Use of laboratory methodologies to confirm an outbreak of acute diarrheal disease caused by multiple pathogens / Uso de metodologias laboratoriais para confirmação de surto de doença diarreica aguda causada por múltiplos patógenos

The aim of this study was to investigate an outbreak caused by protozoa, which occurred in a municipality in the Brazil southern region. The investigations were carried out analyzing 47 fresh stool samples and 26 water samples by parasitological and molecular methods, as well as, direct immunofluorescence. After the filtrations of water samples and purification of stool samples, the concentrates were evaluated microscopically for presence of parasites. Molecular analyses were performed by polymerase chain reaction (PCR) for DNA detection of Giardia spp., Cryptosporidium parvum, C. hominis and Cyclospora cayetanensis. Out of 26 water samples, 30.8% (8/26) had waterborne protozoa and C. cayetanensis was the most prevalent (15.5%). Out of the 47 stool samples, 23.4% (11/47) were infected with C. cayetanensis and Giardia spp. The results showed that backwash water samples from filters of the Water Treatment Station were contaminated with C. cayetanensis, C. hominis and Giardia spp., suggesting the contamination of water sources with human waste brought by sewage. These results show the importance of protozoa investigation in water and stool samples by laboratory methodologies principally in outbreaks causing acute diarrheal disease (AU).

O objetivo do presente estudo foi investigar um surto causado por protozoários, ocorrido em um município da região sul do Brasil. As investigações foram realizadas analisando 47 amostras de fezes frescas e 26 amostras de água por métodos parasitológicos, moleculares e de imunofluorscência direta. Após as filtrações das amostras de água e purificação das amostras de fezes, os concentrados foram avaliados microscopicamente a procura de parasitas. A seguir, foram analisadas, pela reação em cadeia da polimerase (PCR), a detecção de DNA de Giardia spp., Cryptosporidium parvum, C. hominis e Cyclospora cayetanensis. Das 26 amostras de água, 30,8% (8/26) apresentaram protozoários de veiculação hídrica, sendo que, C. cayetanensis foi o mais prevalente (15,5%). Das 47 amostras de fezes, 23,4% (11/47) estavam infectadas por C. cayetanensis e Giardia spp. Os resultados mostraram que as águas de retrolavagem dos filtros da Estação de Tratamento de Água estavam contaminadas com C. cayetanensis, C. hominis e Giardia spp. sugerindo a contaminação dos mananciais com dejetos humanos trazidos pelo esgoto. Estes resultados mostram a importância da investigação de protozoários em água e fezes por metodologias laboratoriais, principalmente em surtos que causam doença diarreica aguda (AU).

Determination of the viability of Toxoplasma gondii oocysts by PCR real-time after treatment with propidium monoazide.

This study aimed to investigate a methodology for discriminating viable and non-viable T. gondii oocysts in water. Analyses included two steps: (i) microscopic investigation with vital dyes; (ii) molecular investigation, using a real time PCR (qPCR), after parasite treatment (or not) with propidium monoazide (PMA). The method was called qPCR-PMA. Oocyst aliquots were incubated (15 min) at 25 ºC or 100 ºC and analyzed by microscopy, after trypan blue and neutral red staining. Microscopic investigation determined viable and non-viable oocysts. For the molecular investigation, both aliquots of oocysts were treated with PMA. Non-viable oocysts, after PMA treatment, exhibited an inhibition of DNA amplification by qPCR. Although analyses were carried out with oocysts treated experimentally, these results suggest that qPCR-PMA can be a useful strategy to distinguish viable and non-viable T. gondii oocysts in water safety testing, showing if water is safe to drink.

Assessment of Consumer Exposure to Salmonella spp., Campylobacter spp., and Shiga Toxin-Producing Escherichia coli in Meat Products at Retail in the City of Sao Paulo, Brazil.

Meat products may be vehicles of bacterial pathogens to humans, and Salmonella spp., Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC) are the most relevant. The aim of this study was to generate data on prevalence of these three pathogens in 552 samples of meat products (hot dogs, pork sausages, raw ground beef, and raw chicken legs) sold at retail in the city of Sao Paulo, Brazil. Salmonella spp. was detected in 5.8% (32/552) of samples, comprising pork sausages 62.5% (20/32) and chicken legs 37.5% (12/32). The counts of Salmonella spp. were low, ranging from < 0.3 to 9.3 × 10 most probable number per gram and the most frequent serovars were Salmonella Typhimurium (28.1%), Salmonella I 4,[5],12:i:- (15.6%), Salmonella Enteritidis (12.5%), Salmonella Derby, and Salmonella Brandenburg (9.4%). Campylobacter spp. was detected in 33 samples (6.0%), comprising chicken legs (82%) and ground beef (18%). All samples were negative for STEC. These results suggest that meat products when subjected to inadequate cooking and/or cross-contamination with other products ready for consumption can lead to occurrence of outbreaks, highlighting the risks associated with them.

PREVALENCE OF DRUG RESISTANCE AND VIRULENCE FEATURES IN Salmonella spp. ISOLATED FROM FOODS ASSOCIATED OR NOT WITH SALMONELLOSIS IN BRAZIL / Prevalência de resistência antimicrobiana e características de virulência em Salmonella spp. isoladas de alimentos associados ou não com salmonelose no Brasil

Salmonella is the most common etiological agent of cases and outbreaks of foodborne diarrheal illnesses. The emergence and spread of Salmonella spp., which has become multi-drug resistant and potentially more pathogenic, have increased the concern with this pathogen. In this study, 237 Salmonella spp., associated or not with foodborne salmonellosis in Brazil, belonging mainly to serotype Enteritidis, were tested for antimicrobial susceptibility and the presence of the virulence genes spvC, invA, sefA and pefA. Of the isolates, 46.8% were sensitive to all antimicrobials and 51.9% were resistant to at least one antimicrobial agent. Resistance to more than one antimicrobial agent was observed in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%) and nalidixic acid (16.9%). No strain was resistant to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxacin and imipenem. The invA gene was detected in all strains. Genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The gene sefA was detected in 31.6% of the strains and only among S. Enteritidis. Resistance and virulence determinants were detected in Salmonella strains belonging to several serotypes. The high rates of antibiotic-resistance in strains isolated from poultry products demonstrate the potential risk associated with the consumption of these products and the need to ensure good food hygiene practices from farm to table to reduce the spread of pathogens relevant to public health.

Salmonella é o agente etiológico mais comumente envolvido em casos e surtos de doenças diarréicas de origem alimentar. A preocupação com este patógeno é, ainda, maior quando se verifica o surgimento e a disseminação de cepas multirresistentes e potencialmente mais patogênicas. Neste estudo, 237 cepas Salmonella spp., associadas ou não com casos ou surtos de salmonelose e pertencentes, principalmente, ao sorovar Enteritidis, foram avaliadas quanto ao perfil de susceptibilidade antimicrobiana e presença dos genes de virulência spvC, invA, sefA e pefA. Entre as cepas avaliadas, 46,8% foram sensíveis a todos os agentes antimicrobianos e 51,9% foram resistentes a pelo menos uma droga. Multirresistência foi observada em 10,5% das cepas. As maiores taxas de resistência foram observadas para estreptomicina (35,9%) e ácido nalidíxico (16,9%). Não foram detectadas cepas resistentes à cefoxitina, cefalotina, cefotaxima, amicacina, ciprofloxaxina e imipenem. O gene invA foi detectado em todas as cepas de Salmonella. Os genes spvC e pefA foram encontrados em 48,1% e 44,3% das cepas, respectivamente. O gene sefA foi detectado em 31,6% das cepas, estando presente somente entre as cepas de S. Enteritidis. Resistência antimicrobiana e marcadores de virulência foram detectados em cepas de Salmonella pertencentes a diversos sorovares. A alta taxa de resistência antimicrobiana verificada em cepas isoladas de frangos e derivados demonstra o potencial risco associado ao consumo destes produtos e a necessidade de se assegurar boas práticas de higiene em toda cadeia produtiva para reduzir a disseminação de patógenos relevantes para a saúde pública.

Prevalence and populations of Listeria monocytogenes in meat products retailed in Sao Paulo, Brazil.

This study evaluated the prevalence of the populations and serotypes of Listeria monocytogenes in 552 refrigerated samples of ground beef, chicken leg, hot dog, and pork sausage collected in supermarkets in the city of Sao Paulo, SP, Brazil, between May 2008 and July 2009. The supermarkets were selected after stratification by geographical region and by random draw. Tests for presence and enumeration of L. monocytogenes were based on ISO 11290-1:1996/Amd.1:2004 and ISO 11290-2:1998 methods, respectively. Listeria spp. were detected in 469 (85.0%) of the studied meat products. The most frequently isolated species was L. innocua (64.1%), followed by L. monocytogenes (48.7%), L. welshimeri (13.4%), L. seeligeri (7.1%), L. ivanovii (0.2%), and L. grayi subspecies murrayi (0.2%). L. monocytogenes was detected in 269 (48.7%) samples, with highest prevalence in ground beef (59.4%) followed by chicken legs (58.0%), pork sausages (39.8%), and hot dogs (37.7%). The populations were <10(2) colony-forming units/g in the majority of samples (62.5%). Prevalence of serotypes varied according to the type of meat product. These data are relevant for estimating the risks of listeriosis associated with consumption of meat products in Sao Paulo, and for establishing science-based intervention strategies aimed at reducing these risks, especially for pregnant women and immunocompromised individuals.

Prevalence of drug resistance and virulence features in Salmonella spp. isolated from foods associated or not with salmonellosis in Brazil.

Salmonella is the most common etiological agent of cases and outbreaks of foodborne diarrheal illnesses. The emergence and spread of Salmonella spp., which has become multi-drug resistant and potentially more pathogenic, have increased the concern with this pathogen. In this study, 237 Salmonella spp., associated or not with foodborne salmonellosis in Brazil, belonging mainly to serotype Enteritidis, were tested for antimicrobial susceptibility and the presence of the virulence genes spvC, invA, sefA and pefA. Of the isolates, 46.8% were sensitive to all antimicrobials and 51.9% were resistant to at least one antimicrobial agent. Resistance to more than one antimicrobial agent was observed in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%) and nalidixic acid (16.9%). No strain was resistant to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxacin and imipenem. The invA gene was detected in all strains. Genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The gene sefA was detected in 31.6% of the strains and only among S. Enteritidis. Resistance and virulence determinants were detected in Salmonella strains belonging to several serotypes. The high rates of antibiotic-resistance in strains isolated from poultry products demonstrate the potential risk associated with the consumption of these products and the need to ensure good food hygiene practices from farm to table to reduce the spread of pathogens relevant to public health.

Pesquisa de bacteriófagos em água suspeita de contaminação por vírus da hepatite A / Study of bacteriophages in water suspected of contamination by hepatitis A virus

Os vírus entéricos, quando presentes no trato gastrointestinal de indivíduos infectados, são eliminados pelas fezes em grandes quantidades podendo contaminar, direta ou indiretamente, as águas destinadas ao consumo humano. O ser humano é o único hospedeiro natural do vírus da hepatite A. A detecção de colifago e de fago de Shigella sonnei em água pode ser indicativa da presença destes vírus. No presente estudo foi realizado o diagnóstico laboratorial presuntivo de um surto de hepatite A, pela pesquisa de bacteriófagos, em uma amostra de água de poço e o estudo da sobrevivência dos fagos de S. sonnei em água. A análise revelou resultado positivo para fagos de Shigella e negativo para colifagos. Quanto aos ensaios de sobrevivência, a presença dos fagos de S. sonnei foi detectada na água do poço naturalmente contaminada até o nono dia de conservação em temperatura ambiente.

The enteric viruses are present in the gastrointestinal tract of infected individuals, being eliminated in feces in large quantities and can directly or indirectly contaminate water intended for human consumption. Humans are the only natural hosts for hepatitis A. Detection of coliphages and phage of Shigella sonnei in water may indicate the presence of these viruses. In the present study was done the presumptive laboratory diagnosis of an outbreak of hepatitis A through search of bacteriophages in a sample of well water and the study of survival of shigelafagos in water. The analysis revealed positive for phages of S. sonnei and negative for coliphages. For the tests of survival, the presence of phages of S. sonnei was detected in naturally contaminated well water until the ninth day of storage at room temperature.

Phylogenetic analysis of a near-full-length sequence of an erythrovirus genotype 3 strain isolated in Brazil.

Human parvovirus B19 is the only member of the genus Erythrovirus that causes human disease. Recent findings of several strains with considerable sequence divergence from B19 have suggested a new classification for parvovirus genotypes as 1 (B19), 2 (A-6 and LaLi) and 3 (V9). In their overall DNA sequence, the three genotypes differ by approximately 10%. Here, we report the isolation of a genotype-3-related strain named BR543 during a prospective study conducted in Sao Paulo, Brazil. Analysis of the nearly full-length genome sequence of BR543 indicates that this B19 variant sequence clusters with Gh2768, a strain from Ghana belonging to subtype 3b, and showed mostly synonymous substitutions.

Comparison of HeLa-I, HEp-2 and NCI-H292 cell lines for the isolation of human respiratory syncytial virus (HRSV).

Generally, laboratory diagnosis of viral respiratory infections utilizes virus isolation in cell culture and immunofluorescence assays. In this study, three cell lines (HEp-2, NCI-H292 and HeLa-I) were used for HRSV isolation of strains obtained from patients admitted at HU-USP with respiratory tract disease. HRSV was isolated in 46% (37) of 80 specimens inoculated in HeLa-I, 48% (39) in HEp-2, and 36.3% (29) in NCI-H292. Immunofluorescence was considered the gold standard and yielded 53% positive (43). The results from both methods combined had better sensitivity (73.2%) compared to either method alone. Comparing results between the cell lines with HEp-2 cells as the benchmark, the greatest sensitivity (72.2%) was observed in HeLa-I. This data shows that HeLa-I is adequate for HRSV isolation, giving results similar to the HEp-2 cells. The combined use of the HEp-2, HeLa-I and NCI-H292 cells improve the detection of HRSV.

The "pressure pan" evolution of human erythrovirus B19 in the Amazon, Brazil.

To understand the evolutionary dynamics of human parvovirus B19, we analyzed VP1 and VP2 gene sequences of B19 sampled from Belém (Amazon), the city of São Paulo, Brazil and globally. Our analysis revealed a strikingly different pattern of evolutionary change for those viral lineages introduced into Belém, which exhibited a higher rate of nonsynonymous substitutions compared to those viruses sampled from other locations. We propose that difference this is due to the high prevalence of B19 in Belém (up to 85%) compared to other locations (prevalences of approximately 50%), which imposes a more intense selection pressure. Hence, those B19 lineages introduced into Belém experienced an elevated rate of amino acid change, driven by positive selection, in order to generate serial re-infections in a small web of transmission, which can be thought of as an evolutionary "pressure pan".

Papular-purpuric "gloves and socks" syndrome caused by parvovirus B19 infection in Brazil: a case report.

Papular-purpuric "gloves and socks" syndrome (PPGSS) is a novel, rare, self-limiting dermatosis caused by human parvovirus B19. It consists of pruritic edema and erythema of the hands and feet in a gloves-and-socks distribution, and it is associated with oral lesions and fever. We present a case of PPGSS in a 22-year-old Brazilian woman. Clinical and laboratory evaluation, including serological tests, PCR and gene sequencing, confirmed the presence of human parvovirus B19.

Papular-purpuric "gloves and socks" syndrome caused by parvovirus B19 infection in Brazil: a case report

Papular-purpuric "gloves and socks" syndrome (PPGSS) is a novel, rare, self-limiting dermatosis caused by human parvovirus B19. It consists of pruritic edema and erythema of the hands and feet in a gloves-and-socks distribution, and it is associated with oral lesions and fever. We present a case of PPGSS in a 22-year-old Brazilian woman. Clinical and laboratory evaluation, including serological tests, PCR and gene sequencing, confirmed the presence of human parvovirus B19.

Testes in vitro como alternativa aos testes in vivo de Draize / In vitro tests used as an alternative to Draize in vivo tests

Os procedimentos descritos por Draize deram origem aos testes de irritação ocular e cutânea adotados internacionalmente para avaliar produtos e substâncias. Entretanto, eles são criticados por motivos éticos, devido a crueldade com os animais, mesmo após diferentes modificações terem sido propostas nos protocolos originais. Metodologias alternativas têm sido estudadas para avaliar a toxicidade de produtos usados em seres humanos. Entre as mais citadas encontram-se as que utilizam organismos inferiores, células vivas de mamíferos, sistemas organotípicos e substâncias inertes, além de bancos de dados informatizados e programas que avaliam a toxicidade pela determinação de relação estrutura-atividade. Os métodos utilizando células vivas têm sido muito utilizados para predizer com segurança a irritação, contribuindo para a redução do número de animais utilizados nos testes in vivo. Até o momento, não existem métodos validados para substituir os ensaios de irritação ocular e cutânea, mas somente para avaliar substâncias corrosivas

Detecçäo de vírus Dengue sorotipos 1 e 2 pela técnica de immunoblotting utilizando proteínas recombinantes / Detection of dengue virus serotypes 1 and 2 by immunoblotting techniques using recombinant proteins

O vírus dengue säo patógenos que vêm afetando milhöes de pessoas durante os dois últimos séculos. No presente trabalho, comparou-se a técnica tradicional de MAC-ELISA e o teste de Immunoblotting, utilizando-se proteínas recombinantes, para pesquisa de anticorpos da classe M, contra os vírus Dengue sorotipos 1 e 2. Foram testadas 100 amostras de soro de pacientes, com suspeita clínica de dengue, por MAC-ELISA e Immunoblotting. Estes soros quando submetidos ao MAC-ELISA resultaram 56 positivos para dengue e 44 negativos. Estas mesmas amostras avaliadas por Immunoblotting erevelaram 56 soros negativos, 36 soros positivos para DEN-1, 6 para DEN-2 e 2 para DEN-1 e 2. Proteínas recombinantes (DEN-1 e DEN-2) utilizadas como antígeno em Immunoblotting possibilitam um diagnóstico diferencial dos sorotipos de dengue circulantes em área, enquanto o teste MAC-ELISA apesar de sensível e rápido näo tipifica o sorotipo de dengue, dado importante durante os períodos de epidemias. (AU)

Dengue viroses (DEN-1 to 4) are human pathogens affeeting millions of people In the last two eenturies. Results of a eomparative study between the traditional MAC-ELISA method and the Immunoblotting teehniques using reeombinant proteins of DEN-l and DEN-2 to deteet vírus speei- fie class M Immunoglobulins, are reported in this paper. One hundred sera samples were studied. Fifty six samples were positive to dengue by MAC-ELISA and 44 were positive by Immunoblotting teehni- que: 36 to DEN-1, 6 to DEN-2 and 2 to DEN-1 and 2 sorotypes. So, the serotype identifieation of the dengue vírus eireulantig in a determined área is possible by using these reeombinant proteins in Immunoblotting teehniques. These data are importante for epidemiologieal surveillanee mainly during the oeeurrenee of an epidemie. (AU)

Detecçäo e tipagem de vírus dengue sorotipos 1 e 2 por multiplex RT-PCR / Multiplex RT-PCR used for detection and tipification of dengue viruses sorotypes 1 and 2

Os vírus Dengue säo arbovírus da família Flaviviridae, com 4 sorotipos antigenicamente distintos. Variaçöes genéticas intraespecíficas entre os mesmos sorotipos, inclusive em uma mesma epidemia, estäo bem estabelecidas. O desenvolvimento da técnica de polimenizaçäo em cadeia (PCR) é uma alternativa na identificaçäo dos vírus Dengue. Neste estudo construímos dois novos pares de primers para os sorotipos 1 e 2, sem a ajuda de programas de computador. Foram utilizadas como modelo as seqüências genômicas de DEN-1 (Western Pacific), Nauru Island e Den-2 New Guinea C. Os primers sense 5' ACA AAA AGT GGA GAC CTG GGC TC 3'(DIS) e complementar 5'GTC TAT TCC AAG TCT CTT GGG 3' (DIA) correspondem respectivamente às posições 769 a 791 e 1607 a 1587 do genoma do vírus DEN-1. Estas seqüência reconhecem parte das regiões de membrana (M) e proteína estrutural (E) do genoma do sorotipos 1 e contem 838 pares de bases. A seqüência de primers de DEN-2 foi 5' TGA AGG GGA CGG TTC TCC ATG 3' (D2S) homólogos aos nucleotídios 1838 a 1859 e 5' GAC TCC CAC CAA TAC TAG TGA CAC 3' (D2A) correspondendo às posições 2312 a 2288. O produto da amplificaçäo foi de 474 pares de bases correspondendo a uma porçäo do genoma responsável pela síntese da proteína E. A especificidade dos primers foi avaliada por multiplex RT-PCR

Presença de vírus endogéno na linhagem celular Aedes albopictus Clone C6/36 näo infectadas / Presence of an endogenous virus in uninfected Aedes albopictus Cell, Clone C6/36

As linhagens celulares obtidas a partir de larvas do mosquito Aedes albopictus apresentam-se naturalmente infectadas por vírus. O clone C6/36 dessas células, obtido por Igarashi em 1978, mostrou estar livre de vírus e tem sido usado para isolamento do vírus Dengue na maioria dos laboratórios especializados. Em nossos estudos com esse clone, após diferentes períodos de incubaçäo, detectamos a presença de um vírus endogéno. Sobrenadante de cultura de células C6/36, após o 12§ dia de repique, analisado por coloraçäo negativa em microscopia eletrônica, revelou a presença de partículas virais sem envoltório, medindo cerca de 20nm. Esse mesmo teste, quando realizado com células após o 2§, 6§ e 10§ dias de incubaçäo, resultou negativo. As células também foram submetidas à imunofluorescência indireta utilizando anti soros para vários grupos de arbovírus. Os resultados foram negativos, descartando a possibilidade de contaminaçäo laboratorial. A presença de vírus endógeno em células C6/36 pode ocorrer em diferentes laboratórios, sem detectada. Nossos resultados mostram que esse vírus endógeno näo influencia o isolamento do vírus Dengue e a replicaçäo das células. Em razäo da ausência de dados, o emprego dessas células para isolamento de outros vírus deve ser cuidadosamente avaliado

Simultaneous infection with dengue 1 and 2 in a brazilian patient

Epidemias de dengue vem ocorrendo em varios estados brasileiros desde 1986 envolvendo os sorotipos 1 (DEN-1) e 2 (DEN-2). Em vista dos poucos casos de dupla infeccao documentados na literatura, relatamos aqui um caso de infeccao simultanea com DEN-1 e DEN-2 em uma paciente residente no municipio de Miranda, Estado de Mato Grosso do Sul, Regiao Centro-Oeste do Brasil. O DEN-1 foi introduzido nesse Estado em 1989 e o DEN-2 em 1996, com a circulacao de ambos em alguns municipios. Esta dupla infeccao foi identificada por isolamento de virus e imunofluorescencia indireta usando anticorpos monoclonais e confirmada pela reacao em cadeia da polimerase (PCR). Este e o primeiro caso de infeccao simultanea com os sorotipos 1 e 2 documentados no Brasil

Dengue: revisäo / Dengue: revision

Este trabalho é uma revisäo sobre vírus Dengue. Numa tentativa de rever os conhecimentos acumulados, apresentando aspectos moleculares, diagnósticos e clínicos

Growth and maintenance of Aedes Albopictus cell line, clone C6/36, in different media

The sensitivity of Aedes albopicts cell line, clone C6/36, to arbovirus isolation and growth has been being shown by several authors. The present work has verified which, among several media under the same conditions, would be the most efficient one for C6/36 cultivation and viral isolation. The L-15 medium has proved to be the best among others (MEM,DMEM and 199) with the quickest viral isolation(DEN-1). Also, in this medium, the samples could be observed until the 14th day, without significative cellular death. The results reccommend L-15 medium as the most efficient and economic one for the purposes