Márcia Paschoal do Espírito-Santo

Hepatitis C virus-associated thrombocytopenia: a controlled prospective, virological study

Chronic hepatitis C virus (HCV) infection has also been associated with the development of several extrahepatic alterations, including thrombocytopenia, and a variety of pathogenic mechanisms are reported to be implicated in this hematological abnormality. Different studies have succeeded in detecting HCV in platelets with discrepant results. Moreover, most of the studies on HCV-associated thrombocytopenia have failed to provide data concerning the infecting genotype, a factor with prognostic implication in chronically HCV-infected patients. To determine whether thrombocytopenia is an extrahepatic alteration dependent on particular HCV genotypes, and to assess the relationship between thrombocytopenia and detection of HCV-RNA (positive strand) in platelets from patients with chronic HCV infection, 106 anti-HCV+/HCV-RNA+ patients (57 thrombocytopenic and 49 non-thrombocytopenic) were prospectively studied. The infecting genotype was analyzed from sera by using direct nucleotide sequencing of the polymerase chain reaction (PCR) products from core region. Genotypes 1a, 1b, and 3a were more prevalent in our patients, and no association between these genotypes and thrombocytopenia was observed (p=0.891). HCV-RNA was detected in platelets by reverse transcriptase (RT)-nested PCR in the 5’ non-coding region with a higher frequency (60%) in thrombocytopenic patients than in non-thrombocytopenic subjects (35%, p=0.017), suggesting that HCV is directly involved in the process that, at least in part, leads to thrombocytopenia.

Epidemic history of Hepatitis C virus in Brazil

Hepatitis C virus (HCV) subtypes 1a, 1b and 3a are the most prevalent strains in Brazil, but very little is known about the epidemic history of these subtypes in the country. A total of 231 HCV NS5B gene sequences (subtype 1a=89, subtype 1b=56, and subtype 3a=86) isolated in Brazil between 1995 and 2007 were analyzed in the present study. Sequences (328-pb) were subjected to phylogenetic analyses and statistical tests of phylogenetic mixing by sampling location and risk group. Our results revealed important variations in the pattern of HCV transmission among subtypes. Transmission of subtype 1a was characterized by dissemination of one major Brazilian lineage with a random virus exchange between different geographical regions but not between IDU and non-IDU populations. Transmission of subtype 1b was characterized by concurrent dissemination of multiple HCV lineages with a restricted virus exchange between country regions and risk groups. Transmission of subtype 3a was characterized by simultaneous spreading of multiple HCV lineages and random phylogenetic mixing by risk group and sampling location. Epidemic histories of major subtypes 1a, 1b and 3a Brazilian clades were estimated using a Bayesian coalescent approach. Our results indicate that all major HCV Brazilian clades probably start to circulate in the country during the second half of the 20th century and displayed roughly similar epidemic histories characterized by an initial phase of exponential expansion and by reduction of growth rates since 1980-1995. This suggests that the expansion of HCV may have been effectively controlled in Brazil.

Identification of two phylogenetic lineages of equine hepacivirus and high prevalence in Brazil

Non-primate hepacivirus (NPHV), as described in horses, is the virus most genetically related to hepatitis C virus (HCV). Although detected worldwide, limited data on genomic variability and distribution of NPHV are available in Latin America. The aim of this study was to investigate the genetic diversity and prevalence of equine NPHV in Brazil. Thirteen percent of 202 equines from three Brazilian states were positive for NPHV genome by reverse transcriptase PCR. Nucleotide sequences of the partial NS5B genome presented the greatest diversity described to date (25.6%), which is comparable to the upper limit of diversity for HCV subtype classification for the same region. Phylogenetic analysis revealed that Brazilian NPHV sequences along with isolates worldwide form two strongly supported clades (pp = 1.0) suggesting the existence of two distinct lineages.

Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples

For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV.

Hepatitis B virus DNA stability in plasma samples under short-term storage at 42°C

We evaluated the stability of hepatitis B virus (HBV) DNA in plasma samples stored at 42°C for external quality assessment (EQA) panels of viral load. To assess the stability of plasma samples containing different concentrations of HBV DNA, serial dilutions of HBV-infected samples with a viral load of 6.40 log(10) IU/mL were made to yield viral loads of 5, 4, and 3 log(10) IU/mL. These were incubated at 42°C for up to 7 days and then frozen at -70°C. Viral load testing for HBV DNA was performed for all samples using COBAS¯ AmpliPrep/COBAS¯ TaqMan¯ HBV Test (v.2.0, Roche, Switzerland). Results were compared with fresh frozen plasma samples as a benchmark to establish acceptable measurements on the days following sample collection. Although the results of this study demonstrated a decrease in HBV DNA viral load ranging from 0.005 to 0.30 log(10) IU/mL after storage at 42°C for up to 7 days, these values did not exceed 0.5 log(10), which is the estimated intra-assay variation for molecular tests. Thus, the insignificant decrease in viral load suggests that shipment of HBV in plasma samples at temperatures of up to 42°C is permissible if they are frozen within 7 days.

Knowledge about viral hepatitis among participants of Gay Pride Event in Brazil.

Male or female homosexual, bisexual and transgender individuals (HBT) can be more exposed to viral hepatitis, but few studies worldwide have been conducted about viral hepatitis knowledge among HBT.1–5 The aim of this study was to evaluate viral hepatitis knowledge and potential risk of HBT and heterosexual individuals attending a Gay Pride Event....(AU)