Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay.

BACKGROUND: The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals. METHODS: We implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George's University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals. RESULTS: Although subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02_AG, and CRF01_AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient's antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George's, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4+ T cell counts in the NHS Lothian patients. CONCLUSIONS: This is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.

High-Throughput Adaptable SARS-CoV-2 Screening for Rapid Identification of Dominant and Emerging Regional Variants.

OBJECTIVES: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant strains can be associated with increased transmissibility, more severe disease, and reduced effectiveness of treatments. To improve the availability of regional variant surveillance, we describe a variant genotyping system that is rapid, accurate, adaptable, and able to detect new low-level variants built with existing hospital infrastructure. METHODS: We used a tiered high-throughput SARS-CoV-2 screening program to characterize variants in a supraregional health system over 76 days. Combining targeted reverse transcription-polymerase chain reaction (RT-PCR) and selective sequencing, we screened SARS-CoV-2 reactive samples from all hospitals within our health care system for genotyping dominant and emerging variants. RESULTS: The median turnaround for genotyping was 2 days using the high-throughput RT-PCR-based screen, allowing us to rapidly characterize the emerging Delta variant. In our population, the Delta variant is associated with a lower cycle threshold value, lower age at infection, and increased vaccine-breakthrough cases. Detection of low-level and potentially emerging variants highlights the utility of a tiered approach. CONCLUSIONS: These findings underscore the need for fast, low-cost, high-throughput monitoring of regional viral sequences as the pandemic unfolds and the emergence of SARS-CoV-2 variants increases. Combining RT-PCR-based screening with selective sequencing allows for rapid genotyping of variants and dynamic system improvement.

Comparison of cytocentrifugation supernatant fluid and formalin-fixed paraffin-embedded tissue for targeted next-generation sequencing.

BACKGROUND: The emergence of less invasive procedures coupled with the growth of molecular testing have created a need for clinical laboratories to optimize workflows to enable tissue preservation and ancillary testing. In the preparation of formalin-fixed paraffin-embedded cell blocks (FFPE CBs), there is a cytocentrifugation step for cell pellet extraction that results in postcentrifugation supernatant fluid (SN). This SN, which in most routine workflows is discarded, has been suggested to contain adequate cellular material for molecular testing. In the current study, the authors describe the use of DNA and RNA extracted from SN for the detection of clinically relevant biomarkers by next-generation sequencing (NGS). METHODS: After cell pellet removal, cytocentrifugation SN from 30 endobronchial fine-needle aspiration rinses that were positive for malignancy on FFPE CB were collected. DNA and RNA were extracted from the SN and tested using an in-house NGS Solid Tumor Focus Assay. The NGS results were compared with findings from corresponding FFPE samples. RESULTS: Testing was successful in all 30 samples. There was 100% concordance between variants observed in the SN and corresponding FFPE specimens, which included 50 single-nucleotide variants, 9 copy number amplifications, 3 structural variants, and 2 indels. Furthermore, there was excellent correlation (correlation coefficient, 0.93) between the variant allele frequency of mutations observed in SN compared with that noted in corresponding FFPE CBs. CONCLUSIONS: Cytocentrifugation SN is a valuable source for NGS, is comparable to FFPE that preserves tissue for other ancillary testing, and can reduce the failure rate of testing that may result from insufficient material being available in the CB.