Implementation of an in-house quantitative real-time polymerase chain reaction method for Hepatitis B virus quantification in West African countries

J Viral Hepat. 2016 Nov;23(11):897-904. doi: 10.1111/jvh.12561. Epub 2016 Jun 29.

Abstract

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 × 108 copies mL-1 ), (ii) very similar in the viral loads detected (R2 = .90), (iii) highly sensitive, as it detected loads as low as 30 copies mL-1 (~5 IU mL-1 ), (iv) cheaper (2- to 3-fold), (v) easier to implement and (vi) more rapid. Based on our experience, we recommend this assay as a reliable alternative to commercial assays, for monitoring HBV viraemia in resource-limited, highly endemic countries to reduce the cost and technical obstacles associated with commercial kits.

Keywords: HBV; Syber Green; real-time quantification; viral load; viral quantification.

Publication types

  • Validation Study

MeSH terms

  • Antiviral Agents
  • Costs and Cost Analysis
  • DNA Primers / genetics
  • DNA, Viral / analysis
  • DNA, Viral / genetics
  • Drug Monitoring / methods
  • Follow-Up Studies
  • Gambia
  • Hepatitis B virus / isolation & purification*
  • Hepatitis B, Chronic / drug therapy
  • Hepatitis B, Chronic / virology*
  • Humans
  • Organic Chemicals / metabolism
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Senegal
  • Sensitivity and Specificity
  • Staining and Labeling / methods
  • Tenofovir / administration & dosage
  • Time Factors
  • Viral Load / methods*

Substances

  • Antiviral Agents
  • DNA Primers
  • DNA, Viral
  • Organic Chemicals
  • SYBR Green I
  • Tenofovir