Detection Of Drug Resistance Mutations Research Articles

Revealing the unseen: next generation sequencing for early detection of drug-resistant cytomegalovirus variants upon letermovir prophylaxis failure.

In allogeneic hematopoietic cell transplant (HCT)-recipients, prophylactic management strategies are essential for preventing CMV-reactivation and associated disease. We report on a 63-year-old male patient with a D-/R+ CMV-serostatus, who showed ongoing low-level CMV-replication post-HCT despite receiving letermovir prophylaxis. Sanger-sequencing failed to detect drug resistance mutations (DRM) until CMV-pneumonitis developed, revealing a UL56-C325R-DRM linked to high-level letermovir resistance. Retrospective analysis with next-generation-sequencing (NGS) revealed the DRM at a low frequency of 6% two weeks prior to detection by Sanger-sequencing. This study highlights the importance of advanced NGS-methods for early detection of CMV-DRMs, allowing for faster adjustments in antiviral treatment strategies.

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis.

Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy. This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing. The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence. The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system's sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics. This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV.

The relevance of ultradeep sequencing for low HIV-1 viral loads and proviruses in the clinical setting.

Improving the therapeutic management of HIV-positive persons is a major public health issue and includes better detection of drug resistance mutations (DRMs). The aim of this study was (i) to explore DRMs in HIV-1-positive persons presenting a blood viral load (VL) < 1000 genomes copies (gc)/mL, including the analyze of cerebrospinal fluid (CSF) and HIV-DNA from peripheral blood mononuclear cells using ultradeep sequencing (UDS) and (ii), to evaluate how these DRMs could influence the clinical practices. For each patient (n = 12), including five low-VL patients (i.e., <1000 gc/mL), HIV-1 UDS targeting the protease, reverse transcriptase and integrase genes was performed on plasma, proviral DNA, and CSF when available. Sequencing discordances or failures were mostly found in samples from low-VL patients. A 5% UDS cut-off allowed to increase the sensitivity to detect DRMs in different compartments, excepted in CSF. Patients with the highest viral quasispecies heterogeneity were naïve of treatment or presented a medical history suggesting low selection pressure or virological failures. When analyzing compartmentalization and following-up patients: low-frequency variants (LFVs) were responsible for 47% (n = 8) and 76% (n = 13) of changes in drug resistance interpretation, respectively. In such cases, we conclude that UDS is a robust technique, which still could be improved by increase the RNA and/or DNA extraction in low-VL samples to detect LFVs. Further studies are needed to define the impact of LFVs on antiretroviral treatments. At last, when considering a DRM, the use of mutational load would probably be more suitable than frequencies.

An ultrasensitive DNA-enhanced amplification method for detecting cfDNA drug-resistant mutations in non-small cell lung cancer with selective FEN-assisted degradation of dominant somatic fragments.

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5'-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing arapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

BackgroundThe successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. MethodFrom a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. ResultsSequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). ConclusionThe study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases.

Bovine respiratory disease (BRD) is a significant veterinary challenge, often exacerbated by pathogen resistance, hindering effective treatment. Traditional testing methods for primary pathogens - Mycoplasma bovis, Pasteurella multocida, and Mannheimia haemolytica - are notably time-consuming and lack the rapidity required for effective clinical decision-making. This study introduces a TaqMan MGB probe detection chip, utilizing fluorescent quantitative PCR, targeting key BRD pathogens and associated drug-resistant genes and sites. We developed 94 specific probes and primers, embedded into a detection chip, demonstrating notable specificity, repeatability, and sensitivity, reducing testing time to under 1 h. Additionally, we formulated probes to detect mutations in the quinolone resistance-determining region, associated with fluoroquinolone resistance in BRD pathogens. The chip exhibited robust sensitivity and specificity, enabling rapid detection of drug-resistant mutations in clinical samples. This methodology significantly expedites the diagnostic process for BRD and sensitive drug screening, presenting a practical advancement in the field.

Current Resistance of HIV-1 Strains Isolated in Volunteer Blood Donors in Gabon.

Detection of drug resistance mutations (DRMs) and HIV-1 subtypes ensures effective therapeutic management for HIV-infected individuals. In Gabon, data on DRMs are very little available in the population of people living with HIV and also among voluntary HIV-positive blood donors. This study aimed to study subtypes and DRMs in HIV-1-positive volunteer blood donors in Gabon. A cross-sectional study was carried out at the National Blood Transfusion Center of Gabon. A purposive sampling method was used to collect 128 HIV-1 seropositive blood samples. Viral RNA was extracted on real-time PCR (Abbott 2000®), and sequencing was performed on ABI 3500 (Hitachi®). SPSS version 21.0 software was used for statistical analysis. Of the 128 seropositive volunteer donors included, men and the 29-39-age group were more representative at 78.9% and 49.2%, respectively. Eighty-two samples were sequenced. The majority strains identified were subtype A, subtype F, subtype G, CRF02_AG, and CRF45_cpx. The resistance mutations identified were K103N, L210W, E138G, V179D, V179T, and M46L. The prevalence of resistant subtypes was 25.6%. CRF02_AG strains exhibited high-level resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), including efavirenz and nevirapine. The study identified major DRMs in reverse transcriptase and protease that confer high-level resistance to most NNRTIs, nucleoside reverse transcriptase inhibitors, and protease inhibitors. CRF02_AG was more predominant, and the frequency of resistant subtypes was high. However, these data will contribute to the therapeutic choice during the initiation of antiretroviral treatment in treatment-naive patients in Gabon.

ClusterV-Web: a user-friendly tool for profiling HIV quasispecies and generating drug resistance reports from nanopore long-read data.

Third-generation long-read sequencing is an increasingly utilized technique for profiling human immunodeficiency virus (HIV) quasispecies and detecting drug resistance mutations due to its ability to cover the entire viral genome in individual reads. Recently, the ClusterV tool has demonstrated accurate detection of HIV quasispecies from Nanopore long-read sequencing data. However, the need for scripting skills and a computational environment may act as a barrier for many potential users. To address this issue, we have introduced ClusterV-Web, a user-friendly web-based application that enables easy configuration and execution of ClusterV, both remotely and locally. Our tool provides interactive tables and data visualizations to aid in the interpretation of results. This development is expected to democratize access to long-read sequencing data analysis, enabling a wider range of researchers and clinicians to efficiently profile HIV quasispecies and detect drug resistance mutations. ClusterV-Web is freely available and open source, with detailed documentation accessible at http://www.bio8.cs.hku.hk/ClusterVW/. The standalone Docker image and source code are also available at https://github.com/HKU-BAL/ClusterV-Web.

Enhanced detection of rifampicin and isoniazid resistance in mycobacterium tuberculosis using AuNP-qPCR: a rapid and accurate method.

To evaluate the resistance of Mycobacterium tuberculosis to Rifampicin (RIF) and Isoniazid (INH) using enhanced qPCR methodologies. This study compared the detection of drug-resistant mutations in the rpoB and katG genes using AuNP-qPCR and No-AuNP-qPCR. Calibration curves were constructed to correlate the amount of template with the Ct values for resistant strains. The AuNP-qPCR method demonstrated high efficacy in detecting RIF resistance with an area under the curve (AUC) of 0.951, sensitivity of 97.92%, specificity of 87.5%, and overall accuracy of 95.31%. Similarly, INH resistance detection by AuNP-qPCR showed an AUC of 0.981, sensitivity of 98.08%, specificity of 94.44%, and accuracy of 97.14%. Comparatively, No-AuNP-qPCR yielded lower performance metrics for RIF resistance (AUC: 0.867, sensitivity: 91.67%, specificity: 75%, accuracy: 87.5%) and INH resistance (AUC: 0.882, sensitivity: 88.46%, specificity: 83.33%, accuracy: 87.14%). AuNP-qPCR exhibits over traditional qPCR methods, making it a promising tool for rapid and precise detection of drug resistance in Mycobacterium tuberculosis. This method's robust performance underscores its potential to improve diagnostic protocols and contribute to more effective management of tuberculosis treatment.

Performance characteristics of Allele-Specific PCR (ASPCR) in detecting drug resistance mutations among non-B HIV-1 Variants

Allele-Specific Polymerase Chain Reaction (ASPCR) is an affordable point-mutation assay whose validation could improve the detection of HIV-1 drug resistance mutations (DRMs) in resource-limited settings (RLS). We assessed the performance of ASPCR onforty-four non-B HIV-1 plasma samples from patients who were ARV treated in failure in N’Djamena-Chad. Viral RNA was reverse-transcribed and amplified using LightCycler® FastStart DNA MasterPLUS SYBR Green I. Detection of six major DRMs (K70R, K103N, Y181C, M184V, T215F, T215Y) was evaluated on Roche LightCycler®480 automated system (with dilutions 0.01–100%). ASPCR-results were compared to Sanger-sequencing (gold-standard). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02–0.28 vs. 0.12–0.37), K103N (0.08–0.42 vs. 0.12–0.37), Y181C (0.12–0.39 vs. 0.31–0.37), M184V (0.13–0.39 vs. 0.23–0.42), T215F (0.05–0.43 vs. 0.04–0.45) and T215Y (0.13–0.41 vs. 0.19–0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02–0.28 vs. 0.12–0.37), K103N (0.08–0.42 vs. 0.12–0.37), Y181C (0.12–0.39 vs. 0.31–0.37), M184V (0.13–0.39 vs. 0.23–0.42), T215F (0.05–0.43 vs. 0.04–0.45) and T215Y (0.13–0.41 vs. 0.19–0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). ASPCR appears more efficient for detecting DRMs on diverse HIV-1 non-B circulating in RLS like Chad.

Detection of Salmonella DNA and drug-resistance mutation by PCR-based CRISPR-lbCas12a system

Salmonella is an important foodborne pathogen, which can cause serious public health problems. Rapid and accurate detection of Salmonella infection and drug resistance mutations in patients will provide timely guidance for clinical treatment and avoid disease progression and other related clinical problems. Here, we established a highly sensitive and quick method for Salmonella and drug resistance mutation detection based on polymerase chain reaction (PCR) and CRISPR-lbCas12a system and evaluated its practicability with clinical samples.Specific CRISPR RNAs (crRNAs) and primers are designed for Salmonella DNA and parC gene S80I mutation diagnosis. CrRNAs with and without phosphorylated modification and different crRNA preparation methods are used to assess the effect on the detection system. After optimization, we detected as low as one copy of Salmonella DNA and drug resistance mutation parC S80I with the Salmonella DNA standard. For 94 clinical samples, this method also showed high sensitivity (100%, 95% CI: 84.98–100%) and specificity (98.48%, 95% CI: 90.73–99.92%) with less time (3 h) than plate culture (16 h) and conventional antimicrobial susceptibility testing (over 16 h). Besides, one parC S80I mutant strain was detected, which is consistent with the result of DNA sequencing. Taken together, we established a highly sensitive and specific method for Salmonella infection and parC S80I drug resistance mutation detection with fewer reagents and ordinary instruments. This assay has wide application prospects for fast detection of pathogen (bacterium and virus) infection, drug resistance determination, and proper treatment guidance.

Prevalence of HIV-1 drug resistance mutations in proviral DNA in the Swiss HIV Cohort Study, a retrospective study from 1995 to 2018.

Genotypic resistance testing (GRT) is routinely performed upon diagnosis of HIV-1 infection or during virological failure using plasma viral RNA. An alternative source for GRT could be cellular HIV-1 DNA. A substantial number of participants in the Swiss HIV Cohort Study (SHCS) never received GRT. We applied a method that enables access to the near full-length proviral HIV-1 genome without requiring detectable viraemia. Nine hundred and sixty-two PBMC specimens were received. Our two-step nested PCR protocol was applied to generate two overlapping long-range amplicons of the HIV-1 genome, sequenced by next-generation sequencing (NGS) and analysed by MinVar, a pipeline to detect drug resistance mutations (DRMs). Six hundred and eighty-one (70.8%) of the samples were successfully amplified, sequenced and analysed by MinVar. Only partial information of the pol gene was contained in 82/681 (12%), probably due to naturally occurring deletions in the proviral sequence. All common HIV-1 subtypes were successfully sequenced. We detected at least one major DRM at high frequency (≥15%) in 331/599 (55.3%) individuals. Excluding APOBEC-signature (G-to-A mutation) DRMs, 145/599 (24.2%) individuals carried at least one major DRM. RT-inhibitor DRMs were most prevalent. The experienced time on ART was significantly longer in DRM carriers (P = 0.001) independent of inclusion or exclusion of APOBEC-signature DRMs. We successfully applied a reliable and efficient method to analyse near full-length HIV-1 proviral DNA and investigated DRMs in individuals with undetectable or low viraemia. Additionally, our data underscore the need for new computational tools to exclude APOBEC-related hypermutated NGS sequence reads for reporting DRMs.

Prevalence of HIV-1 infection and associated characteristics in a Brazilian indigenous population: a cross-sectional study

Despite significant progress in the areas of prevention, diagnosis, and treatment, HIV continues to result in a substantial number of fatalities on a global scale each year. Gaining insights from epidemiological data can prove instrumental in the development of health promotion strategies, particularly within vulnerable populations, such as indigenous groups. Consequently, our study aimed to investigate the prevalence of HIV infection within the indigenous population residing in the second-largest region of Brazil. Additionally, we sought to explore the subtypes of HIV-1 and detect any drug-resistance mutations present within this population. In this cross-sectional study, we aimed to evaluate the prevalence of HIV-1 infection and explore its associated characteristics within the indigenous population residing in the villages of Jaguapiru and Bororó, located in the Dourados area of Mato Grosso do Sul (MS), Brazil. Blood samples were collected for rapid HIV screening, serological tests, nucleic acid amplification, and HIV subtyping. Additionally, the HIV-1 viral load and CD4+ T lymphocyte count of the people living with HIV (PLHIV) were assessed at the time of recruitment and 24 weeks later. Out of the 2190 invited individuals, 1927 (88%) were included in this study. The average age of the participants was 34.2 (±13.8) years, with a majority of 74% being female. Moreover, 68.44% of the participants identified themselves as belonging to the Guarani-Kaiowa ethnic group. HIV seroprevalence was 0.93% (18/1927), and 73.22% (1411/1927) were unaware of their serological status. The prevalence of HIV-1 was higher in single indigenous people [10/617 (1.62%)], who received government benefits [14/1021 (1.37%)], had less than five years of formal education [11/685 (1.61%)], had sexual intercourse with users of injectable drugs [2/21 (9.52%)], with history of sexually transmitted infections (STIs) [10/62 (16.2%)] and incarceration [3/62 (4.84%)]. Of 18 positive samples, 44.4% (8/18) were successfully amplified, and HIV-1 subtype C was prevalent. Furthermore, we identified HIV-1 drug resistance mutations in four patients, specifically from the classes of Protease Inhibitor, Nucleoside Reverse Transcriptase Inhibitor, and Non-Nucleoside Reverse Transcriptase Inhibitor. Notably, three of these patients exhibited a high viral load even after 24 weeks of undergoing antiretroviral therapy. Out of the 18 PLHIV, 66.66% (12/18) had a viral load below 1000 copies/mL, while 50% (9/18) had a CD4+ T lymphocytes count greater than 350cells/mL after 24 weeks of treatment. Despite the concerted efforts to control HIV infection, the prevalence observed in the indigenous population under study surpassed that reported in other Brazilian indigenous groups. This disparity highlights the disproportionate impact of the disease on this particular group. The detection of drug-resistance mutations further emphasizes the critical need to expand diagnostic coverage, closely monitor treatment strategies, and maintain ongoing molecular surveillance. These measures are imperative for enhancing HIV management within this vulnerable population. This study was partially funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Secretaria do Estado de Saúde (SES) of Governo do Estado de Mato Grosso do Sul, and Universidade Federal da Grande Dourados (UFGD).

An optimized GATK4 pipeline for Plasmodium falciparum whole genome sequencing variant calling and analysis

BackgroundAccurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here a falciparum variant calling pipeline based on GATK version 4 (GATK4) was optimized and applied to 6626 public Illumina WGS samples.MethodsControl WGS and accurate PacBio assemblies of 10 laboratory strains were leveraged to optimize parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs. From these controls, a high-quality training dataset was generated to recalibrate the raw variant data.ResultsOn current high-quality samples (read length = 250 bp, insert size = 405–524 bp), the optimized pipeline shows improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). Its sensitivity on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. The resulting combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. Finally, increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection.ConclusionsOverall, this study provides an optimized falciparum GATK4 pipeline resource for variant calling which should help improve genomic studies of malaria.

Full-spectrum HIV drug resistance mutation detection by high-resolution complete pol gene sequencing.

Drug resistance mutation testing is a key element for HIV clinical management, informing effective treatment regimens. However, resistance screening in current clinical practice is limited in reporting linked cross-class resistance mutations and minority variants, both of which may increase the risk of virological failure. To address these limitations, we obtained 358 full-length pol gene sequences from 52 specimens of 20 HIV infected individuals by combining microdroplet amplification, unique molecular identifier (UMI) labeling, and long-read high-throughput sequencing. We conducted a rigorous assessment of the accuracy of our pipeline for precision drug resistance mutation detection, verifying that a sequencing depth of 35 high-throughput reads achieved complete, error-free pol gene sequencing. We detected 26 distinct drug resistance mutations to Protease Inhibitors (PIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and Integrase Strand Transfer Inhibitors (INSTIs). We detected linked cross-class drug resistance mutations (PI+NRTI, PI+NNRTI, and NRTI+NNRTI) that confer cross-resistance to multiple drugs in different classes. Fourteen different types of minority mutations were also detected with frequencies ranging from 3.2% to 19%, and the presence of these mutations was verified by Sanger reference sequencing. We detected a putative transmitted drug resistance mutation (TDRM) in one individual that persisted for over seven months from the first sample collected at the acute stage of infection prior to seroconversion. Our comprehensive drug resistance mutation profiling can advance clinical practice by reporting mutation linkage and minority variants to better guide antiretroviral therapy options.

Quantification of Isoniazid-Heteroresistant Mycobacterium tuberculosis Using Droplet Digital PCR.

The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.

SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 – Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software.

Drug-resistance mutations were mostly detected using capillary electrophoresis sequencing, which does not detect minor variants with a frequency below 20%. Next-Generation Sequencing (NGS) can now detect additional mutations which can be useful for HIV-1 drug resistance interpretation. The objective of this study was to evaluate the performances of CE-IVD assays for HIV-1 drug-resistance assessment both for target-specific and whole-genome sequencing, using standardized end-to-end solution platforms. A total of 301 clinical samples were prepared, extracted, and amplified for the three HIV-1 genomic targets, Protease (PR), Reverse Transcriptase (RT), and Integrase (INT), using the CE-IVD DeepChek® Assays; and then 19 clinical samples, using the CE-IVD DeepChek® HIV Whole Genome Assay, were sequenced on the NGS iSeq100 and MiSeq (Illumina, San Diego, CA, USA). Sequences were compared to those obtained by capillary electrophoresis. Quality control for Molecular Diagnostics (QCMD) samples was added to validate the clinical accuracy of these in vitro diagnostics (IVDs). Nineteen clinical samples were then tested with the same sample collection, handling, and measurement procedure for evaluating the use of NGS for whole-genome HIV-1. Sequencing analyzer outputs were submitted to a downstream CE-IVD standalone software tailored for HIV-1 analysis and interpretation. The limits of range detection were 1000 to 106 cp/mL for the HIV-1 target-specific sequencing. The median coverage per sample for the three amplicons (PR/RT and INT) was 13,237 reads. High analytical reproducibility and repeatability were evidenced by a positive percent agreement of 100%. Duplicated samples in two distinct NGS runs were 100% homologous. NGS detected all the mutations found by capillary electrophoresis and identified additional resistance variants. A perfect accuracy score with the QCMD panel detection of drug-resistance mutations was obtained. This study is the first evaluation of the DeepChek® Assays for targets specific (PR/RT and INT) and whole genome. A cutoff of 3% allowed for a better characterization of the viral population by identifying additional resistance mutations and improving the HIV-1 drug-resistance interpretation. The use of whole-genome sequencing is an additional and complementary tool to detect mutations in newly infected untreated patients and heavily experienced patients, both with higher HIV-1 viral-load profiles, to offer new insight and treatment strategies, especially using the new HIV-1 capsid/maturation inhibitors and to assess the potential clinical impact of mutations in the HIV-1 genome outside of the usual HIV-1 targets (RT/PR and INT).

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

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. 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. 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%. 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.

HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads.

Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401-999 copies/mL) due to the use of a threshold of VL ≥1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV. We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401-999 copies/mL, and 46 had ≥1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test. The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI- and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI- and NRTI-associated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥1000 copies/mL. The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.