Application of real-time PCR to quantify hepatitis B virus DNA in chronic carriers in The Gambia.
OBJECTIVE
The study aimed at developing a real-time quantitative PCR assay to monitor HBV serum virus load of chronic carriers enrolled in therapeutic trials.


METHODS
Quantitative real-time PCR assay was carried out using SYBR-Green signal detection and primers specific to the S gene. Thermal cycling was performed in an ABi 5700 sequence detection system. The assay was calibrated against an international HBV DNA standard and inter- and intra-assay reproducibility determined. Levels of viral load were monitored for 1-year in lamivudine treated carriers. Correlation between HBV DNA levels and HBeAg sero-status was determined in untreated carriers.


RESULTS
The qPCR assay showed good intra- and inter-assay reproducibility over a wide dynamic range (1.5 x 103 to 1.5 x 108 copies/mL) and correlated well with those from a commercial assay (r = 0.91, (p < 0.001). Viral load levels dropped dramatically but temporarily during and after a short course of lamivudine therapy. HBV DNA was a more reliable indicator of the presence of virus than HBe antigen and was detected in 77.0% (161/209) of HBeAg negative and in all HBeAg positive carriers.


CONCLUSIONS
This method is reliable, accurate, and reproducible. HBV DNA Quantification by qPCR can be used to monitor the efficacy of HBV therapy and useful in understanding the natural history of HBV in an endemic area.
Mendy
Maimuna E
ME
Medical Research Council, Atlantic Boulevard, Fajara, P O Box 273, Banjul, The Gambia. mmendy@mrc.gm
Kaye
Steve
S
van der Sande
Marianne
M
Rayco-Solon
Pura
P
Waight
Pauline A
PA
Shipton
Deborah
D
Awi
Dorka
D
Snell
Paul
P
Whittle
Hilton
H
McConkey
Samuel J
SJ
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D000998:Antiviral Agents / Q000008:administration & dosage / Q000627:therapeutic use; D002353:Carrier State / Q000188:drug therapy / Q000821:virology*; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000097:blood*; D005714:Gambia; D006510:Hepatitis B Antibodies / Q000097:blood; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006513:Hepatitis B e Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D007223:Infant; D019259:Lamivudine / Q000008:administration & dosage / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000008:administration & dosage / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine