Frequency matters: comparison of drug resistance mutation detection by Sanger and next-generation sequencing in HIV-1.

Balakrishna, Suraj; Loosli, Tom; Zaheri, Maryam; Frischknecht, Paul; Huber, Michael; Kusejko, Katharina; Yerly, Sabine; Leuzinger, Karoline; Perreau, Matthieu; Ramette, Alban; Wymant, Chris; Fraser, Christophe; Kellam, Paul; Gall, Astrid; Hirsch, Hans H; Stoeckle, Marcel; Rauch, Andri; Cavassini, Matthias; Bernasconi, Enos; Notter, Julia; ... (2023). Frequency matters: comparison of drug resistance mutation detection by Sanger and next-generation sequencing in HIV-1. The journal of antimicrobial chemotherapy, 78(3), pp. 656-664. Oxford University Press 10.1093/jac/dkac430

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BACKGROUND

Next-generation sequencing (NGS) is gradually replacing Sanger sequencing (SS) as the primary method for HIV genotypic resistance testing. However, there are limited systematic data on comparability of these methods in a clinical setting for the presence of low-abundance drug resistance mutations (DRMs) and their dependency on the variant-calling thresholds.

METHODS

To compare the HIV-DRMs detected by SS and NGS, we included participants enrolled in the Swiss HIV Cohort Study (SHCS) with SS and NGS sequences available with sample collection dates ≤7 days apart. We tested for the presence of HIV-DRMs and compared the agreement between SS and NGS at different variant-calling thresholds.

RESULTS

We included 594 pairs of SS and NGS from 527 SHCS participants. Males accounted for 80.5% of the participants, 76.3% were ART naive at sample collection and 78.1% of the sequences were subtype B. Overall, we observed a good agreement (Cohen's kappa >0.80) for HIV-DRMs for variant-calling thresholds ≥5%. We observed an increase in low-abundance HIV-DRMs detected at lower thresholds [28/417 (6.7%) at 10%-25% to 293/812 (36.1%) at 1%-2% threshold]. However, such low-abundance HIV-DRMs were overrepresented in ART-naive participants and were in most cases not detected in previously sampled sequences suggesting high sequencing error for thresholds <3%.

CONCLUSIONS

We found high concordance between SS and NGS but also a substantial number of low-abundance HIV-DRMs detected only by NGS at lower variant-calling thresholds. Our findings suggest that a substantial fraction of the low-abundance HIV-DRMs detected at thresholds <3% may represent sequencing errors and hence should not be overinterpreted in clinical practice.

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