.57 mg/L) against Lymnaea acuminata is lower than the crude powder of Canna indica root (359.02 mg/L)40, Thuja orientalis leaf powder (250.55 mg/L), Thuja orientalis fruit powder (255.12 mg/L)31, Zingiber officinale rhizome (273.80 mg/L), Allium cepa bulb (253.27 mg/L)34.

9. Tyrosinase inhibition and free radical scavenging: CHEN et al.2 first evaluated that the extracts of Sapindus mukorossi seeds using methanol (MeOH), ethyl acetate (EA) or hexane as solvents show tyrosinase inhibition, free radical scavenging, antimicrobial and anticancer properties. Sapindus mukorossi extracts showed strong specific inhibition activities on the proliferation of human melanoma and lung cell lines. The data exhibited the high potential of applying Sapindus mukorossi extracts in medical cosmetology, food supplementation, antibiotics and chemotherapy.

10. Fungicidal activity: The crude extract of Sapindus mukorossi exhibits a strong growth inhibition against the pathogenic yeast Candida albicans, which causes cutaneous candidiasis. Extracts from the dried pericarp of Sapindus L. (Sapindaceae) fruits were investigated for their antifungal activity against clinical isolates of yeasts Candida albicans and Candida non-albicans from vaginal secretions of women with Vulvovaginal Candidiasis. Four clinical isolates of C. albicans, a single clinical isolate of each of the species C. parapsilosis, C. glabrata, C. tropicalis, and the strain of C. albicans ATCC 90028 were used. The hydroalcoholic extract was bioactivity-directed against a clinical isolate of C. parapsilosis, and showed strong activity. The n-BuOH extract and one fraction showed strong activity against all isolates tested41. The saponin fraction inhibited the dermatophytic fungi Trichophyton rubrum, Trichophyton mentagrophytes, Sabouraudites canis and Epidermophyton floccosum37.

Fig R1 R2

25 Ara2-Rha3-Ara3-OAC H

26 Ara2-Rha3-Rha4-OAC H

2-OAC

27 Ara2-Rha3-Xyl H

3-OAC

2-OAC

28 Ara2-Rha3-Xyl H

4-OAC

3-OAC

29 Ara2-Rha3-Xy H

4-OAC

30 Ara2-Rha3-Xyl4-OAC H

31 Ara2-Rha3-Xyl Glc2-Glc

32 Ara2-Rha3-Xyl Glc2-Glc

33 Ara2-Rha Glc2-Glc

34 Ara2-Rha H

35 Ara2-Rha3-Xyl H

36 Ara2-Rha3-Xyl4-Glc H

6-Rha

37 Ara2-Rha3-Xyl4-Glc H

2-Glc

6-Rha

38 Ara2-Rha3-Xyl Ara2-Rha3-Xyl4-Glc 2-Glc

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11. Anti-inflammatory activity: TAKAGI et al.35 reported that crude saponin and hederagenin isolated from Sapindus mukorossi inhibited the development of carrageen-induced edema in the rat hind paw as well as on granuloma and exudates formations induced by croton oil in rats. The effects of these agents on vascular permeability and acetic acid induced writhing in mice were also examined. Anti-inflammatory activity on carrageenin edema was observed after intraperitoneal and oral administration of crude saponin, whilst hederagenin and the other agents showed activity only when administered.

12. Piscicidal activity: Effects of Sapindus mukorossi have been studied on fish. Pericarp of Sapindus mukorossi is the most toxic parts yielding 100% mortality within 12 hours and mean survival time was found to be 1.18 hours. LD10, LD50, LD100 ranging between 3.5 ppm and 10 ppm at 48 hrs and possess high potential for fish eradication. Sapindus mukorossi fruit pericarp can be used as a selective eradicant for horny fish like Heteropneustes fossils and channa punctuate44.

13 Anti-platelet aggregation activity: HUANG and co workers demonstrated that five new tirucallane type saponins, sapinmusasaponins from the galls of Sapindus mukorossi, showed moderate activity in a 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay25.

DISCUSSION

Sapindus mukorossi is a versatile and exceptionally valuable medicinal plant. It is known by such regional names as soapnut, soapberry, washnut, reetha and dodan. The phytochemical screening of the plant extract showed the presence of saponins (10.1%) present in the pericarp of the fruit. The use of Sapindus mukorossi in folk medicine worldwide30 is validated by scientific studies that have demonstrated the efficacy of the extracts in various experimental models. Pharmacological effects of Sapindus mukorossi have been reported like anti-bacterial19, insecticidal12,13,26, spermicidal11,27, anti-trichomonas11,39, anti-tumor22,29,36, hepatoprotective18, anxiolytic1, molluscicidal16,42, fungicidal37,41, anti-inflammatory35 and piscicidal44 activities and are being employed for the treatment of different ailments in the indigenous system of medicine. Although a number of phytochemicals present in Sapindus mukorossi have been isolated and identified by researchers working in different laboratories, their pharmacological/biological studies in human welfare has not been studied so far.

Most of the scientific study is confined to the elaboration of traditional practices of Sapindus mukorossi. There is a long list of saponins present in Sapindus mukorossi. It needs individual attention so that they can be explored in different pharmacological studies. The literature reviewed gives a limited picture of pharmacological effects of Sapindus mukorossi. There is a need for much additional research regarding pharmacological effects of Sapindus mukorossi at molecular level to explain their mode of action.

CONCLUSION

Sapindus mukorossi is a tropical tree whose numerous economic applications and whose facility of propagation are arousing international interest. It needs to be widely cultivated in most of the areas where climatic conditions favor its optimum growth. In this way, a maximum

yield of its different usable parts could be achieved to derive the maximal amount of commodities of a multifarious nature for the welfare of mankind. This plant has been used as traditional medicine for various ailments. The earlier reports on chemical investigation and pharmacological evaluation showed that Sapindus mukorossi contains a number of bio-active novel compounds. As literature illustrates, many biological and pharmacological activities are shown by fractions of crude extracts and isolated substances. Furthermore, the detailed chemical analysis is required to isolate bio-active constituents from Sapindus mukorossi and to trace out their biological activities. Thus, it can be concluded that Sapindus mukorossi can play an important role in modern medical system in near future.

RESUMO

Efeitos farmacológicos do Sapindus mukorossi

Sapindus mukorossi é planta medicinal extremamente valiosa distribuída nas regiões tropical e subtropical da Ásia. O propósito da presente revisão é uma compilação curta da composição fitoquímica e das propriedades farmacológicas desta árvore que apresenta múltiplos propósitos. O principal fitoconstituinte isolado e identificado das diferentes partes desta planta são as saponinas triterpenoidais do tipo da oleana, damarana e tiruculana. A estrutura e o nome químico de todos os tipos de saponinas triterpenoidais encontrados no Sapindus mukorossi estão incluídos nesta revisão. Muitas pesquisas tem sido conduzidas para provar o potencial desta planta como espermaticida, contraceptivo, hépato-protetor, emético, anti-inflamatório e anti-protozoário. A presente revisão exalta alguns principais usos farmacológicos do Sapindus mukorossi.

ACKNOWLEDGEMENT

One of the authors Aparna Upadhyay is thankful to Department of Science and Technology, New Delhi for financial assistance (Inspire Fellowship number- IF10296).

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6. Chirva V, Kintya P, Sosnovskii VA, Krivenchuk PE, Zykova NY. Triterpene glycosides of Sapindus mukorossi. II. The structure of Sapindoside A & B. Chem Nat Compounds. 1970;6:213-5.

7. Chopra R, Ghosh S. Poisonous plants of India. Delhi: The Manager of Publishers; 1946. p. 308.

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UPADHYAY, A. & SINGH, D.K. - Pharmacological effects of Sapindus mukorossi. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 273-80, 2012.

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9. Dwivedi AK, Chaudhry M, Sarin JPS. Standardization of a new spermicidal agent Sapindus saponin and its estimation in its formulation. Indian J Pharm Sci. 1990;52:165-7.

10. Francis G, Kerem Z, Makkar H, Becker K. The biological action of saponins in animal systems: a review. Br J Nutr. 2002;88:587-605.

11. Garg S, Taluja V, Upadhyay M, Talwar GP. Studies on contraceptive efficacy of Praneem polyherbal cream. Contraception. 1993;48:591-6.

12. Geyter ED, Geelen D, Smagghe G. First results on the insecticidal action of saponins. Commun Agric Appl Biol Sci. 2007;72:645-8.

13. Geyter ED, Lambert E, Geelen D, Smagghe G. Novel advances with plant saponins as natural insecticides to control pest insects. Pest Tech. 2007;1:96-105.

14. Huang HC, Tsai WJ, Liaw CC, Wu SH, Wu YC, Kuo YH. Anti-platelet aggregation triterpene saponins from the galls of Sapindus mukorossi. Chem Pharm Bull (Tokyo). 2007;55:1412-5.

15. Huang HC, Wu MD, Tsai WJ, Liao SC, Liaw CC, Hsu LC, et al. Triterpenoid saponins from the fruits and galls of Sapindus mukorossi. Phytochemistry. 2008;69:1609-16.

16. Huang HC, Liao SC, Chang FR, Kuo YH, Wu YC. Molluscicidal saponins from Sapindus mukorossi, inhibitory agents of Golden Apple snails Pomacea canaliculata. J Agric Food Chem. 2003;51:4916-9.

17. Huang HC, Tsai WJ, Morris-Natschke SL, Tokuda H, Lee KH, Wu YC, et al. Sapinmusaponins F-J, bioactive tirucallane-type saponins from the galls of Sapindus mukorossi. J Nat Prod. 2006;69:763-7.

18. Ibrahim M, Khaja MN, Aara A, Khan AA, Habeeb MA, Devi, YP, et al. Hepatoprotective activity of Sapindus mukorossi and Rheum modi extracts: in vitro and in vivo studies. World J Gastroenterol. 2008;14:2566-71.

19. Ibrahim M, Khan AA, Tiwari SK, Habeeb MA, Khaja MN, Habibullah CM. Anti-microbial activity of Sapindus mukorossi and Rheum modi extracts against Helicobacter pylori: in vitro and in vivo studies. World J Gastroenterol. 2006;12:7136-42.

20. Kasai R, Fujino H, Kuzuki T, Wong WH, Goto C, Yata N, et al. Acyclic sesquiterpene oligoglycosides from pericarps of Sapindus mukorossi. Phytochemistry. 1986;25:871-6.

21. Kirtikar KR, Basu BD. Indian medicinal plants. Allahabad: B.L.M. Publication; 1991.

22. Man S, Gao W, Zhang Y, Huang L, Liu C. Chemical study and medical application of saponins as anti-cancer agents. Fitoterapia. 2010;81:703-14.

23. Nakayama K, Fujino H, Kasai R, Mitoma Y, Yata N, Tanaka O. Solubilizing properties of saponins from Sapindus mukorossi Gaertn. Chem Pharm Bull (Tokyo). 1986;34:3279-83.

24. Ni W, Hua Y, Liu HY, Teng RW, Kong YC, Hu XY, et al. Tirucallane-type triterpenoid saponins from the roots of Sapindus mukorossi. Chem Pharm Bull (Tokyo). 2006;54:1443-6.

25. Ni W, Hua Y, Teng RW, Kong YC, Chen CX. New tirucallane-type triterpenoid saponins from Sapindus mukorossi. J Asian Nat Prod Res. 2004;6:205-9.

26. Rahman SS, Rahman M, Begum SA, Khan MMR, Bhuiyan MH. Investigation of Sapindus mukorossi extracts for repellency, insecticidal activity and plant growth regulatory effect. J Appl Sci Res. 2007;3:95-101.

27. Rastogi RP, Mehrotra BN. Compendium of Indian medicinal plants. New Delhi: CDRI Publication; 1999. p. 609-10.

28. Saxena D, Pal R, Dwivedi AK, Singh S. Characterization of sapindosides in Sapindus mukorossi saponin (Reetha saponin) and quantitative determination of sapindoside B. J Sci Ind Res. 2004;63:181-6.

29. Sengupta A, Basu SP, Saha S. Triglyceride composition of Sapindus mukorossi seed oil. Lipids. 1975;10:33-40.

30. Sharma A, Sati SC, Sati OP, Sati D, Maneesha Kothiyal SK. Chemical constituents and bioactivities of genus Sapindus. Int J Res Ayurveda Pharm. 2011;2:403-9.

31. Singh A, Singh VK. Molluscicidal activity of Saraca asoca and Thuja orientalis against the fresh water snail Lymnaea acuminata. Vet Parasitol. 2009;164:206-10.

32. Singh DK, Agarwal RA. In vivo and in vitro studies on synergism with anticholinesterase pesticides in the snail Lymnaea acuminata. Arch Environ Contain Toxicol. 1983;12:483-7.

33. Singh N, Kaur A, Yadav K. A reliable in vitro protocol for rapid mass propagation of Sapindus mukorossi Gaertn. Nat Sci. 2010;8:41-7.

34. Singh S, Singh VK, Singh DK. Molluscicidal activity of some common spice plants. Biol. Agric. Hortic. 1997;14:237-49.

35. Takagi K, Park EH, Kato H. Anti-inflammatory activities of hederagenin and crude saponin isolated from Sapindus mukorossi Gaertn. Chem Pharm Bull(Tokyo). 1980;28:1183-8.

36. Takechi M, Tanaka Y. Structure-activity relationships of the saponin α-hederin. Phytochemistry. 1990;29:451-2.

37. Tanaka O, Tamura Y, Masuda H, Mizutani K. Application of saponins in food and cosmetics: saponins of Mohova Yucca and Sapindus mukorossi Gaertn, saponins used in food and agriculture. New York: Plenum Press; Waller GR and Yamasaki K; 1996. p.1-11.

38. Teng RW, Ni W, Hau Y, Chen CX. Two new tirucallane-type triterpenoid saponins from Sapindus mukorossi. Acta Botanica Sinica. 2003;45:369-72. [Article in Chinese].

39. Tiwari P, Singh D, Singh MM. Anti-Trichomonas activity of Sapindus saponins, a candidate for development as microbicidal contraceptive. J Antimicrob Chemother. 2008;62:526-34.

40. Tripathi SM, Singh DK. Molluscicidal activity of Punica granatum bark, Canna indica root. Braz J Med Biol Res. 2000;33:1351-5.

41. Tsuzuki JK, Svidzinski TIE, Shinobu CS, Silva LFA, Rodrigues-Filho E, Cortex DAG, et al. Antifungal activity of the extracts and saponins from Sapindus saponaria L.. An Acad Bras Cienc. 2007;79:577-83.

42. Upadhyay A, Singh DK. Molluscicidal activity of Sapindus mukorossi and Terminalia chebula against the freshwater snail Lymnaea acuminata. Chemosphere. 2011;83:468-74.

43. Vincken JP, Heng L, de Groot A, Gruppen H. Saponins, classification and occurrence in the plant kingdom. Phytochemistry. 2007;68:275-97.

44. Virdi, GS. The piscicidal properties of Acorus calamus, Sapindus mukorossi and Xeromphis spinosa on 7 species of fishes of North India. Indian J Phys Nat Sci. 1982;2:28-35.

45. Yao HK, Hui CH, Li-Ming YK, Ya-Wen H, Kuo-Hsiung L, Fang-Rong C, et al. New dammarane-type saponins from the galls of Sapindus mukorossi. J Agric Food Chem. 2005;53:4722 -7.

Received: 17 December 2011Accepted: 21 March 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):281-286, September-October, 2012doi: 10.1590/S0036-46652012000500008

(1) Department of Pharmacognosy, Faculty of Pharmacy, Cairo University.(2) Department of Clinical Parasitology, Research Institute of Ophthalmology, Giza, Egypt.(3) Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Egypt.Correspondence to: Eman E. Taher. E-mail: [email protected]

MOLLUSCICIDAL AND MOSQUITOCIDAL ACTIVITIES OF THE ESSENTIAL OILS OF Thymus capitatus HOFF. ET LINK. AND Marrubium vulgare L.

Maha M. SALAMA(1), Eman E. TAHER(2) & Mohamed M. EL-BAHY(3)

SUMMARY

Steam distillation of essential oils of aerial parts of Thymus capitatus and Marrubium vulgare L. collected at North cost of Egypt yielded 0.5% and 0.2%, respectively. Results of Gas chromatography-mass spectrometry analyses of the two samples identified 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare, respectively. The two oil samples appeared dominated by the oxygenated constituents (88.22% for T. capitatus and 57.50% for M. vulgare), composed of phenols, mainly carvacrol (32.98%) and thymol (32.82%) in essential oil of T. capitatus, and thymol (34.55%) in essential oil of M. vulgare. It was evaluated the molluscicidal activity of T. capitatus and M. vulgare essential oils on adult and eggs of Biomphalaria alexandrina as well as their mosquitocidal activity on Culex pipiens. The LC

50 and LC

90 of T. capitatus essential oil against adult snails was 200 and 400 ppm/3hrs, respectively,

while for M. vulgare it was 50 and 100 ppm/3hrs, respectively. Moreover, M. vulgare showed LC100

ovicidal activity at 200 ppm/24 hrs while T. capitatus oil showed no ovicidal activity. It was verified mosquitocidal activity, with LC

50 and LC

90 of 100 and 200 ppm/12hrs

respectively for larvae, and 200 and 400 ppm/12hrs respectively for pupae of C. pipiens.

KEYWORDS: Vector control; Plant products; Thymus capitatus; Marrubium vulgare.

INTRODUCTION

Vector borne diseases are major sources of illness and death worldwide. Mosquitoes are primary vectors for many dreadful and fatal diseases such as dengue, malaria, yellow fever and filariasis. It can transmit diseases to more than 700 million people each year8,33. Lymphatic filariasis infects 120 million people in 73 countries worldwide and continues to be a worsening problem, especially in Africa and the Indian subcontinent28. Control of such diseases is becoming increasingly difficult because of increasing resistance to synthetic insecticides29.

Schistosomiasis affects more than 200 million people worldwide and is considered as the world’s most widespread parasitic disease19. The life cycle of the parasite in Egypt includes Biomphalaria and Bulinus snails as intermediate hosts12. Interruption of the parasite’s life cycle, via control of the snail’s population, is one of the strategies to combat schistosomiasis38. Molluscicidal agents thus interrupt the life cycle of the causative parasite and prevent human infection21.

Using natural products of plant origin (botanical derivatives) is an alternative and recent approach for mosquito and snail control. Despite their toxicity to pests and snails, they are readily biodegradable and usually lack toxicity to higher animals so they are eco-friendly4,5. Essential oils of plants are outstanding candidates, since they are; in some cases;

highly active, readily available in tropical countries and economically viable21. Essential oils molluscicidal and mosquitocidal activities have been reported by many studies23,24,27.

Thymus capitatus Hoff. et Link. and Marrubium vulgare L. are aromatic plants belonging to the family Lamiaceae. They are distributed along the Mediterranean area, at the North coast of Egypt. Few reports dealt with mosquitocidal activity of T. vulgaris7,18 and nothing was traced concerning the mosquitocidal activity of M. vulgare or the molluscicidal activity of both plants. In this respect, the present work is an attempt to characterize the different constituents of oils hydro distilled from T. capitatus and M. vulgare and to evaluate the effect of these essential oils of both plants on B. alexandrina snails and their egg masses as well as on larvae and pupae of Culex pipiens.

MATERIAL AND METHODS

Plant material: The aerial parts of T. capitatus and M .vulgare were collected from the North coast of Egypt during April 2010. Authentication of the plant was established in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Egypt. Voucher specimens (No. T-12 and M-22).

Preparation of the essential oil: Fresh aerial parts (500 g) of both

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SALAMA, M.M.; TAHER, E.E. & EL-BAHY, M.M. - Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 281-6, 2012.

282

plants under investigation were separately subjected to hydrodistillation (HD) in a Clevenger-type apparatus and the oil obtained from each plant was dried over anhydrous sodium sulfate and stored in a refrigerator till analysis. The percentage yield for each sample was determined. The specific gravity and refractive index for each oil sample were also determined13.

Analysis of the oils: Investigation of the prepared oils was carried out on an Agilent (USA) Gas Chromatography-Mass Spectrometry system (GC-MS), model 6890, fitted with an Agilent mass spectroscopic detector (MSD), model 5937, as well as a 30 m long, cross-linked 5% phenyl polysiloxane (HP-5MS, Hewlett Packard, USA) fused-silica column (i. d. 0.25 mm, film thickness 0.25 µm). The initial temperature was 80 °C, kept isothermal for three min, then increased to 260 °C at 8 °C/min, and the final temperature was kept isothermal for 15 min. The ion source temperature was 230 °C and the quadrupole temperature was 150 °C. The

carrier gas was helium adjusted at a flow rate of 0.1 mL/min. Ionization energy was 70 eV, and scan range was 40 - 500 m/z at 3.62/scan.

Identification of the oil components: Search for identification of the oil components was carried out using the Willey 275 L GC-MS library data base. A series of authentic n-alkanes (C8-C22, Poly Science Inc., Niles, USA) was subjected to Gas Liquid Chromatography (GLC) analysis under the same experimental conditions. The retention indices (Kovat’s indices, KI) of the volatile constituents were computed by logarithmic interpolation between bracketing alkanes17. Identification of the individual components was confirmed by comparison of their retention indices and MS fragmentation patterns with published data2. Relative percentages were calculated from the Total Ion Chromatograms by the computerized integrator .Results of GC/MS analysis and for the relative percentages are shown in Tables 1 and 2 respectively.

Table 1Results of GC/MS analysis of the essential oil of Thymus capitatus Hoff. et Link. and Marrubium vulgare L.

No. Rt (min.) KI Identified compoundPercentage

T. capitatus M. vulgare

1 9.08 1167 Borneol 9.15 ------------

2 11.24 1293 Thymol 32.82 34.55

3 11.67 1298 Carvacrol 32.98 4.35

4 12.1 1335 δ-Elemene --------- 2.16

5 12.37 1347 α-Terpinyl acetate --------- 0.51

6 12.41 1355 Thymol acetate 3.27 ---------

7 12.71 1368 Carvacrol acetate 1.8 ---------

8 12.98 1403 Caryophyllene 6.15 ---------

9 13.42 1431 γ-Elemene ------- 1.24

10 13.45 1443 Aromandrene 0.43 --------

11 13.72 1451 α-Humulene 0.3 1.89

12 13.80 1479 Germacrene D -------- 0.74

13 13.90 1480 α-Amorphene ----- 2.39

14 13.93 1509 γ-Cadinene ------ 17.68

15 13.98 1516 δ-Cadinene 0.38 2.21

16 14.05 1525 α-Cadinene --------- 2.97

17 14.12 1559 α-Muurolene -------- 1.20

18 14.45 1566 Germacrene D-4-ol -------- 6.37

19 15.90 1568 Spathulenol 1.2 ---------

20 15.96 1580 Caryophyllene oxide 3.45 1.74

21 16.75 1636 Caryophylla-4(14),8,(15)-diene-5-β-ol 1.17 2.55

22 17.03 1640 Alloaromadendrene 1.01 ------

23 17.32 1652 α-Cadinol --------- 5.39

24 17.41 1671 α-Bisabolol 0.64 ------

25 18.12 1740 Cucurmenol -------- 0.95

26 18.76 1768 Pentadecanol 0.2 1.09

27 20.33 1881 n-Hexadecanol 0.53 -------

28 22.25 2015 n-Heneicosane 0.48 -------

29 24.39 2292 n-Tricosane 0.31 0.21Rt, retention time; KI, Kovat’s index.

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SALAMA, M.M.; TAHER, E.E. & EL-BAHY, M.M. - Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 281-6, 2012.

283

Collection and laboratory maintenance of snails: Biomphalaria alexandrina snails and egg masses were collected and identified from irrigation canals in Giza Governorate9. Snails were screened for natural infection with larval trematodes. Uninfected snails were maintained in the laboratory conditions for seven days before being used in the study in dechlorinated tap water and fed daily on green lettuce. Tests were carried out at room temperature (26 +1 ºC). In each step, a fine mesh was placed over the container to prevent snails crawling out of the container.

Molluscicidal bioassay: The bioassay of molluscicidal activity against the B. alexandrina was evaluated according to the established procedures36. Five adult snails (8-14 mm in diameter) and snail’s egg masses (three days old) were placed, separately, in a beaker containing 200 mL of essential oil water solution of T. capitatus and M. vulgare at a series of concentrations ranging from 75-1000 parts per million (ppm) for each tested plant oil. Each experiment was set in triplicate. Snails and egg masses remained in dechlorinated water during the experiment served as control group. Immersion technique was adopted according to WHO35. Adult snails and egg masses were exposed for 24h at room temperature. After 24h, snails were rinsed twice with aerated tap water. At the end of this period; tested snails and egg masses were examined to assess mortality. Mortality was evaluated using crushing technique (5% sodium hydroxide solution)35. Egg masses were examined under the microscope for detecting the embryos and its vitality.

Snails were considered dead if they remained motionless, did not respond to the presence of food or if the shell looked discolored. The number of dead snails was expressed as percent mortality. The lethal concentration to 50% (LC

50) and 90% (LC

90) for snails and 100% (LC

100)

for the eggs was calculated following the method of Finney15. Samples that caused no mortality at 1000 ppm were considered inactive and were not investigated.

Mosquitocidal bioassay: Eggs of C. pipiens were obtained from The Medical Research Institute of Insects, Giza, Egypt were soaked in dechlorinated tap water to develop into first instar larvae. Larvae were reared in the same aquarium until the development of third instar larvae and pupae. Twenty C. pipiens third instar larvae as well as pupae were picked up from the aquarium and located in a 200 mL beaker. The bioassay was done according to WHO guidelines with slight modifications37. Essential oils of both T. capitatus and M. vulgare were

tested at the same concentrations as those applied for snails for each tested plant. Stock solution of the essential oil was prepared in Tween 80. From this stock solution, concentrations of 12.5, 25, 50, 100 and 200 ppm were prepared and replicated three times for each concentration. Mosquitoes were exposed to essential oils for 24h at room temperature, and were kept under normal laboratory conditions at 26 ± 2 ºC and 60 ± 10% relative humidity with 12:12 D/L photoperiod. Mortality was recorded after 24 hours of continuous exposure during which no food was offered to the test organisms. The LC

50 and LC

90 of tested plants,

95% confidence interval and their slopes of probit regression line were determined to probit analysis program to compare their effectiveness30.

RESULTS

Steam distillation of the essential oils of T. capitatus and M. vulgare yielded 0.5% and 0.2%, respectively. The specific gravity and refractive index (at 25 °C) for T. capitatus were 0.8561 and 1.5213, respectively while those of M. vulgare were 0.9562 and 1.6705, respectively. Results of GC/MS analyses of the two samples are displayed in Table 1. Numbers of the identified components in both oils were 18 and 19, amounting to 96.27% and 90.19% of the total oil composition for T. capitatus and M. vulgare respectively. Constituents identified under the adopted operating conditions of the essential oils under investigation were 29. Detected components in both samples viz., thymol, carvacrol, δ -cadinene, caryophyllene oxide, caryophylla-4(14), 8,(15)-diene-5-β-ol, n-tricosane, pentadecanol and humulene. The rest of constituents appeared, however, unevenly distributed in the analyzed oils.

The identified amount of oxygenated constituents were 88.22% and 57.50% for T. capitatus and M. vulgare, respectively (Table 2) while the amount of identified hydrocarbons was 8.05% and 32.69%, respectively. The overall chromatographic profile of the two oil samples was dominated by the oxygenated constituents. These were mainly composed of phenols among which carvacrol (32.98%) and thymol (32.82%) were the major constituents in T. capitatus oil while in M. vulgare oil thymol (34.55%) was the major constituent. Borneol, bicyclic monoterpenoid alcohol, was only present in T. capitatus oil (9.15%). Sesquiterpenes were the major class of hydrocarbons in the two oil samples amounted to 7.26% and 32.48% while non-terpenoid hydrocarbons were only 0.79% and 0.21% for T. capitatus and M. vulgare oils, respectively. The major sesquiterpene hydrocarbon in T. capitatus was z-caryophyllene (6.15%) while γ-cadinene was the major sesquiterpene hydrocarbon in M. vulgare oil (17.68%).

Molluscicidal activity of T. capitatus and M. vulgare essential oils against adult B. alexandrina and egg masses was evaluated at different concentrations ranging from 75-1000 ppm. After screening; the LC

50 and

LC90

of T. capitatus essential oil versus adult snails were 200 and 400 ppm/3hrs, respectively while of M. vulgare essential oil were 50 and 100 ppm/3hrs, respectively (Table 3). Furthermore, M. vulgare oil showed 100% snail ovicidal activity against B. alexandrina eggs at 200 ppm/24 hrs. However, T. capitatus oil showed no snail ovicidal activity (Table 3).

Concerning mosquitocidal activity, both T. capitatus and M. vulgare oils gave 50% and 90% larvicidal activity at 100 and 200 ppm/12hrs, respectively. Moreover, both samples showed 50% and 90% pupicidal activity at 200 and 400 ppm/12hrs, respectively (Table 4).

Table 2Percentages of the different classes of constituents identified by GC/MS in the

essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L.

ClassPercentage

T. capitatus M. vulgare

Hydrocarbons 8.05 32.69

• Sesquiterpenes 7.26 32.48

• Non-terpenoid hydrocarbons 0.79 0.21

Oxygenated constituents 88.22 57.5

• Monoterpenes 9.15 0.51

• Sesquiterpenes 7.67 18.09

• Other oxygenated constituents 71.4 38.9

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SALAMA, M.M.; TAHER, E.E. & EL-BAHY, M.M. - Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 281-6, 2012.

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DISCUSSION

Among the most promising advances in the field of drug development is discovering new molecules or novel uses of the already available compounds with known safety and without any side effects. Thymol is a naturally occurring phenolic monoterpene, known for its antioxidant, anti-inflammatory, antimicrobial, antileishmanial, antimalarial, antiprotozoal, insecticidal and molluscicidal activities6,14. Phenolic compounds were proved to be useful in a variety of molluscicidal applications21. Thymol showed also considerable molluscicidal effect against B. alexandrina, Bulinus truncatus and Lymnaea natalensis14. An isomer of thymol, namely carvacrol, showed in vitro antifilarial activity but to a lesser extent than thymol25. Furthermore, some studies revealed that carvacrol has an antibacterial, antifungal, antiparasitic and antioxidants activities31. According to these results, two members of the Lamiaceae cultivated in Egypt (T. capitatus and M. vulgare) were screened for the presence of thymol in their essential oils. Both oils contain thymol in high content so they are screened for their molluscicidal and insecticidal activities as a way to relate these activities with the previous findings of thymol itself.

Both oils showed promising molluscicidal activity against B. alexandrina snails and mosquitocidal activity versus C. pipiens larvae and pupae. These activities may be attributed to thymol or its isomer carvacrol which agreed with other studies32. Volatile oils caused significant behavioral changes in snails with the most obvious sign of distress being muscular and spiral twisting of the body followed by crawling on one another. The nature and rapid onset of these responses showed that these oils probably contain neurotoxins that might be active at the neuromuscular system of exposed animals39. Similar metabolic disorders on life were revealed including egg laying, egg hatchability, hepatic cells damages, lack of smooth transmission at nerve junction, loss of muscular coordination and convulsions, then snails’ death1.

The results of the present study might be attributable also to the high sesquiterpene content of Marrubuim (50.57%) as compared to oil of Thymus (14.93%). Sesquiterpenoids are credited with various biological actions. They may kill snails as well as snail egg masses via contact poisoning resulting in killing the early egg embryos11. These findings are in accordance with other studies that mentioned sesquiterpenes as promising skin penetration enhancers with specific P-glycoprotein modulators that can reverse cellular multidrug resistance by inhibiting the drug efflux process26,34. Other studies also stated that antimicrobial properties of essential oils from M. vulgare are associated with their high contents of oxygenated compounds (46.21%)41. Researchers also reported mono and sesquiterpenoids as the major components of essential oils which are phenolic in nature40. It is therefore reasonable to assume that their antimicrobial activity might be related to the abundance of phenolic compounds21.

It was reported that methanolic extracts from many plants had the maximum larvicidal activity against C. pipiens larvae; moreover, plant extracts have been shown to have good effective control properties on mosquitoes besides being environmental friendly bio-pesticide20. On the contrary, other studies revealed that methanolic extracts were less effective than the extracts of other essential oils3. Researchers suggested that larvicides affect mosquito in one of three possible mechanisms; by physical flooding of the tracheal system or by toxicity specially that of volatile components and by interference with surface forces10. Different sensitivities of mosquito species towards different volatile oils has been also recorded3. Thymol, one of the major components of Lippia sidoides, was identified as an active component of the larvicidal action against Aedes aegypti causing 100% larval mortality16.

In conclusion, the essential oils of both M. vulgare and T. capitatus are biologically active agents against mosquitoes and snails, and effect showed significant time and dose dependant. Our results suggest that the essential oils of these plants may have a promising role in this regard.

RESUMO

Atividades moluscicida e mosquitocida de óleos essenciais de Thymus capitatus Hoff. et Link. e de Marrubium vulgare L.

A destilação por arraste a vapor dos óleos essenciais de partes aéreas de Thymus capitatus Hoff. et Link. e de Marrubium vulgare L. coletadas na costa norte do Egito resultaram em rendimento de 0,5% e 0,2%, respectivamente. Resultados de análises por cromatografia gasosa acoplada à espectrometria de massas de ambas as amostras possibilitaram a identificação de 96,27% e 90,19% dos constituintes químicos respectivamente de T. capitatus e M. vulgare. Verificou-se predomínio de constituintes oxigenados (88,22% para T. capitatus e 57,50% para M. vulgare, principalmente fenóis, como carvacrol (32,98%) e timol (32,82%) no óleo essencial de T. capitatus, e timol (34,55%) no óleo essencial de M. vulgare. Avaliou-se a atividade dos óleos essenciais obtidos contra adultos e ovos de Biomphalaria alexandrina, bem como em larvas e pupas de Culex pipiens. A CL

50 e CL

90 do óleo essencial de T. capitatus em

moluscos adultos foi respectivamente 200 e 400 ppm/3hrs, enquanto para o óles essencial de M. vulgare verificou-se CL

50 e CL

90 de 50 e 100 ppm/3hrs,

respectivamente. Além disso, M. vulgare apresentou atividade ovicida, com CL 100 de 200 ppm/24 horas, enquanto o óleo essencial de T. capitatus não demonstrou atividade ovicida. Verificou-se ainda atividade mosquitocida,

Table 4Results of larvicidal activity of the essential oils of Thymus capitatus

Hoff. et Link. and Marrubium vulgare L. against Culex pipiens after 12 h exposure period

Essential oilLarvae Pupae

LC50

LC90

LC50

LC90

T. capitatus 100 ppm 200 ppm 100 ppm 200 ppm

M. vulgare 200 ppm 400 ppm 200 ppm 400 ppm

Table 3Results of molluscicidal activity of essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. against Biomphalaria alexandrina after 3hr

exposure period

Essential oilAdult snails Eggs

LC50

LC90

LC100

T. capitatus 200 ppm 400 ppm No effect

M. vulgare 50 ppm 100 ppm 200ppm*

* After 24 hr

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SALAMA, M.M.; TAHER, E.E. & EL-BAHY, M.M. - Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. et Link. and Marrubium vulgare L. Rev. Inst. Med. Trop. Sao Paulo, 54(5): 281-6, 2012.

285

com CL50

e CL90

de 100 e 200 ppm/12hrs respectivamente para larvas, e 200 e 400 ppm/12hrs contra pupas de C. pipiens.

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11. Cornwell PA, Barry BW. Sesquiterpene components of volatile oils as skin penetration enhancers for the hydrophilic permeant 5-fluorouracil. J Pharm Pharmacol. 1994;46:261-9.

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13. Egyptian Pharmacopeia. General Organization for Governmental Printing office. Cairo: Egyptian Government; 2005. Available from: http://www.egypt.gov.eg/arabic/default.asp

14. El-Din AT. Molluscicidal effect of three monoterpenes oils on schistosomiasis and fascioliasis vector snails in Egypt. J Egypt Soc Parasitol. 2006;36:599-612.

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16. Govindarajan M, Sivakumar R, Rajeswari M, Yogalakshmi K. Chemical composition and larvicidal activity of essential oil from Mentha spicata (Linn) against three mosquito species. Parasitol Res. 2012;110:2023-32.

17. Jennings W, Shibamoto T. Qualitative analysis of flavor and fragrances volatiles by glass capillary gas chromatography. 2nd ed. New York: Academic Press; 1980.

18. Khanuja SPS, Srivastava S, Shasney AK, Darokar MP, Kumar TRS, Agarwal KK, et al. Inventors. Formulation comprising thymol useful in the treatment of drug resistant bacterial infections. United States Patent 6,824,795. 2004 Nov 30.

19. King CH, Dangerfield-Cha M. The unacknowledged impact of chronic schistosomiasis. Chronic Illn. 2008;4:65-79.

20. Kovendan K, Murugan K, Panneerselvam C, Mahesh Kumar P, Amerasan D, Subramaniam J, et al. Laboratory and field evaluation of medicinal plant extracts against filarial vector, Culex quinquefasciatus Say (Diptera: Culicidae). Parasitol Res. 2012;110:105-15.

21. Lahlou M. Study of the molluscicidal activity of some phenolic compounds: structure-activity relationship. Pharm Biol. 2004;42:258-61.

22. Lemos TLJ, Monte FJQ, Matos FJA, Barbosa RCB, Lima EO. Chemical composition and antimicrobial activity of essential oils from Brazilian plants. Fitoterapia, 1992;63:266-8.

23. Mansour SA, Messeha SS, El-Gengaihi SE. Botanical biocides. 4. Mosquitocidal activity of certain Thymus capitatus constituents. J Nat Toxins. 2000;9:49-62.

24. Mansour SA, El-Sharkawy AZ, Ali AR. Botanical biocides.12.Mosquitocidal activity of citrus peel oils with respect to their limonene content. Egypt J Nat Toxins. 2004;1:111-34.

25. Mathew N, Misra-Bhattacharya S, Perumal V, Muthuswamy K. Antifilarial lead molecules isolated from Trachyspermum ammi. Molecules. 2008;13:2156-68.

26. Muñoz-Martinez F, Lu P, Cortés-Selva F, Pérez-Victoria JM, Jiménez IA, Ravelo AG, et al. Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance. Cancer Res. 2004;64:7130-8.

27. Oparaocha ET, Iwu I, Ahanakuc JE. Preliminary study on mosquito repellent and mosquitocidal activities of Ocimum gratissimum (L.) grown in eastern Nigeria. J Vector Borne Dis. 2010;47:45-50.

28. Ottesen EA, Duke BO, Karam M, Behbehani K. Strategies and tools for the control/elimination of lymphatic filariasis. Bull World Health Organ. 1997;75:491-503.

29. Ranson H, Rossiter L, Ortelli F, Jensen B, Wang X, Roth CW, et al. Identification of a novel class oil of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochem J. 2001;359(pt 2):295-304.

30. Raymond M. Présentation d’un programme d’analyse log-probit pour micro-ordinnateur. Cah ORSTOM Sér Ent Méd Parasitol. 1985;23:117-21.

31. Singh B, Patial J, Sharma P, Chandra S, Kaul P, Maity S. Role of acidity for the production of carvacrol from carvone over sulfated zircona. Indian J Chem Technol. 2011;18:21-8.

32. Tasdemir D, Kaiser M, Demirci F, Baser KHC. Essential oil of Turkish Origanum onites L. and its main components, carvacrol and thymol show potent antiprotozoal activity without cytotoxicity. Planta Med. 2006;72:1006.

33. Taubes GA. Mosquito bites back. New York Times Magazine. 1997;August 24:40-6.

34. Williams AC, Barry BW. Terpenes and the lipid-protein-partitioning theory of skin penetration enhancement. Pharm Res. 1991;8:17-24.

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39. Yadav RP, Singh A. Efficacy of Euphorbia hirta latex as plant derived molluscicides against freshwater snails. Rev Inst Med Trop Sao Paulo. 2011;53:101-6.

40. Youssif, RS, Shaalan EA. Mosquitocidal activity of some volatile oils against Aedes caspius mosquitoes. J Vector Borne Dis. 2011;48:113-5.

41. Zarai Z, Kadri A, Ben Chobba I, Ben Mansour R, Bekir A, Mejdoub H, et al. The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia. Lipids Health Dis. 2011;10:161.

Received: 23 December 2011Accepted: 3 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):287-292, September-October, 2012doi: 10.1590/S0036-46652012000500009

Department of Epidemiology, Faculdade de Saúde Pública, University of São Paulo, São Paulo, SP, Brazil.Correspondence to: Mauro Toledo Marrelli. E-mail: [email protected]

gENETIC CONTROL OF MOSQUITOES: POPULATION SUPPRESSION STRATEgIES

André Barretto Bruno WILKE & Mauro Toledo MARRELLI

SUMMARY

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito’s great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

KEYWORDS: Mosquito; Genetic control; SIT; RIDL; Field tests.

INTRODUCTION

Arthropods act as vectors for many human agents that cause several diseases30. Over the last two decades, morbidity and mortality from malaria, dengue fever, West Nile Virus among other pathogens are an increasing Public Health problem. Malaria has been increasing due to deteriorating health systems, increased mosquito resistance to insecticides, parasite resistance to anti-malaria drugs and slow progress in vaccine development28. In 2008 malaria was endemic in 109 countries and almost 3.3 billion people were at risk worldwide. It is estimated that in 2008 there were 863,000 deaths from about 243 million cases40. Dengue fever, including dengue hemorrhagic fever and dengue shock syndrome, is a rapidly emerging arthropod-borne viral disease threatening one-third of the world’s population with an estimated 50-100 million new infections per year13,18,33,41. Aedes aegypti, the main dengue vector, is an invasive species spread inadvertently around the world by human trade and travel and it is now distributed widely in tropical and subtropical regions, most notably in urban environments where it has adapted to breed in artificial containers and refuse. Finding and treating sufficient numbers of mosquito breeding sites as a measure of control is extremely

challenging or impossible even for the most well-funded and organized programs.

The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There is currently no vaccine or specific therapeutic drug available for dengue; therefore, control focuses on the mosquito. Bed nets are largely ineffective against this day biting mosquito, making source reduction and space spraying the mainstays of control. New approaches and vector control tools are urgently needed11,24,26,31,39.

Mosquito control measures have failed to achieve their goals, mostly because of the mosquito’s great reproductive capacity and genomic flexibility36. These two characteristics are exemplified by two observations. First of all, mosquito and other insects are well-known for developing resistance to insecticides and its resistance has been reported in most major insect disease vectors and against every class of chemical insecticide and insecticidal crops30,34, and chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental

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impacts22,37. The second is the existence of a variety of closely related species that form complexes of cryptic species, some of which seem to be undergoing speciation in the process of adapting to an environment modified by man10. Resistance to insecticides has led to serious mosquito control problems, contributing to the resurgence of mosquito-borne diseases.

In light of this type of problem, other strategies for mosquito control must be considered, including genetic control (use of sterile mosquitoes and related techniques). The sterile insect technique is a species-specific, effective and environmentally friendly technique of insect control that has been widely used, and has succeeded in controlling agricultural pests and, in certain cases, mosquito vectors12,35,42. The aim of this review is to revisit strategies of SIT-based genetic control of mosquito populations and the recent advances in molecular biology and field tests that promise to control vectored diseases.

STERILE INSECT TECHNIQUE (SIT)

The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population control24,32. In 1955, KNIPLING proposed the concept of introducing sterile insects into the population as a form of controlling pests with agricultural importance. SIT is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects into a given target area33 (Fig. 1). Any successful mating with the sterile insect will result in no offspring. If enough sterile insects are released the population will decline24,43. Reduction or elimination of vector populations will tend to reduce or eliminate transmission of vector-borne diseases and has been an effective method of disease control in many regions31.

One of the major advantages of SIT over other techniques, such as insecticides, larvicides, and breeding site removal is that the males are very good at seeking out females of the same species and the technique becomes more effective as the population is reduced3,5,42. The Sterile Insect Technique is amongst the most non-disruptive pest control methods. Unlike some other biologically-based methods, it is species specific, does not release exotic agents into new environments and does not even introduce new genetic material into existing populations as the released organisms are not self-replicating21. The paradigm for this methodology was the successful elimination of Cochliomyia hominivorax (the causing agent of myiasis) from Southern United States, Mexico,

and Central America. This area is currently protected from reinvasion from South-American flies by means of a barrier in Panama consisting of only a few sterile flies.

SIT ISSUES

A major difficulty with SIT as currently practiced is that the released sterile insects are required to compete for mates with wild insects8. The production process, however, and in particular the need to sterilize the insects by irradiation, causes a dramatic loss of competitive mating ability relative to wild type5,27. The magnitude of this problem varies from one species to another but the combination of these effects leads to a tenfold or greater reduction in effectiveness for some species1.

Another problem is that SIT relies on the release of large numbers of sterile insects, but in some cases the adult females may themselves be unwanted or even hazardous. Mass rearing facilities initially produce equal numbers of the two sexes, but generally try to separate and discard females before release5,15.

Due to the possibility of preferential mating between released sterile insects and the fact that released sterile females do not diminish populations, bisexual releases are far less effective and more expensive than male-only releases in introducing sterility into wild populations. For agricultural pests in which females cause no damage, sex separation systems are not essential, but highly desirable in terms of increased efficiency. For the early SIT programs for mosquitoes, especially Aedes aegypti, sexes were separated using differences in pupal size, principally to increase efficiency. Since released sterile females may repeatedly feed on humans and thus contribute to disease transmission, tolerance for females in releases by programs targeting vector species is likely to be much lower than for agricultural pests. For these vectors, SIT can, therefore, only be applied if some highly efficient way to exclude females is developed. It is surprising that, in the earliest comprehensive description of SIT against mosquito vectors, developing sex separation methods is given almost no mention. Several inventive methods for sex separation of mosquitoes based solely on naturally occurring biological differences between males and females have been used. These have had varying degrees of success and were implemented at different scales.

Highly successful, area-wide SIT programs have eliminated or suppressed a range of major veterinary and agricultural pests around the world. These programs can succeed on very large scales - the largest rearing facility alone produces around two billion sterile male Mediterranean fruit flies per week (~20 tons/week), primarily for use in California and Guatemala. For these pests, SIT is a proven, cost-effective strategy for eradication or suppression of target populations, or to protect areas against invasion or re-invasion.

For mosquitoes, the situation is much different, with variable successes and problems. About 20 field trials during the 1970s and 1980s demonstrated that the SIT could also be made to work against mosquitoes10,23. For example, Anopheles albimanus was successfully controlled in a field trial in El Salvador, using chemo-sterilized mosquitoes25. The fundamental properties of SIT are still highly attractive for mosquito control. This has led to a resurgence of interest in recent years, with several research groups trying to circumvent some of the technical limitations which prevented conventional SIT from becoming

Fig. 1 - Conventional SIT schedule: mass rearing of mosquitoes followed by manual sex

separation to assure that exclusively males are to be sterilized by ionizing radiation and

further released to mate with wild females resulting in no progeny. This procedure must be

repeated each and every time.

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a widespread approach following the early trials.

The use of SIT against mosquitoes is problematic, due mainly to the fitness costs and operational difficulty of irradiation, and the density-dependent nature of the target mosquito populations4,31. Distribution of mosquitoes may also be more problematic than for current SIT target species, for example, Ceratitis capitata (Medfly) and Cochliomyia hominivorax (New World Screwworm), because adult mosquitoes are less robust and more likely to suffer damage during transit and release3,32,38.

IMPROVING SIT: THE RIDL SYSTEM

It is clear that an effective Genetic System Mechanism (GSM) would reduce the cost and increase the efficiency of a SIT program. Various female-killing and sex-sorting genetic systems have been developed. So far, all the GSMs brought into use in factory mass production have relied on the linking of a dominant selectable marker to the male determining chromosome5. These issues could be mitigated by the use of insects engineered to carry specific unique traits, including conditional genetic sterilization or lethality that removes the need for irradiation15. The ability to use recombinant DNA to molecularly engineer insects opens the door to a wide array of techniques to control pests and improve beneficial species and, in particular, create strains to improve biocontrol methods such as the sterile insect technique (SIT)19.

The RIDL (Release of Insects Carrying a Dominant Lethal Gene) system proposed by THOMAS et al. (2000) consists of introducing a lethal dominant gene that could be under control of a female-specific promoter, such as that of vitellogenin gene. Expression of the lethal gene could be inactivated by treatment with tetracycline, allowing a colony to be maintained. When male and female separation is required, tetracycline is removed from the system, causing the death of all females (Fig. 2). The RIDL system is centered on the expression of tTA, a fusion protein that combines sequence-specific tetracycline-repressible binding to tRe, a tetracycline-response element, to a eukaryotic transcriptional activator. In the absence of tetracycline, this protein will bind to the tRe sequence, activating transcription from a nearby minimal promoter5

(Fig. 2).

When preparing mosquitoes for release, the repressor is inactivated and the lethal gene is expressed, causing the death of all females. When mating with wild females, males homozygous for the lethal gene will produce heterozygous progenies, of which only males will

survive. Releasing of Insects carrying a Dominant Lethal gene (RIDL technology) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility4. Transgenic males are homozygous for a dominant lethal gene. Mating with indigenous population results in offspring that are heterozygous for the lethal gene leading to the death of all females and hence eventual suppression of the population due to a decrease in its reproductive capacity (Fig. 3)20,38. Genetic control aims to achieve universal coverage by taking advantage of the male insect’s efficiency in locating and mating with females of the same species44.

Insects engineered to carry a female-specific lethal (or otherwise incapacitating) gene could be used to remove females prior to release3,14,23,38. A system based on a lethal gene (RIDL)38 that acts late in development would prevent mosquitoes from becoming adults, the only harmful life stage, yet enable them to survive and compete at the larval stage, when density-dependent competition occurs. Modeling this system predicts that fewer male mosquitoes of a late-lethal strain need to be released as compared to those carrying an early-lethal gene or irradiated strain to achieve an equivalent level of control of a target population7,14,16,17,32,38.

A female-lethal version of RIDL, with insects homozygous for one or more female-specific dominant lethal genetic constructs, has been constructed in several species14. F1 progeny of RIDL males and wild females inherit a dominant female-specific lethal gene; the F1 females die, thereby reducing the reproductive potential of the wild population, but the F1 males are viable and fertile. This provides a genetic sexing mechanism facilitating male only release, either by employing the female-lethal version of RIDL and withdrawing the repressor from the generation prior to release, or by combining a bisex-lethal system with female lethality (with an independent means of repressing or inducing lethality) to permit male only release of bisex-lethal strains designed to kill progeny of both sexes in the field (Fig. 3)5.

ECONOMIC DEPENDENCE

Economic cost-benefit analysis, which is needed to support use of novel interventions, is difficult because of lack of reliable data on the economic burden of disease for dengue and other neglected tropical diseases, and because of uncertainty around development and

Fig. 3 - RIDL system: once a stable strain of genetic modified mosquitoes with female specific

lethal gene is obtained, all that is need is to mass rear and remove the genetic repressor

(tetracycline), the lethal gene will kill all females leaving males ready to be released and

mate with wild females.

Fig. 2 - tTA and the tetracycline-repressible expression system. The tetracycline-repressible

transcriptional activator (tTA) protein is placed under a promoter control. When expressed,

the tTA protein binds to a specific DNA sequence, tetO, driving expression from an adjacent

minimal promoter which leads to expression of any sequence (the effector gene) placed

under the control of this minimal promoter. The combined effect is that the effector gene is

expressed in essentially the pattern of the promoter driving tTA. However, in the presence of

low concentrations of tetracycline, the tTA protein does not bind DNA and so expression of

the effector gene is prevented. (Modified from ALPHEY 2002)5.

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implementation costs. Ideally it would be possible to analyze not only the cost-effectiveness of the stand-alone novel strategy, but also to compare it with existing alternatives and to model its incorporation in integrated vector management (IVM) programs, and indeed integrated disease management programs including drugs and vaccines, where available40.

RIDL FIELD TESTS

Several different genetic approaches to mosquito control are being developed. These include approaches that are: Self-Limiting - repeated or recurrent releases are necessary to maintain the genetic construct in the target population. To have a significant epidemiological effect it will usually be necessary to release relatively large numbers of mosquitoes (inundative releases). Self-Sustaining - releases need to occur only once or a few times, and the construct will increase in frequency of its own accord and maintain itself at high frequency. Releases can often be of relatively fewer mosquitoes (inoculative releases)40.

As genetics-based population suppression (self-limiting) moves from laboratory to field, the lack of a clear regulatory framework for field use of modified mosquitoes is a significant challenge. This issue is not restricted to developing countries, or to strategies dependent on the use of recombinant DNA technology. Once regulatory frameworks are in place, risk assessments and public consultation also will be lengthy processes due the novelty of technologies and lack of experience by regulating agencies. The route to implementation of control programs based on these technologies is not obvious40.

Some issues must be fulfilled before a large scale mosquito release: (a) adult males must be known to mate with wild females at the release site, since laboratory cage fitness and competitiveness experiments of males does not ensure mating with wild type females; (b) sex separation lines improve efficiency and the effect of releasing programs, and are essential for mosquito vectors; (c) releasing methods must be suitable for all weather conditions anticipated, and are established and tested before control release programs start; (d) while female sterility provides an indicator of mating frequency, vector density is a more relevant indicator of the control effect on disease transmission; (e) mass rearing levels must be stable before releases begin; (f) releases must be programmed for maximum effect into suppressed populations; (g) wide control areas with similar monitoring must be available for comparison during suppression strategies development; (h) dispersion and mating characteristics are essential factors for quality control to assess the fitness of release material; (i) isolation of the test areas must be conducted and demonstrated conclusively for all weather conditions reasonably expected; (j) independent monitoring is essential to demonstrate effectiveness before release programs; (k) political stability, and healthy relationships with the public, press and political entities are essential for sustainability of control efforts10.

The self-limiting nature of sterile insects (whether sterilized by radiation, Wolbachia/CI (IIT), or RIDL genetic engineering) tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies (including Wolbachia - and gene driver-based replacement strategies). They are also closer to field use, so might be appropriate to consider first. WHO/TDR funding for capacity-building and guidance

development and this technical consultation are all steps in the right direction40.

There have been significant advances in population method using strains of Aedes aegypti homozygous for a dominant lethal genetic system1,5. Efficacy testing of RIDL strains has already been carried out in laboratory and/or contained “semi-field” conditions in Brazil, Malaysia, Mexico and Cayman Islands9.

Under an envisioned RIDL release program, it would be important to detect any resistance in the pest population that was capable of having a significant detrimental impact on the program’s effectiveness in time to take remedial action. Our population dynamic models predict a clear change in the pattern of pest as such resistance begins to spread. A properly designed monitoring strategy would be alert to such changes. Wild-caught individuals could then be used to screen for cross-resistance against a panel of available RIDL strains that use different lethal mechanisms, so that operations (or further trials, if this occurred before full implementation) could switch to deploying an appropriate alternative RIDL line before significant lasting loss of efficacy could result2,7.

PERSPECTIVES

The prospect of genetic control methods against mosquito vectored human diseases is rapidly approaching a reality. With the potential of a promising additional method for dengue and or malaria disease control, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

A potential concern about releasing GM insects into the wild is that the inserted DNA may have unforeseen consequences. This has particularly been raised regarding strategies that aim to replace a wild population, for example with a version engineered to be unable to transmit a pathogen. One advantage claimed for genetic SIT strategies over population replacement strategies is that autocidal engineered insects are programmed to die and therefore the lethal genetic construct should die out if releases cease. This relies on the construct having some fitness costs even if it does not retain its efficacy. It has been shown that resistance to the construct might significantly increase its frequency.

In theory, there are combinations of genetic properties of resistance for which the construct could eventually be driven to fixation; that scenario is highly unlikely to be played out in real life because the release program could be stopped if substantial resistance were detected, long before the RIDL construct became common, and the wild type would reinvade through immigration6.

Endemic disease countries need international guidance to assess the risks and benefits of using genetically modified mosquitoes. Since they may reach different conclusions about using these mosquitoes the World Health Organization (WHO) and its partners are in process to provide best practice guidance to the endemic countries on these issues (Mosqguide)29. Communicating mosquito and disease biology, and risks and benefits associated with specific novel control strategies, is resource intensive and has no obvious endpoint40.

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RESUMO

Controle genético de mosquitos: estratégias de supressão de populações

Ao longo das duas últimas décadas, morbidade e mortalidade da malária e dengue e outros patógenos tem se tornado cada vez mais um problema de Saúde Pública. O aumento na distribuição geográfica de seus respectivos vetores é acompanhada pela emergência de doenças em novas áreas. Não estão disponíveis drogas específicas suficientes e não há vacinas específicas para imunizar as populações alvo. As medidas de controle de mosquitos atuais falharam em atingir os objetivos propostos, principalmente devido à grande capacidade reprodutiva dos mosquitos e alta flexibilidade genômica. O controle químico se torna cada vez mais restrito devido a sua potencial toxicidade aos seres humanos, mortalidade de organismos não alvos, resistência a inseticida além de outros impactos ambientais. Novas estratégias de controle são necessárias. A técnica do inseto estéril (SIT) é um método de supressão populacional espécie específico e ambientalmente amigável, baseia-se na criação em massa, esterilização mediante irradiação e liberação de um grande número de insetos machos. Liberar insetos carregando um gene letal dominante (RIDL) oferece uma solução a muitas limitações impostas pela técnica do inseto estéril (SIT) que limitaram sua aplicação em mosquitos e ainda assim mantém suas características de ambientalmente amigável e espécie específica. A natureza auto-limitante de mosquitos estéreis tende a deixar alguns empecilhos para uso no campo, de certa forma, menos desafiadores quando comparados a sistemas auto-propagação, característicos de estratégias de substituição de população. Sistemas auto-limitantes estão mais próximos para uso no campo, portanto pode ser apropriado considerá-lo primeiro. A perspectiva de métodos de controle genéticos contra mosquitos vetores de doenças que acometem humanos está rapidamente se tornando uma realidade, muitas decisões terão de ser tomadas em âmbito nacional, regional e internacional com relação a aspectos étnicos, sociais, culturais e de biossegurança para o uso e liberação destes métodos de controle de vetores.

ACKNOWLEDGMENTS

The authors would like to thank FAPESP for the financial support to MTM and MLC laboratory. ABBW is fellow of FAPESP.

REFERENCES

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15. Fu G, Lees RS, Nimmo D, Aw D, Jin L, Gray P, et al. Female-specific flightless phenotype for mosquito control. Proc Natl Acad Sci USA. 2010;107:4550-4.

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20. Heinrich JL, Scott MJ. A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc Natl Acad Sci USA. 2000;97:8229-32.

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25. Lofgren CS, Dame DA, Breeland SG, Weidhaas DE, Jeffery G, Kaiser R, et al. Release of chemosterilized males for the control of Anopheles albimanus in El Salvador. 3. Field methods and population control. Am J Trop Med Hyg. 1974;23:288-97.

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27. Marrelli MT, Moreira CK, Kelly D, Alphey L, Jacobs-Lorena M. Mosquito transgenesis: what is the fitness cost? Trends Parasitol. 2006;22:197-202.

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28. Moreira LA, Ghosh AK, Abraham EG, Jacobs-Lorena M. Genetic transformation of mosquitoes: a quest for malaria control. Int J Parasitol. 2002;32:1599-605.

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40. WHO/TDR. Planning meetings on Progress and Prospects for the Use of Genetically Modified Mosquitoes to Prevent Disease Transmission: Meeting 1. Technical Consultations on the Current Status and Planning for Future Development. Geneva, May 4-6, 2009.

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42. Wilke AB, Nimmo DD, St John O, Kojin BB, Capurro ML, Marrelli MT. Mini-review: genetic enhancements to the sterile insect technique to control mosquito populations. AsPac J Mol Biol Biotechnol. 2009;17:65-74.

43. Wilke AB, Gomes AC, Natal D, Marrelli MT. Control of vector populations using genetically modified mosquitoes. Rev Saude Publica. 2009;43:869-74.

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Received: 31 October 2011Accepted: 17 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):293-297, September-October, 2012doi: 10.1590/S0036-46652012000500010

(1) Department of Gastroenterology, Hospital Nossa Senhora da Conceição, Porto Alegre, RS, Brazil.(2) Department of Pathology, Hospital Nossa Senhora da Conceição, Porto Alegre, RS, Brazil.Correspondence to: Marcelo Campos Appel-da-Silva, R. Comendador Caminha 250/902, 90430-030 Porto Alegre, RS, Brasil. Tel./Fax: +5551-3222-6891. E-mail: [email protected]

CASE REPORT

WHIPPLE’S DISEASE: RARE DISORDER AND LATE DIAgNOSIS

Viviane Plasse RENON(1), Marcelo Campos APPEL-DA-SILVA(1), Rafael Bergesch D’INCAO(1), Rodrigo Mayer LUL(1), Luciana Schmidt KIRSCHNICK(2) & Bruno GALPERIM(1)

SUMMARY

Whipple’s disease is a rare systemic infectious disorder caused by the bacterium Tropheryma whipplei. We report the case of a 61-year-old male patient who presented to emergency room complaining of asthenia, arthralgia, anorexia, articular complaints intermittent diarrhea, and a 10-kg weight loss in one year. Laboratory tests showed the following results: Hb = 7.5 g/dL, albumin = 2.5 mg/dL, weight = 50.3 kg (BMI 17.4 kg/m2). Upper gastrointestinal endoscopy revealed areas of focal enanthema in the duodenum. An endoscopic biopsy was suggestive of Whipple’s disease. Diagnosis was confirmed based on a positive serum polymerase chain reaction. Treatment was initiated with intravenous ceftriaxone followed by oral trimethoprim-sulfamethoxazole. After one year of treatment, the patient was asymptomatic, with Hb = 13.5 g/dL, serum albumin = 5.3 mg/dL, and weight = 70 kg (BMI 24.2 kg/m2). Whipple’s disease should be considered a differential diagnosis in patients with prolonged constitutional and/or gastrointestinal symptoms. Appropriate antibiotic treatment improves the quality of life of patients.

KEYWORDS: Whipple’s disease; Tropheryma whipplei; Malabsorption syndromes.

INTRODUCTION

Whipple’s disease is a rare multisystemic infection caused by Tropheryma whipplei - a gram-positive bacterium belonging to the phylum Actinobacteria and a member of the order Actinomycetales5. It is recognized as an important bacterial cause of malabsorption, mostly affecting middle-aged Caucasian men. Its classic clinical course has three stages: (1) nonspecific prodromal symptoms, including migratory polyarthralgia (mainly in the large joints); (2) typical abdominal symptoms: pain, diarrhea, weakness, and weight loss; and (3) generalized stage, including steatorrhea, cachexia, lymphadenopathy, hyperpigmentation, and cardiovascular, pulmonary, and neurological dysfunctions11,14. Laboratory tests may provide several nonspecific findings that in combination can be suggestive of diagnoses such as: hypoalbuminemia, elevated erythrocyte sedimentation rate, and anemia8. Diagnosis is based on the presence of typical signs and symptoms and identification of Tropheryma whipplei in the histopathological examination of duodenal biopsies. When there is clinical suspicion without histological findings, the use of molecular biology tests is recommended, especially polymerase chain reaction (PCR)6. Treatment consists of induction of antibiotic therapy followed by a maintenance regimen for a prolonged period11.

CASE REPORT

Sixty-one-year-old Caucasian male patient admitted in March 2010 to the emergency room for investigation of 10-kg weight loss, asthenia, anemia, and intermittent diarrhea, two to three bowel movements a day, without blood, mucus or pus for about one year. His previous medical history included 10 years of prodromal symptoms associated with depression and migratory arthralgia. During this period, the patient was seen by several physicians, who could not establish a diagnosis. At admission, the patient was thin, weighing 50.3 kg (BMI = 17.4 kg/m2), and presenting with lower limb edema and little ascites. Laboratory tests showed iron-deficiency anemia (Hb = 7.5 g/dL, ferritin = 111 ng/mL, transferrin saturation = 7.8%, serum iron = 14 ug/dL) and hypoalbuminemia (2.5 g/dL). In relation to the imaging studies, a chest X-ray revealed pericardial calcification and bilateral pleural effusion. An abdominal ultrasound showed hepatomegaly, distention of hepatic veins and inferior vena cava, and a small amount of free fluid in the abdominal cavity. A Doppler ultrasound demonstrated an ejection fraction of 40%, left ventricular contractile dysfunction, thickened aortic valve with moderate regurgitation and mild mitral valve regurgitation. Anti-HIV, HBsAg, anti-HCV, antinuclear antibodies, rheumatoid factor, and TSH were investigated and showed negative or within normal limit results. An upper gastrointestinal endoscopy showed focal enanthema of the duodenal mucosa. Biopsies were performed and

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revealed spongy macrophages in the lamina propria using hematoxylin-eosin staining and periodic acid-Schiff (PAS) staining. These findings are suggestive of Whipple’s disease (Fig. 1). Given the abnormal results described above, serum and CSF PCR were requested to investigate the presence of Tropheryma whipplei. Serum PCR was positive for Tropheryma whipplei, whereas CSF PCR was negative for this bacterium. Treatment was started with intravenous ceftriaxone 2 g/day for two weeks, followed by oral trimethoprim-sulfamethoxazole 800/160 mg twice daily for a period of 12 months. It is noteworthy that after 14 days of treatment, the patient had normalization of bowel habits and started to show progressive weight increase.

After hospital discharge, the patient received outpatient follow-up for 16 months. There was significant clinical and laboratory improvement after one year of treatment. At the last follow-up visit, five months after treatment completion, the patient remained asymptomatic, with albumin level of 5.3 g/dL, Hb level of 13.5 g/dL, and weighing 70 kg.

DISCUSSION

Whipple’s disease is a rare disorder. Its estimated annual prevalence is 1:1,000,000, mostly affecting middle-aged Caucasian men5. Some studies have shown a higher prevalence among rural residents8,13. Tropheryma whipplei is found in the soil (which could explain a higher prevalence among farmers), in sewage contaminated water, in the oral cavity and feces of healthy individuals (although there is no evidence of interpersonal transmission)13. There is evidence that this organism may be ubiquitous in humans, since there are studies using PCR amplification of Tropheryma whipplei from samples of saliva, gastric juice, and duodenal biopsies of patients without Whipple’s disease5.

After infection, the bacterium invades the whole body, including the intestinal epithelium, lymphatic and capillary endothelium, synovium, heart, lungs, liver, brain, eyes, and skin. There is failure of immune response to Tropheryma whipplei in these sites, suggesting that such

deficiency has a role in the occurrence of the disease8. Several immune system abnormalities have been associated with Whipple’s disease. These alterations may be transient during the exacerbation period or even typical of the host of the bacterium. Among the innate abnormalities, the association of Whipple’s disease with the human leukocyte antigen (HLA) has been described since 197915,24, when the association with HLA B27 was demonstrated even in the absence of ankylosing spondylitis15. Approximately 26% of patients have the HLA class I histocompatibility antigen and HLA B27 three to four times higher than expected, although this characteristic is not found in all populations studied8,9,13. Because of the low incidence of the disease, this association has been studied only in small case series, generating conflicting data1.

This immune deficiency appears to be specific, because the patients are not predisposed to infection with other bacteria. In addition, IgG antibodies against Tropheryma whipplei are detected in approximately 70% of healthy individuals. Whipple’s disease is rare, but apparently Tropheryma whipplei is not8.

The classic disease can be divided into three stages: (1) nonspecific prodromal symptoms, mainly joint manifestations (e.g., arthritis, arthralgia, migratory polyarthralgia); (2) gastrointestinal symptoms such as diarrhea, weight loss, and weakness; and (3) generalized symptoms, including anemia, steatorrhea, hypoalbuminemia, and neurological and/or cardiovascular manifestations11,14. The most frequent extra-intestinal manifestations are joint disease and constitutional symptoms (mainly weight loss, which is present in more than 2/3 of cases in some series). However, the following systems may also be affected in some way during the course of the disease in order of frequency: central nervous system (CNS), cardiovascular system, mucocutaneous system, pleuropulmonary system, and vision13. The mean time between the onset of prodromal symptoms and the advance stage is approximately six years8,10. However, despite this being the most common sequence, there are cases of isolated Whipple’s disease without gastrointestinal disorders5. Approximately 15% of patients do not have typical symptoms and signs of the disease8,10.

Fig. 1 - (A) Biopsy of the second portion of the duodenum, stained with periodic acid-Schiff (PAS), 10x magnification; and (B) stained with hematoxylin-eosin, 40x magnification, showing

macrophages in the lamina propria.

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Therefore, the Whipple’s disease should be considered a differential diagnosis in many clinical situations: malabsorption with involvement of the small intestine (tropical sprue, celiac disease, sarcoidosis, and lymphoma), inflammatory rheumatic disease (seronegative arthritis), Addison’s disease, conjunctive tissue disease, and a variety of neurological diseases. Patients receiving immunosuppressive therapy, such as corticosteroids and tumor necrosis factor antagonists, may have a faster clinical progression of Whipple’s disease8.

The central nervous system (CNS) may be affected in up to 50% of patients in combination with the gastrointestinal tract or even alone5,12,17. CNS manifestations are characterized by slowly progressive dementia, ophthalmoplegia, headache, myoclonus, hypothalamic dysfunction, and a pathognomonic movement - oculofacial-skeletal myorhythmia, dementia, ophthalmoplegia, and myoclonus are the most common1,17. Some of the most unusual symptoms are seizures and signs similar to ischemic brain syndrome2,22. Despite the intense gastrointestinal symptoms, our patient did not have any neurological manifestations or positive CSF PCR.

The most common gastrointestinal symptom of Whipple’s disease is weight loss, often associated with diarrhea. Abdominal pain, hepatosplenomegaly, and, occasionally, hepatitis may occur. Ascites has been reported in 5% of patients8.

Joint involvement is the most common extra-intestinal symptom, occurring in approximately 65-90% of patients with classic disease, and it usually precedes gastrointestinal symptoms in up to 63% of affected individuals8,19. Intermittent migratory arthralgia and/or arthritis are the most common manifestations, usually in combination with polyarthritis, affecting the peripheral joints8,20.

Cardiac involvement is a common manifestation in Whipple’s disease and it is usually present with endocardial or valvular disease or, rarely, congestive heart failure13. After a long duration of nonspecific symptoms, our patient reached the generalized stage of the disease with uncommon manifestations such as pleural effusion, ascites, and heart failure.

In the present case report, the patient had clinical and laboratory manifestations consistent with Whipple’s disease, and the biopsy of the second portion of the duodenum showed typical findings, which confirmed the diagnosis.

Because of its broad spectrum of clinical manifestations, Whipple’s disease is very similar to other chronic inflammatory diseases5,8. This factor added to its low incidence in the general population makes the diagnosis difficult, usually leading to a late diagnosis. In the case described herein, our patient had prodromal symptoms for a period of 10 years. His diagnosis was only established after the onset of the abdominal symptoms and the malabsorption syndrome.

Diagnosis is often made based on a biopsy of small intestine or proximal jejunum, as these regions are commonly affected in symptomatic patients, even in the early stage of the disease. The infiltration of the lamina propria of the small intestine by macrophages filled with bacilliform bodies, positive PAS, and diastase-resistant structures, accompanied by dilated lymphatic ducts, are specific and diagnostic aspects of Whipple’s disease5.

In some cases, the diagnosis is established without the presence of classic signs when typical histological lesions are found in duodenal biopsies stained with PAS10. Nevertheless, the presence of macrophages with positive PAS material is not completely specific for Whipple’s disease, since macrophages are also found in patients with infections caused by Mycobacterium avium-intracellulare, Rhodococcus equi, Bacillus cereus, Corynebacterium, Histoplasma, or certain types of fungi. Failure to obtain positive intestinal biopsies does not invalidate the diagnosis because the disease can be restricted to the submucosa and, therefore, it may not be diagnosed by a biopsy of the mucosa13.

Thus, the diagnosis should be made based on clinical and endoscopic suspicion, and confirmed by duodenal biopsy, associated with molecular biology methods (PCR), immunohistochemistry and serological methods5,24. PCR is an important diagnostic tool for this disease because it has high sensitivity and specificity and it is useful mainly in typical cases and/or when there is the possibility of histological confirmation of the diagnosis23. Nevertheless, PCR for Tropheryma whipplei in the serum is not a useful test for the diagnosis once a negative result does not exclude the diagnosis. On the other hand, false positive results in PCR can be seen in cases of intestinal colonization by T. whipplei. That’s why some authors recommend that the diagnosis should be established based on the combination of two positive tests, usually the histological analysis of duodenal biopsy with PAS staining, along with PCR or immunohistochemistry25.

Electron microscopy has contributed in a decisive way, since 1961, for the detection of the bacillus. Although electron microscopic examination is considered “gold standard” for confirmation of diagnosis8, it is a more expensive and demanding method since it involves complex laboratory procedures, which are not always available. Therefore, electron microscopy is only used to clarify those cases where PCR and/or histology provide doubtful results23.

Immunohistochemistry is a tool that can help establish the diagnosis with good sensitivity8,23. This technique improves the histologic examination using T. whipplei-specific antibodies showing greater sensitivity than PAS staining, being able to identify T. whipplei using immunohistochemistry on tissues in which PAS staining provided negative results4,16.

When the diagnosis of Whipple’s disease is established, the cerebrospinal fluid should be tested with PCR, even in the absence of neurological signs, it has important implications for therapy and prognosis8,24. In patients with CNS involvement, CSF is usually normal or shows mild pleocytosis, while the PCR result is usually positive18,26.

Antibiotic therapy should be started early, preferred medications that have good penetration into the central nervous system, preventing neurologic relapses, given the frequent involvement of the CNS and the fact that this is the most frequent site of relapses5,8,9,23,24. If not treated, Whipple’s disease can have a fatal outcome5,8,11,24. Thus, the antibiotic selection was based on the fact that trimethoprim/sulfamethoxazole is an antibiotic that crosses the blood-brain barrier, with probability of being effective in the CNS involvement. Various antibiotic regimens have been tried, from chloramphenicol to tetracyclines, penicillin alone, penicillin and streptomycin, ampicillin, erythromycin, third generation cephalosporins5,8,23,24. Some studies suggest initial treatment

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with penicillin and streptomycin intravenously for two weeks. Another possible regimen is ceftriaxone (2 g intravenous/day) during the first two weeks followed by oral administration of trimethoprim/sulfamethoxazole for one year5,8,24.

Whipple’s disease is an infectious disease with an excellent clinical response within a few weeks after initiation of antibiotic therapy5,8,23. The typical evolution of the treatment is the improvement of the classic symptoms (such as arthralgia and diarrhea) within two weeks after appropriate treatment is initiated. However, about 9-15%15 of patients develop failure during or relapse after treatment with trimethoprim-sulfamethoxazole8. Jarisch-Herxheimer reaction has been reported after initiation of antibiotic therapy, with symptoms similar to systemic inflammatory response syndrome, especially in patients with CNS disease or receiving immunosuppressive therapy17.

According to different studies, the decision to discontinue therapy seems to depend on clinical and laboratory remission, as well as on decreased amount of PAS-positive macrophages and absence of free bacilli in follow-up biopsies after apparent clinical remission5,23. Also in relation to this fact, there are doubts related to the number of biopsies needed for a strict follow-up, as well as regarding the time interval between each procedure. Usually, if there is good clinical response, biopsy may be repeated six and twelve months after diagnosis, although some studies suggest longer time intervals5.

Conversely, there is evidence that PAS-positive macrophages may remain in the lamina propria for several years after complete clinical remission, representing degraded bacterial material, which might cause a positive test result after treatment show a false positive result instead of a relapse5,23. Thus its validity in monitoring the disease is controversial. However, it has a 100% negative predictive value, meaning that the most important use of PCR seems to be to confirm the diagnosis after histology and exclude disease relapse after apparently effective therapy despite histological changes. Thus a negative PCR may exclude disease relapse (100% negative predictive value), whereas the visualization of intact bacilli by means of electron microscopy may be a sign of active disease5. In cases with neurological manifestations, antibiotic therapy should only be discontinued when the PCR is negative in the CSF and the duodenum23.

In addition, all patients should be monitored for life because relapses may occur after long-term remission with severe involvement of the CNS1,15.

Relapses of Whipple’s disease may appear several years after cessation of therapy, even when the initial treatment was considered effective. Even with an appropriate clinical treatment, there are reports of clinical relapse in about 2-33% of cases after a mean period of five years, and the CNS is the most frequent site involved8. It occurs most frequently in patients with CNS involvement and in patients treated with a single type of antibiotic or with antibiotics that do not cross the blood brain barrier (such as tetracycline or oral penicillin)1,23.

In cases of failure or relapse, other antibiotic regimens are suggested. In patients without neurological involvement, doxycycline (100 mg twice a day) in combination with hydroxychloroquine (600 mg/day) without induction could be used. In patients with neurologic manifestations or

positive CSF PCR, the regimen mentioned above may be associated with sulfadiazine7,24.

RESUMO

Doença de Whipple: patologia rara e de diagnóstico tardio

Doença de Whipple é uma rara infecção sistêmica causada pelo Tropheryma whipplei. Caracteriza-se por fase prolongada de sintomas inespecíficos, levando longo período até o seu diagnóstico. Sem tratamento, pode ser grave e fatal, mas com antibioticoterapia tem ótima resposta clínica e laboratorial. Relatamos o caso de paciente masculino, 61 anos, internado por astenia, anorexia, diarréia intermitente e perda de 10 kg em um ano. Apresentava-se com hemoglobina (Hb) 7,5 g/dL, albumina de 2,5 mg/dL, peso 50,3 kg (IMC 17,4). Endoscopia digestiva alta com áreas de enantema focal da mucosa duodenal e biópsia compatível com doença de Whipple. O diagnóstico foi confirmado com PCR sérica positiva, sendo instituído tratamento com ceftriaxone seguido de sulfametoxazol-trimetropim. Após um ano de tratamento, encontrava-se assintomático, com Hb 13,5 g/dL, albumina sérica de 5,3 mg/dL e peso de 70 kg. Doença de Whipple deve fazer parte da lista de diagnósticos diferenciais em pacientes com sintomas constitucionais e/ou com queixas gastrointestinais com evolução prolongada. O tratamento antibiótico pode curar a infecção, recuperando a qualidade de vida do paciente.

REFERENCES

1. Abreu P, Azevedo E, Lobo L, Moura CS, Pontes C. Doença de Whipple e o sistema nervoso central. Acta Med Port. 2005;18:199-208.

2. Anderson M. Neurology of Whipple’s disease. J Neurol Neurosurg Psychiatry. 2000;68:2-5.

3. Bai JC, Mota AH, Mauriño E, Niveloni S, Grossman F, Boerr LA, et al. Class I and class II HLA antigens in a homogeneous Argentinian population with Whipple’s disease: lack of association with HLA-B 27. Am J Gastroenterol. 1991;86:992-4.

4. Baisden BL, Lepidi H, Raoult D, Argani P, Yardley JH, Dumler JS. Diagnosis of Whipple disease by immunohistochemical analysis: a sensitive and specific method for the detection of Tropheryma whipplei (the Whipple bacillus) in paraffin-embedded tissue. Am J Clin Pathol. 2002;118:742-8.

5. Carneiro AC, Lima P, Barbosa IP, Chaves FC. Doença de Whipple: desafio diagnóstico. Acta Med Port. 2004;17:481-6.

6. Cosme Á, Ojeda E, Muñagorri AI, Gaminde E, Bujanda L, Larzabal M, et al. Diagnosis of Whipple’s disease using molecular biology techniques. Rev Esp Enferm Dig. 2011;103:213-7.

7. Dobbins WO III. HLA antigens in Whipple’s disease. Arthritis Rheum. 1987;30:102-5.

8. Fenollar F, Puéchal X, Raoult D. Whipple’s disease. N Engl J Med. 2007;56:55-66.

9. Feurle GE, Junga NS, Marth T. Efficacy of ceftriaxone or meropenem as initial therapies in Whipple’s disease. Gastroenterology, 2010;138:478-86.

10. Feurle GE, Dörken B, Schöpf E, Lenhard V. HLA-B27 and defects in the T-cell system in Whipple’s disease. Eur J Clin Invest. 1979,9:385-9.

11. Freeman, HJ. Tropheryma whipplei infection. World J Gastroenterol. 2009;15:2078-80.

12. Gerard A, Sarrot-Reynauld F, Liozon E, Cathebras P, Besson G, Robin C, et al. Neurologic presentation of Whipple disease: report of 12 cases and review of the literature. Medicine (Baltimore). 2002;81:443-57.

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13. Iqbal T, Karovitch A, Veinot J, Saginur R, Beauchesne L. Whipple’s disease with constrictive pericarditis: a rare disease with a rare presentation. Can J Cardiol. 2009;25:e89-e91.

14. Jackuliak P, Koller T, Baqi L, Plank L, Lasabova Z, Minarik G, et al. Whipple’s disease-generalized stage. Dig Dis Sci. 2008;53:3250-8.

15. Lagier JC, Fenollar F, Lepidi H, Raoult D. Failure and relapse after treatment with trimethoprim/sulfamethoxazole in classic Whipple’s disease. J Antimicrob Chemother. 2010;65:2005-12.

16. Lepidi H, Fenollar F, Gerolami R, Mege JL, Bonzi MF, Chappuls M, et al. Whipple’s disease: immunospecific and quantitative immunohistochemical study of intestinal biopsy specimens. Hum Pathol. 2003;34:589-96.

17. Longman RS, Moreira RK, Littman DR, Green PHR, Sethi A. A rare intestinal infection with systemic effects. Gastroenterol Hepatol (N Y). 2012;8:60-3.

18. Louis ED, Lynch T, Kaufmann P, Fahn S, Odel J. Diagnostic guidelines in central nervous system Whipple’s disease. Ann Neurol. 1996;40:561-8.

19. Marth T, Strober W. Whipple’s disease. Semin Gastrointest Dis. 1996;7:41-8.

20. Marth T. Raoult D. Whipple’s disease. Lancet. 2003;361:239-46.

21. Martinetti M, Biagi F, Badulli C, Feurle GE, Müller C, Moos V, et al. The HLA alleles DRB1*13 and DQB1*06 are associated to Whipple’s disease. Gastroenterology. 2009;136:2289-94.

22. Naegeli B, Bannwart F, Bertel O. An uncommon cause of recurrent strokes: Tropheryma whippelii endocarditis. Stroke. 2000;31:2002-3.

23. Oliveira L, Gorjao R, Deus JR. Doença de Whipple. J Port Gastrenterol. 2010;17:69-77.

24. Schijf LJ, Becx MC, de Bruin PC, Van der Vegt SG. Whipple’s disease: easily diagnosed, if considered. Neth J Med. 2008;66:392-5.

25. Schneider T, Moos V, Loddenkemper C, Marth T, Fenollar F, Raoult D. Whipple’s disease: new aspects of pathogenesis and treatment. Lancet Infect Dis. 2008;8:179-90.

26. Von Herbay A, Ditton HJ, Schuhmacher F, Maiwald M. Whipple’s disease: staging and monitoring by cytology and polymerase chain reaction analysis of cerebrospinal fluid. Gastroenterology, 1997;113:434-41.

Received: 10 January 2012Accepted: 14 May 2012

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Rev. Inst. Med. Trop. Sao Paulo54(5):298, September-October, 2012doi: 10.1590/S0036-46652012000500011

LETTER TO THE EDITOR

ANALOgIES IN MEDICINE: PICTURE FRAME AND TAPIR’S NOSE

Belo Horizonte, August 2012

Dear Sir

Picture frame and tapir’s nose. The tapir (Brazilian - from Tupi language) is any of several large hoglike mammals of the genus Tapirus, of tropical America (South America and Central America) and the Southeast Asia - Malayan peninsula. Tapirs have a long flexible snout (proboscis), and four toes to the fore feet and three to the hind ones. They feed on plants and move about night. Due to its large size, the tapir has few natural predators in its environment. The human is believed to be the most common predator of the tapir as they have been hunted for food and even domesticated in some areas. The four species of tapirs are: the Brazilian or South American tapir, Tapirus terrestris, the Malayan tapir, Baird’s tapir and the Mountain tapir. All four are classified as endangered or vulnerable. Their closest relatives are the other odd-toed ungulates, including horses and rhinoceroses. The South American tapir is the size of a small ass, with a brown, nearly naked skin.

The proboscis of the tapir is a highly flexible structure, able to move in all directions, allowing the animals to grab foliage that would otherwise be out of reach. The length of the proboscis varies among species; Malayan tapirs have the longest snouts and Brazilian tapirs have the shortest. The evolution of tapir proboscises, made up almost entirely of soft tissues rather than bony internal structures, gives the Tapiridae skull a unique form in comparison to other perissodactyls.

The American Tegumentary Leishmaniasis (ATL) is an infectious disease, caused by Leishmania of the gender protozoa, and the main species are Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) amazonensis. All species of Leishmania are transmitted by the sting of female mosquitoes called phlebotomines, which belong to the Lutzomyia and Phlebotomus genders, and this transmission is made by inoculation of the promastigote forms in the skin of the vertebrate host. The ATL occurs in the Americas from the South of the United States up to the north of Argentina2-4.

The initial lesion appears at the site of the bite of the sandfly vector and is small, erythematous, solid and elevated, and its diameter increases gradually. Frequently it ulcerates and then is covered by a crust. The ulcerated lesion develops and reaches a diameter of three to 12 cm, with a very characteristic infiltrated/elevated border, as in a picture frame, that is, a frame, often ornamental, for forming a border around a picture2-4. An ulcer of such aspect is almost diagnostic in regions where ATL is found.

Months to years after healing of the primary lesion, an ulcer develops at a mucocutaneous junction, most often in the nasal septum. The reason why leishmaniasis patients develop mucosal involvement is not fully clear. Progression of the disease from cutaneous to nose lesions, appears to take place through lymphatic system and blood vessels. The nasal lesion is slowly progressive, highly destructive and disfiguring, eroding the mucosal surfaces and cartilage. Destruction of the nasal septum results in a free-hanging collapsed nose, with enlargement and flattening, resulting in the so called tapir’s nose deformity (Port. nariz de tapir or focinho de anta)1,2,3. Some hypotheses try to clarify the reason of such a preference. The most consistent hypothesis states that the Leishmania needs lower temperatures for its growth. Thus, since the front area of the nasal septum is more cooled due to the inspiration air current, there would be preference for the proliferation of the parasites2.

José de Souza ANDRADE-FILHOFaculdade de Ciências Médicas de Minas Gerais

Belo Horizonte, Minas Gerais, Brasil E-mail: [email protected]

REFERENCES

1. Andrade-Filho JS, Pena GP. Analogies in medicine. Int J Surg Pathol. 2001;9:345-6. 2. Palheta Neto FX, Rodrigues AC, Silva LL, Palheta ACP, Rodrigues LG, Silva FA. Otorhinolaryngologic manifestations relating American Tegumentary Leishmaniasis: literature review.

Int Arch Otorhinolaryngol. 2008;12:531-7. 3. Pena GP, Andrade-Filho JS. Analogies in medicine: valuable for learning, reasoning, remembering and naming. Adv Health Sci Educ Theory Pract. 2010:15:609-19. 4. Sampaio SAP, Rivitti EA. Dermatologia. São Paulo: Editora Artes Médicas; 1998. p. 565-571.

Fig. 1 - Brazilian Tapir