sybr-green-i and Hepatitis-B--Chronic

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. HBV infection is diagnosed by serological tests, while real-time polymerase chain reaction (qRT-PCR) assays are used to quantify viral load, which is a crucial parameter to determine viral replication and to monitor antiviral treatments. However, measuring viral load in resource-limited countries remains nonsystematic, due to the high cost of commercial kits. Here, we describe the development, validation and implementation of a low-cost, in-house qRT-PCR assay to monitor HBV viral load in chronic carriers enrolled in the PROLIFICA programme in the Gambia and Senegal. Over 1500 HBsAg-positive patients, including 210 chronically infected HBV patients, who were given antiviral treatment (tenofovir), were monitored by qRT-PCR using the SYBR Green- and HBV-specific primers. Twenty-four tenofovir-treated patients were followed up and their viral load was tested every 3 months over the 12-month experimental time course. Compared to commercial assays, our in-house assay was shown to be (i) highly reliable, with good intra- and interassay reproducibility over a wide range (45-4.5 × 10

Topics: Antiviral Agents; Benzothiazoles; Costs and Cost Analysis; Diamines; DNA Primers; DNA, Viral; Drug Monitoring; Follow-Up Studies; Gambia; Hepatitis B virus; Hepatitis B, Chronic; Humans; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Senegal; Sensitivity and Specificity; Staining and Labeling; Tenofovir; Time Factors; Viral Load

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10(3)-10(9) IU/ml (2.28 x 10(3) to 2.28 x 10(9) copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within +/-1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.

Topics: Benzothiazoles; Diamines; DNA, Viral; Hepatitis B virus; Hepatitis B, Chronic; Humans; Molecular Sequence Data; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reagent Kits, Diagnostic; Sensitivity and Specificity; Viral Load