Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

Abstract

f Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had lowabundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

Deep sequencing: Becoming a critical tool in clinical virology

“…Using deep sequencing, a series of studies have demonstrated the ability to detect minority HIV drug resistant variants at levels as low as 0.1% to 1% of the virus population [26, 106, 158–162]. Most of these studies used the 454™ platform [106, 158, 159, 163, 164], but recently other deep sequencing technologies such as Illumina® [165] and Ion Torrent™ [162, 166] have proved to be useful in the identification of low-level drug resistant HIV variants. Deep sequencing has been used in HIV drug resistance surveillance [167, 168], to study transmission of HIV drug resistant viruses [101, 160, 169–175], and to evaluate the impact of minority variants on treatment efficacy [160, 176–183].…”

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library

“…The laboratory manipulations necessary for library generation are recognised as potential sources of artefactual recombination. Several papers have addressed the issue of minimising PCR artefacts [ 1 - 3 ] and there is growing interest in optimising RT-PCR techniques to enable accurate analysis of patient RNA virus load and early detection of the emergence of drug resistant quasi-species [ 3 - 6 ].…”

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

“…However, and although this is still uncertain, drug-resistant HIV-1 minority variants (i.e., those present in as low as 1% of the viral population) have been suggested to be clinically relevant, as they have a high chance of selection under antiretroviral drug pressure conditions (49)(50)(51)(52)(53)(54)(55)(56)(57). For that reason, a series of ultrasensitive assays have been developed to detect drug-resistant HIV-1 minority variants, e.g., allele-specific PCR (49,58), oligonucleotide ligation assays (33,59), and deep (next-generation) sequencing (60)(61)(62). On the other hand, as described above, the adoption of genotypic HIV-1 tropism assays in the clinical setting has been hampered by the limited sensitivities of the population-based sequencing assays to detect minor non-R5 variants.…”