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

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 × 10 copies mL ), (ii) very similar in the viral loads detected (R = .90), (iii) highly sensitive, as it detected loads as low as 30 copies mL (~5 IU mL ), (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.

Hepatitis B testing and treatment in HIV patients in The Gambia—Compliance with international guidelines and clinical outcomes

“…Demographic and clinical information such as age, gender, past medical history, duration of HIV infection and antiretroviral drug exposure were collected, and a clinical examination and bedside ultrasonography performed. Liver transaminases (Vitros 350 Analyzer, Ortho Clinical Diagnostics, USA), haemoglobin and platelet count (CELL-DYN 3700 sample loader, Abbott, USA), HIV RNA (Abbott Real Time HIV-1 assay, 40copies/mL limit of detection), CD4+ T-cell count (flow cytometry) and in-house HBV DNA viral load quantification [16] were measured for all patients.…”

“…HBV DNA was quantified was by TaqMan based quantitative PCR assay with lower limit of detection of 50IU/L [16], using probe (200nM) sequence with forward and reverse primer (400nM) sequences and respectively [19]. Hepatitis B polymerase gene sequence was assessed in all participants with HBV DNA viral load ≥20,000IU/L.…”

Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes

“…Absolute and relative quantification were performed using complete thermocycling parameters previously described [23] using the iQ SYBR Green Supermix kit (Bio-Rad). All samples were assessed in duplicates.…”

“…HBV DNA from HBV particles production was extracted using the High Pure Viral Nucleic Acid Kit (ROCHE diagnostics). Absolute quantification was performed as previously described [23] using a standard curve by serial dilution of a cloned HBV (ayw, genotype D in pTriEx vector) plasmid and the following primers HBV FW: and HBV R: (Amplicon length: 98 bp). For HBV RNA analysis, total RNA was extracted with the MasterPure RNA Purification Kit (Epicentre) following manufacturer’s instructions.…”

Hepatitis B Core-related Antigen: An Alternative to Hepatitis B Virus DNA to Assess Treatment Eligibility in Africa

“…In addition, the program also recruited symptomatic patients with chronic liver disease referred from health facilities throughout the country [21]. After informed consent, HBsAg-positive participants systematically underwent following clinical evaluation: fasting transient elastography (FibroScan 402, Echosens, France) [22], abdominal ultrasonography, hematology and biochemistry tests, HBeAg (ETI-EBK Plus, Diasorin, Italy), and HBV DNA (inhouse real-time PCR, limit of detection: 50 IU/ml) [23]. All these laboratory analyses were performed locally.…”