Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery

OBJECTIVES: Bacterial and aseptic meningitis after neurosurgery can present similar clinical signs and symptoms. The aims of this study were to develop and test a molecular method to diagnose bacterial meningitis (BM) after neurosurgery. METHODS: A 16S ribosomal RNA gene PCR-based strategy was developed using artificially inoculated cerebrospinal fluid (CSF) followed by sequencing. The method was tested using CSF samples from 43 patients who had undergone neurosurgery and were suspected to suffer from meningitis, and from 8 patients without neurosurgery or meningitis. Patients were classified into five groups, confirmed BM, probable BM, possible BM, unlikely BM, and no meningitis. RESULTS: Among the samples from the 51 patients, 21 samples (41%) were culture-negative and PCR-positive. Of these, 3 (14%) were probable BM, 4 (19%) were possible BM, 13 (62%) were unlikely BM, and 1 (5%) was meningitis negative. Enterobacterales, non-fermenters (Pseudomonas aeruginosa and Acinetobacter baumannii), Staphylococcus haemolyticus, Granulicatella, Variovorax, and Enterococcus cecorum could be identified. In the group of patients with meningitis, a good agreement (3 of 4) was observed with the results of cultures, including the identification of species. CONCLUSION: Molecular methods may complement the diagnosis, guide treatment, and identify non-cultivable microorganisms. We suggest the association of methods for suspected cases of BM after neurosurgery, especially for instances in which the culture is negative.

Detection of HIV-1 infections in blood donors during the pre-seroconversion window period in São Paulo, Brazil

Abstract By decreasing the pre-seroconversion window period, nucleic acid testing (NAT) has improved the safety of blood products and reduced the risk of transfusion-transmitted infections. Between 2011 and 2017, NAT determinations for approximately 898,202 donations were performed at Fundação Pró-Sangue/Hemocentro de São Paulo (FPS-HSP). Three seronegative HIV-viremic donations were detected. The NAT yield rate per million donations was 3.34 for HIV, and the acute HIV-1 infections detected are described, followed by a brief review of the situation in Brazil.

Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil

ABSTRACTBACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo.METHODS:A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program).RESULTS:Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r = 0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r = 0.32). An evident exponential growth (r = 0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s.CONCLUSIONS:After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city / Distribuição dos genótipos do vírus da hepatite B e níveis de carga viral em pacientes brasileiros cronicamente infectados na cidade de São Paulo

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68 percent samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

O objetivo do presente estudo foi avaliar a carga viral no soro de pacientes cronicamente infectados pelo vírus da Hepatite B (HBV) e investigar a distribuição de genótipos HBV na cidade de São Paulo. PCR quantitativo do HBV e genotipagem ganharam importância para a previsão de progressão da doença, empregada para avaliar a infectividade, para tratamento e acompanhamento e para detectar o aparecimento de resistência aos anti-retrovirais. Vinte e nove pacientes brasileiros com suspeita de hepatite B crônica foram estudados, utilizando PCR em tempo real para a determinação da carga viral e seqüenciamento direto para determinação do genótipo. A sorologia revelou que 22 estavam, de fato, cronicamente infectados pelo HBV. O HBV-DNA foi positivo em 68 por cento das amostras (15/22). Em sete casos, HBV-DNA foi indetectável por PCR quantitativo. A análise filogenética mostra que onze pacientes foram infectados com hepatite B genótipo A, dois com genótipo F e dois com genótipo D. Desta forma, o genótipo A foi o mais prevalente em nosso estudo.

Serological, epidemiological and molecular aspects of hepatitis C virus infection in a population from Londrina, PR, Brazil, 2001-2002

Aspectos sorológicos, epidemiológicos e moleculares da infecção pelo vírus da hepatite C (HCV) foram avaliados em 183 indivíduos da região de Londrina, Paraná. Amostras soropositivas para anti-HCV pelo enzimaimunoensaio de micropartículas (MEIA), provenientes de oito pacientes com hepatite C crônica, 48 doadores de sangue e 127 indivíduos infectados pelo vírus da imunodeficiência humana (HIV), foram submetidas ao enzimaimunoensaio (ELISA) e a reação em cadeia da polimerase (PCR). Em 78,7% das amostras, verificou-se resultado reagente no ELISA, ocorrendo maior proporção de resultados discordantes entre doadores de sangue (70,8%). O mesmo ocorreu com a pesquisa do RNA viral, na qual 111/165 (67,3%) amostras foram positivas com PCR, verificando-se maior positividade entre indivíduos HIV soropositivos e pacientes com hepatite crônica do que em doadores de sangue. Em 61 amostras com viremia detectável, realizou-se a genotipagem do HCV, encontrando-se os genótipos 1 (77,1%), 3 (21,3%) e 2 (1,6%). Por fim, foram avaliados os fatores epidemiológicos em indivíduos com infecção ativa, nos quais o uso de drogas injetáveis foi o principal fator de risco encontrado em indivíduos co-infectados pelo HIV/HCV e a transfusão sangüínea foi o mais comum em indivíduos sem infecção pelo HIV. O presente estudo contribuiu para o conhecimento do perfil da infecção pelo HCV em indivíduos da nossa população e da distribuição dos genótipos do HCV nesta região.

Genotype distribution of the GB virus C in citizens of Säo Paulo City, Brazil

There has been several studies worldwide on phylogenetics and genotype distribution of the GB-virus C / Hepatitis G virus (GBV-C/HGV). However, in their great majority, those investigations were based on some epidemiologically linked group, rather than on a representative sampling of the general population. The present is a continuation of the first study in Brazil with such a population; it addresses the GBV-C/HGV phylogenetics and genotype distribution based on samples identified among more than 1,000 individuals of the city of Säo Paulo. For this purpose, a 728 bp fragment of the 5 non-coding region (5 NCR) of the viral genome, from 24 isolates, was sequenced and subjected to phylogenetic analysis. Genotypes 1, 2a and 2b were found at 8.3 percent (2/24), 50 percent (12/24) and 41.7 percent (10/24), respectively. In conclusion Säo Paulo displays a genotype distribution similar to the published data for other States and Regions of Brazil, endorsing the notion that types 1 and 2 would have entered the country with African and European people, respectively, since its earliest formation

New prevalence estimate of TT virus (TTV) infection in low- and high-risk population from Säo Paulo, Brazil

The prevalence of TT virus (TTV) infection was investigated by Polymerase Chain Reaction (PCR) in low- (blood donors and healthy children/adolescents) and high-risk (hemophiliacs) groups from Säo Paulo, Brazil. Primers based on the untranslated region (UTR) of the viral genome proved to be much more ubiquitous, leading to much higher frequencies for both groups ( > or = 81 percent) than the earlier N22-PCR directed to the open reading frame 1 (blood donors, 5.5 percent, and hemophiliacs, 42.3 percent). The UTR-PCR also revealed an interesting profile for healthy children/adolescents: very high prevalence at the early years and significant decrease in male teenagers. The N22-PCR, in turn, demonstrated higher frequency in hemophiliacs treated with fresh blood products (58 percent), than in those treated with virus-inactivated clotting factors (9.4 percent) and blood donors (5.5 percent)