MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

QUANTITATIVE REAL-TIME PCR (Q-PCR) FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

Objective: To assess quantitative real-time polymerase chain reaction (q-PCR) for the sputum smear diagnosis of pulmonary tuberculosis (PTB) in patients living with HIV/AIDS with a clinical suspicion of PTB.Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard.Results: Of the 140 sputum samples, 47 (33.6%) were positive with the gold standard. q-PCR was positive in 42 (30%) of the 140 patients. Only one (0.71%) did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93%) of the 42 q-PCR positive cases, the CT (threshold cycle) was equal to or less than 37.Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

Carga proviral do HTLV-1 e HTLV-2: um método simples através da PCR quantitativa em tempo real

Os vírus linfotrópicos de células T humanas, quando integrados ao genoma da célula hospedeira, provírus, têm como marcador de replicação seu DNA proviral. A carga proviral parece ser um importante fator no desenvolvimento de patologias associadas a estes retrovírus. Neste estudo foi desenvolvida uma metodologia para quantificação absoluta da carga proviral dos HTLV-1 e HTLV-2 através da PCR em tempo real. Cinqüenta e três amostras de doadores de sangue com teste de ELISA reagente foram submetidas à metodologia, que utilizou o sistema TaqMan® para três seqüências alvo: HTLV-1, HTLV-2 e albumina. A quantificação proviral absoluta foi determinada através da proporção relativa entre o genoma do HTLV e o genoma da célula hospedeira, levando em consideração o número de leucócitos. O método apresentado é sensível (215 cópias/mL), prático e simples para quantificação proviral, além de eficiente e adequado para confirmação e discriminação da infecção pelos tipos virais.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Malacological survey of Biomphalaria snails in municipalities along the Estrada Real in the southeast of the State of Minas Gerais, Brazil

INTRODUCTION: The increasing practice of ecotourism and rural tourism in the State of Minas Gerais, Brazil, highlights the importance of studies concerning the occurrence of potential intermediate hosts of Schistosoma mansoni. This study aimed to identify species of Biomphalaria snails in municipalities along the Estrada Real, an important Brazilian tourism project. METHODS: The specimens were collected in different water collections of 36 municipalities along the Estrada Real in the southeast of the State of Minas Gerais. Biomphalaria species were characterized using both morphological and molecular approaches. The research was conducted between August 2005 and September 2009 and all the sites visited were georeferenced using GPS. RESULTS: Six Biomphalaria species were found in 30 of the 36 municipalities studied: glabrata, tenagophila, straminea, peregrina, occidentalis and schrammi. The first three species of Biomphalaria, recognized as intermediate hosts of S. mansoni, were present in 33.3%, 47.2% and 8.3% of the municipalities studied, respectively. The mollusks were found in different types of water collections and no infection by S. mansoni was detected. The highest occurrence of Biomphalaria concentration was verified in the area covered by the Caminho Novo route (Diamantina/MG to Rio de Janeiro/RJ). CONCLUSIONS: Considering the occurrence of schistosomiasis in the State of Minas Gerais and the socioeconomic repercussions involved in the Estrada Real Project, this work focuses on the vulnerability of water collections due to the presence of Biomphalaria mollusks and emphasizes the need for epidemiological surveillance and sanitary and educational measures integrated with the local community and tourism sectors.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

Demonstração do mecanismo da terapia de ressincronização ventricular com ecocardiografia tridimensional em tempo real em paciente com insuficiência cardíaca

Relatamos caso de homem de 66 anos de idade portador de insuficiência cardíaca classe funcional (NYHA) IV que foi submetido a terapia de ressincronização cardíaca por implante de marcapasso biventricular. O paciente foi avaliado antes e 48 horas após o implante do marcapasso com o emprego da ecocardiografia tridimensional transtorácica em tempo real. A utilização da ecocardiografia tridimensional contribuiu para o entendimento do mecanismo envolvido na ressincronização cardíaca através da demonstração da melhor sincronização dos segmentos cardíacos, o que resultou em melhora clínica do paciente.