QuasiFlow: a Nextflow pipeline for analysis of NGS-based HIV-1 drug resistance data.

SUMMARY: Next-generation sequencing (NGS) enables reliable detection of resistance mutations in minority variants of human immunodeficiency virus type 1 (HIV-1). There is paucity of evidence for the association of minority resistance to treatment failure, and this requires evaluation. However, the tools for analyzing HIV-1 drug resistance (HIVDR) testing data are mostly web-based which requires uploading data to webservers. This is a challenge for laboratories with internet connectivity issues and instances with restricted data transfer across networks. We present QuasiFlow, a pipeline for reproducible analysis of NGS-based HIVDR testing data across different computing environments. Since QuasiFlow entirely depends on command-line tools and a local copy of the reference database, it eliminates challenges associated with uploading HIV-1 NGS data onto webservers. The pipeline takes raw sequence reads in FASTQ format as input and generates a user-friendly report in PDF/HTML format. The drug resistance scores obtained using QuasiFlow were 100% and 99.12% identical to those obtained using web-based HIVdb program and HyDRA web respectively at a mutation detection threshold of 20%. AVAILABILITY AND IMPLEMENTATION: QuasiFlow and corresponding documentation are publicly available at https://github.com/AlfredUg/QuasiFlow. The pipeline is implemented in Nextflow and requires regular updating of the Stanford HIV drug resistance interpretation algorithm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Advances online

High Levels of Acquired HIV Drug Resistance Following Virological Nonsuppression in HIV-Infected Women from a High-Risk Cohort in Uganda.

HIV drug resistance (HIVDR) is of increasing health concern, especially among key populations. We investigated the prevalence of virological suppression (VS), prevalence and correlates of HIVDR in HIV-infected women, enrolled in a high-risk cohort. We enrolled 267 women initiated on first-line antiretroviral therapy (ART) between 2015 and 2018. Participants' plasma samples were analyzed for HIV RNA viral load (VL) and genotypic resistance testing was performed on those with VL nonsuppression (defined as VL ≥1,000 copies/mL). We used the Stanford HIVDR database-algorithm to assess HIVDR mutations and logistic regression to assess risk factors for VL nonsuppression and HIVDR. We observed an overall VS prevalence of 76.0% (203/267) and detected respective acquired drug resistance prevalence to non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) of 81.3% [confidence interval (CI) 67.4-91.1] and 45.8% (CI 31.4-60.8) among the 48 successfully genotyped VL nonsuppressors. NNRTI mutations were observed in 81.3% (39/48) of the genotyped participants and 45.8% (22/48) had both NRTI and NNRTI mutations. The mutation K103N was detected in 62.5% (30/48) of participants, 41.7% (20/48) had M184V/I, 14.6% had K65R, and 12.5% (6/48) had thymidine analog mutations (TAMs). None of the analyzed potential risk factors, including age and duration on ART, was significantly correlated with VL nonsuppression or HIVDR. Although high levels of NNRTI mutations support the transition to dolutegravir, the presence of NRTI mutations, especially TAMs, may compromise dolutegravir-based regimens or other second-line ART options. The moderate VS prevalence and high HIVDR prevalence therefore call for timely ART switching and intensive adherence counseling

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants

Spike Protein Cleavage-Activation in the Context of the SARS-CoV-2 P681R Mutation: an Analysis from Its First Appearance in Lineage A.23.1 Identified in Uganda.

Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences. IMPORTANCE During the course of the SARS-CoV-2 pandemic, viral variants have emerged that often contain notable mutations in the spike gene. Mutations that encode changes in the spike S1/S2 (furin) activation site have been considered especially impactful. The S1/S2 change from proline to arginine at position 681 (P681R) first emerged in the A.23.1 variant in Uganda, and subsequently occurred in the more widely transmitted Delta variant. We show that the A.23.1 spike is more readily activated by the host cell protease furin, but that this is not reproduced in an original SARS-CoV-2 spike containing the P681R mutation. Changes to the S1/S2 (furin) activation site play a role in SARS-CoV-2 infection and spread, but successful viruses combine these mutations with other less well identified changes, occurring as part of natural selection