Abstract
BackgroundDrug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research.MethodsWe used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method.ResultsSixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%).ConclusionsWe developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.
Highlights
Since combination antiretroviral therapy was introduced, the prognosis of patients with HIV-1 infection has improved dramatically [1,2]
Rates of transmitted HIV-1 drug resistance (DR) have remained limited in resource-limited settings; limitation of the first-line and subsequent regimens would be a concern. combination antiretroviral therapy (cART) consisting of two nucleotide reverse transcriptase inhibitor (NRTI) and one non-nucleoside reverse transcriptase inhibitors (NNRTI), most often zidovudine (AZT) + lamivudine (3TC) or stavudine (d4T) + 3TC plus nevirapine (NVP) or efavirenz (EFV), has been widely used as the treatment regimen in the resource-limited settings [5,6]; Drug resistance (DR) might become a larger public health challenge in the developing countries
Sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more convenient and suitable for public health research [8,9]
Summary
Since combination antiretroviral therapy (cART) was introduced, the prognosis of patients with HIV-1 infection has improved dramatically [1,2]. Nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) resistance has declined over time in resource-rich settings, presumably reflecting the improvement of treatment regimens [3,4]. CART consisting of two NRTIs and one non-nucleoside reverse transcriptase inhibitors (NNRTI), most often zidovudine (AZT) + lamivudine (3TC) or stavudine (d4T) + 3TC plus nevirapine (NVP) or efavirenz (EFV), has been widely used as the treatment regimen in the resource-limited settings [5,6]; DR might become a larger public health challenge in the developing countries. M184V is highly associated with 3TC and emtricitabine (FTC) resistance, and reduce the susceptibility to 3TC by 200-fold The monitoring of these six DR mutations should be important for molecular epidemiologic study estimating the efficacy of anti-HIV drugs especially in resource limited settings. Sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research
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