New Aspects of the Virus Life Cycle and Clinical Utility of Next Generation Sequencing based HIV-1 Resistance Testing in the Genomic, the Proviral, and the Viral Reservoir of Peripheral Blood Mononuclear Cells.

BACKGROUND

Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load < 500 copies/ml), resistance testing by population-based sequencing is very difficult.

OBJECTIVE

Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative.

MATERIAL AND METHODS

EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors.

RESULTS

A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA.

CONCLUSION

Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus.