Abstract
The usefulness of ultra-deep pyrosequencing (UDPS) for the diagnosis of HIV-1 drug resistance (DR) remains to be determined. Previously, we reported an explosive outbreak of HIV-1 circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan in 2004. The goal of this study was to characterize the DR of CRF07_BC strains using different assays including UDPS. Seven CRF07_BC isolates including 4 from early epidemic (collected in 2004–2005) and 3 from late epidemic (collected in 2008) were obtained from treatment-naïve patient’s peripheral blood mononuclear cells. Viral RNA was extracted directly from patient’s plasma or from cultural supernatant and the pol sequences were determined using RT-PCR sequencing or UDPS. For comparison, phenotypic drug susceptibility assay using MAGIC-5 cells (in-house phenotypic assay) and Antivirogram were performed. In-house phenotypic assay showed that all the early epidemic and none of the late epidemic CRF07_BC isolates were resistant to most protease inhibitors (PIs) (4.4–47.3 fold). Neither genotypic assay nor Antivirogram detected any DR mutations. UDPS showed that early epidemic isolates contained 0.01–0.08% of PI DR major mutations. Furthermore, the combinations of major and accessory PI DR mutations significantly correlated with the phenotypic DR. The in-house phenotypic assay is superior to other conventional phenotypic assays in the detection of DR variants with a frequency as low as 0.01%.
Highlights
Combination antiretroviral therapy, known as highly active antiretroviral therapy (HAART) can decrease the morbidity and mortality of HIV-1/AIDS patients [1,2,3]
ultra-deep pyrosequencing (UDPS) showed that early epidemic isolates contained 0.01–0.08% of protease inhibitors (PIs) drug resistance (DR) major mutations
In-house phenotypic assay showed that early epidemic CRF07_BC isolates from treatment naïve patients had phenotypic DR to most PIs, while genotypic assay and Antivirogram showed that none of them had DR mutations in the HIV-1 protease region
Summary
Combination antiretroviral therapy (cART), known as highly active antiretroviral therapy (HAART) can decrease the morbidity and mortality of HIV-1/AIDS patients [1,2,3]. Genotypic assay uses direct PCR amplification of the HIV-1 pol region followed by Sanger sequencing ( called bulk sequencing). It is widely used in the clinical laboratory diagnosis of HIV-1 DR since it is less expensive and has a short processing time [6]. There are two types of phenotypic assays: commercially available phenotypic assays generate chimeric viruses by homologous recombination of PCR-derived sequences and culture with cells in different drug concentrations [10, 11] and in-house phenotypic assay use peripheral blood mononuclear cells (PBMCs) to isolate HIV-1 and incubate them in target cells (MAGIC-5 cells) with different drug concentrations [12, 13]. It has been reported that phenotypic drug resistance using recombinant virus assay was limited to detect low-frequency viral quasispecies below than 50% [14]. There is no data on the sensitivity of the in-house phenotypic assay which uses primary isolates from the patients directly
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