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EDITORIAL BOARD Alan L. de Melo (Belo Horizonte, MG) Fernando Montero‑Gei (San José, Costa Rica) Maria I. S. Duarte (S. Paulo, SP) Alberto Duarte (S. Paulo, SP) Flair J. Carrilho (S. Paulo, SP) Maria L. Higuchi (S. Paulo, SP) Angela Restrepo M. (Medellin, Colombia) Gil Benard (S. Paulo, SP) Mario Mariano (S. Paulo, SP) Anna Sara S. Levin (S. Paulo, SP) Gioconda San-Blas (Caracas, Venezuela) Mirian N. Sotto (S. Paulo, SP) Antonio A. Barone (S. Paulo, SP) Govinda Visvesvara (Atlanta, USA) Moisés Goldbaum (S. Paulo, SP) Antonio Carlos Nicodemo (S. Paulo, SP) Heitor F. Andrade Jr. (S. Paulo, SP) Moysés Mincis (S. Paulo, SP) Antonio Sesso (S. Paulo, SP) Henrique L. Lenzi (Rio de Janeiro, RJ) Moysés Sadigursky (Salvador, BA) Antonio W. Ferreira (S. Paulo, SP) Hiro Goto (S. Paulo, SP) Myrthes T. Barros (S. Paulo, SP) Barnett L. Cline (New Orleans, USA) Ises A. Abrahamsohn (S. Paulo, SP) Nilma Cintra Leal (Recife, PE) Carlos F. S. Amaral (Belo Horizonte, MG) João Carlos Pinto Dias (Belo Horizonte, MG) Paulo C. Cotrim (São Paulo, SP) Celso Granato (S. Paulo, SP) João Renato Rebello Pinho (Sao Paulo, SP) Paulo M. Z. Coelho (Belo Horizonte, MG) Cesar A. Cuba Cuba (Brasília, DF) José Eduardo Levi (S. Paulo, SP) Regina Abdulkader (S. Paulo, SP) César Naquira V. (Lima, Peru) José M. R. Zeitune (Campinas, SP) Ricardo Negroni (B. Aires, Argentina) Clarisse M. Machado (S. Paulo, SP) Julia Maria Costa-Cruz (Uberlândia, MG) Robert H. Gilman (Baltimore, USA) Claudio S. Pannuti (S. Paulo, SP) Julio Litvoc (S. Paulo, SP) Roberto Martinez (Rib. Preto, SP) Cláudio Santos Ferreira (S. Paulo, SP) Luiz Carlos Severo (P. Alegre, RS) Semíramis Guimarães F. Viana (Botucatu, SP) Dalton L. F. Alves (Belo Horizonte, MG) Luiz Jacintho da Silva (Campinas, SP) Silvino A. Carvalho (S. Paulo, SP) Eridan Coutinho (Recife, PE) Luiz T. M. Figueiredo (Rib. Preto, SP) Silvio Alencar Marques (Botucatu, SP) Ernesto Hofer (Rio de Janeiro, RJ) Lygia B. Iversson (S. Paulo, SP) Sumie Hoshino‑Shimizu (S. Paulo, SP) Euclides A. Castilho (S. Paulo, SP) Marcello Fabiano de Franco (S. Paulo, SP) Tsutomu Takeuchi (Tokyo, Japan) Eufrosina S. Umezawa (S. Paulo, SP) Marcos A. Rossi (Ribeirão Preto, SP) Venâncio A. F. Alves (S. Paulo, SP) Fan Hui Wen (S. Paulo, SP) Marcos Boulos (S. Paulo, SP) Vicente Amato Neto (S. Paulo, SP) Fernando A. Corrêa (S. Paulo, SP) M. A. Shikanai‑Yasuda (S. Paulo, SP) Zilton A. Andrade (Salvador, BA)
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UNIVERSIDADE DE SÃO PAULO - BRAZIL FACULDADE DE MEDICINA Instituto de Medicina Tropical de São Paulo Director: Prof. Dr. Paulo C. Cotrim I The purpose of the “Revista do Instituto de Medicina Tropical de São Paulo” (Journal of the São Paulo Institute of Tropical Medicine) is to publish the results of researches which 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
1959-2013, 1-55 1973-2002 (supl. 1-12) 2003 (supl. 13 - on-line only) 2005-2012 (supl. 14-18) 2014, 56 (1)
ISSN 0036-4665 ISSN 1678-9946 on line
II Impact Factor: 0.959
ISSN 0036-4665 ISSN 1678-9946 on line
Rev. Inst. Med. Trop. Sao Paulo Vol. 56 No. 1 P. 1-92 January-February, 2014
CONTENTS
REVIEW Animal models for the study of leishmaniasis immunology - E.N. LORÍA-CERVERA & F.J. ANDRADE-NARVÁEZ...... 1 PLANT ANTIMICROBIAL ACTIVITY Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado - J.M. CHAVASCO, B.H.M. PRADO E FELIPHE, C.D. CERDEIRA, F.D. LEANDRO, L.F.L. COELHO, J.J. SILVA, J.K. CHAVASCO & A.L.T. DIAS...... 13 LEISHMANIASIS Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model - C.O. VASCONCELOS, Z.C.B. COÊLHO, C.S. CHAVES, C.R. TEIXEIRA, M.M.L. POMPEU & M.J. TEIXEIRA...... 21 MICROBIOLOGY Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates - C.F. OLIVEIRA, T.G.S. PAIM, K.C. REITER, A. RIEGER & P.A. D’AZEVEDO...... 29 PARASITOLOGY Fasciola hepatica in bovines in Brazil: data availability and spatial distribution - S.C. BENNEMA, R.G.C. SCHOLTE, M.B. MOLENTO, C. MEDEIROS & O.S. CARVALHO...... 35
In vitro antigiardial activity of the cysteine protease inhibitor E-64 - T.B. CARVALHO, T.C.G. OLIVEIRA-SEQUEIRA & S. GUIMARÃES...... 43
Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo - E.B. DAVID, M. PATTI, S.T. CORADI, T.C.G. OLIVEIRA-SEQUEIRA, P.E.M. RIBOLLA & S. GUIMARÃES...... 49 URINARY TRACT INFECTION Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, Southeast Nigeria - U.M.E. DIBUA, I.S. ONYEMERELA & E.I. NWEZE...... 55 ANIMAL ENVENOMATION Coral snake antivenom produced in chickens (Gallus domesticus) - I. AGUILAR, E.E. SÁNCHEZ, M.E. GIRÓN, A. ESTRELLA, B. GUERRERO & F.A. RODRIGUEZ-ACOSTA...... 61 NOSOCOMIAL INFECTIONS First report of metallo-β-lactamases producing Enterobacter spp. strains from Venezuela - D. MARTÍNEZ, H.E. RODULFO, L. RODRÍGUEZ, L.E. CAÑA, B. MEDINA, M. GUZMAN, N. CARREÑO, D. MARCANO & M. DE DONATO...... 67
Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units - I. BOSZCZOWSKI, G.V.B. PRADO, M.F. DALBEN, R.C.P. TELLES, M.P. FREIRE, T. GUIMARÃES, M.S. OLIVEIRA, J.F. ROSA, R.E. SOARES, P.E.D. LLACER, F.L. DULLEY, S.F. COSTA & A.S. LEVIN...... 71
Enterococcus faecium and Enterococcus faecalis in blood of newborns with suspected nosocomial infection - I. FURTADO, P.C.N. XAVIER, L.V.M. TAVARES, F. ALVES, S.F. MARTINS, A.S. MARTINS & D.B. PALHARES...... 77 CASE REPORT First case of autochthonous human visceral leishmaniasis in the urban center of Rio de Janeiro: case report - G.A.R. SILVA, T.O. BOECHAT, F.R.A. FERRY, J.F.C. PINTO, M.C.V.M. AZEVEDO, R.S. CARVALHO, R.N. MOTTA & M.F. VERAS...... 81
Biopsy proven acute tubular necrosis due to rhabdomyolysis in a dengue fever patient: a case report and review of literature - L.P. REPIZO, D.M. MALHEIROS, L. YU, R.T. BARROS & E.A. BURDMANN...... 85
Multiple pulmonary nodules caused by Corynebacterium striatum in an immunocompetent patient - C.B. SEVERO, L.S. GUAZZELLI, M.B. BARRA, B. HOCHHEGGER & L.C. SEVERO...... 89 LETTER TO THE EDITOR Analogies in Medicine: white clay-pipestem “cirrhosis” - J.S. ANDRADE FILHO...... 92
ADDRESS SUBSCRIPTIONS INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO FOREIGN COUNTRIES Av. Dr. Enéas de Carvalho Aguiar, 470 One year (six issues)...... U$ 200.00 05403-000 São Paulo, SP - Brazil Single issue...... U$ 50.00 Phone/Fax: 55.11.3062.2174; 55.11.3061-7005 e-mail: [email protected] III Impact Factor: 0.959
ISSN 0036-4665 ISSN 1678-9946 on line
Rev. Inst. Med. Trop. Sao Paulo Vol. 56 No. 1 P. 1-92 Janeiro-Fevereiro, 2014
CONTEÚDO
REVISÃO Modelos animales para el estudio de la inmunología de la leishmaniosis - E.N. LORÍA-CERVERA & F.J. ANDRADE-NARVÁEZ...... 1
ATIVIDADE ANTIMICROBIANA DE PLANTAS Avaliação das atividades antimicrobiana e citotóxica de extratos de plantas do Cerrado do Sul de Minas Gerais - J.M. CHAVASCO, B.H.M. PRADO E FELIPHE, C.D. CERDEIRA, F.D. LEANDRO, L.F.L. COELHO, J.J. SILVA, J.K. CHAVASCO & A.L.T. DIAS...... 13
LEISHMANIOSE Migração celular distinta induzida por Leishmania infantum chagasi e saliva de Lutzomyia longipalpis em um modelo de pool hemorrágico - C.O. VASCONCELOS, Z.C.B. COÊLHO, C.S. CHAVES, C.R. TEIXEIRA, M.M.L. POMPEU & M.J. TEIXEIRA...... 21
MICROBIOLOGIA Avaliação de quatro métodos diferentes de extração de DNA em isolados clínicos de estafilococos coagulase negativos (SCoN) - C.F. OLIVEIRA, T.G.S. PAIM, K.C. REITER, A. RIEGER & P.A. D’AZEVEDO...... 29
PARASITOLOGIA Fasciola hepatica em bovinos no Brasil: disponibilidade de dados e distribuição espacial - S.C. BENNEMA, R.G.C. SCHOLTE, M.B. MOLENTO, C. MEDEIROS & O.S. CARVALHO...... 35
Atividade in vitro do inibidor de cisteína-proteases E-64 sobre trofozoítos de Giardia - T.B. CARVALHO, T.C.G. OLIVEIRA-SEQUEIRA & S. GUIMARÃES...... 43
Genotipagem de isolados de Giardia duodenalis de primatas não humanos mantidos em zoológico do Brasil - E.B. DAVID, M. PATTI, S.T. CORADI, T.C.G. OLIVEIRA-SEQUEIRA, P.E.M. RIBOLLA & S. GUIMARÃES...... 49
INFECÇÕES DO TRATO URINÁRIO Perfil da frequência, análise urinária e suscetibilidade de patógenos causando infecções do trato urinário no estado de Enugu, Sudeste da Nigéria - U.M.E. DIBUA, I.S. ONYEMERELA & E.I. NWEZE...... 55
VENENOS ANIMAIS Antiveneno de serpiente coral producido en gallinas (Gallus domesticus) - I. AGUILAR, E.E. SÁNCHEZ, M.E. GIRÓN, A. ESTRELLA, B. GUERRERO & F.A. RODRIGUEZ-ACOSTA...... 61
INFECÇÕES HOSPITALARES Primer reporte de cepas de Enterobacter spp productoras de metalobetalactamasas de Venezuela - D. MARTÍNEZ, H.E. RODULFO, L. RODRÍGUEZ, L.E. CAÑA, B. MEDINA, M. GUZMAN, N. CARREÑO, D. MARCANO & M. DE DONATO...... 67
Surto policlonal de infecção de corrente sanguínea causada pelo complexo Burkholderia cepacia em unidades de hospital-dia de hematologia e transplante de medula óssea - I. BOSZCZOWSKI, G.V.B. PRADO, M.F. DALBEN, R.C.P. TELLES, M.P. FREIRE, T. GUIMARÃES, M.S. OLIVEIRA, J.F. ROSA, R.E. SOARES, P.E.D. LLACER, F.L. DULLEY, S.F. COSTA & A.S. LEVIN...... 71
Enterococcus faecium e Enterococcus faecalis no sangue de recém-nascidos com suspeita de infecção nosocomial - I. FURTADO, P.C.N. XAVIER, L.V.M. TAVARES, F. ALVES, S.F. MARTINS, A.S. MARTINS & D.B. PALHARES...... 77
RELATOS DE CASOS Primeiro caso de leishmaniose visceral humana de transmissão autóctone no centro urbano do Rio de Janeiro: relato de caso - G.A.R. SILVA, T.O. BOECHAT, F.R.A. FERRY, J.F.C. PINTO, M.C.V.M. AZEVEDO, R.S. CARVALHO, R.N. MOTTA & M.F. VERAS...... 81
Necrose tubular aguda comprovada por biópsia em paciente com dengue e rabdomiólise - L.P. REPIZO, D.M. MALHEIROS, L. YU, R.T. BARROS & E.A. BURDMANN...... 85
Nódulos pulmonares múltiplos causados por Corynebacterium striatum numa paciente imunocompetente - C.B. SEVERO, L.S. GUAZZELLI, M.B. BARRA, B. HOCHHEGGER & L.C. SEVERO...... 89
CARTA AO EDITOR Analogies in Medicine: white clay-pipestem “cirrhosis” - J.S. ANDRADE FILHO...... 92
ENDEREÇO INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO Av. Dr. Enéas de Carvalho Aguiar, 470 05403-000 São Paulo, SP - Brasil Fone/Fax: 55.11.3062.2174; 55.11.3061-7005 IV e-mail: [email protected] Rev. Inst. Med. Trop. Sao Paulo 56(1):1-11, January-February, 2014 doi: 10.1590/S0036-46652014000100001
REVIEW
ANIMAL MODELS FOR THE STUDY OF LEISHMANIASIS IMMUNOLOGY
Elsy Nalleli LORÍA-CERVERA & Fernando José ANDRADE-NARVÁEZ
SUMMARY
Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers (“low” 1x102 and “high” 1x106) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease.
KEYWORDS: Animal models; Leishmania; Immune response.
INTRODUCTION the ten vector-borne diseases that have the greatest potential to affect European inhabitants101. Leishmaniasis encompasses a group of diseases which are caused by infection with protozoan parasites of the Leishmania (Kinetoplastida: The disease is transmitted to humans by sand flies and displays Trypanosomatidae) genus. They are still a major worldwide public different clinical manifestations, ranging from asymptomatic or health problem considering they are endemic in 98 countries or subclinical infection to disfiguring forms of cutaneous and mucosal territories, with more than 350 million people at risk6. Moreover, it leishmaniasis or potentially fatal visceral disease35,36,78,93. This is estimated visceral leishmaniasis (VL) causes over 50,000 deaths polymorphic outcome has been considered to depend largely on the annually, a rate only surpassed, among parasitic diseases, by malaria, virulence of the infecting parasite strain, immunoregulatory effects of and 2,357,000 disability-adjusted life years lost, placing leishmaniasis sand fly saliva, as well as the host’s genetic background and immune ninth in a global analysis of infectious diseases6,28. Despite this response31,60,62. In summary, the leishmaniases remain as unpreventable very strong data, leishmaniasis is largely ignored in discussions of and uncontrollable diseases; moreover, their epidemiological profile is tropical disease priorities and is one of the most neglected tropical shifting towards an increased prevalence, and therefore novel instruments diseases51,52. It has been pointed out that this consignment to critical and approaches to reach their control are urgently necessary. oblivion possibly “results from its complex epidemiology and ecology, the lack of simple, easily-applied tools for case management and the Leishmaniasis is most likely to be controlled by a successful paucity of current incidence data, and often results in a failure on the vaccination program. The relatively uncomplicated leishmanial life part of policy-makers to recognize its importance5,14. Political and cycle and the fact that recovery from a primary infection renders the host socioeconomic changes may have an even more important role than resistant to subsequent infections indicate that a vaccine is feasible55. global warming on the changing epidemiology of the leishmaniases, Many immunological aspects of the disease have been studied in as has been argued for tick-borne diseases in Europe91. The European experimental animal models, such as mice, hamsters, domestic dogs Centre for Disease Prevention and Control lists the leishmaniases among and non-human primates. Although most experimental models of
Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Mérida, Yucatán, México. Correspondence to: Elsy Nalleli Loría-Cervera, Universidad Autónoma de Yucatán, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Laboratorio de Inmunología, Ave. Itzáes No. 490 x 59-A, Mérida, Yucatán, México. Phone: 52-999 9246412 ext. 1155. Fax: 52-999 9236120. E-mail: [email protected] LORÍA-CERVERA, E.N. & ANDRADE-NARVÁEZ, F.J. - Animal models for the study of leishmaniasis immunology. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 1-11, 2014.
leishmaniasis have the major advantage of allowing control over the healer phenotype has been shown to be associated with a parasite-specific genetics of both the parasite and the host, none of them, in any way, Th2 response characterized by the enhanced expression of deactivating reproduces the outcome of human infection by Leishmania spp49. macrophage cytokines such as interleukin 4 (IL-4), interleukin 10 (IL- 10) and transforming growth factor-β (TGF-β)11,94,98,. Studies of mouse Among the main factors contributing to differences between humans models of leishmaniasis have provided important insights into the and animal models are the size and nature of the inocula, the infection response of the host to infection. However, the use of different parasite route and the strain of host or parasite37,60,70. Currently, small numbers of species, tissue targets (mice footpad, ear, or base of tail) and doses (105 in vitro-derived metacyclic promastigotes together with strongly bioactive to 107) of metacyclic promastigotes has generated a wide variety of saliva, intradermal infection and host reservoirs as experimental animal experiments that do not reproduce the natural infection and cannot be models are used to mimic the clinical and immunological features found extrapolated to human disease.’ in human disease49. These approaches could contribute to developing improved experimental models for studying leishmaniasis and identifying In natural infections, the sand fly introduces into the skin a very small possible targets to evaluate vaccine candidates. number (possibly as few as 100 to 1,000) of metacyclic promastigotes together with strongly bioactive saliva, whereas in laboratory infections The present review describes the most common animal experimental thousands to millions of culture-derived promastigotes or tissue-derived models which have been employed to study the immune response to amastigotes are injected with a saline solution or culture medium57. Leishmania spp. and includes wild rodents. The main purpose is to discuss the concept of experimental animal models to study leishmaniasis In order to have a better understanding of natural Leishmania immunology. infection in the laboratory, investigators are now using small numbers of in vitro-derived metacyclic promastigotes and intradermal rather than MOUSE MODEL subcutaneous infection into the ears of mice49.
The laboratory mouse owes much of its popularity as a model It was demonstrated, for example, that in mice inoculated with a organism in biomedical research to the existence of a large collection of mixture of phlebotomine saliva and L. major promastigotes, the lesions inbred strains that represent an immortal population of genetic clones grew faster and were bigger than those of mice inoculated only with derived by repeated brother sister mating. Because mice from each strain promastigotes13. are genetically identical it is possible to collect and combine biological data over time and space leading to a depth of phenotype characterization The ability of saliva to enhance Leishmania infection has been rarely achieved in other mammalian systems. Furthermore, the existence attributed to modulation of the host immune system, potentially of a definite set of genetic differences among inbred strains allows through anti-inflammatory properties such as down regulation of scientists to explore the effect of genetic diversity on almost any antigen presentation, co-stimulatory molecule expression, and nitric phenotype of interest107. Another advantage of the murine model is the oxide production13, 81. However, vaccination by pre-exposure to the simplicity of keeping, breeding and reproducing them47. bites of uninfected sand flies, with whole saliva or with defined salivary proteins has shown to protect against cutaneous L. major infection97. During the past 40 years, murine models of the human disease The frequent exposure to sand fly bites leads to the production of cutaneous leishmaniasis have been extensively employed to elucidate the neutralizing antibodies against salivary proteins and also to the activation cell types, cytokines, signal transduction cascades and antileishmanial of cellular mechanisms that may have an adverse effect on Leishmania effector mechanisms that are necessary for the control of parasites, as establishment. In this perspective, characterization of immune responses well as for the clinical resolution of disease, resistance to a secondary against sand fly saliva can help estimate both risk of infection and, to some infection, and vaccine development18,73. Experimental infection of degree, anti-parasite immunity. Although this hypothesis has been proven mice with L. major promastigotes has allowed understanding of the in animal models, additional large-scale clinical studies are necessary immunologic mechanisms governing resistance (C57BL/6 strain) and to validate it in humans15. Although the regulation of host immune susceptibility (BALB/c strain) to infection2. response to Leishmania has been well defined in cutaneous L. major infection of inbred mice, many studies have demonstrated that the host Susceptibility has been correlated with the development of lesions responses within the same mouse strain could vary according to different associated with a Th2 type of immune response, while the healing of Leishmania species. Different virulence factors have been identified for lesions in resistant mice has been correlated with the development of a distinct Leishmania species, and there are profound differences in the Th1 type of immune response98. The resolution of lesions in C57BL/6 immune mechanisms that mediate susceptibility/resistance to infection mice has been shown to involve several factors contributing to the killing and in the pathology associated with disease66. For example, C57BL/6 or of L. major within macrophages. The most efficient mechanism of C3H mice, which heal from L. major infection, develop chronic disease parasite killing involves the production of gamma interferon (IFN-γ) and when infected with either L. (L.) amazonensis or L. (L.) mexicana. tumor necrosis factor alpha (TNF-α) by CD4+ Th1 cells, which stimulate the synthesis of inducible nitric oxide synthase (iNOS), generating the The characteristic chronic lesions of L. (L.) amazonensis infection production of nitric oxide (NO), a potent cytotoxin involved in the in C57BL/6 and C57BL/10 mice are independent of IL-4 expression clearance or inhibition of Leishmania parasites61,65,77. and corresponding Th2 response1,54. In addition, L. (L.) amazonensis infection in C3H mice results in low levels of production of IL-12 and In contrast, susceptible BALB/c mice develop severe and uncontrolled IFN-γ by antigen-specific CD4+ T cells54. However, lesion development lesions that lead to progressive disease and eventual death. This non- and parasite burden have been shown to be exacerbated in the presence
2 LORÍA-CERVERA, E.N. & ANDRADE-NARVÁEZ, F.J. - Animal models for the study of leishmaniasis immunology. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 1-11, 2014.
of CD4+ T cells, demonstrating that T cells are activated during L. Eventually, splenic replication is controlled but parasites are usually (L.) amazonensis infection and that they contribute significantly to the maintained for life58. Parasite persistence in mice is accompanied by immunopathology of chronic disease103. failure of granuloma formation and splenomegaly39.
Recently, it was demonstrated that susceptibility to L. (L.) Due to the fact that visceral infection in BALB/c mice is chronic amazonensis in the mouse model of cutaneous leishmaniasis does not but not fatal, it may be more appropriate to use it as a model for depend only on the expression of IL-10. L. (L.) amazonensis parasites studying self-healing or subclinical infection. Although experimental persistent in IL-10-deficient mice; even in the presence of an enhanced murine models of VL do not allow exact extrapolations with subclinical Th1 response during the early stages of infection and in the presence infection in humans they have been useful to identify genes and predict of antigen-specific cells primed for Th1 effectors function during the their functional roles in the protective immune response. Genetically chronic phase53. resistant mice have the functional NRAMP1 gene which is involved in macrophage activation16. The NRAMP1 gene encodes a protein expressed Although the requirements for effective intracellular killing of L (L.) on the membrane of infected macrophages and exerts an enhanced effect amazonensis by activated macrophages are relatively unknown, it has on iNOS expression and generation of NO, restricting intracellular been demonstrated that the presence of both superoxide and nitric oxide is Leishmania multiplication17. In this context, visceral infection in BALB/c necessary for efficient killing of amastigotes within LPS/IFN-γ–activated mice provides a good model for the evaluation of candidate vaccines. bone marrow-derived macrophages generated from C3H mice74. HAMSTER MODEL Control of the closely related L. (L.) mexicana in C57BL/6 mice has been shown to be IFN-γ and STAT4-dependent and surprisingly The Syrian golden hamster (Mesocricetus auratus) is highly independent of IL-12 production while the presence of IL-4, STAT6 susceptible to infection with visceralizing Leishmania species (L. and, perhaps as a consequence, the ability to generate Th2 responses, donovani, L. infantum) and is considered the best experimental are essential for the rapid lesion growth and nonhealing responses21,100,106. model to study visceral leishmaniasis (VL) because it reproduces the Later on, it was demonstrated that endogenous IL-12 is only critical for clinicopathological features of human disease. However, the wide use of controlling the late but not the early stage of L. (L.) mexicana infection hamsters is still limited due to the scarcity of reagents (e.g., antibodies, in C57BL/6 mice; however, they fail to resolve lesions, in contrast to L. cell markers and cytokines) of defined specificity available to study the major infection3. role of the immune response in disease pathology48,68,113.
As an evasion mechanism, L. (L.) mexicana promastigotes mediate In 1978, CHANG & DWYER provided quantitative evidence the phosphorylation of specific transcription factors to enhance iNOS, indicating an avid ingestion of L. donovani amastigotes by hamster COX-2 and arginase-1 expression in LPS induced macrophages via macrophages and supported the early findings that lysosome-phagosome TLR-4. The activities associated with all three enzymes are the main fusion ocurrs26. factors leading to the downregulation of the IL-12 production in L. (L.) mexicana infected macrophages102. In order to understand the immune response to L. donovani infection the nucleotide sequences of several hamster cytokine genes (IL-2, IL-4, The significant differences in the immune response between the INF-γ, TNF-α, IL-10, IL-12 and TGF-β) were cloned and used to analyze Old World (L. major) and New World (L. mexicana/L. amazonensis) their expression in a model of visceral infection. In this hamster model Leishmania species not only point to interesting features of the there was a pronounced expression of the Th1 cytokine mRNAs (IL-2 host‑pathogen interaction and immunobiology of this genus of parasitic and IFN-γ), with transcripts being detected as early as one week post- protozoa, but also have important implications for immunotherapy infection. Surprisingly, although the basal expression of IL-4 was detected and vaccine development. A view of leishmaniasis that only considers in uninfected hamsters, their expression did not increase in response to mouse model infection with L. major misses a wealth of interesting infection with L. donovani. IL-12 transcript expression was detected at low immunobiology associated with other species of Leishmania66. Therefore, levels starting seven days post-infection and its expression paralleled that of our understanding of the mechanisms involved in mucocutaneous and IFN-γ. Additionally, the mRNA for TNF-α was increased within one week cutaneous diseases caused by these organisms remains limited. of infection but levels did not increase further during the first month of infection. Expression of IL-10, a potent macrophage deactivator, increased Mouse models have also been used to study visceral leishmaniasis in splenic tissue over the first four weeks after infection, suggesting that caused by both L. donovani and L. infantum. Although the outcome of this cytokine could contribute to progressive disease in hamsters. These murine VL infection is genetically determined, most susceptible mouse studies provided the first description of the molecular immunopathogenesis strains including BALB/c are able to control visceral disease75. Following of disease in hamsters and indicated that progressive disease in this model of Leishmania infection, BALB/c mice develop an organ-specific immune VL is not associated with early polarization of the splenic cellular immune response113. During the first weeks of infection, the parasites multiply response toward a Th2 phenotype and away from a Th1 phenotype, offering rapidly in the liver; however four weeks later, the mice develop an important insights into human disease67. effective Th1 immune response, clear the parasites and become resistant to reinfection76. The hepatic resistance to Leishmania infection in these During progressive disease in the hamster model of VL, uncontrolled mice is associated with the development of a granulomatous reaction in parasite replication in the liver, spleen, and bone marrow occurred despite the liver12. While pathology in the liver is limited, the parasites persist the high activation of the immune response and the strong Th1-like in the spleen and the infection progresses for a longer period of time. cytokine microenvironment. The failure in the control of VL could be
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partially explained by the lymphoproliferative suppression process which IFN-γ at the site of the bite could partly explain the protection observed in occurs during active disease45. Visceral infection caused by L. donovani the viscera of LJM19-immunized hamsters through direct parasite killing led to a gradual impairment of the proliferative response to parasite and/or priming of anti-Leishmania immunity. These findings reinforce antigens in hamsters79. The latter dysfunction has been attributed to the the concept of using components of arthropod saliva in vaccine strategies inability of the infected antigen-presenting cells (APCs) to stimulate against vector-borne diseases44. specific T cells, the production of TGF-β which triggers the apoptotic death of lymphocytes and the downregulation of protein kinase C To better understand the hamster immune response to important activity9,72,95. Interestingly, the antigen-dependent immunosuppression pathogens such as Leishmania, a duplex real-time reverse transcriptase (RT) observed in L. chagasi-infected hamsters with active visceral disease is PCR assay was developed for the relative quantification of the mRNAs of not related to the cytokine profile41. hamster cytokines, chemokines, and related immune response molecules. The application of this assay to a biological model was demonstrated Furthermore, the fatal outcome of the disease in the hamster model in a cutaneous hamster model by comparing mRNA expression in skin has been related to the loss of macrophages effector functions. Indeed, and lymph node tissues between uninfected and L. panamensis infected throughout the course of infection, inducible NO synthase (iNOS, NOS2) hamsters. As a result, there was a relatively greater basal expression in mRNA or enzyme activity in liver or spleen tissue was not detected. the LN compared to the skin for most transcripts (IL-4, CCR4, IL-21, Thus, although a Th1-like cytokine response was prominent, the major TNF-α, TGF-β, IFN-γ, IL-12p40, IL-10, and Foxp3). Conversely, the assay antileishmanial effector mechanism that is responsible for control of identified that the basal expression of CCL22 and CCL17 mRNAs was infection in mice was absent throughout the course of progressive VL significantly greater in the normal skin compared to the LN. At an early in the hamster68. stage of infection(one week p.i. ) there was concomitant upregulation of the type 1 (IFN-γ and IL-12p40) and type 2 (IL-4, IL-10, IL-13, and IL-21) Later on, it was shown that the lack of NO production was due to cytokines at the site of cutaneous infection, suggesting that a balanced type a defect in the transcriptional activation of NOS2. Luciferase reporter 1 and type 2 cytokine response contributes to the chronicity of the disease assays demonstrated that the hamster NOS2 promoter, like the human caused by L. panamensis in hamsters40. NOS2 promoter, has reduced basal and IFN-γ/LPS-induced activity compared with the mouse promoter. The mechanism described above Undoubtedly, the hamster model may be helpful for understanding is the most probable reason for the inability of hamsters to control the immunological mechanisms involved in the pathogenesis of Leishmania infection84. visceral leishmaniasis. However, it is necessary to continue the efforts of producing specific reagents (i.e. cytokine-specific and cell surface The role played by infected macrophages in the development of the markers of monoclonal antibodies) and develop more sensitive techniques cellular unresponsiveness present in visceral leishmaniasis has been that allow the study of the immunopathogenesis of the disease, which studied. Adherent spleen cells from infected hamsters were unable to has important implications for the generation of therapeutic and vaccine present L. donovani antigens to antigen specific T cells, however they targets. were able to present KLH. Conversely, T cells from infected animals did not respond to parasite antigens even when these antigens were presented DOG MODEL by normal syngeneic macrophages. Interestingly, lymphocytes from inguinal lymph nodes of infected animals sensitized in their footpad Wild canines and domestic dogs are the main reservoirs of zoonotic with parasite antigens proliferated well when stimulated in vitro with visceral leishmaniasis caused by L. infantum in the Mediterranean area, L. donovani antigens. These results suggest that the defect in the cellular Middle-East, Asian countries and Latin America. The role of dogs as the immune response of the L. donovani infected hamsters is a consequence main reservoir of visceral leishmaniasis has led to an increased interest in of a selective inability of their antigen presenting cells to process and studying the immune response and finding Leishmania antigens implicated present parasite antigens to T cells95. in protective cellular immunity in canine visceral leishmaniasis. Recent research has provided new insights on the epidemiology, pathology To date, progressive disease in hamsters has been mostly achieved and immunology of canine leishmaniasis and its genetic basis. These by the injection of a large number of parasites via the i.v., intracardial, new findings have led to better understanding of the disease, and have or i.p. routes. However, these routes of infection do not mimic natural also helped in the development of new diagnostic methods and control transmission by sand fly bite where the parasites are delivered into the measures against the infection, such as insecticide-impregnated collars skin of a mammalian host in the presence of saliva. Recently, it was for dogs, new drugs, and second generation vaccines4,10. demonstrated that a salivary protein of the sand fly vectorLutzomia longipalpis protects against the fatal outcome of visceral leishmaniasis Canine visceral leishmaniasis is a multisystemic disease with variable caused by L. infantum in a hamster model. Immunization with 16 DNA clinical signs. Infected dogs may develop symptomatic infection resulting plasmids coding for salivary proteins of Lu. longipalpis resulted in the in death, while others remain asymptomatic, or develop one or more identification of LJM19, a novel 11-kDa protein, that protected hamsters mild symptoms and are classified as oligosymptomatic27. The typical against the fatal outcome of VL. LJM19-immunized hamsters maintained histopathological finding in the skin, liver and spleen, is a granulomatous a low parasite load that correlated with an overall high IFN-γ/TGF-β inflammatory reaction associated with the presence of Leishmania ratio and iNOS expression in the spleen and liver up to five months amastigotes within macrophages10. postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-γ was also noted in the skin of LJM19-immunized Studies on experimentally infected dogs have demonstrated that hamsters 48 hours after exposure to uninfected sand fly bites. Induction of three years after infection, asymptomatic or resistant dogs responded to
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L. infantum antigen both in lymphocyte proliferation assays in vitro and been implicated in murine and human leishmaniasis, the involvement in delayed-type hypersensitivity reaction, whereas no serum antibodies of these cells in canine visceral leishmaniasis has not been explored. to parasite antigen were shown. In contrast, symptomatic or susceptible animals failed to respond to the parasite antigen in cell-mediated Few studies have demonstrated the involvement of CD8+ assays both in vitro and in vivo and showed considerably higher serum lymphocytes in resistance to canine VL. These lymphocytes were detected antibodies to leishmanial antigens, which are not immunoprotective. In in asymptomatic dogs experimentally infected with L.infantum but not in addition, peripheral mononuclear cells from asymptomatic dogs produced symptomatic animals, suggesting that direct lysis of L. infantum-infected significantly higher levels of IL-2 and TNF-α than symptomatic and macrophages by cytotoxic T lymphocytes represents an additional control uninfected dogs. Similar results were observed with a group of effector mechanism in resistance to VL86. In dogs naturally infected mixed-breed dogs with natural Leishmania infections, also grouped as with L. infantum, a reduction in both CD4+ and CD8+ populations was asymptomatic or symptomatic on the basis of clinical signs of canine observed, while restoration of these cells occurred after drug treatment19. visceral leishmaniasis85. The use of dogs as experimental models to study visceral leishmaniasis The main effector mechanism involved in the protective immune has led to elucidate the role of immune cells and their principal products response of dogs infected with L. infantum is the activation of to better understand the possible mechanisms mediating immune response macrophages by IFN-γ and TNF-α to kill intracellular amastigotes via during Leishmania infection, which may contribute to the development the L-arginine nitric oxide pathway, as has been observed following of vaccines or immunotherapy. successful chemotherapy of L. infantum-infected dogs112. NO production and anti-leishmanial activity has also been detected in a Natural infection of domestic dogs with L. (V.) braziliensis, L. (V.) canine macrophage cell line infected with L.infantum after incubation peruviana, L. (V.) panamensis, L. (V.) colombiensis and L. (L.) mexicana with IFN-γ, TNF-α and IL-288, as well as in macrophages from dogs has been reported in Latin America30. To date, there is no solid evidence immunized with killed L.infantum promastigotes82. Later on, it was that dogs act as reservoir hosts for the domestic transmission of CL92,99. demonstrated that NO production may be involved in the long-term Most studies are designed to determine the prevalence of CL in dogs, protection of dogs against natural Leishmania infection and in the clinical however, little is known about the parasitologic and immunologic course presentation of canine leishmaniasis83. of infection.
The local tissue cytokine response of dogs naturally infected with L. NON-HUMAN PRIMATE MODEL infantum has been evaluated. The analysis revealed an enhanced INF-γ mRNA accumulation in infected dogs which was positively correlated Non-human primates are valuable models for biomedical research with humoral, (IgG1) but not with lymphoproliferative, responses to the because of their similarities to humans in anatomy, immunology and Leishmania antigen. However, infected dogs with detectable IL-4 mRNA physiology. However, they are expensive laboratory animals that are had significantly more severe symptoms90. A balanced production of Th1 difficult to obtain and to handle. Availability of a non-human primate and Th2 cytokines was detected in the spleen of L. infantum infected dogs, model of leishmaniasis would facilitate the study of different aspects with a predominant accumulation of mRNA for IL-10 and IFN-γ that was of this disease and would accelerate the development of vaccines and related to the parasitic load and to clinical progression59. Additionally, a testing of new drug candidates. mixed cytokine profile with high levels of expression of IFN-γ, TNF-α and IL-13 was determined in the skin of asymptomatic dogs naturally The Asian rhesus macaques (Macaca mulatta) are quite susceptible infected with L. infantum. Moreover, the levels of transcription factors to Leishmania infection: they develop a human-like disease, exhibit GATA-3 and FOXP3 were correlated with the asymptomatic disease. antibodies to Leishmania and parasite-specific T-cell mediated immune These results indicate that in addition to the mixed cytokine profile, responses both in vivo and in vitro, and can be protected effectively the enhanced expression of their associated transcription factors plays by vaccination46. Distinct histopathological patterns were observed in an important role in the clinical status of Leishmania infected dogs69. Macaca mulatta lesions at biopsy, but healing lesions contained more The role of Th2 type cytokines in canine VL has not yet been defined. organized epithelioid granulomas and activated macrophages, followed Evidence for Th1 and Th2 mixed responses has been reported in antigen- by fibrotic substitution in response to L. (L.) amazonensis infection7. stimulated PBMC from asymptomatic dogs experimentally infected Interestingly in L. (V.) braziliensis infection, the presence of antigen- with L. infantum, which displayed IL-2, IFN-γ and IL-10 mRNA specific IFN-γ or TNF-α-producing CD4+ and CD8+ cells are likely transcripts. However, IL-2 and IFN-γ predominated in asymptomatic important for the immunological effectiveness of granulomas. However, dogs and the development of symptomatic infections could not be their resolution can be attributed to the concomitant recruitment of IL- related to IL-10 expression25. IL-10 mRNA transcripts were detected in 10-producing CD4+CD25+ regulatory T cells that suppress the effector Con A-stimulated PBMC derived from dogs with clinical signs of VL87. T-cell mediated inflammatory response32,105. The progression and All of these results are in agreement with experiments in which PBMC resolution of skin lesions caused by both Leishmania species appears obtained from symptomatic VL dogs were stimulated by a recombinant to be very similar to that observed in humans, confirming the potential L. infantum cysteine proteinase and high levels of IL-10 were detected for this monkey as a viable surrogate to study the immune response in by an ELISA assay. In contrast, low or undetectable concentrations of human cutaneous leishmaniasis7. this cytokine were found in PBMC supernatants from oligosymptomatic and asymptomatic animals, respectively89. Macaques have also been used to explore immune response against L. major infection. Infected animals develop a simple cutaneous lesion Although IL-10 secreted by CD25+ CD4+-regulatory T cells has which progresses spontaneously to ulceration and complete resolution
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within about three months which is associated with a non-specific WILD RODENTS chronic inflammation and/or tuberculoid-type granulomatous reaction. Additionally, macaques develop varying levels of resistance against Classical laboratory inbred strains of mice have been extremely homologous re-infection as it happens in humans. Thus, the importance helpful for research in immunology and oncology. Unfortunately, because of this model in experimental CL lies in the reproduction of clinical and they all derive from a relatively small pool of ancestors, their genetic histopathological features that are common in L. major-infected humans polymorphism is rather limited47. and in the resistance to secondary infection, indicating the development of an acquired immunity8. A new approach to study host-parasite relationships has been the use of wild rodents, particularly primary reservoirs, as experimental New World primates, such as owl monkeys (Aotus trivirgatus), animal models. They are, as the human being, genetically polymorphic squirrel monkeys (Saimiri sciureus), and marmosets (Callithrix jaccus and represent an emerging system for the genetic analyses of the jaccus) have been considered potential hosts for studying visceral physiological and behavioral bases of habitat adaptation47. Laboratory leishmaniasis. Owl monkeys develop a visceral disease characterized by studies using natural hosts as experimental models provide a suitable weight loss, anemia and hepatosplenomegaly. Its high susceptibility to indication of the importance of these hosts as reservoirs, since it allows a L. donovani infection suggest it may be useful for the study of VL20. In better understanding of the dynamics of infection, especially concerning contrast, squirrel monkeys develop a visceral disease when infected with the ability to retain the infection and amplify parasite populations in a L. donovani but are able to recover from disease and became resistant given environment, due to features that favor parasite transmission (e.g., to reinfection34. presence of parasites in the skin). Moreover, the study of these rodents could allow the understanding of the mechanisms involved in immune Although little is known about immune response to Leishmania activation during nonpathogenic and pathogenic infections, to clarify infection in monkeys, they are frequently used as models for preclinical how the reservoir immune response regulates Leishmania infection and testing of Leishmania candidate vaccines. The safety, immunogenicity, how the parasites evade a sterilizing immune response. and efficacy of a vaccine combining heat-killed L. (L.) amazonensis with human rIL-12 (rhIL-12) and alum (aluminium hydroxide gel) The role of several species of rodents as wild reservoirs of Leishmania as adjuvants was evaluated in rhesus macaques. The single s.c. species is well known24,33,96,110. However, there are only a few studies that vaccination was found to be safe and immunogenic, although a small followed up experimentally infected wild hosts by Leishmania species, transient s.c. nodule developed at the vaccination site. Groups receiving mostly due to the difficulties of managing wild mammals in captivity. rhIL-12 had an augmented in vitro Ag-specific IFN-γ response after To date the host-parasite interactions involved in persistent infections in vaccination, as well as increased production of IgG. Furthermore, different Leishmania reservoirs are unknown. intradermal forehead challenge infection with 107 metacyclic L. (L.) amazonensis promastigotes at four weeks demonstrated protective Sigmodon hispidus has been identified asLeishmania reservoir, immunity in all monkeys receiving rhIL-12 with alum and Ag. Thus, however no studies of experimental infection have been carried out with a single dose vaccine with heat-killed Leishmania using rhIL-12 and this pathogen33. Currently Sigmodon hispidus is used as a model for alum as adjuvants was safe and fully protective in a primate model of the study of various infectious diseases, mainly caused by viruses and cutaneous leishmaniasis56. bacteria, due to its high susceptibility to a wide variety of pathogens80. A large number of cytokine and chemokine genes have been cloned and Successful vaccination has been achieved against visceral sequenced and monoclonal antibodies have been generated in order to leishmaniasis by intradermal inoculation of alum-precipitated autoclaved facilitate its use as an experimental animal model. Recently, low levels of L. major with BCG (bacile Calmette-Guérrin) and autoclaved L. donovani NO production and iNOS expression similar to human macrophages were with BCG in Indian langurs. Vaccinated animals show a delayed found in Sigmodon hispidus infected with bacteria23. These similarities protection and significant lymphoproliferative response with high levels could explain the high susceptibility of this rodent to human pathogens. of IFN-γ and IL-238,71. Thrichomys laurentius is a South American caviomorph rodent Attempts were made to reproduce the spectrum of human visceral formerly included in a monospecific genus, in which the importance of leishmaniasis due to L. donovani in Vervet monkeys (Chlorocebus the retention of infection and transmission of Leishmania species has pygerythrus). Both symptomatic and asymptomatic/cryptic infections been established. These rodents were found infected with Leishmania were observed. However asymptomatic infected animals had competent species of different complexes – L. (L.) mexicana and L. donovani – in humoral and cellular responses to homologous parasites43. an endemic area of both visceral and tegumentary leishmaniasis in Brazil. Thrichomys laurentius was adapted to captivity and experimental The development of a non-human primate model of leishmaniasis, patterns of L. infantum and L. braziliensis infections were identified in which largely mimics the human situation, is described for studies of this rodent. Both Leishmania species demonstrated the ability to invade different aspects of the disease that would not be possible in humans and persist in the viscera and skin of T. laurentius, yet no rodent displayed for ethical reasons. However, for financial and ethical reasons, the use skin lesions, histological changes in skin, spleen or liver, nor clinical of primates in biomedical research is limited. Studies involving these evidence of infection96. animals have, therefore, been tailored to solve questions that cannot be answered in other animals. Monkeys are normally the final experimental In the Yucatan peninsula of Mexico, Peromyscus yucatanicus has been animals to be used in studies of the safety and efficacy of vaccines and identified as primary reservoir ofL. (L.) mexicana22,109. It has been adapted drugs developed in other laboratory animals48. to the laboratory and a colony was established for experimental studies.
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P. yucatanicus inoculated with 106 promastigotes of L. (L.) mexicana on flagellated promastigotes penetrate into the macrophage, transform into the base of the tail reproduced both the clinical and histopathological amastigotes and multiply. The infected macrophage eventually bursts and picture of CL in humans, supporting its utility as a novel experimental the released parasites are able to infect new phagocytic cells. When the model to study CL caused by L. (L.) mexicana104. Moreover 100% of P. infected host is bitten by another female sand fly, parasites are ingested yucatanicus inoculated with 102 (“low inoculum”) developed subclinical and the life cycle continues. The course of the disease is variable ranging infection (absence of clinical signs and evidence of parasite´s DNA at from spontaneous healing to chronicity, but most infected individuals the site of inocula) and when immunosupressed with cyclophosphamide remain asymptomatic or subclinical. Therefore, there is a wide infection a reactivation with the appearance of lesions was observed29. Nitric spectrum as a result of the parasite inoculation. As a consequence it is oxide production was documented in co-cultured macrophages and necessary to study the significance of subclinical infection in humans and lymphocytes from P. yucatanicus with clinical and subclinical infection other hosts. Therefore, when building a “good” animal model to study caused by L. (L.) mexicana64. Although NO production was observed in leishmaniasis immunology, should we consider all the possible outcomes these wild rodents, they were unable to clear the infection, which differs of the Leishmania spp. infection, particularly subclinical infection? with the response observed in murine models where the generation of NO is the main effector mechanism involved in the control of L. With reference to the suggested requirement to “mimic immunological major infection. Similarly, the role of this cytotoxic molecule in the responses observed in humans” when exposed to a particular Leishmania antileishmanial activity of human macrophages remains controversial42. spp., the problem becomes more complex if we consider that it varies depending on multiple factors according to present knowledge of the Recently cDNAs of Th1, Th2 and Th17 cytokines have been amplified host-parasite interaction. In a recent work of the Working Group on from P. yucatanicus spleen cells by PCR using P. maniculatus primers research Priorities for Development of Leishmania Vaccines, a good cloned into TOPO TA cloning vector and sequenced63,111. These results review was made on vaccine trials in the last three decades, the profile strongly support employing P. maniculatus specific primers to study the of strategies, and animal models used in leishmaniasis trials108. The main kinetics of cytokines involved in the immune response against clinical and questions raised encompassed issues concerning all of the leishmaniases. subclinical L. (L.) mexicana infection in P. yucatanicus. This approach They have addressed the employment of live attenuated or genetically will allow the quantifying and analyzing of the expression of important modified parasites, the role of vectors, and elucidation of protective cytokines, transcription factors and cellular markers involved in the immunity. Regarding the last issue, they considered it crucial to test immune response to L. (L.) mexicana infection in a specific manner. It will vaccine candidates in different models using different species, and to also permit to determine the immune response leading to the clinical and test the effects of including salivary proteins of vectors. The major subclinical infection in Yucatan deer mice and compare with the immune challenge is the absence of an experimental animal model that mimics response observed in humans, in order to confirm its importance as an the whole picture of human leishmaniasis, i.e. different subclinical and experimental model to study LCL caused by L. (L.) mexicana. clinical outcomes and protective immune response. This situation leads to the necessary development of research studies focused mainly on CONCLUSIONS building new animal models capable of evaluating the same criteria in both models and humans. First of all, there is a worldwide agreement regarding the concept that experimental animal models are expected to mimic the pathological The use of wild rodents, primary reservoirs, as experimental models features and immunological responses observed in humans when for studying Leishmania infection could be very useful to elucidate their exposed to a variety of Leishmania spp. with different pathogenic role as reservoirs so as to improve our knowledge about the parasite- characteristics50. This approach deserves to be re-analyzed based on vector and parasite-host relationship, in order to understand what happens updated studies. in human Leishmania infections.
What does it mean to “mimic the patholgical features”? It is clear FINAL CONSIDERATIONS AND PERSPECTIVES to date that the outcomes of the infection depend on a variety of factors in each particular laboratory animal including: a) the Leishmania spp. The use of experimental animal models remains a good alternative inoculated; b) the virulence of the parasite isolate used; c) the parasite for designing immunological studies that, for ethical reasons, cannot stage, via, size, and route of the inoculum. In addition, the nature of be performed in humans. Certainly, the increased interest in studying each laboratory animal, i.e. the genetic makeup that relates to the the immune response against Leishmania infection in different immunological background, which plays an important role in host- animal models has contributed to our understanding of parasite-host parasite realtionships. Moreover, when we say “leishmaniasis” as so “the relationship. However no model can develop all the possible outcomes leishmaniases”, we are referring to a group of diseases that are caused of the Leishmania infection or entirely reproduce the disease in humans. by different species of protozoan parasites of the genus Lesihmania. Thus, there are still so many questions to answer in order to find control We have been trying (as it has been done in other pathologies such as strategies or a successful vaccination program. “cancer”) to include different diseases as a single pathological entity. Therefore, shouldn’t we develop a different experimental animal model Although mouse model has widely contributed to the understanding for each leishmaniasis? of immune response against L. major infection many studies have demonstrated profound differences in the immune mechanisms related It is well known that infection begins when an infected female sand to infections with New World Leishmania species. Furthermore, fly takes a blood meal from a human host in a leishmaniasis endemic visceral infection in mice does not mimic the pathological features area. Following inoculation into the skin by the sand fly bite, the and immunological responses observed in human cases. The hamster
7 LORÍA-CERVERA, E.N. & ANDRADE-NARVÁEZ, F.J. - Animal models for the study of leishmaniasis immunology. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 1-11, 2014.
result is a better model to study the progressive disease of visceralizing ACKNOWLEDGMENTS Leishmania spp. The lack of reagents for immunological analysis and the strong immunosuppression of the lymphoproliferative response in To Dr. Bruno Travi for editing the manuscript. hamsters make difficult its use for the evaluation of vaccine candidates. The increased interest of researchers to use dogs as experimental REFERENCES models lies in the possibility of studying the immune response in natural infection. Since dogs are important reservoirs of visceralizing 1. Afonso LC, Scott P. Immune responses associated with susceptibility of C57BL/10 Leishmania, vaccination of these animals would constitute a major step mice to Leishmania amazonensis. Infect Immun. 1993;61:2952-9. towards the control of human infections. Finally, the use of monkeys has 2. Aguilar-Torrentera F, Carlier Y. Immunological factors governing resistance and been explored for testing vaccine candidates, however little is known as susceptibility of mice to Leishmania major infection. Rev Latinoam Microbiol. to whether the immune response to Leishmania infection is similar to 2001,43:135-42. that observed in humans. 3. Aguilar-Torrentera F, Laman JD, Van Meurs M, Adorini L, Muraille E, Carlieri, Y. Endogenous interleukin-12 is critical for controlling the late but not the early stage In order to obtain more meaningful data regarding immune response of Leishmania mexicana infection in C57BL/6 mice. Infect Immun. 2002;70:5075-80. that parallels human disease it is very important to continue with the efforts in developing strategies to mimic natural transmission such as 4. Alvar J, Cañavate C, Molina R, Moreno J, Nieto J. Canine leishmaniasis. Adv Parasitol. the use of low infectious doses, bioactive saliva and natural reservoir 2004;57:1-64. hosts to have a better approximation of the dynamics of natural infection. 5. Alvar J, Yactayo S, Bern C. Leishmaniasis and poverty. Trends Parasitol. 2006;22:552-7. These approaches could contribute to developing improved experimental models for studying leishmaniasis and identifying possible targets to 6. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide evaluate vaccine candidates. and global estimates of its incidence. PLoS One. 2012;7:e35671.
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FAPESP/BIREME Project on Scientific Electronic Publications Latin American and Caribbean Center on Health Sciences Information Rua Botucatu 862 – 04023-901 São Paulo, SP – Brazil Tel. (011) 5576-9863 [email protected] Rev. Inst. Med. Trop. Sao Paulo 56(1):13-20, January-February, 2014 doi: 10.1590/S0036-46652014000100002
EVALUATION OF ANTIMICROBIAL AND CYTOTOXIC ACTIVITIES OF PLANT EXTRACTS FROM SOUTHERN MINAS GERAIS CERRADO
Juliana Moscardini CHAVASCO, Bárbara Helena Muniz PRADO E FELIPHE, Claudio Daniel CERDEIRA, Fabrício Damasceno LEANDRO, Luiz Felipe Leomil COELHO, Jéferson Junior da SILVA, Jorge Kleber CHAVASCO & Amanda Latercia Tranches DIAS
SUMMARY
The antimicrobial activity of plant hidroethanolic extracts on bacteria Gram positive, Gram negative, yeasts, Mycobacterium tuberculosis H37 and Mycobacterium bovis was evaluated by using the technique of Agar diffusion and microdilution in broth. Among the extracts evaluated by Agar diffusion, the extract of Bidens pilosa leaf presented the most expressive average of haloes of growth inhibition to the microorganisms, followed by the extract of B. pilosa flower, of Eugenia pyriformis’ leaf and seed, of Plinia cauliflora leaf which statistically presented the same average of haloes inhibitory formation on bacteria Gram positive, Gram negative and yeasts. The extracts of Heliconia rostrata did not present activity. Mycobacterium tuberculosis H37 and Mycobacterium bovis (BCG) appeared resistant to all the extracts. The susceptibility profile of Candida albicans and Saccharomyces cerevisiae fungi were compared to one another and to the Gram positive Bacillus subtilis, Enterococcus faecalis and the Gram negative Salmonella typhimurium bacteria (p > 0.05). The evaluation of cytotoxicity was carried out on C6-36 larvae cells of the Aedes albopictus mosquito. The extracts of stem and flower of Heliconia rostrata, leaf and stem of Plinia cauliflora, seed of Anonna crassiflora and stem, flower and root of B. pilosa did not present toxicity in the analyzed concentrations. The highest rates of selectivity appeared in the extracts of stem of A. crassiflora and flower of B. pilosa to Staphylococcus aureus, presenting potential for future studies about a new drug development.
KEYWORDS: Plant extracts; Antimicrobial activity; Mycobacterium tuberculosis; Mycobacterium bovis; Cytotoxicity.
INTRODUCTION gastroenteritis and hepatitis6, in buccal antissepsia and anti-jaundiced33. Some compound classes like flavonoids and polyacetylenes were isolated The indiscriminate use of antimicrobials by population in general from Bidens pilosa30. has been causing serious public health problems all over the world due to the fact that the microorganisms have the ability to develop resistance Eugenia pyriformis Cambess, popularly known as uvaia, has a yellow to these therapeutic agents29. fruit, is edible and may be used to make juices, vinegar and wine1,16. According to STIEVEN et al., (2009)40 the fruit of Eugenia pyriformis Bacteria which belong to the Mycobacterium type, mainly the has bacteriostatic activity when compared to isolates of Escherichia coli, Mycobacterium tuberculosis has been presenting resistance to the Staphylococcus aureus and Enterococcus faecalis. antibiotics, probably, due to the long treatment time and to the adverse drug effects, factors which make patients decline the treatment42. Annona crassiflora Mart is a fruitlike species and exclusive from the Brazilian cerrado, known as araticum and marolo25. Phytochemical study Nowadays, there is a great interest from the pharmaceutical conducted by GONÇALVES et al., (2009)20 with the stem of Anonna industries in the use of biodiversity as a source of new medicine4, and the crassiflora Mart resulted in obtaining alkaloids with antimicrobial pharmaceutical industries’ integration with universities that are studying activity. bioprospective natural resources in search of new medicines has stood out as being increasingly more important26. Plinia cauliflora Berg, known as the jabuticaba tree, is a fruit tree belonging to the Myrtaceae family, spontaneously growing in large Among the plants found in the southern cerrado of Minas Gerais, parts of Brazil. The bark is astringent, useful against diarrhea and skin Bidens pilosa Linné, known as Picão preto, is widely used in the irritations. We also have indications in folk medicine as anti-asthmatic traditional medicine as an anti-flu, for diabetes control, as a treatment for medicine, for bowels inflammation and hemoptysis27.
Biomedical Science Institute, Microbiology and Immunology Department, Federal University of Alfenas, Alfenas, MG, Brazil. Correspondence to: Prof. Amanda L.T. Dias. Rua Gabriel Monteiro da Silva 700, 37130-000 Alfenas, MG, Brasil. Phone: +55.35.32991305. E-mail: [email protected] CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
There have been few studies surrounding the Heliconia genus and The preparation of the plant extracts: Extracts were prepared from the number of existing species is still not certain23. Among the various fresh plant parts (leaves, flowers, roots, stems and fruits) in ethyl alcohol species, there is the Heliconia rostrata also known as banana garden tree. 70% w/v. After being soaked for seven days, the extracts were filtered, subjected to concentration on rotary evaporator to a negative pressure The need for new antimicrobial substances reinforces the growing of 500 mmHg at 60 °C and, subsequently frozen and lyophilized. Upon investigation of the therapeutic potential of medicinal plants, where some use, the lyophilized extracts were relifted in distilled water to obtain of its compounds with the antimicrobial property such as flavonoids, concentrations of 100 mg/mL and then sterilized by filtration. alkaloids, sterols and saponins have been the subject of interest in the treatment of various human infections34,43. Microbial strains: The strains studied are recommended by other authors and they are representative of a wide variety of microorganisms COWAN (1999)13 reported, based on studies of this literature, that that belong to the main bacterial and fungal groups. the antimicrobial activity of flavonoids is possibly due to the ability of this group to complex with extracellular and soluble proteins and also Yeasts: Candida albicans ATCC 10231; Saccharomyces cerevisiae with the bacterial cell wall. The mechanism of the antimicrobial action ATCC 2601; of tannins can be explained by three hypotheses. The first assumes tannins inhibiting bacterial and fungal enzymes and/or complexing with Gram positive bacteria: Bacillus subtilis ATCC 6633, Bacillus cereus the enzyme substrates and, the second includes the action of tannins on ATCC 11778, Micrococcus luteus ATCC 9341, Enterococcus faecalis the cell membranes of the microorganisms, altering metabolism, and the ATCC 51299 and Staphylococcus aureus ATCC 6538; third is based on the complexation of macromolecules with metal ions, reducing the availability of ions essential for microbial metabolism36. Gram negative bacteria: Escherichia coli ATCC 25922, Serratia marcescens LMI-UNIFAL, Pseudomonas aeruginosa ATCC 27853, The ability of saponins to form complexes with steroids, proteins Proteus mirabilis ATCC 25922, Salmonella typhimurium ATCC 14028 and phospholipids of cell membranes may change its permeability or Enterobacter cloacae LMI-UNIFAL. even lead to its destruction37. Mycobacteria: Mycobacterium bovis (BCG strain) ATCC 27289 and Alkaloids are complex compounds, basic in nature, defined by the Mycobacterium tuberculosis (H37) ATCC 27294. amine function, which provides its constituents, chemical properties that are related to a high toxicity and a remarkable pharmacological Evaluation of antimicrobial activity of the extracts: The activity. Isolated compounds and plant extracts rich in alkaloids, have antimicrobial activity was evaluated by agar diffusion through the method demonstrated antimicrobial activity in several studies5. defined in document M7A6 (CLSI, 2003)9 for bacteria, M24A2 (CLSI, 2008)11 for Mycobacterium spp. and M44A2 (CLSI, 2009)8 for fungi. With the development of this research, we aimed to study the Mueller Hinton agar was used for bacteria and Mueller Hinton agar feasibility of using the plant extracts found in Southern cerrado area of supplemented with 2% glucose was used for yeasts. Minas Gerais State as probable antimicrobial agents with potential action on bacteria and fungi, besides the knowledge of the cytotoxic potential The activity on Mycobacterium bovis and Mycobacterium of these extracts. This study will significantly contribute to national and tuberculosis was determined by agar diffusion method in Middlebrook international research aimed at the bio-prospecting of new bioactive 7H10 agar medium with Middlebrook OADC Enrichment®. The herbal plants, safe in accordance with the clinical point of view, which have extracts, 100 mg/mL, in a volume of 10 µL, were placed on filter paper the potential to be future drug candidates for their use in antimicrobial disks of 10 mm diameter and dried at 37 °C. The Middlebrook 7H10 therapeutic regimens. agar was inoculated with a suspension of M. bovis and M. tuberculosis with a turbidity corresponding to the Mac Farland 0.5 range. The disks MATERIALS AND METHODS containing the extracts were placed on the surface of the culture medium. Cultures were incubated at 37 ºC for 28 days. Plant collection and identification: The Bidens pilosa samples were collected in January 2011, at Jatobá Neighborhood, in Pouso Alegre city - The minimum inhibitory concentration (MIC) was assessed through MG (22° 27’ 75’’ S, 18° 45’ 90’’ W). The collection of stem samples, leaves broth microdilution according to the methodology proposed in document and fruits of adult Eugenia pyriformis was conducted in Alfenas countryside M27A3 (CLSI, 2008)10 and with concentration range from 50 mg/mL to - MG (21° 25’ 44’’ S and 45° 56’ 49’’ W) in January 2011. Samples of leaf, 0.010 mg/mL. The MIC for M. bovis and M. tuberculosis was determined stem and fruit of adult Plinia cauliflora were collected in January 2011 in in Middlebrook 7H10 agar dilution in concentrations of 50 mg/mL, the city of Alfenas - MG (21° 25’ 44’’ S, 45° 56’ 49” W). The collections of 25 mg/mL and 12.5 mg/mL. The tests were performed in triplicate at leaves, flowers, pseudostem and rhizome of Heliconia rostrata were held different days. in Areado city - MG (21º 24’ 39.44’’ S and 46º 08’ 53.81’’ W) in January 2011. A fruit sample, stem and leaf of Annona crassiflora was held in Evaluation of the cytotoxic activity of the plant extracts Alterosa city, (21° 8’ 50.18’’ S and 46° 5’ 06.13” W) in March 2011. The on cell culture: Cytotoxicity was assessed by MTT method [3 - plant samples are deposited in the Herbarium of the Federal University (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. In this of Alfenas (UNIFAL-MG), receiving the following tumble numbers: test 1x104 cells (derived from Aedes albopictus larvae) were seeded Bidens pilosa (1745), Eugenia pyriformis (1459), Plinia cauliflora (1637), per well in 96-well plates, containing L-15 medium. 0.1 mL of L-15 Heliconia rostrata (1636), Annona crassiflora (1401). medium containing 1% fetal bovine serum with decreasing dilutions of
14 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
the extracts (from 5 mg/mL to 0.039 mg/mL) was added to the culture. 25 mg/mL. S. aureus was shown to be the most susceptible to the extract After incubation, 10 µL of MTT at a concentration of 5 mg/mL was added of flower whose MIC was 1.56 mg/mL (Table 1). In phytochemical and incubated for four hours at room temperature to a MTT incorporation tests, the root extract showed tannin and saponin in its composition. and the formation of formazan crystals. Spectrophotometric analysis was The stem extracts showed alkaloid, tannin and saponin. The leaf and performed in microplate reader at 600 nm. The percentage of cytotoxicity flower extracts showed alkaloid, flavonoid, tannin and saponin (Table was calculated using the [(AB) / A X 100] formula, where A and B are 2). Results similar to OLIVEIRA et al., (2004)30 and MARTINS et al., values of optical densities of treated and controlled cells, respectively. (2000)24, without specifying the source of the extract, found flavonoids The values of cytotoxic concentration (CC50 and CC90)2 were stablished. and tannins, respectively. Considering the wealth of constituents that these extracts present, antimicrobial activity may be related to the presence of Determination of the plant extract selectivity index: The compounds like tannins, saponins, flavonoids and alkaloids found, yet selectivity index of the extracts was calculated by evaluating the ratio these compounds have demonstrated antimicrobial activity. between the CC50 and MIC5021. Values greater than 1 were considered promising for the selection of extracts for further studies. Evaluation of antimicrobial activity of Eugenia pyriformis Cambess extracts: According to the results presented in Table 1 we Evaluation of phytochemical profile of the extracts: In see that the leaf extract of E. pyriformis inhibited the growth of all phytochemical screening the following groups of active ingredients microorganisms, with the exception of M. bovis and M. tuberculosis. were surveyed: flavonoids, the Shinoda reaction technique and alkalis The largest diameter of inhibition zone in terms of absolute value was hydroxide reaction; tannins through precipitation method with iron salts, observed for M. luteus. The diameter of inhibition haloes was compared lead acetate, alkaloids, gelatin and copper acetate; alkaloids through among Gram positive and Gram negative bacteria. There were also precipitation method with Mayer, Bertrand, and Dragendorff Bouchardat inhibition haloes against the S. cerevisiae and C. albicans yeasts. The reactives; steroids, the Liebermann-Burchard reaction; saponins, stem extract inhibited the growth of ten microorganisms (67%). The through the stir of aqueous extract with persistent foam formation and largest diameter of inhibition zone was observed for M. luteus, while anthraquinones through direct Borntrager reaction12. for S. marcescens and P. aeruginosa the lowest diameters were found. The fruit extract inhibited the growth of nine microorganisms (60%). Statistical analysis: The data were statistically considered by The extract showed no activity against P. mirabilis, E. coli, C. albicans, variance analyses (ANOVA) and Scott-Knott test at 5% significance S. cerevisiae, M. bovis and M. tuberculosis. (Sisvar Software version 5.3, 2010). The seed extract inhibited the growth of 13 (87%) of the 15 RESULTS AND DISCUSSION microorganisms. The extract did not show activity against S. marcescens. The largest diameter of inhibition zone was verified to M. luteus. The Evaluation of antimicrobial activity of Bidens pilosa extracts: extract showed antifungal activity against S. cerevisiae and C. albicans. The stem extract showed activity against four microorganisms: S. aureus, In our study, the fruit extract showed antimicrobial activity against M. luteus, C. albicans and S. cerevisiae. The largest inhibition zone, in E. faecalis and S. aureus and P. aeruginosa, unlike STIEVEN et al., absolute value was observed for M. luteus. The flower and leaf extracts (2009)40, who found activity against E. coli and found no activity against inhibited the growth of 10 microorganisms and the largest inhibition P. aeruginosa. This difference may be related to the origin of the plant zone, in absolute figure was observed forS. cerevisiae. The leaf extract extracts, because the ATCC strain that we evaluated was the same as of Bidens pilosa showed the most significant average haloes of growth Stieven group. inhibition when compared among the microorganisms (p < 0.05). The largest zones of inhibition were observed at S. aureus, M. luteus, S. The leaf extracts showed alkaloid, flavonoid, tannin and saponin. typhimurium, B. cereus, E. coli, C. albicans and S. cerevisiae. The leaf The extracts showed stem tannin and saponin in their composition. The extract showed no activity against P. aeruginosa, B. subtilis, E. faecalis. seed extracts showed alkaloids, tannins and saponins (Table 1). The The smallest halo of growth inhibition was observed for S. marcescens. results of this study are similar to ARMSTRONG’s (2011)3 which found The stem extract showed no activity against Gram negative bacteria. The flavonoids, tannins and saponins in the extracts. Considering the presence smallest inhibition zones were observed for C. albicans and S. cerevisiae. of compounds such as tannins, saponins, flavonoids and alkaloids, which The flower extract showed no activity against P. aeruginosa, B. subtilis, present proven antimicrobial activity, it is concluded that the antimicrobial E. faecalis, M. bovis and M. tuberculosis. According to the results shown activity of the extracts may be related to the presence of these compounds. in Table 1 we can see that the root extract of B. pilosa did not inhibit the Regarding the MIC the values ranged from 12.50 to 50 mg/mL. growth of microorganisms. Evaluation of antimicrobial activity of Annona crassiflora Mart MOTSEI et al., (2003)28 and DEBA et al., (2008)15, without specifying extracts: In Table 1 we can see that the leaf extract of A. crassiflora the source of the extract showed that B. pilosa has antifungal activity, inhibited the growth of six (40%) of the 15 microorganisms tested: S. similar to our results, however both yeasts showed no susceptibility to the aureus, M. luteus, P. mirabilis, B. subtilis, E. faecalis and B. cereus. root extract of B. pilosa. ROJAS et al., (2006)33 also without specifying Comparing the size of inhibition haloes, in absolute values, we conclude the source of the extract, showed that B. pilosa presents growth inhibitory that the Gram positive had a higher susceptibility than the Gram negative. activity on B. cereus, E. coli and S. aureus, similar to our results, verifying The leaf extract did not show antimicrobial activity on yeasts. The stem that these bacteria weren’t susceptible to the root extract of B. pilosa. extract inhibited the growth of seven (47%) of the 15 microorganisms tested. The extract did not show activity against E. cloacae, P. aeruginosa, Related to MIC determination, the values varied from 1.56 mg/mL to S. marcescens, E. coli, C. albicans, S. cerevisae, M. bovis and M.
15 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
Table 1 Evaluation of antimicrobial activity (agar diffusion and broth microdilution), cytotoxicity and selectivity index of plant extracts from cerrado in the south of Minas Gerais.
Microorga Plant Heliconia rostrata Plinia cauliflora Anonna crassiflora Bidens pilosa Eugenia pyriformis Controls nism/Test Fraction Ca Fl Fo Riz CF Fo Ca CF Ca Se Po Fo Ra Ca Fl Fo Fo Ca Fr Se Cl Ri Ag Results MH 0 0 0 0 14 14 9 15.3 10 0 9 13 0 0 17.3 18 15 11.5 12.3 14.7 13 ---- 0 Bacillus MIC N N N N 50 50 25 50 50 N 50 25 N N 12.5 12.5 25 12.5 12.5 25 ------cereus SI NA NA NA NA 0.026 NA NA 0.0412 0.1172 NA 0.0536 0.1544 NA NA 0.2256 NA 0.3822 NA 0.1688 0.0504 ------MH 0 0 0 0 10 11.7 7.3 11.3 10.3 0 0 11.3 0 0 0 0 13.7 11.3 10.3 13 15.3 ------0 Bacillus MIC N N N N 50 50 50 50 25 N N 50 N N N N 25 25 50 50 ------subtilis SI NA NA NA NA 0.026 NA NA 0.0412 0.2344 NA NA 0.0722 NA NA NA NA 0.3824 NA 0.0422 0.0252 ------MH 0 0 0 0 13.7 12.7 7.3 15 8.3 0 9 14.3 0 0 0 0 13.7 10 10.3 12 14 ------0 Enterococ- MIC N N N N 50 50 25 25 25 N 50 25 N N N N 50 50 50 50 ------cus faecalis SI NA NA NA NA 0.026 NA NA 0.0824 0.2344 NA 0.0536 0.1544 NA NA NA NA 0.1912 NA 0.0422 0.0252 ------MH 0 0 0 0 16.3 18.3 18.3 19 15 0 13 13.7 0 15.7 23.3 23.7 18 14.7 19.3 18 20 ------0 Micrococcus MIC N N N N 50 25 25 12.5 25 N 50 50 N 25 25 25 50 50 50 25 ------luteus SI NA NA NA NA 0.026 NA NA 0.1648 0.2344 NA 0.0536 0.0772 NA 0.1564 0.1128 NA 0.1912 NA 0.0422 0.0504 ------MH 0 0 0 0 15.3 16.7 13 18 12 0 16 12 0 14.3 22 26.3 12 11.3 12 13.7 16.3 ------0 Staphylococ- MIC N N N N 25 50 6.25 6.25 1.56 N 12.5 25 N 25 1.56 25 25 25 12.5 25 ------cus aureus SI NA NA NA NA 0.052 NA NA 0.3296 3.76 NA 0.1072 0.1544 NA 0.1564 1.81 NA 0.3824 NA 0.1688 0.0504 ------MH 0 0 0 0 7.3 10.3 8 0 0 0 0 0 0 0 10.3 12.7 12 0 12 10.3 13.7 ------0 Enterobacter MIC N N N N 50 25 25 N N N N N N N 25 50 25 N 12.5 50 ------cloacae SI NA NA NA NA 0.026 NA NA NA NA NA NA NA NA NA 0.1128 NT 0.3824 NA 0.1688 0.0252 ------MH 0 0 0 0 11.3 10.3 12.3 0 0 0 0 0 0 0 12 16.3 11.3 0 0 9.7 14 ------0 Escherichia MIC N N N N 25 25 50 N N N N N N N 25 25 12.5 N N N ------coli SI NA NA NA NA 0.052 NA NA NA NA NA NA NA NA NA 0.1128 NT 0.7648 NT NA 0.0252 ------MH 0 0 0 0 8.3 13.7 10.3 16 10 0 12 13.3 0 0 9 11 13.4 11.3 0 11 6 ------0 Proteus MIC N N N N 50 50 25 50 50 N 50 25 N N 50 50 50 50 N 50 ------mirabilis SI NA NA NA NA 0.026 NA NA 0.0412 0.1172 NA 0.0536 0.1544 NA NA 0.0564 NA 0.1912 NA NA 0.0252 ------Pseudo- MH 0 0 0 0 9 13.7 9.7 0 0 0 0 0 0 0 0 0 10 8 9.7 10.3 12 ------0 monas MIC N N N N 50 50 50 N N N N N N N N N 50 50 50 50 ------aeruginosa SI NA NA NA NA 0.026 NA NA NA NA NA NA NA NA NA NA NA 0.1912 NA 0.0422 0.0252 ------MH 0 0 0 0 7.3 7.7 8 8 7.3 0 0 0 0 0 16 24 10 14.3 10.3 12 12.3 ------0 Salmonella MIC N N N N 50 50 50 50 50 N N N N N 25 25 50 50 50 50 ------typhimurium SI NA NA NA NA 0.026 NA NA 0.0412 0.1172 NA NA NA NA NA 0.1128 NA 0.1912 NA 0.0422 0.0252 ------MH 0 0 0 0 10 8.7 8 0 0 0 0 0 0 0 11.7 10.7 10 8 8 0 3.7 ------0 Serratia MIC N N N N 25 25 25 N N N N N N N 25 25 25 50 25 N ------Marcescens SI NA NA NA NA 0.052 NA NA NA NA NA NA NA NA NA 0.1128 NA 0.3824 NA 0.0844 NA ------MH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ----- 26.3 0 Mycobacte- MIC N N N N N N N N N N N N N N N N N N N N ------rium bovis SI NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA ------Mycobac- MH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ----- 26.3 0 terium MIC N N N N N N N N N N N N N N N N N N N N ------tuberculosis SI NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT ------MH 0 0 0 0 0 11.7 0 0 0 0 0 0 0 13.3 24 28 10.3 10.3 0 15 15.3 ------0 Candida MIC N N N N N 50 N N N N N 0 N 25 25 12.5 12.5 50 N 25 ------albicans SI NA NA NA NA NA NA NA NA NA NA NA NA NA 0.1584 0.1128 NT 0.7648 NA NA 0.0504 ------MH 0 0 0 0 11.5 8.3 8.7 0 0 0 0 0 0 13.3 26.7 29.3 12.3 0 0 14 18.7 ------0 Saccharomy- MIC N N N N 50 50 25 N N N N N N 25 12.5 12.5 25 N N 50 ------ces cerevisae SI NA NA NA NA 0.026 NA NA NA NA NA NA NA NA 0.1564 0.2256 NA 0.3824 NA NA 0.0252 ------
CC50 NT NT 1.4 2.15 1.3 NT NT 2.06 5.86 NT 2.88 3.86 NT 3.91 2.82 NT 9.56 NT 2.11 1.26 ------
CC90 NT NT 2.6 6.9 2.55 NT NT 9.38 10.77 NT 5.98 8.03 NT 7.27 5.41 NT 19 NT 4.25 3.44 ------AH: Halo Average; MIC: Minimal Inhibitory Concentration; CC50: Cytotoxic Concentration for 50% of the cells; CC90: Cytotoxic Concentration for 90% of the cells; SI: Selectivity Index (CC 50 / MIC), S: Stem, Fl: Flower; Lf: Leaf; Riz: Rhizome, FP: Fruit Peel, Fr: Fruit, Sd: Seed; Pl: Pulp, Rt: Root, Cl.: chlorhexidine 0.12%; Ri: Rifamycin 30 µg; Dw: distilled water, NT = non-toxic in the concentrations used in the experiment, NA: Not Applicable, N = not detected values at the concentrations tested.
16 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
Table 2 Determination of chemical compounds of Anonna crassiflora, Bidens pilosa, Eugenia pyriformis, Heliconia rostrataand Plinia cauliflora plant extracts by phytochemical analysis
Plant Extrat Alkaloids Anthraquinones Flavonoids Tannins Saponins Fruit Peel + - - + - Stalk - - - + - Anonna crassiflora Seed - - - - - Pulp + - - - + Leaf + - + - - Root - - - + + Stalk + - - + + Bidens pilosa Flower + - + + + Leaf + - + + + Leaf + - + + + Stalk - - - + + Eugenia pyriformis Fruit - - - - - Seed + - - + + Stalk - - - + + Flower - - - - + Heliconia rostrata Leaf - - + + - Rizoma - - + - - Fruit Peel + - - - + Plinia cauliflora Leaf - - + + + Stalk - - - + + + : presence in reactions; - : absence. tuberculosis microorganisms. The pulp extract inhibited the growth of five Evaluation of antimicrobial activity of Plinia cauliflora extracts: (33%) of the 15 microorganisms tested. The seed extract did not inhibit All tested microorganisms were susceptible to P. cauliflora extracts, the growth of any of the microorganisms. The bark extract of the fruit however, C. albicans was susceptible only to the leaf extract except for inhibited the growth of seven of the 15 microorganisms tested. The extract M. bovis and M. tuberculosis. Among the evaluated microorganisms, M. showed no activity against E. cloacae, P. aeruginosa, S. marcescens, E. luteus showed greater halo inhibition in absolute value when compared coli, C. albicans, S. cerevisiae, M. bovis and M. tuberculosis. The bark to other microorganisms. In the studies of MACEDO et al. (2009)22 , the extract had greater activity against Gram positive bacteria. SILVA et leaf extract showed antimicrobial action on five strains of Streptococcus al., (2001)38 conducted experiments with leaf extracts of A. crassiflora spp., similar to our results whose leaf extract was active on all Gram investigating its activity over 52 strains of C. albicans from clinical origin, positive with MIC values among 6.25 mg/mL and 50 mg/mL. which showed susceptibility to them. In our study, the evaluated extracts didn’t show growth inhibitory activity against C. albicans. This difference Alkaloid and saponin were found in extracts of the fruit skin. in results can probably be explained by the technique of obtaining the Flavonoid, tannin and saponin were found in leaf extracts and tannins extract, or by the origin of the plant or by the diversity of strains when and saponins were found in stem extracts (Table 2). In the phytochemical isolated from clinical specimens. In relation to MIC the values ranged study conducted by REYNERTSON et al. (2006)32, without specifying from 1.56 to 50 mg/mL. the extract source, tannin was found in its composition, that is similar to this work result. The antimicrobial activity of P.cauliflora may be related The extracts of fruit showed alkaloid and tannin in their composition. to the presence of flavonoids, alkaloids, saponins and tannins, which have The stem extracts presented tannin in their composition. The fruit pulp proven antimicrobial activity. extracts showed saponin, while the leaf extracts showed flavonoid and alkaloid (Table 2). Similar results were found by GONÇALVES et Evaluation of antimicrobial activity of Heliconia rostrata extracts: al., (2009)20 that found alkaloid in phytochemical screening. As these The results show that H. rostrata extracts did not inhibit the growth of the compounds have antimicrobial activity, the obtained results may be microorganisms, as described in Table 1. According to Table 2, the stem related to the presence of the same. extract presented tannin and saponin in its composition. The flower extracts
17 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
presented saponin. The leaf extracts presented flavonoid and tannin, and fungi C. albicans and S. cerevisiae were comparable to the Gram positive rhizone extracts presented flavonoids. Studies about the phytochemical B. subtilis, E. faecalis and Gram negative bacteria S. typhimurium (p > composition of the extracts and antimicrobial activity of H. rostrata were 0.05). From microorganisms which showed some susceptibility to any not found in literature to be compared to ours. As there is no growth of the extracts, M. bovis, P. aeruginosa, S. marcescens, E. cloacae and inhibition we can assume that the substances are found in low concentration E. coli were statistically also less susceptible. Only the leaf extracts of B. or that the active compounds are not soluble in liquid extractor, although, pilosa showed statistically significant equality over the average inhibitory many authors have suggested the use of 70% ethanol as a liquid extractor effectiveness when compared to the positive control Chlorhexidine. for plant extracts. Many negative results can also be influenced by external factors intrinsic to the extraction process as the degradation of compounds Evaluation of the cytotoxic activity of the extracts on cell culture: with antimicrobial activity during the extraction process. Another point According to results shown in Table 1, the stem and flower extracts of H. to be considered is the influence of external factors in the production of rostrata, leaf and stem of P.cauliflora, seed of A. crassiflora, stem, flower secondary metabolites, such as nutrient availability, interactions with pests, and root of B. pilosa showed no toxicity on the concentrations tested soil, circadian cycle, seasonality, postharvest life and humidity. Despite on cell culture. This is the first study that evaluated the antimicrobial some extracts do not present antimicrobial activity in agar diffusion tests, and cytotoxic activities of H. rostrata species, which is considered a they were subjected to MIC determination since discrepancies can occur member of a family of plants reported to have antiophidic activities among the results obtained by these methods, with alterations of the results (ESTRADA et al., 2010)17. Our results to B. pilosa are in accordance with when the test is performed in a liquid environment. the results of GARCIA et al. (2011)19 that found the extracts of B. pilosa didn’t present significant cytotoxic activity against a human pulmonary In general, the hydroethanolic extracts of tested plants showed tumor cell line A549. SOUZA-MOREIRA et al. (2011)39 evaluated in activity against the Gram positive, Gram negative bacteria and yeasts, vitro the cytotoxicity of fruit and leaf extracts of P. cauliflora with the however, the MIC values were greater than 1000 mg/mL. According to fibroblast cell line SIRC CCL 60, and leaf extract showed a considerable DALL’AGNOL et al. (2003)14; FABRY et al. (1998)18 and TANAKA et 50% inhibitory concentration of 0.48 μg/mL. In the study of SANTOS al. (2005)41 plant extracts are considered to be a promising inhibitory PIMENTA et al. (2003)35, the ethanolic extracts of A. crassiflora leaves potential and demonstrate antimicrobial activity at concentrations up to did not show cytotoxic activity, but the fractions resulting from solvent 100 mg/mL, a moderate inhibitory activity of 100-500 mg/mL, a weak extraction showed good brine shrimp larvicidal activity. In our study, the activity of 500-1000 mg/mL and inactive extracts when MICs are higher cytotoxic concentration for 50% of cells (CC50) of the extracts ranged than 1000 mg/mL. Therefore, according to this established pattern, the from 1.30 mg/mL to 9.56 mg/mL and the cytotoxic concentration for 90% hydroethanolic extracts tested were ineffective. of cells (CC90) of the extracts ranged from 2.55 mg/mL to 19.00 mg/mL.
However, new assessment tests with extracts prepared with other KLEYMANN & WERLING (2004)21 propose the activity selectivity solvents should be conducted. It is known that the chemical composition assay format as a new standard in anti-infective drug discovery and of an extract is the result of chemical reagents and methods used to clinical development. According to our results, the selectivity index values obtain it, and that the active components that are usually found in low (SI) for A. crassiflora stem and B. pilosa flower for S. aureus showed concentrations and in crude extract may be more diluted (CECHINEL values greater than 1, respectively, 3.76 and 1.86, showing a promising FILHO, 2000)7. As there were some different results from those reported potential for evaluations like antimicrobial drug candidates in infections in the literature, we conclude that several factors may have influenced treatment caused by S. aureus. the study, from climatic conditions and location of geographic species to the diffusion of the extract used in the experiment as well as the genetic RESUMO variation of some microbial strains. These findings emphasize the need to standardize and develop the several evaluation methodologies, in order Avaliação das atividades antimicrobiana e citotóxica de extratos to confirm and ensure the reliability of results obtained in antimicrobial de plantas do Cerrado do Sul de Minas Gerais assays with plant extracts. Foi avaliada a atividade antimicrobiana de extratos hidroetanólicos The development of new studies using other methods of plant de plantas sobre bactérias Gram positiva, Gram negativa, leveduras, extract diffusion is suggested as well as the use of extracts obtained in Mycobacterium tuberculosis H37 e Mycobacterium bovis pela técnica de the presence of other solvents besides other bacterial and fungal strains, difusão em Agar e microdiluição em caldo. Dentre os extratos avaliados when coupled with toxicity tests. pelo método de difusão em Agar, o extrato da folha de Bidens pilosa apresentou a mais expressiva média de halos de inibição de crescimento Among the extracts evaluated by agar diffusion method, the leaf frente aos microrganismos, seguido pelo extrato da flor de B. pilosa, da extract of B. pilosa showed the most significant average of growth folha e semente de Eugenia pyriformis, da folha de Plinia cauliflora inhibition haloes when compared to the microorganisms used in this que apresentaram estatisticamente a mesma média de formação de study, followed by the flower extracts of B. pilosa, leaves of E. pyriformis halos inibitórios sobre bactérias Gram positivas, Gram negativas e and P. cauliflora, and E. pyriformis seed which statistically showed leveduras. Os extratos de Heliconia rostrata não apresentaram atividade. the same average formation of inhibitory haloes. Among the tested Mycobacterium tuberculosis H37 e Mycobacterium bovis (BCG) microorganisms, the Gram positive bacteria M. luteus and S. aureus mostraram-se resistentes a todos os extratos. O perfil de sensibilidade dos showed the same susceptibility profile (p > 0.05). Gram negative bacteria fungos Candida albicans e Saccharomyces cerevisiae foram comparáveis E. coli, E. cloacae, S. marcescens and P. aeruginosa also showed the entre si e entre as bactérias Gram positivas Bacillus subtilis, Enterococcus same susceptibility profile (p > 0.05). The susceptibility profile of the faecalis e Gram negativa Salmonella typhimurium (p > 0.05). A avaliação
18 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
da citotoxicidade foi realizada sobre células C6-36 de larvas de mosquito 16. Delgado LF, Barbedo CJ. Tolerância à dessecação de sementes de espécies de Eugenia. Aedes albopictus. Os extratos de caule e flor de H. rostrata, folha e caule Pesq Agropec Bras. 2007;42:265-72. de P. cauliflora, semente de Anonna crassiflora e caule, flor e raiz de 17. Estrada GS, Jiménez SI, Alarcon JC, Vargas LJ. Application of ultrasound in the dissolution B. pilosa não apresentaram toxicidade nas concentrações avaliadas. Os of potential antiophidian compounds from two ethanolics extracts of two species of maiores índices de seletividade foram apresentados pelos extratos de Heliconias. Ultrason Sonochem. 2010;17:756-9. caule de A. crassiflora e flor de B. pilosa para Staphylococcus aureus, apresentando potencial para estudos como futuros candidatos a fármacos. 18. Fabry W, Okemo PO, Ansorg R. Antibacterial activity of East African medicinal plants. J Ethnopharmacol. 1998;60:79-84.
ACKNOWLEDGMENT 19. Garcia AD, Sánchez HR, Lizama RS. Citotoxicidad de extractos de plantas medicinales sobre la línea cellular de carcinoma de pulmón humano A549. Rev Cubana Farm. We thank Professor Dr. Marcelo Polo for plant identification and 2011;45:101-8. The Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ- 20. Gonçalves MA, Lara TA, Pimenta LP. Alcaloides oxaporfínicos da madeira em Annona 01684/08; 02782/10, 01413/12). crassiflora Mart. In: 25a Reunião Anual da Sociedade Brasileira de Química; 20-23 de maio de 2002; Poços de Caldas, MG. REFERENCES 21. Kleymann G, Werling HOA. A generally applicable, high-throughput screening- 1. Andrade RNB, Ferreira AG. Germination and storing of uvaia seeds (Eugenia pyriformis compatible assay to identify, evaluate, and optimize antimicrobial agents for drug Camb.) - Myrtaceae. Rev Bras Sementes. 2000;22:118-25. therapy. J Biomol Screen. 2004;9:578-87.
2. Araújo SAC, Teixeira MFS, Dantas TVM, Miranda AM, Lima FES, Melo VSP, et al. 22. Macedo-Costa MR, Diniz DN, Carvalho CM, Pereira MS, Pereira JV, Higino PJS. Eficácia Avaliação in vitro da atividade citotóxica de drogas antivirais em fibroblastos caprinos. do extrato de Myrciaria cauliflora (Mart.) O. Berg. (Jabuticabeira) sobre bactérias Ci Animal. 2008;18:25-31. orais. Braz J Pharmacogn. 2009;19(2B):565-71.
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6. Chang JS, Chiang LC, Chen CC, Liu LT, Wang KC, Lin CC. Antileukemic activity of 26. Miranda RRS. Estudo fitoquímico e avaliação do potencial farmacológico de Maytenus Bidens pilosa L. var, minor (Blume) Sherff and Houttuynia cordata Thunb. Am J salicifolia Reissek. [tese]. Belo Horizonte: Universidade Federal de Minas Gerais; Chin Med. 2001;29:303-12. 2007.
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9. Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial 28. Motsei ML, Lindsey KL, Van Staden J, Jãger AK. Screening of traditionally used South susceptibility tests for bacteria that grow aerobically. Approved standard. 6th ed. African plants for antifungal activity against Candida albicans. J Ethnopharmacol. Wayne, PA: CLSI; 2003. p. M7-A6. 2003;86:235-41.
10. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution 29. Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts antifungal susceptibility testing of yeasts. Approved Standard. 3rd ed. Wayne, PA: and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol. 2000;31:247- CLSI; 2008. p. M27-A3. 56.
11. Clinical and Laboratory Standards Institute (CLSI). Susceptibility testing of Mycobacteria, 30. Oliveira FQ, Andrade-Neto V, Krettli AU, Brandão MGL. New evidencies of antimalarial Nocardiae, and other aerobic Actinomycetes. Approved Standard. 2nd ed. M24-A2; activity of Bidens pilosa roots extract correlated with polyacetylene and flavonoids. 2008. J Ethnopharmacol. 2004;93:39-42.
12. Costa AF. Isolation and identification of the vegetables. In: Pharmacognosy. Lisboa: 31. Rajendran NK, Ramakrishnan J. In vitro evaluation of antimicrobial activity of crude Calouste Gulbenkian; 1982. v. 3, p. 926-62. extracts of medicinal plants against multi drug resistant pathogens. Biyoloji Bilimleri Araptyrma Dergisi. 2009;2:97-101. 13. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12:564-82. 32. Reynertson KA, Wallace AM, Adachi S, Gil RR, Yang H, Basile MJ, et al. Bioactive 14. Dall’Agnol R, Ferraz A, Bernardi AP, Albring DC, Sarmento L, Lamb L, et al. depsides and anthocyanins from Jabuticaba (Myrciaria cauliflora). J Nat Prod. Antimicrobial activity of some Hypericum species. Phytomedicine. 2003;10:511-6. 2006;69:1228-30.
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19 CHAVASCO, J.M.; PRADO E FELIPHE, B.H.M.; CERDEIRA, C.D.; LEANDRO, F.D.; COELHO, L.F.L.; SILVA, J.J.; CHAVASCO, J.K. & DIAS, A.L.T. - Evaluation of antimicrobial and cytotoxic activities of plant extracts from Southern Minas Gerais Cerrado. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 13-20, 2014.
34. Santos SC, Ferreira FS, Damião AO, Barros TF, Rossi-Alva JC, Fernandez LG. Avaliação 39. Souza-Moreira TM, Severi JA, Santos E, Silva VYA, Vilegas W, Salgado HRN, et al. da atividade antibacteriana dos extratos de Avicennia schaueriana Stapf & Leechm. Chemical and antidiarrheal studies of Plinia cauliflora. J Med Food. 2011;14:1590-6. ex Moldenke, Verbenaceae. Rev Bras Farmacogn. 2010;20:124-9. 40. Stieven AC, Moreira JJS, Silva CF. Essential oils of uvaia (Eugenia pyriformis 35. Santos Pimenta LP, Pinto GB, Takahashi JA, Silva LGF, Boaventura MAD. Biological Cambess):evaluation of microbial and antioxidant activities. Eclet Quim. screening of annonaceous Brazilian medicinal plants using Artemia salina (Brine 2009;34(3):7-13. shrimp test). Phytomedicine. 2003;10:209-12. 41. Tanaka JCA, Silva CC, Dias Filho BD, Nakamura CV, Carvalho JE, Foglio MA. 36. Scalbert A. Antimicrobial properties of tannins. Phytochemistry. 1991;30:3875-83. Constituintes químicos de Luehea divaricata Mart. (Tiliaceae). Quím Nova. 2005;5:834-7. 37. Schenkel EP, Gosmann G, Athayde ML. Saponinas. In: Simões CM, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR, org. Farmacognosia: da planta ao 42. Trabulsi LR, Althertum F. Microbiology. 4 ed. SãoPaulo: Atheneu; 2005. medicamento. 3 ed. Porto Alegre: UFRGS, Florianópolis: UFSC; 2002. p. 597-619. 43. Verdi LG, Brighente IMC, Pizzolatti MG. Gênero Baccharis (Asteraceae): aspectos 38. Silva MV, Costa TR, Costa MR, Ferreira EC, Fernandes OFL, Santos SC, et al. Growth químicos, econômicos e biológicos. Quím Nova. 2005;28:85-94. inhibition effect of Brazilian cerrado plant extracts on Candida species. Pharm Biol. 2001;39:138-41. Received: 12 January 2013 Accepted: 29 April 2013
20 Rev. Inst. Med. Trop. Sao Paulo 56(1):21-27, January-February, 2014 doi: 10.1590/S0036-46652014000100003
DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL
Camila Oliveira VASCONCELOS(1), Zirlane C. Branco COÊLHO(1), Cristina de Souza CHAVES(1), Clarissa Romero TEIXEIRA(2), Margarida M. Lima POMPEU(1) & Maria Jania TEIXEIRA(1)
SUMMARY
Recruitment of a specific cell population afterLeishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host’s blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host’s blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.
KEYWORDS: L. infantum chagasi; Sand fly saliva; Leukocytes; Inflammation.
INTRODUCTION pathways7. After C3 opsonization, promastigotes undergo an immune adherence reaction and bind to CR1 erythrocyte receptors9. Opsonization In the New World, visceral leishmaniasis is caused by protozoan of Leishmania metacyclic promastigotes with complement is rapid and parasites of the genus Leishmania (L. chagasi, synonymous L. infantum) lysis, via the membrane, attack complex (C5b-C9 complex) that begins and transmitted by the female phlebotomine sand fly, Lutzomyia 60 s after serum contact7. This results in efficient killing of approximately longipalpis, during blood repast. To obtain a blood meal, sand flies locate 90% of all inoculated parasites within a few minutes. Upon encounter of blood by introducing their mouthparts into the skin, tearing tissues, macrophages, parasites bind to CR3 on their surface that facilitates the lacerating capillaries and creating hemorrhagic pools upon which they uptake of parasites into their main host cell11. feed26. During this process, salivary gland contents are injected together with Leishmania promastigotes into the host’s skin32. In an attempt to It has also known that cell number and composition of the cellular probe and feed sand flies must first circumvent the host’s homeostatic infiltrate in the initial stages after infection greatly influences innate system, and the innate and acquired immune responses2. It has been well immune response and the development of acquired immune response. documented that to overcome these obstacles, sand flies evolved within Along gradients of C3a and C5a and several chemokines, such as their salivary secretion an array of potent pharmacological components, MIP-1β and MIP-2, inflammatory cells, monocytes/macrophages and such as anticoagulants, anti-platelet, vasodilators and, importantly, polymorphonuclear leukocytes (PMN) migrate to the inoculation site in immunomodulator and anti-inflammatory molecules2. the skin31. Once in the dermis, Leishmania can penetrate granulocytes, and macrophages or dendritic cells13,24,25. Macrophages are the reservoir Analysis of early promastigote-host cell interactions indicates that cells for parasite replication and regulate the infection by their ability to after inoculation of Leishmania into the dermis, the promastigotes interact potentially phagocytose and kill the parasite if it can avoid Leishmania- rapidly with serum components. Metacyclic promastigotes of Leishmania mediated functional inhibition21. Neutrophils and eosinophils possess have been shown to activate complement in both classical and alternative leishmanicidal activity that restrains parasite progression at the initial
(1) Faculdade de Medicina, Universidade Federal do Ceará, Rua Alexandre Baraúna 949, 60430-160 Fortaleza, CE, Brazil. (2) Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA. Correspondence to: Maria Jania Teixeira, Phone: +55-85-33668311, Fax: +55-85-33668316. E-mail: [email protected] VASCONCELOS, C.O.; COÊLHO, Z.C.B.; CHAVES, C.S.; TEIXEIRA, C.R.; POMPEU, M.M.L. & TEIXEIRA, M.J. - Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 21-7, 2014.
step of the infection13,24. However, it was demonstrated that Leishmania salivary glands were disrupted by ultra-sonication and the supernatant can survive transiently within neutrophils5, and following infection with collected after centrifugation at 15,000 g for two minutes. Salivary glands Leishmania, the lifespan of neutrophils can be increased to two days, were lyophilized in groups of 10 pairs. Each 10 pairs of glands were through the inhibition of procaspases processing in the infected cells1. resuspended with sterile distilled water and before the use were diluted in sterile saline in the concentration of one pair of gland per injection. Here, we investigated the cell number and composition in the initial response to infection induced by L. chagasi combined or not with the Air pouch and in vivo cell migration: Air pouches were induced host’s blood or saliva from Lu. longipalpis in hamster employing an air on the back of anesthetized hamsters (5-6 animals per group for each pouch model of inflammation. There are same reports using this model, time) by injection of 5 mL of sterile air, as described elsewhere19, and but only in mice, where the authors evaluated the cellular recruitment immediately inoculated with 100 µL either one of the following stimuli induced by L. major and L. donovani15, or by L. braziliensis30, as well (all prepared in endotoxin-free saline): endotoxin-free saline alone; as reports of Lu. longipalpis saliva in macrophage recruitment29, and stationary-phase L. chagasi promastigotes (107 parasites); salivary gland the chemotactic effects of Lu. intermedia combined or not with L. of Lu. longipalpis (equivalent to one pair of salivary glands/animal); braziliensis18. However, migration of cells induced by L. infantum chagasi heparinized blood collected from the animal itself; L. chagasi plus associated with host’s blood and Lu. longipalpis saliva in the hamster heparinized blood; L. chagasi plus salivary gland; or L. chagasi plus model has not been described. The hamster air pouch model was chosen heparinized blood and salivary gland. One hundred to 150 µL of blood here because it is the animal model for visceral leishmaniasis that best of each animal was obtained via orbital plexus after light anesthesia mimics the various aspects of human disease17. with ketamine and xylazine at the moment of the experiment, and used to inoculate the animal itself. The number of leukocytes present in the The complex microenvironment where the parasite, vector saliva, and blood was determined in a Neubauer hemocytometer before inoculation the host’s blood components encounter for the first time can determine (each 100 µL of blood injected contained 40-50 x 104 cells). After 6, 12 the outcome of infection. The description of the initial inflammatory and 24 h, animals were lethally anesthetized, and the pouches washed response is important especially when the three major components, with a total of 10 mL of endotoxin-free saline to collect leukocytes from parasite, saliva and blood, are analyzed together. the exudates. Lavage fluids (approximately 6 to 8 mL) were washed, and cell pellets were resuspended in saline plus 10% BSA, stained MATERIALS AND METHODS in Turk’s solution, and counted in a Neubauer hemocytometer. Cells were cytoadhered to glass slides using Shandon cytospin2 and stained Animals and study design: Two-month-old Syrian hamsters with hematoxylin and eosin to determine proportions of monocytes/ (Mesocricetus auratus) of both sexes were obtained from the central macrophages, neutrophils, eosinophils, basophils, and lymphocytes. animal facility of Departamento de Patologia e Medicina Legal of In all stimuli with blood, the cell values that were determined at 12 h Universidade Federal do Ceará (DPML/UFC), and held under specific were subtracted from the values of the cells found in the blood that was pathogen-free conditions. For the experiments, the animals were divided injected initially (average of 45 x 104 cells). The values of monocytes/ in the following groups (15 to 30 animals per group): Group 1, inoculated macrophages, neutrophils, eosinophils, basophils, and lymphocytes found with saline sterile; Group 2, inoculated with L. chagasi; Group 3, in the experiments were also subtracted from normal blood values found inoculated with the heparinized blood collected from each animal; Group in hamsters. The viability of the cells obtained from the exudates was 4, inoculated with vector saliva; Group 5, inoculated with L. chagasi verified by trypan blue exclusion (0.1%). associated with heparinized blood; Group 6, inoculated with L. chagasi associated with vector saliva; and Group 7, inoculated with L. chagasi Histological analysis: The pouches lining tissues were dissected associated with heparinized blood and vector saliva. The Animal Care and and fixed in 10% neutral buffered formalin. Tissues were mounted in Utilization Committee from UFC approved all experimental procedures paraffin blocks, sectioned at 5-µm intervals, and stained with hematoxylin (process No. 029/2008). and eosin for histological analysis. The alterations were observed by analyzing 50 microscopic fields per section in the pouch lining tissue, Parasite culture: L. infantum chagasi (MHOM/BR/BA-262) on two sections from each animal (four to five hamsters per group), promastigotes were grown in Schneider’s Drosophila medium (Sigma- using a 40 x objective, taking into account the following parameters: Aldrich, St. Louis, MO) at 25 oC supplemented with 20% heat-inactivated hemorrhage, edema, hyperemia and fibrin. Each parameter was evaluated fetal calf serum, 2 mM L-glutamine, 200 U/mL of penicillin and 200 g/ according to the intensity of the event through scores: 0 (absence), 1 mL streptomycin (all from Sigma-Aldrich), and 2% sterile human urine. (rare), 2 (moderate) and 3 (accentuated). Cellular analysis was evaluated The infectivity of parasites was maintained with regular passage through in five fields, using the increase of 1000 x. The population of cells from hamsters, and for use in the experiments, parasites were expanded for each animal corresponds to the sum of the fields analyzed. The cellular several days to reach stationary growth phase. population observed was: macrophages, neutrophils, eosinophils, and lymphocytes. Sand fly salivary glands: Salivary glands of females of Lu. longipalpis (Jacobina strain) were obtained from Jesus G. Valenzuela, Statistical analysis: Statistically significant differences of the results Laboratory of Malaria and Vector Research (LMVR), Vector Molecular were determined using nonparametric statistical tests: t-Student for Biology Section (NIAID-NIH),USA. Twenty females of Lu. longipalpis comparisons between two groups; one-way ANOVA for comparisons reared at LMVR (NIAID-NIH) were used for dissection of salivary between three or more groups. Statistical analysis and graphs were glands 5-8 days post-eclosion; salivary glands were dissected and performed using GraphPad prism version 5.0 (GraphPad Software, San stored in sterile PBS (pH 7.4) at -70 0C. To obtain the homogenate, Diego, USA). Values of p < 0.05 were considered significant.
22 VASCONCELOS, C.O.; COÊLHO, Z.C.B.; CHAVES, C.S.; TEIXEIRA, C.R.; POMPEU, M.M.L. & TEIXEIRA, M.J. - Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 21-7, 2014.
RESULTS macrophages as compared to L. chagasi alone, and L. chagasi plus saliva (Fig. 2B). Leukocyte recruitment in air pouch exudates: Leukocyte migration reached a maximal peak at 12 h after injection and then declining over a Types of cells that migrated in the pouch lining tissue: 24-h period into the air pouch (Fig. 1A). L. chagasi and saliva induced Macrophages were the predominant cell type at 12 h and 24 h post- significantly more cells into air pouches at 12 h after stimulation when stimulation in the pouch lining tissue, followed by neutrophils. It was compared with saline (Fig. 1A). Of interest, migration induced by saliva observed significantly almost 4-fold more macrophages than neutrophils was 3-fold more than that induced by Leishmania alone (Fig. 1A). In at 12 h after stimulation by L. chagasi (Fig. 3A), however after 24h relation to stimuli containing blood, is noteworthy that the number of macrophages number decreased while neutrophils almost doubled (Fig. cells in the blood injected containing an average of 45 x 104 cells, while 3B). L. chagasi associated with blood or L. chagasi plus blood plus the number of cells collected after 12 h of blood stimulation was 100 x saliva induced similarly cell migration at 12 h and 24 h post-stimulation 104 cells, which means that the number of cells that migrated induced (Fig. 3A and 3B). The combination of L. chagasi with blood and saliva by blood was in fact 55 x 104 cells (Fig. 1A). This number of cells was showed that the number of neutrophils dropped considerably after 24h, greater than those induced by L. chagasi or saline. Also, in Fig. 1B, the while the number of macrophages did not change (Fig. 3B). After 12 number of cells collected after 12 h of stimulation with Leishmania h of stimulation with L. chagasi and saliva was possible to observe a plus blood and Leishmania associated with blood and saliva was 144 smaller number of cells in the pouch lining tissue, as compared with other x 104 cells and 250 x 104 cells, respectively, meaning a real number of stimuli, also showing a virtual absence of neutrophils and presence of migration of 99 x 104 cells for Leishmania plus blood, and 205 x 104 a small number of eosinophils, however, 24 h after stimulation with L. cells for Leishmania plus saliva plus blood. These data were significant chagasi associated with saliva, there was a slight increase in macrophages in relation to stimulation with Leishmania alone. Interestingly, L. chagasi (Fig. 4B). associated with saliva resulted in a reduced cellular migration, 55 x 104 cells (Fig. 1B), when compared with stimulation with saliva alone (76 Histopathological findings in the pouch lining tissue: All stimuli x 104 cells) (Fig. 1A). alone presented a slight edema and hyperemia (Fig. 4A). Edema and hyperemia also appeared in all groups with association of stimuli, ranging Types of cells that migrated in air pouch exudates: All stimuli from rare to moderate (Fig. 4B). The major inflammatory changes were induced migration of a mixed population of leukocytes with a observed when Leishmania was associated with blood and saliva (Fig. predominance of neutrophils at 12 h (Fig. 2A and 2B). Neutrophils 4B). Hemorrhage was observed in a more pronounced manner following migration induced by saliva was more expressive than that induced in stimulation with L. chagasi associated with blood and saliva, when response to blood, L. chagasi or saline at 12 h after stimulation (Fig. compared with other groups (Fig. 3B). 2A). Saliva also induced a considerable number of eosinophils after 12 h, as compared to all stimuli (Fig. 2A). L. chagasi associated to DISCUSSION saliva was a great inducer of cell migration into the exudate; however the combination of L. chagasi with blood and saliva was even better In this study we found that L. infantum chagasi proved to be a inducer of all the types of cell to the inflammatory site (Fig. 2B). This poor inducer of cellular migration into air pouch exudate, although combination induced migration of a large number of neutrophils and there was migration of a diverse population of cells. The data observed
Fig. 1 - Number of leukocytes accumulating in air pouch exudate in response to (A) L. chagasi, blood, or Lu. longipalpis saliva; (B) L. chagasi plus blood, L. chagasi plus Lu. longipalpis saliva, or L. chagasi plus blood and plus Lu. longipalpis saliva. Air pouches were raised on the backs of male hamsters. One milliliter of endotoxin-free saline, L. chagasi (107 promastigotes), salivary gland of Lu. longipalpis (one pair of salivary glands/animal); blood; L. chagasi plus blood; L. chagasi plus salivary gland; or L. chagasi plus blood and salivary gland were injected into pouches, and exudate was collected at 6, 12, and 24 h after inoculation. Leukocytes were enumerated microscopically. Data are mean ± SE of 5 hamsters. (A) *p < 0.05, Leishmania- or blood- or saliva-stimulated hamster vs. saline-stimulated hamster. (B) **p < 0.05, Leishmania plus blood- or Leishmania plus blood plus saliva- stimulated hamster vs. Leishmania-stimulated hamster. The data are representative of three independent experiments.
23 VASCONCELOS, C.O.; COÊLHO, Z.C.B.; CHAVES, C.S.; TEIXEIRA, C.R.; POMPEU, M.M.L. & TEIXEIRA, M.J. - Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 21-7, 2014.
Fig. 2 - Total number of neutrophils, macrophages, eosinophils, and lymphocytes accumulated in air pouches in response to (A) L. chagasi, Lu. longipalpis saliva, or blood; (B) L. chagasi, L. chagasi plus blood, L chagasi plus Lu. longipalpis saliva, or L.chagasi plus blood plus saliva, at 12 h after stimulation. Stimulations were done as described in the legend to Figure 1. Exudate were placed onto microscope slides by use of cytospin and stained with Diff-Quik solution; proportion of neutrophils, macrophages, eosinophils, and lymphocytes/200 cells were enumerated and relative cell numbers were calculated from total exudate leukocytes. Data are mean ± SE of five hamsters. Differences observed for neutrophils, and macrophages in Leishmania-, saliva-, and blood-inoculated hamsters were significant (p < 0.05; n = 5), compared with saline control. Differences observed for eosinophils in Leishmania-, and saliva-inoculated hamsters were significant (p < 0.05; n = 5), compared with saline control. The data are representative of three independent experiments.
Fig. 3 - Number of leukocytes accumulating in the pouch lining tissue in response to L. chagasi, L. chagasi plus blood, L chagasi plus Lu. longipalpis saliva, or L.chagasi plus blood plus saliva at 12 h (A) or 24 h (B) after inoculation. Stimulations were done as described in the legend to Figure 1, and the pouches lining tissues were dissected at 12 and 24 h after inoculation. The data were obtained by analyzing 50 microscopic fields per section in the pouch lining tissue, on two sections from each animal and from five hamsters per group, using a 100 x objective. The cellular population observed was: macrophages, neutrophils, eosinophils, and lymphocytes. (A) *p < 0.05 (neutrophils: L. chagasi plus blood- or L. chagasi plus saliva plus blood-stimulated hamster vs. L. chagasi-stimulated hamster); **p < 0.05 (macrophages: L. chagasi- or L. chagasi plus blood- or L. chagasi plus saliva plus blood-stimulated hamster vs. L. chagasi plus saliva- stimulated hamster). (B) *p < 0.05 (eosinophil: L. chagasi-stimulated hamster vs. L. chagasi plus blood- or L. chagasi plus saliva or L. chagasi plus saliva plus blood-stimulated hamster. The data are representative of three independent experiments. in pouch lining tissue suggest that most of these cells do not seem to VCAM-1 on endothelial cells, suggesting the ability of L. donovani to have migrated to the inflammatory exudate, remaining in lining tissue. prevent transendothelial migration of monocytes14. Thus, it is possible Corroborating these findings, a study demonstrated that infection with that infection with viscerotropic Leishmania parasites did not induce a other viscerotropic species of Leishmania, such as L. donovani, did not strong recruitment of leukocytes in the exudate because cells are recruited induce a strong recruitment of leukocytes in the exudate15. Maybe one but can fail to migrate to air punch exudate, suggesting the blockade of possible explanation to these findings is the fact that promastigotes of transendothelial cell migration. However, this fact needs to be clarified Leishmania have a large quantity of lipophosphoglycan (LPG) on their with further studies. surface, which has potent inhibitory cell activity33. Studies have showed that LPG of L. donovani blocks expression of E-selectin, ICAM-1, and On the other hand, L. chagasi associated with blood and saliva recruited
24 VASCONCELOS, C.O.; COÊLHO, Z.C.B.; CHAVES, C.S.; TEIXEIRA, C.R.; POMPEU, M.M.L. & TEIXEIRA, M.J. - Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 21-7, 2014.
Fig. 4 - Inflammatory scores in the pouch lining tissue at 12 h of stimulation with(A) L. chagasi, saline, blood, or Lu. longipalpis saliva; (B) L. chagasi, L. chagasi plus blood, L.chagasi plus saliva, or L. chagasi plus blood plus saliva. Stimulations were done as described in the legend to Figure 1, and the pouches lining tissues were dissected at 12 and 24 h after inoculation. The data were obtained by analyzing 50 microscopic fields per section in the pouch lining tissue, on two sections from each animal and from five hamsters per group, using a 40 x objective. Inflammatory scores: 0 (absence), 1 (rare), 2 (moderate) and 3 (accentuated). more leukocytes than L. chagasi did alone. Leishmania is an intracellular with Leishmania, particularly complement components. The complement parasite that has adopted several strategies to survive and to replicate inside system exerts a strong selective pressure on the survival of the parasite, the host, and saliva may represent one such strategy, as has been suggested and most of the parasites enter permissive monocytes rapidly to prevent by several studies2,10. Herein we observed that sand fly saliva induces a their death7. Promastigotes opsonized by C3b bind to erythrocytes and significant influx of leukocytes into air punch exudate. The chemotactic are more readily phagocytized by PMN and macrophages9. In humans, effect for leukocytes, mainly macrophages, induced by sandy fly saliva the serum cytotoxic activity against promastigotes is the major effector was previously described in models of cell migration in vitro with Lu. mechanism during the initial invasion by Leishmania8,9. The immune longipalpis saliva, and other sand fly species asP. duboscq and P. papatasi10. adherence of promastigotes can facilitate the progression of infection Other study suggests that Lu. longipalpis saliva induces an important and by the transfer of parasites attached to red cells for blood phagocytes. diffuse inflammatory infiltrate characterized by neutrophils, eosinophils, Activation of the classical complement pathway by Leishmania in the and macrophages which are observed after 48 h in the dermis of the ear of deposition of C3 on the surface of parasites produces the C5 convertase BALB/c mice exposed to bites of uninfected sand flies27. Sand fly saliva that initiates the lytic cascade with the death of the parasite8. Phagocytosis has also been previously described as capable of exacerbating L. major of promastigotes by host cells during this period allows the Leishmania infection resulting from an increased production of Th2 cytokines due by evade lysis by complement7. This is believed to be the most efficient P. papatasi saliva, indicating that saliva can modulate the host immune mechanism of invasion of promastigotes in the host. This fact can response16. Other studies have shown that sand fly saliva modulates the explain the importance of blood to carry a greater induction of cells response of macrophages to a phenotype more permissive to the survival as a mechanism of escape and infection by Leishmania promastigotes. 30 of Leishmania . Sand fly saliva is also able to suppress NO, 2H O2, and antigen presentation by macrophages20. Maybe this anti-inflammatory Herein, L. chagasi was able to induce an inflammatory response activity of Lu. longipalpis saliva may partly explain the inhibition that characterized by the influx of neutrophils, macrophages and eosinophils. occurred in cell recruitment when saliva was associated with Leishmania. In addition, the association of Leishmania with blood and saliva induced Another explanation would be to consider some sort of modulation of the the recruitment of more cells than did L. chagasi alone. Previous inflammatory response made by parasites as a strategy to increase your works demonstrated that Leishmania elicits the recruitment of a mixed chance of survival in the animal model used in this study. It has been population of inflammatory cells that can vary between species and reported that hamsters are more susceptible to all species of viscerotropic strains14,29. Neutrophils were the main cells recruited after the stimuli in Leishmania than mice17, the model used in all studies cited above. air pouch exudate, especially when L. chagasi was associated with saliva or blood and saliva. Studies showed that PMN are the first leukocytes to Besides the effect observed with sand fly saliva, Leishmania appear at the site of inflammation where they phagocytose the parasites, associated with blood also shown to have an important role in modulating some of which are able to survive within these first host cells6,23,35. the inflammatory response, suggesting synergism between these stimuli. Leishmania is able to delay apoptosis of neutrophils, a mechanism Some studies have shown that inflammatory mediators present in blood that involves inhibition of caspase-3, which is known as an inducer of may regulate the initial inflammatory response that develop after infection apoptosis in PMN1. Parasites that are ingested but not killed by PMN, at
25 VASCONCELOS, C.O.; COÊLHO, Z.C.B.; CHAVES, C.S.; TEIXEIRA, C.R.; POMPEU, M.M.L. & TEIXEIRA, M.J. - Distinct cellular migration induced by Leishmania infantum chagasi and saliva from Lutzomyia longipalpis in a hemorrhagic pool model. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 21-7, 2014.
this early stage, can benefit from this early accumulation of neutrophils Histopathological analysis in punch lining tissue showed Leishmania to the site of infection, as has been shown for L. major28. The influx associated with saliva and blood induced some important inflammatory of neutrophils in the first 24 h modifies the T cells response, via IL-4 changes, although mild to moderate, such as edema, hyperemia, production, and the susceptibility to infection by L. major, by developing hemorrhage and fibrin. Previous reports have showed that saliva from a Th2 response28. Furthermore, it was demonstrated that Leishmania Lu. intermedia could induce inflammatory changes such as edema promastigotes induce migration of PMN by releasing Leishmania and hyperemia in the ear dermis of BALB/c18. The mild or moderate chemotactic factor (LCF), and that the coincubation of Leishmania inflammation induced by L. chagasi in this study also suggests the parasite com PMN inhibits the chemokine CXCL10, suggesting that Leishmania has developed a strategy to minimize the initial inflammatory response, inhibits the activity of Th1 or NK cells and, consequently, interferes allowing an unlimited progression within the host. with the development of the protective immune response35, which may facilitate its progression within the host. More detailed studies of leukocyte populations that migrate to the initial site of Leishmania inoculation, cytokine and chemokine Also, the combination of L. chagasi with blood and saliva was production, as well as characterization of cellular phenotype, can clarify capable of inducing an influx of many eosinophils to air pouch exudate. new aspects involved in the survival of L. chagasi in the host. This Other species of Leishmania such as L. major, L. donovani and L. work reinforces the importance of studies on the salivary components braziliensis are also able to induce migration of eosinophils15,30. It is of sand fly vectors of leishmaniasis in the transmission process and the known that eosinophils, like neutrophils, are also able to phagocyte and establishment of the infection. kill Leishmania13,24. Eosinophils can also participate in the vasodilatation facilitating the blood meal and at the same time creating an inhospitable RESUMO environment for pathogens transmitted by vector4. Sand fly saliva was also an important inducer of eosinophil migration to air pouch. In Migração celular distinta induzida por Leishmania infantum dogs, inoculation of Lu. longipalpis saliva resulted in an inflammatory chagasi e saliva de Lutzomyia longipalpis em um modelo de pool response with the presence of an intense eosinophilia22. Eosinophils hemorrágico were also observed in the inflammatory process developed at the site of immunization with recombinant 15 kDa protein from saliva of P. O recrutamento de uma população de células específicas após infecção papatasi34. In studies of sand fly saliva was observed that the exacerbation por Leishmania pode influenciar o resultado da doença. A migração celular of infection has been linked to inhibition of production of Th1 cytokine em resposta a Leishmania ou saliva do vetor tem sido reportada utilizando and increased production of Th2 cytokines caused by saliva from P. o modelo da bolsa de ar subcutânea, entretanto, a migração celular induzida papatasi, indicating that Lu. longipalpis saliva may have influenced por Leishmania associada com o sangue do hospedeiro e saliva do vetor the type of immune response, since the saliva promotes the recruitment neste modelo ainda não foi descrita. Neste trabalho foi investigada a of eosinophils, possibly by the production of eotaxin, a chemokine migração celular no modelo da bolsa de ar subcutânea em hamster após characteristic of the Th2 immune response16. The presence of eosinophils a estimulação com a combinação de L. chagasi, sangue do hospedeiro e suggests a Th2 immune response induced by saliva of the vector, and saliva de Lutzomyia longipalpis. A migração induzida por saliva foi três this type of response would facilitate the survival of Leishmania in the vezes maior do que a induzida por L. chagasi sozinha. Adicionalmente, early stage of infection. L. chagasi associada com sangue e saliva induziu significativamente ainda mais leucócitos no exsudato inflamatório do que o estímulo com Macrophage was the second predominant cell type in air pouch Leishmania sozinha. L. chagasi recrutou uma população de células exudate, especially when the stimulus was with L. chagasi associated distintas, no entanto, a maioria dessas células parece não ter migrado para with blood and saliva. In the early phase of infection, the ability of o exsudato inflamatório, permanecendo no tecido da bolsa de ar. Estes macrophages to respond to activation signals of Th1 against intracellular resultados indicam que L. chagasi pode reduzir o acúmulo de leucócitos pathogens is important in determining proliferation or elimination of para o local inicial da infecção e que quando associada à saliva do vetor e na the parasite. The recruitment of a small number of macrophages has presença de componentes do sangue aumenta o influxo de mais neutrófilos been associated with smaller lesions in a model of immunodeficient do que macrófagos, sugerindo que o parasito desenvolveu uma estratégia mice3. The intracellular killing of Leishmania by macrophages depends para minimizar a resposta inflamatória inicial, permitindo uma progressão on the production of ROS and NO, as already shown11. The proportion ilimitada dentro do hospedeiro. Este trabalho reforça a importância de mais of neutrophils and macrophages changes during the first weeks of estudos sobre os componentes da saliva dos vetores das leishmanioses no infection, and as expected, the number of neutrophils decreases soon processo de transmissão e no estabelecimento da infecção. with a concomitant increase of macrophages. Studies by15 showed that the number of macrophages increased approximately 50% after 48 h ACKNOWLEDGMENTS of infection with L. donovani. In the present study, we observed that macrophages were more predominant cells in the punch lining tissue This work received financial support from the Coordenação de than neutrophils at 12 h after stimulation with L. chagasi. As expected, Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil. the number of neutrophils increased after 24 h of stimulation. However, in the pouch lining tissue stimulated by L. chagasi associated with saliva REFERENCES and blood the macrophages were the predominant cells with both 12 h and 24 h post-inoculation, suggesting that these cells were recruited, 1. Aga E, Katschinski DM, Van Zandbergen G, Laufs H, Hansen B, Muller K, et al. but may not have migrated into punch exudate, as discussed above, or Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J Immunol. 2002;169:898-905. actually migrated only later (after 24 h).
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18. Moura TR, Oliveira F, Rodrigues GC, Carneiro MW, Fukutani KF, Novais FO, et al. 35. Van Zandbergen G, Hermann N, Laufs H, Solbach W, Laskay T. Leishmania Immunity to Lutzomyia intermedia saliva modulates the inflammatory environment promastigotes release a granulocyte chemotactic factor and induce interleukin-8 induced by Leishmania braziliensis. PLoS Negl Trop Dis. 2010;4:e712. release but inhibit gamma interferon-inducible protein 10 production by neutrophil granulocytes. Infect Immun. 2002;70:4177-84. 19. Muller K, Van Zandbergen G, Hansen B, Laufs H, Jahnke N, Solbach W, et al. Chemokines, natural killer cells and granulocytes in the early course of Leishmania Received: 7 February 2012 major infection in mice. Med Microbiol Immunol. 2001;190:73-6. Accepted: 6 May 2013
27 LIBRARY OF THE SÃO PAULO INSTITUTE OF TROPICAL MEDICINE
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EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES
Caio Fernando de OLIVEIRA(1,2), Thiago Galvão da Silva PAIM(1,2), Keli Cristine REITER(1,2), Alexandre RIEGER(3,4) & Pedro Alves D’AZEVEDO(1,2,5)
SUMMARY
Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase- negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (–x = 1,018.2 ng/µL), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it’s storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.
KEYWORDS: DNA extraction; Coagulase-negative Staphylococci (CoNS); Spectrophotometry.
INTRODUCTION The gram-positive cell wall is composed of a complex reticulate of peptideoglycan, teichoic acid, polysaccharides, and other proteins, In recent years, numerous methods developed by the molecular whereas the gram-negative cell wall is thinner with a simpler reticular biology have been applied on the scientific research in clinical pattern13. Methods used for genomic DNA isolation from gram-negative microbiology. On those methods, the genetic material most widely used bacteria are not always successful with gram-positive2,6,11. This is is DNA. Therefore, for an effective DNA extraction there are several important because a suitable method of DNA isolation and recovery is methods based on different principles4. However, the amount and the one of the most important prerequisites for molecular tests18,19. quality of the DNA obtained for each one of those methods are variable, becoming an important factor in the type of molecular method that will be The aim of this study was to select a method that provides the best used, and on the subsequent storage of this material18. Moreover, to access DNA from culture strains of CoNS clinical isolates. The main parameters bacterial DNA it is necessary to disrupt the cell wall, which presents considered were those related to storage for future testing in genomic distinct characteristics among different types of microorganisms13. libraries and application in molecular biology tests for scientific research or clinical diagnosis. To achieve this, the quality and the quantity of the Coagulase-negative staphylococci (CoNS) are gram-positive cocci DNA extracted from CoNS cultures through four different techniques often considered as culture contaminants, since many species are members were analyzed. The quality and amount of DNA was determined by of the human skin microbiota and mucous membranes. However, CoNS are spectrophotometry. The visualization of pattern bands in agarose gel increasingly being recognized as agents of clinically important infections. electrophoresis was performed to evaluate the DNA integrity and a PCR In some recent studies they have been considered the main responsible for to test the possibility of amplification of the different types of materials. infections related to medical devices and surgical sites9,12. Furthermore, those microorganisms are also common as agents of the bovine mastitis, MATERIALS AND METHODS as well as infections in humans related to contaminated animal products7,16. 267 CoNS from infections in patients admitted at the “Santa CoNS have a rigid cell wall that can be difficult to lyse, and special Casa de Misericórdia de Porto Alegre Medical Center” were selected. enzymes and methods have been developed to address this problem2,18. Those microorganisms were obtained from 2002 to 2004 mainly
(1) Health Sciences Post-graduate Program, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). (2) Laboratory of Gram-positive Cocci, UFCSPA; 245 Sarmento Leite Street, 90050-170 Porto Alegre, RS, Brazil. (3) Laboratory of Biotechnology and Genetic, Universidade de Santa Cruz do Sul (UNISC). (4) Department of Biology and Pharmacy, UNISC, 2293 Independência Av., 96815-900, Santa Cruz do Sul. (5) Department of Microbiology and Parasitology, UFCSPA, Rio Grande do Sul, Brazil. Correspondence to: Pedro Alves d’Azevedo. Laboratório de Cocos Gram-positivos, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite 245, 90050-170 Porto Alegre, RS, Brasil. Phone: 55 (51) 3303-8742; Fax: 55 (51) 3303-8740. E-mail: [email protected] OLIVEIRA, C.F.; PAIM, T.G.S.; REITER, K.C.; RIEGER, A. & D’AZEVEDO, P.A. - Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 29-33, 2014.
from bacteremia, but also from other types of infections such as After extraction procedures, we verify the presence and integrity surgical wounds, and infections related to indwelling devices. Species of the DNA on the extracted material in agarose gel electrophoresis. identification was previously performed by SECCHI et al. and published Five microliters of DNA and five µL of loading buffer (buffer type IV14) in 200815 (Table 1). Each isolate was stored on skimmed milk (Difco were mixed in a 0.2 mL tube. This mixture was placed into the gel wells Skim Milk, Becton Dickinson) and was cultivated in tryptone soy agar and subjected to a voltage of six volts/cm for 20 min in a horizontal (TSA) (Tryptone Soy Agar, Oxoid) for 24 hours prior to the extraction electrophoresis system (PowerPac Basic, Bio-Rad). After that, DNA procedures. One or two isolated colonies were then grown in two mL visualization and photo documentation were performed by the system brain heart infusion broth (BHI, Merck) for 16 hours; one mL of this MF-Chemi BIS (BioAmerica, Inc.). The extracted material of each method suspension was centrifuged at 7000 x g/5 min and the bacterial pellet was also analyzed by spectrophotometry. Two µL of DNA extracted from obtained used as a template in all methods. each sample were placed directly on the spectrophotometer (NanoDrop, 2000, Thermo Cientific) and measures in 230, 260 and 280 nm were Four methods were performed, the first one, popularly known performed. The system software provides the DNA concentration in ng/µL as boiling, was based on ALEXOPOULOU et al.1 with no relevant and automatically calculates the absorption ratio 260/280 (A260/280) and modifications. This procedure was performed on 50 randomly selected 260/230 (A260/230). Lastly, we performed amplification by PCR using the samples. The second, phenol-chloroform, it was based on SILVA pair of primers described by JENSEN et al.8: G1 (GAA GTC GTA ACA & SILVA16 with just one relevant modification: 20 µL of lysozyme AGG) and L1 (CAA GGC ATC CAC CGT) (Integrated DNA Technologies, (100 mg mL-1) (Sigma) were added to the enzymatic lysis suspension. The Coralville, USA). This pair of primers was selected from conserved phenol-chloroform used was Equilibrated Phenol, pH 8.0, purchased from sequences from the adjacent 16S and 23S genes8. The amplification reaction USB Corporation. This procedure was applied to 70 randomly selected was performed on a LifePro Thermal Cycler (Hangzhou Bioer Technology samples. The third method was based on extraction columns and was Co. Ltda, Hangzhou, China). The reagent concentrations and the reaction performed according to the kit manual (QIAamp DNA mini kit, Qiagen). conditions were the same ones used by COUTO et al.3. Based on the results Lysozyme and lysostaphin were used together on the enzymatic lyses of DNA quantification found by spectrophotometry, approximately 50 ng step. One modification was introduced: after the deproteinization step of DNA were used on each PCR reaction. The amplification was checked with proteinase K, 4 µL of RNase A (100 mg mL-1) (BioAmerica) were in agarose gel electrophoresis under the same conditions described above. added to it and then incubated at 70 °C for 10 min. All the samples (n = Each sample was tested once. The major costs of reagents per sample are 267) were submitted to this extraction method. Finally, we also tested a shown in Table 2. salting out method following the kit manual (Wizard® Genomic DNA Purification Kit, Promega) recommendations for gram-positive bacteria To obtain values of mean and standard deviation, calculations of without modifications. This procedure was performed on 50 randomly descriptive measures were used. The quantitative variables were tested selected samples. The usual bacterial amount used in all methods had for normality by the Kolmogorov-Smirnov test. Mann-Whitney U test a cell growth incubation period of 16 hours in one mL of brain heart was used to compare results among species. Values of the quantity and infusion broth (BHI) (Merck). quality of the DNA from the four extraction methods were compared
Table 1 Species and mean yield of DNA quantity (ng/µL) of each method
Extraction method Specie No. of isolates (%) Boiling Phenol-chloroform Extraction column Salting out S. epidermidis 107 (40.1) 1173.3 67.3 100.1 30.1 S. haemolyticus 91 (34.2) 1181.8 202.1 35.1 16.4 S. hominis 27 (10.1) 989.4 61.6 34.5 14.0 S. warneri 15 (5.6) 1002.2 NT 12.9 86.9 S. capitis 7 (2.6) 1016.5 89.2 15 21.1 S. saprophyticus 6 (2.2) 1052* 50.6 54.3 NT S. cohnii 4 (1.5) 825* 28* 41.7 18.9* S. caprae 3 (1.1) NT 40* 28.8 NT S. xylosus 3 (1.1) NT 23 31.6 NT S. sciuri 2 (0.7) NT NT 86.5 NT S. lugdunensis 1 (0.4) NT NT 32* NT S. auricularis 1 (0.4) NT NT 41* NT Total 267 (100.0) 1018.2 87.8 65.5 28.2 * Just one sample tested; NT = not tested.
30 OLIVEIRA, C.F.; PAIM, T.G.S.; REITER, K.C.; RIEGER, A. & D’AZEVEDO, P.A. - Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 29-33, 2014.
Table 2 Main cost of reagents per sample of each method in U. S. dollars (US$)
Method Kit Lysostaphin Lisozyme Proteinase K Equilibrated phenol Total Boiling NR NR NR 0.51 NR 0.51 Phenol-chloroform NR 13.80 0.08 0.50 0.15 14.53 Column 6.15 24.85 0.14 * NR 31.14 Salting out 2.79 20.71 0.03 NR NR 23.53 NR: not required; *: provided in the kit. by the Kruskal-Wallis test; multiple comparisons between methods higher than by extraction column, 87.8 and 65.5 ng/µL, respectively, were analyzed using Dunn’s test. Kruskal-Wallis and Dunn’s test were but this was not statistically significant (p > 0.05). Likewise, according performed with the support of the program GraphPad Prism (GraphPad, to Kruskall-Wallis test, the average A280/230 and A260/230 differences version 5) and other tests, with the program Statistical Package for the was statistically significant between all four methods (p < 0.0001); except Social Sciences (SPSS Statistics, version 17). The significance level for average A260/280 between extraction column and salting out and the used was 5%. average A260/230 between phenol-chloroform and salting out (p > 0.05) (Table 3). Finally, all four methods provided effective DNA for PCR RESULTS AND DISCUSSION amplification with the pair of primers used. The percentage of positive amplification results in one single reaction performed was 94% for the The increasing use of molecular methods on clinical microbiology extraction column method, 91% for the salting out, 83% for the boiling, further emphasizes the necessity of efficient DNA purification, free of and 71% for the phenol-chloroform. proteins and high molecular weight cellular debris5,10. Several studies have shown the importance of the parameter “quality” in obtaining better The bands visualized by the four extraction methods in agarose gel results on techniques like RAPD (Random Amplification of Polymorphic electrophoresis showed the same quality parameters. The great majority DNA) and RFLP (Restriction Fragment Length Polymorphism)1,4,11,17. of the bands were well defined and not fragmented. However, some Furthermore, differences between gram-positive and gram-negative samples, mainly the ones extracted by boiling, phenol-chloroform, bacteria should also be considered when choosing the DNA extraction and salting out showed no visible bands on the gel. Surprisingly, in the method10, a fact that prevents the use of many low-cost protocols indicated spectrophotometry those samples showed to hold an amount of DNA for any type of microorganisms11. This study aimed to identify the most detectable using the method of agarose gel electrophoresis. Regarding suitable method for DNA extraction for CoNS evaluating mainly the agarose gel electrophoresis, similar results were found by NOGUEIRA quality of the material obtained from four different methodologies. et al.11 and as reported by them the absence of bands on the gel was not determinant for obtaining a successful PCR. On the boiling method, The agarose gel electrophoresis showed better results for the samples the high protein contamination can lead to an overestimation of the extracted by the method of extraction column. Although some samples real concentration of DNA14. Besides that, DNA fragmentation by the evidenced DNA fragmentation, on almost all samples extracted by this high temperatures used on this method can be responsible for the low method it was possible to visualize the DNA. Samples extracted by visualization of bands on agarose gels. boiling, phenol-chloroform and salting out showed the same quality range, but inconstant presence. On the other hand, comparing the four methods Analysis by spectrophotometry can reveal important information by spectrophotometry, samples extracted by boiling demonstrated more about the DNA extracted from one sample. Besides determining the DNA DNA than the others (–x = 1,018.2 ng/µL) (Table 1). The average amount amount (ng/µL), the A260/280 can be used to assess the DNA purity of DNA extracted by the phenol-chloroform method was a little bit concerning the protein contamination. Pure DNA preparations have a
Table 3 Mean results of each extraction method regarding parameters evaluated
Parameter Boiling Phenol-chloroform Extraction column Salting out p Agarose gel electrophoresis Variable* Variable* Positive Variable* Amount of DNA in ng/µL 1018.2 (168.8) 87.8 (70.2) 65.5 (64.5) 28.2 (32.5) < 0.0001 A260/280 0.42 (0.03) 1.42 (0.15) 1.95 (0.26) 1.82 (0.44) < 0.0001 A260/230 2.99 (0.72) 0.66 (0.31) 1.34 (0.66) 0.81 (0.45) < 0.0001 PCR Positive Positive Positive Positive Yeld 200 µL 100 µL 200 µL 100 µL * Some samples showed no visible bands; ( ) = Standard deviation.
31 OLIVEIRA, C.F.; PAIM, T.G.S.; REITER, K.C.; RIEGER, A. & D’AZEVEDO, P.A. - Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 29-33, 2014.
A260/280 of 1.8. Similarly, significant absorbance at 230 nm decreases hope that the results presented in this study could help other researches the values of A260/230 and indicates contamination with polyphenols or professionals to make this important decision. and organic compounds14. So, although the boiling method presented the greatest average amount of DNA extracted, this may not be true since the RESUMO very low value of A260/280 (0.42) evidences a high protein contamination (Table 3). In the same way, looking for A260/230 we also can see a low Avaliação de quatro métodos diferentes de extração de DNA em value (0.66) for the phenol-chloroform extraction method. In this case, isolados clínicos de estafilococos coagulase negativos (SCoN) this high presence of organic solvents can be responsible for the low PCR amplification (71%). The extraction column method showed the Atualmente, para extrair o DNA bacteriano, existem diversos best results regarding quality and purity of the DNA extracted, which métodos baseados em diferentes princípios. Entretanto, a quantidade partially explains the better results obtained on the PCR amplification. e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos Calculations of the standard deviation values demonstrated that coagulase-negativos (CoNS), por exemplo, possuem parede celular the quality of DNA obtained in each one of the four methods did not espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade change much (normal distribution). In contrast, the quantitative values e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando on phenol-chloroform, extraction column, and salting out had higher quatro metodologias diferentes: dois protocolos caseiros e dois kits variation resulting on a significant standard deviation (Table 2). The comerciais. A determinação da quantidade e da qualidade do DNA foi reason for this is the different amount of DNA obtained in each species. realizada por espectrofotometria. O DNA extraído também foi analisado Comparing each quantitative result with the respective specie, some em eletroforese em gel de agarose e por PCR. A concentração média interesting observations can be made. The mean amount of DNA extracted de DNA foi mais alta no método de lise térmica (–x = 1.018,2 ng/µL). by the extraction column method from Staphylococcus epidermidis was Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza 100.1 ng/µL, greater than the overall average of other species um método de extração baseado em uma coluna de separação apresentou (p < 0.001) (Table 1). In the specific case of this method, this finding melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas is important because Staphylococcus epidermidis is the most common testadas forneceram DNA passível de amplificação por PCR, porém CoNS clinical isolate12. Nevertheless, the boiling method didn’t show com diferentes rendimentos. A qualidade do DNA extraído de bactérias significant differences among species. On the other hand, Staphylococcus é importante, pois possibilita a realização de maior número de técnicas haemolyticus and Staphylococcus warneri had the largest average number de biologia molecular e também armazenamento do material por maior by phenol-chloroform and salting out, respectively and Staphylococcus período de tempo. Neste sentido, a técnica de extração por coluna de capitis, the lower by extraction column. However, our results cannot separação apresentou melhor desempenho frente aos CoNS. explain those findings and further studies are needed to investigate the reason of those variations. ACKNOWLEDGMENTS
Using the pair of primers described by JENSEN et al.8 and the DNA The authors thanks to Coordenação de Aperfeiçoamento de Pessoal obtained by the methods used in this study we were able to perform de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Rio the proposed PCR. However, using similar methods of boiling and Grande do Sul (FAPERGS) for financial support. phenol-chloroform, NOGUEIRA et al.11 found no positive results in a RAPD-PCR for the gram-positive samples, only for the gram-negative. AUTHORS CONTRIBUTIONS Nevertheless, ALEXOPOULOU et al.1 published a study in 2006 in which they successfully used the same boiling method used by us and KCR and TGSP participated in the discussions during the study, performed a CoNS identification by RFLP-PCR. elaboration of statistical analyses, figure and tables, and helped to draft the manuscript. CFO designed the study, carried out the molecular biology Currently there are numerous in-house protocols and commercial procedures and drafted the manuscript. AR participated in the design of kits based on different principles for DNA extraction from bacteria. the study, elaboration of statistical analyses, figure and tables and helped Nevertheless, the number of comparative studies between these methods to carry out the spectrophotometry analyses. PAd’A participated in the is still scarce5,10. Taking the difficulty to lyse the CoNS cell wall into design and coordination of the study. All authors read and approved the account, we evaluated four methods based on different principles and final manuscript. found significant differences between the quality and the amount of the DNA extracted. Quality of the DNA extracted is important when choosing REFERENCES the extraction method to be used for those species, especially if we consider posterior sequencing, long term storage or the construction of 1. Alexopoulou K, Foka A, Petinaki E, Jelastopulu E, Dimitracopoulos G, Spiliopoulou genomic libraries for future tests. Considering these parameters extraction I. Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci. column method has proven to be the best alternative when extracting Lett Appl Microbiol. 2006;43:450-4. DNA from CoNS. However, when working with CoNS the choice of the best method should be made thinking about both the purpose of the 2. Cheng HR, Jiang N. Extremely rapid extraction of DNA from bacteria and yeasts. investigation and the future perspectives, taking into consideration the Biotechnol Lett. 2006;28:55-9. methods yield and the resources available. In-house protocols are cheaper 3. Couto I, Pereira S, Miragaia M, Sanches IS, de Lencastre H. Identification of clinical and can be used mainly for quick determinations, but the quality of the staphylococcal isolates from humans by internal transcribed spacer PCR. J Clin material makes commercial kits worthwhile in deeper investigations. We Microbiol. 2001;39:3099-103.
32 OLIVEIRA, C.F.; PAIM, T.G.S.; REITER, K.C.; RIEGER, A. & D’AZEVEDO, P.A. - Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 29-33, 2014.
4. Forbes BA. Introducing a molecular test into the clinical microbiology laboratory – 13. Salton MR. Studies of the bacterial cell wall. IV. The composition of the cell development, evaluation, and validation. Arch Pathol Lab Med. 2003;127:1106-11. walls of some Gram-positive and Gram-negative bacteria. Biochim Biophys Acta. 1953;10:512-23. 5. Giraffa G, Rossetti L, Neviani E. An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria. J Microbiol Methods. 2000;42:175-84. 14. Sambrook J, Russel DW. Quantitation of nucleic acids. In: Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. New York: Cold Spring Harbor 6. Goldenberger D, Perschil I, Ritzler M, Altwegg M. A simple “universal” DNA extraction Laboratory Press, 2001. p. A8.19-A8.24. procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Res. 1995;4:368-70. 15. Secchi C, Antunes AL, Perez LR, Cantarelli VV, d’Azevedo PA. Identification and detection of methicillin resistance in non-epidermidis coagulase-negative 7. Huber H, Ziegler D, Pflüger V, Vogel G, Zweifel C, Stephan R. Prevalence and staphylococci. Braz J Infect Dis. 2008;12:316-20. characteristics of methicillin resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons. BMC 16. Silva ER, Silva N. Coagulase gene typing of Staphylococcus aureus isolated from cows Vet Res. 2011;7:6. with mastitis in southeastern Brazil. Can J Vet Res. 2005;69:260-4.
8. Jensen MA, Webster JA, Straus N. Rapid identification of bacteria on the basis of 17. Tyler KD, Wang G, Tyler SD, Johnson WM. Factors affecting reliability and reproducibility polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl of amplification based DNA fingerprinting of representative bacterial pathogens. J Environ Microbiol. 1993;59:945-52. Clin Microbiol. 1997;35:339-46.
9. Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative 18. Wilfinger W. Extended bactozol enzyme solutionTM lysis procedure for the isolation of staphylococci. Clin Microbiol Rev. 1994;7:117-40. genomic DNA from recalcitrant gram-positive bacteria. Molecular Research Center Technical Bulletin No. 8. [Cited 2012 October]. Available from: URL: http://mrcgene. 10. McOrist AL, Jackson M, Bird AR. A comparison of five methods for extraction of bacterial com/tb8-grampositive.htm DNA from human faecal samples. J Microbiol Methods. 2002;50:131-9. 19. Yang G, Erdman DE, Kodani M, Kools J, Bowen MD, Fields BS. Comparison of 11. Nogueira CAM, Momesso CAS, Machado RLD, de Almeida MTG, Rossit ARB. commercial systems for extraction of nucleic acids from DNA/RNA respiratory Desempenho de kits comerciais e protocolos laboratoriais para a extração de DNA pathogens. J Virol Methods. 2011;171:195-9. genômico bacteriano. Rev Panam Infectol. 2004;6:35-8. Received: 16 January 2013 12. Rogers KL, Fey PD, Rupp ME. Coagulase-negative staphylococcal infections. Infect Accepted: 19 April 2013 Dis Clin North Am. 2009;23:73-98.
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Fasciola hepatica IN BOVINES IN BRAZIL: DATA AVAILABILITY AND SPATIAL DISTRIBUTION
Sita C. BENNEMA(1), Ronaldo Guilherme Carvalho SCHOLTE(1), Marcelo Beltrão MOLENTO(2), Camilla MEDEIROS(1) & Omar dos Santos CARVALHO(1)
SUMMARY
Fasciolosis is a disease of importance for both veterinary and public health. For the first time, georeferenced prevalence data of Fasciola hepatica in bovines were collected and mapped for the Brazilian territory and data availability was discussed. Bovine fasciolosis in Brazil is monitored on a Federal, State and Municipal level, and to improve monitoring it is essential to combine the data collected on these three levels into one dataset. Data were collected for 1032 municipalities where livers were condemned by the Federal Inspection Service (MAPA/SIF) because of the presence of F. hepatica. The information was distributed over 11 states: Espírito Santo, Goiás, Minas Gerais, Mato Grosso do Sul, Mato Grosso, Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Santa Catarina and São Paulo. The highest prevalence of fasciolosis was observed in the southern states, with disease clusters along the coast of Paraná and Santa Catarina and in Rio Grande do Sul. Also, temporal variation of the prevalence was observed. The observed prevalence and the kriged prevalence maps presented in this paper can assist both animal and human health workers in estimating the risk of infection in their state or municipality.
KEYWORDS: Fasciola hepatica; Spatial distribution; Cattle; Zoonosis; Brazil.
INTRODUCTION have been described in Brazil, most of them in Paraná3,4,14,27,37,40. Due to poor awareness about human fasciolosis, this number is likely to be an Fasciola hepatica is a trematode that parasitizes the liver of the underestimation. final host and can infect several species including ruminants, equines, pigs, several wild mammals and humans. To complete its lifecycle, F. Knowledge about the spatial distribution of fasciolosis in cattle hepatica needs a snail intermediate host of the Lymnaeidae, in Brazil in Brazil can contribute to the identification of risk areas for infection Pseudosuccinea columella, Galba viatrix, Galba cubensis, Galba of both animals and humans. Focusing parasite control programs on truncatula and Lymnaea rupestris. Except for L. rupestris, the other these areas will lead to an increase of the cost-effectiveness of control. species have proved to be susceptible to infection by F. hepatica. Previous studies on the spatial distribution of F. hepatica in cattle have concentrated on the southern states15,17,50. Therefore, in this paper we Fasciolosis is responsible for a decrease in animal welfare and discuss the data availability for bovine fasciolosis in Brazil and provide significant economic losses in the cattle and sheep rearing sector54. It the first description of the spatial distribution of bovine fasciolosis in the has been recognized as an emerging zoonosis and is included in the list entire Brazilian territory. of neglected tropical diseases. The disease mostly affects inhabitants of rural areas endemic for animal fasciolosis, who are at risk of ingesting MATERIAL AND METHODS metacercariae through consumption of contaminated water or freshwater plants16,34,49,56. Study area: The Brazilian territory comprises 8,514,215.3 km2 and is divided in five regions, 27 states and 5567 municipalities26. According to The spatial distribution of F. hepatica depends strongly on the Köppen climate classification system, the climate varies from equatorial the presence of the intermediate hosts, and thus on climatic and and tropical in the north to semiarid in the northeast, highland tropical at environmental factors providing a suitable habitat for these snails. In the highlands of Brasília, Belo Horizonte and São Paulo and subtropical Brazil, the area most known for the presence of F. hepatica in cattle is or even temperate in the South. In 2006, the total cattle herd in Brazil was the South region8,17,23,50, but bovine fasciolosis has also been noted in the 205.9 million heads24. Cattle production takes place in the entire Brazilian states of Rio de Janeiro, São Paulo, Espírito Santo, Minas Gerais and territory, but is concentrated in the central west region, predominantly in Goiás6,9,11,20,22,25,29,30,36,48,51,52,55. So far, around 50 cases of human fasciolosis the states of Mato Grosso, Mato Grosso do Sul and Goiás24.
(1) Laboratório de Helmintologia e Malacologia Médica, Centro de Pesquisas René Rachou, Fiocruz, Av. Augusto Lima 1715, Barro Preto, 30190-002 Belo Horizonte, Minas Gerais, Brazil. (2) Laboratório de Doenças Parasitárias, Setor de Ciências Agrárias, Universidade Federal do Paraná, Rua dos Funcionários 1540, Cabral, 80035-050 Curitiba, Paraná, Brazil. Correspondence to: Omar dos Santos CARVALHO, Laboratório de Doenças Parasitárias, Setor de Ciências Agrárias, Univ. Federal do Paraná, R. dos Funcionários 1540, Cabral, 80035-050 Curitiba, PR, Brasil. E-mail: [email protected] BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
Data collection: For this study, liver inspection data were used of cluster statistic and describes the autocorrelation between the values of cattle slaughtered in establishments registered with the Federal Inspection a variable in a certain location with and the values of this same variable Service (SIF) of the Ministry of Agriculture, Livestock and Supply in neighboring locations5. The Moran’s I was calculated using inverse (Ministério da Agricultura, Pecuária e Abastecimento, MAPA). These distance weighing and the bandwidth was set at 750 km (the bandwidth data were collected from the period of 2002 to 2011. The data were at which the highest Z-score was obtained). registered on the municipality of origin. The clusters were then mapped using Hot Spot Analysis. The Hot The databank of SIF only includes the municipalities where livers Spot Analysis tool in ArcGIS calculates a local Getis-Ord Gi* for each have been condemned, and no ‘absence data’ was available. Absence feature in the sample. This statistic compares the sum of a feature’s value of data for municipalities can therefore either mean the absence of F. and that of its neighbors to the sum of the rest of the features, and areas hepatica, or the absence of inspection data. where this sum this is statistically different (higher or lower), are defined as hot or cold spots. In this analysis a fixed bandwidth of 750 km was used. In order to distinguish between the absence of F. hepatica and the absence of data, figures of the Brazilian Institute of Geography As another way of obtaining insight in the spatial distribution of the and Statistics (IBGE) on the number of slaughtered cattle and type of prevalence of F. hepatica, the prevalence was kriged using the spatial inspection per state were used to construct maps displaying the percentage analyst tool in ArcGIS. Centroids of the municipalities were calculated of cattle slaughtered under federal, state and municipal inspection25. and these point data were used for the kriging. This map was made using the IBGE figures for 2010. In Figure 1, establishments registered with the SIF were marked at the centroids of RESULTS the municipalities. Data availability: Data were available for 19,696,469 slaughtered bovines distributed over 1032 municipalities (18.5% of total) of 11 states. Figure 1 shows the number of years the data were available per municipality. It can be observed that the municipalities where F. hepatica was detected are concentrated in the southern and central states, whereas in the north and northeast no infected municipalities were detected. Also, in the South, data were available for a longer period of time.
Using the data of IBGE, maps were made displaying the percentage of cattle slaughtered under federal, state and municipal inspection (Fig. 2). Federal inspection is common in the Southern and central states, whereas in the Northeast municipal inspection is more important. State inspection is common practice in Rio de Janeiro, Santa Catarina and Rio Grande do Sul, and to a lesser extent in the Northeast.
Prevalence: Entire Brazil and per state: The total number of cattle in the database was 19,696,469 and the total number of livers condemned Fig. 1 - Data availability per municipality on bovine fasciolosis in Brazil in the period of due to F. hepatica was 1,244,123. The total prevalence of F. hepatica 2002-2011. The colors show the number of years for which prevalence data was available. over the period of 2002 to 2011 in Brazil was therefore 6.32% (95% Geographic Coordinate System, Horizontal Datum: WGS84. Confidence Interval (CI): 6.31-6.33). In Rio Grande do Sul the highest prevalence (14.39%) was observed, followed by Santa Catarina (4.50%). Descriptive statistics and exploratory spatial analysis: Descriptive In the other states the observed prevalence ranged between 0 and 3% statistics were calculated using Statistica 6.0 (Statsoft; Tulsa, OK, USA) (see Table 1). and R 2.15.046. The temporality of the prevalence is described in Figure 3. The The prevalence of F. hepatica was mapped using ArcGIS 9.3 (ESRI; prevalence in RS increased to a maximum of 19.18% in 2006, and then Redlands, CA, USA). A single database was constructed containing the declined to 12.87% in 2011. The state of Paraná also saw a maximum prevalence data per municipality for the years 2002 to 2011. This database prevalence in 2006, of 9.37%. During the other years the prevalence in was joined with the map of municipalities available from the IBGE. Paraná was just above zero. Santa Catarina had a prevalence peak F. hepatica in 2010 (8.26%) and a sharp decline in 2011. In the other states The geographical distribution of F. hepatica was further studied prevalence was stable and just above zero. using exploratory spatial statistics in ArcGIS. The spatial distribution of the sample was assessed using Ripley’s K. This is a statistic that is The prevalence in Brazil ranged between 4-8%, with a peak of used to detect deviations from spatial homogeneity. It describes the 11.55% in 2006 due to the increased prevalence in RS and SC. number of observations within a certain distance of a typical point in the sample, and the function of the sample is compared to the function of Prevalence: per municipality the random Poisson distribution7. The data were analyzed for presence of ‘disease clusters’ calculating the Moran’s I. The Moran’s I is a global Descriptive statistics: F. hepatica was detected in 1032 municipalities
36 BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
Fig. 2 - The percentage of slaughtered cattle covered by the Federal (A), State (B) and Municipal (C) Meat Inspection Services in Brazil. The black triangles in A represent the municipalities of the abattoirs under Federal Inspection. Geographic Coordinate System, Horizontal Datum: WGS84.
Table 1 Description per state of the total number of municipalities with presence of bovine fasciolosis, the number of animals slaughtered, the number of animals diagnosed with Fasciola hepatica and the prevalence (%) based on these numbers in Brazil
95% CI* State Municipalities (n) Animals (n) Infected (n) Prevalence (%) Lower Upper ES 1 2416 52 2.15 1.61 2.81 GO 50 1929432 581 0.03 0.03 0.03 MG 25 44269 43 0.10 0.07 0.13 MS 32 3931245 86 0.00 0.00 0.00 MT 8 1327983 40 0.00 0.00 0.00 PA 1 82548 1 0.00 0.00 0.00 PR 108 959510 734 0.08 0.07 0.08 RJ 1 360 4 1.11 0.30 2.82 RS 409 8427727 1212966 14.39 14.37 14.42 SC 205 609416 27395 4.50 4.44 4.55 SP 192 2381563 2221 0.09 0.09 0.10 * CI, Confidence Interval.
the distribution was skewed to the left, indicating the presence of many municipalities with a low prevalence. The prevalence ranged from 0.00 to 70.90%.
Exploratory spatial statistics: In Figure 5, the spatial distribution of F. hepatica in Brazil as estimated from our data was mapped. It can be observed that F. hepatica is most densely present in the southern states of RS and SC, followed by SP and PR. Also, a higher prevalence (> 20%) can be seen in RS and along the coast of SC and PR. Fasciolosis appears to have a more or less even spatial distribution in the states of GO and MS, whilst in MG all infected municipalities present in the database were located in the south of the state. In MT, PA, ES and RJ only a few municipalities with cattle infected with F. hepatica were registered. Fig. 3 - The annual prevalence of Fasciola hepatica per state and for the whole of Brazil in the period of 2002 to 2011 (error bars: binomial 95% Confidence Interval). The Ripley’s K values were higher than expected in a randomly distributed dataset, indicating spatial clustering of the sample. distributed over 11 states. The average prevalence per municipality was 7.1%, while the median was 2.69%: the histogram (Fig. 4) shows that The Moran’s I (0.37, p < 0.01) confirmed presence of spatial clusters
37 BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
Fig. 6 - The spatial distribution of the prevalence of Fasciola hepatica (%) in livers of Fig. 4 - Histogram of the prevalence of Fasciola hepatica on municipality level during 2002 slaughtered cattle per municipality in the period of 2002 to 2011 in Brazil, interpolated using to 2011 in Brazil. Ordinary Kriging in the spatial analysis tool in ArcGIS. Geographic Coordinate System, Horizontal Datum: WGS84.
regularly, while in the South region, state and federal inspection are combined. The Northeast is the only region where the municipalities have a large share in the inspection, along with the state. In the northern states of Amazonas and Roraima data is very scarce. These large regional differences in type of inspection cause an unequal spatial distribution of the data used in the present study, as only federal inspection data were included. As for the North and Northeast of Brazil no federal inspection data are available, this study fails to provide information about those regions. The lack of data for these areas is due to the scattered data collection and the absence of a unified databank combining the different levels (federal, state and municipal) and a change in this system is essential to improve monitoring of F. hepatica and other animal and zoonotic diseases of importance for the veterinary public health; e.g. echinococcosis and cysticercosis.
Fig. 5 - The spatial distribution of the prevalence of Fasciola hepatica (%) in livers of Notwithstanding possible bias due to nonuniformity in sampling, slaughtered cattle per municipality in the period of 2002 to 2011 in Brazil. Geographic possible inter-abattoir differences in inspection and uncertainty of the Coordinate System, Horizontal Datum: WGS84. origin of the cattle (cattle migration is not taken into account), the present study provides valuable information about a large area of Brazil, based on in the prevalence of F. hepatica. Maps of these clusters (figures not many observations and including important livestock regions. Confirming shown) showed a significant hot spot in RS and SC and a significant cold previous literature, the southern states of RS and SC showed the highest spots in PR, SP, MS and GO. Kriging provided a risk map displaying the prevalence of F. hepatica infections17,23. In SP, the Vale do Paraíba region prevalence of F. hepatica (Fig. 6) and confirmed the clustering observed was described as an important focus of F. hepatica50. The presence of with the hot spot analysis and in Figure 4. fasciolosis in this valley was confirmed by our study.
DISCUSSION The presence of F. hepatica in RJ and ES is also confirmed by previous literature, although those studies reported a higher number of For the first time, georeferenced presence data of F. hepatica in municipalities infected with F. hepatica. For RJ, the only municipality bovines were collected and mapped for entire Brazil. F. hepatica is an included as positive in our database is Barra Mansa, situated in the RJ part emerging zoonosis and although transmission to humans is also related of the Vale do Paraíba. The first record of F. hepatica in Brazil describes to social factors such as dietary habits, mapping the distribution of this the presence of F. hepatica in Três Rios, a municipality close to this trematode in cattle and other final hosts can give indications of the region31, and later studies confirmed presence of F. hepatica in various possible risk areas for zoonotic endemicity. municipalities located there21,43,48,51. Fasciolosis was also described in the Norte Fluminense, East and South Region, the Metropolitan Region and Monitoring of F. hepatica in cattle is therefore essential. In Brazil, the Lagos Region22,29,42,44,51. In southern ES all 10 municipalities studied this happens through inspection at slaughter, but currently, inspection by ALVES et al. (2011) were found positive using coproparasitologic data are not centralized. When mapping the levels of inspection, regional tests and 23 municipalities were predicted to be at risk by a prediction differences were seen: in central Brazil federal inspection is carried out model based on climatic and geographic factors32. BERNARDO et
38 BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
al. (2011) found a prevalence of bovine fasciolosis in Southern ES of metacercariae should be strictly controlled16,28,35,49. Currently, a lack of higher than 15% in 2006-2009. The federal inspection data used in our monitoring policies exists which hampers the identification of potentially paper included only the municipality of Cachoeiro de Itapemirim, also contaminated vegetables and drinking water. For an adequate control and located in the south. monitoring and a deeper insight in the epidemiology of this increasingly important zoonosis, the development of a laboratory protocol to test We found F. hepatica in cattle from the southern part of MG for metacercariae is required, so that routine tests can be performed on (specifically, in the South-Southwest Region and the regions of Zona vegetables and drinking water. da Mata, Campo das Vertentes and Triângulo Mineiro/Alto Paranaíba), agreeing with the results of SERRA-FREIRE et al. (1995) and LIMA et Combining the prevalence data presented in this paper with climatic, al. (2009). However, LIMA et al. (2009) found no presence of F. hepatica geographical and herd-management data, the distribution of F. hepatica in Campo das Vertentes and did detect F. hepatica in Metalurgica. in Brazil can be modeled and the presence of risk areas can be explained. Itajubá and Careaçú, municipalities in the South of MG known to This could provide a deeper insight in the factors of importance for the be endemic areas for bovine fasciolosis13,18,40,51, were not included as distribution of F. hepatica in Brazil and render methods to predict the infected municipalities in the used database, as well as Viçosa, the first prevalence in similar areas where no data is available. municipality of MG to have a case of bovine fasciolosis described12. In GO, the presence of fasciolosis had previously been studied by ARAÚJO Considerable inter annual differences in prevalence were observed et al. (2007) who, also using data of MAPA, found a similar distribution in the present study. These might be due to variation in climatic as seen in the present study. In MT and MS, the presence of F. hepatica circumstances. On a global scale, there has been an increase in human in cattle, to the authors’ knowledge, has not been described before, cases since the 80’s, and human and animal fasciolosis is seen as an although reports of cases of human fasciolosis47 and of the intermediate emerging disease. This emergence might be related to climate and host41 indicated the presence of the trematode in the region. For the environmental changes19,35,38,39,45,53. Studying temporal trends in the North and Northeast, not enough data were available since federal prevalence of fasciolosis in cattle will attribute to the understanding of inspection is non-existent or scarce in those regions. Although the high these relations, and can be used to predict eventual geographic shifts and temperature in the North and North East might make a high prevalence of changes in infection rates. F. hepatica unlikely, an outbreak of human fasciolosis has been described in Amazonas and intermediate hosts have been found at several locations RESUMO in the North and North-East1,40. Fasciola hepatica em bovinos no Brasil: disponibilidade de dados e The maps presented in this paper, combined with knowledge from distribuição espacial previous literature and if possible with information from state and municipal inspection, could serve to assist both animal sanitary programs A fasciolose é doença de alta importância para a saúde tanto and human health workers in estimating the risk of infection in their state veterinária quanto humana. Pela primeira vez, dados georreferenciados or municipality. According to ROBINSON & DALTON (2009) the human da prevalência de Fasciola hepatica em bovinos foram coletados e population at risk of F. hepatica infections is, most commonly, people mapeados para o território brasileiro e a disponibilidade desses dados living in rural areas endemic for animal fasciolosis, sharing water sources discutida. Fasciolose bovina no Brasil é monitorado em nível Federal, with the animals and consuming contaminated vegetables. Although Estadual e Municipal, e para melhorar esse monitoramento é preciso high correlation is not always present between a high prevalence of juntar os dados dos três níveis para construir um único banco de dados. fasciolosis in cattle and in humans33, in Brazil an overlap between the As informações foram coletadas de 1032 municípios onde fígados disease in both species was seen, as well as an overlap with the presence bovinos foram condenados por causa de F. hepatica pelo Serviço de of Lymnaeidae species10. In areas where fasciolosis was traditionally Inspeção Federal (MAPA/SIF). Onze estados foram representados: present in cattle, control programs focused on treating livestock and Espírito Santo, Goiás, Minas Gerais, Mato Grosso do Sul, Mato Grosso, changing management practices on the farms; e.g. fencing wetlands. The Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Santa Catarina e latter strategy was suggested to limit the access of cattle into grazing São Paulo. A prevalência mais alta da fasciolose foi observada nos areas where F. hepatica positive-snails were present. The treatment estados do Sul, com presença de focos da doença ao longo do litoral do with triclabendazole as a preventive strategy is necessary to reduce the Paraná e Santa Catarina e no Rio Grande do Sul. Variação temporal da infection burden, reducing egg shedding in the suitable environment54. prevalência também foi observada. Os mapas de prevalência observada Unfortunately these measurements are considered to be inefficient to e de krigeagem aqui apresentados podem auxiliar a profissionais da control the zoonotic spread of the disease in areas where health workers área da saúde veterinária e humana a estimar o risco de infecção nos are not properly trained to recognize possible symptoms of fasciolosis seus estados e/ou municípios. in humans and to carry out the adequate diagnostic techniques used to confirm the infection. In the areas where fasciolosis in bovines is endemic, ACKNOWLEDGEMENTS epidemiological studies need to be conducted in humans, to obtain insight into the importance of fasciolosis as a zoonosis in Brazil. In areas The authors are grateful to Dr. Carlos R. Conti Naumann from the endemic for human disease, if present, the population should be informed Federal Inspection Service 2354, MAPA (Colombo, PR) for supplying about the danger and transmission of the disease and educated about the data from the SIF database. DMV Lew Kan Sprenger is thanked for the preventive methods (e.g. cooking or freezing before consumption, his help in obtaining the data. The work of R. Scholte was sponsored separation of grazing grounds and cultivation areas). Consumption of by the National Council of Technological and Scientific Development unsafe drinking water and freshwater plants possibly contaminated with (Process 150386/2012-5).
39 BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
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9. Bernardo CC, Carneiro MB, Avelar BR, Donatele DM, Martins IVF, Pereira MJS. 29. Lessa CSS, Scherer PO, Vasconcellos MC, Freire LS, Santos JAA, Freire NMS. Prevalence of liver condemnation due to bovine fasciolosis in Southern Espírito Santo: Registro de Fasciola hepatica em eqüinos (Equus caballus), caprinos (Capra hircus) temporal distribution and economic losses. Rev Bras Parasitol Vet. 2011;20:49-53. e ovinos (Ovis aries) no município de Itaguaí, Rio de Janeiro, Brasil. Rev Bras Ci Vet. 2000;1:63-4. 10. Busetti ET. Informações adicionais sobre a fasciolose hepática em Curitiba (Estado do Paraná, Brasil). Rev Inst Med Trop Sao Paulo. 1982;24:104-6. 30. Lima WS, Soares LRM, Barçante TA, Guimarães MP, Barçante JMP. Occurrence of Fasciola hepatica (Linnaeus, 1758) infection in Brazilian cattle of Minas Gerais, 11. Bruno SF, Mattos Junior DG, Silva EV, Francis M, Brito DB. Fasciola hepatica Brazil. Rev Bras Parasitol Vet. 2009;18:27-30. (Linnaeus 1758) em bovinos do município de Cachoeiras de Macacu. Estado do Rio de Janeiro, Brasil. Parasitol Día. 1995;19:65-8. 31. Lutz A. Sobre a ocorrência de Fasciola hepatica no estado do Rio de Janeiro. Bol Inst Oswaldo Cruz. 1921;1:9-13. 12. Carvalho JCM. Contribuição para o conhecimento da fauna helmintológica de Minas Gerais. Ceres (Viçosa). 1940;5:411-23 32. Martins IVF, de Avelar BR, Pereira MJS, da Fonseca AH. Application of a geographical information system approach for risk analysis of fascioliasis in southern 13. Coelho LHL, Lima WS. Population dynamics of Lymnaea columella and its natural Espírito Santo State, Brazil. Geospat Health. 2012;6:S87-S93. infection by Fasciola hepatica in the State of Minas Gerais, Brazil. J Helminthol. 2003;77:7-10. 33. Mas-Coma S, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bull World Health Organ. 1999;77:340-6. 14. Coral RP, Mastalir ET, Mastalir FP. Retirada de Fasciola hepatica da via biliar principal por coledocoscopia. Rev Col Bras Cir. 2007;34:70-1. 34. Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005;35:1255-78. 15. Cunha FOV, Marques SMT, Mattos MJT. Prevalência de Fasciola hepatica em ovinos no Rio Grande do Sul, Brasil. Parasitol Latinoam. 2007;62:188-91. 35. Mas-Coma S, Valero MA, Bargues MD. Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis. Vet Parasitol. 2009:163:264-80. 16. Dorny P , Praet N, Deckers N, Gabriel S. Emerging food-borne parasites. Vet Parasitol. 2009;163:196-206. 36. Mattos MJT, Ueno H, Gonçalves PC, Almeida JEM. Ocorrência estacional e bioecologia de Lymnaea columella Say, 1817 (Mollusca, Lymnaeidae) em habitat 17. Dutra LH, Molento MB, Naumann CRC, Biondo AW, Fortes FS, Savio D, et al. natural no Rio Grande do Sul. Rev Bras Med Vet. 1997;19:248-52. Mapping risk of bovine fasciolosis in the south of Brazil using Geographic Information Systems. Vet Parasitol. 2010;169:76-81. 37. Mezzari A, Antunes HBB, Coelho N, Cauduro PF, Brodt TC. Fasciolíase humana no Brasil diagnosticada por colangiografia endoscópica retrógrada. J Bras Patol. 18. Faria RN, Cury MC, Lima WS. Prevalence and dynamics of natural infection with 2000;36:93-5. Fasciola hepatica (Linnaeus, 1758) in Brazilian cattles. Rev Méd Vét. 2005;156:85-6. 38. Mitchell GB. Update on fascioliasis in cattle and sheep. In Pract. 2002;24:378-85. 19. Fox NJ, White PC, McClean CJ, Marion G, Evans A, Hutchings MR. Predicting impacts of climate change on Fasciola hepatica risk. PLoS One. 2011;6:e16126. 39. Mitchell GB, Somerville DK. Effects of climate change on helminth diseases in Scotland. Ayr: SAC Veterinary Centre Auchincruive; 2005. p. 1-11.
40 BENNEMA, S.C.; SCHOLTE, R.G.C.; MOLENTO, M.B.; MEDEIROS, C. & CARVALHO, O.S. - Fasciola hepatica in bovines in Brazil: data availability and spatial distribution. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 35-41, 2014.
40. Oliveira AA, Nascimento AS, Santos TAM, Carmo GMI, Dimech CPN, Alves RMS, et 49. Robinson MW, Dalton JP. Zoonotic helminth infections with particular emphasis on al. Estudo da prevalência e fatores associados à fasciolose no Município de Canutama, fasciolosis and other trematodiases. Philos Trans R Soc Biol Sci. 2009;364:2763-76. Estado do Amazonas, Brasil. Epidemiol Serv Saúde. 2007;16:251-9. 50. Serra-Freire NM, Nuernberg S. Geopolitical dispersion of the occurrence of Fasciola 41. Paraense WL. Lymnaea rupestris sp. n. from Southern Brazil (Pulmonata: hepatica in the state of Santa Catarina, Brazil. Mem Inst Oswaldo Cruz. 1992;87(Suppl Lymnaeidae). Mem Inst Oswaldo Cruz. 1982;77:437-43. 1):263-9.
42. Pile E, Lessa CSS, Scherer PO, Albuquerque Dos Santos JA, de Vasconcellos MC. 51. Serra-Freire NM, Bordin EL, Lessa CSS, Scherer PO, Farias MT, Malacco A, et Ocurrencia de fasciolosis bovina en Itaguaí, Rio de Janeiro, Brasil. Parasitol Día. al. Reinvestigação sobre a distribuição da Fasciola hepatica no Brasil. Hora Vet. 1999;23:3-4. 1995;1(Edição extra):19-21.
43. Pile E, Gazeta G, Santos JAA, Coelho B, Serra-Freire NM. Ocorrência de fascioliasis 52. Serra-Freire NM. Fasciolose hepática no Brasil: análise retrospectiva e prospectiva. humana no município de Volta Redonda, RJ, Brazil. Rev Saúde Pública. 2000;34:413- Cad Téc-Cient Esc Med Vet. 1999;1:9-70. 4. 53. Thomas C, Jacquiet P, Dorchies P. La prévalence des helminthoses bovines a-t-elle 44. Pile E, Santos JAA, Pastorello T, Vasconcellos, M. Fasciola hepatica em búfalos été modifiée par la canicule de l’été 2003 dans le Sud-Ouest de la France? Parasite. (Bubalus bubalis) no município de Maricá, Rio de Janeiro, Brasil. Braz J Vet Res 2007;14:265-8. Anim Sci. 2001;38:42-3. 54. Torgerson P, Claxton J. Epidemiology and control. In: Dalton JP, editor. Fasciolosis. 45. Pritchard GC, Forbes AB, Williams DJL, Salimi-Bejestami MR, Daniel RG. Wallingford: CABI Publishing; 1999. p. 113-49. Emergence of fasciolosis in cattle in East Anglia. Vet Rec. 2005;157:578-82. 55. Ueno H, Gutierres VC, Mattos MJT, Muller G. Fascioliasis problems in ruminants 46. R Development Core Team. R: a language and environment for statistical computing. in Rio Grande do Sul, Brazil. Vet Parasitol. 1982;11:185-91. Vienna: R Foundation for Statistical Computing; 2008. 56. WHO. Working to overcome the global impact of neglected tropical diseases. First 47. Rey L. Primeiro encontro de ovos de Fasciola hepatica em inquérito helmintológico WHO report on neglected tropical diseases. Geneva: World Health Organization; de populações brasileiras (Campo Grande, Mato Grosso do Sul). Rev Paul Med. 2010. 1958;53:60. Received: 20 December 2012 48. Rezende HEB, Araujo JLB, Gomes PAC, Nuernberg S, Neto MP, Oliveira GP, et al. Accepted: 23 April 2013 Notas sobre duas espécies de Lymnaea Lamark, 1799, hospedeiros intermediários da Fasciola hepatica no estado do Rio de Janeiro. (Mollusca, Gastropoda, Basommatophora, Lymnaeidae). Arq Univ Fed Rural. 1973;3:21-3.
41 LIBRARY OF THE SÃO PAULO INSTITUTE OF TROPICAL MEDICINE
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In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64
Thaís Batista de CARVALHO, Teresa Cristina Goulart OLIVEIRA-SEQUEIRA & Semíramis GUIMARÃES
SUMMARY
The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.
KEYWORDS: Giardia duodenalis; Protease inhibitor; Trophozoites; Growth; Adherence; Viability.
INTRODUCTION with cure rates ranging from 80 to 95%21. Despite its efficiency, treatment shows undesirable side effects, failures are common and evidences Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), a suggest the emergence of drug resistance2,9,14. Therefore, there is an worldwide zoonotic intestinal protozoan, is one of the major causes of increasing interest for new antigiardial agents that can be effective non-viral diarrheal disease in humans. Despite giardiasis has a global against the parasite, less harmful for the host and may provide options distribution, higher infection rates (20-60%) have been reported for for refractory cases, especially for children, who may require treatment developing countries, mainly among socially and economically deprived often because of reinfections. populations22. Since in developed areas, infections rates are lower and G. duodenalis is often involved in numerous outbreaks that have been Nowadays, the quest for new antiparasitic alternatives has led attributed to an inappropriate water treatment1. Considering the infection researchers to base their studies on insights into biology, host- impact on health and on socio-economic improvements of developing parasite interactions and pathogenesis. In light of this, advances in nation’s population, Giardia was recently included in the WHO Neglected the understanding of the biochemistry and molecular biology of Diseases Initiative, a group including pathogens that have a common link parasites have led to a better understanding of molecules which play with poverty20. In these areas, giardiasis is a common cause of diarrhea in a role in biological, metabolic and physiological processes. Among nursery and school children22, especially in undernourished individuals, these molecules, the proteolytic enzymes or proteases have excited which in turn, gives rise to nutritional deficiencies leading to growth the researcher’s interest, once they have been identified as important failure and cognitive impairment20. virulent factors as well as potential chemotherapeutic targets in parasites. Considering that proteolytic activity is naturally regulated by specific So far, drug intervention has been recommended as a preventive inhibitors, the selective inhibition of crucial proteases can be a promising strategy to limit and reduce the transmission of giardiasis. Currently, strategy to develop new antiparasitic therapies19. In this context, it is some therapeutic measures have been available in clinical practice for important to emphasize that proteases have been validated as druggable the treatment of giardiasis, highlighting acridine, 5-nitroimidazole, targets as evidenced by the use of protease inhibitors as effective therapy 5-nitrofurans, 5-nitrothiazoles and benzimidazole derivates27. For for hypertension, diabetes, osteoporosis, certain cancers and AIDS17,28. decades, the most prescribed antigiardial drug is the metronidazole, a In relation to parasites, several protease targets have been validated by 5-nitroimidazole compound that has a recognized antigiardial activity genetic or chemical knockout in protozoan parasites, and many other
Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, São Paulo, Brazil. Correspondence to: Semíramis Guimarães, Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), 18618-970 Botucatu, SP, Brasil. Phone: 55 14 3880-0539; Fax: 55 14 3815-2838; E-mail: [email protected] CARVALHO, T.B.; OLIVEIRA-SEQUEIRA, T.C.G. & GUIMARÃES, S. - In vitro antigiardial activity of the cysteine protease inhibitor E-64. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 43-7, 2014.
enzymes appear promising as targets but they require more investigations Adherence assay: The E-64 effect on adherence of Giardia for validation, or to identify viable drug leads. So, interesting results trophozoites was analyzed as previously described by EDLIND et have been reported in studies with parasitic infections such as malaria, al.10, with slight modifications. An inoculum of 105 organisms was leishmaniasis and Chagas disease and they have provided important incubated in TYI-S-33 medium for four hours at 37 ºC. The medium evidences that these substances selectively inhibit parasite proteases with unattached cells was replaced with one supplemented with E-64 at without affecting host homologues17. 10, 50 and 100 µM. After incubation for 24 and 48 h at 37 ºC, adherent cells were dislodged by a 10 min on ice, centrifuged at 250xg and the With respect to Giardia, notwithstanding recent studies have shed number of attached cells was determined using a haemocytometer. light on trophozoites proteases and their involvement in metabolism and Control assays were performed under similar experimental conditions physiologic processes4,5,12,23 relatively little is known about the effect with cultures in the absence of E-64 and cultures treated with of protease inhibitors on vital processes of this parasite. To date, as far metronidazole at 40 µg/mL15. For each inhibitor concentration, three as we know, there are few studies that have evaluated the in vitro and tubes were assayed and the effect on adhesion capacity was evaluated in vivo performance of protease inhibitors on multiplication8,11 and on comparing the number of organisms in E-64-treated cultures with their encystation/excystation7,23,24 of the parasite. Thus, considering the aspects number in control cultures. The results were expressed as number of above, here we report the in vitro effects of E-64, a specific cysteine attached cells (percentage of control). protease inhibitor, on axenic trophozoites growth and adherence abilities, and viability. The results assembled here may open perspectives to pursue Cell viability assay: The effect of E-64 on trophozoites viability the development of further investigations in order to assess protease was evaluated by the quantitative colorimetric MTT-tetrazolium salt inhibitors as potential antigiardial agents. [3-(4,5-dimethilthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] technique, as previously described by PONCE-MACOTELA et al.18. MATERIALS AND METHODS Briefly, trophozoites (105) exposed to each E-64 concentration for 48 and 72 h were transferred to 1.5 Eppendorf® tubes, washed with PBS Parasite and cultivation conditions: G. duodenalis axenic (pH7.4) and incubated for 60 min at 37 ºC in a solution containing 40 trophozoites, BTU-11 strain, were axenically cultivated in filter-sterilized µL of MTT (5 mg/mL; Sigma) and 20 µL of the catalyst phenazine- TYI-S-33 (Tryptcase, yeast extract, iron serum) medium, modified by methosulfate (PMS 2.5 mg/mL; Sigma). Then, the cells were pelleted KEISTER13 in 5 mL Vacutainer® tubes at 37 ºC. The strain isolated in by centrifugation and the purple product obtained by the conversion Brazil at the Giardiasis Laboratory (IB/UNESP) in Botucatu, São Paulo of MTT to the formazan was resuspended in isopropanol/hydrochloric was established from cysts of a symptomatic patient presenting diarrhea, acid. The assay was performed in triplicate and aliquots of 150 µL were flatulence, abdominal cramps and resistance to conventional therapy. collected and the optical densities of each sample were measured at 540 Trophozoites harvested in log-phase growth 72 h postinoculation, after nm. The parasite viability was calculated regarding the untreated cultures chilling in bath ice for 10 min, were collected by centrifugation at 250×g (100% viability). for 15 min at 4 °C. Pooled cells were resuspended in sterile phosphate- buffered saline (PBS; pH 7.2) and the total number of trophozoites was Statistical analysis: To evaluate E-64 effect on the growth, adherence counted microscopically in a haemocytometer (Neubauer cell-counter and viability of Giardia trophozoites, a factorial design layout was chamber) and adjusted to an inoculum of 105 parasites. employed for counting data following multiple comparisons. Data were submitted to analyze the variance (ANOVA) and the means were Preparation of inhibitor: E-64 [(L-trans-epoxysuccinyl-L- compared by Tukey’s test. All analyses were conducted in SAS 9.2 for leucylamido-(4-guanidino)-butane], a commercially available cysteine Windows (SAS Institute Inc., Cary, NC) and p-values less than 0.05 protease inhibitor was purchased from Sigma. The inhibitor was prepared were deemed statistically significant. The inhibitory concentrations at one mM in aqueous solution and stored at -20 ºC, until required. The at 50% (IC50) were calculated from dose-response curves by linear inhibitor stock solution was diluted in sterile phosphate buffered saline regression analysis. (pH 7.2) at final concentrations of 10, 50 and 100 µM and sterilized in 0.22 µm filter. The assayed concentrations were established according RESULTS AND DISCUSSION to inhibitor effective concentration3. As many efforts have been made to search new alternatives for Growth inhibition assay: Firstly, an inoculum of 105 trophozoites Giardia infection treatment, the assessment of a drug potential requires were incubated in TYI-S-33 medium (4.5 mL/tube) containing the suitable models that allow identifying the biological processes on which inhibitor at different concentrations (10, 50 and 100 µM) for 24, 48 they can interfere. For this purpose, assays for in vitro screening are a and 72 h at 37 ºC. After incubation, the tubes were cooled for 10 min, preliminary step and advances in trophozoites axenic cultivation have centrifuged at 250xg (10 min, 4 ºC) and the population density of cultures allowed the investigation of antigiardial activity of a range of compounds, were estimated by counting in a haemocytometer. As controls, cultures especially, in relation to their ability to exert effect on trophozoites containing only the parasites and cultures treated with metronidazole at growth, adherence and viability. The present study led to some interesting 40 µg/mL15 were included in all the assays and submitted to the same findings concerning the in vitro activity of the cysteine protease inhibitor experimental conditions. For each E-64 concentration assessed, three E-64 on axenic trophozoites. tubes were screened and the protease inhibitor activity on the parasite growth was evaluated comparing the number of organisms in inhibitor- As shown in Figure 1, E-64 exerted inhibitory activity on parasite treated cultures with their number in controls. The results were expressed multiplication in a dose dependent manner. Considering the assayed as the trophozoites number and growth inhibition percentage. concentrations of 10, 50 and 100 µM, growth inhibition was observed in
44 CARVALHO, T.B.; OLIVEIRA-SEQUEIRA, T.C.G. & GUIMARÃES, S. - In vitro antigiardial activity of the cysteine protease inhibitor E-64. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 43-7, 2014.
cultures exposed to the highest of them, in which the number of parasites As in the growth and adherence assays, E-64 at 100 µM showed a recovered was significantly lower than that obtained in untreated cultures very significant reduction of viability rate in comparison to non-treated (p < 0.05). The inhibitory concentrations at 50% (IC50) were 144.29 cultures after 48 h (Fig. 3) (p < 0.05). The highest rates were detected µM for 24 h, 53.72 µM for 48 h and 27.02 µM for 72 h. At both 48 and after 48 h, when the inhibitor at 50 and 100 µM was able to kill 80% and 72 h, growth reduction by ≥ 50% and ≥ 70% were observed in cultures 93% of the trophozoites, respectively (p < 0.05). However, this effect treated with 50 and 100 µM, respectively (p < 0.05). A significant level was lower than that obtained by exposure to metronidazole (p < 0.05). of inhibition (~80%) was detected in 72 h cultures treated with the 100 µM, but the reduction rate was still lower than that obtained in metronidazole-treated cultures. In adherence assays (Fig. 2), only the concentrations of 50 and 100 µM were able to promote detachment of trophozoites. The IC50 values after 24 and 48 h were 128.65 µM and 33.80 µM respectively. The greater activity was induced by the inhibitor at 100 µM after 48 h (p < 0.05) when only 10% of trophozoites remained attached. Despite E-64 markedly diminished parasite adherence at 100 µM, the rates of metronidazole inhibition were higher. In both growth and adherence assays, E-64 exhibited a dose-response effect, however by light microscope observations, this activity was not accompanied by marked changes in trophozoites morphology as body swollen and reduction of flagellar beating frequency.
Fig. 3 - Viability (MTT assay) of G. duodenalis after incubation with different concentrations of E-64 inhibitor (µM). Cultures treated with metronidazole (MTZ) at 40 µg/mL were included as control. The values correspond to mean ± standard deviation (SD) in assays performed in triplicate.
So far, the most widely used drug against Giardia infection is the metronidazole, a 5’-nitroimidazole derivate, although there are some problems related to resistance and toxicity. Once many antiparasitic drugs currently in use show the same disadvantages, recently, investigations have identified promising antiparasitic drug candidates as well as potential targets in parasites. In this context, proteases and Fig. 1 - In vitro effect of E-64 inhibitor (µM) on the growth of G. duodenalis trophozoites, their inhibitors are focused, respectively, as druggable targets and new after incubation for 24, 48 and 72 hours. Non-treated cultures (NTC) and metronidazole- therapy alternatives. treated cultures (MTZ) were included in all assays. Data expressed as means of trophozoites number (105) ± standard deviation (SD) in assays performed in triplicate. In the last years, several investigations have focused the potential of protease inhibitors in important parasitic infections such as malaria, Chagas’ disease, leishmaniasis and toxoplasmosis17 and they have revealed promising insights on the use of these substances, especially those against thiol or cysteine proteases. Regarding to Giardia, advances in the study of its proteases had become clear that this protozoan is actively proteolytic and contain multiple proteases, with the predominance of the cysteine peptidases that are responsible for the main proteolytic activity4,5,12,26.
In the present study, although metronidazole was very effective, the cysteine protease inhibitor E-64 was shown to be also efficient in affecting the parasite growth, adherence and viability. These biological parameters were inhibited by ≥ 50% at concentrations of 50 and 100 µM. With respect to Giardia, to date, there are only two studies that evaluate the performance of inhibitors on the life forms of the parasite. In one of these investigations8, antiretroviral protease inhibitors (Kaletra®, ritonavir and saquinavir) were assessed in vitro for their activity on trophozoite ® Fig. 2 - In vitro effect of E-64 inhibitor (µM) on the adherence of G. duodenalis trophozoites, proliferation and the results revealed that Kaletra was the most effective after incubation for 24 and 48 hours. Non-treated cultures (NTC) and metronidazole-treated of them. More recently, in vitro and in vivo assays to evaluate the cultures (MTZ) were included in all assays. Data expressed as means of attached trophozoites antigiardial potential of E-64 showed a significant inhibitory effect on number (105) ± standard deviation (SD) in assays performed in triplicate. parasite growth and on the efficiency of encystation11.
45 CARVALHO, T.B.; OLIVEIRA-SEQUEIRA, T.C.G. & GUIMARÃES, S. - In vitro antigiardial activity of the cysteine protease inhibitor E-64. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 43-7, 2014.
Interestingly, E-64 was also able to inhibit trophozoite adherence and presente estudo, embora o metronidazol tenha se apresentado bastante a marked detachment of 90% was observed in cultures exposed to the efetivo, o E-64 mostrou ser capaz de inibir o crescimento, a aderência substance at 100 µM. It is important to emphasize that the attachment e a viabilidade em taxas superiores a 50%, especialmente nos cultivos of Giardia trophozoites to intestinal mucosa is an essential factor for expostos à concentração de 100 µM. A despeito de preliminares, esses their colonization and survival in the host as well as for giardiasis resultados demonstram que o inibidor E-64 pode interferir em processos pathogenesis. According to some authors, the adherence process has primordiais para a sobrevivência do parasita, além do que, abrem novas been considered as a potential target for chemotherapeutic alternatives perspectivas para investigações futuras a fim de se avaliar o real potencial that induce detachment and/or prevent the adhesion of parasite on the giardicida dos inibidores de proteases. intestinal epithelium16. Another point to make is that substances that are capable of altering microtubule assembly of the trophozoites have the ACKNOWLEDGEMENTS potential to affect a range of biological processes such as cytokinesis, locomotion, adhesion and detachment, situations that can enhance The authors are grateful to the student’s fellowship granted by the reduction in cell viability16. Brazilian agency CAPES.
Despite the E-64 in vitro effect, light microscope observations do REFERENCES not reveal marked alterations in trophozoites morphology. Although E-64 is a very useful cysteine inhibitor, it is known that its hydrophilic 1. Adam RD. Biology of Giardia lamblia. Clin Microbiol Rev. 2001;14:447-75. character is a property that can interfere in its cell permeability and 25 2. Barat LM, Bloland PB. Drug resistance among malaria and other parasites. Infect Dis impair the effective penetration through cell membranes . In contrast to Clin North Am. 1997;11:969-87. E-64, its synthetic analogues E-64c and E-64d act as potent membrane- permeable cysteine protease inhibitors that can permeate an intact 3. Beynon RJ, Bond JS, editors. Proteolytic enzymes: a practical approach. Oxford: Oxford cell. Recently, in a study on the structure and function of the Giardia University Press: 1989. endomembrane system and the cysteine proteases, DUBOIS6 reported 4. Carvalho TB, David EB, Coradi ST, Guimarães S. Protease activity in extracellular that growth was not affected by E-64d up to 50 µM. In addition, products secreted in vitro by trophozoites of Giardia duodenalis. Parasitol Res. according to this author, the survival of trophozoites in the presence 2008;104:185-90. of this inhibitor did not differ from that observed in the untreated cultures. In view of these findings with E-64d and considering the 5. Coradi ST, Guimarães S. Giardia duodenalis: protein substrates degradation by trophozoite observations in the present study when E-64 was able to reduce parasite proteases. Parasitol Res. 2006;99:131-6. growth by ≥ 50% at concentrations of 50 and 100 µM, future studies 6. Dubois KN. Trafficking and biological function of Giardia cysteine proteases. are a prerequisite to elucidate in more detail the effects of E-64 and its [dissertation]. San Francisco: University of California; 2007. derivatives on Giardia biological processes. 7. Dubois KN, Abodeely M, Sakanari J, Craik CS, Lee M, McKerrow JH, et al. Identification The findings assembled herein lead us to suggest that E-64 can of the major cysteine protease of Giardia and its role in encystation. J Biol Chem. 2008;283:18024-31. interfere in some crucial process in relation to the parasite survival, but until today little is known about the efficacy and safety of this protease 8. Dunn LA, Andrews KT, McCarthy JS, Wright JM, Skinner-Adams TS, Upcroft P, et al. inhibitor in vivo. So, it is suitable that the concentrations assessed in vitro The activity of protease inhibitors against Giardia duodenalis and metronidazole- must be tested in vivo before a definitive statement can be made on its resistant Trichomonas vaginalis. Int J Antimicrob Agents. 2007;29:98-102. antiparasitic potential. Even so, our results provide insights on protease 9. Dunn LA, Burgess AG, Krauer KG, Eckmann L, Vanelle P, Crozet MD, et al. A new- inhibitors targeting parasite cysteine proteases open perspectives for generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia future investigations in order to confirm the real antigiardial potential lamblia in vitro. Int J Antimicrob Agents. 2010;36:37-42. of E-64 and other potent inhibitors of cysteine proteases. 10. Edlind TD, Hang TI, Chakraborty PR. Activity of the anthelmintic benzimidazoles against RESUMO Giardia lamblia in vitro. J Infect Dis. 1990;162:1408-11. 11. Hussein EM, Dawoud HA, Salem AM, Atwa MM. Antiparasitic activity of cystine protease Atividade in vitro do inibidor de cisteína-proteases E-64 sobre inhibitor E-64 against Giardia lamblia excystation in vitro and in vivo. J Egypt Soc trofozoítos de Giardia Parasitol. 2009;39:111-9.
As cisteína-proteases estão entre os alvos mais promissores para o 12. Jiménez JC, Uzcanga G, Zambrano A, Di Prisco MC, Lynch NR. Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia desenvolvimento de novos agentes terapêuticos, visto que participam intestinalis. J Parasitol. 2000;86:859-62. de eventos fundamentais do ciclo de vida de muitos microorganismos, inclusive Giardia. Como a atividade das proteases pode ser controlada 13. Keister DB. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with por inibidores específicos, essas substâncias têm sido avaliadas quanto ao bile. Trans R Soc Trop Med Hyg. 1983;77:487-8. potencial antiparasitário. Diante disso, o presente estudo teve por objetivo 14. Lemée V, Zaharia I, Nevez G, Rabodonirina M, Brausser P, Ballet JJ, et al. Metronidazole avaliar o efeito in vitro do inibidor de cisteína-proteases E-64 sobre o and albendazole susceptibility of eleven clinical isolates of Giardia duodenalis from crescimento, a aderência e a viabilidade de trofozoítos de cepa de Giardia France. J Antimicrob Chemother. 2000;46:819-21. isolada em Botucatu. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado microscopicamente em hemocitômetro, 15. McAllister TA, Annett CB, Cockwill CL, Olson ME, Wang Y, Cheeke PR. Studies on the enquanto que a viabilidade celular foi avaliada pelo método do MTT. No use of Yucca schidigera to control giardiosis. Vet Parasitol. 2001;97:85-99.
46 CARVALHO, T.B.; OLIVEIRA-SEQUEIRA, T.C.G. & GUIMARÃES, S. - In vitro antigiardial activity of the cysteine protease inhibitor E-64. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 43-7, 2014.
16. Machado M, Dinis AM, Salgueiro L, Custódio JBA, Cavaleiro C, Sousa MC. Anti-Giardia 23. Touz MC, Nores MJ, Slavin I, Carmona C, Conrad JT, Mowatt MR, et al. The activity of activity of Syzygium aromaticum essencial oil and eugenol: effects on growth, viability, a developmentally regulated cysteine proteinase is required for cyst wall formation adherence and ultrastructure. Exp Parasitol. 2011;127:732-9. in the primitive eukaryote Giardia lamblia. J Biol Chem. 2002;277:8474-81.
17. McKerrow JH, Rosenthal PJ, Swenerton R, Doyly P. Development of protease inhibitors 24. Ward W, Alvarado L, Rawlings ND, Engel JC, Franklin C, McKerrow JH. A primitive for protozoan infections. Curr Opin Infect Dis. 2008;21:668-72. enzyme for a primitive cell: the protease required for excystation of Giardia. Cell. 1997;89:437-44. 18. Ponce-Macotela M, Rufino-González Y, de la Mora-de la Mora JI, González-Maciel A, Reynoso-Robles R, Martínez-Gordillo MN. Mortality and morphological changes in 25. Wilcox D, Mason RW. Inhibition of cysteine proteinases in lysosomes and whole cells. Giardia duodenalis induced by exposure to ethanolic extracts of Justicia spicigera. Biochem J. 1992;285:495-502. Proc West Pharmacol Soc. 2001;44:151-2. 26. Williams AG, Coombs GH. Multiple protease activities in Giardia intestinalis 19. Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol. trophozoites. Int J Parasitol. 1995;25:771-8. 2002;120:1-21. 27. Yarden NT, Millman M, Lauwaet T, Davids BJ, Gillin FD, Dunn L, et al. Impaired parasite 20. Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium join the ‘Neglected attachment as fitness cost of metronidazole resistance in Giardia lamblia. Antimicrob Diseases Initiative’. Trends Parasitol. 2006;22:203-8. Agents Chemother. 2011;55:4643-51.
21. Thompson RC, Reynoldson JA, Mendis AH. Giardia and giardiasis. Adv Parasitol. 28. Zucca M, Savoia D. Current developments in the therapy of protozoan infections. Open 1993;32:71-160. Med Chem J. 2011;5:4-10.
22. Thompson RC. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Received: 6 December 2012 Int J Parasitol. 2000;30:1259-67. Accepted: 16 May 2013
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FAPESP/BIREME Project on Scientific Electronic Publications Latin American and Caribbean Center on Health Sciences Information Rua Botucatu 862 – 04023-901 São Paulo, SP – Brazil Tel. (011) 5576-9863 [email protected] Rev. Inst. Med. Trop. Sao Paulo 56(1):49-54, January-February, 2014 doi: 10.1590/S0036-46652014000100007
MOLECULAR TYPING OF Giardia duodenalis ISOLATES FROM NONHUMAN PRIMATES HOUSED IN A BRAZILIAN ZOO
Érica Boarato DAVID(1), Mariella PATTI(2), Silvana Torossian CORADI(2), Teresa Cristina Goulart OLIVEIRA-SEQUEIRA(1), Paulo Eduardo Martins RIBOLLA(1) & Semíramis GUIMARÃES(1)
SUMMARY
Giardia infections in captive nonhuman primates (NHP) housed at a Brazilian zoo were investigated in order to address their zoonotic potential. Fresh fecal samples were collected from the floors of 22 enclosures where 47 primates of 18 different species were housed. The diagnosis of intestinal parasites after concentration by sedimentation and flotation methods revealed the following parasites and their frequencies: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); Oxyurid (4.5%) and Strongylid (4.5%). Genomic DNA extracted from all samples was processed by PCR methods in order to amplify fragments of gdh and tpi genes of Giardia. Amplicons were obtained from samples of Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. Clear sequences were only obtained for the isolates from Ateles belzebuth (BA1), Alouatta fusca (BA2) and Alouatta caraya (BA3). According to the phenetic analyses of these sequences, all were classified as assemblage A. For the tpi gene, all three isolates were grouped into sub-assemblage AII (BA1, BA2 and BA3) whereas for the gdh gene, only BA3 was sub- assemblage AII, and the BA1 and BA2 were sub-assemblage AI. Considering the zoonotic potential of the assemblage A, and that the animals of the present study show no clinical signs of infection, the data obtained here stresses that regular coproparasitological surveys are necessary to implement preventive measures and safeguard the health of the captive animals, of their caretakers and of people visiting the zoological gardens.
KEYWORDS: Giardia duodenalis; Molecular characterization; Nonhuman primates; Zoo; Zoonosis
INTRODUCTION can be clustered into at least eight groups, referred to as genotypes A to H17,23, but only assemblages A and B are known to infect humans, as well Today, in view of the increasing interest for biodiversity conservation, as a variety of other mammals. Based on the data on the prevalence of wildlife species have been subject to many investigations; however the giardiasis in different hosts and on existing knowledge of the genotypes search for knowledge goes beyond the behavioral and ecological aspects of G. duodenalis, THOMPSON proposed the existence of transmission of these animals and includes research on their infectious diseases and cycles that keep the infection among the mammals31. Even so, it has their role in public health. For this purpose, studies have been carried not been fully elucidated how these cycles interact and how often the out on the occurrence of enteric parasites in a range of wild mammals’ transmission of zoonotic assemblages occurs. species, many of them kept in zoological gardens, conservation parks or sanctuaries, where captivity may raise their exposure to conditions Despite the interest of researchers, as yet, genotyping data from that facilitate the spread of these pathogens4,5,7,9,19,21. In this context, it is wildlife mammals’ species are scarce. Lately, APPELBEE et al. important to stress that a wide range of captive species may be important highlighted that the infection of free-living and captive wildlife mammals reservoirs for zoonotic parasites acting as potential sources of human by both host-adapted and zoonotic isolates of Giardia can provide insights and animal infections and environmental contamination. into the host range and zoonotic potential of this parasite1. An overview of recent studies enables us to ascertain that almost all mammalian The enteric protozoan Giardia duodenalis (syn. G. intestinalis, G. orders include free-living and captive species that can harbor Giardia lamblia) is one of the most common pathogens of zoonotic importance isolates2,18,26. Among these groups, Giardia isolates have been obtained that infect humans and domesticated animals, in addition to many other from nonhuman primates’ species, however, until now, few studies have mammalian wildlife species, including free-living and captive animals1. addressed the molecular characterization of these isolates and discussed Currently, genetic analyses have shown that human and animal isolates their role in public health3,5,15,20,22,32.
(1) Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, São Paulo, SP, Brazil. (2) Departamento de Ciências Biológicas e da Saúde, Universidade do Sagrado Coração (USC), Bauru, São Paulo, SP, Brazil. Correspondence to: Semíramis Guimarães, Departamento de Parasitologia/IBB, Universidade Estadual Paulista (UNESP), 18618-97 Botucatu, São Paulo, SP, Brasil. Phone: + 55-14-38800539, Fax: +55-14-38153744. E-mail: [email protected] DAVID, E.B.; PATTI, M.; CORADI, S.T.; OLIVEIRA-SEQUEIRA, T.C.G.; RIBOLLA, P.E.M. & GUIMARÃES, S. - Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 49-54, 2014.
Thus, we focused our attention here to verify the occurrence of the The samples were assessed for helminthes and protozoans by Giardia infection in nonhuman primates’ fauna of the Bauru Municipal conventional coprological techniques of spontaneous sedimentation and Zoological Garden (São Paulo State, Brazil). centrifuge-flotation with zinc sulfate, according to SLOSS et al.27. For Cryptosporidium diagnosis, a part of each stool sample was previously MATERIALS AND METHODS concentrated by centrifugation (500 xg, 10 min) and the sediment obtained was used to prepare smears stained by a modified Ziehl-Neelsen Study site and sampling: The Bauru Municipal Zoological Garden, technique13. An aliquot of this sediment was also stored at -20 °C for located in the city of Bauru in São Paulo State, covers an area of 43.5ha subsequent DNA extraction. and houses 880 animals of 210 species, comprising mammals, birds and reptiles. Before the start of the study, approval was obtained from the DNA extraction: The genomic DNA was extracted using Research Ethics Committee at Sacred Heart University (105/10 CEP-USC) the QIAamp® Stool Mini Kit (Qiagen, Germany) following the as well as by the zoo’s administration. From September to November 2009, manufacturer’s instructions. Prior to extraction, the samples were fecal samples from forty-seven primates of 18 species, belonging to three subjected to three freeze/thaw cycles as follows: two cycles in liquid families of New World monkeys (Atelidae, Callitrichidae and Pitheciidae) nitrogen for five min and at 70 °C for five min and concluding in liquid and one of Old World (Cercopithecidae) were analyzed. The animals were nitrogen for five min and thawed at 95 °C for five min. For molecular either kept individually (five animals) or in monospecific groups of 2-7 characterization of Giardia, DNA was extracted from all the samples individuals (42 animals) sharing the same enclosures (Table 1). regardless the coprological diagnosis.
Due to the group housing, all the feces discharged by the animals PCR assays and sequencing: Molecular analysis was carried out in each pen were recovered from the floor and pooled in labeled plastic by amplifying gdh and tpi fragment genes. A nested PCR procedure was vials containing 2.5% potassium dichromate. For those animals kept used to amplify a 530 bp region of the tpi fragment using the primers alone in the enclosure, individual sampling was possible. Collection of AL3543 and AL3546 for the first step, and AL3544 and AL3545 for the stool was carried out in the morning, after the feeding of animals, on second one29. For gdh, a semi-nested PCR was used to amplify a 432 bp three alternate days. fragment with the primers GDHeF and GDHiR in the primary reaction
Table 1 Intestinal parasites identified in nonhuman primates at Bauru zoo and G. duodenalis PCR analysis
No. of animals (no. enclo- Primate specie Common name Coprological analysis Giardia PCR sures) Alouatta caraya Black howler monkey 4 (1) Giardia, Entamoeba spp + Alouatta fusca Brown howler monkey 3 (1) Giardia + Alouatta seniculus Red howler monkey 4 (2) Giardia, Entamoeba spp, + Endolimax nana Ateles belzebuth White-fronted spider monkey 2 (1) Giardia, Iodamoeba bütschilii + Ateles fusciceps Brown-headed spider monkey 2 (1) - - Ateles paniscus Red-faced spider monkey 5 (1) Oxyurid eggs - Callicebus nigrifrons Black-fronted titi monkey 2 (1) - - Callithrix argentata Silvery marmoset 2 (1) - - Callithrix geoffroyi Geoffroy's tufted-ear marmoset 1 - - Lagothrix lagotricha Woolly monkey 5 (3) - - Leontopithecus chrysomelas Golden-headed lion tamarin 7 (2) - - Leontopithecus rosalia Golden lion tamarin 2 (1) Strongyle-type eggs - Mandrillus sphinx Mandrill 2 (1) Entamoeba spp - Papio hamadryas Hamadryas baboon 1 - - Papio papio Guinea baboon 2 (1) - - Saguinus bicolor Brazilian bare-faced tamarim 1 - - Saguinus fuscicollis Brown-headed tamarin 1 - - Saguinus melanoleucus White saddleback tamarin 1 Entamoeba spp - - negative samples
50 DAVID, E.B.; PATTI, M.; CORADI, S.T.; OLIVEIRA-SEQUEIRA, T.C.G.; RIBOLLA, P.E.M. & GUIMARÃES, S. - Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 49-54, 2014.
and GDHiF and GDHiR in the secondary25 one. In all PCR reactions, nuttalli5. However, through direct microscopic identification of the positive (DNA from axenic Giardia culture) and negative controls were parasite, it is impossible to distinguish between E. histolytica and the included. The amplified products were submitted to 1.5% agarose gel non-pathogenic species E. dispar and E. moshkovskii, once these amoebae electrophoresis, stained with ethidium bromide. are morphologically indistinguishable. It is important to emphasize that, although Giardia and Entamoeba spp. are commonly found in stools of Bands of the predicted sizes were excised from agarose gel, then, wild and captive NHP and can be a significant cause of diarrhea, in the DNA was purified using the Ultrafree® DA kit (Millipore Corp., USA) present survey, all fecal samples had a normal consistency and animals and sequenced in both directions in Macrogen Inc. (Seoul, Korea). The showed no symptoms of infection, even when both protozoan were nucleotide sequences obtained were manually corrected and multiple detected in animals kept in the same pen (Table 1). alignments for each locus were performed using CLUSTAL X version 1.830 and queried against known sequences of GenBank database using Given that Cryptosporidium is an important pathogenic protozoa that BLAST. The phenetic analyses were performed with the MEGA software has been detected in a range of captive mammals’ species 7,11,15,19,21,24, it was (http://www.mega-software.net) and the sequences obtained in the present expected that this parasite could be detected in the animals of the present study were deposited in the GenBank data base under accession numbers study. It is noteworthy that the occurrence of Cryptosporidium infection JN172997-JN172999 for gdh sequences, and JN410841-JN410843 for is still underestimated, since its diagnosis depends on the identification tpi sequences. of oocysts in fecal smears stained, being a more laborious method that is generally not included in many surveys. This was not the point in our The DNA sequences were translated into amino acids using the online study since smears were prepared and examined, but a likely reason for software Emboss gui - European Molecular Biology Open Software Suite negative results of Cryptosporidium could be the analysis of pooled stool (http://bips.u-strasbg.fr/EMBOSS/). samples, despite efforts to concentrate the material.
RESULTS AND DISCUSSION Although the increasing interest to gain insight into the role of nonhuman primates as hosts of Giardia, molecular characterization of The microscopic examination of stool samples revealed that enteric isolates from New World species is still limited, especially in Brazil, parasites pathogenic and/or non pathogenic were detected in samples which is the world’s richest country in primate species diversity. from 36% (8/22) of the enclosures. Among the 18 species of primates, intestinal parasites were found in feces from animals belonging to the Herein, the four Giardia-positive samples produced PCR products of genera Ateles, Alouatta, Leontopithecus, Mandrillus and Saguinus the expected size in the gdh and tpi genes, but no amplicon was obtained (Table 1). Giardia cysts were found in the feces collected in four from the eighteen Giardia cyst-negative samples assessed. Clear sequences enclosures (18%) where animals of the following species were kept: of amplicons were only obtained for the isolates from Ateles belzebuth Ateles belzebuth, Alouatta seniculus, Alouatta caraya and Alouatta fusca (BA1), Alouatta fusca (BA2) and Alouatta caraya (BA3) (Tables 1 and (Table 1). Other intestinal protozoa were identified and their frequencies 2). A comparison of these sequences with the homologous gdh and tpi were as following: Entamoeba spp (18%), Iodamoeba butschlii (4.5%) deposited in GenBank showed that all three isolates were assigned to and Endolimax nana (4.5%). No Cryptosporidium oocysts were found assemblage A (Table 2; Fig. 1). Taking as reference the comparative on fecal smears of all assessed samples. Parasitism by helminthes was evaluation of G. duodenalis sequence data carried out by WIELINGA detected in animals from two enclosures, whose fecal samples were & THOMPSON, the alignment between these three sequences and other positive for strongylid and oxyurid nematodes eggs (Table 1). ones from humans and nonhuman primates revealed that in tpi locus, the isolates BA1 (JN410841), BA2 (JN410842), and BA3 (JN410843) were The occurrence of both protozoa and helminthes have been reported subtype AII, whereas in the gdh locus, only the isolate BA3 (JN172999) in captive nonhuman primates of different regions5,7,9,16,19,24, with the was classified as AII and the isolates BA1 (JN172997) and BA2 (JN172998) predominance of the former7,19,20. According to some authors, the most corresponded to sub-assemblage AI (Fig. 1; Table 2). frequent occurrence of protozoa infections can be explained by factors such as the short prepatent period, being immediately infective when Interestingly, in regards to the analysis of polymorphisms in the excreted and the low infective dose required19 for infection. Besides, nucleotide sequences (SNPs) obtained for the gdh gene, the BA2 the restricted and collective environments conditions where the animals (JN172998) isolate did show 100% homology with reference strains are kept facilitate protozoan transmission, because these organisms can for sub-assemblage AI while the isolates BA1 (JN172997) and BA2 be acquired through direct contact, which may be favored by primates’ (JN172998) revealed at least one SNP (Table 2). Thus, the isolate BA1 highly social nature. In the present study, besides the occurrence of the showed one SNP at position 666, and the BA3 showed the same SNP protozoa Giardia and species of amoebae, the detection of oxyurid eggs at position 666 and a novel one at position 621(Table 2). Among the tpi reinforces these aspects, since unlike most other nematodes, this helminth sequences, the isolates BA2 (JN410842) and BA3 (JN410843) did show is already able to infect the host when passed in feces. 100% homology with reference strains for sub-assemblage AII whereas the isolate BA1 (JN410841) reveled a novel SNP at position 107 (Table Likewise as previously reported in the literature5,19, herein, Giardia 2). In all sequences retrieved, nucleotide substitutions were characterized and Entamoeba spp. were the most frequent parasites diagnosed in NHP. as transitions and the SPNs were synonymous, resulting in no change Nevertheless, the clinical relevance of these infections is difficult to in the amino acids encoded. So, only in the tpi sequence of isolate BA1 ascertain, especially regarding to Entamoeba, because several species (JN410841), a non-synonymous SNP in position 634 determined a change are considered non-pathogenic5. So, the known species considered in the deduced amino acid sequence leading to a replacement of a serine pathogenic for NHP include E. histolytica and its virulent variant E. by a phenylalanine.
51 DAVID, E.B.; PATTI, M.; CORADI, S.T.; OLIVEIRA-SEQUEIRA, T.C.G.; RIBOLLA, P.E.M. & GUIMARÃES, S. - Molecular typing of Giardia duodenalis isolates from nonhuman primates
housed in a Brazilian zoo. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 49-54, 2014. 1266
------G 1080
------T 902
. - - - - - C 894
. - - - - - C 870
. - - - - T C 867
. - - - - T C 861
. . - - - - T 831
. . - - - - C 807
. . - - - - C 786
. - - - - T C 771
. - - - - A C 756
. - - - - T C 753
. . - - - - C 723
. - - - - T C 720
. - - - - T C 699
. - - - - T C 693
. - - - - T C 687
. - - - - T C 672
. - -
- - T C
666 453
......
- T T T C T C C
636 399
......
- T T C T T C T C
633 394
...... - T T C
C
621 Nucleotide position 393
......
- T C C A
603 352
......
T T T C C C T C
570 268
......
T C G A
534 231
......
T C G A
531 189
......
T C G A
525 174
......
T C G A
522 162
...... Table 2 Table .
C G A G
510 144
......
T C A G
396 133
...... - -
- - T G G A
390 129
. . . - -
- - T T C T C C C C
372 126
...... - -
- - T A C C
309 120
...... - -
- - T T C C
270 117
...... - -
- - T T C C
258 111
...... - -
- - C A G G
249 108
...... - -
- - T T C C
246 107
...... - -
- - T C C
237 93 . . . . ------T T C C Host/Country Fallow deer/ Italy Fallow Roe deer/The Netherlands monkey/Brazil monkey/Brazil monkey/Brazil monkey/Brazil monkey/Brazil Human/ The Netherlands monkey/Brazil Human/ USA B.-c.squirrel monkey/ The Netherlands B.-c. squirrel monkey/ The Netherlands B.-c. squirrel monkey/ The Netherlands - Human/ Afega nistan Human/Australia TM tpi gdh gdh and tpi genes* of intra-sub-assemblage substitutions in the A: isolates, positions and breakdowns duodenalis assemblage Giardia GenBank NLR118 JN172999 JN172998 DQ650648 JN172997 JN410841 JN410842 AY826195 JN410843 U57897 FJ890951 FJ890961 FJ890962 L02120 M84604 BA3 BA2 _ BA1 BA1 BA2 NLH45 BA3 JH SQ678,681 SQ678,681 SQ694 Isolate WB Portl 1 AI AI AII AII AIII AIII Assemblage *The isolates of the present study are highlighted in bold as well as the substitution patterns proposed by WIELINGA & THOMPSON (2007). Novel substitution positions are highlighted in bold highlighted positions are substitution Novel (2007). THOMPSON & WIELINGA by patterns proposed the substitution as as well in bold are highlighted study the present isolates of *The for comparison are indicated by (-). And nucleotide sequences not available italics. Nucleotide sequences identical to reference isolates are indicated by (.)
52 DAVID, E.B.; PATTI, M.; CORADI, S.T.; OLIVEIRA-SEQUEIRA, T.C.G.; RIBOLLA, P.E.M. & GUIMARÃES, S. - Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 49-54, 2014.
Concerning subtypes of this assemblage, AI and AII are the two most frequently involved in the infections, even though they appear to differ in host preference, since there is evidence that humans are more commonly infected with AII whereas animals are more commonly infected with AI10. However, both subgroups have been detected in humans and animals.
Finally, despite the low number of tested isolates, the results presented herein outline some pertinent aspects about the occurrence of G. duodenalis assemblages in captive nonhuman primates’ species and also provide insights for future elucidation of Giardia epidemiology in endemic areas. All things considered, this kind of investigation highlights the importance of coproparasitological surveys as part of practice in zoos in order to implement preventive measures and safeguard the health of captive animals and people near them (animal keepers and visitors). In this context, it is appropriate to take into account that the transmission of pathogens between humans and nonhuman species has to be considered in either direction, so that recognizing the links between human, animal and ecosystem health can provide an effective approach to understanding the transmission of pathogens among these hosts8.
RESUMO
Genotipagem de isolados de Giardia duodenalis de primatas não humanos mantidos em zoológico do Brasil Fig. 1 - Dendrograms of Giardia duodenalis based on nucleotide sequences of gdh and tpi genes. Trees were constructed using the A pesquisa de infecções por Giardia e a caracterização genotípica neighbor-joining method implemented by the computer program Mega deste protozoário foi realizada em primatas não humanos (PNH) 4.1. Bootstrap values were calculated by the analysis of 1000 simulations mantidos em Zoológico a fim de avaliar o seu potencial zoonótico. As (only bootstrap values >50% are shown). The reference sequences for amostras dos animais consistiram de fezes colhidas do piso de 22 baias both genes are underlined. onde eram mantidos 47 primatas de 18 diferentes espécies. Exames coproparasitológicos foram realizados pelos métodos de concentração In the present study, the finding of infection within assemblage A, por sedimentação e centrífugo-flutuação e revelaram a presença dos confirms previous reports that have detected the same genotype in either seguintes parasitas e suas respectivas frequências: Giardia (18%); the gdh or the tpi locus20,22 as well as in β-giardin20,32 and SSUrRNA12 Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); genes. Additionally, in these studies, assemblage A subtypes were oxiurídeos (4.5%) e estrongilídeos (4.5%). O DNA extraído de todas identified as either AI or AII. as amostras fecais foi submetido à técnica de PCR para a amplificação dos genes gdh e tpi de Giardia, porém, só foram obtidos amplicons das Recently, both assemblages A and B were found in New World quatro amostras positivas provenientes de Ateles belzebuth, Alouatta monkeys14,20,28,32, including species that live in South American forests28,32. caraya, Alouatta fusca and Alouatta seniculus. O seqüenciamento dos In Brazil, the occurrence of genotype A in NHP was first reported by fragmentos amplificados foi possível apenas para as amostras oriundas de VOLOTÃO and colleagues, who detected only isolates belonging to Ateles belzebuth (BA1), Alouatta fusca (BA2) e Alouatta caraya (BA3), the AI assemblage in samples from southern brown howler monkeys cuja análise fenética de ambos os genes revelou pertencerem ao genótipo (Alouatta clamitans)32. In contrast, SOARES et al. recently identified A. As análises das sequências de tpi revelaram que todas as amostras assemblage B in all the samples from 20 howler monkeys Alouatta fusca pertencem ao subgenótipo AII. No que se refere ao gene gdh as análises housed in a wild animal center27. revelaram uma amostra pertencente ao subgenótipo AII (BA3) e duas ao subgenótipo A1 (BA1 e BA2). Considerando o potencial zoonótico With respect to the separation of isolates into sub-assemblages, herein, do genótipo A e o fato de que os animais não apresentavam sintomas de the inconsistency observed among the loci assessed is not an unusual infecção, os dados do presente trabalho salientam a importância de se situation. According to authors BONHOMME et al., inconsistent results realizar, periodicamente, exames coproparasitológicos dos animais de are sometimes generated when different loci are targeted, especially zoológico, para implementação de medidas preventivas para resguardar regarding the subtypes. Thus, in spite of the possibility of assemblage a saúde dos animais em cativeiro, a de seus tratadores e dos visitantes swapping, explaining different assemblages or sub-assemblages in de parques zoológicos. different loci in the same isolate has not been an easy task. ACKNOWLEDGEMENTS Despite the finding of two different sub-assemblages in the same isolate, as we can see, the infection of captive nonhuman primates within The authors wish to thank the administrative members of the Bauru assemblage A has an epidemiological relevance, since this genetic group is Zoological Garden for giving permission to use the animals’ feces and, to zoonotic and the most common non-host specific assemblage in animals10. all animal keepers for providing assistance during fecal samples collection.
53 DAVID, E.B.; PATTI, M.; CORADI, S.T.; OLIVEIRA-SEQUEIRA, T.C.G.; RIBOLLA, P.E.M. & GUIMARÃES, S. - Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 49-54, 2014.
CONFLICT OF INTEREST 17. Lasek-Nesselquist E, Welch DM, Sogin ML. The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol. 2010;40:1063-74. The authors declare that there is no conflict of interest. 18. Lebbad M, Mattsson JG, Christensson B, Ljungström B, Backhans A, Andersson JO, et REFERENCES al. From mouse to moose: multilocus genotyping of Giardia isolates from various animal species. Vet Parasitol. 2010;168:231-9. 1. Appelbee AJ, Thompson RC, Olson ME. Giardia and Cryptosporidium in mammalian wildlife: current status and future needs. Trends Parasitol. 2005;21:370-6. 19. Levecke B, Dorny P, Geurden T, Vercammen F, Vercruysse J. Gastrointestinal protozoa in non-human primates of four zoological gardens in Belgium. Vet Parasitol. 2. Ash A, Lymbery A, Lemon J, Vitali S, Thompson RC. Molecular epidemiology of Giardia 2007;148:236-46. duodenalis in an endangered carnivore. The African painted dog. Vet Parasitol. 2010;174:206-12. 20. Levecke B, Geldhof P, Claerebout E, Dorny P, Vercammen F, Cacciò SM, et al. Molecular characterisation of Giardia duodenalis in captive non-human primates reveals 3. Beck R, Sprong H, Bata I, Lucinger S, Pozio E, Cacciò SM. Prevalence and molecular mixed assemblage A and B infections and novel polymorphisms. Int J Parasitol. typing of Giardia spp. in captive mammals at the zoo of Zagreb, Croatia. Vet Parasitol. 2009;39:1595-601. 2011;175:40-6. 21. Lim YA, Ngui R, Shukri J, Rohela M, Mat Naim HR. Intestinal parasites in various 4. Beck R, Sprong H, Lucinger S, Pozio E, Cacciò SM. A large survey of Croatian wild animals at a zoo in Malaysia. Vet Parasitol. 2008;157:154-9. mammals for Giardia duodenalis reveals a low prevalence and limited zoonotic potential. Vector Borne Zoonotic Dis. 2011;11:1049-55. 22. Martínez-Díaz RA, Sansano-Maestre J, Martínez-Herrero MD, Ponce-Gordo F, Gómez-Muñoz MT. Occurrence and genetic characterization of Giardia duodenalis 5. Berrilli F, Prisco C, Friedrich KG, Di Cerbo P, Di Cave D, De Liberato C. Giardia from captive nonhuman primates by multi-locus sequence analysis. Parasitol Res. duodenalis assemblages and Entamoeba species infecting non-human primates in an 2011;109:539-44. Italian zoological garden: zoonotic potential and management traits. Parasit Vectors. 2011;4:199. 23. Monis PT, Andrews RH, Mayrhofer G, Ey PL. Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect Genet Evol. 6. Bonhomme J, Le Goff L, Lemée V, Gargala G, Ballet JJ, Favennec L. Limitations of 2003;3:29-38. tpi and bg genes sub-genotyping for characterization of human Giardia duodenalis isolates. Parasitol Int. 2011;60:327-30. 24. Muriuki SM, Murugu RK, Munene E, Karere GM, Chai DC. Some gastro-intestinal parasites of zoonotic (public health) importance commonly observed in old world 7. Cordón PG, Prados AH, Romero D, Moreno MS, Pontes A, Osuna A, et al. Intestinal non-human primates in Kenya. Acta Trop. 1998;71:73-82. parasitism in the animals of the zoological garden “Peña Escrita” (Almuñecar, Spain). Vet Parasitol. 2008;156:302-9. 25. Read CM, Monis PT, Thompson RC. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect Genet 8. Epstein JH, Price JT. The significant but understudied impact of pathogen transmission Evol. 2004;4:125-30. from humans to animals. Mt Sinai J Med. 2009;76:448-55. 26. Robertson LJ, Forberg T, Hermansen L, Hamnes IS, Gjerde B. Giardia duodenalis cysts 9. Fagiolini M, Lia RP, Laricchiuta P, Cavicchio P, Mannella R, Cafarchia C, et al. isolated from wild moose and reindeer in Norway: genetic characterization by PCR- Gastrointestinal parasites in mammals of two Italian zoological gardens. J Zoo Wildl RFLP and sequence analysis at two genes. J Wildl Dis. 2007;43:576-85. Med. 2010;41:662-70. 27. Sloss, MW, Zajac AN, Kemp, RL. Parasitologia clínica veterinária. 6 ed. São Paulo: 10. Feng Y, Xiao L. Zoonotic potencial and molecular epidemiology of Giardia species and Manole; 1999. giardiasis. Clin Microbiol Rev. 2011;24:110-40. 28. Soares RM, de Souza SL, Silveira LH, Funada MR, Richtzenhain LJ, Gennari SM. 11. Geurden T, Goossens E, Levecke B, Vercammen F, Vercruysse J, Claerebout E. Occurrence Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals and molecular characterization of Cryptosporidium and Giardia in captive wild kept in captivity in Brazil. Vet Parasitol. 2011;180:344-8. ruminants in Belgium. J Zoo Wildl Med. 2009;40:126-30. 29. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate 12. Graczyk TK, Bosco-Nizeyi J, Ssebide B, Thompson RC, Read C, Cranfield MR. isomerase gene characterization and potential zoonotic transmission of Giardia Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitats duodenalis. Emerg Infect Dis. 2003;9:1444-52. of free-ranging human-habituated gorillas, Uganda. J Parasitol. 2002;88:905-9. 30. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The Clustal_X 13. Henriksen A, Pohlenz JFL. Staining of Cryptosporidia by a modified Ziehl-Neelsen windows interface: flexible strategies for multiple sequence alignment aided by technique. Acta Vet Scand. 1981;22:594-6. quality analysis tools. Nucleic Acids Res. 1997;25:4876-82.
14. Itagaki T, Kinoshita S, Aoki M, Itoh N, Saeki H, Sato N, et al. Genotyping of Giardia 31. Thompson, RC. The zoonotic significance and molecular epidemiology ofGiardia and intestinalis from domestic and wild animals in Japan using glutamate dehydrogenase giardiasis. Vet Parasitol. 2004;126:15-35. gene sequencing. Vet Parasitol. 2005;133:283-7. 32. Volotão AC, Souza Júnior JC, Grassini C, Peralta JM, Fernandes O. Genotyping of 15. Kowalewski MM, Salzer JS, Deutsch JC, Raño M, Kuhlenschmidt MS, Gillespie TR. Giardia duodenalis from Southern Brown Howler Monkeys (Alouatta clamitans) Black and gold howler monkeys (Alouatta caraya) as sentinels of ecosystem health: from Brazil. Vet Parasitol. 2008;158:133-7. patterns of zoonotic protozoa infection relative to degree of human-primate contact. Am J Primatol. 2011;73:75-83. 33. Wielinga CM, Thompson, RCA. Comparative evaluation of Giardia duodenalis sequence data. Parasitology. 2007;134:1795-821. 16. Labes EM, Hegglin D, Grimm F, Nurcahyo W, Harrison ME, Bastian ML, et al. Intestinal parasites of endangered orangutans (Pongo pygmaeus) in Central and East Kalimantan, Received: 25 July 2012 Borneo, Indonesia. Parasitology. 2010;137:123-35. Accepted: 5 June 2013
54 Rev. Inst. Med. Trop. Sao Paulo 56(1):55-59, January-February, 2014 doi: 10.1590/S0036-46652014000100008
FREQUENCY, URINALYSIS AND SUSCEPTIBILITY PROFILE OF PATHOGENS CAUSING URINARY TRACT INFECTIONS IN ENUGU STATE, SOUTHEAST NIGERIA
Uju M.E. DIBUA(1), Ifeoma S. ONYEMERELA(1) & Emeka I. NWEZE(1)
SUMMARY
Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture. Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch’ method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin. Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.
KEYWORDS: Urinary tract infection; Pathogen; Southeast Nigeria; Etiology; Antibiotic susceptibility.
INTRODUCTION are required in monitoring the trend and gaining useful knowledge about the type of pathogens responsible for UTIs. The etiology and resistance Urinary tract infections (UTIs) are among the most common patterns of community- acquired UTI pathogens have not been studied infections diagnosed in both hospitalized and outpatients12. About 150 extensively in many settings8. Moreover, even in areas where a few studies million UTIs are reported worldwide every year9 costing the world have been done, there is need to continue to monitor the trend in terms economy over six billion US dollars2. UTIs are reportedly the most of the etiology and antibiotic resistance patterns of these uropathogens. common bacterial infections in women and account for significant Although a few studies have been carried out previously in Southeast morbidity and increase in the cost of healthcare. In young sexually active Nigeria5,6,14, most of them investigated UTIs in association with other women, the incidence of UTI exceeds 0.5 episodes per year with about diseases in certain specific populations. Thus, there is not much recent 30% of women experiencing recurrent infections. In developed countries data specifically on the etiology and antimicrobial resistance patterns such as the USA, UTIs alone account for over eight million physician of uropathogens among individuals specifically presenting with UTIs visits per year7. In developing countries, especially in many resource in Enugu State, Southeast Nigeria. This information will be very useful poor settings, the situation is worse and many clinicians often initiate to clinicians so as to facilitate empirical treatment and management of empiric antibiotic treatment even before the final laboratory diagnostic patients with symptoms of UTIs. We therefore carried out this study to test results are available. This is occasioned by the long duration, high ascertain the current etiology and antimicrobial susceptibility patterns cost and non-availability of experienced personnel and equipment needed of uropathogens in Enugu State, Nigeria. for reporting accurate diagnosis by culture and susceptibility testing. Currently, there is an increasing problem of resistance to antibiotics MATERIALS AND METHODS especially in developing countries, prompting the need for a good knowledge of antibiotic resistance patterns of these pathogens so as to Sample collection and analysis: Urine samples were collected from ensure efficient treatment and eradication. Moreover, area-specific studies three randomly selected centers: Bishop Shanahan Hospital, Nsukka,
(1) Department of Microbiology, University of Nigeria, Nsukka, Nigeria. Correspondence to: Emeka I. Nweze, PhD, AIMLS, Department of Microbiology, University of Nigeria, Nsukka, Nigeria. Phone: +2348068535841. E-mail: [email protected] DIBUA, U.M.E.; ONYEMERELA, I.S. & NWEZE, E.I. - Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, Southeast Nigeria. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 55-9, 2014.
McChuks Medical Diagnostic Laboratory, Enugu and Dynamic Medical phosphate crystals (with no microbial growth); and another seven had Diagnostic Laboratory, Enugu all located in Enugu State, Southeast numerous epithelial cells, also without any growth. Urinalysis carried Nigeria. The patients who came for consultation and whose urine samples out with combi-9 (Medi-Test) showed 17 urine samples (10 with growth were collected were all outpatients. They were given sterile wide-mouthed and seven without growth) had protein; seven urine samples (all with containers and instructed on how to collect clean, mid-stream, voided growth) were nitrite positive and three urine samples had moderate to high urine samples. Those who were unable to collect their own samples were glucose concentration. Fifty-four urine samples (36.2%) from females aided. Consent approvals were obtained from all those who participated and twelve urine samples (19.4%) from males showed significant growth. in the study. The study was approved in each center where samples were The isolates were identified using Gram stain and biochemical tests as collected. Patients who had no symptoms suggestive of UTI at the time of Escherichia coli (17), Staphylococcus species (12), Staphylococcus observation were excluded from the study. Those suffering from diabetes aureus (7), Proteus species (8) Pseudomonas aeruginosa (6), Candida mellitus, renal disorders, HIV positivity or on corticosteroid or antibiotic species (7), Enterobacter species (5), Klebsiella species (3), and Candida therapy were also excluded. Those who refused to give consent or were albicans (2). non-cooperative were not included in the study. From the above isolates, 43 strains - Escherichia coli (12), Urinalysis: The urine samples were examined macroscopically Staphylococcus species (10), Proteus species (5), Staphylococcus aureus for color and turbidity. They were then centrifuged and the sediment (4), Candida species (4), Klebsiella species (3), Pseudomonas aeruginosa examined microscopically for the presence of red blood cells, white blood (3), and Candida albicans (2) were used for the study. cells (pus cells), protozoa, Schistosoma eggs, yeasts and crystals. They were inspected for pH and the presence of glucose, protein and nitrite Antibiotic susceptibility profile of the recovered urine isolates: using Combi 9 (Medi-Test, Machery-Nagel GmbH, Duren) dipstick. Out of the 43 clinical bacterial isolates used in the study, 30 (81.08%) were resistant to four or more antibiotics with the highest resistance Isolation and identification of bacterial isolates from urine shown by E. coli (76.67%), followed by Klebsiella species (63.33%), S. samples: A loopful of each fresh urine sample was inoculated onto aureus (57.5%), Pseudomonas aeruginosa (53.33%), Staphylococcus Cysteine Lactose Electrolyte-Deficient (CLED) agar (Oxoid, UK) and species (51%), and the least was Proteus species (38%). All the incubated for 18-24hrs at 37 °C. Urine samples, that had yeast cells upon Gram-negative isolates were resistant to Ampiclox, Cefuroxime and microscopic examination, were inoculated onto Sabouraud Dextrose Agar Amoxacillin. However, most isolates were less resistant to Ciprofloxacin (SDA) (Oxoid, UK) and incubated at 37 °C for 18-24h. Discrete colonies and it was thus used as the antibiotic control (Tables 1 and 2). were placed in slants and kept in the refrigerator and characterized as previously described4. DISCUSSION
Antibiotic Susceptibility Test: Sterile Mueller-Hinton Agar plates Microscopy of the centrifuged urine samples showed presence of were seeded with 0.1mL of 0.5 MacFarland (approximately 107CFU/ moderate to many pus cells (white blood cells) in fewer urine samples mL) standardized isolates, spread out with a bent sterile glass rod and which showed significant microbial growth on culture; presence of left to dry on the bench for 30 minutes. Antibiotic discs were placed crystals such as calcium oxalates, tyrosine, cystine and triple phosphate; aseptically on the plates using sterile forceps. Plates were then incubated epithelial cells and Schistosoma eggs with numerous red blood cells. at 37 °C for 16-18h. The diameters of the zones of inhibitions were Pyuria, which is the presence of pus cells in this quantity, is an indication measured, recorded and interpreted according to CLSI approved standard of microbial infections since pus cells are white blood cells that have guidelines13. The antibiotics tested against Gram positive organisms were succumbed in defense of the body against pathogens that invade it. Few Septrin (30 μg), Erythromycin (10 μg), Pefloxacin (10 μg),Gentamycin are singly excreted in normal urine but in clumps in urinary infections. (10 μg), Ampiclox (30 μg), Zinnacef (20 μg), Amoxacillin (30 μg), Pyuria with significant microbial growth ≥( 105 organisms/mL) in 16 urine Rocephin (30 μg) and Ciprofloxacin (10 μg) while those tested against samples is a good indication of urinary tract infection while significant Gram negative organisms were septrin (30 μg), Chloramphenicol (30 microbial growth (bacteriuria) without pyuria in 54 urine samples μg), Sparfloxacin (10 μg), Ciprofloxacin (10 μg), Amoxacillin (30 μg), screened in this study may be an indication of diabetes, enteric fever, Augmentin (30 μg), Gentamycin (10 μg), Pefloxacin (30 μg), Tarivid (10 bacterial endocarditis or contaminants from the perineum. μg) and Streptomycin (30 μg). Crystals are formed from chemicals in normal urine and are usually RESULTS refractive in appearance. The crystal types found indicate the presence or absence of a disease. Calcium oxalate crystals are formed from oxalic Isolation and characterization of microbial isolates: A total of acid abundance in diet rich in leafy vegetables or coffee and calcium 211 urine samples (149 from females, 62 from males) were collected excreted into urine due to chronic dehydration or hyperparathyroidism. from all three centers located in Enugu State, Nigeria. Microscopy of The crystals usually form stones (calculi) in the kidneys, ureters or centrifuged urine samples showed 16 (11 females and five males) had bladders. Therefore, the presence of calcium oxalate crystals might be an significant number of pus cells (pyuria) while 54 (46 female and eight indication of calculi in the urinary tract, a predisposing factor to urinary males) had fewer pus cells. Calcium oxalate crystals were found in 14 tract infection. Tyrosine crystals presence might indicate a possible urine samples (four with growth and 10 without growth). One male urine severe liver disease while cystine crystals are found in cystinuria, a rare sample contained tyrosine crystals; another contained cysteine crystals congenital metabolic disorder. Presence of numerous epithelial cells with microbial growth on inoculation, while another had Schistosoma may indicate inflammation of the urinary tract or vaginal contamination eggs and a lot of red blood cells. One female urine sample had triple of the specimen; triple phosphate crystals in urine is not harmful but
56 DIBUA, U.M.E.; ONYEMERELA, I.S. & NWEZE, E.I. - Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, Southeast Nigeria. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 55-9, 2014.
Table 1 Antibiotic susceptibility test result against Gram - positive UTI isolates
ANTIBIOTIC TESTED Urine isolates (No.) % resistance S SXT E PEF CN APX Z AM R CPX S. aureus (1) + + + + + + + + + - 90 S. aureus (2) + - - - - + + + - - 40 S. aureus (3) - + + - +/- + + + - - 60 S. aureus ( 4) - - + - - + + + - - 40 Staphylococcus spp (1) - - + + - + + + + + 70 Staphylococcus spp ( 2) - - + - - + + + - - 40 Staphylococcus spp (3) - - - - - + + + + - 40 Staphylococcus spp (4) +/- + + - - + + + - - 60 Staphylococcus spp (5) + + + + + + + + + - 90 Staphylococcus spp (6) - - - - - + + + - - 30 Staphylococcus spp (7) + - + - - + + + - - 50 Staphylococcus spp (8) - - - - - +/- + + - - 30 Staphylococcus spp (9) - + + - +/- + + + - - 60 Staphylococcus spp (10) - - + - - + + + - - 40 Total resistance (%) 35.71 35.71 71.43 21.43 28.57 100 100 100 28.57 7.14 - = susceptible; +/- = intermediate; + = resistant; sp = species; S = Septrin 30 μg, E = Erythromycin 10 μg, PEF = Pefloxacin 10 μg, CN = Gentamycin 10 μg, APX = Ampiclox 30 μg, Z = Zinnacef 20 μg, AM = Amoxacillin 30 μg, R = Rocephin 30 μg and CPX = Ciprofloxacin 10 μg. indicates the pH is highly alkaline while presence of Schistosoma cells have been said to be common cause of nosocomial infections3,4. We also and numerous red blood cells indicates the infection, schistosomiasis10,11. isolated these organisms though the percentage of Klebsiella species Urinalysis results showed the presence of protein, nitrite and moderate to (4.3%) was smaller than those reported above. Staphylococcus species high glucose concentration in urine samples. Presence of protein in urine and Staphylococcus aureus are normal body flora and must have come known as proteinuria is usually an indication of kidney disorders including from the environment. The fact that more urine samples from females glomerulonephritis and urinary tract infection. Nitrates are found in (36.2%) showed significant microbial growth as against males (19.4%) is normal urine and are as a result of feeding on a diet containing vegetables. in line with the findings of AIYEGORO et al.1 who reported bacteriuria They are reduced to nitrites by pathogens such as E. coli, Proteus species only in urine from females. KWOK et al.10 sampled children aged 0-18 and Klebsiella species when in sufficient concentration in the urine. On years and showed that UTI in girls was (34.4 episodes/1000 persons) the other hand, presence of glucose in urine is an indication of diabetes as against males (4.4 episodes/1000 persons) and this agrees with our mellitus11,19. The different urine isolates recovered were Escherichia finding. The nature and proximity of the urinary system and the anus in coli (24. 6%), Staphylococcus species (17.4%), Staphylococcus aureus women may be the reason why women had more bacteria growth in their (11.6%), Proteus species (11.6%), Pseudomonas aeruginosa (10.1%), samples than men since many organisms from feces can easily infect or Candida species (10.1%), Enterobacter species (7.2%), Klebsiella species contaminate the urinary system in females. (4.3%) and Candida albicans (2.9%). This agrees with the findings of LAUPLAUD et al.11 which gave the percentage of E. coli implicated in The antibiotic susceptibility profile of the urine isolates showed urine of patients in Calgary Health Region Intensive Care Unit in Canada that E. coli (76.7%), Klebsiella species (63.3%), S. aureus (57.5%), as 23%. Only 2.9% of Candida albicans were recovered compared to P. aeruginosa (53.3%), Staphylococcus (51%) and Proteus (38%) 20% found by LAUPLAUD and co-workers11 suggesting that they were species were resistant (not susceptible) to conventional antibiotics used. not nosocomial infections. However, the percentage of E. coli (24.6%) BROOKS et al.3 stated that antibiotics such as derivatives of penicillin disagrees with the findings of AIYEGORO et al.1 and ZEIGHAMI (ampicillin and amoxicillin), sulphonamides, and the quinolones (2008)21 which were respectively 52.6% and 55.9%. AIYEGORO et al.1 (nitrofurantoin and nalidixic acid) are effective urinary antiseptics. working with out-patients aged 5-18 years visiting Obafemi Awolowo The genera Escherichia, Klebsiella and Pseudomonas from urine show University Teaching Hospital Complex Ile-Ife in Nigeria also isolated multiple resistances to antibiotics with Klebsiella species and E. coli Klebsiella spp. (25%), Proteus spp. (3.9%) and P. aeruginosa (2.8%). noted to be resistant to broad-spectrum β-lactam drugs like penicillins ZEIGHAMI (2008)21 working with urine of patients that had kidney and third generation cephalosporins20. All S. aureus, Klebsiella species, transplant and asymptomatic bacteriuria also isolated Klebsiella spp. P. aeruginosa and 83.33% of E. coli in our study were resistant to four (22.4%), P. aeruginosa (8.6%), and Proteus spp. (5.2%). These organisms or more antibiotics. Among the E. coli, 83.33% were resistant to septrin
57 DIBUA, U.M.E.; ONYEMERELA, I.S. & NWEZE, E.I. - Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, Southeast Nigeria. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 55-9, 2014.
Table 2 Antibiotic susceptibility test result against Gram – negative UTI isolates
ANTIBIOTIC TESTED Urine isolates (No.) % resistance SXT CH SP CPX AM AU CN PEF OFX S E. coli 1 + + + + + + +/- + + +/- 100 E. coli 2 +/- - +/- - +/- - - - - +/- 40 E. coli 3 - - + ------10 E. coli 4 + + + +/- + + +/- + +/- + 100 E. coli 5 + + + + + + - + + + 90 E. coli 6 + + + + + + + + + +/- 100 E. coli 7 + + + + + + + + + +/- 100 E. coli 8 + + + + + + +/- + + +/- 100 E. coli 9 - - + + + - - - - - 30 E. coli 10 + + + - - - - +/- +/- +/- 60 E. coli 11 + - + + + + + + + +/- 90 E. coli 12 + + + + + + + + + +/- 100 Klebsiella spp 1 + - - - +/- + + - - + 50 Klebsiella spp 2 + + + +/- + + +/- - - +/- 80 Klebsiella spp 3 + + + - + + - - - + 60 Proteus spp 1 - - - - - + - - - - 10 Proteus spp 2 + + + - + +/- - + + - 70 Proteus spp 3 + + + - + + - - - - 50 Proteus spp 4 +/- + - - - + - - - - 30 Proteus spp 5 +/- + - - - + - - - - 30 P. aeruginosa 1 + + + - - + - - - - 40 P. aeruginosa 2 + + + - - + - - - - 40 P. aeruginosa 3 + + + + + + - + + - 80 % resistance 87.0 73.9 82.6 47.8 69.6 82.6 39.1 47.8 47.8 56.5 - = susceptible; +/- = intermediate; + = resistant; spp = species, SXT = 30 μg septrin; CH = 30 μg chloramphenicol; SP = 10 μg sparfloxacin; CPX = 10 μg ciprofloxacin; AM = 30 μg Amoxacillin; AU = 30 μg Augmentin; CN = 10 μg Gentamycin; PEF = 30 μg perfloxacin; OFX = 10 μg tarivid; S = 30 μg streptomycin.
(cotrimoxazole), amoxicillin and streptomycin; 75% to ciprofloxacin while that of P. aeruginosa to amoxicillin/clavulanate (Augumentin) (a quinolone mostly prescribed currently), tarivid and perfloxacin (also and cotrimoxazole is supported by findings of AIYEGORO et al.1. quinolones) etc. These findings validate the data from WILLEY et al.20. Our findings and those of others seem to contradict the statements of It also falls in line with the findings of SAHM et al.18 where 95.8% of BROOKS et al.3 who reported that excretion of high percentage of E. coli isolated was resistant to ampicillin and 92.8% to trimethoprim- ampicillin and amoxicillin in urine as a basis for their preference in sulfamethoxazole (drug of choice in United States). The 83.3% E. coli treatment of acute urinary tract infections. This contradiction might have resistant to cotrimoxazole and amoxicillin is supported by the data from resulted from abuse of these drugs in the environment leading to urinary ODUYEBO et al.15 where all E. coli showed resistance to amoxicillin- pathogens becoming more resistant to them. clavulanic acid and 87.5% to cotrimoxazole and also in OLSON et al.16 in which a total of 80.1% E. coli tested was resistant to ampicillin. In conclusion, we observed high resistances of isolates to antibiotics which is consistent with the information in literature. Furthermore, test All S. aureus isolates were resistant to Amoxicillin Clavulanate. In results with broad spectrum antibiotics showed that Gram-negative ONANUGA et al.17, all S. aureus tested were resistant to ampicillin. isolates are showing higher resistances than Gram-positives. Similarly, Klebsiella species were all resistant to cotrimoxazole and augmentin while all P. aeruginosa were also resistant to cotrimoxazole, Some of the limitations of our study include the non-determination chloramphenicol and augmentin. High resistance of Klebsiella species to of the MICs of the tested antibiotics and the non- inclusion of control cotrimoxazole was observed by ODUYEBO et al.15 where it was 87.5% strains in the susceptibility studies because they were not available.
58 DIBUA, U.M.E.; ONYEMERELA, I.S. & NWEZE, E.I. - Frequency, urinalysis and susceptibility profile of pathogens causing urinary tract infections in Enugu State, Southeast Nigeria. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 55-9, 2014.
RESUMO 5. Chukwu BF, Okafor HU, Ikefuna AN. Asymptomatic bacteriuria in children with sickle cell anemia at the University of Nigeria teaching hospital, Enugu, South East, Nigeria. Ital J Pediatr., 2011;37:45. Perfil da frequência, análise urinária e suscetibilidade de patógenos causando infecções do trato urinário no estado de 6. Eyong ME, Ikepeme EE, Ekanem EE. Relationship between Schistosoma Enugu, Sudeste da Nigéria haematobium infection and urinary tract infection among children in South Eastern, Nigeria. Niger Postgrad Med J. 2008,15:89-93. Objetivo: O estudo teve como objetivo determinar a frequência e 7. Gonzalez CM, Schaeffer AJ. Treatment of urinary tract infection: what’s old, what’s agentes causadores das infecções do trato urinário (UTIs) em indivíduos new, and what works. World J Urol. 1999;6:372-82. com sintomas desta infecção no estado de Enugu, Sudeste da Nigéria e determinar a suscetibilidade antibiótica dos agentes microbianos isolados 8. Hooton TM, Scholes D, Hughes JP, Winter C, Roberts PL, Stapleton AE, et al. A de cultura da urina. Métodos: O estudo envolveu 211 indivíduos (149 prospective study of risk factors for symptomatic urinary tract infection in young mulheres e 62 homens) clinicamente suspeitos para UTI. Amostras women. N Engl J Med. 1996,335:468-74. urinárias foram coletadas pelo método de meia corrente “clean catch” e 9. Karlowsky JA, Lagacé-Wiens PR, Simner PJ, DeCorby MR, Adam HJ, Walkty A, et testados por procedimentos standards. Suscetibilidade aos antibióticos al. Antimicrobial resistance in urinary tract pathogens in Canada from 2007 to 2009: dos patógenos isolados foi testada usando a técnica de Kirby-Bauer e de CANWARD surveillance study. Antimicrob Agents Chemother. 2011;55:3169-75 acordo com as diretrizes do “Clinical and Laboratory Standards Institute” (CLSI). Resultados: Microscopia das amostras de urina centrifugadas 10. Kwok W, Kwaadsteniet MC, Harmsen M, van Suijlekom-Smit LW, Schellevis FG, van der Wouden JC. Incidence rates and management of urinary tract infections mostraram que 16 pacientes tinham piúria enquanto que 54 tinham células among children in Dutch general practice: results from a nation-wide registration do pus. Cristais de oxalato de cálcio foram encontrados em 14 amostras. study. BMC Pediatr. 2006,6:10. Análise de amostras da urina mostraram que 17 tinham proteína; sete eram positivas para nitrito e três tinham concentração de glicose de 11. Laupland KB, Magshaw SM, Gregson DB, Kirkpatrick AW, Ross J, Church DL. moderada para alta. Cinquenta e quatro amostras de urina (36.2% de Intensive care unit-acquired urinary tract infections in a regional critical care system. Crit Care. 2005;9:60-5. mulheres) e 12 (19.45% de homens) mostraram crescimento significante após cultura. Coloração pelo Gram e testes bioquímicos identificaram 12. Muvunyi CM, Masaisa F, Bayingana C, Mutesa L, Musemakweri A, Muhirwa G, nove organismos diferentes com a Escherichia coli sendo a mais comum et al. Decreased susceptibility to commonly used antimicrobial agents in bacterial das espécies isoladas. Quarenta e três espécies selecionadas ao acaso pathogens isolated from urinary tract infections in Rwanda: need for new antimicrobial foram testadas posteriormente para sua suscetibilidade contra um painel guidelines. Am J Trop Med Hyg. 2011;84:923-8. de antibióticos. Trinta amostras isoladas (81.08%) foram resistentes a 13. National Committee for Clinical Laboratory Standards. Methods for disk susceptibility quatro ou mais antibióticos sendo que a maior resistência foi da E. coli test for bacteria that grow aerobically. 7th ed. Wayne: National Committee for Clinical (76.67%). Todas as amostras Gram negativas isoladas foram resistentes Laboratory Standards. (NCCLS Document M2-A7). a Ampicilox, Cefuroxime e Amoxicilina. 14. Obiogbolu CH, Okonko IO, Anyamere CO, Adedeji AO, Akanbi AO, Ogun AA, et al. Incidence of Urinary Tract Infections (UTIs) among pregnant women in Akwa ACKNOWLEDGEMENTS metropolis, Southeastern Nigeria. Sci Res Essays. 2009,4:820-4
The authors wish to thank the authorities of the three centers where 15. Oduyebo OO, Daso MA, Uti RA, Ketiku KK. Prevalence of urinary tract infection samples were collected for their interest in clinical research and their in patients undergoing pelvic radiotherapy at a teaching hospital in Lagos, Nigeria. help during sample collection. We also wish to thank all the persons who J Nigerian Infect Control Assoc. 2001,4:6-10. gave consent and submitted their urine samples for this study. 16. Olson RP, Harrell LJ, Kaye KS. Antibiotic resistance in urinary isolates of Escherichia coli from college women with urinary tract infections. Antimicrob Agents Chemother. CONFLICT OF INTEREST 2009,53:1285-6.
Authors have no conflict of interest to declare. 17. Onanuga A, Oyi AR, Olayinka BO, Onaolapo JA. Prevalence of community-associated multi-resistant Staphylococcus aureus among healthy women in Abuja, Nigeria. Afr J Biotech. 2005;4:942-5. REFERENCES 18. Sahm DF, Thornsberry C, Mayfield DC, Jones ME, Karlowsky JA. Multi-drug resistant 1. Aiyegoro OA, Igbinosa OO, Ogunmwonyi IN, Odjadjare EE, Igbinosa OE, Okoh AI. urinary tract isolates of Escherichia coli: prevalence and patient demographics in the Incidence of urinary tract infection (UTI) among children and adolescents of Ile-Ife United States in 2000. Antimicrob Agents Chemother. 2001;45:1402-6. Nigeria. Afr J Microbiol Res. 2007;1:13-9. 19. The British Medical Association. Illustrated medical dictionary. London: Dorling 2. Akoachere JF, Yvonne S, Akum NH, Seraphine EN. Etiologic profile and antimicrobial Kindersley; 2002. susceptibility of community-acquired urinary tract infection in two Cameroonian towns. BMC Res Notes. 2012;5:219. 20. Willey JM, Sherwood LM, Woolverton JC. In: Prescott, Harley and Klein’s Microbiology. 7th ed. Boston: McGraw; 2008. 3. Brooks GF, Carroll KC, Butel JS, Morse SA. In: Jawetz, Melnick, Adelberg’s medical Microbiology. New York: McGraw Hill; 2007. 21. Zeighami H. Urinary tract infections in renal transplantation patient. Res J Biol Sci. 2008;3:1194-6. 4. Cheesbrough, M. District laboratory practice in tropical countries. Part 2. 2nd ed. Cambridge: University Press; 2004. p. 105-14. Received: 16 January 2013 Accepted: 18 April 2013
59 LIBRARY OF THE SÃO PAULO INSTITUTE OF TROPICAL MEDICINE
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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.
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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. Rev. Inst. Med. Trop. Sao Paulo 56(1):61-66, January-February, 2014 doi: 10.1590/S0036-46652014000100009
CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus)
Irma AGUILAR(1)§, Elda E. SÁNCHEZ(2)§, María E. GIRÓN(1,3), Amalid ESTRELLA(1,3), Belsy GUERRERO(4) & F. Alexis RODRIGUEZ-ACOSTA(1,3)
SUMMARY
The production of anti-snake venom from large mammal’s blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in
the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake anti- venom prepared in birds.
KEYWORDS: Coral antivenom; Elapidae; IgY immunoglobulins; Micrurus; venom.
INTRODUCTION and regulations for physicians to improve antivenom use must also be addressed. Even though coral snake envenomations could be handled Snake envenomation produces tissue effects such as local swelling using medications that act on presynaptic and postsynaptic receptors, and necrosis, neurotoxicity and hemostatic disorders. Considerable since some patients may only survive under intensive therapy treatment advancement has been made in understanding the pathophysiology of such as respiratory support23, specific treatment with antivenoms ophidic accidents, motivating transformations in treatment procedures. continues to be the elected method for treating these incidents, which can Latest advances, including the production of new antivenoms using new efficiently deactivate all systemic activities of the venom. Nevertheless, processes1-4,19, have encouraged developing coral snake antivenoms with there are some collateral effects of antivenom such as anaphylaxis and attractive protocols. serum sickness5. The majority of these alterations seem to be caused by the action of high concentrated proteins, which are not immunoglobulins, Nearly two hundred species from the Elapidae family are dispersed but contaminating polyvalent antivenoms. However, the benefits of across the Southeastern and Southwestern United States, as well as all of antivenom treatment may be more important than its risks. Mexico, Central America and South America, and are also established in the African, Asian and Oceanic continents3. On the American continent, The specific therapeutics for coral snake envenomations is the use more than 120 species and subspecies have been described, separated of heterologous antivenom, and to date, this type of antivenom is not into three genera: Leptomicrurus with three species, Micruroides, with available in Venezuela. In light of the information that coral snake venom one, and Micrurus, with approximately 70 species21,22. can reveal a multiplicity of composition and toxic activities, we have included the most important venom species occurring in Venezuela and The production of safe, efficient and reasonably priced antivenoms United States in the immunization protocol. At this time, we present a is a priority. Alternative progress in the therapeutics of coral snake study on the production of a specific coral snake (Micrurus) antivenom bite victims in Venezuela requires an answer to the production of new and its purification of immunoglobulins from the egg yolk of immunized animal models, logistical, financial, marketing, delivery and storage hens, with the purpose of providing a more efficient antivenom for difficulties related to the supply of antivenom. Furthermore, the norms therapeutic treatments.
(1) Immunochemistry Section, Tropical Medicine Institute, Universidad Central de Venezuela, Caracas, Venezuela. (2) National Natural Toxins Research Center and the Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas, U.S.A. (3) Sección de Ultraestructura Toxinológica, Instituto Anatómico, Universidad Central de Venezuela, Caracas, Venezuela. (4) Laboratorio de Fisiopatología, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela. §Authors contributed equally to this work. Correspondence to: Alexis Rodríguez-Acosta MD Ph.D. Apartado 47423, Caracas 1041, Venezuela. Tel: +58-416-3333361. E-mail: [email protected] AGUILAR, I.; SÁNCHEZ, E.E.; GIRÓN, M.E.; ESTRELLA, A.; GUERRERO, B. & RODRIGUEZ-ACOSTA, F.A. - Coral snake antivenom produced in chickens (Gallus domesticus). Rev. Inst. Med. Trop. Sao Paulo, 56(1): 61-6, 2014.
MATERIALS AND METHODS 0.8 mg/kg) was injected into the tail vein of 18-20 g female BALB/c mice. A comparable volume of PBS was injected as a negative control group. Ethical statement: All the experimental events concerning the use of live animals were carried out by specialized staff. The relevant Sodium dodecyl sulphate-polyacrylamide gel electrophoresis regulations of Venezuela as well as institutional guidelines, according (SDS-PAGE) of coral snake’s crude venom: Pools of different coral to protocols approved by the Institute of Anatomy of the Universidad snakes’ crude venom under non-reduced conditions were electrophoresed Central de Venezuela, and the norms obtained from the guidelines for using a MINIPROTEAN II (BioRad, USA) chamber. SDS-PAGE was the care and use of laboratory animals13 were followed. performed according to the Laemmli method (1970)14 using 15% gels. Wide range molecular weight markers (Bio-Rad) were run in parallel Coral snakes (Micrurus) venom: Venom from a collection (twenty and gels were stained with Comassie blue (National Diagnostic, USA). specimens) of different Micrurus species from Venezuela and the United States were used in the immunization protocol. The venom of Venezuelan Immunization: A pool was made with concentrations of venom, coral snakes consisted of (Micrurus isozonus (Calabozo, Guárico State), corresponding to the LD50 median. A sub-lethal dose was used for Micrurus isozonus (Caracas, D.C), Micrurus isozonus (La Boyera, immunizations. Four-month-old egg-laying hens, weighing ~1 kg were Miranda State), and Micrurus isozonus (Maracay, Aragua State) which maintained pathogen-free and immunized with the pool of coral snakes’ were supplied by the Serpentarium of the Tropical Medicine Institute of venom. Venom (0.24 mg/kg in 0.1 mL) was taken into an Eppendorf tube the Universidad Central de Venezuela, Caracas, Venezuela. The venom and then mixed with an equal volume of Freund’s complete adjuvant, from the U.S. consisted of Micrurus fulvius fulvius (Eastern, USA), whereas the second doses consisted of venom emulsified with Freund’s and Micrurus tener tener (Western, USA), purchased from the National incomplete adjuvant (GIBCO, USA). The third venom doses were mixed Natural Toxins Research Center, Texas A&M University-Kingsville, with a saline solution. All doses were administered subcutaneously, via Kingsville, Texas, USA. the deltoid region in four different places, alternating right and left every two weeks for eight weeks. One week after the last dose, the hens’ blood Fifteen days prior to venom extraction, the coral snakes were fed and was obtained for the detection of immunoglobulins that could recognize made to fast to guarantee enough venom in their glands. The venom was and precipitate the coral snake venom. collected through a 50-mL plastic centrifuge tube transversely cut and covered on the top with Parafilm® (Millipore Corp, USA). The snake Isolation of immunoglobulin: The modified method of SVENDSON was forced to bite the Parafilm. Venom was collected by glass capillaries et al. (1995)30 using the freeze and thaw principle to remove lipids was used. through the excretory conduit in the base of the fangs, centrifuged, and For a brief period, the egg yolk was diluted 10 times with distilled water, supernatants were placed in Eppendorf® (Eppendorf Int, USA) tubes and and the diluted egg yolk was frozen at -80 ºC overnight, and permitted to stored at -30 °C until use. Stock solutions were prepared in phosphate- thaw at an increased temperature rate of ~ 2 ºC (six min) by maintaining buffered saline (PBS) (10 Mm sodium phosphate containing 150 Mm it at 2-4 ºC. The egg yolk was then centrifuged at 18,000 g for 1h at 4 ºC NaCl, Ph 7.2) at 1.0 mg/mL. and the supernatant gathered was cleared by filtration on Whatman (Nº. 1) filter paper (Whatman, USA). To precipitate the immunoglobulins, the Mice: Female mice (INH strain) weighing 18-20 g were obtained filtered supernatant was then precipitated by 40% ammonium sulfate at 4 from the Instituto Nacional de Higiene “Rafael Rangel”, Caracas, °C. Afterwards, the pellet was re-suspended in 0.01M Tris-HCl (pH 8.0) Venezuela. The colony of mice was kept in plastic boxes (Tecniplast, to a volume equal to half of the supernatant. Following centrifugation for Italy) at six mice per cage, in a room maintained at 23 °C on a 12/12-hr 15 min at 13 000 g, the pellet was washed twice with 40% ammonium light/dark cycle. sulfate (ReAgent, UK). The solution was dialyzed carefully (three buffer changes, at least 150 times volume) against 10 mM phosphate buffer, pH Hens: Six egg-laying, red hens (Gallus domesticus) of Rhode 7.0, for 24 to 48h in a dialyzing tube with molecular cut off weight of 14 Island strain, approximately 16 weeks old, obtained from a poultry KDa to remove the ammonium sulfate5,19. farm from Lagunita town, Miranda State, Venezuela, were located in individual henhouses (Centenosuplidores C.A, Venezuela)(one hen per Antibody activity and purity were determined using a SDS-PAGE14, 18 henhouse) prior to the commencement of the production of eggs. Hens double gel diffusion test and a serum protection test (ED50), respectively. were maintained on 12/12-hr light/dark cycle at 23 °C with food and water ad libitum. Gel diffusion assay using specific IgY against coral snake venom: To show the specific IgY immunoglobulin activity, a 1% agarose (Sigma- Determination of protein concentration: Protein determination Aldrich Co. USA) double gel diffusion test was used18. The immunizing was established by the method of LOWRY et al. 15. pool of coral snake crude venom at 10 mg/mL was placed in the central well, while different coral snake venoms and PBS were placed in the Micrurus’ venom lethality: Lethality of crude venom was outer wells and incubated at 37 °C. determined by intravenous injections into mice and the LD50 value 31 calculated according to the Spearman-Karber method . The venom was Serum protection test (Median effective dose = ED50): For antivenom diluted in a phosphate-buffered saline solution (PBS). The endpoint of effectiveness, five groups of eight mice were challenged with a mixture of lethality of the mice was determined after 48h. All solutions during the varying concentrations of IgY antivenom containing three LD50 of venom. experiments were stored at 4 ºC and warmed to 37 ºC prior to being The antivenom/venom mixture was prepared at 0 °C and incubated for injected into mice. The lethal toxicity was determined in five groups 30 min at 37 °C prior to injection. Each mouse was injected with 0.2 mL containing five mice. A total of 0.2 mL of venom (dosages from 0.05 to of venom/antivenom mixture into the tail vein. The mice were observed
62 AGUILAR, I.; SÁNCHEZ, E.E.; GIRÓN, M.E.; ESTRELLA, A.; GUERRERO, B. & RODRIGUEZ-ACOSTA, F.A. - Coral snake antivenom produced in chickens (Gallus domesticus). Rev. Inst. Med. Trop. Sao Paulo, 56(1): 61-6, 2014.
for 48 h and the survival percentage and ED50 were calculated according Immunization: The LD50s for the venoms were carried out according to Spearman and Karber31. Saline controls and antivenom controls were to the SÁNCHEZ et al.23 method. A sub-lethal dose of the pool of venoms used. Neutralizing capability was expressed as the 50% effective dose made with concentrations corresponding to the LD50 median of 0.3 mg/ (the amount of antivenom that protects 50% of the population) (Table 2). kg, was used to immunize the hens (Table 1).
Specificity of coral snake antivenom (IgY) to various snake venom Sodium dodecyl sulphate-polyacrylamide gel electrophoresis via Western blot: To explore the specificity of the immunoglobulins (SDS-PAGE) of coral snake crude venom: The SDS-PAGE (15%) against coral snake venom, the antibodies were also assayed with Crotalus protein profiles of Micrurus isozonus venoms were analyzed. The and Bothrops venom. A total of nine venom (22 µg/each) consisting of individual venom differed in composition, quantity and intensity of bands M. isozonus, M. surinamensis, M. f. fulvius, M. t. tener, Naja kaouthia, (Fig. 1). The M. isozonus venom from Calabozo contained more protein Naja pallida, Bothrops colombiensis, Crotalus durissus cumanensis bands above 31.kDa (Fig. 1; lane 2) than all other M. isozonus venom. and C. vegrandis were electrophoresed on a 10-20% Tricine SDS gel The M. isozonus venom from Caracas and Maracay both contained a using a XCell SureLock™ system at 150V (Bio-Rad PowerPac Basic) similar protein band between 66.2 and 97.4 kDa (Fig. 1; lanes 3 and 5, for one hour. The proteins were transferred onto a 0.2 µm nitrocellulose respectively). The sample from La Boyera (Fig. 1; lane 4) contained all membrane (Millipore) using a Trans Blot SD system (BioRad) at 100 protein bands below 21.5 kDa. mA for one hour. The primary antibody (chicken anti-coral snake venom IgY) was diluted to 1/200, the secondary antibody (rabbit anti-chicken IgY-alkaline phosphatase) (Sigma-Aldrich Co. USA) was diluted to 1/50,000. SIGMA FAST™ BCIP/NBT tablets were used to visualize the bands on the blot and SimplyBlue (Life Technologies, USA) was used to visualize the bands on the gel. SeeBluePlus2 (Life Technologies, USA) markers were used as standards.
Antivenom: The antivenom passed the standard tests for neutralizing potency, miocrobiological purity, lack of pyrogenicity, appropriate protein concentration, lack of abnormal toxicity, sterility, and pH (6.9)11.
RESULTS
Lethality assay: The LD50 calculated as a median from the mixture of coral snake venom used for immunization was 0.58 mg/kg. The LD50 values were different among Micrurus venoms tested, with LD50 of 0.32 mg/kg (Micrurus fulvius), 0.5 mg/kg (Micrurus isozonus) and 0.78 mg/ kg (Micrurus tener) (Table 1). Fig. 1 - SDS-polyacrylamide gel electrophoresis (15%) of individual M. isozonus venoms Table 1 from Venezuela. A total of 20 µg of venom sample was applied to the gel. Lanes: 1) Markers;
LD50s of Venezuelan and United States coral snake venoms. Lethality of crude 2) M. isozonus (Calabozo); 3) M. isozonus (Caracas); 4) M. isozonus (La Boyera); 5) M.
venom was determined by intravenous injections into mice and the LD50 value isozonus (Maracay). The gel was stained with Comassie blue. calculated according to Spearman-Karber method31 Isolation of immunoglobulin: This method30 involved a gently a synchronized -70 ºC freeze and 4 ºC thaw cycle, consequently giving Species Pool LD50 ± SD a clear egg yolk solution. The total protein concentration in one egg La Boyera, Miranda M. isozonus 0.5 ± 0.012 yolk was around 1.3 ± 0.5 g. In this method, a larger component of the State (Venezuela) additional proteins was excluded through lipid elimination of egg yolk Caracas, Capital M. isozonus 0.5 ± 0.012 by the freeze and thaw cycle. IgY was reduced with β-Mercaptoethanol District (Venezuela) (Sigma-Aldrich Co. USA) showing a heavy chain fragment of 68 kDa Calabozo, Guárico and light chain of 27 kDa (Fig. 3). The material, after precipitation, M. isozonus 0.8 ± 0.016 State (Venezuela) contained about 90% pure IgY. Maracay, Aragua State M. isozonus 0.6 ± 0.02 (Venezuela) Gel diffusion assay using specific IgY against coral snake venom: The existence of antivenom IgY in egg yolk was tested by Ouchterlony’s Kingsville, Texas State M. tener tener 0.78 ± 0.14 immunodiffusion assay18 using crude venom as the antigen (Fig. 2). (United States) The protein concentration in the wells (1-12) was 1 mg/ mL, and 20 µL Tampa, Florida State M. fulvius 0.32 ± 0.12 was added to each well. Antivenom added to the middle well was (United States) 20 µL, containing a concentration of 1 mg/mL Lowry’s protein. Several a precipitin lines at each location pointed out the polyvalent character of The LD50 is the concentration of venom required to kill 50% of a mouse population after 48 h. Results are expressed in mg venom/kg body weight. Mean the antibodies. The antibody titer augmented after the booster dose, and
LD50 = 0.58(mg/kg). antibodies were present 180 days after the first injection.
63 AGUILAR, I.; SÁNCHEZ, E.E.; GIRÓN, M.E.; ESTRELLA, A.; GUERRERO, B. & RODRIGUEZ-ACOSTA, F.A. - Coral snake antivenom produced in chickens (Gallus domesticus). Rev. Inst. Med. Trop. Sao Paulo, 56(1): 61-6, 2014.
Fig. 3 - Gel diffusion assay using specific IgY against coral snake venom. 1. Micrurus isozonus (Calabozo, Guárico State); 2. PBS; 3. Micrurus isozonus (Caracas, D.C.); 4. Micrurus isozonus (La Boyera, Miranda State); 5. PBS; 6. Micrurus isozonus (Maracay, Aragua State); 7. Micrurus surinamensis (Amazonas State); 8. PBS; 9. Micrurus dissoleucus dissoleucus (Paraguaná, Fig. 2 - SDS-PAGE analysis (15% gel concentration, under reducing conditions) of IgY Falcón State); 10. Micrurus tener (Western, USA); 11 PBS; Micrurus fulvius (Eastern, USA). Antibodies after precipitation with ammonium sulfate and dialysis. 1) Molecular weight markers. 2) IgY. resulting from accidents with coral snakes, the research and investment of funds for the production of antivenoms is considered insufficient. On
Serum protection test (Median effective dose = ED50): Coral the other hand, biochemical studies relating to Micrurus venoms are snake antivenom (IgY) was able to effectively neutralize the pool of all limited, due to the complexity of accurately identifying the species, the Micrurus isozonus coral snake venom used in the immunization with a difficulties in maintaining them in captivity, and the difficulty in obtaining mean ED50 of 477.8 mg/kg (Table 2). a desirable amount of venom; in addition, venom varies between intra and interspecies in composition, associated with their age, gender, geographic Specificity of coral snake antivenom (IgY) to various snake venom distribution, and diet4,10. via Western blot: The coral snake antivenom was able to recognize, to some degree, all the coral snake venoms used in this study in addition to In this study, the competence of hens to make antibodies against two cobra snake venoms and three Venezuelan pit vipers. The antivenom small quantities of antigens was studied. In some conditions, there was most specific to M. f. fulvius followed by M. isozonus, M. t. tener and may be problems with getting adequate amounts of antigen to make M. surinamensis. The two Naja spp. had similar reactions to each other, immunizations. In this experiment the hens were immunized with 0.1- and the two Crotalus spp. also showed similarities. Bothrops colombiensis 100 μg of antigen with booster immunizations. Every two weeks for venom had a different reactivity pattern than the other Venezuelan venom. eight weeks, the IgY response in the yolk demonstrated a similar picture Micrurus surinamensis venom had the least reaction with the antivenom as that in the serum. The study confirmed that it is possible to obtain compared to all the venom tested. No protein bands below 12 kDa were a good immune response with less amounts of antigen than is usually detected for any venom. recommended to immunize sheep or horses. Studies by some groups have established that antibody responses to foreign antigens are genetically DISCUSSION controlled. It has been feasible to breed hens that are high and low antibody responders intramuscularly20 or by intravenous9,16 injections. Despite the significant pressure due to death and grave consequences
Table 2
Effective dose fifty (ED50=477 mg/kg/3 LD50) assay of yielded antibodies (IgY coral snake antivenom neutralizing lethal toxic activity of coral snake venom
Antivenom concentration Total protein of Number of mice Percentage of deaths (%) (mg/mL) antivenom (mg/mouse) Died Lived Total 86 17.2 0 8 8 0 43 8.6 4 4 8 50 21.5 4.3 8 0 8 100 10.7 21.5 8 0 8 100 5.3 5.3 8 0 8 100 a ED50 = 477 mg/kg
31 The ED50 was calculated according to Spearman and Karber :
ED50= the 50% effective dose. log X100 = log dose giving 100% survival and having 100% survival for all higher doses. log Fd = the log dilution factor. N = # mice used a at each dose level. T = #mice alive at each dose level. Σ = the sum of mice surviving at every dose level. The ED50 is the effective dose of IgY that will protect 50% of the mouse population when injected with 3LD50s.
64 AGUILAR, I.; SÁNCHEZ, E.E.; GIRÓN, M.E.; ESTRELLA, A.; GUERRERO, B. & RODRIGUEZ-ACOSTA, F.A. - Coral snake antivenom produced in chickens (Gallus domesticus). Rev. Inst. Med. Trop. Sao Paulo, 56(1): 61-6, 2014.
The production of IgY from small amounts of antigen required for immunization, the ease in collecting the eggs, and the uncomplicated purification techniques to enhance the function of immunological assays, make the use of IgY attractive and profitable.
Normally, antivenoms are achieved by immunizing horses with increasing doses of venom to obtain a high-quality antibody titer8. A range of side effects exists in the administration of antivenom; for instance, anaphylaxis and serum sickness27. The elevated concentrations of proteins, which are not antibodies, existing in many common antivenom probably produce most of these symptoms. With the aim of eliminating the unpleasant effects of the antivenom treatment, it is necessary to achieve antivenom immunoglobulins in a reasonable purification state. Investigators24,25,30 have proposed that birds are a suitable and economical supply of IgY immunoglobulins. Chicken egg yolks have IgY, which is a species-specific immunoglobulin with a molecular weight of 190kDa26. Our laboratory has produced hen antibodies against Scolopendra gigantea toxins with high titers19. Others have reported the production of immunoglobulins against different types of protein antigens, synthetic peptides, etc.29. We have improved the production of polyclonal antibodies by means of crude coral snake venom injected in hens with an alternative protocol, from that of the classical method used for horse immunoglobulin production. In our current work, we were capable of obtaining a good antibody titer with immunized hens.
Due to the high incidences of coral snake envenomations by M. isozonus in Venezuela, this particular snake venom was used to carry out the in vivo efficacy test of coral snake IgY antivenom, which resulted in ED50 of 477 mg/kg body weight (Table 2). This value falls within acceptable ED 50 Fig. 4 - Gel electrophoresis and Western blot analysis of various snake venoms with ranges of other antivenoms tested on different snake venoms7. A Western IgY antibodies. A total of nine venoms (22 µg/each) consisting of M. isozonus (Mi), M. blot was done to determine the specificity of the IgY antivenom to different surinamensis (Ms), M. f. fulvius (Mff), M. t. tener (Mtt), Naja kaouthia (Nk), Naja pallida coral snake venoms as well as other Venezuelan venomous species. It (Np), Bothrops colombiensis (Bs), Crotalus durissus cumanensis (Cdc) and C. vegrandis appears that the IgY coral snake venom antivenom was able to recognize (Cv) were electrophoresed on a 10-20% Tricine SDS gel using a XCell SureLock™ system all four coral snake venoms used in the immunization protocol with M. at 150V (Bio-Rad PowerPac Basic) for 1h. The proteins were transferred onto a 0.2 µm isozonus, M. surinamensis, M. f. fulvius and M. t. tener having the highest nitrocellulose membrane (Millipore) using a Trans Blot SD system (BioRad) at 100 mA for recognition. The venom of M. surinamensis was the least reactive (Fig. 1 h. SIGMA FAST™ BCIP/NBT tablets were used to visualize the bands on the blot and 4). In addition to those venoms used to produce the IgY antivenom, two SimplyBlue (Life Techonologies) was used to visualize the bands on the gel. SeeBluePlus2 cobra venoms (N. kaouthia and N. pallida), and three Venezuelan Viperidae (Life Techonologies) markers were used as standards. species (B. colombiensis, C. d. cumanensis and C. vegrandis) were also detected by the antivenom. This cross reactivity suggests there are similar la serpiente coral en huevos de gallinas inmunizadas. La técnica consistió venom toxins found in species of snakes located in distinct geographical en la eliminación de lípidos de las yemas del huevo, diluidas en agua y locations. The fact that M. surinamensis had the least reactivity of all en una secuencia de congelación-descongelación. Las inmunoglobulinas venoms, including those not used in the immunization protocol, indicates específicas estuvieron presentes en la yema de huevo hasta 180 días después the uniqueness of the proteins found in that venom. The possible use of IgY de la inmunización. La unión del antígeno a las inmunoglobulinas se detectó in therapy and/or diagnostic assays either in humans or animals provides mediante un ensayo de inmunodifusión doble. Los títulos de anticuerpos en an invaluable field of interesting and useful applications12,17,28. la yema fueron estimados con un ensayo de protección (dosis efectiva media = ED50). Dado que las gallinas reproductoras son económicamente viables, RESUMEN la recolección de huevos es no invasiva y la purificación de anticuerpos IgY es rápida y fácil, la inmunización de la gallina es una excelente alternativa Antiveneno de serpiente coral producido en gallinas (Gallus para la producción de anticuerpos policlonales. A nuestro entender, esta es domesticus) el primer anti-veneno contra serpiente de coral preparado en aves.
La producción de antiveneno de serpiente usando sangre de grandes ACKNOWLEDGEMENTS mamíferos se ha encontrado que es de bajo rendimiento y de trabajo arduo, en consecuencia, las inmunoglobulinas antiveneno para el tratamiento se Foundation Project: This research was supported by grants from obtienen generalmente, como suero polivalente. Hemos estandarizado una the FONACIT (Venezuela)(G-2005000400), Shell Venezuela S.A, técnica poco exigente para la fabricación de inmunoglobulina purificada Ministry of the Popular Power for Science and Technology (LOCTI IgY, que consistió en generar anticuerpos policlonales contra el veneno de program), CDCH/UCV PG: 09-8760-2013/1, and the Natural Toxins
65 AGUILAR, I.; SÁNCHEZ, E.E.; GIRÓN, M.E.; ESTRELLA, A.; GUERRERO, B. & RODRIGUEZ-ACOSTA, F.A. - Coral snake antivenom produced in chickens (Gallus domesticus). Rev. Inst. Med. Trop. Sao Paulo, 56(1): 61-6, 2014.
Research Center and Department of Chemistry at Texas A&M University- 13. Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. th Kingsville, Kingsville, Texas, USA. 8 ed. Washington: National Research Council, National Academies Press; 2011. 14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of AUTHOR CONTRIBUTIONS bacteriophage T4. Nature. 1970;227:680-5.
15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, Protein measurement with the Folin Irma Aguilar is a Ph.D student who worked on the immunization of phenol reagent. J Biol Chem. 1951;193:265-75. animals and purified the IgYs. She was involved in all the experiments 16. Martin A, Dunnington EA, Gross WB, Briles WE, Briles RW, Siegel PB. Production done. Elda E. Sánchez developed the LD50 and ED50 assays, the gel electrophoresis and Western blot of the various snake venoms with the traits and alloantigen systems in lines of chickens selected for high or low antibody responses to sheep erythrocytes. Poult Sci. 1990;69:871-8. IgG antibodies as well as collaborated with the writing of the manuscript. María E. Girón and Amalid Estrella are Research Assistants who did the 17. Meenatchisundaram S, Parameswari G, Michael A, Ramalingam S. Studies on general experiments. Belsy Guerrero carried out the electrophoresis and pharmacological effects of Russell’s viper and Saw-scaled viper venom and its neutralization by chicken egg yolk antibodies. Int Immunopharmacol. 2008;8:1067- Western blot as well as collaborated with the writing of the manuscript. 73. Alexis Rodriguez-Acosta designed and supervised the project and wrote the manuscript. He is the thesis tutor of MSc. Aguilar. 18. Ouchterlony O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1-78.
CONFLICT OF INTEREST STATEMENT 19. Parrilla P, Navarrete LF, Girón M.E, Aguilar I, Rodríguez-Acosta A. Use of chicken egg yolk-derived immunoglobulin against Scolopendra venom as an alternative to treat We declare that we have no conflict of interest. Authors transfer the scolopendrism. Rev Cient FCV-LUZ. 2008;18:385-92. copyright of the article to the publisher. 20. Pinard MH, van Arendonk JA, Nieuwland MG, van der Zijpp AJ. Divergent selection for immune responsiveness in chickens: estimation of realized heritability with an REFERENCES animal model. J Anim Sci. 1992;70:2986-93.
21. Prieto da Silva AR, Yamagushi IK, Morais JF, Higashi HG, Raw I, Ho PL, et al. Cross 1. Almeida CM, Kanashiro MM, Rangel Filho FB, Mata MF, Kipnis TL, da Silva WD. reactivity of different specific Micrurus antivenom sera with homologous and Development of snake antivenom antibodies in chickens and their purification from heterologous snake venoms. Toxicon. 2001;39:949-53. yolk. Vet Rec. 1998;143:579-84. 22. Roze JA. Coral snakes of the Americans. Biology, identification, and venoms. Florida: 2. Araújo AS, Lobato ZI, Chávez-Olórtegui C, Velarde DT. Brazilian IgY-Bothrops Krieger Publishing Co; 1996. antivenom: studies on the development of a process in chicken egg yolk.Toxicon. 2010; 55:739-44. 23. Sánchez EE, Lopez-Johnston JC, Rodríguez-Acosta A, Pérez JC. Neutralization of two North American coral snake venoms with United States and Mexican antivenoms. 3. Campbell JA, Lamar WW. The venomous reptiles of Latin America. Ithaca: Cornell Toxicon. 2008;51:297-303. University Press; 1989. 24. Sells PG, Laing GD, Theakston RD. An in vivo but insensate model for the evaluation 4. Chippaux JP, Williams V, White J. Snake venom variability methods of study, results and of antivenoms (ED50) using fertile hens’ eggs. Toxicon. 2001;39:665-8. interpretation. Toxicon. 1991;29:1279-303. 25. Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR. 5. Christensen PA. Production and standardization of antivenin. In: Lee CY, editor. Handbook Chicken egg yolk antibodies (IgY-technology): a review of progress in production and of experimental pharmacology. New York: Springer-Verlag; 1979. p. 1-825. use in research and human and veterinary medicine. Altern Lab Anim. 2005;33:129- 54. 6. Consroe P, Egen NB, Russell FE, Gerrish K, Smith DC, Sidki A, et al. Comparison of a new ovine antigen binding fragment (Fab) antivenin for United States Crotalidae 26. Schade R, Schniering A, Hlinak A. Polyclonal avian antibodies extracted from egg with the commercial antivenin for protection against venom-induced lethality in mice. yolk as an alternative to the production of antibodies in mammals: a review. ALTEX. Am J Trop Med Hyg. 1995;53:507-10. 1992; 9:43-56. 7. Devi CM, Bai MV, Lal AV, Umashankar PR, Krishnan LK. An improved method for 27. Schellekens H. How to predict and prevent the immunogenicity of therapeutic proteins. isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Biotechnol Annu Rev. 2008;14:191-202. Methods. 2002;51:129-38. 28. Spillner E, Braren I, Greunke K, Seismann H, Blank S, du Plessis D. Avian IgY 8. Estrada R, Chaves F, Robles A, Rojas E, Segura E, Gutiérrez JM. Valores hematológicos antibodies and their recombinant equivalents in research, diagnostics and therapy. y de enzimas séricas en caballos inoculados com venenos de serpientes para la Biologicals. 2012;40:313-22. producción de antivenenos en Costa Rica. Rev Biol Trop. 1992;40:95-9. 29. Stuart CA, Pietrzyk RA, Furlanetto RW, Green A. High affinity antibody from hen’s 9. Gross WG, Siegel PB, Hall RW, Domermuth CH, DuBoise RT. Production and persistence eggs directed against the human insulin receptor and the human IGF-I receptor. Anal of antibodies in chickens to sheep erythrocytes. 2. Resistance to infectious diseases. Biochem. 1988;173:142-50. Poult Sci. 1980;59:205-10. 30. Svendson L, Crowley A, Ostergaard LH, Stodulski G, Hau J. Development and comparison 10. Gutiérrez JM, Chaves F, Rojas R, Bolaños R. Efectos locales inducidos por el veneno de of purification strategies for chicken antibodies from egg yolk. Lab Anim Sci. la serpiente coral Micrurus nigrocinctus en Ratón Blanco. Toxicon. 1980;18:633-9. 1995;45:89-3. 11. Gutiérrez JM, Rojas E, Quesada L, Léon G, Núñez J, Laing GD, et al. Pan-African 31. World Health Organization. Progress in the characterization of venoms and standardization polyspecific antivenom produced by caprylic acid purification of horse IgG: an of antivenoms. Who Offset Publ. 1981;58:1-44. alternative to the antivenom crisis in Africa. Trans R Soc Trop Med Hyg. 2005;99:468- 75. Received: 9 April 2013 Accepted: 6 June 2013 12. Hernández-Campos FJ, Brito-De la Fuente E, Torrestiana-Sánchez B. Purification of egg yolk immunoglobulin (IgY) by ultrafiltration: effect of pH, ionic strength, and membrane properties. J Agric Food Chem. 2010;58:187-93.
66 Rev. Inst. Med. Trop. Sao Paulo 56(1):67-69, January-February, 2014 doi: 10.1590/S0036-46652014000100010
FIRST REPORT OF METALLO-β-LACTAMASES PRODUCING Enterobacter spp. STRAINS FROM VENEZUELA
Dianny MARTÍNEZ(1,2), Hectorina E. RODULFO(1), Lucy RODRÍGUEZ(2), Luisa E. CAÑA(2), Belkis MEDINA(2), Militza GUZMAN(3), Numirin CARREÑO(1), Daniel MARCANO(4) & Marcos DE DONATO(1)
SUMMARY
Clinical strains of Enterobacter were isolated from Cumana’s Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of
blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed
blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.
KEYWORDS: Carbapenemase; Metallobetalactamase; VIM; Enterobacter; Carbapenems.
INTRODUCTION classification of Enterobacter species tests for the fermentation of glucose and lactose in Kligler medium, use of citrate, arginine and malonate, use Species of the genus Enterobacter have been reported as an important of MIO, LIA, and methyl red and Voges-Proskauer medium according source of intrahospital infections, especially those showing resistance to to standard biochemical tests established for Enterobacteriaceae8,16. betalactams by the production of enzymes like extended spectrum beta- lactamases (ESBL) such as TEM, SHV, CTX, VEB, and carbapenemases Antimicrobial susceptibility was assessed by Kirby-Bauer disk such as VIM, KPC and GES14. This represents an important therapeutic diffusion susceptibility test following the recommendations of the challenge because of the few remaining treatment options, which gives Clinical and Laboratory Standards Institute (CLSI). For tigecycline rise to morbimortality and hospital expenses. (TGC), we used the cutoff of the attached insert (Pfizer, INC).
There are reports of Enterobacter strains producing metallo-b- Screening of extended spectrum beta-lactamases (ESBL), were lactamase (MBL) in different parts of the world, such as E. cloacae in carried out using the synergy effect between the antimicrobial disks Japan, Taiwan, Korea and Italy, as well as E. aerogenes in Japan and CAZ, FEP, CRO, ATM and CTX surrounding AMC as well as the France2. However, no MBL-producing strains of Enterobacter have been confirmatory test for ESBL was carried out using the combined disc test7. reported anywhere in the Americas, except in Mexico11 and Argentina4. Additionally, in order to detect the presence of ESBL enzymes we used the modification suggested by SONG et al. (2007), in order to avoid the METHODS masking effect that a derepressed AmpC gene could produce15. For this, disks containing CAZ (30 μg) with and without clavulanic acid (10 μg) During August 2010 and March 2011, clinical strains of Enterobacter were added 3-aminophenyl boronic acid with a final amount of 400 μg. were isolated in the University Hospital Antonio Patricio de Alcala in Cumana, Venezuela. The use of the strains was approved by the patients The phenotypic detection of MBLs was carried out using IPM or their relatives, according to the recommendations of the Bioethics and MER disks on each side of a disk with ethylendiaminotetracetic and Biosecurity Committee of IIBCA, Universidad de Oriente, Cumana, acid-sodium mercaptoacetate (EDTA-SMA, 0.5 µmoles-3 µg) and the Venezuela. combined disc test (IMP, IMP-EDTA and MER, MER-EDTA)6.
Isolated strains were inoculated in BHI broth, incubated for 12 hours DNA extraction was carried out using the Wizard Genomic DNA at 37 ºC and later in MacConkey agar for 24 hours, in order to evaluate kit (Promega) from the strains isolated after incubation in LB broth the morphological characteristics of the colonies to verify purity. For the for 20 hours at 37 ºC. The blaVIM gene was detected by PCR according
Part of the results in this manuscript was presented at the 60th annual meeting of the American Society of Tropical Medicine and Hygiene, held in Philadelphia, PA, USA, in December 4-8, 2011. (1) Lab. Genetica Molecular, IIBCAUDO, Universidad de Oriente, Cumana, Venezuela. (2) Lab. Bacteriologia, Hospital Universitario Antonio Patricio de Alcalá, Cumaná, Venezuela. (3) Lab. de Bacteriologia Molecular, Dpto. Bioanalisis, Universidad de Oriente, Cumaná, Venezuela. (4) Instituto Nacional de Higiene Dr Rafael Rangel, Caracas, Venezuela. Correspondence to: Hectorina Rodulfo, Av. Universidad, Cerro del medio, IIBCAUDO, Universidad de Oriente, Nucleo de Sucre, Cumaná, Venezuela. Tel.: +58-293-4175285. Fax:+58-293- 4521297. E-mail: [email protected] MARTÍNEZ, D.; RODULFO, H.E.; RODRÍGUEZ, L.; CAÑA, L.E.; MEDINA, B.; GUZMAN, M.; CARREÑO, N.; MARCANO, D. & DE DONATO, M. - First report of metallo-β-lactamases producing Enterobacter spp. strains from Venezuela. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 67-9, 2014.
10 to MENDEZ et al. . Additionally, we detected the type 1 and 2 blaVIM 3 according to FIETT et al. . The genes blaCTX-M (EDELSTEIN et al. unpublished results, http://www.antibiotic.ru/en/pdfs/006-51.pdf), blaTEM 12 and blaSHV were also detected. Finally, in order to determine the clonality of the three Enterobacter strains showing a phenotype consistent with MBL by the IMP/MER/EDTA synergy test, we used ERIC-PCR13.
RESULTS
The 33 strains of Enterobacter isolated were classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three were not possible to classify.
Ten of the strains were from infections acquired outside the hospital and most of the intrahospitalary strains were isolated in ICU (5), internal medicine areas A and B (4), soft surgery hall (4), pediatric area (3) and neonatology (3). Fig. 1 - Antimicrobial susceptibility of the Enterobacter strains isolated in the Cumana hospital of Venezuela. CAZ: ceftazidime, CRO: ceftriaxone, FEP: cefepime, ATM: aztreonam, Antimicrobial susceptibility tests show high levels of resistance IPM: imipenem, MER: meropenem, PIP: piperacillin, TZP: piperacillin/tazobactam, SXT: in most of the strains, with resistance to SXT (58.1%), CRO (48.8%), trimethoprim-sulfamethoxazole, GM: gentamicin, AK: amikacin, CIP: ciprofloxacin and CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%) being the TGC: tigecycline. highest (Fig. 1). However, all the strains were sensitive to TGC. Results of the synergy test between CAZ/FEP/CRO/CTX/ATM with AMC in Sequencing of the fragment of the blaVIM gene, amplified using
16 of the strains were compatible with ESBL enzymes. We found that primers for type 2, produced sequences 100% homologous to blaVIM-2 these strains showed the typical phenotypic effect for ESBL enzymes reported in the GenBank in P. aeruginosa and other bacteria. Also, the when using the combined disc test as a confirmatory. Additionally, three sequences of the blaTEM gene were 100% homologous to type 1 reported of the Enterobacter strains (two E. cloacae and one Enterobacter sp.) for many Enterobacteria. In addition, the fragment of the blaCTX-M gene were resistant to cabapenems showing also synergy between IPM/MER sequenced showed 100% homology with CTX-M-15 found in E. coli and EDTA, typical of MBL enzymes (Table 1). ERIC-PCR patterns and other Enterobacteria. show no similarities among these strains. They showed resistance to multiple families of antibiotics (MDRs) and two of them also showed DISCUSSION presence of ESBL by the synergy assay. These strains amplified for blaVIM-2 fragments (801 bp). Furthermore, both strains of E. cloacae According to our phenotypic tests, BLEA and ESBL-type of amplified the typical fragment of the blaTEM gene (1080 bp), but only enzymes was very prevalent. On the other hand, we are not aware one of them amplified the fragment of the blaCTX-M gene (543 bp), while of previous reports of the presence of VIM-producing Enterobacter no blaSHV gene was detected. strain in any South American country. In Mexico, strains of E. cloacae
Table 1
Resistance pattern and epidemiological data of the three strains of Enterobacter showing blaVIM type 2 MBLs
Species Isolation date Hospital area Type of sample Resistance pattern Synergy tests Detected genes
E. cloacae August 2010 Soft Surgery catheter CAZ, FEP, IPM, ESBL, blaVIM-2,
MER, TZP, PIP, MBL blaTEM-1,
CRO, ATM, SXT, blaCTX-M-15 AK, GM
Enterobacter sp. March 2011 ICU bronchial CAZ, FEP, IPM, AmpC, blaVIM-2 secretion MER, TZP, PIP, MBL CRO, ATM, SXT, CIP
E. cloacae April 2011 Internal urine CAZ, FEP, IPM, ESBL, blaVIM-2,
Medicine B MER, TZP, PIP, MBL blaTEM-1 CTX, ATM, SXT, CIP, AK ESBL: extended-spectrum betalactamase, MBL: metallo-b-lactamase, AmpC: de-repression of the chromosomal AmpC gene. ICU: intensive care unit. Acronyms of antibiotic as shown in the legend of Fig. 1.
68 MARTÍNEZ, D.; RODULFO, H.E.; RODRÍGUEZ, L.; CAÑA, L.E.; MEDINA, B.; GUZMAN, M.; CARREÑO, N.; MARCANO, D. & DE DONATO, M. - First report of metallo-β-lactamases producing Enterobacter spp. strains from Venezuela. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 67-9, 2014.
11 2. Falcone M, Mezzatesta ML, Perilli M, Forcella C, Giordano A, Cafiso V, et al. Infections have been shown to produce MBL . These strains produced blaVIM-2 MBLs, the same type we found in this study. In Argentina, one strain with VIM-1 metallo-beta-lactamase-producing Enterobacter cloacae and their correlation with clinical outcome. J Clin Microbiol. 2009;47:3514-9. of E. cloacae was reported containing blaIMP gene, along with blaPER 4 and genes that confer resistance to aminoglycosides and quinolones . 3. Fiett J, Baraniak A, Mrówka A, Fleischer M, Drulis-Kawa Z, Naumiuk Ł, et al. Molecular This type of gene has been reported in Venezuela but only in strains epidemiology of acquired-metallo-beta-lactamase-producing bacteria in Poland. of Pseudomonas aeruginosa and Klebsiella pneumoniae5,9. VIM- Antimicrob Agents Chemother. 2006;50:880-6. producing P. aeruginosa strains were previously found in Cumana 4. Gomez S, Rapoport M, Togneri A, Viegas-Caetano J, Faccone D, Corso A, et al. Emergence hospital (ENSONY TOVAR & MARCOS DE DONATO, unpublished of metallo-β-lactamases in Enterobacteriaceae from Argentina. Diagn Microbiol Infect results). It seems very likely that the gene found in P. aeruginosa could Dis. 2011;69:94-7. have been transferred through mobile elements such as plasmid and/ or integrons between these two species which are sharing the same 5. Guevara A, de Waard J, Araque M. Detección del gen blaVIM-2 en cepas de Pseudomonas environment, as previously reported14, making possible the spread aeruginosa productoras de metalo β-lactamasa aisladas en una unidad de cuidados intensivos en Ciudad Bolívar, Venezuela. Rev Chil Infect. 2009;26:336-41. of this gene to many other bacteria species causing infection in this hospital environment. 6. Lee K, Lim Y, Yong D, Yum J, Chong Y. Evaluation of the Hodge test and the imipenem- EDTA double-disk synergy test for differentiating metallo-ß-lactamase-producing The presence of multiple mechanisms of resistance in the bacteria isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol. 2003;41:4623- isolated in the Cumana hospital causing intrahospital infections, 9. especially in species of Enterobacter, which have natural resistance 7. Lezameta L, Gonzáles-Escalante E, Tamariz JH. Comparación de cuatro metodos to several antibacterial drugs, suggests that more efficient preventive fenotipicos para la detección de beta-lactamasas de espectro extendido. Rev Peru measures must be put in place in this hospital to avoid the survival and Med Exp Salud Publica. 2010;27:345-51. transmission of these strains. However, all the strains were susceptible to tigecycline, making it a suitable treatment for infections caused 8. MacFaddin JE. Biochemical tests for identification of medical bacteria. 3rd ed. Bloomington: Lippincott Williams & Wilkins; 2000. by MBL-producing enzymes in this hospital. This result agrees with numerous reports describing the use of tigecycline to treat infections 9. Marcano D, Pasterán F, Rapoport M, Faccone D, Ugarte C, Salgado N, et al. First isolation caused by multidrug resistant bacteria, including those producing of a VIM-producing Klebsiella pneumoniae from a seven-year-old child in Venezuela. carbapenemases14. J Infect Dev Ctries. 2008;2:241-4.
10. Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, et al. Rapid AUTHOR CONTRIBUTIONS detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol. 2007;45:544-7. DM, LR and LC isolated the strains. HER, DM and MDD carried out the molecular analysis, BM, MG and NC helped in the bacteriological 11. Morfin-Otero R, Rodriguez-Noriega E, Deshpande LM, Sader HS, Castanheira M. analysis. DM, HER and MDD wrote the manuscript and everyone Dissemination of a bla(VIM-2)-carrying integron among Enterobacteriaceae species in Mexico: report from the SENTRY Antimicrobial Surveillance Program. Microb reviewed the manuscript. Drug Resist. 2009;15:33-5.
RESUMEN 12. Quinteros M, Radice M, Gardella N, Rodriguez MM, Costa N, Korbenfeld D, et al. Extended-spectrum β-lactamases in Enterobacteriaceae in Buenos Aires, Argentina, Primer reporte de cepas de Enterobacter spp productoras de public hospitals. Antimicrob Agents Chemother. 2003;47:2864-7.
metalobetalactamasas de Venezuela 13. Reboli AC, Houston ED, Monteforte JS, Wood CA, Hamill RJ. Discrimination of epidemic and sporadic isolates of Acinetobacter baumannii by repetitive element PCR-mediated Cepas clínicas de Enterobacter fueron aisladas del Hospital central DNA fingerprinting. J Clin Microbiol. 1994;32:2635-40. de Cumaná en Venezuela, y se clasificaron como E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) y 3 sin clasificar. 14. Sader HS, Castanheira M, Mendes RE, Toleman M, Walsh TR, Jones RN. Dissemination and diversity of metallo-β-lactamases in Latin America: report from the SENTRY Las cepas mostraron altos niveles de resistencia, especialmente a SXT Antimicrobial Surveillance Program. Int J Antimicrob Agents. 2005;25:57-61. (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and
ATM (43.3%). Este es el primer reporte de América del Sur de blaVIM-2 15. Song W, Bae I, Lee YN, Lee CH, Lee SH, Jeong SH. Detection of extended-spectrum en dos cepas de E. cloacae y una de Enterobacter sp., las cuales también β-lactamases by using boronic acid as AmpC β-lactamase inhibitor in clinical isolates mostraron múltiples mecanismos de resistencia. Ambas especies de E. of Klebsiella spp. and Escherichia coli. J Clin Microbiol. 2007;45:1180-4. cloacae mostraron genes bla , pero solo una mostro el gen bla , TEM-1 CTX-M-15 16. Winn WC, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, et al. Koneman’s th mientras que blaSHV no fue detectado. color atlas and textbook of diagnostic microbiology. 6 ed. Bloomington: Lippincott Williams & Wilkins; 2005. REFERENCES Received: 13 December 2012 1. Betriu C, Rodríguez-Avial I, Gómez M, Culebras E, López F, Alvarez J, et al. Accepted: 29 May 2013 Antimicrobial activity of tygecicline against clinical isolates from Spanish medical centers. Second multicenter study. Diagn Microbiol Infect Dis. 2006;56:437-44.
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FAPESP/BIREME Project on Scientific Electronic Publications Latin American and Caribbean Center on Health Sciences Information Rua Botucatu 862 – 04023-901 São Paulo, SP – Brazil Tel. (011) 5576-9863 [email protected] Rev. Inst. Med. Trop. Sao Paulo 56(1):71-76, January-February, 2014 doi: 10.1590/S0036-46652014000100011
POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS
Icaro BOSZCZOWSKI(1), Gladys Villas Boas do PRADO(1), Mirian F. DALBEN(1), Roberto C. P. TELLES(2), Maristela Pinheiro FREIRE(1), Thaís GUIMARÃES(1), Maura S. OLIVEIRA(1), Juliana F. ROSA(2), Robson E. SOARES(2,3), Pedro Enrique Dorlhiac LLACER(4), Frederico Luiz DULLEY(5), Silvia F. COSTA(2) & Anna S. LEVIN(1,2)
SUMMARY
Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc) in bone marrow transplant and hematology outpatients. Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers’ (HCW) hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC) care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. Findings:14 patients had B. multivorans (one patient had also B. cenopacia), six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology); in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock- therapy. Eight patients (30%) were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication), no new cases occurred. Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A) of multi-dose vials.
KEYWORDS: Burkholderia cepacia complex; Bloodstream infection; Nosocomial infection; Hematology; Bone marrow transplant.
INTRODUCTION affiliated to the University of São Paulo, during the period from January 1 to February 29, 2008. The hospital is divided into six institutes and Burkholderia cepacia complex (Bcc) comprises at least 15 different transfer of patients between institutes occurs. The outbreak involved two species or genomovars based on phenotypic and genotypic analyses16,17. outpatient units: one for patients with hematological malignancies and Identification to species level has been troublesome in the clinical the other for bone marrow transplant patients. These units were adjacent laboratory. Automated methods seem to perform poorly in identifying and independent but shared one room which contained a laminar flow species of Bcc19,25. Molecular methods are more appropriate8,12,16. cabinet used to prepare chemotherapy and a counter used to prepare Clinically, Bcc mainly cenocepacia and multivorans species are important intravenous medication. In these units, the patients were admitted for colonizers and cause respiratory tract illness among cystic fibrosis a few hours at a time and received medication including intravenous patients1. In the hospital, the importance of Bcc is due to its potential therapy such as chemotherapy, antimicrobial drugs and hydration through to spread from person to person and to survive in moist environments. central venous catheters. Multiple healthcare-associated outbreaks have been described involving contaminated water11, prefabricated moist washcloths13, contaminated The outbreak of bacteremia caused by B. cepacia was suspected on medication14, nebulization solution15, antiseptic solution9, heparin24, February 15, 2008 and an investigation was triggered. A case was defined as moisturizing body milk2, mouthwash solution1. We report here an a patient with B. cepacia bacteremia occurring in January or February, 2008 outbreak involving different species of Bcc and the dissemination of a who received care or medication prepared in these units. An evaluation of all predominant clone suggesting heavy contamination by a common source. positive blood cultures in patients hospitalized in the entire hospital during these two months and during the previous 12 months was also undertaken. METHODS Visits to both affected outpatient units were made for evaluation.
The outbreak occurred in Hospital das Clínicas, a 2000-bed hospital The hospital records of all case patients were evaluated and the
(1) Infection Control Department, Hospital das Clinicas, Universidade de São Paulo, SP, Brazil. (2) Department of Infectious Diseases and LIM-54, University of São Paulo, São Paulo, SP, Brazil. (3) Pontifícia Universidade Católica de São Paulo, Brazil. (4) Hematology Unit, Hospital das Clínicas, University of São Paulo, São Paulo, SP, Brazil. (5) Chief of Bone Marrow Transplant Unit, Hospital das Clínicas, University of São Paulo, São Paulo, SP, Brazil. Correspondence to: Anna S. Levin, Rua Banibas 618, 05460-010 São Paulo, SP, Brasil. Phone/fax: 55.11.2661-7066. E-mail: [email protected] BOSZCZOWSKI, I.; PRADO, G.V.B.; DALBEN, M.F.; TELLES, R.C.P.; FREIRE, M.P.; GUIMARÃES, T.; OLIVEIRA, M.S.; ROSA, J.F.; SOARES, R.E.; LLACER, P.E.D.; DULLEY, F.L.; COSTA, S.F. & LEVIN, A.S. - Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 71-6, 2014. following factors were recorded: sex; age; underlying diseases; the cepacia selective agar) and then inoculated in a 600,000 UI/L presence and the type of central venous catheter (CVC), insertion date, polymyxin B, vancomycin 2.5 mg/L and gentamicin 10 mg/L BHI type of dressing used (gauze or transparent), type of antiseptic used broth (brain heart infusion). They were incubated at 10-32 °C up to during CVC handling; the use of IV medications, including heparin, and seven days. Growing colonies were then submitted to Gram staining. of blood products; the use of chemotherapy; dates of the last three uses Glucose fermenting was tested by OF (oxidation-fermentation) of the CVC before the first positive culture and the healthcare workers open and closed tubes and oxidase tests. After non-fermenting who had cared for each patient. Cases were treated with meropenem. confirmation, biochemical tests for B. cepacia complex identification were performed: OF-glucose, OF-maltose, OF-xylose, OF-lactose, Infection Control Measures: Two visits on different days and OF-sucrose and/or lysine decarboxylation; meetings with all the staff of the affected units were held in order to - Water samples were collected (1 L of both cold and hot water) in evaluate practices in infection control and managing of infusion therapy. sterile containers. Samples were divided into two aliquots of 500 The main findings were the following: mL each and filtered in an acetate cellulose membrane with 0.25 µm - Multi-dose heparin, methylprednisolone and saline solution for pores and then plated on BCSA and MacConkey agar. intravenous infusion were routinely used. The containers and vials were not left with needles inserted in them but it was not clear how The hospital’s clinical microbiology laboratory used an automatized many patients used each vial nor was there a control of when they system for identification (Vitek, bioMerrieux). Reidentification of B. should be discarded. These multi-use items were placed on the shared cepacia isolates was performed using the phenotypic method API ® 20 counter; NE (bioMérieux SA, Lyon, France). The isolates that were phenotypically - Nurses frequently prepared prescribed medications in the morning defined as belonging to the B. cepacia complex were submitted to for use during the whole day in order to “save time”; molecular identification using multiplex polymerase chain reaction - Syringes with aspirated solution without identification (patient name, with the primers and technique as described elsewhere12. The fur gene type of solution or time of preparation) were placed on the counter was amplified for various strains using primers JD490 and JD491 and and used for flushing catheters during the day; the DNA sequences were determined by direct sequencing from the - There was no exclusive sink inside the room used for the preparation amplicons using the MegaBACE 1000 DNA Sequencer. The sequences of infusion therapy; were analyzed using the software Sequence Analyzer with the Base Caller - There were empty vials of alcohol-based solution for hand hygiene; Cimarron 3.12 and compared with those in GenBank (www.ncbi.nlm. - There was no evidence of systematic cleaning of the laminar flow nih.gov). This method can identify the following species: B. cepacia, B. cabinet and the refrigerator used for medication storage as well as multivorans, B. cenocepacia, B. dolosa, B. vietnamiensis, B. ambifaria, controls of refrigerator temperature; B. stabilis, B. antina, Bcc group K and B. pyrrocinia. Isolates were typed - There was no evidence of systematic cleaning of surfaces with a using pulsed-field gel electrophoresis (PFGE)18 using the restriction disinfectant product. enzyme SpeI and interpreted according to criteria by TENOVER et al.20 . Minimum inhibitory concentrations (MIC) for ceftazidime, meropenem, Based on these findings, the following control measures were minocycline, levofloxacin and sulfamethoxazole-trimethoprim were implemented: determined and interpreted using the agar microdilution technique4. - Heparin, methylprednisolone and hydrocortisone vials were defined for a single patient and each vial received a label with the patient’s RESULTS name; - Syringes with medication were left in the laminar flow cabinet until Based on the case definition, 27 patients were identified but we could the moment of administration to patient and medication other than only retrieve clinical information pertaining to 24 due to failure to retrieve chemotherapy was to be prepared immediately before administration; complete records for three patients. Revision of the infection control - Batches of antiseptic chlorhexidine (soap and alcohol-based) were database could not identify any cases of bacteremia by B. cepacia from replaced; January through December 2007 in our hospital (Fig. 1). - Cleaning of laminar flow cabinet and control of temperature of refrigerators were reinforced; The main symptoms of cases were fever, present in 21 (88%) patients, - Cleaning of the areas in which medication was prepared with a chills in 14 (58%), coughing in two and hypotension and septic shock in chlorine-based product was reinforced; one patient each (Table 1). Ten patients had their CVC used on February - Hand hygiene was emphasized and the number of alcohol rub 11 and/or 12 and, in nine of these patients, this had been their last CVC dispensers in the units was increased; use before the positive blood culture. None of the HCWs could be directly - Blood cultures of all patients with a CVC that had been handled in implicated as the source of the outbreak, especially because each HCW the affected units during the outbreak period were collected; only worked in one of the affected units (Fig. 2). - Lock with ceftazidime for all long-term CVCs was instituted; - Active surveillance for new cases was implemented. Two patients were hospitalized in Hematology inpatient unit when they acquired the Burkholderia infection. However, they were included Microbiologic study: The following items were cultured between in the outbreak because they received chemotherapy prepared in the February 21 and 28: outpatient unit. Fifteen patients (56%) had their catheters removed, on - Swabs from taps, sink drains, counters, laminar flow cabinet and average 17.8 days after the positive blood culture (range: 1-70, median: healthcare workers hands (nurses, pharmacists) and other patients in 14). Eight patients (30%) were hospitalized, and one patient died within direct contact with cases. Swabs were plated on BCSA (Burkholderia thirty days after the positive culture of causes not related to the infection.
72 BOSZCZOWSKI, I.; PRADO, G.V.B.; DALBEN, M.F.; TELLES, R.C.P.; FREIRE, M.P.; GUIMARÃES, T.; OLIVEIRA, M.S.; ROSA, J.F.; SOARES, R.E.; LLACER, P.E.D.; DULLEY, F.L.; COSTA, S.F. & LEVIN, A.S. - Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 71-6, 2014.
Table 1 Characteristics of 24 patients involved in an outbreak of bloodstream infections caused by Burkholderia cepacia in two outpatient units (Hematology and Bone Marrow Transplant). Hospital das Clínicas, University of São Paulo, Brazil. January-February, 2008
Characteristics Male sex 10 (42%) Age (years) Mean (SD) 34 (16.4) Median (range) 30 (2-64) Outpatient unit Hematology 13 (54%) Bone marrow transplant 11 (46%) Underlying disease Fig. 1 - Distribution of cases over time and molecular types during an outbreak of bloodstream Hodgkin’s lymphoma 8 (33%) infections caused by Burkholderia cepacia in two outpatient units (Hematology and Bone Non-Hodgkin’s lymphoma 7 (29%) Marrow Transplant). Hospital das Clínicas, University of São Paulo, Brazil. 2007 to March, Aplastic anemia 3 (13%) 2008. (Only the first isolate from each patient is depicted). Chronic myelocytic leukemia 2 (8%) Myelofibrosis 1 Acute myelocytic leukemia 1 Acute lymphocytic leukemia 1 Primitive neuroectodermal tumor 1 Type of CVC Totally implanted 13 (54%) Partially implanted 11 (46%) Type of dressing used on CVC site Transparent film 17 (71%) Gauze 7 (29%) Use of chlorhexidine at CVC site 24 (100%) Under chemotherapy 8 (33%) Products administered through CVC Heparin 24 (100%) Dexamethasone 7 (29%) Fig. 2 - Number of case patients cared for by each healthcare worker during an outbreak of Teicoplanin 7 (29%) bloodstream infections caused by Burkholderia cepacia in two outpatient units. (Hematology Ondansetron 6 (25%) Unit: black bars; Bone Marrow Transplant Unit: grey bars). Hospital das Clínicas, University Cefepime 5 (21%) of São Paulo, Brazil. January-February, 2008. Dipirone 5 (21%) Meropenem 5 (21%) Filgrastim 4 (17%) After the implementation of the control measures no new cases Ceftriaxone 3 (13%) occurred. Platelet concentrate 3 (13%) Fluconazole 3 (13%) None of the environmental cultures or HCW hand swabs was positive Levofloxacin 3 (13%) for Burkholderia cepacia complex. Acyclovir 2 Red blood cell concentrate 2 Twenty-three isolates from 21 patients were available for molecular Dimenidrate 2 identification and typing and for determination of MICs. 14 isolates were CVC was used on February 11 and/or 12, 2008 10 (42%) identified as B. multivorans and six belonged to the B. cepacia complex Days between CVC insertion and positive blood culture1 but could not be identified to the species level. One isolate did not belong Mean (SD) 276.5 (257) to the B. cepacia complex, and could not be completely identified. The Median (range) 189 (16-853) distribution of molecular types based on PFGE can be seen in Figure 1. 2 PFGE types were named A through F. The predominant type, called A Days between last use of CVC and positive blood culture and identified as B. multivorans, was only present starting on February 12, Mean (SD) 6.4 (6.6) Median (range) 6 (1-32) 2008 and all infections occurred in patients belonging to the Hematology Unit. PFGE type D was B. cenocepacia and type E was B. multivorans. SD: standard deviation; CVC: central venous catheter; 1 data available for 20 Isolates PFGE types B and C were the B. cepacia complex ones with patients; 2 data available for 21 patients.
73 BOSZCZOWSKI, I.; PRADO, G.V.B.; DALBEN, M.F.; TELLES, R.C.P.; FREIRE, M.P.; GUIMARÃES, T.; OLIVEIRA, M.S.; ROSA, J.F.; SOARES, R.E.; LLACER, P.E.D.; DULLEY, F.L.; COSTA, S.F. & LEVIN, A.S. - Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 71-6, 2014. species level not identified. They only occurred during the last three days reported. In an outbreak of CVC associated bloodstream infection by of the outbreak. The isolate presenting PFGE type F was the one that did B. cepacia and Myroides odoratus, commercial ampoules of sterile not belong to the B. cepacia complex. water intrinsically contaminated were found to be the common source7. The contamination of Ringer lactate solution used as multiple-dose Two patients presented more than one isolate. One patient had two vial to flush peripheral venous catheters was the cause of an outbreak different species, initially B. multivorans (PFGE type E), and then B. of bloodstream infections by clonal B. cepacia6. The contamination of cenocepacia 25 days after the first episode (PFGE type D). The other a heparin flush solution used for catheters in an oncology unit caused patient had two isolates with different PFGE types (B and E) that were a B. cepacia (formerly Pseudomonas cepacia) outbreak already back identified from cultures collected five days apart. in 199121. In a systematic review of HAI related to contaminated substances23, Bcc ranked first as contaminating pathogen in substances
MIC50 was 4 µg/mL; 1; 2; 2 and 1 µg/mL for ceftazidime, other than blood. levofloxacin; minocycline, meropenem and sulfamethoxazole/ trimethoprim, respectively. MIC90 was 8 µg/mL; 16; 8; 4; and 8 µg/mL The contamination of multi-dose medication does not fully respectively for the same antimicrobial drugs. explain the outbreak. Although most outbreaks of Bcc described have been clonal6,10,11, an outbreak caused by contaminated bromopride in DISCUSSION 2006 was shown to be polyclonal14. Bcc are bacteria largely present in the natural environment, such as soil, water and rhizosphere, Our study describes an outbreak of Burkholderia cepacia complex which is the environment adjacent to the roots of plants22, in which bloodstream infections in Hematologic and Bone Marrow Transplant different species may co-exist. Most species belonging to Bcc have outpatient units with multiple clones and species. No other cases of Bcc also been shown to produce biofilm5 which may be important in the infections were diagnosed in the entire hospital during the outbreak pathogenesis of human disease. Patients with cystic fibrosis have been period or in the previous year, fitting the definition of outbreak and described to carry different species of Bcc3. Thus it is possible that strongly pointing to a localized problem in the two affected outpatient in the hospital, environmental contamination by Bcc be polyclonal, units. Although evidence points to a common source, this could not thus causing polyclonal infections. It is possible that a contaminated be completely proven and the fact that there are multiple clones and environmental source, such as the laminar flow cabinet, was involved species makes interpretation difficult. During the entire outbreak in the outbreak. This cabinet was the only common feature between the period, inadequate practices in preparation and storage of intravenous two units and inadequate maintenance of the cabinets was observed. It medication probably favored contamination leading to bloodstream is possible that biofilm formed within the cabinet contained multiple infections. This hypothesis is reinforced by the fact that two case patients species of Burkholderia which would explain the two cases with B. were hospitalized and their only epidemiologic link to the other cases multivorans found in BMT patients that did not belong to the same was that they had received medication prepared in the outpatient area. clone, B. cenocepacia and the cases caused by unidentified species No healthcare worker could be directly implicated as the source of the of Burkholderia. Unfortunately, this is only a hypothesis as cultures outbreak especially because, although the unit shared areas, each worker obtained from the cabinet were negative. Measures to improve cabinet belonged strictly to one of the units and there was no sharing of healthcare maintenance and cleaning were implemented among others to control workers between the units. the outbreak.
We hypothesized that two distinct events took place. There seems Identification to the species level of Burkholderia cepacia poses to clearly have been a contamination by B. multivorans (molecular a problem to the clinical laboratory because phenotypical tests are type A) and probably the source was the use of intravenous drugs not reliable to differentiate between species within Bcc. Reference or solutions prepared from multi-dose vials in the Hematology unit. laboratories are usually needed for identification that depends on Although we could not definitely demonstrate a common source of B. molecular techniques. Thus, managing clinical and epidemiological multivorans, the case distribution and the fact that ten patients had had issues, such as outbreaks, at the point of care is a problem. New methods their CVC manipulated on February 11 and/or 12 makes it probable that allow the search for species-specific biomarkers such as matrix- that contaminated solution was administered. As there were no cases in assisted laser desorption ionization-time of flight mass spectrometry other units of the hospital, contamination during industrial production (MALDI-TOF MS) are promising for Bcc16. Molecular typing such as is improbable and it must have occurred during manipulation of the PFGE, used in our outbreak, matched the results of species identification. multi-dose vials. Solutions under suspicion were saline used for central However typing was useless to explain the outbreak. In fact it could even catheter flushing and heparin. All twelve patients who presented with have been misleading as finding multiple clones may have weakened the type A-B. multivorans in blood cultures had received heparin through hypothesis of an outbreak. their catheters and it is not possible to ascertain whether they received saline from a multi-dose vial as this was not routinely registered in the A limitation of our study was that medications from multi-dose vials patients’ records. Saline flushing of the CVC was a common practice in were not available for culturing and that the laminar flow cabinet was the units. Among the recommendations on good practices in intravenous not entirely dismantled for culturing. therapy, multi-dose vials should be avoided and intravenous solutions should not be stored for long periods of time after preparation. To our In summary, the outbreak of bloodstream infections caused by knowledge there have not been reports of outbreaks of B. multivorans Burkholderia spp. was interrupted by reinforcing good practices, probably due to the contamination of multi-dose vials of intravenous medication eliminating the contamination of multi-dose vials and the common source. although reports of such contamination by other agents have been The finding of different species was an original feature of this outbreak.
74 BOSZCZOWSKI, I.; PRADO, G.V.B.; DALBEN, M.F.; TELLES, R.C.P.; FREIRE, M.P.; GUIMARÃES, T.; OLIVEIRA, M.S.; ROSA, J.F.; SOARES, R.E.; LLACER, P.E.D.; DULLEY, F.L.; COSTA, S.F. & LEVIN, A.S. - Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 71-6, 2014.
RESUMO 6. De Smet B, Veng C, Kruy L, Kham C, van Griensven J, Peeters C, et al. Outbreak of Burkholderia cepacia bloodstream infections traced to the use of Ringer lactate solution as multiple-dose vial for catheter flushing, Phnom Penh, Cambodia. Clin Surto policlonal de infecção de corrente sanguínea causada pelo Microbiol Infect. 2013;19:832-7. complexo Burkholderia cepacia em unidades de hospital-dia de hematologia e transplante de medula óssea 7. Douce RW, Zurita J, Sanchez O, Cardenas Aldaz P. Investigation of an outbreak of central venous catheter-associated bloodstream infection due to contaminated water. Infect O objetivo foi descrever um surto de infecções da corrente sanguínea Control Hosp Epidemiol. 2008;29:364-6. por complexo B. cepacia (Bcc) nos ambulatórios de hematologia e 8. Drevinek P, Vosahlikova S, Dedeckova K, Cinek O, Mahenthiralingam E. Direct culture- transplante de medula óssea. Métodos: Em 15/02/2008, um surto de Bcc independent strain typing of Burkholderia cepacia complex in sputum samples from foi suspeitado. 24 casos foram identificados. Os dados demográficos e patients with cystic fibrosis. J Clin Microbiol. 2010;48:1888-91. clínicos foram avaliados. Mãos de profissionais da saúde e ambiente foram cultivadas. Espécies foram determinadas e tipadas. Reforço 9. Heo ST, Kim SJ, Jeong YG, Bae IG, Jin JS, Lee JC. Hospital outbreak of Burkholderia stabilis bacteremia related to contaminated chlorhexidine in hematological da higiene das mãos, cuidados com cateteres, terapia de infusão e malignancy patients with indwelling catheters. J Hosp Infect. 2008;70:241-5. manutenção da câmara de fluxo laminar foram realizadas. 16 profissionais de saúde (PS) diferentes manipularam os cateteres. Heparina multidoses 10. Lo Cascio G, Bonora MG, Zorzi A, Mortani E, Tessitore N, Loschiavo C, et al. A napkin- e soro eram preparadas em um balcão comum a ambas as unidades. associated outbreak of Burkholderia cenocepacia bacteraemia in haemodialysis Resultados: 14 pacientes tiveram B. multivorans (um paciente teve patients. J Hosp Infect. 2006;64:56-62. também B. cenopacia), 6 Bcc não-multivorans e um teve um agente não 11. Lucero CA, Cohen AL, Trevino I, Rupp AH, Harris M, Forkan-Kelly S, et al. Outbreak of pertencente a Bcc. Clone A de B. multivorans ocorreu em 12 pacientes Burkholderia cepacia complex among ventilated pediatric patients linked to hospital (da Hematologia), em 10 o cateter havia sido utilizado nos dias 11 ou sinks. Am J Infect Control. 2011;39:775-8. 12 de fevereiro. Culturas ambientais e de PS foram negativos. Todos os pacientes foram tratados com meropenem e selo de ceftazidima. Oito 12. Lynch KH, Dennis JJ. Development of a species-specific fur gene-based method for identification of the Burkholderia cepacia complex. J Clin Microbiol. 2008;46:447-55. pacientes (30%) foram hospitalizados. Não ocorreram mortes. Após as medidas de controle, nenhum novo caso ocorreu. Conclusões: Este surto 13. Martin M, Christiansen B, Caspari G, Hogardt M, von Thomsen AJ, Ott E, et al. policlonal pode ser explicado por uma fonte comum contendo várias Hospital-wide outbreak of Burkholderia contaminans caused by prefabricated moist espécies de Bcc, talvez a câmara de fluxo laminar comum a ambas as washcloths. J Hosp Infect. 2011;77:267-70. unidades. Pode ter havido contaminação por B. multivorans (clone A) 14. Martins IS, Pellegrino FL, Freitas A, Santos M da S, Ferraiuoli GI, Vasques MR, et de frascos multi-dose. al. Case-crossover study of Burkholderia cepacia complex bloodstream infection associated with contaminated intravenous bromopride. Infect Control Hosp FUNDING Epidemiol. 2010;31:516-21.
This study was funded by Hospital das Clínicas, University of São 15. Memish ZA, Stephens G, Balkhy HH, Cunningham G, Francis C, Poff G. Outbreak of Burkholderia cepacia bacteremia in immunocompetent children caused Paulo, Brazil. RCPT received a student grant from Fundação de Auxílio by contaminated nebulized salbutamol in Saudi Arabia. Am J Infect Control. à Pesquisa do Estado de São Paulo (FAPESP 2010/09714-8) 2009;37:431-2.
CONFLICTS OF INTEREST 16. Mott T, Soler M, Grigsby S, Medley R, Whitlock GC. Identification of potential diagnostic markers among Burkholderia cenocepacia and B. multivorans supernatants. J Clin Microbiol. 2010;48:4186-92. The authors declare no conflicts of interest involving this study. 17. Pegues DA, Carson LA, Anderson RL, Norgard MJ, Argent TA, Jarvis WR, et al. REFERENCES Outbreak of Pseudomonas cepacia bacteremia in oncology patients. Clin Infect Dis. 1993;16:407-11. 1. Abe K, D’Angelo MT, Sunenshine R, Noble-Wang J, Cope J, Jensen B, et al. Outbreak of Burkholderia cepacia bloodstream infection at an outpatient hematology and 18. Pfaller MA, Hollis RJ, Sader HS. Molecular biology – PFGE analysis of chromosomal oncology practice. Infect Control Hosp Epidemiol. 2007;28:1311-3. restriction fragments. In: Isenberg HD, editor. Clinical microbiology procedures handbook. Washington: ASM Press; 1992. p. 10.5.c.1-10.5.c.11. 2. Alvarez-Lerma F, Maull E, Terradas R, Segura C, Planells I, Coll P, et al. Moisturizing body milk as a reservoir of Burkholderia cepacia: outbreak of nosocomial infection 19. Snyder JW, Munier GK, Johnson CL. Direct comparison of the BD Phoenix system with in a multidisciplinary intensive care unit. Crit Care. 2008;12:R10. the MicroScanWalkAway system for identification and antimicrobial susceptibility testing of Enterobacteriaceae and nonfermentative Gram-negative organisms. J Clin 3. Carvalho GMV, Carvalho APD, Folescu TW, Higa L, Teixeira LM, Plotkowski MCM, et Microbiol. 2008;46:2327-33. al. Transient isolation of Burkholderia multivorans and Burkholderia cenocepacia from a Brazilian cystic fibrosis patient chronically colonized with Burkholderia 20. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. vietnamiensis. J Cystic Fibrosis. 2005;4:267-70. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strains typing. J Clin Microbiol. 1995;33:2233-9. 4. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: nineteenth informational supplement. CLSI document M100 21. Vanlaere E, Baldwin A, Gevers D, Henry D, De Brandt E, LiPuma JJ, et al. Taxon K, - S20. Wayne: Clinical and Laboratory Standards Institute; 2010. a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov. Int J Syst 5. Conway BA, Venu V, Speert DP. Biofilm formation and acyl homoserine lactone Evol Microbiol. 2009;59:102-11. production in the Burkholderia cepacia complex. J Bacteriol. 2002;184:5678-85.
75 BOSZCZOWSKI, I.; PRADO, G.V.B.; DALBEN, M.F.; TELLES, R.C.P.; FREIRE, M.P.; GUIMARÃES, T.; OLIVEIRA, M.S.; ROSA, J.F.; SOARES, R.E.; LLACER, P.E.D.; DULLEY, F.L.; COSTA, S.F. & LEVIN, A.S. - Polyclonal outbreak of bloodstream infections caused by Burkholderia cepacia complex in Hematology and Bone Marrow Transplant outpatient units. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 71-6, 2014.
22. Vial L, Chapalain A, Groleau M-C, Déziel E. The various lifestyles of the Burkholderia 25. Zbinden A, Böttger EC, Bosshard PP, Zbinden R. Evaluation of the colorimetric VITEK cepacia complex species: a tribute to adaptation. Environm Microbiol. 2011;13:1-12. 2 card for identification of Gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing. J Clin Microbiol. 2007;45:2270-3. 23. Vonberg RP, Gastmeier P. Hospital-acquired infections related to contaminated substances. J Hosp Infect. 2007;65:15-23. Received: 4 March 2013 Accepted: 23 May 2013 24. Yang CJ, Chen TC, Liao LF, Ma L, Wang CS, Lu PL, et al. Nosocomial outbreak of two strains of Burkholderia cepacia caused by contaminated heparin. J Hosp Infect. 2008;69:398-400.
76 Rev. Inst. Med. Trop. Sao Paulo 56(1):77-80, January-February, 2014 doi: 10.1590/S0036-46652014000100012
Enterococcus faecium AND Enterococcus faecalis IN BLOOD OF NEWBORNS WITH SUSPECTED NOSOCOMIAL INFECTION
Isabela FURTADO(1), Paula Cristhina Niz XAVIER(1), Luciana Venhofen Martinelli TAVARES(2), Fabiana ALVES(3), Sarah Fonseca MARTINS(3), Almir de Sousa MARTINS(3) & Durval Batista PALHARES(4)
SUMMARY
Enterococci are Gram-positive cocci saprophyte of the human gastrointestinal tract, diners who act as opportunistic pathogens. They can cause infections in patients hospitalized for a long time or who have received multiple antibiotic therapy. Enterococcus faecalis and Enterococcus faecium are the most common species in human infections. To evaluate the possibility of rapid detection of these species and their occurrence in the blood of newborns with suspected nosocomial infection, blood samples were collected from 50 newborns with late infections, admitted to the Neonatal Care Unit of the University Hospital Federal de Mato Grosso do Sul (UFMS- HU), from September 2010 to January 2011. The samples were subjected to conventional PCR and real time PCR (qPCR) to search for Enterococcus faecium and Enterococcus faecalis, respectively. The PCR results were compared with respective blood cultures from 40 patients. No blood cultures were positive for Enterococci, however, eight blood samples were identified as genomic DNA of Enterococcus faecium by qPCR and 22 blood samples were detected as genomic DNA of Enterococcus faecalis by conventional PCR. These findings are important because of the clinical severity of the evaluated patients who were found positive by conventional PCR and not through routine microbiological methods.
KEYWORDS: Enterococcus faecium; Enterococcus faecalis; Prematurity; PCR.
INTRODUCTION bacteremia caused by Enterococcus faecalis were documented, 83.2% with nosocomial origin, 85.3% associated with invasive procedures, Neonatal sepsis is the most frequent nosocomial infection in neonatal 9.5% in neonates and 41.1% that had previously received broad spectrum intensive care units and its incidence is increasing due to the increased antibiotics. In Brazil, a study by TITZE-DE-ALMEIDA et al. 200419, survival of infants with very low birth weights13,15. The greatest risk factors with results of phenotypic and molecular analysis showed rates of around for late onset sepsis in neonates include low birth weight, prematurity, 95% in both Enterococcus faecalis and Enterococcus faecium. long periods of hospitalization and invasive procedures3. The late sepsis usually manifests as septicemia and pneumonia, occurring 72 hours Blood culture is the gold standard for diagnosis of sepsis, but has low after birth20, caused by pathogens found in the nosocomial environment, sensitivity and the results are available in no less than 48 to 72 hours9. The including enterococci among the main etiological agents3,11. PCR methods are shown to be fast and specific8,14, but when we compare qPCR with conventional PCR, it is known that even though it consists of The genus Enterococcus is comprised of gram-positive cocci in pairs a more stringent and sensitive technique, in practice it demands higher or short chains6,9, considered saprophytes of the human gastrointestinal costs than conventional PCR. tract. They can survive on inanimate objects such as thermometers and stethoscopes, as well as on the hands of health professionals for long Thus, the objective of this study was to identify through the periods18. technique of qPCR and conventional PCR, respectively, the presence of Enterococcus faecium and Enterococcus faecalis in the blood of Symptoms of neonatal infection are not specific, even for the different newborns, suspected of infection and admitted to the Intensive Care Unit. agents, with a need for more sensitive and specific microbiological and molecular tests10. MATERIAL AND METHODS
In a study in Spain by FERNANDEZ et al., 20042, 95 episodes of The study included infants who were suspected of nosocomial
(1) Program Graduate Health and Development in the Midwest Region, Federal University of Mato Grosso do Sul, Campo Grande, Brazil. (2) Department of Physical Therapy, Dom Bosco Catholic University and the Federal University of Mato Grosso do Sul, Campo Grande, Brazil. (3) Department of Physiology and Biofísica-ICB/UFMG, Belo Horizonte, Brazil. (4) Department of Pediatrics, University Hospital, Federal University of Mato Grosso do Sul, Campo Grande, Brazil. Correspondence to: Paula Cristina Niz Xavier. Av. Senador Filinto Muller, Vila Ipiranga. Laboratório de Pesquisas Pediátricas. Departamento de Pediatria. 79080-190 Campo Grande, Mato Grosso do Sul, MS, Brasil. Phone: +55-67-3345-3221. E-mail: [email protected]. FURTADO, I.; XAVIER, P.C.N.; TAVARES, L.V.M.; ALVES, F.; MARTINS, S.F.; MARTINS, A.S. & PALHARES, D.B. - Enterococcus faecium and Enterococcus faecalis in blood of newborns with suspected nosocomial infection. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 77-80, 2014.
infection, admitted to the Neonatal Intensive Care Unit, University The amplified fragments were photographed after electrophoresis Hospital, Federal University of Mato Grosso do Sul, with at least 72 hours on 6% of poliacrilamide gel (Ludwig Biotec) stained with silver nitrate. of admission or those with clinical worsening or a change of antibiotics, ATCC 19433 was used as a positive control for Enterococcus faecalis, in a five month follow up, from September 2010 to January 2011. autoclaved ultrapure water was used as a negative control.
This research was initiated only after approval by the Ethics The comparison between the groups of newborns evaluated in Committee on Human Research of the Federal University of Mato Grosso relation to blood PCR was performed using the nonparametric chi-square do Sul (Protocol 1520). contingency table with 2x2 cells and with a value greater than five. The other results of the variables evaluated in this study were presented as The peripheral blood samples were collected with all accuracy descriptive statistics or graphs. Statistical analysis was performed using aseptically as usually taken for routine under medical request. Samples the SigmaStat version 2.0, whereas there were significant differences for blood cultures were sent to the Central Laboratory of the HU-UFMS, for values of p < 0.0516. where they were processed and analyzed by the automated system, BACTTECTM FX (BD, New Jersey, USA) and the results compared with RESULTS those obtained by conventional PCR. We evaluated 50 newborns of both genus; the mean gestational age The DNA extraction was performed using the GE Health Care was 31 weeks, and the mean birth weight was equal to 1.745 kg. extraction Kit and the blood samples of 50 infants, identifying Enterococcus faecium by qPCR and Enterococcus faecalis, by With respect to invasive procedures, all newborns in which we conventional PCR. detected the presence of genetic material of bacteria studied were subjected to some type of vascular catheter. The use of mechanical For qPCR reaction, the reagents kit from: SYBR Green PCR ventilation and parenteral nutrition represent important features among Biosystems (Applied Biosystems, Warrington, UK) was used. these patients. Designed oligonucleotide primers selected within the PBP5 (penicillin-binding protein) genome region were FAEFOR-1 = 5’-ggt Of the 50 patients studied, 40 had blood culture and none of the acaacccgattacttcgtcccat-3’ and FAEFOR-2 = 5’-ggtacaacccgattactttgt cultures observed Enterococcus faecalis or Enterococcus faecium. For cccat-3’; FAEREV-1 = 5’–tctgccgtctacttcttggatggt-3’ and FAEREV-2 the other ten blood cultures, the results were not included in the medical = 5’-tctgccgtctacttcttgaatggt-3’, to obtain a specific target fragment of record. 94 bp. The results showed 16% (n = 8) positive for Enterococcus faecium The thermocycling conditions for qPCR were those standardized in in peripheral blood of the newborn evaluated by qPCR (Fig. 1) and 44% the ABI Prism 7000 unit (52 °C / 2 min, 95 °C / 10 min, 45 cycles of: 95 (n = 22) were positive for Enterococcu faecalis, by simple conventional to 60 °C / 15 ° C / 15 s) with a final dissociation curve for each sample. PCR (Fig. 2). Among all the infants evaluated, 12% (n = 6) were positive Primers (1.5 pmol each), DNA (50-100 ng) and Syber Green PCR Master for both types of bacteria, Enterococcus faecalis being more frequent Mix (Applied Biosystems, Warrington, UK) were combined and qPCR (p = 0.004). carried out under conditions recommended by the manufacturer. All positive and negative tests had controls included. A human s26 normalizer DISCUSSION was used as internal control. The cumulative CT values (cycle threshold) were collected in qPCR for each positive sample amplified in triplicate. Prematurity and low birth weights are described as neonatal factors relevant to the development of late onset sepsis, since it is the main Standard conventional PCR was used for the detection of cause of admission of neonates in intensive care units4. We also observed Enterococcus faecalis12. The specific oligonucleotides primers prematurity (46%) as an important characteristic of this population. designed and selected to amplify an amplicon of 87 bp, within the target region EntC2 (enterocin peptide precursor) of the genome of In this study, among the blood samples in which at least one of the Enterococcus faecalis, were FAECFOR (5’-gcaattactggtggaggacctgg-3’) microorganisms described here was detected, the use of central venous and FAECREV (5’-tccaattccttttgcaagacctgc-3’). Primers were catheters has been the procedure with the highest correlation with the designed based on sequences selected in the Genbank (program presence of bacteria (54%), followed by mechanical ventilation (32%) NCBI-BLASTn). and parenteral nutrition (32%).
PCR consisted of: 9.75 μL of water sterile filtered, 1.5 µL 10x PCR This data agrees with HERRMANN et al.4, who in 2008 also found buffer, 1.2 μL of dNTP mix (200 mM each), 0.4 μL of 50 mM MgCl2, 0.5 that the use of invasive procedures such as venous catheters, parenteral µL of each primer (10 pmol / microl), 0.15 μL (5 U / microl) of Taq DNA nutrition and mechanical ventilation had an important relationship with polymerase (Ludwig Biotec, Alvorada, Brazil) and 1 μL of genomic DNA the development of late neonatal sepsis. (200 ng) in a total volume of 15 μL reaction. The reaction was carried out in a MJ Research PTC 100 Thermal Cycler with the following program: Some practices may be adopted as mitigation measures for late sepsis. 95 °C / 5 min, followed by 05 cycles of 94 °C / 1 min, 56 °C / 1 min, Knowing that the use of vascular catheters and mechanical ventilation are 72 °C / 1 min, followed by 40 cycles: 92 °C / 1 min, 60 °C / 1 min, closely related to the presence of infection, time of use must be reduced 72 °C / 1 min, and final extension at 72 ° C / 4 min. with safe and hygienic measures in the handling of equipment, and proper
78 FURTADO, I.; XAVIER, P.C.N.; TAVARES, L.V.M.; ALVES, F.; MARTINS, S.F.; MARTINS, A.S. & PALHARES, D.B. - Enterococcus faecium and Enterococcus faecalis in blood of newborns with suspected nosocomial infection. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 77-80, 2014.
Fig. 1 - Samples positive for Enterococcus faecium by Real-Time PCR.
Fig. 2 - Conventional PCR for Enterococcus faecalis present in blood of newborns with suspected nosocomial infection. (M) 50 bp ladder marker (1µg); (C+) positive control for E. faecalis, ATCC 19433. (C–) negative control; 22 positive samples = (11,13,14,16,17,18,20,21,25,28,29,30,31,32,35,38,40,42,43,44,46,50). hand washing are important measures in preventing infection nosocomial4. are distinguished by the rapidity and specificity of their results, which are essential for the prevention and control of transmission of infection, The use of parenteral nutrition has been associated with late infection being all PCR based methods. and delaying the start of enteral feeding. Other authors suggest that the enteral route should be initiated as early as possible to reduce the time of Further studies are needed to reveal in more detail the late onset of parenteral nutrition, which is associated with a high risk of infection4,7,11. sepsis and its risk factors among newborns. Even though the presence of genomic DNA by conventional PCR and qPCR does not necessarily For genomic detection of bacteria Enterococcus faecium and mean sepsis, possibly just colonization, adding to the patients symptoms Enterococcus faecalis in the blood of newborns with risk factors for and clinical observations, especially prematurity conditions, together infection, we used qPCR and conventional PCR, respectively, for being lead the physician to take proper action or medical procedures in favor rapid and specific methods. We also compared these results with blood of children’s treatment, avoiding deadly sepsis. High prematurity, culture. associated with Enterococcus faecium and Enterococcus faecalis in the blood of newborns, reinforces the usage of conventional PCR and qPCR There was no positive result for the bacteria studied by means of as additional tools for clinics. blood cultures. Other studies have reported lower sensitivity of blood cultures1,5,21, despite being the gold standard for diagnosis of sepsis, is CONCLUSIONS a time consuming technique, whose results are ready in no less time than 48 to 72 hours and several authors have questioned its credibility5. The present finding is important due to the clinical severity of the evaluated patients who were positive by methods PCR and were not Several methods for the molecular detection of Enterococcus faecium detected in routine microbiological methods. These methods were more and Enterococcus faecalis have been reported as a powerful tool in the effective compared to blood cultures that did not show any positive case investigation of outbreaks of nosocomial infections12. Such techniques for enterococci.
79 FURTADO, I.; XAVIER, P.C.N.; TAVARES, L.V.M.; ALVES, F.; MARTINS, S.F.; MARTINS, A.S. & PALHARES, D.B. - Enterococcus faecium and Enterococcus faecalis in blood of newborns with suspected nosocomial infection. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 77-80, 2014.
RESUMO 6. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC Jr, Cury AE. Diagnóstico microbiológico: texto e atlas colorido. 5. ed. Rio de Janeiro: MEDSI; 2001. p. 589-659. Enterococcus faecium e Enterococcus faecalis no sangue de recém- nascidos com suspeita de infecção nosocomial 7. Leal YA, Álvarez-Nemegyei J, Velázquez JR, Rosado-Quiab U, Diego-Rodríguez N, Paz-Baeza E, et al. Risk factors and prognosis for neonatal sepsis in southeastern Os enterococos são cocos Gram-positivos saprófitas do trato Mexico: analysis of a four-year historic cohort follow-up. BMC Pregnancy Childbirth. gastrointestinal humano, atuam como patógenos oportunistas. Podem 2012;12:48. causar infecções em pacientes: hospitalizados por um longo tempo 8. Miglioli AMD. DNA genômico de Estreptococos e Escherichia coli em sangue e aspirado ou que receberam antibioticoterapia múltipla. Enterococcus faecalis traqueal e gástrico de recém-nascidos intubados imediatamente após o nascimento e Enterococcus faecium são as espécies mais comuns em infecções [tese]. Campo Grande: Universidade Federal do Mato Grosso do Sul; 2009. humanas. Para avaliar a possibilidade de detecção rápida dessas espécies e sua ocorrência no sangue de recém-nascidos com suspeita de infecção 9. Murray PR, Resenthal KS, Kobayashi GS, Pfaller MA. Microbiologia médica. 4. ed. Rio de Janeiro: Guanabara Koogan; 2004. p. 220-3. hospitalar, foram coletadas amostras de sangue de 50 recém-nascidos, com infecção tardia, internados na Unidade de Terapia Neonatal do 10. Nourse C, Byrne C, Murphy H, Kaufmann ME, Clarke A, Butler K. Eradication of Hospital Universitário da Universidade Federal de Mato Grosso do Sul vancomycin resistant Enterococcus faecium from a paediatric oncology unit and (UFMS-HU), no período de setembro de 2010 a janeiro de 2011. As prevalence of colonization in hospitalized and community-based children. Epidemiol amostras foram submetidas a PCR convencional e PCR em tempo real Infect. 2000;124:53-9.
(qPCR) para pesquisa de Enterococcus faecium e Enterococcus faecalis, 11. Opilla M. Epidemiology of bloodstream infection associated with parenteral nutrition. respectivamente. Os resultados da PCR foram comparados com culturas Am J Infect Control. 2008;36:S173-8. de sangue respectivos de 40. Nenhuma hemocultura foi positiva para enterococos, no entanto, em oito amostras de sangue foi identificado 12. Sader HS, Pignatari AC, Hollis RJ, Jones RN. Evaluation of interhospital spread of DNA genômico de Enterococcus faecium através da técnica de reação methicillin-resistant Staphylococcus aureus in São Paulo, Brazil, using pulsed field gel electrophoresis of chromosomal DNA. Infect Control Hosp Epidemiol. em cadeia da polimerase em tempo real, e em 22 amostras de sangue, 1994;15:320‑3. foram detectados DNA genômico de Enterococcus faecalis, através de PCR convencional. A descoberta é importante por causa da gravidade 13. Sader HS, Sampaio JLM, Zocolli C, Jones RN. Results of 1997 SENTRY antimicrobial clínica dos pacientes avaliados que foram positivos por PCR convencional surveillance program in three Brazilian medical centers. Braz J Infect Dis. e não foram detectados na rotina por métodos microbiológicos. 1999;3:63‑79.
14. Saiki RK,Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer directed ACKNOWLEDGEMENTS enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487-91. To the team of NICU HU-UFMS for openness and collaboration in the blood samples and provided data and access to research patient’s records. 15. Schrag SJ, Cutland CL, Zell ER, Kuwanda L, Buchmann EJ, Velaphi SC, et al. Risk factors for neonatal sepsis and perinatal death among infants enrolled in the prevention of perinatal sepsis trial, Soweto, South Africa. Pediatr Infect Dis J. 2012;31:821-6. FINANCIAL SUPPORT 16. Shott S. Statistics for health professionals. London: W.B. Saunders Company; 1990. This research was supported by the National Council for Scientific and Technological Development (CNPq) and Foundation for Development 17. Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Late-onset sepsis in very low birth weight neonates: a report from the National Institute of Child Health Support of Education, Science and Technology (FUNDECT). There was and Human Development Neonatal Research Network. J Pediatr. 1996;129:63-71. no conflict of interest between the authors of this research. 18. Teixeira LM, Facklam RR. Enterococcus. In: Murray PR, Baron EJ, Jorgensen JH, REFERENCES Pfaller MA, Yolken RH, editors. Manual of clinical microbiology. 8. ed. Washington: American Society for Microbiology; 2003. p. 422-33. 1. Brozanski BS, Jones JG, Krohn MJ, Jordan JA. Use of polymerase chain reaction as a diagnostic tool for neonatal sepsis can result in a decrease in use of antibiotics and 19. Titze-de-Almeida R, Rollo Filho M, Nogueira CA, Rodrigues IP, Eudes Filho J, total neonatal intensive care unit length of stay. J Perinatol. 2006;26:688-92. Nascimento RS, et al. Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units. Braz J Infect Dis. 2. Fernández Fernández FJ, de la Fuente Aguado J, Rubianes González M, Pérez Fernández 2004;8:197-205. S, Alvarez Férnandez M, Nodar Germiñas A, et al. Enterococcus faecalis bacteremia. Rev Clin Esp. 2004;204:244-50. 20. Vergnano S, Sharland M, Kazembe P, Mwansambo C, Health PT. Neonatal sepsis: an international perspective. Arch Dis Child Fetal Neonatal Ed. 2005;90:220-4. 3. Haque KN. Neonatal sepsis in the very low birth weigth preterm infants: part 1. Review of patho-physiology. J Med Sci. 2010;3:1-10. 21. Yadav AK, Wilson CG, Prasad PL, Menon PK. Polymerase chain reaction in rapid diagnosis of neonatal sepsis. Indian Pediatr. 2005;42:681-5. 4. Herrmann DMML, Amaral LMB, Almeida SC. Fatores de risco para o desenvolvimento de sepse neonatal tardia em uma unidade de terapia intensiva. Pediatria(São Paulo). Received: 25 January 2013 2008;30:228-36. Accepted: 6 June 2013
5. Honest H, Sharma S, Khan KS. Rapid tests for group B. Streptococcus colonization in laboring women: a systematic review. Pediatrics. 2006;117:1055-66.
80 Rev. Inst. Med. Trop. Sao Paulo 56(1):81-84, January-February, 2014 doi: 10.1590/S0036-46652014000100013
FIRST CASE OF AUTOCHTHONOUS HUMAN VISCERAL LEISHMANIASIS IN THE URBAN CENTER OF RIO DE JANEIRO: CASE REPORT
Guilherme Almeida Rosa da SILVA(1), Thiago de Oliveira BOECHAT(2), Fernando Raphael de Almeida FERRY(1), Jorge Francisco da Cunha PINTO(1), Marcelo Costa Velho Mendes de AZEVEDO(1), Ricardo de Souza CARVALHO(1), Rogerio Neves MOTTA(1) & Mariana Ferreira VERAS(3)
SUMMARY
Visceral leishmaniasis is an anthropozoonosis that is caused by protozoa of the genus Leishmania, especially Leishmania (Leishmania) infantum, and is transmitted to humans by the bite of sandflies of the genus Lutzomyia, such as Lutzomyia longipalpis. There are many reservoirs, including Canis familiaris. It is a chronic infectious disease with systemic involvement that is characterized by three phases: the initial period, the state period and the final period. The main symptoms are fever, malnutrition, hepatosplenomegaly, and pancytopenia. This article reports a case of a patient diagnosed with visceral leishmaniasis in the final period following autochthonous transmission in the urban area of Rio de Janeiro. The case reported here is considered by the Municipal Civil Defense and Health Surveillance of Rio de Janeiro to be the first instance of autochthonous visceral leishmaniasis in humans in the urban area of this city. The patient was discharged and is undergoing a follow-up at the outpatient clinic, demonstrating clinical improvement.
KEYWORDS: Visceral leishmaniasis; Transmission; Epidemiology.
INTRODUCTION Visceral leishmaniasis is a chronic infectious disease with systemic involvement that is characterized by three phases: the initial period, Visceral leishmaniasis is an anthropozoonosis that is caused by the state period and the final period. The incubation period can range protozoa of the genus Leishmania, especially Leishmania (Leishmania) from 10 days to 24 months, with only a small proportion of infected infantum, and is transmitted to humans by the bite of sandflies of the genus individuals manifesting the disease. The initial period includes fever, Lutzomyia, such as Lutzomyia longipalpis. There are many reservoirs, pallor and hepatosplenomegaly, which are occasionally accompanied by including Canis familiaris in urban areas, foxes and marsupials in rural coughing and diarrhea. Certain cases may be oligosymptomatic. The state areas. period is characterized by intermittent fever, weight loss and increased hepatosplenomegaly, as well as the deterioration of the individual’s The first case of visceral leishmaniasis in Brazil was recorded in 1913 general condition. The final period of disease development occurs when in Porto Esperança, Mato Grosso do Sul. Afterward, in the 1930s, a major the individual experiences severe malnutrition, pancytopenia, jaundice investigation in the northeast part of the country confirmed 41 cases of and ascites. Death is typically the result of opportunistic infections and visceral leishmaniasis during autopsies of suspected cases of yellow fever. bleeding2. The following years were characterized by new cases emerging in rural areas in more than ten states. After the 1940s, with Vargas government’s This article reports a case of a patient diagnosed with visceral investment in the urbanization of the country, cases were also recorded in leishmaniasis in the final period following autochthonous transmission areas of rural-urban transition. L. longipalpis seems to be well adapted in the urban area of Rio de Janeiro. The case reported here is considered to peridomiciles, but the many factors that influence this organism’s by the Municipal Civil Defense and Health Surveillance of Rio de Janeiro sporadic presence in urban areas are still poorly understood. However, to be the first instance of autochthonous visceral leishmaniasis in humans the transmission of visceral leishmaniasis in areas close to major urban in the urban area of this city. centers in Brazil has been reported1,4. In 1979, the first autochthonous case in Rio de Janeiro was reported by SALAZAR and confirmed by SOUZA CASE REPORT in 1981, with the demonstration of the presence of the L. longipalpis and enzootic cases of visceral leishmaniasis in dogs at this region11. Since The patient was female, 29 years old and single; born and living in the then, there are about 87 cases of autochthonous visceral leishmaniasis municipality of Rio de Janeiro; resident of Cajú neighborhood, working as confirmed in peri-urban areas in the municipality of Rio de Janeiro7. a cleaner in the cemetery of Cajú, RJ. She denied any trips outside of the
(1) UNIRIO, Professor of Internal Medicine, Rio de Janeiro, RJ, Brazil. E-mails: [email protected]; [email protected]; [email protected]; [email protected]; ricardocarvalho@ gmail.com; [email protected] (2) HEMORIO, Resident physician, Hematology, Rio de Janeiro, RJ, Brazil. E-mail: [email protected] (3) UNIRIO, Student of Medicine, Rio de Janeiro, RJ, Brazil. E-mail: [email protected] Correspondence to: Guilherme Almeida Rosa da Silva, Hospital Universitário Gaffrée e Guinle/Universidade Federal do Estado do Rio de Janeiro, 10ª Enfermaria, R. Mariz e Barros 775, Tijuca, 20550-110 Rio de Janeiro, Brasil. Phone: +55.21.8186-8189. SILVA, G.A.R.; BOECHAT, T.O.; FERRY, F.R.A.; PINTO, J.F.C.; AZEVEDO, M.C.V.M.; CARVALHO, R.S.; MOTTA, R.N. & VERAS, M.F. - First case of autochthonous human visceral leishmaniasis in the urban center of Rio de Janeiro: case report. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 81-4, 2014.
city limits of Rio de Janeiro throughout her life. Five months before the hospitalization, the patient experienced an intermittent night fever of 39 ºC that was not improved by antipyretics and was accompanied by poor appetite, postprandial fullness, nausea and vomiting episodes. There was no history of liver disease or alcohol abuse. Between the months from October to December 2012, she gradually noticed yellow sclera and skin, dark urine, and weight loss of approximately 10 kg, increased abdominal size and a soft and cold edema of the lower limbs up to the knees. The patient sought treatment in emergency units on several occasions but only received symptomatic prescriptions. In January 2013, she was admitted for diagnostic investigation to the 10th ward of the Gaffrée and Guinle University Hospital, UNIRIO.
A physical examination revealed poor general health, malnutrition, numbness, pallor +3/+4, dehydrated +1/+4, jaundice +2/+4, cyanosis, Fig. 1 - Abdominal examination revealing hepatomegaly and splenomegaly. 98 x 73 mm fever, tachycardia and eupnea. An abdominal examination revealed (150 x 150 DPI). moderate ascites, hepatomegaly that was 7 cm from the right costal margin and splenomegaly that was palpable 2 cm below the navel (Fig. 1). count showing severe pancytopenia, an extended prothrombin time, a Additionally, the lower limbs showed bilateral soft edema up to the knees. significant elevation of the aminotransferase levels, hypoalbuminemia and hypergammaglobulinemia accompanied by severe jaundice with a Laboratory examinations (Table 1) revealed a complete blood cholestatic pattern. A total abdominal ultrasound evaluation revealed
Table 1 Laboratorial exams during internation
Parameter References 04/01/13 10/01/13 21/01/13 06/02/13 20/02/13 Hemoglobin (11.0-18.8) 6.44 5.40 8.37 11.5 11.4 Ht (35.0-55.0) 19.8% 15.9% 18.3% 36.7% 36.9% MCV (80.0-100) 76.8 77.5 85.0 86.5 84.6 MCH (26.0-34.0) 25.0 26.3 26.1 27.2 26.1 MCHC (31.0-35.0) 32.5 34.0 30.6 31.4 30.8 RDW (11.0-15.0) 20.3% 19.8% 18.3% 16.6% 15.7% WBC (4000-11000) 435 548 3210 5250 4360 Platelets (150000-400000) 30100 46400 98400 172000 106000 HSV (< 15) 28 - - 10 - INR (1.00) 3.07 1.379 - 1.584 - TTP(Rel) (1.00) Incoag 1.80 - 1.05 - Creatinine (0.7-1.3) 1.02 2.58 1.03 1.23 1.45 Urea (10-50) 48 75 36 45 55 AST (< 38) 1557 137 111 42 18 ALT (< 41) 329 154 82 37 24 Alcalin Fosfatase (65-300) 136 545 1280 532 429 GGT (11-50) 34 284 694 486 467 Direct Bilirubin (< 0.3) 9.39 5.26 2.02 1.21 0.73 Indirect Bilirubin (< 0.8) 5.23 2.04 0.94 0.76 0.39 Albumin (3.5-4.8) 1.3 2.3 3.5 3.8 3.4 Globulin (1.4-3.2) 4.7 3.9 3.8 3.8 3.3 K+ (3.6-5.5) 4.0 2.95 2.68 3.00 2.34 Na+ (134-149) 138 148 146 153 145 Cl- (94-112) 105 113 102 106 103 Ca++ (8.5-10.5) 6.5 (8.7) 6.7 (9.1) 8.0 (8.4) 8.9 9.5 (9.9) Mg++ (1.7-2.5) 1.7 1.7 1.5 1.5 1.5 P (2.5-4.5) 3.1 3.1 2.5 4.1 3.7
82 SILVA, G.A.R.; BOECHAT, T.O.; FERRY, F.R.A.; PINTO, J.F.C.; AZEVEDO, M.C.V.M.; CARVALHO, R.S.; MOTTA, R.N. & VERAS, M.F. - First case of autochthonous human visceral leishmaniasis in the urban center of Rio de Janeiro: case report. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 81-4, 2014.
the presence of free fluid in the peritoneal cavity, hepatomegaly of patient was discharged in February 2013 and is undergoing a follow up approximately 20 cm, splenomegaly of 25 cm and gallstones. at the outpatient clinic, demonstrating clinical improvement.
The diagnostic hypotheses contemplated disseminated tuberculosis DISCUSSION and visceral leishmaniasis. Other hypotheses covered dengue, yellow fever, prolonged septicemic salmonellosis, brucellosis, malaria, Chagas The presence of the vector L. longipalpis in the state of Rio de disease and lymphoproliferative malignancies. An evaluation by thick Janeiro has been documented in the southern part of the country, on and thin blood smear, blood culture, stool tests, bone marrow biopsy Marambaia Island, in the municipality of the cities of Mangaratiba and aspiration and serology for dengue, yellow fever, brucellosis and and Saquarema3,9. Cases of the autochthonous transmission of canine Chagas disease were requested. A diagnosis of AIDS was refuted by HIV visceral leishmaniasis were reported in the southern part of the city testing. The bone marrow aspirate confirmed the presence of Leishmania of Rio de Janeiro, in the Laranjeiras neighborhood, and in the city amastigotes (Fig. 2). The case was reported by telephone immediately, of Angra dos Reis5,10. Moreover, sporadic cases of tegumentary followed by an investigation by the Municipal Civil Defense and Health leishmaniasis have been reported in the country, especially in Pau Surveillance. Infected dogs were found in the cemetery in Cajú and were da Fome in the Jacarepagua neighborhood6, and periurban cases of sacrificed. The Municipal Civil Defense and Health Surveillance then visceral leishmaniasis have been reported in the Maciço de Pedra implemented a plan to combat the vector, to improve environmental Branca (Serra do Barata in Realengo and Rio da Prata in the district sanitation and to search for new cases, which initially yielded two new of Campo Grande)7,8,12 and the continental slope of the Maciço de suspected cases. Gericinó7. However, the case reported in this article is considered by the Municipal Civil Defense and Health Surveillance of Rio de Janeiro to be the first report of autochthonous visceral leishmaniasis in humans in the urban area of Rio de Janeiro. Measures for vector control, reservoir targeting and the improvement of environmental sanitation as well as a search for new cases are now underway to prevent further transmission.
According to information collected from the patient and the employees of the Cajú cemetery, dogs belonging to people buried in the cemetery are often abandoned beside the graves of their owners. Certain cases involved people from the south of the state or other regions with canine leishmaniasis who potentially owned infected dogs. A few cemetery employees took care of the abandoned dogs, providing food and water and thus maintaining the presence of the animals in the cemetery. L. longipalpis has been found in the area of the Cajú cemetery, so it is likely that this peculiar situation facilitated the urban transmission of Fig. 2 - The bone marrow aspirate confirming the presence of Leishmania amastigotes. visceral leishmaniasis in this region. 282 x 211 mm (72 x 72 DPI). The absence of visceral leishmaniasis cases recorded in the urban area For the patient, a therapeutic regimen was initiated with amphotericin of Rio de Janeiro certainly influenced the delay to diagnose the patient, B at 0.25 mg/kg/day diluted in 500 mL 5% dextrose and 1000 IU who was diagnosed in the final phase of the disease. heparin and infused for 6 h. We routinely administered a dose of 100 mg hydrocortisone IV and one g dipyrone IV prior to infusion to reduce From a clinical perspective, this case was a typical manifestation of the probability of an allergic reaction to amphotericin B. The dose of visceral leishmaniasis in the final period. We chose to use amphotericin amphotericin B was increased by approximately 2.5 mg every 3-5 days B for treatment because antimonials are contraindicated in cases of liver if there were no complications, such as severe hypokalemia, cardiac failure, and the patient presented cholestatic jaundice, hypoalbuminemia arrhythmia or a significant increase in creatinine. Adjustments and and an increased INR2. Amphotericin B was used at a cumulative dose of suspension of the amphotericin B treatment were necessary for a few 0.25 mg/kg/day diluted with 500 mL 5% dextrose and 1000 IU heparin days because of increased creatinine levels and hypokalemia. Electrolyte and infused for six hours. The patient had an estimated dry weight replacements were also needed, as well as therapy to treat iron deficiency- of 45 kg. The administration of a dose of 100 mg hydrocortisone IV related anemia and the extension of the prothrombin time, which included and one g dipyrone IV, prior to infusion, was routinely performed to 300 mg ferrous sulfate orally twice daily, 5 mg folic acid PO daily and reduce the probability of an allergic reaction to amphotericin B. The 10 mg vitamin K SC in a single dose. dose of amphotericin was increased by approximately 2.5 mg every 3-5 days if there were no complications, such as severe hypokalemia, Over a period of seven weeks (January-February 2013), a cardiac arrhythmia or a significant increase in creatinine. The treatment cumulative dose of 700 mg amphotericin B was administered. Fever proved to be effective at a cumulative dose of 700 mg, which required reduction, an improvement in the level of consciousness, a noticeable approximately seven weeks of treatment. The increased creatinine levels improvement in general health and nutrition and a significant reduction and hypokalemia were controlled during hospitalization. At the end of of hepatosplenomegaly were noted during the treatment. Laboratory tests the therapy, the patient’s splenomegaly was reduced by approximately showed pancytopenia and the progressive improvement of dyscrasia, 60%, the fever was gone, and the pancytopenia was completely reversed. the normalization of liver enzyme levels and cholestasis (Table 1). The This presentation dismissed any more tests for parasitologic control.
83 SILVA, G.A.R.; BOECHAT, T.O.; FERRY, F.R.A.; PINTO, J.F.C.; AZEVEDO, M.C.V.M.; CARVALHO, R.S.; MOTTA, R.N. & VERAS, M.F. - First case of autochthonous human visceral leishmaniasis in the urban center of Rio de Janeiro: case report. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 81-4, 2014.
RESUMO 4. Cavalcanti AT, Medeiros Z, Lopes F, Andrade LD, Ferreira VM, Magalhães V, Miranda- Filho DB. Diagnosing visceral leishmaniasis and HIV/AIDS co-infection: a case series study in Pernambuco, Brazil. Rev Inst Med Trop Sao Paulo. 2012;54:43-7. Primeiro caso de leishmaniose visceral humana de transmissão autóctone no centro urbano do Rio de Janeiro: relato de caso 5. Figueiredo FB, Barbosa Filho CJ, Schubach EY, Pereira SA, Nascimento LD, Madeira MF. Relato de caso autóctone de leishmaniose visceral canina na zona sul do município A leishmaniose visceral é uma antropozoonose causada por do Rio de Janeiro. Rev Soc Bras Med Trop. 2010;43:98-9. protozoários do gênero Leishmania, principalmente Leishmania 6. Kawa H, Sabroza PC, Oliveira RM, Barcellos C. A produção do lugar de transmissão da (Leishmania) infantum e transmitida ao homem pela picada do leishmaniose tegumentar: o caso da Localidade Pau da Fome na cidade do Rio de flebotomíneo do gênero Lutzomyia, destacando-se no Brasil a Lutzomyia Janeiro, Brasil. Cad Saúde Publica. 2010;26:1495-507. longipalpis. Os animais reservatórios são muitos, tendo o cão doméstico (Canis familiaris) como principal reservatório. Trata-se de uma 7. Marzochi MC, Fagundes A, Andrade MV, Souza MB, Madeira MF, Mouta-Confort E, doença infecciosa crônica, de envolvimento sistêmico e caracterizado et al. Visceral leishmaniasis in Rio de Janeiro, Brazil: eco-epidemiological aspects and control. Rev Soc Bras Med Trop. 2009;42:570-80. por três fases: período inicial, período de estado e período final. As principais manifestações são febre, hepatoesplenomegalia, desnutrição 8. Marzochi MC, Sabroza PC, Toledo LM, Marzochi KB, Tramontano NC, Rangel-Filho e pancitopenia. Este artigo tem como objetivo relatar o caso de paciente F. Leishmaniose visceral na cidade do Rio de Janeiro, Brasil. Cad Saúde Pública. diagnosticada com leishmaniose visceral em período final, de transmissão 1985;1:5-17. autóctone na área urbana da cidade do Rio de Janeiro. O caso relatado 9. Novo SPC. Levantamento da fauna de flebotomíneos, vetores de leishmanioses, na Ilha de neste artigo é considerado, após investigação, pela Secretaria Municipal Marambaia, município de Mangaratiba, Rio de Janeiro. [dissertação]. Rio de Janeiro: de Saúde e Defesa Civil do Rio de Janeiro como o primeiro caso autóctone Escola Nacional de Saúde Pública Sergio Arouca; 2011. de leishmaniose visceral em humanos na área urbana da cidade do Rio de Janeiro. O tratamento oferecido foi eficaz e a paciente encontra-se 10. Souza MB, Carvalho RW, Machado RNM, Wermelinger ED. Flebotomíneos de áreas em acompanhamento ambulatorial. com notificações de casos autóctones de leishmaniose visceral canina e leishmaniose tegumentar americana em Angra dos Reis, Rio de Janeiro, Brasil. Rev Bras Entomol. 2009;53:147-50. REFERENCES 11. Souza MA, Sabroza PC, Marzochi MC, Coutinho SG, Souza WJ. Leishmaniose visceral 1. Alencar JE. Expansão do Calazar no Brasil. Ceará Méd. 1983;5:86-102. no Rio de Janeiro. Flebotomíneos da área de procedência de caso humano autóctone. Mem Inst Oswaldo Cruz. 1981;76:161-8. 2. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Manual de vigilância e controle da leishmaniose visceral. Brasília: Ministério da Saúde, Secretaria de 12. Toledo LM. Leishmaniose tegumentar e visceral em área peri-urbana no município do Vigilância em Saúde; 2006. p.13-36. Rio de Janeiro. [dissertação]. Rio de Janeiro: Fundação Oswaldo Cruz; 1987.
3. Brazil RP, Pontes MCQ, Passos WL, Fuzari AA, Brazil BG. Lutzomyia longipalpis Received: 20 March 2013 (Diptera: Psychodidae: Phlebotominae) in the region of Saquarema: potential area Accepted: 23 May 2013 of visceral leishmaniasis transmission in the state of Rio de Janeiro, Brazil. Rev Soc Bras Med Trop. 2012;45:120-1.
84 Rev. Inst. Med. Trop. Sao Paulo 56(1):85-88, January-February, 2014 doi: 10.1590/S0036-46652014000100014
BIOPSY PROVEN ACUTE TUBULAR NECROSIS DUE TO RHABDOMYOLYSIS IN A DENGUE FEVER PATIENT: A CASE REPORT AND REVIEW OF LITERATURE
Liliany P. REPIZO, Denise M. MALHEIROS, Luis YU, Rui T. BARROS & Emmanuel A. BURDMANN
SUMMARY
Renal histology results are very scarce in dengue-associated rhabdomyolysis patients developing acute kidney injury (AKI). We report a case of dengue fever-induced AKI associated to rhabdomyolysis with a renal biopsy showing acute tubular necrosis (ATN) and renal deposition of myoglobin. A 28-year-old patient who presented dengue fever (DF) complicated by severe AKI and rhabdomyolysis is described. The patient required hemodialysis for three weeks. A renal biopsy revealed ATN with positive staining for myoglobin in the renal tubuli. The patient was discharged with recovered renal function. In conclusion, this case report described a biopsy proven ATN associated to DF-induced rhabdomyolysis, in which renal deposition of myoglobin was demonstrated. We suggest that serum creatine phosphokinase should be monitored in DF patients to allow for an early diagnosis of rhabdomyolysis and the institution of renal protective measures.
KEYWORDS: Dengue fever; Acute kidney injury; Acute tubular necrosis; Renal histology; Myoglobin; Rhabdomyolysis; Creatine phosphokinase.
INTRODUCTION rhabdomyolysis associated with DF. A renal biopsy revealed acute tubular necrosis and positive staining for myoglobin in the renal tubuli. Dengue is currently the most important infectious viral mosquito- The literature on dengue-associated renal injury and rhabdomyolysis is borne disease in the world. In recent years, there has been an explosive reviewed and discussed. outbreak of this infectious disease, mainly affecting tropical countries, but also the warmer areas of developed countries, such as the southern United CASE REPORT States. In fact, the number of cases worldwide that are annually reported to the World Health Organization (WHO) increased from approximately A 28-year-old married male painter, from Sao Paulo, SP, Brazil, 900 in the 1950s to almost one million at present41. previously healthy, presented a 20-day history of fever, myalgia, muscle weakness, cough, nausea and vomiting, diarrhea, epigastric Dengue is a multifaceted disease that can manifest as an pain and lower limb edema. The patient history did not disclose any undifferentiated fever, classical dengue fever (DF), dengue hemorrhagic epidemiological clue for a specific infectious disease nor had he receive fever (DHF) and dengue shock syndrome (DSS)41. any nephrotoxic drug.
Different renal injuries have been described in dengue patients, Laboratory blood analysis at the time of the first hospital admission such as an increase in serum creatinine, proteinuria, glomerulonephritis, revealed the following: creatinine (SCr) 11.6 mg/dL, urea 277 mg/dL, hemolytic-uremic syndrome and acute kidney injury (AKI)4,10,16,37,39. sodium 124 mEq/L, potassium 6.6 mEq/L, bicarbonate 15.6 mEq/L, Most cases of AKI have been associated with DHF or DSS6,7,20-23,25,27,34,40, SGOT 1,206 IU/L, SGPT 853 IU/L, amylase 121 IU/L, total bilirubin but AKI has also been described in DF13-15,30,31, albeit less commonly. 0.38 mg/dL, alkaline phosphatase 68 IU/L, gamma-glutamyltransferase Similarly, rhabdomyolysis with1,5,7,19 and without AKI9,24,28 has been 104 IU/L, hemoglobin 14.2 g/dL, leukocytes 10,200/mm3, platelets described in dengue patients. Renal histology results from patients with 292,000/mm3 and C-reactive protein (CRP) 1.95 mg/dL. The urinalysis dengue-associated AKI are scarce and to the best of our knowledge, there disclosed a specific gravity of 1,027, pH 5.0, protein 3+, glucose traces, were no previous cases of biopsy proven ATN with renal myoglobin red blood cells > 1 million/mL, leukocytes > 1 million/mL and no casts. deposition due to rhabdomyolysis associated with DF31,37,39. Renal replacement therapy (RRT) was initiated.
We report a case of a young patient who developed AKI and After one week, he was transferred to Hospital das Clinicas, University
Division of Nephrology, University of Sao Paulo Medical School, Sao Paulo, Brazil Correspondence to: Emmanuel A. Burdmann; Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo 455, 3º Andar, sala 3310 (LIM 12), 01246-903 São Paulo, SP, Brasil. Tel.: 55.11.30617343, Fax: 55.11.30882267. E-mail: [email protected] REPIZO, L.P.; MALHEIROS, D.M.; YU, L.; BARROS, R.T. & BURDMANN, E.A. - Biopsy proven acute tubular necrosis due to rhabdomyolysis in a dengue fever patient: a case report and review of literature. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 85-8, 2014.
of Sao Paulo Medical School, Sao Paulo, Brazil. At admission, he was pale, had a blood pressure of 140/90 mmHg and oliguric (150 m/24 h), with a dialysis catheter in his right internal jugular vein and had no evidence of systemic hemorrhagic phenomena. His physical exam was otherwise unremarkable. Laboratory tests performed at admission revealed: SCr 14.8 mg/dL, serum urea 323 mg/dL, hemoglobin 11.8 g/dL, leukocytes 9,400/mm3, platelets 469,000/mm3, albumin 3.6 g/L and CRP 2.5 mg/ dL. The patient presented serum creatine phosphokinase (CK) 4,063 IU/L, serum lactic dehydrogenase 657 IU/L, uric acid 13.4 mg/dL and Fig. 1 - A. Light microscopy of renal tissue stained with Masson’s trichrome (20X). Note the phosphorus 9.2 mg/dL, suggesting rhabdomyolysis. The urinalysis showed presence of diffuse acute tubular necrosis and preserved glomeruli. In the interstitial area, protein > 1 g/L, red blood cells > 100/field, and leukocytes 20/field. A thin fibrosis lines and edema are evident. B. Immunostaining of renal tissue. Note the positive subsequent 24 h proteinuria showed 0.83 g of protein/24 h. Anti-nuclear immunostaining for myoglobin in the cytoplasm of the tubular cells. factor, antineutrophil cytoplasmic antibodies, C3 and C4 complement fractions, rheumatoid factor and anticardiolipin antibody levels were 3 RBCs. After three weeks, he returned to the outpatient facility with no normal. A renal ultrasound disclosed normal kidneys. Serology for HIV, complains and an unremarkable physical examination. Laboratory tests leptospirosis, hepatitis C, hepatitis B and syphilis were negative. Patient performed at that time revealed a SCr of 0.98 mg/dL. tested positive for hepatitis A and cytomegalovirus immunoglobulin G. Five days after admission, a positive test for dengue was obtained (IgM, DISCUSSION ELISA assay). As the patient had a clinical picture and serology consistent with dengue infection without hemorrhagic phenomena, low platelet count To the best of our knowledge, the current case is the first biopsy or evidence of plasma leakage, he was diagnosed with a case of dengue proven description of rhabdomyolysis and AKI associated with DF in fever complicated by rhabdomyolysis and AKI. which ATN and myoglobin staining in the renal tubuli was demonstrated.
After two weeks on RRT, due to non-recovery of renal function, There are only four previously reported cases of dengue-induced a percutaneous renal biopsy was performed. The biopsy included 11 rhabdomyolysis and AKI (Table 1), but renal biopsy was not performed glomeruli with normal volume and cellularity, dilated tubules with on those patients1,7,15,19. All of them presented elevated CK levels, three foci of epithelial cellular necrosis, interstitial area with focal fibrosis studies reported myalgia, two reported muscle weaknesses and three (approximately 10%) and normal arterioles. Immunofluorescence studies presented dark urine, positive urinary myoglobin and oliguria. assays for IgA, IgG, IgM, C1q, C3 and fibrinogen were negative. Renal replacement therapy was performed in two cases. The pathological diagnosis was acute tubular necrosis. Myoglobin immunohistochemistry staining was positive in the renal tubuli (Fig. 1 There are also case descriptions of dengue-associated rhabdomyolysis A and B). with myalgia, dark urine and highly elevated CK levels without AKI development5,7,9,24. In addition, AKI was not reported in series of patients Three weeks after admission, RRT was interrupted and patient was with dengue-induced quadriparesis or myositis and high CK levels28,33. discharged with SCr 2.4 mg/dL, normal urine output and urinalysis with Although rhabdomyolysis is a well-known risk factor for AKI, the pH 5.5, specific gravity 1015, blood +, protein 0.3 g/dL, 12 WBCs and presence of other simultaneous injury factors, such as hypovolemia or
Table 1 Comparison of the current case with the cases of dengue with rhabdomyolysis and AKI previously described
Gender, Type of Muscle Dark CK Cr Renal Author, year, country Myalgia UMyo Oliguria RRT Outcome age dengue weakness urine (IU/L) (mg/dL) biopsy Gunasekera et al, Female, NR yes yes + + yes >5,000 8.8 no PD, HF recovery 2000, Ceylon (ref 14) 28 y Davis & Bourke, Male, 2004, East Timor DHF NR NR NR NR NR 17,548 NR no NR death 33 y (ref 7) Karakus et al, 2007, Male, DSS yes NR + + yes 156,900 3.6 no NR death Suriname (ref 19) 66 y Acharya et al, 2010, Male, NR yes yes + + yes 29,000 2.6 no NR NR India (ref 1) 40 y Current case, 2013, Male, DF yes ND yes 4,063 14.8 ATN HD recovery Brazil 28 y UMyo: urinary myoglobin; CK: serum creatine phosphokinase; Cr: serum creatinine; RRT: renal replacement therapy; NR: not reported; PD: peritoneal dialysis; HF: hemofiltration; DHF: dengue hemorrhagic fever; DSS: dengue shock syndrome; DF: dengue fever; ND: not done; ATN: acute tubular necrosis; HD: hemodialysis.
86 REPIZO, L.P.; MALHEIROS, D.M.; YU, L.; BARROS, R.T. & BURDMANN, E.A. - Biopsy proven acute tubular necrosis due to rhabdomyolysis in a dengue fever patient: a case report and review of literature. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 85-8, 2014.
dehydration, acidosis and aciduria, must be present to cause clinically of the written consent is available for review by the Series Editor of relevant myoglobinuria-induced renal injury. this journal.
AKI has also been reported in DHF, DSS and DF without RESUMO rhabdomyolysis. Hemodynamic instability, hemolysis, glomerular injury and direct action of viral particles on renal tissue have been considered Necrose tubular aguda comprovada por biópsia em paciente com as possible injury mechanisms6,10,25,30,32,34,37. In fact, the current patient dengue e rabdomiólise presented proteinuria but the renal biopsy did not disclose glomerular injury. Resultados de histologia renal são muito escassos em pacientes com rabdomiólise e injúria renal aguda (IRA) associada a dengue. Myositis and rhabdomyolysis have been considered as rare Descrevemos caso de dengue complicado por rabdomiólise e IRA no qual complications of dengue infection but few authors have systematically a biópsia renal mostrou necrose tubular aguda (NTA) e deposição renal assessed their occurrences. MALHEIROS et al.26 performed muscle de mioglobina. Paciente de 28 anos que apresentou dengue complicado biopsies in 15 patients with DF and myalgia during an epidemic outbreak por IRA grave e rabdomiólise é descrito. Ele necessitou de diálise por in Brazil. Only three patients presented mild increased CK levels. They três semanas. A biópsia renal mostrou NTA, com imunohistoquímica found mononuclear infiltrates in 12 biopsies, lipid accumulations in 11 fortemente positiva para mioglobina nos túbulos renais. O paciente and rare foci of myonecrosis in three (one case had an elevated CK level). recebeu alta com recuperação da função renal. Em conclusão, Seven out of 16 patients with serologically proven dengue infections in descrevemos caso de dengue complicado por IRA e rabdomiólise, em India presented muscle weakness and increased CK levels18. A muscle que a biópsia renal mostrou NTA e deposição de mioglobina. Sugerimos biopsy was performed in one patient, revealing myositis18. SAID et al. que creatinofosfoquinase deve ser monitorizada em pacientes com dengue prospectively assessed CK levels and the incidence of myositis in 101 para permitir o diagnóstico precoce de rabdomiólise e a instituição de patients with serologically proven dengue in Saudi Arabia35. In that study, medidas protetoras para o rim. 91% of the patients had elevated CK, 63.4% complained of myalgia and 2.9% had muscle weakness. MISRA et al. evaluated 39 patients with REFERENCES positive dengue serology during a dengue outbreak in Northern India29. Transient muscle dysfunction with CK levels of approximately 1,000 U/L 1. Acharya S, Shukla S, Mahajan SN, Diwan SK. Acute dengue myositis with was observed in 11 (73.3%) patients with DF, in 18 (81.8%) with DHF and rhabdomyolysis and acute renal failure. Ann Indian Acad Neurol. 2010;13:221-2. in the two patients with DSS. This collection of data suggests that muscle 2. Barreto DF, Takiya CM, Paes MV, Farias-Filho J, Pinhão AT, Alves AM, et al. injury is not uncommon in dengue and has probably been underreported. Histopathological aspects of dengue-2 virus infected mice tissues and complementary virus isolation. J Submicrosc Cytol Pathol. 2004;36:121-30. The pathogenesis of dengue-associated muscle injury is unclear. Different mechanisms have been hypothesized, such as direct viral invasion 3. Basílio-de-Oliveira CA, Aguiar GR, Baldanza MS, Barth OM, Eyer-Silva WA, Paes MV. Pathologic study of a fatal case of dengue-3 virus infection in Rio de Janeiro, of and immune-mediated injury to the muscle fibers. Striated skeletal Brazil. Braz J Infect Dis. 2005;9:341-7. muscles from mice inoculated with dengue type 2 viruses exhibited myofibril destruction, sarcoplasm involution, mitochondrial changes and 4. Basu G, Chrispal A, Boorugu H, Gopinath KG, Chandy S, Prakash JA, et al. Acute aggregates of electron-dense material and cytoplasmic glycogen particles32. kidney injury in tropical acute febrile illness in a tertiary care centre - RIFLE criteria The dengue virus has demonstrated high efficiency in infecting and validation. Nephrol Dial Transplant. 2011;26:524-31. 38 replicating in human primary muscle satellite cells and muscle cells were 5. Beauvais P, Quinet B, Richardet JM. Dengue. A propos of 2 cases. Arch Fr Pediatr. demonstrated to be highly susceptible to in vitro infection by the dengue 2 1993;50:905-7. virus. The infected cells exhibited increased expression of inflammatory genes and the IP-10 protein and elevated intracellular calcium36. Dengue 6. Chacko B, John GT, Jacob CK, Vijayakumar TS. Dengue shock syndrome in a renal virus infection has also been associated with increased production of transplant recipient. Transplantation. 2004;77:634-5. 11,12 inflammatory cytokines which may cause muscle injury . 7. Davis JS, Bourke P. 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CONSENT 11. Gagnon SJ, Mori M, Kurane I, Green S, Vaughn DW, Kalayanarooj S, et al. Cytokine gene expression and protein production in peripheral blood mononuclear cells of Written informed consent was obtained from the patient for the children with acute dengue virus infections. J Med Virol. 2002;67:41-6. publication of this Case report and any accompanying images. A copy
87 REPIZO, L.P.; MALHEIROS, D.M.; YU, L.; BARROS, R.T. & BURDMANN, E.A. - Biopsy proven acute tubular necrosis due to rhabdomyolysis in a dengue fever patient: a case report and review of literature. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 85-8, 2014.
12. Gandini M, Reis SR, Torrentes-Carvalho A, Azeredo EL, Freire M da S, Galler R, et 28. Mishra UK, Kalita J. Spectrum of neurological manifestations of dengue in India. al. Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting Dengue Bull. 2006;30:107-13. in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles. Mem Inst Oswaldo Cruz. 2011;106:594-605. 29. Misra UK, Kalita J, Maurya PK, Kumar P, Shankar SK, Mahadevan A. Dengue- associated transient muscle dysfunction: clinical, electromyography and 13. George R, Liam CK, Chua CT, Lam SK, Pang T, Geethan R, et al. Unusual clinical histopathological changes. Infection. 2012;40:125-30. manifestations of dengue virus infection. Southeast Asian J Trop Med Public Health. 1988;19:585-90. 30. Mohsin N, Mohamed E, Gaber M, Obaidani I, Budruddin M, Al Busaidy S. Acute tubular necrosis associated with non-hemorrhagic dengue fever: a case report. Ren 14. Gunasekera HH, Adikaram AV, Herath CA, Samarasinghe HH. Myoglobinuric acute Fail. 2009;31:736-9. renal failure following dengue viral infection. Ceylon Med J. 2000;45:181. 31. Nair VR, Unnikrishnan D, Satish B, Sahadulla MI. Acute renal failure in dengue 15. Hommel D, Talarmin A, Reynes JM, Hulin A. Acute renal failure associated with fever in the absence of bleeding manifestations or shock. Infect Dis Clin Pract. dengue fever in French Guiana. Nephron. 1999;83:183. 2005;13:142-3.
16. Hutspardol S, Prommalikit O, Upiya N, Chataroopwijit J, Khemakanok K, 32. Nath P, Agrawal DK, Mehrotra RM. Ultrastructural changes in skeletal muscles in Assadamongkol K. Heavy proteinuria following dengue hemorrhagic fever. Southeast dengue virus-infected mice. J Pathol. 1982;136:301-5. Asian J Trop Med Public Health. 2011;42:579-82. 33. Paliwal VK, Garg RK, Juyal R, Husain N, Verma R, Sharma PK, et al. Acute dengue 17. Jessie K, Fong MY, Devi S, Lam SK, Wong KT. Localization of dengue virus in virus myositis: a report of seven patients of varying clinical severity including two naturally infected human tissues, by immunohistochemistry and in situ hybridization. cases with severe fulminant myositis. J Neurol Sci. 2011;300:14-8. J Infect Dis. 2004;189:1411-8. 34. Radakovic-Fijan S, Graninger W, Muller C, Honigsmann H, Tanew A. Dengue 18. Kalita J, Misra UK, Mahadevan A, Shankar SK. Acute pure motor quadriplegia: is hemorrhagic fever in a British travel guide. J Am Acad Dermatol. 2002;46:430-3. it dengue myositis? Electromyogr Clin Neurophysiol. 2005;45:357-61. 35. Said SM, Elsaeed KM, Alyan Z. Benign acute myositis in association with acute 19. Karakus A, Banga N, Voorn GP, Meinders AJ. Dengue shock syndrome and dengue viruses’ infections. Egypt J Neurol Psychiat Neurosurg. 2008;45:193-200. rhabdomyolysis. Neth J Med. 2007;65:78-81. 36. Salgado DM, Eltit JM, Mansfield K, Panqueba C, Castro D, Vega MR, et al. Heart and 20. Kuo MC, Lu PL, Chang JM, Lin MY, Tsai JJ, Chen YH, et al. Impact of renal failure skeletal muscle are targets of dengue virus infection. Pediatr Infect Dis J. 2010;29: on the outcome of dengue viral infection. Clin J Am Soc Nephrol. 2008;3:1350-6. 238-42.
21. Laoprasopwattana K, Pruekprasert P, Dissaneewate P, Geater A, Vachvanichsanong P. 37. Upadhaya BK, Sharma A, Khaira A, Dinda AK, Agarwal SK, Tiwari SC. Transient Outcome of dengue hemorrhagic fever-caused acute kidney injury in Thai children. IgA nephropathy with acute kidney injury in a patient with dengue fever. Saudi J J Pediatr. 2010;157:303-9. Kidney Dis Transpl. 2010;21:521-5.
22. Lee IK, Liu JW, Yang KD. Clinical and laboratory characteristics and risk factors 38. Warke RV, Becerra A, Zawadzka A Schmidt DJ, Martin KJ, Giaya K, et al. Efficient for fatality in elderly patients with dengue hemorrhagic fever. Am J Trop Med Hyg. dengue virus (DENV) infection of human muscle satellite cells upregulates type I 2008;79:149-53. interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells. J Gen Virol. 2008;89:1605-15. 23. Lee IK, Liu JW, Yang KD. Clinical characteristics, risk factors, and outcomes in adults experiencing dengue hemorrhagic fever complicated with acute renal failure. 39. Wiersinga WJ, Scheepstra CG, Kasanardjo JS, de Vries PJ, Zaaijer H, Geerlings SE. Am J Trop Med Hyg. 2009;80:651-5. Dengue fever-induced hemolytic uremic syndrome. Clin Infect Dis. 2006;43:800-1.
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25. Lima EQ, Gorayeb FS, Zanon JR, Nogueira ML, Ramalho HJ, Burdmann EA. Dengue 41. World Health Organization (WHO) and the Special Programme for Research and haemorrhagic fever-induced acute kidney injury without hypotension, haemolysis or Training in Tropical Diseases (TDR). Dengue: guidelines for diagnosis, treatment, rhabdomyolysis. Nephrol Dial Transplant. 2007;22:3322-6. prevention and control. Geneva: WHO; 2009.
26. Malheiros SM, Oliveira AS, Schmidt B, Lima JG, Gabbai AA. Dengue. Muscle Received: 26 March 2013 biopsy findings in 15 patients. Arq Neuropsiquiatr. 1993;51:159-64. Accepted: 11 June 2013
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88 Rev. Inst. Med. Trop. Sao Paulo 56(1):89-91, January-February, 2014 doi: 10.1590/S0036-46652014000100015
MULTIPLE PULMONARY NODULES CAUSED BY Corynebacterium striatum IN AN IMMUNOCOMPETENT PATIENT
Cecília Bittencourt SEVERO(1), Luciana Silva GUAZZELLI(1), Marinez Bizarro BARRA(2), Bruno HOCHHEGGER(3) & Luiz Carlos SEVERO(4)
SUMMARY
Nondiphtherial corynebacteria are ubiquitous in nature and commonly colonize the skin and mucous membranes of humans, however they rarely account for clinical infection. We present the first reported case of multiple pulmonary nodules caused by Corynebacterium striatum. The infection occurred in a 72-year-old immunocompetent female, and the diagnosis was obtained by Gram’s stain and culture of lung biopsy. C. striatum should be recognized as a potential pathogen in both immunocompromised and normal hosts in the appropriate circumstances.
KEYWORDS: Multiple nodules; Corynebacterium striatum.
INTRODUCTION under CT guidance and revealed lung parenchyma with elastofibrosis and anthracosis. The Corynebacteria are a group of aerobic, gram-positive, non- sporulating, predominantly non-motile rods. Due to their saprophytic Examination of the lung fragment by direct microscopy showed nature most diphtheroid isolates in man are ignored and attributed to multiple non-acid-fast (Ziehl-Neelsen), gram-positive rods (Fig 2). contamination from skin or mucous membrane sources. In the last The searches for malignant (H&E) and fungus (Grocott) cells were decade, an increasing number of studies have reported different species negative. Culture on blood agar yielded pure growth of an organism of Corynebacterium causing infections in underlying immunosuppressive identified as Corynebacterium striatum (99.7% probability) by the conditions. However, we report the first of multiple pulmonary nodules API-CORYNE system (BioMérieux Marcy l’Etoile, France). Laboratory caused by Corynebacterium striatum, in a 72-year-old immunocompetent investigations revealed hemoglobin concentrations of 9.3 g/d, a total female. white blood cell count of 12 x 109/L with 80% neutrophils, 7% bands, 9% lymphocytes, and 3% monocytes; platelets of 452 x 109/L, and a CASE REPORT erythrocyte sedimentations rate of 55 mm/hour. Routine blood chemistry was normal. Creatinine was 1.6 mg/dL, alkaline phosphatase was 82 U/L, A 72-year-old woman was admitted to our hospital with right-sided aspartate aminotransferase was 42 U/L, and alanine aminotransferase 30 pleuritic chest pain, cough with purulent sputum, fatigue and shortness U/L. Antibiotic susceptibility was tested by the disk diffusion method of breath. Her pulse was 90 beats per minute, blood pressure was 120/80 (Oxoid SA, Spain) in Mueller-Hinton agar supplemented with 5% blood mmHg, respiration was 22/min, and temperature was 37 °C. On physical for all antibiotics tested including penicillin (10U), gentamicin (10 µg), examination, the patient appeared thin but healthy. There was no history vancomycin (30 µg), erythromycin (15 µg), and tetracycline (30 µg). of smoking tobacco. Her past medical history was remarkable only for The susceptibility criteria of the CLSI for Staphylococcus spp. were coronary disease and hypertension, which were subsequently treated with used for all antibiotics, which result in sensitivity for erythromycin and appropriate drugs. An HIV test was negative and no other underlying vancomycin, and resistance for all the others. The patient was treated disease was detected. A computed tomography (CT) scan revealed multiple with erythromycin (500 mg/qds) and rifampin (600 mg/daily), both pulmonary nodules in the lower lobes. The CT scan also demonstrated some administered orally. Despite therapy, relentless deterioration continued, cavitary lung lesions with a feeding vessel sign in the lower lobes (Fig 1). resulting in death 12 days after respiratory failure.
Microscopic examinations of sputum were negative for acid-fast DISCUSSION bacilli and fungi; cytologic examination showed no malignant tumor cells. Blood cultures were negative. Needle biopsy of the lung was performed The genus Corynebacterium forms a group of 81 validly described
(1) Mycology Laboratory, Santa Casa Hospital Complex, Porto Alegre, Rio Grande do Sul, Brazil. E-mail: [email protected]; [email protected] (2) Pathology Laboratory, Santa Casa Hospital Complex, Porto Alegre, Rio Grande do Sul, Brazil. E-mail: [email protected] (3) Radiology Service, Santa Casa Hospital Complex, Porto Alegre, Rio Grande do Sul, Brazil. E-mail: [email protected] (4) Department of Internal Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. E-mail: [email protected] Correspondence to: Luiz Carlos Severo, MD, PhD, Laboratório de Micologia, Hospital Santa Rita, Santa Casa Complexo Hospitalar, Annes Dias 285, 90020-090 Porto Alegre, RS, Brasil. Phone: 55 51.32148410. Fax: 55.51.32148435. E-mail: [email protected] SEVERO, C.B.; GUAZZELLI, L.S.; BARRA, M.B.; HOCHHEGGER, B. & SEVERO, L.C. - Multiple pulmonary nodules caused by Corynebacterium striatum in an immunocompetent patient. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 89-91, 2014.
Fig. 2 - Imprint smear of lung tissue. Gram’s stain showing clusters of gram-positive bacilli of Corynebacterium striatum.
present the first reported case of multiple pulmonary nodules due to C. striatum in a previously healthy patient, diagnosed by lung biopsy.
Reports of C. striatum are extremely rare. It is part of the normal microbiota of skin and cutaneous membranes and very often is considered contaminant. However, C. striatum was included in a variety of different types of infection: pneumonia and empyema, CSF-shunt infection, endocarditis, peritonitis, arthritis, keratitis, intra-uterine infections, wound infection, breast abscess, osteomyelitis11. Many cases of infection were hospital-acquired, namely wound infections. The discrimination between colonization and infection is difficult in some cases, especially nosocomial infection. Serious infections with C. striatum have been reported only sporadically1.
A pulmonary cavity is a gas-filled area of the lung in the center of a nodule or area of consolidation; it may be clinically observed by a plain chest radiography or CT. Cavities are present in a wide variety of infectious and noninfectious processes. A cavity can be the result of any of a number of pathological processes, including suppurative necrosis (e.g., pyogenic lung abscess), caseous necrosis (e.g., tuberculosis), ischemic necrosis (e.g., pulmonary infarction), cystic dilatation of lung structures (e.g., ball valve obstruction and Pneumocystis pneumonia), or displacement of lung tissue by cystic structures (e.g., Echinococcus). In Fig. 1 - A computed tomography scan revealing multiple pulmonary nodules (arrow in A) in addition, malignant processes may cavitate because of treatment-related the lower lobes. The scan also demonstrates some cavitary lung lesions (arrowhead in B) with necrosis, internal cyst formation, or internal desquamation of tumor cells feeding vessel sign in the lower lobes. These imaging findings suggest secondary vascular with subsequent liquefaction4. implants that could be of infectious or neoplastic etiology. In CT, the feeding vessel sign consists of a distinct vessel leading species, 50 of which can cause clinical disease in humans5. C. striatum directly into the center of the nodule. This sign has been considered is ubiquitous and colonizes the skin and mucous membranes of normal highly suggestive of septic embolism, the prevalence varying from 67 to hosts and hospitalized patients8,14. C. striatum has been one of the more 100% in various series7. However, the feeding vessel sign also occurs in commonly isolated coryneform bacteria in the clinical microbiology about 20% of pulmonary metastases13. These imaging findings suggest laboratory10. Reports of true infection, confirmed by isolation of C. secondary vascular implants that could be of infectious or neoplastic striatum from a sterile site, are rare and have been reported mainly in etiology. This is the first reported case of multiple pulmonary nodules patients with indwelling devices or immunosuppression6. We herein resulting from infection by C. striatum. The presence of multiple
90 SEVERO, C.B.; GUAZZELLI, L.S.; BARRA, M.B.; HOCHHEGGER, B. & SEVERO, L.C. - Multiple pulmonary nodules caused by Corynebacterium striatum in an immunocompetent patient. Rev. Inst. Med. Trop. Sao Paulo, 56(1): 89-91, 2014.
pulmonary nodules with a negative tissue diagnosis of malignancy was 2. Bowstead TT, Santiago SM. Pleuropulmonary infection due to Corynebacterium striatum. the key to a presumptive diagnosis of another fatal pulmonary infection2,9 Br J Dis Chest. 1980;74:198-200. 3 with this emerging respiratory pathogen . 3. Díez-Aguilar M, Ruiz-Garbajosa P, Fernández-Olmos A, Guisado P, Del Campo R, Quereda C, et al. Non-diphtheria Corynebacterium species: an emerging respiratory The learning point of this fatal pulmonary infection by C. striatum pathogen. Eur J Clin Microbiol Infect Dis. 2013;32:769-72. with isolation of a pure culture, diagnosed by lung biopsy, in the absence of other recognized respiratory pathogens and knowing the high levels of 4. Dodd GD, Boyle JJ. Excavating pulmonary metastases. Am J Roentgenol Radium Ther Nucl Med. 1961;85:277-93. antibiotic resistance, including to erythromycin and rifampicin, is that the empiric use of these drugs despite the susceptibility to the erythromycin, 5. Funke G, Graevenitz A, Clarridge JE 3rd, Bernard KA. Clinical microbiology of was not adequate. coryneform bacteria. Clin Microbiol Rev. 1997;10:125-59.
Finally, the vancomicin could be candidate to the first-line therapeutic 6. Funke G, Bernard KA. Corynebacterium gram-positive. In: Versalovic J, ed. Manual of clinical microbiology. Washington: ASM Press; 2011. p. 413-22. option for treatment of C. striatum5, but reports of true infections 12 confirmed by isolation from a sterile site, like our case, are rare . 7. Kuhlman JE, Fishman EK, Teigen C. Pulmonary septic emboli: diagnosis with CT. Furthermore, the threat of emerging resistance of available anti-C. Radiology. 1990;174:211-3. striatum drugs highlights the need for the continuing investigation of the biology of this organism as the usefulness of these drugs can only 8. Martinez-Martinez L, Suárez AI, Rodríguez-Baño J, Bernard K, Munián MA. Clinical significance of Corynebacterium striatum isolated from human samples. Clin be assessed in randomized clinical trials. Microbiol Infect. 1997;3:634-9.
RESUMO 9. Martinez-Martinez L, Suárez AI, Ortega MC, Rodríguez-Jiménez R. Fatal pulmonary caused by Corynebacterium striatum. Clin Infect Dis. 1994;19:806-7. Nódulos pulmonares múltiplos causados por Corynebacterium 10. Martinez-Martinez L, Suárez AI, Winstanley J, Ortega MC, Bernard K. Phenotypic striatum numa paciente imunocompetente characteristics of 31 strains of Corynebacterium striatum isolated from clinical samples. J Clin Microbiol. 1995;33:2458-61. Bacilos não diftéricos são ubiquitários na natureza e comumente colonizam a pele e as membranas mucosas humanas, contudo eles 11. Martin MC, Mélon O, Celada MM, Alvarez J, Méndez FJ, Vásquez F. Septicaemia dueto raramente acarretam doença clínica. Apresentamos o primeiro relato to Corynebacterium striatum: molecular confirmation of entry via the skin. J Med Microbiol. 2003;52:599-602. de múltiplos nódulos causados por Corynebacterium striatum. A infecção ocorreu numa mulher imunocompetente de 72 anos de idade 12. Meyer DK, Reboli AC. Other coryneform bacteria and rhodococci. In: Mandell GL, e o diagnóstico foi obtido pela coloração de Gram e cultivo de biópsia Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett`s principles and practice pulmonar. C. striatum deve ser reconhecido como potencial patógeno of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier; 2010. vol. tanto em pacientes imunodeprimidos como em hospedeiros normais, em 2. p. 2695-2706. circunstâncias apropriadas. 13. Murata K, Takahashi M, Mori M, Kawaguchi N, Furukawa A, Ohnaka Y, et al. Pulmonary metastatic nodules: CT-pathologic correlation. Radiology. 1992;182:331-5. REFERENCES 14. Watkins DA, Chahine A, Creger RJ, Jacobs MR, Lazarus HM. Corynebacterium striatum: 1. Brandenburg AH, van Belkum A, van Pelt CV, Bruining HA, Mouton JW, Verbrugh HA. a diphtheroid with pathogenic potential. Clin Infect Dis. 1993;17:21-5. Patient-to-patient spread of a single strain of Corynebacterium striatum causing infections in a surgical intensive care unit. J Clin Microbiol. 1996;34:2089-94. Received: 20 March 2013 Accepted: 11 June 2013
91 Rev. Inst. Med. Trop. Sao Paulo 56(1):92, January-February, 2014 doi: 10.1590/S0036-46652014000100016
LETTER TO THE EDITOR
ANALOGIES IN MEDICINE: WHITE CLAY-PIPE STEM “CIRRHOSIS”
October, 2013
Dear Sir,
Schistosomiasis infects approximately 200 million persons and kills approximately 280,000 annually. In the case of a severe S. mansoni infection - hepatosplenic schistosomiasis - the external surface of the liver may be smooth, macronodular, or micronodular, but broad tracts of portal fibrosis are obvious upon gross examination. The cut surfaces reveal a widespread fibrosis portal enlargement without distortion of the intervening parenchyma by regenerative nodules. There is usually no hepatic parenchymal cell damage and hepatic function usually remains normal. Many of the portal triads lack a vein lumen, causing presinusoidal portal hypertension and severe congestive splenomegaly, esophageal Schistosomiasis of the liver, showing the peculiarly heavy fibrosis of the larger portal tracts 1-3 varices, and ascites . that is commonly referred to as ‘pipe-stem fibrosis’ or ‘clay pipe fibrosis’. The fibrosis is the The English pathologist William St. Claire Symmers (1863-1937), end-result of the granulomatous inflammation evoked by the eggs of Schistosoma mansoni who was working at the Kass-El-Ainy Hospital in Cairo, described for the (Symmers, W. St. C., J. Pathol. Bacteriol. 1904,9:237). first time in 1904 a new form of cirrhosis of the liver caused by eggs of schistosoma. From post-mortem examinations of diseased livers, Symmers described lesions which he likened to “white clay-pipe stems” at various angles throughout the liver. What we call liver fibrosis nowadays, Symmers called cirrhosis and made the further mistake, understandable at the time, of describing what were surely S. mansoni eggs presenting lateral spicules in the hepatic tissue as “the ova of Bilharzia haematobia”. Spindle cells arranged concentrically around the eggs were ringed with fibrous tissue that formed “periportal cirrhosis”1. Symmers’ original description was as follows: “When a portal canal is cut transversely, the mouths of the contained vessels and bile duct are seen embedded in the center of a circular or slightly oval area of white connective tissue, the diameter of the mass being, on average, between a sixth to a quarter of an inch; whereas longitudinal sections of the canal reveals elongated masses of similar appearance and thickness, so that the cut surface of the liver looks as if a number of white clay-pipe stems have been thrust at various angles through the organ”4. (Port. Fibrose em haste de cachimbo de barro branco. Esp. Fibrosis en boquilla de pipa). Clay is a soft, sticky earth, which can be molded into different forms and then hardened in ovens. Bricks, pottery, and tile are made of clay. Pipestem is the long, slender stem of a tobacco pipe through which the smoke is drawn. Subsequently pathologists in various countries, mainly Egypt, Puerto Rico and Brazil have used the terminology coarse periportal fibrosis, axial fibrosis, pipe stem fibrosis, or the best-known term, Symmers’ fibrosis, in dealing with the hepatopathy of advanced Bilharziasis1. Was Dr. Symmers a pipe smoker? We do not know. But, we believe, he was influenced by an old-fashioned white pipe to make this analogy of the liver damaged by schistosomiasis. Anyway, all the textbooks and papers published in the Western World on advanced stage of schistosomiasis always mention the pipe stem fibrosis. It is a consecrated term3.
José de Souza ANDRADE-FILHO Faculdade de Ciências Médicas de Minas Gerais Belo Horizonte, Minas Gerais, Brasil E-mail: [email protected]
REFERENCES
1. Coutinho AD. A new dynamic approach to the diagnosis of Symmers’ fibrosis in schistosomiasis by ultrasound. Rev Inst Med Trop Sao Paulo. 1990;32:73-7. 2. Kumar V, Abbas AK, Fausto N. Robbins and Cotran pathologic basis of disease. 7th ed. Philadelphia: Elsevier Saunders; 2005. p. 408-9. 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. Symmers WSC. Note on a new form of liver cirrhosis due to the presence of the ova of bilharzia haematobia. J Pathol Bacteriol. 1904;9:237-9.