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

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

Author information

1
INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France.
2
La Dantec, Dakar, Senegal.
3
MRC, The Gambia Unit, Banjul, The Gambia.
4
Department of Hepatology, Imperial College London, London, UK.
5
Laboratory Services and Biobank Group (DIR/LSB), IARC, Lyon, France.
6
Department of Hepatology, Hospices Civils de Lyon, Lyon, France.
7
INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France. Isabelle.chemin@inserm.fr.

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 (R = .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

PMID:
27353593
DOI:
10.1111/jvh.12561
[Indexed for MEDLINE]

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