The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications. Here we compared the performance of five NGS HIVDR pipelines using proficiency panel samples from NIAID Virology Quality Assurance (VQA) program. Ten VQA panel specimens were genotyped by each of six international laboratories using their own in-house NGS assays. Raw NGS data were then processed using each of the five different pipelines including HyDRA, MiCall, PASeq, Hivmmer and DEEPGEN. All pipelines detected amino acid variants (AAVs) at full range of frequencies (1~100%) and demonstrated good linearity as compared to the reference frequency values. While the sensitivity in detecting low abundance AAVs, with frequencies between 1~20%, is less a concern for all pipelines, their specificity dramatically decreased at AAV frequencies <2%, suggesting that 2% threshold may be a more reliable reporting threshold for ensured specificity in AAV calling and reporting. More variations were observed among the pipelines when low abundance AAVs are concerned, likely due to differences in their NGS read quality control strategies. Findings from this study highlight the need for standardized strategies for NGS HIVDR data analysis, especially for the detection of minority HiVDR variants.Genotypic HIV drug resistance (HIVDR) testing not only guides effective clinical care of HIV-infected patients but also serves to provide surveillance of transmitted HIVDR in the population. Treatment guidelines in resource-permitted settings advocate the use of HIVDR monitoring both prior to ART initiation and when treatment failure is suspected 1,2 . There is increasing evidence showing that the presence of minority resistance variants open Scientific RepoRtS | (2020) 10:1634 | https://doi.org/10.1038/s41598-020-58544-z www.nature.com/scientificreports www.nature.com/scientificreports/ (MRV) in the HIV quasispecies (i.e., a swarm of highly-related but genotypically different viral variants) may be clinically significant and increase the risk of virological failure, impair immune recovery, lead to accumulation of drug resistance, increase risk of treatment switches and death [3][4][5][6][7][8] . A nationwide study in Mexico focusing on pretreatment drug resistance (PDR) found that lowering the detection threshold for PDR to 5% versus the conventional 20% improved the ability to identify patients with virological failure 6 . In addition, a European wide study found that pre-existing minority drug-resistant HIV-1 variants more than doubled the risk of virological failure to first-line NNRTI-based ART 9 . A more recent African study also reported similar findings, suggesting lowering the threshold below 20% improved the ability to i...

f Limited access to HIV drug resistance testing in low-and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n ‫؍‬ 759) and from a Ugandan study cohort (n ‫؍‬ 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log 10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.A dvances in highly active antiretroviral therapy (HAART) in recent decades have resulted in sustained decreases in HIVrelated morbidity and mortality rates. HIV-infected individuals who receive treatment now have nearly normal life expectancies, such that HIV is now considered a manageable chronic disease (1, 2); antiretroviral therapy (ART) not only provides benefits at the individual patient level but also results in a population-level advantage through HAART-induced suppression of HIV replication and the inherent prevention of onward transmission of the virus, termed "treatment as prevention" (3-6).Drug resistance testing is an essential complement to HAART, enabling clinicians to identify patients infected with drug-resistant HIV and to prescribe the appropriate antiretroviral regimens (7). Lack of access to HIV drug resistance testing acts as a major barrier to long-term treatment success, either through prescription of ineffective regimens in the case of transmitted resistance or through decreased ability of physicians to identify causes of treatment failure (7-9). Cases of unsuppressed viremia allow continued transmission and can compromise the management of HIV on both the patient and population levels. These challenges are particula...

SARS-CoV-2 Omicron infections are common among individuals who are vaccinated or have recovered from prior variant infection, but few reports have immunologically assessed serial Omicron infections. We characterized SARS-CoV-2 humoral responses in an individual who acquired laboratory-confirmed Omicron BA.1.15 ten weeks after a third dose of BNT162b2, and BA.2 thirteen weeks later. Responses were compared to 124 COVID-19-naive vaccinees. One month post-second and -third vaccine doses, the participant’s wild-type and BA.1-specific IgG, ACE2-displacement and virus neutralization activities were average for a COVID-19-naive triple-vaccinated individual. BA.1 infection boosted the participant’s responses to the cohort ≥95th percentile, but even this strong “hybrid” immunity failed to protect against BA.2. Reinfection increased BA.1 and BA.2-specific responses only modestly. Though vaccines clearly protect against severe disease, results highlight the continued importance of maintaining additional protective measures to counteract the immune-evasive Omicron variant, particularly as vaccine-induced immune responses naturally decline over time.