Pyrosequencing method for sensitive detection of HBV drug resistance mutations.

Hepatitis B (HBV) drug resistance assay is important for guiding therapy after the development of virologic breakthrough for patients receiving nucleoside/-tide analog therapy. However, the existing genotyping tools are either costly or lack sensitivity to detect mixed genotypes, and an improved method of resistant mutation detection is needed. An assay protocol for clinical application using pyrosequencing method was developed, capable of detecting all known validated HBV polymerase gene mutations that impart resistance to lamivudine, adefovir, tenofovir, and entecavir. Sixty-eight serum samples with known HBV resistance genotypes, previously tested with either Sanger sequencing assay or commercial line probe assay, were used for validation. Where there were discrepancies between the two methods, clonal sequencing by Sanger's method was used for confirmation. The modified pyrosequencing method accurately identified all the cloned polymerase genotypes and was able to distinguish as little as 5% of the mutant populations. This assay can be performed on serum sample with HBV DNA as low as 13.5 IU/mL. The cost per test was less than existing commercial assay. HBV drug resistance pyrosequencing assay was accurate, more sensitive and cheaper compared with the existing methods. It can detect minor populations of drug-resistant clones earlier, before the drug resistant clones become dominant, allowing the opportunity for an earlier change of therapy.

Clinical evaluation of a low cost, in-house developed real-time RT-PCR human immunodeficiency virus type 1 (HIV-1) quantitation assay for HIV-1 infected patients.

OBJECTIVES: HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access. MATERIALS AND METHODS: A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays. RESULTS: The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100-1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A-H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate. CONCLUSIONS: With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.