Utility of Next-Generation Sequencing for HIV Antiretroviral Resistance Genotyping in a Pragmatic Clinical Laboratory Setting

Abstract

Routine HIV antiviral resistance genotyping is an important tool in the clinical management of HIV infections and interest is growing among clinical laboratories for the use of next-generation sequencing (NGS) in HIV drug resistance testing. Although resistance genotyping has traditionally been conducted using Sanger sequencing methods, NGS is emerging as a powerful tool due to its ability to detect lower frequency alleles. This study compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 141 sequences (103 protease-reverse transcriptase tests and 38 integrase tests) in the pragmatic clinical laboratory setting of the University of Washington (UW) Clinical Virology Laboratory. NGS was found to be technically equivalent to Sanger for the detection of high frequency drug resistant variants. NGS detected more variants associated with higher levels of drug resistance in 18% of protease-reverse transcriptase sequences and 10% of integrase sequences, compared to Sanger, due to its capacity to detect low frequency variants. Clinical follow-up of sixty-nine patients for almost two years found some evidence to suggest that low frequency drug-resistant variants increase patients’ risk of unsuppressed plasma HIV RNA (RR = 1.56; 95% CI: 0.75-3.22) and indicates the need for a larger study to confirm the association.