Serotipos de Streptococcus pneumoniae responsables de enfermedad invasiva en niños cubanos / Streptococcus pneumoniae serotypes responsible for invasive disease affecting Cuban children

Introducción: Streptococcus pneumoniae es causa importante de morbilidad y mortalidad a nivel mundial, fundamentalmente en niños < 5 años. En Cuba aún no se introdujo la vacunación antineumocócica, pero desde 2014, con el propósito de sentar las bases para la evaluación de su impacto, se lleva a cabo un protocolo de vigilancia centinela de la enfermedad neumocócica invasiva en niños ≤ 5 años. Objetivos: notificar los serotipos de S. pneumoniae responsables de enfermedad neumocócica invasiva en la población pediátrica cubana, y valorar la contribución de ese protocolo a la vigilancia. Métodos: se determinaron los serotipos de todos los aislamientos pediátricos invasivos y los recuperados de otitis media aguda, recibidos en el Laboratorio Nacional de Referencia de Neumococo, del Instituto de Medicina Tropical Pedro Kourí, entre 2013-2015. Se utilizó el método de hinchazón capsular empleando el juego de reactivos Pneumotest. Resultados: se notificaron 141 aislamientos invasivos en edad pediátrica. Predominaron los responsables de neumonías (76 vs. 49 aislamientos meníngeos) y la mayoría de estos fueron aportados por los hospitales involucrados en la vigilancia centinela (75 por ciento; 57/76). El 85,8 por ciento de los aislamientos quedaron contenidos en siete serotipos, que por orden de frecuencia fueron: 14, 19A, 6A, 19F, 6B, 3 y 23F. La cobertura serotípica de las diferentes vacunas neumocócicas multivalentes con posibilidades de ser empleadas se estimó entre 54 y 90 por ciento. Conclusiones: tras la introducción de la vacunación cabría esperar una reducción de la enfermedad neumocócica invasiva debida a los serotipos contenidos en las vacunas conjugadas disponibles, pero se insiste en la necesidad de fortalecer la vigilancia clínico-epidemiológica que se hace hoy de esta entidad en el país(AU)

Introduction: Streptococcus pneumoniae is a significant cause of morbidity and mortality worldwide mainly in children younger than 5 years. The pneumococcal vaccination has not been yet put into practice in Cuba; however, since 2014 a protocol of sentinel surveillance of the invasive pneumococcal disease in children aged 5 years or less is being implemented to lay the foundations for the evaluation of the impact of this vaccine. Objectives: to report on the S. pneumoniae serotypes responsible for the invasive pneumococcal disease in the Cuban pediatric population and to assess the contribution of this protocol to surveillance. Methods: the serotypes of all the invasive pediatric isolates and the recovered ones of acute otitis media were determined by the national laboratory of pneumococcal reference of Pedro Kourí Institute of Tropical Medicine from 2013 to 2015. The capsular swelling method was used with the Penumotest reagent set. Results: one hundred and forty one invasive isolates were reported at pediatric ages. The isolates causing pneumonia predominated (76 vs. 49 meningeal isolates) and most of them were provided by hospitals involved in the sentinel surveillance project (57 out of 76; 75 percent). In this regard, 85.8 percent of isolates belonged to seven serotypes that were in order of frequency the following: 14, 19A, 6A, 19F, 6B, 3 and 23F. The serotype coverage of the various multivalent pneumococcal vaccines of possible use was estimated at 54-90 percent. Conclusions: after the introduction of the vaccinations, one might expect that a reduction of the invasive pneumococcal disease occurs due to the serotypes included in the available conjugate vaccines, but emphasis is made on the need of strengthening at present the clinical and epidemiological surveillance system for this disease nationwide(AU)

Método, software e banco de dados para sorotipagem molecular de Streptococcus pneumoniae visando o monitoramento da eficácia do programa de vacinação no Brasil

Noventa e dois sorotipos de Streptococcus pneumoniae já foram descritos, mas a vacina pneumocócica conjugada (PCV10) introduzida no calendário básico de vacinação do Brasil, em 2010, cobre somente os mais prevalentes no país. A substituição dos sorotipos vacinais após imunização em massa é uma grande preocupação e monitorar esse fenômeno requer métodos de sorotipagem eficientes e acessíveis. A Sorotipagem clássica de pneumococos baseada em antissoros produzidos em animais é trabalhosa, restrita a poucos laboratórios de referência, e não pode tipar isolados não capsulados. Alternativamente, os métodos de sorotipagem molecular avaliam os polimorfismos dos genes do cluster cps, que codificam enzimas chave para a síntese do CPS em Streptococcus pneumoniae. Em uma abordagem apropriada, cps-RFLP, os loci cps amplificados por PCR são digeridos com uma endonuclease gerando perfis únicos à eletroforese em gel de agarose, permitindo assim a identificação do sorotipo. Neste trabalho, nós combinamos abordagens in silico e in vitro para demonstrar que XhoII é a endonuclease mais discriminante para o método cps-RFLP, e para construir um banco de dados de perfis sorotipo-específico que acomodou a diversidade genética do lócus cps de 91 conhecidos sorotipos de pneumococos. O banco de dados de perfis cps-RFLP foi integrado ao Molecular Serotyping Tool (MST), software anteriormente publicado baseado em web-based para sorotipagem molecular. Usando XhoII, o método cpsRFLP obteve especificidade de 84,6% para sorotipagem e 100 % para sorogrupagem de pneumococos. Esta nova ferramenta pode representar uma colaboração relevante para vigilância epidemiológica em tempo real da diversidade de pneumococos em resposta a programas de imunização em massa.

Método, software e banco de dados para sorotipagem molecular de Streptococcus pneumoniae visando o monitoramento da eficácia do programa de vacinação no Brasil / Method, software and database for molecular serotyping of Streptococcus pneumoniae aimed at monitoring the effectiveness of the vaccination program in Brazil

Noventa e dois sorotipos de Streptococcus pneumoniae já foram descritos, mas a vacina pneumocócica conjugada (PCV10) introduzida no calendário básico de vacinação do Brasil, em 2010, cobre somente os mais prevalentes no país. A substituição dos sorotipos vacinais após imunização em massa é uma grande preocupação e monitorar esse fenômeno requer métodos de sorotipagem eficientes e acessíveis. A Sorotipagem clássica de pneumococos baseada em antissoros produzidos em animais é trabalhosa, restrita a poucos laboratórios de referência, e não pode tipar isolados não capsulados. Alternativamente, os métodos de sorotipagem molecular avaliam os polimorfismos dos genes do cluster cps, que codificam enzimas chave para a síntese do CPS em Streptococcus pneumoniae. Em uma abordagem apropriada, cps-RFLP, os loci cps amplificados por PCR são digeridos com uma endonuclease gerando perfis únicos à eletroforese em gel de agarose, permitindo assim a identificação do sorotipo. Neste trabalho, nós combinamos abordagens in silico e in vitro para demonstrar que XhoII é a endonuclease mais discriminante para o método cps-RFLP, e para construir um banco de dados de perfis sorotipo-específico que acomodou a diversidade genética do lócus cps de 91 conhecidos sorotipos de pneumococos. O banco de dados de perfis cps-RFLP foi integrado ao Molecular Serotyping Tool (MST), software anteriormente publicado baseado em web-based para sorotipagem molecular. Usando XhoII, o método cpsRFLP obteve especificidade de 84,6% para sorotipagem e 100 % para sorogrupagem de pneumococos. Esta nova ferramenta pode representar uma colaboração relevante para vigilância epidemiológica em tempo real da diversidade de pneumococos em resposta a programas de imunização em massa...

Molecular serovar characterization of Leptospira isolates from animals and water in Colombia / Caracterización molecular de serovariedades de Leptospira spp. aisladas de muestras de animales y agua en Colombia

Introduction: Leptospirosis is a bacterial disease transmitted directly or indirectly from animals to humans that may result in severe hemorrhagic, hepatic/renal and pulmonary disease. There are 20 known Leptospira species and hundreds of serovars, some of which belong to different species. It is essential to identify pathogenic Leptospira serovars and their potential reservoirs to prepare adequate control strategies. Objective: To characterize the Leptospira serovars isolated from rodents, dogs, pigs and water samples in Colombia. Materials and methods: Leptospira organisms were isolated and cultured, and pathogenic strains were identified using a polymerase chain-reaction (PCR). Leptospira DNA and Salmonella Braenderup H9812 (molecular weight standard) DNA were cleaved using NotI and subjected to pulsed-field gel electrophoresis (PFGE). The PFGE patterns were analyzed based on bacterial strain-typing criteria and Dice coefficients (DCs) between these isolates and over 200 Leptospira organisms isolated from other parts of the world. Results: All of the isolates were pathogenic strains, and five were genetically characterized. The P275 (84% DC) and P282 (95% DC) pig isolates were related to the Leptospira interrogans Pomona serovar; the I15 (DC: 100%) rat isolate was identical to the Leptospira interrogans Icterohameorrhagiae or Copenhageni serovars, while the C67 (64% DC) dog and A42 (60% DC) water isolates were not related (< 73.7% DC) to any of the 200 reference serovars; the closest serovars were the Leptospira noguchii Nicaragua and Orleans serovars, respectively. Conclusion: This was the first molecular characterization of Colombian Leptospira spp isolates; these isolates will be used to develop a Colombian diagnostic panel.

Introducción. La leptospirosis es una infección bacteriana transmitida directa o indirectamente de animales a humanos, la cual puede resultar en una enfermedad hemorrágica grave, hepática o renal y pulmonar. Hay 20 especies de Leptospira conocidas y cientos de serovariedades, algunas de las cuales pertenecen a diferentes especies. Es esencial identificar las serovariedades patógenas y sus reservorios potenciales para enfocar estrategias de control. Objetivo. Caracterizar las serovariedades de Leptospira aisladas de muestras de roedores, perros, cerdos y agua en Colombia. Materiales y métodos. Las cepas de leptospiras aisladas fueron identificadas como patógenas usando la reacción en cadena de la polimerasa (PRC). Sus ADN y el ADN de Salmonella Braenderup H9812 (marcador de peso molecular) fueron cortados con NotI y corridos en electroforesis de campo pulsado. Los patrones de la ECP se analizaron con base en los criterios de tipificación para cepas bacterianas y el coeficiente de Dice, cuando se compararon con 200 cepas aisladas en otras partes del mundo. Los perfiles de ADN con un coeficiente de Dice entre 73,7 % y 100 % se consideraron pertenecientes a la misma especie. Resultados. Todos los aislamientos fueron cepas patógenas y cinco se caracterizaron genéticamente. El aislamiento P275 (coeficiente de Dice: 84 %) y el P282 (coeficiente de Dice: 95 %) de cerdos, se relacionaron con Leptospira interrogans de serovariedad Pomona; el aislamiento de rata (I15) fue indistinguible de Leptospira interrogans de serovariedades Icterohaemorrhagiae o Copenhageni (coeficiente de Dice: 100 %), mientras que los aislamientos de perro (C67) y agua (A42) no se relacionaron (coeficiente de Dice <73,7 %) con ninguna de las 200 cepas de referencia; las más cercanas fueron Leptospira noguchii de serovariedades Nicaragua (coeficiente de Dice: 63 %) y Orleans (coeficiente de Dice: 60 %). Conclusiones. Esta fue la primera caracterización molecular de serotipos de aislamientos colombianos, los cuales serían los primeros miembros de un panel diagnóstico colombiano.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Correlation between the RhD genotyping and RhD serotyping in isoimmunized pregnancies

Alloimmunisation was one of the most important causes of perinatal mortality and morbidity by the middle of the last century. The objective of the present study was to investigate the presence of the RHD gene in fetal cells [amniocytes] obtained from amniotic fluid by genotyping to compare it with the RhD serotyping. Also to correlate the presence of RhD gene with the neonatal outcome. This work was carried out at Maternity hospital and Medical Genetics center, while PCR testing was done at the Medical Research center, Faculty of Medicine, Ain Shams University in the period from 2008 to 2010. The present study included recruiting of 20 RhD negative [sensitized to the RhD antigen] pregnant mothers. The entire study group was subjected to complete general, obstetric and a detailed obstetric ultrasonographic examination. Rh typing and indirect Coomb's test were also done. Amniocentesis was performed with a 20-gauge needle under continuous ultrasound guidance. RhD serotyping of the fetuses showed that, 14 fetuses [70%] were positive and six fetuses [30%] were negative. While using RhD gentyping 13 cases [65%] were positive and seven cases [35%] were negative [P value = 0.002]. Among fetuses positive for RhD genotyping six fetuses [46%] had received postnatal treatment, while among fetuses negative for RhD genotyping, neither of them had received postnatal treatment [P value = 0.032], which is statistically significant. From the present study we can conclude that, the identification of an antigen-negative fetus on the basis of the blood group genotype provides significant advantages in managing the pregnancy at risk for HDFN

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis / Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique

INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.

INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.

Triagem neonatal da infecção pelo Trypanosoma cruzi em Minas Gerais, Brasil: transmissão congênita emapeamento das áreas endêmicas / Neonatal screening program for the infection by Trypanosoma Cruzi in Minas Gerais, Brazil: congenital transmission and tracking of the endemic areas

No Brasil, uma vez controlada a transmissão pelas vias vetorial e transfusional, a via vertical adquiriu maior importância na transmissão da doença de Chagas (DC). A alta possibilidade de cura da doença de Chagas congênita (DCC) faz com que seu diagnóstico seja imperativo.Visando definir o risco de transmissão vertical e mapear áreas de risco, realizou-se inquérito sorológico em 63.673 neonatos do Programade Triagem Neonatal de Minas Gerais. A prevalência de DC em puérperas foi 0,5 por cento (IC95 por cento 0,37-0,54) e, as prevalências mais elevadas foram observadas na região norte do estado, variando de 2,3 por cento a 23 por cento. O risco de transmissão vertical foi 0,2 por cento (IC95 por cento 0,00-0,55) e a incidência de DCC foi 1,6 em cem mil nascidos vivos (IC95 por cento 0,00-5,00). A sorologia demonstrou ser eficiente ferramenta para o diagnóstico da DCC, e propõe-se que deva ser incluída no Programa de Triagem Neonatal nas áreas consideradas endêmicas. No estudo, a IgG materna persistiu positiva, em 17 crianças, entre seis e nove meses de idade. Portanto, filhos assintomáticos de mães chagásicas devem ser submetidos à sorologia após seis meses e, se positiva, deve ser repetida aos nove meses, antes de intervenção terapêutica.

In Brazil, once vectoral and transfusional transmissions are under control, congenital transmission of Chagas disease has become the main form. Treatment of congenital infection is often successful, so early detection becomes a relevant issue of public health. To determine the risk of congenital Chagas disease (CCD), were studied 63. 673 newborns enrolled at Neonatal Screening Program, at Minas Gerais, Brazil. The prevalence of Chagas disease in pregnant women was 0.5 per cent(IC95 per cent 0,37-0,54), varying from 2,3 to 23 per cent, with higher rates found in the northern state region. Transmission risk was estimated at 0.2 per cent(IC95 per cent 0,00-0,55) leading to an incidence rate of 1.6 per 100,000 live births(IC95 per cent 0,00-5,00) Serology survey was shown to be an efficient diagnostic tool, it should be included at neonatal screening programs in endemic areas. Asymptomatic children from infected mothers should be tested at six months of age, and if positive, serology should be repeated at nine months of age, before initiating therapeutic interventions.

Rapid and real-time detection technologies for emerging viruses of biomedical importance.

The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/or detection of antibodies,which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. We have developed two real-time assays ie., SYBR green I based real time reverse transcription polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (LAMP) assay for rapid detection as well as typing of some of the emerging viruses of biomedical importance viz. dengue, Japanese encephalitis, chikungunya, west Nile, severe acute respiratory syndrome virus (SARS) etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity, specificity. One of the most important advantages of LAMP is its field applicability, without requirement of any sophisticated equipments. Both these assays have been extensively evaluated and validated with clinical samples of recent epidemics from different parts of India. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.

Serotype and mating type characterization of Cryptococcus neoformans by Multiplex PCR

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (Aalpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

Cryptococcus neoformans é levedura encapsulada, agente etiológico da criptococose. As espécies são comumente associadas com fezes de pombos e material vegetal. O objetivo do presente trabalho foi verificar a presença de leveduras em fezes de pombos e identificar os isolados em relação aos sorotipos e "mating types". Dez amostras de fezes de pombos foram coletadas na zona rural da cidade de Alfenas, Brasil. As amostras foram inoculadas em agar Niger e 22 isolados com características de C. neoformans foram obtidos. Os sorotipos e "mating types" foram determinados pela PCR multiplex e os sorotipos foram identificados também pelo Kit Crypto Check. Dentre as 22 amostras avaliadas, oito foram identificadas como C. neoformans através dos testes clássicos. Estas amostras foram caracterizadas como sorotipo A pelo Kit Crypto check e como sorotipo A MATalfa pela PCR multiplex. O presente estudo reforça a evidência de que as fezes de pombos constituem reservatório para C. neoformans e confirma a prevalência de C. neoformans var. grubii (Aalfa) nos isolados ambientais. PCR multiplex é uma alternativa aceitável para análise do sorotipo porque reduz os custos de cada reação e analisa simultaneamente os sorotipos e "mating type".

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping.

PURPOSE: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. METHODS: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. RESULTS: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciprofloxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identified. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. CONCLUSIONS: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.

Characterization of dengue virus serotype 1 in epidemics in Porto Velho, Rondônia, in 2001-2003

The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universal primers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98 percent similarity to sequences from Southeast Asia that were obtained from GenBank.

A primeira epidemia de febre do dengue no Estado de Rondônia, Região Ocidental do Brasil foi registrado em 1997, sem confirmação laboratorial. Em seguida, houve uma epidemia descrita em 1998, em Manaus, no vizinho Estado do Amazonas, onde os vírus DENV-1 e DENV-2 foram isolados de pacientes. No presente artigo, foi descrito a caracterização do sorotipo do vírus dengue isolado de pacientes com suspeitas clinicas de dengue em Porto Velho, Rondônia, entre 2001 a 2003. Foram coletadas 150 amostras de sangue, entre primeiro e quinto dia de sintomas. Setenta amostras de vírus isolados foram submetidas a identificação do dengue pela RT-PCR usando primers universais para gene da NS1 do DENV que amplifica um fragmento de 419pb. O amplicon obtidos foram submetidos a digestão enzimática para caracterização do sorotipo viral. Todas as amostras analisadas foram DENV-1. A seqüência nucleotídica de um dos amplicons aleatoriamente selecionada também DENV-1 demonstrou 98 por cento similaridade com as seqüências do Sudeste Asiático obtidas no GenBank.

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in São Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5 percent) and one to serogroup Sejroe (2.5 percent). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.

Double infection of heteroserotypes of dengue viruses in field populations of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and serological features of dengue viruses found in patients in southern Thailand.

In order to understand more about the epidemiology of DHF, a study of the type of dengue viruses and vectors under natural conditions was carried out. Mosquito vectors in the field and the serum of DHF patients in southern Thailand were examined. The two mosquito species are abundant and DHF incidence remains high in this region. Dengue viruses were examined in field-caught mosquitoes by RT-PCR technique. The mosquitoes were caught in 4 provinces: Krabi, Phuket, Phang-Nga and Surat Thani during the late dry season until the early rainy season in 2005. Three dengue serotypes (DEN-2, DEN-3, DEN-4) were detected in Ae. aegypti males and females, and 2 (DEN-2, DEN-3) were detected in Ae. albopictus females. Double infection with 2 serotypes of dengue viruses (DEN-2 and DEN-3) were detected in Ae. aegypti males and females and Ae. albopictus females. DEN-2 and DEN-1 were the most prevalent serotypes found in the serum of the patients in this area, followed by DEN-4 and DEN-3. The prevalence of the predominant dengue serotype varied from province to province. Detection of viruses in adult male mosquitoes reveals the role of transovarial transmission of dengue viruses in field populations of DHF vectors and elucidates circulation of dengue viruses in vectors in the natural environment of endemic areas. The incidence of multiple serotypes of dengue virus in Ae. aegypti and Ae. albopictus in the same area points toward a high risk for an epidemic of DHF. These findings provide greater understanding of the relationship among mosquito vectors, virus transmission and DHF epidemiology in endemic areas.

Poliovirus environmental surveillance in sistan-balouchestan province, isolation and identification of viruses by cell culture and microneutralization, and intratypic differentiation by ELISA and probe hybridization

In some countries, wild polioviruses have been isolated from environment despite the absence of viruses being recovered from clinical cases, therefore to confirm of final Polio eradication, WHO has recommended environmental surveillance using sewage specimens and surface water. During the present study, in order to assure the polio eradication in Iran, Sistan-Balouchestan province was chosen as the target area. During a 12-month period, 86 specimens from 2 sewage disposal systems and 5 hospitals, as well as surface water from several rural areas were collected by Grab Sampling and tested for polioviruses using direct and concentrated specimens with 2 concentration methods: Pellet and Two-phase. Then the isolated viruses were serotyped by microneutralization method and differentiated intratypically by ELISA and probe hybridization techniques. Of all studied specimens, 18 [20.9%] were identified as poliovirus, none of which were wild virus, fortunately. Among these, 2 [2.3%], 8 [9.3%] and 13 [15.1%] were isolated from direct specimens, Pellet and Two-phase concentrated specimens, respectively. The most frequent viruses were Polio 2 [72.2%] and Polio 3 [27.8%]. Results have revealed the efficacy of immunization coverage in Iran. Meanwhile, sufficient surveillance programs have been observed during the recent years

Determination of dengue virus serotypes in Thailand using PCR based method.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and a single-tube multiplex PCR assay was modified for typing of dengue virus in different geographical areas of Thailand during 2000-2001. A set of primers (D1 and D2) was used to generate the RT-PCR product of 511 bp in size which subsequently underwent a single-tube multiplex PCR amplification using the highly specific primers for each of the dengue virus serotypes (D1, TS1, TS2, TS3 and DEN4). The PCR products of 482, 119, 290 and 392 bp in size were generated for dengue virus serotypes 1, 2, 3, and 4, respectively. Each set of specific primers showed no amplification of non-specific and non-target PCR products from human genomic DNA. The method was applied for investigation of 637 human blood samples in Thailand during 2000-2001 and found that 71, 43, 28, and 43 patients were classified as having a single infection with serotypes 1, 2, 3, and 4, respectively. Multiple infections with two or more dengue virus serotypes were also detected.

Emergência de Streptococcus pneumoniae resistente à penicilina em Salvado, Bahia: um estudo epidemiológico e molecular / Emergence of Streptococcus pneumoniae resistant to penicillin Salvado, Bahia: an epidemiological study and molecular

As infecções pelo S. pneumoniae persistem como uma das principais causas de morbimortalidade, apesar da disponibilidade de antibioticoterapia apropriada. Em 1995, foi implantado uma rede de vigilância para identificar pneumococos não susceptível à penicilina em dois diferentes grupos populacionais: 1) pacientes com meningite pneumocócica e 2) indivíduos saudáveis na comunidade. No período de dezembro de 1995 a agosto de 2002, o Hospital Couto Maia atendeu 477 pacientes com meningite pneumocócica identificados através da cultura de líquor positiva para S. pneumoniae, 453 (95%) tiveram os isolados caracterizados quanto à susceptibilidade e sorotipagem. Cepas não susceptíveis à penicilina (CIM 0,1 - 1,0 μg/ml) foram isoladas em 59 (13%) indivíduos com meningite pneumocócica. Os sorotipos mais prevalentes foram 14 (14%), 3 (10%), 19F (8%), 6B (8%), 6A (7%), 23F (6%), 4 (5%), 18C (5%) e 8 (4%), sendo que reduzida sensibilidade à penicilina foi associada aos sorogrupos 14 (38), 6 (9), 23 (7) e 19 (5). A provável taxa de cobertura da vacina heptavalente conjugada foi de 70% entre pacientes <5 anos e 91% entre aqueles com isolados não susceptíveis à penicilina. Tipagem de cepas pela reação de polimerase em cadeia (PCR) do elemento repetitivo BOX A, demonstou que os isolados de sorotipo 14 não susceptíveis à penicilina tinham um padrão clonal relacionado e quando comparados com isolados de outras cidades brasileiras tiveram um padrão similar. O segundo grupo populacional foram residentes de 39 domicílios selecionados randomicamente em uma comunidade urbana. Neste grupo, a colonização nasofaringeana foi investigada em 262 indivíduos, 95 (36%) estavam colonizados por pneumococos. Destes, 9 (9,5%) tiveram isolados não susceptíveis à penicilina. Os sorotipos mais prevalentes foram 19F (13%), e 6A (11%), seguidos do sorotipo 14 (7%), 23F (7%), 18C (6%) e 19A (5%). A cobertura teórica da vacina conjugada heptavalente nesta comunidade seria de 49% para todos...