Applying Next-Generation Sequencing to Track HIV-1 Drug Resistance Mutations Circulating in Portugal.

BACKGROUND: The global scale-up of antiretroviral treatment (ART) offers significant health benefits by suppressing HIV-1 replication and increasing CD4 cell counts. However, incomplete viral suppression poses a potential threat for the emergence of drug resistance mutations (DRMs), limiting ART options, and increasing HIV transmission. OBJECTIVE: We investigated the patterns of transmitted drug resistance (TDR) and acquired drug resistance (ADR) among HIV-1 patients in Portugal. METHODS: Data were obtained from 1050 HIV-1 patient samples submitted for HIV drug resistance (HIVDR) testing from January 2022 to June 2023. Evaluation of DRM affecting viral susceptibility to nucleoside/tide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs) was performed using an NGS technology, the Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay. RESULTS: About 71% of patients were ART naïve and 29% were experienced. Overall, 20% presented with any DRM. The prevalence of TDR and ADR was 12.6% and 41.1%, respectively. M184V, T215S, and M41L mutations for NRTI, K103N for NNRTI, and M46I/L for PIs were frequent in naïve and treated patients. E138K and R263K mutations against INSTIs were more frequent in naïve than treated patients. TDR and ADR to INSTIs were 0.3% and 7%, respectively. Patients aged 50 or over (OR: 1.81, p = 0.015), originating from Portuguese-speaking African countries (PALOPs) (OR: 1.55, p = 0.050), HIV-1 subtype G (OR: 1.78, p = 0.010), and with CD4 < 200 cells/mm3 (OR: 1.70, p = 0.043) were more likely to present with DRMs, while the males (OR: 0.63, p = 0.003) with a viral load between 4.1 to 5.0 Log10 (OR: 0.55, p = 0.003) or greater than 5.0 Log10 (OR: 0.52, p < 0.001), had lower chances of presenting with DRMs. CONCLUSIONS: We present the first evidence on TDR and ADR to INSTI regimens in followed up patients presenting for healthcare in Portugal. We observed low levels of TDR to INSTIs among ART-naïve and moderate levels in ART-exposed patients. Regimens containing PIs could be an alternative second line in patients with intermediate or high-level drug resistance, especially against second-generation INSTIs (dolutegravir, bictegravir, and cabotegravir).

Characterization of NS5A and NS5B Resistance-Associated Substitutions from Genotype 1 Hepatitis C Virus Infected Patients in a Portuguese Cohort.

This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the NS5 coding region, which is followed by Sanger population sequencing. NS5A RASs were present in 28.4% (23/81) of all GT1-infected patients with 9.9% (8/81) having the Y93C/H mutation. NS5B RASs showed a prevalence of 14.8% (12/81) and were only detected in GT1b. Overall 38.3% (31/81) of all GT1 HCV-infected patients presented baseline RASs. The obtained data supports the usefulness of resistance testing prior to treatment since a statistically significant association was found between treatment failure and the baseline presence of specific NS5 RASs known as Y93C/H (p = 0.04).

Discordant predictions of residual activity could impact dolutegravir prescription upon raltegravir failure.

BACKGROUND: Dolutegravir is approved for the treatment of HIV-1 patients exposed to other integrase inhibitors, but the decision to use dolutegravir in this setting should be informed by drug resistance testing. OBJECTIVES: This study determined the extent of disagreement in predicted residual dolutegravir activity after raltegravir use, and identified individual mutational patterns for which uncertainty exists among HIV-1 expert systems. STUDY DESIGN: Mutation patterns were classified in raltegravir signature pathways including positions 143, 148 and 155, and interpreted into clinically informative resistance levels using genotypic drug resistance interpretation systems ANRS v24, HIVdb v7.0 and Rega v9.1.0, and instructions of dolutegravir use as approved by the Food and Drug Administration and the European Medicines Agency. RESULTS: In 216HIV-1 patients failing raltegravir-therapy, 87% patients displayed mutations associated with resistance towards integrase inhibitors. A total of 141 unique mutational patterns were observed, with N155H (25.4%), Q148H (16.2%) and Y143R (8.3%) the most prevalent signature mutations. The Q148 pathway occurred almost exclusively in HIV-1 subtype B viruses. Concordances in predicted dolutegravir susceptibility scores among 5 systems were obtained in 57.8% of patients, and concordant intermediate resistant and concordant resistant scores were only observed in 6.5% and 0.9% of patients, respectively. However, systems individually scored higher levels of dolutegravir intermediate resistance and resistance, ranging from 4.2% to 10.2% and from 14.8% to 22.7% of patients, respectively. A consensus on interpreting the extent of residual activity was lacking in 34.7% of patients and was highly resistance pathway-specific. CONCLUSIONS: Dolutegravir may potentially be effective in the majority of HIV-1 patients failing raltegravir, but concern over the uncertainty in predicted residual activity could withhold clinicians from prescribing dolutegravir during its clinical assessment.

A retrospective observational study of low-level viraemia and its immunological and virological significance: which outcome to expect.

INTRODUCTION: Low-level viraemia (LLV) is observed in some patients with HIV-1 infection on stable antiretroviral therapy (ART). The significance of these findings remains controversial as it conflicts with traditional optimal clinic outcome. This study aims to evaluate the effect of LLV on the establishment of virological failure (VF) and immune deterioration. METHODS: Retrospective observational study of a cohort of HIV-1 infected patients of an Infectious Diseases Clinic, who presented an HIV-1 viral load of 20 to 200 cp/mL, during the year 2012. Patients who were not on ART or non-adherent in the previous 6 months were excluded. Compliance was quantified by clinical and pharmaceutical records. Adherence was defined as ≥95% compliance rate. Demographic, clinical, immunological and therapeutic data were collected from clinical records. LLV was defined as a range of 20-200 cp/mL and stratified as transient (T-LLV): only one measurement, persistent (P-LLV): 2 consecutive measurements with an interval ≥3 months and recurrent (R-LLV): ≥1 T-LLV during an 18-month follow-up. Statistical analysis was performed with Microsoft Office® - Excel 2012. Kolmogorov-Smirnov test, t-test and chi-square test were performed for a significant p value <0.05. RESULTS: During 2012, 2161 HIV-1 infected patients were evaluated at our Clinic, 93% of which were on ART. LLV was documented in 378 (19%), adherence was verified in 151 (52%). The analysis of this cohort (n=151) revealed: 77 (51%) T-LLV, 13 (8.6%) R-LLV and 61 (40%) P-LLV. Mean viral load was 46 cp/mL. Mean TCD4 count was 665 cells/µL with a variation of +63 cells/µL during the study period. There was no VF documented. ART regimens were switched in 16 (11%) patients. Gastrointestinal disturbance was found in 13 (9%). Analysis showed no statistical differences between the analyzed variables (CD4 variation, time of diagnosis and treatment, duration of LLV persistence (less than or more than one year), number of ART regimens, ART regimen and type of NRTI backbone) for all groups (T-LLV, R-LLV, P-LLV), except for mean viral load that showed significant superiority in the T-LLV(38 cp/mL) and R-LLV(36 cp/mL) vs P-LLV(58 cp/mL) (p=0.01 and p<0.01, respectively). CONCLUSIONS: The absence of significant differences in immunological and virological outcomes in this cohort and the absence of VF in all groups, suggests a scarce impact of LLV in patient's prognosis. Prospective studies, with longer follow-up could bring more accurate information.

Comparison of the NucliSENS EasyQ HIV-1 v2.0 with Abbott m2000rt RealTime HIV-1 assay for plasma RNA quantitation in different HIV-1 subtypes.

Quantitation of HIV-1 RNA levels in plasma has significant prognostic value since high viral load concentrations in plasma are associated with a faster disease progression. Viral load testing became one the most important tools for monitoring HIV patients. New real time methodologies to quantify HIV viral load had arisen in the last decade. HIV is a virus with a high genetic variability, with the potential to negatively affect the performance of the viral load assays. Consequently, any new assay should be challenged against, at least, the most prevalent HIV-1 genetic variants. In the present study, the new version of NucliSENS EasyQ(®) HIV-1 (Version 2.0) quantitative assay was compared with another ultra-sensitive test--Abbott RealTime HIV-1--using 175 plasma samples from patients infected with several HIV-1 subtypes and recombinant forms: subtype B (41, 23%), subtype C (19, 11%), subtype G (76, 44%), and CRF02_AG (39, 22%). Overall, there was agreement between the assays in 95.43% of the samples. Both assays have a very good dynamic range [1.4-6.9] and [1.60-7.0] log10 copies/mL and excellent correlation in samples with various subtypes. Based on the fact that no clinically significant differences were observed in the viral load measurements by these two assays, HIV-1 subtypes are quantified equally by both assays. However due to HIV diversity, mainly in regions were non B subtypes are predominant more evaluations are needed, so we do not recommend to switch platform during longitudinal viral load monitoring.

Effect of human immunodeficiency virus type 1 protease inhibitor therapy and subtype on development of resistance in subtypes B and G.

Europe is currently observing a significant rise in non-B subtypes. Consequently, the effect of genetic variability on therapy response or genotypic resistance interpretation algorithms is an emerging concern. The purpose of this study is to investigate the amino acid substitutions selected under drug pressure in the protease of human immunodeficiency virus type 1 (HIV-1) subtypes B and G, and determine if there are any significant differences. We investigated therapy-related and subtype-related substitutions in the protease, considering subtype, overall protease inhibitor treatment and individual drug exposure. Many mutations were significantly related to protease inhibitor (PI) therapy, with mutations exclusive to subtype B or subtype G. Some mutations are at positions related to resistance in both subtypes, but the amino acid substitution is different. Other mutations were significantly associated with subtype and PI selective pressure (p<0.05), pointing towards a differential selective pressure in both subtypes. We confirmed previous reports on the subtype-dependent selection of D30N and 89I, and identified a new mutation with such differential selective pressure: 37D was preferentially selected by lopinavir in subtype B. Other novel mutations found under therapy pressure were 13A, 35N, K55R, I66F, I72L/T, T74S, 82M and 89I/V. Our study indicates that even though in general, drug selective pressure and resistance pathways are relatively similar between subtypes B and G, some differences do occur, leading to subtype-dependent substitutions.

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes.

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.