Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay.

BACKGROUND: With a conservatively estimated 1 million cases of leptospirosis worldwide and a 5-10% fatality rate, the rapid diagnosis of leptospirosis leading to effective clinical and public health decision making is of high importance, and yet remains a challenge. METHODOLOGY: Based on parallel, population-based studies in two leptospirosis-endemic regions in Brazil, a real-time PCR assay which detects lipL32, a gene specifically present in pathogenic Leptospira, was assessed for the diagnostic effectiveness and accuracy. Patients identified by active hospital-based surveillance in Salvador and Curitiba during large urban leptospirosis epidemics were tested. Real-time PCR reactions were performed with DNA-extracted samples obtained from 127 confirmed and 23 unconfirmed cases suspected of leptospirosis, 122 patients with an acute febrile illness other than leptospirosis, and 60 healthy blood donors. PRINCIPAL FINDINGS: The PCR assay had a limit of detection of 280 Leptospira genomic equivalents/mL. Sensitivity for confirmed cases was 61% for whole blood and 29% for serum samples. Sensitivity was higher (86%) for samples collected within the first 6 days after onset of illness compared to those collected after 7 days (34%). The real-time PCR assay was able to detect leptospiral DNA in blood from 56% of serological non-confirmed cases. The overall specificity of the assay was 99%. CONCLUSIONS: These findings indicate that real-time PCR may be a reliable tool for early diagnosis of leptospirosis, which is decisive for clinical management of severe and life-threatening cases and for public health decision making.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9 percent) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9 percent), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3 percent), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil.

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9%) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9%), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3%), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Molecular epidemiology of the human group A rotavirus in the Paraná State, Brazil

From January/2000 to December/2003, 550 diarrheic fecal samples from the children and adults were collected in several geographical regions of Paraná State, Brazil. The enzyme immunoassay showed 120 (21.8 percent) samples positive for the group A rotaviruses. One hundred and fourteen samples were genotyped by multiplex-nested-PCR assay. The highest frequency (77.5 percent) of the positive samples (n=93) was observed in the children under 5 years old. Rotavirus diarrhea was more frequent in the cold and dry seasons of the four evaluated years. The most frequent genotypes were: G1 (50.9 percent), G4 (9.6 percent), G9 (7.0 percent), G2 (1.7 percent), G3 (0.9 percent), P[ 8] (71.9 percent), and P[ 4] (3.5 percent). The P[ 8] G1 (46.5 percent) and P[ 8] G4 (9.6 percent) were the main combinations found to P and G genotypes. The mixed infections, characterized by the rotaviruses with more than one genotype G or P, and nontypeable rotavirus were observed in 8.8, 3.5, and 16.7 percent of the samples, respectively. The identification of the G9 genotype in the rotavirus strains tested along the four years of studies ratifies the emergency of this genotype also in Paraná State, South region of Brazil, as the worldwide.

No período de janeiro de 2000 a dezembro de 2003 foram colhidas, em várias regiões geográficas do Estado do Paraná, 550 amostras fecais de crianças e adultos com quadro clínico de diarréia aguda. Por meio de ensaio imunoenzimático comercial, 120 (21,8 por cento) amostras foram positivas para o rotavírus grupo A. Dessas, 114 amostras foram genotipadas por meio da multiplex-nested-PCR. A maior freqüência (77,5 por cento) de amostras positivas (n=93) foi observada em crianças menores de cinco anos de idade. A maior concentração de casos positivos para o rotavírus ocorreu nos meses frios e secos dos quatro anos avaliados. Os genotipos de maior ocorrência foram: G1 (50,9 por cento), G4 (9,6 por cento), G9 (7,0 por cento), G2 (1,7 por cento), G3 (0,9 por cento), P[ 8] (71,9 por cento) e P[ 4] (3,5 por cento). P[ 8] G1 (46,5 por cento) e P[ 8] G4 (9,6 por cento) foram as associações de genotipos P e G mais encontradas. Infecções mistas, caracterizadas por estirpes de rotavírus com mais de um genotipo G, P e amostras não-genotipadas foram observadas, respectivamente, em 8,8, 3,5 e em 16,7 por cento das amostras. De forma semelhante ao que vem ocorrendo em várias partes do mundo a identificação do genotipo G9 em estirpes de rotavírus identificadas ao longo dos quatro anos do estudo ratifica a emergência desse genotipo também no Estado do Paraná, Brasil.

Enterovirus meningitis in Brazil, 1998-2003.

Acute viral infections of the central nervous system (CNS) such as acute flaccid paralysis, meningitis, and encephalitis, are responsible for a high morbidity, particularly in children. Non-Polio enteroviruses (NPEV) are known to be responsible for over 80% of viral meningitis in which the etiologic agent is identified. In the present study, we show the frequency of enterovirus meningitis in Brazil from December 1998 to December 2003. Enterovirus were isolated from 162 (15.8%), of a total of 1,022 cerebrospinal fluid (CSF) specimens analyzed. Echovirus 30 was identified in 139 of these isolates (139/162-85.2%). Other identified enteroviruses were: Coxsackievirus B5 (3.7%), Echovirus 13 (3.7%), Echovirus 18 (3%), Echovirus 6 (1.2%), Echovirus 25 (1.2%), Echovirus 1 (0.6%), and Echovirus 4 (0.6%). Patients's age ranged from 28 days to 68 years old. The most frequent symptoms were fever (77%), headache (69.5%), vomiting (71.3%), neck stiffness (41.3%), convulsion (7.1%), and diarrhea (3.7%). Although, the majority of the patients recovered without any complication or permanent squeal, five deaths occurred. Throughout the surveillance period, five viral meningitis outbreaks were confirmed: four in the Southern Brazil and one in the Northeast Brazil. Echovirus 30 was responsible for four out of the five outbreaks while Echovirus 13 caused the fifth one. Besides the outbreaks, 734 sporadic cases were also identified during the study period and 59 of these were positive for virus isolation (8%). Echovirus 30 accounted for 70% of the isolates. Our results showed that Echovirus 30 was the most prevalent etiological agent of viral meningitis in Brazil, causing both outbreaks and sporadic cases.

Clinical survey of hantavirus in southern Brazil and the development of specific molecular diagnosis tools.

Hantavirus pulmonary syndrome (HPS) is an emerging disease caused by an increasing number of distinct hantavirus serotypes found worldwide. It is also a very severe immune disease. It progresses quickly and is associated with a high mortality rate. At the prodrome phase, hantavirosis symptoms can resemble those of other infectious diseases such as leptospirosis and influenza. Thus, prognosis could be improved by developing a rapid and sensitive diagnostic test for hantavirus infection, and by improving knowledge about clinical aspects of this disease. This study describes clinical features and laboratory parameters throughout the course of HPS in 98 patients. We report the seasonality and regional distribution of this disease in Paraná State, Brazil during the last seven years. In addition, we evaluated a specific molecular diagnostic test based on a nested reverse transcriptase-polymerase chain reaction for the detection of hantaviruses circulating in Brazil.

Avaliacao da Protecao Induzida pela vacina Oral contra a Poliomielite, realizada em Curitiba, Parana / Evaluation of the protection induced by oral vaccine counter the poliomyelitis, realized in Curitiba,Parana

Foi estudada a protecao induzida pela vacina oral antipoliomielite em 171 criancas de 2 a 24 meses de idade (amostras pareadas). A determinacao dos titulos de anticorpos especificos antipoliovirus 1, 2 e 3, pre e pos-vacinacao foi realizada pela tecnica de soroneutralizacao. As criancas foram agrupadas de acordo com a dose de vacina que estava agendada para receber no momento em que o estudo foi realizado. Os grupos foram constituidos da seguinte forma: A - criancas que estavam recebendo a 1 dose da vacina oral antipoliomielite; B - que estavam recebendo a 2 dose; C - que estavam recebendo a 3 dose e D - queestavam recebendo a 4 dose. Observou-se que um percentual elevado de criancas jaapresentava titulo de anticorpos protetores, antes mesmo de receber a 1 dose da vacina (58,7 por cento para poliovirus tipo 1; 55,6 por cento para poliovirus tipo 2 e 31,7 por cento para poliovirus tipo 3). As taxas de soropositivos obtidas apos a vacinacao, em cada grupo estudado, foram: A - 86,7 por cento ; 86,7 por cento e 71,1 por cento para os poliovirus 1, 2 e 3 respectivamente; B - 100 por cento ; 95,7 por cento e 91,5 por cento para os poliovirus 1, 2 e 3 respectivamente; C - 100 por cento para os tres tipos de poliovirus; D - 100 por cento ; 100 por cento e 97,6 por cento para os poliovirus 1, 2 e 3 respectivamente. Esses resultados confirmam que a vacina utilizada em nosso meio e eficaz, sendo que as taxas encontradas sao semelhantes as obtidas em paises desenvolvidos

Induçäo de anticorpos específicos pela vacina anti-sarampo: estudo em crianças nutridas e desnutridas de 6 a 24 meses / Induction of specific antibodies by measles vaccine: study in normal and undernourished infants from 6 to 24 months

Foi estudada a induçäo de anticorpos anti-sarampo em 223 crianças nutridas e desnutridas vacinadas entre 6 e 24 meses e naquelas que permaneceram soronegativas com 1 dose da vacina realizada antes dos 12 meses que foram revacinadas após os 12 meses. A determinaçäo de anticorpos anti-sarampo foi realizada pelas técnicas de inibiçäo da hemaglutinaçäo e de soroneutralizaçäo. Observamos que a taxa de soroconvençäo aumentou progressivamente com a idade, sendo de 43% aos 6 meses e de 80% aos 15 meses. A taxa de soroconversäo em crianças marasmáticas foi semelhante à obtida em crianças nutridas, concordando com a literatura que vem demonstrando que crianças desnutridas näo apresentam alteraçäo na capacidade de resposta humoral à vacina. Utilizando os dados de mortalidade por sarampo e as taxas de soroconversäo à vacina, idealizou-se um modelo hipotético para a avaliaçäo da aplicaçäo da vacina em diferentes idades e suas conseqüências em têrmos de mortalidade, e observou-se que quando a vacina é realizada em idades precoces (6-7 meses) o número de óbito esperado é inferior ao esperado quando a vacina é realizada em idades posteriores (9-11 meses), indicando que a proposta atual do Ministério da Saúde de aplicar dose única aos 9 meses, teoricamente aumentaria o risco de mortalidade. Considerando o número de crianças protegidas com 1 dose de vacina aplicada antes dos 12 meses e o número de crianças protegidas com a revacinaçäo a taxa de soroconversäo foi de 84,3%

Inquérito sorológico de sarampo em crianças de 5 a 15 meses de idade, em Curitiba e Säo José dos Pinhais, Estado do Paraná, Brasil, 1983 / Serological survey of measles in children from 5 to 15 months, in Curitiba and Sao Jose dos Pinhais, State of Parana, Brazil, 1983

Inquérito sorológico, realizado para avaliar os motivos da alta incidência e surtos freqüentes de sarampo nas cidades de Curitiba e Säo José dos Pinhais - Paraná (Brasil), revela que entre 7 e 10 meses de idade só 44% das crianças vacinadas estäo protegidas e que entre o 11o. e 15o. mês o índice de proteçäo se eleva para 65,1%. As näo vacinadas, ao 5o., 6o. e 7o. mês de idade apresentaram percentuais de proteçäo de 56,7, 28,6 e 14,8, respectivamente. As crianças que foram vacinadas ao 7o. e 8o. mês apresentaram percentuais de proteçäo de 50,8 e as vacinadas a partir do 9o. mês de 75,75%. As médias geométricas dos títulos de anticorpos também foram menores nas vacinas antes dos 9 meses, em relaçäo as vacinadas a partir desta idade