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.

Evaluation of dried blood spot samples for hepatitis C virus detection and quantification.

BACKGROUND: Dried blood spots (DBS) could be an excellent alternative for HCV diagnosis, since it is less invasive and can be stored and transported without refrigeration. OBJECTIVES: The aim of this study was to optimize quantitative and qualitative methods for HCV detection in DBS. STUDY DESIGN: DBS and serum samples were collected from 99 subjects (59 anti-HCV/HCV RNA positive and 40 seronegative samples). Seven extraction methods and different PCR parameters were evaluated in DBS samples in the quantitative RT-PCR (qRT-PCR) developed to amplify the 5' noncoding region of HCV. A qualitative PCR for amplification of NS5B region of HCV was also valued and the nested-PCR sequenced. RESULTS: The qRT-PCR showed good correlation to commercial assay for HCV viral measurement in serum. To quantify HCV RNA in DBS, it was necessary to increase reverse transcriptase and cDNA concentration. HCV RNA quantification in DBS demonstrated sensitivity of 65.9%, 100% of specificity and kappa statistic of 0.65. The median viral load of DBS samples was 5.38 log10 copies/ml (minimum value=1.76 and maximum value=10.48 log10 copies/ml). HCV RNA was detected in NS5B regions and nucleotide sequences obtained in 43 serum and 11 DBS samples. The presence of the same subtype was observed in paired serum and DBS samples. CONCLUSIONS: In this study, it was possible to demonstrate that, despite the low sensitivity, the optimized protocol was able to determine the viral load, as well as, the infecting HCV genotype, validating the usefulness of DBS for viral load determination and molecular epidemiology studies of HCV.

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.

Detection of hepatitis C virus in platelets: evaluating its relationship to antiviral therapy outcome.

BACKGROUND/AIMS: Detection of HCV has been documented in extrahepatic sites such as platelets. However, its influence on antiviral therapy outcome is unknown. In this study, we investigated the relationship between the detection of HCV in platelets from a cohort of 48 chronically HCV-infected patients and response to antiviral therapy. METHODOLOGY: This study comprised of 19 males and 29 females, mean age 54.9 +/- 8.72 years, followed-up in Rio de Janeiro, Brazil, between August 2004 and October 2006. HCV-RNA was detected in serum and platelets (pre-treatment, end-of-treatment and 24 weeks after completion of therapy) by reverse transcription-nested polymerase chain reaction. Patients with genotype 1 or 4 were treated with peginterferon-alfa/ribavirin for 48 weeks, and patients with genotype 3 received interferon-alfa/ribavirin for 24 weeks. RESULTS: Baseline detection of HCV in platelets was found not to be related to therapy outcome. However, significant associations between detection rates of HCV in platelets and serum at the end-of-treatment (p = 0.0203), and 24 weeks after completion of therapy (p = 0.0016) were observed. Interestingly, HCV was detected in platelets from two patients with normal ALT who lost detectable serum HCV at the end-of-treatment and, after 24 weeks of followup, relapsed virologically in serum. CONCLUSIONS: Our data suggest that patients with HCV persistence in platelets by the end-of-treatment appear to be at an increased risk of recurrent HCV infection.

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.