Research Article: Characterization of the Drug Resistance Profiles of Patients Infected with CRF07_BC Using Phenotypic Assay and Ultra-Deep Pyrosequencing

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Szu-Wei Huang, Wei-You Li, Wen-Hung Wang, Yu-Ting Lin, Chih-Hung Chou, Marcelo Chen, Hsien-Da Huang, Yen-Hsu Chen, Po-Liang Lu, Sheng-Fan Wang, Shinichi Oka, Yi-Ming Arthur Chen, Dimitrios Paraskevis.


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%.

Partial Text

Combination antiretroviral therapy (cART), also known as highly active antiretroviral therapy (HAART) can decrease the morbidity and mortality of HIV-1/AIDS patients [1–3]. However, the emergence of HIV-1 drug resistance (DR) may lead to cART failure [4, 5]. Therefore, detection of DR viruses is important for clinical management of HIV-1/AIDS. Two assays have been designed for the detection of HIV-1 DR: genotypic and phenotypic assays [6]. Genotypic assay uses direct PCR amplification of the HIV-1 pol region followed by Sanger sequencing (also 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]. However, the results of these assays do not always represent the clinical outcome because resistance is predicted by mutations that had been previously observed [7]. In addition, the specimens need to contain at least 20% of the DR quasispecies or variants [8, 9]. In contrast, phenotypic assays measure HIV-1 viral replication in cells cultured in different drug concentrations. There are two types of phenotypic assays: commercially available phenotypic assays generate chimeric viruses by homologous recombination of PCR-derived sequences and then 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 then 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]. However, there is no data on the sensitivity of the in-house phenotypic assay which uses primary isolates from the patients directly.

In this study, 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. 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 phenotypic PI DR.




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