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.

Hepatitis C virus infection in patients with tuberculosis in Central Brazil.

SETTING: Goiânia City, Goiás State, Brazil. OBJECTIVES: To determine the prevalence of hepatitis C virus (HCV) infection, risk factors, HCV genotype/ subtype, HCV viral load and human immunodeficiency virus (HIV) status in patients with tuberculosis (TB) in Central Brazil. DESIGN: A cross-sectional study was carried out with 402 patients who were under tuberculosis (TB) treatment in the reference hospital for infectious diseases in Goiânia, Goiás, Central Brazil. RESULTS: The prevalence rates of HCV and HIV were respectively 7.5% and 27.6%. Two thirds of the HCV-infected patients (20/30) were HIV-positive. Age, injecting drug use (IDU) and HIV status were factors independently associated with HCV infection. HCV RNA was detected in 23 serum samples; HCV RNA levels were measured in 22/23 samples. HCV RNA level was slightly higher in HCV-HIV co-infected patients than in HCV monoinfected patients. Genotypes 1 (n = 17) and 3 (n = 6) were determined by LiPA. Using phylogenetic tree analysis of the NS5B region, subtypes 1a (n = 12), 1b (n = 2) and 3a (n = 6) were identified. CONCLUSION: These data indicate that patients with TB may benefit from integrated HIV and HCV screening, which may have an important impact upon TB management and treatment.

Autoimmune thrombocytopenia related to chronic hepatitis C virus infection.

Since the identification of hepatitis C virus (HCV) in 1989 as a causative agent for a number of the extrahepatic alterations related to HCV infection an underlying immune mediated pathogenetic mechanism has been postulated. HCV-associated thrombocytopenia may be considered complex and multifactorial in origin, since different mechanisms have been implicated in its pathophysiology. With respect to autoimmune thrombocytopenia in chronic HCV infection, the detection of specific antibodies against platelet glycoproteins have been reported only in a few studies, but no systematic study has been carried out. We examined the clinical, laboratory, and virological characteristics of a case series of 10 patients with autoimmune thrombocytopenia (platelet count <150.0 x 10(9)/L) related to chronic HCV infection. Cases, six males and four females, aged 57.1 +/- 12.6 years, presented high titers of antibodies against platelet glycoprotein (GP) IIb/IIIa, GP Ia/IIa, and/or GP Ib/IX, and no other mechanism involved in the pathogenesis of HCV-associated thrombocytopenia was identified. Furthermore, cases were not associated with particular HCV genotype. Complete platelet response was observed in two patients treated with pegylated interferon plus ribavirin, and partial platelet response was seen in two patients treated with anti-D Ig and one patient treated with corticosteroids. These findings indicate that an autoimmune mechanism may play a role in the pathogenesis of HCV-associated thrombocytopenia in a proportion of these patients.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis.

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4 percent) and 3 (7.6 percent) were found. Subtype 1a (65.7 percent) was the most prevalent, followed by subtypes 1b (26.7 percent) and 3a (7.6 percent). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4 percent) were classified as genotype 1, subtypes 1a (72.6 percent) and 1b (19.8 percent), and 8 sequences (7.6 percent) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2 percent of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1 percent for subtype 1a and 95.2 percent for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.