Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation.

In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes.

Comparison of serum HBsAg quantitation by four immunoassays, and relationships of HBsAg level with HBV replication and HBV genotypes.

BACKGROUND: The decline in hepatitis B virus surface antigen (HBsAg) may be an early predictor of the viral efficacy of Hepatitis B virus (HBV) therapy. The HBsAg levels obtained by different immunoassays now need comparing and the relationships between levels of HBsAg and HBV DNA alongside HBsAg and genotype must be evaluated. METHODOLOGY/PRINCIPAL FINDINGS: HBsAg levels were compared among 80 patients using the Abbott Architect assay, a commercial immunoassay approved for HBsAg detection and quantitation, and three other assays derived from immunoassays approved for HBsAg detection (manufactured by Diasorin, Bio-Rad and Roche). Good correlation was found between the Abbot vs. Diasorin, Bio-Rad and Roche assays with narrow 95% limits of agreement and small mean differences: -0.06 to 0.11, -0.09 log(10) IU/mL; -0.57 to 0.64, -0.04 log(10) IU/mL; -0.09 to 0.45, -0.27 log(10) IU/mL, respectively. These agreements were not affected by genotypes A or D. HBsAg was weakly correlated with HBV DNA, whatever the HBsAg assay used: Abbott, ρ = 0.36 p = 0.001, Diasorin ρ = 0.34, p = 0.002; Bio-Rad ρ = 0.37, p<0.001; or Roche ρ = 0.41, p<0.001. This relationship between levels of HBsAg and HBV DNA seemed to depend on genotypes. Whereas HBsAg (Abbott assay) tended to correlate with HBV DNA for genotype A (ρ = 0.44, p = 0.02), no such correlation was significant for genotypes D (ρ = 0.29, p = 0.15). CONCLUSION/SIGNIFICANCE: The quantitation of HBsAg in routine clinical samples is comparable between the reference assay and the adapted assays with acceptable accuracy limits, low levels of variability and minimum discrepancy. While HBsAg quantitation is not affected by HBV genotype, the observed association between levels of HBsAg and HBV DNA seems genotype dependent.

Dried blood spot for hepatitis C virus serology and molecular testing.

UNLABELLED: We investigated the performance of dried blood spots (DBS) in hepatitis C virus (HCV) diagnosis using modified commercial tests. Paired DBS and serum samples were collected from 200 patients: 100 patients with anti-HCV antibodies (anti-HCV), including 62 patients with detectable serum HCV RNA, and 100 patients without anti-HCV. The DBS sample consisted of three drops of approximately 50 microL of whole blood applied to a paper card, which was then stored at -20 degrees C within 48 hours of collection. Using the Ortho HCV 3.0 enzyme-linked immunosorbent assay kit on DBS, we observed both a specificity and sensitivity of 99% in detecting anti-HCV. HCV RNA was detected on DBS in 60/62 (97%) patients with detectable serum HCV RNA, which was then successfully quantified in 55 samples (89%) using the Cobas TaqMan HCV test. A good correlation was observed between the DBS HCV RNA concentration and the serum level (r(2) = 0.95; P < 0.001). HCV genotyping was successfully performed on DBS samples, with a full concordance between the 14 paired DBS and serum samples (genotypes 1-4). CONCLUSION: This study presents DBS as a reliable alternative to serum specimens for detecting anti-HCV, quantifying HCV RNA and genotyping HCV. DBS may increase the opportunities for HCV testing and treatment follow-up in hard-to-reach individuals.

Impact of hepatitis C virus (HCV) genotypes on quantification of HCV RNA in serum by COBAS AmpliPrep/COBAS TaqMan HCV test, Abbott HCV realtime assay [corrected] and VERSANT HCV RNA assay.

The VERSANT HCV RNA 3.0 (bDNA), COBAS AmpliPrep/COBAS TaqMan HCV, and Abbott ART HCV RealTime assays were compared for hepatitis C virus RNA quantification in 158 clinical specimens (genotypes 1 to 5). RNA values differed significantly between methods (P < 0.0001), and mean titer differences ranged from 0.01 to 0.50 log(10) IU/ml depending on the genotypes.

High Prevalence of Torque teno (TT) virus in classical Kaposi's sarcoma.

Human herpes virus 8 infection is the primary and necessary factor in the development of Kaposi's sarcoma, but is not sufficient per se to trigger the onset of the disease. In order to search for virological cofactors associated with the occurrence of the disease, we investigated the prevalence of active infection by two newly discovered viruses, hepatitis G virus and TT virus, among patients with classical Kaposi's sarcoma. Serum of 24 patients with Mediterranean Kaposi's sarcoma was investigated using polymerase chain reaction and compared with that of 68 healthy subjects. Cutaneous samples from patients with Kaposi's sarcoma and healthy subjects were investigated for TT virus DNA. No patient had serum markers for hepatitis G virus. TT virus DNA was present in the serum of 21/24 (87.5%) patients and 32/68 (47%) controls (p=0.002). TT virus DNA was present in the lesional skin of 5/18 patients with Kaposi's sarcoma (27.7%), but not in the skin of controls. TT virus might play a role as a cofactor in the clinical emergence of Kaposi's sarcoma in patients infected with Human herpes virus 8, perhaps by immunosuppressive effects or by a common transmission pathway for these two viruses.