title: 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 × 108copies 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.
authors.affiliation: INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France.
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: La Dantec, Dakar, Senegal.
authors.forename: A
authors.initials: A
authors.lastname: Sow
authors.affiliation: INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Guillot
authors.affiliation: MRC, The Gambia Unit, Banjul, The Gambia.
authors.forename: A
authors.initials: A
authors.lastname: Jeng
authors.affiliation: MRC, The Gambia Unit, Banjul, The Gambia.|Department of Hepatology, Imperial College London, London, UK.
authors.forename: G
authors.initials: G
authors.lastname: Ndow
authors.affiliation: MRC, The Gambia Unit, Banjul, The Gambia.|Laboratory Services and Biobank Group (DIR/LSB), IARC, Lyon, France.
authors.forename: R
authors.initials: R
authors.lastname: Njie
authors.affiliation: MRC, The Gambia Unit, Banjul, The Gambia.
authors.forename: S
authors.initials: S
authors.lastname: Toure
authors.affiliation: La Dantec, Dakar, Senegal.
authors.forename: M
authors.initials: M
authors.lastname: Diop
authors.affiliation: La Dantec, Dakar, Senegal.
authors.forename: S
authors.initials: S
authors.lastname: Mboup
authors.affiliation: La Dantec, Dakar, Senegal.
authors.forename: C T
authors.initials: CT
authors.lastname: Kane
authors.affiliation: Department of Hepatology, Imperial College London, London, UK.
authors.forename: M
authors.initials: M
authors.lastname: Lemoine
authors.affiliation: Department of Hepatology, Imperial College London, London, UK.
authors.forename: M
authors.initials: M
authors.lastname: Thursz
authors.affiliation: INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France.|Department of Hepatology, Hospices Civils de Lyon, Lyon, France.
authors.forename: F
authors.initials: F
authors.lastname: Zoulim
authors.affiliation: Laboratory Services and Biobank Group (DIR/LSB), IARC, Lyon, France.
authors.forename: M
authors.initials: M
authors.lastname: Mendy
authors.affiliation: INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon Université Claude Bernard, Lyon, France. Isabelle.chemin@inserm.fr.
authors.forename: I
authors.initials: I
authors.lastname: Chemin
mesh_terms: D000998:Antiviral Agents; D052160:Benzothiazoles; D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D003959:Diamines; D016903:Drug Monitoring / Q000379:methods; D005500:Follow-Up Studies; D005714:Gambia; D006515:Hepatitis B virus / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012675:Senegal; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D000068698:Tenofovir / Q000008:administration & dosage; D013997:Time Factors; D019562:Viral Load / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D000068698:Tenofovir
keywords: 
  HBV
  ; Syber Green; real-time quantification; viral load; viral quantification