HIV-1 Subtype Research Articles

HIV-1 transmission clusters and drug resistance in men who have sex with men in Portugal

Abstract Introduction In 2022, 45.8% of diagnoses in the EU/EEA with known route of transmission were in Men who have Sex with Men (MSM). We aim to characterise HIV transmission clusters and transmission of drug resistance (TDR), as well as its determinants, using integrated sociodemographic, behavioural, clinical, and viral genomic data of MSMs newly diagnosed in Portugal between 2014 and 2019. Methods This study included data from 340 MSM who were diagnosed with HIV-1 infection. These individuals were newly diagnosed at 17 hospitals in Portugal between September 2014 and December 2019. Phylogenies were constructed and transmission clusters were identified with branch support ≥ 90% and 1.5% genetic distance. Logistic regression models were computed to examine the factors associated with clusters of transmission and with the presence of TDR. Results We identified 38 transmission clusters with 104 MSM, which included 26.6% of the total 305 MSM from our database used for cluster analysis. The overall prevalence of TDR was 8.2%. Only subtype C was significantly associated with TDR. 10.5% of the clusters had at least 1 drug resistance mutation (K103N, L90M or N88S). There was no significant difference in the prevalence of TDR between MSM inside and outside clusters. 50% of the clusters were composed of portuguese native MSM only, whereas 16% had exclusively migrant MSMs. No significant difference was found in the proportion of portuguese and migrant MSM inside and outside clusters. Factors associated with HIV-1 transmission clusters were age at diagnosis, district of residence, unprotected anal or vaginal sex with a woman, HIV testing frequency, presenter status and subtype. 87% of men engaged in sexual activity only with other men, while 13% with both men and women. Conclusions Specific subgroups of MSM are contributing to HIV dissemination in Portugal. Direct and precise prevention measures based on molecular epidemiology should be developed. Key messages • Specific subgroups of MSM are contributing to HIV dissemination in Portugal. • Direct and precise prevention measures based on molecular epidemiology should be developed.

Comparison of the Performance of Commercially Available Quantitative Viral Load Assays Using Clinical Samples from Patients from Regions Where Distinct HIV-1 Subtypes Co-Circulate: Potential Implications for Patient Management.

HIV RNA plasma viral load (VL) is the standard surrogate marker to monitor response to antiretroviral treatment (ART). We compared the linearity, repeatability, and concordance of six commercially available HIV RNA VL platforms using clinical samples from patients from Brazilian sites where different HIV-1 subtypes co-circulate. A total of 150 plasma samples from each city were collected in Curitiba, Southern Brazil (subtype C), São Paulo (subtype B), and Santos (BF recombinants), Southeast Brazil. Platforms were VERSANT® Siemens HIV RNA 1.0 (kPCR); VERSANT® Siemens HIV-1 RNA 3.0 (bDNA); Abbott Real-Time HIV-1; NucliSens EasyQ® HIV-1 v2.0 Biomerieux; COBAS® TaqMan®, Roche; and artus HIV Virus-1 RT-PCR, QIAGEN. OptiQuant HIV-1 RNA quantification panel was used to compare VL linearity, using samples containing 50, 500,5,000, 50,000, 500,000, and 5,000,000 HIV copies/mL. HIV RNA panels with subtypes A, B, C, D, F, G, H, circulating recombinant form (CRF)1, and CRF2 were utilized. A high degree of linearity and repeatability was demonstrated for all platforms. When compared with a subtype B reference sample, 17 of 54 (31.48%) samples diverged by more than 0.5 log10 copies/mL. Except for the Roche platform, all platforms underestimated subtype C VLs. A total of 743 (82.6%) valid results were obtained with samples from São Paulo, 707 (78.6%) from Santos, and 673 (74.8%) from Curitiba (São Paulo vs. Santos, p = .03; São Paulo vs. Curitiba, p = .00006; Santos vs. Curitiba, p = .06). The number of discordant samples between different methodologies when VL was undetectable in one method and detectable in the other ranged from 1.25% (Abbot vs. Siemens) to 44.8% (Abbott vs. Biomerieux). Finding samples with undetectable VL in one method and a high VL in another might have important individual and public health consequences. Standardization of VL measurements, particularly for non-B subtypes infections, especially subtype C, is necessary to maximize the individual and public health benefits of ART globally.

Determination of the mean duration of recent infection and false recency rate for the HIV triplex multiplex bead assay.

We developed the HIV Triplex multiplex bead assay to identify and serotype HIV infection with high sensitivity and specificity; and distinguish recent from long-term HIV-1 infections. It can facilitate accurate incidence estimation, while reducing the number of tests and blood collected, which is highly desirable for use in future studies and surveys. Using previously collected, treatment-naive longitudinal seroconversion HIV-1 positive panels and specimens from individuals infected for >12 months, we determined the assay's mean duration of recent infection (MDRI) and false-recency rate (FRR) respectively, at various mean fluorescent intensity (MFI) cutoffs. We tested seroconversion specimens (N = 814) from 142 individuals infected with HIV-1 subtypes B, C, or AE, and 1341 cross-sectional specimens from individuals infected >12 months. The MFI cutoffs of 1000 to 2000 were evaluated for recency classification, including an MFI of 1250 corresponding to the limiting antigen avidity enzyme immunoassay (LAg-EIA) cutoff of 1.5 normalized optical density for MDRI and FRR. We used four statistical methods: Methods 1 and 2 used the empirically balanced observation time approach. Method 2 MFI values were raised to power = 1.33, based on a repeated measures model to linearize the relationship between MFI and time points, whereas Method 1 was not linearized. Methods 3 and 4 employed quadratic and linear interpolations for each seroconversion panel. FRR was calculated by dividing the number of specimens misclassified as recent by the total number of specimens tested. MDRI values ranged from 135-146 days at MFI = 1000 to 229-279 days at MFI = 2000 by the 4 methods. FRR varied from 0.15%-1.27% with increasing MFI cutoff. At MFI = 1250, the average MDRI of 4 methods was 169 days and ranged from 159-183 with overlapping 95% CIs and FRR = 0.52%. The HIV Triplex assay demonstrates a longer dynamic range compared to current HIV recency assays with a low FRR for cutoffs examined. With a longer dynamic range and low FRR, the MDRI for recent infection can be extended as appropriate to detect more recent infections, increasing the value of incidence assays benefiting public health surveillance and future surveys.

Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection.

Background Persisting HIV reservoir viruses in resting CD4 T cells and other cellular subsets are the main barrier to cure efforts. Antiretroviral therapy (ART) intensification by early initiation has been shown to enable post-treatment viral control in some cases but the underlying mechanisms are not fully understood. We hypothesized that ART initiated during the hyperacute phase of infection before peak will affect the size, decay dynamics and landscape characteristics of HIV-1 subtype C viral reservoirs. Methods We studied 35 women at high risk of infection from Durban, South Africa identified with hyperacute HIV infection by twice weekly testing for plasma HIV-1 RNA. Study participants included 11 who started ART at a median of 456 (297-1203) days post onset of viremia (DPOV), and 24 who started ART at a median of 1 (1-3) DPOV. We used peripheral blood mononuclear cells (PBMC) to measure total HIV-1 DNA by ddPCR and to sequence reservoir viral genomes by full length individual proviral sequencing (FLIP-seq) from onset of detection of HIV up to 1 year post treatment initiation. Results Whereas ART in hyperacute infection blunted peak viremia compared to untreated individuals (p<0.0001), there was no difference in total HIV-1 DNA measured contemporaneously (p=0.104). There was a steady decline of total HIV DNA in early treated persons over 1 year of ART (p=0.0004), with no significant change observed in the late treated group. Total HIV-1 DNA after one year of treatment was lower in the early treated compared to the late treated group (p=0.02). Generation of 697 single viral genome sequences revealed a difference in the longitudinal proviral genetic landscape over one year between untreated, late treated, and early treated infection: the relative contribution of intact genomes to the total pool of HIV-1 DNA after 1 year was higher in untreated infection (31%) compared to late treated (14%) and early treated infection (0%). Treatment initiated in both late and early infection resulted in a more rapid decay of intact (13% and 51% per month) versus defective (2% and 35% per month) viral genomes. However, intact genomes were still observed one year post chronic treatment initiation in contrast to early treatment where intact genomes were no longer detectable. Moreover, early ART reduced phylogenetic diversity of intact genomes and limited the seeding and persistence of cytotoxic T lymphocyte immune escape variants in the reservoir. Conclusions Overall, our results show that whereas ART initiated in hyperacute HIV-1 subtype C infection did not impact reservoir seeding, it was nevertheless associated with more rapid decay of intact viral genomes, decreased genetic complexity and immune escape in reservoirs, which could accelerate reservoir clearance when combined with other interventional strategies.

Integrase strand transfer inhibitors resistance-associated mutations in HIV-infected pregnant women.

To date, no data exist regarding the prevalence of integrase inhibitor (INSTI) resistance-associated mutations (HIVDRM) in HIV-infected pregnant women (HPW) in Latin America. We describe the prevalence and transmissibility of integrase HIVDRM in a historical cohort of INSTI-naïve HPW from Argentina (n=56) with Next Generation Sequencing (NGS). Bioinformatics analysis was performed by HyDRA software for 20%, 10%, 5%, 2%, and 1% sensitivity thresholds. We calculated the mutational viral load for each INSTI-HIVDRM, considering those with >1000 c/mL as of high risk of transmissibility. The predominant HIV subtype was BF (78.5%). Major HIVDRM were not detected with the population sequencing 20% filter. With a 1% threshold, the prevalence increased to 8.9%; Y143C/S, E92G, E138K, and T66I mutations were found. The median (range) mutational load (expressed in c/mL) was: 355 (50.2-11705); with only 1 case >1000 c/mL Accessory mutations (G163R/K, T97A) were detected mostly with a 20% sensitivity threshold with an overall prevalence of 23.2%; the median (IQR) mutational load was: 23929 (4009-63158) c/mL; all of them above 1000 c/mL. Our results show evidence of the presence of major INSTI-HIVDRM as aleatory mutations and a high frequency of accessory mutations with potential transmissibility in HPW.

Detection of antiretroviral drug-resistant mutations and HIV-1 subtypes in circulation among men who have sex with men, SEM females and female sex workers: results of Vietnam's HIV Sentinel Surveillance Plus (HSS+) system, 2018 - 2020.

HIV drug resistance (HIVDR) can reduce the effectiveness of antiretroviral (ARV) drugs in preventing morbidity and mortality, limit options for treatment, and prevention. Our study aimed to assess HIV-1 subtypes and HIVDR among key populations in HIV Sentinel Surveillance Plus Behavior (HSS+) in 2018 and 2020. One-stage venue-based cluster sampling was used to recruit participants at hotspots identified for Men who have sex with men (MSM) in 7 provinces and SEM females and female sex worker (FSW) in 13 provinces. Participants completed a standard questionnaire about risk and preventive behaviors, and ART history, and provided intravenous blood for HIV testing. HIVDR testing was conducted on HIV-positive samples with VL >1,000 copies/ml. A total of 185/435 (42.5%) HIV-positive samples had viral load ≥1,000 copies/ml, of which 130/136 from MSM and 26/49 from FSW, were successfully sequenced. Six HIV-1 subtypes were detected (CRF01_AE, A, CRF07/08_BC, B, C, CRF25_cpx), with CRF01_AE (82.7%, 129/156) the most common. Drug resistance mutations were detected in 16.7% of participants overall (26/156), in 15.4% (20/130) of MSM, and in 23.1% (6/26) of FSW. Mutations associated with resistance to NNRTI were the most frequently detected (73.1%, 19/26). The high level of resistance was presented in NNRTI and NRTI classes. There are 10 major resistance mutations detected with NRTI (M184VI-25.0%, K65KR-50.0%, Y115F-25%), NNRTI (K103N-21.1%, E138A-10.5%, V106M-5.3%, K101E-5.3%, G190A-5.3%) PI (L33F-40.0%, M46L-20.0%). Vietnam's HSS+ system identified an emerging strain of HIV-1 and mutations associated with resistance to multiple drug classes among MSM and FSW.

Characterizing HIV-1 transmission by genetic cluster analysis among newly diagnosed patients in the China-Myanmar border region from 2020 to 2023

ABSTRACT Cluster analysis of HIV sequence can provide insights into viral transmission patterns in border regions. This study aims to illuminate the HIV-1 subtype distribution and transmission dynamics among newly diagnosed individuals in Dehong prefecture, a region along the China-Myanmar border. Among 948 participants with pol gene sequences, 36 HIV-1 subtypes were identified, with URFs (18.8%, 178/948) being the dominant strain, followed by CRF01_AE (18.5%, 175/948) and CRF07_BC (10.9%, 103/948). Additionally, 287 sequences (30.3%, 287/948) were grouped into 91 clusters, 31 of which contained both Chinese and Burmese individuals. Multivariable logistic regression indicated that men who have sex with men (MSM), CD4 + cell count of 200∼499 cells/μl, and 500 cells/μl and above, and CRF01_AE were risk factors for entering the network. Through Chord diagram, we found frequent transmission relationships among heterosexual China male group, especially those over 35 years of age. Additionally, the correlation between heterosexual Myanmar female group and heterosexual China male group among cross-risk groups deserved to be emphasized. Furthermore, the network exhibited a growing trend over time, with the largest active transmission cluster identified in Ruili county. In conclusion, the HIV-1 subtype landscape in Dehong has become increasingly complex, and the region has faced risks of transmission from both domestic and international sources. Targeted intervention strategies should be implemented for MSM, heterosexual Chinese middle-aged and elderly men, and heterosexual Burmese young adults to mitigate these risks. These findings provided evidence-based insights for local government to formulate coordinated transnational intervention approaches.

Insights into the molecular network characteristics of major HIV-1 subtypes in developed Eastern China: a study based on comprehensive molecular surveillance data.

This study aimed to conduct a comprehensive molecular epidemiology study of major HIV-1 subtypes in developed Eastern China (Zhejiang Province). Plasma samples and epidemiological information were collected from 4180 newly diagnosed HIV-1 positive patients in Zhejiang Province in 2021. Pol sequences were obtained to determine the subtypes via multiple analytical tools. HIV-1 molecular networks were constructed on the basis of genetic distances to analyze transmission patterns among major subtypes. Furthermore, the birth-death skyline (BDSKY) model was utilized to estimate the transmission risks associated with large clusters (LCs). In 4180 patients, 3699 (88.49%) pol sequences were successfully obtained and classified into four subtype groups. In the networks under an optimal genetic distance of 0.01 substitutions/site, the majority of links (74.52%, 1383/1856) involved individuals within the same city, highlighting the predominant role of local transmission in driving the HIV-1 epidemic. In the CRF07_BC, CRF01_AE, and others/URFs networks, men who have sex with men (MSM) were the primary sexual transmission population, with the younger MSM group (< 30 years old) exhibiting higher linkage frequencies. Within the CRF08_BC network, 93.98% of individuals were infected primarily through heterosexual contact and had a significantly greater risk of localized clustering than other subtypes did. Moreover, fifteen identified LCs were predominantly transmitted through commercial heterosexual contact (CHC), exhibiting localized clustering and high potential for sustained diffusion. Overall, our findings reveal a diverse and heterogeneous distribution of HIV-1 subtypes in Zhejiang Province, with noticeable variations in hotspots across different geographic areas and populations.

K103N, V106M and Y188L Significantly Reduce HIV-1 Subtype C Phenotypic Susceptibility to Doravirine.

Doravirine (DOR) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with efficacy against some NNRTI-resistant mutants. Although DOR resistance mutations are established for HIV-1 subtype B, it is less clear for non-B subtypes. This study investigated prevalent NNRTI resistance mutations on DOR susceptibility in HIV-1 subtype C. Prevalent drug resistance mutations were identified from a South African genotypic drug resistance testing database. Mutations, single or in combination, were introduced into replication-defective pseudoviruses and assessed for DOR susceptibility in vitro. The single V106M and Y188L mutations caused high-level resistance while others did not significantly impact DOR susceptibility. We observed an agreement between our in vitro and the Stanford HIVdb predicted susceptibilities. However, the F227L mutation was predicted to cause high-level DOR resistance but was susceptible in vitro. Combinations of mutations containing K103N, V106M or Y188L caused high-level resistance, in agreement with the predictions. These mutations are frequently observed in patients failing efavirenz- or nevirapine-based first-line regimens. However, they are also observed in those failing a protease inhibitor-based second-line regimen, as we have observed in our database. Genotypic drug resistance testing is therefore vital prior to the initiation of DOR-based treatment for those previously exposed to efavirenz or nevirapine.

Characterization of Two Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Forms Among Men Who Have Sex with Men and Mother-to-Child Transmission Cases in Baoding City, China.

CRF01_AE and CRF07_BC are predominant circulating HIV-1 subtypes in China. In this study, we report two novel HIV-1 CRF01_AE/CRF07_BC recombinant forms isolated from one man who has sex with men (MSM) (BDD027) and one mother-to-child transmission (MTCT) case (BDL123) in Baoding City, Hebei Province, China. The recombination breakpoint analysis showed that the recombination pattern of the near-full-length genome of BDD027 consisted of two CRF07_BC fragments inserted into a CRF01_AE backbone, while the recombination pattern of the near-full-length genome of BDL123 consisted of one CRF01_AE fragment inserted into a CRF07_BC backbone. This study demonstrates the importance of strengthening the monitoring of HIV-1 molecular epidemiological characteristics and emphasizes the urgent need to reduce the HIV-1 epidemic among MSM and MTCT populations in China.

HIV drug resistance: analysis of viral genotypes and mutation loci in people living with HIV in Chongqing, China (2016–2023)

BackgroundLarge-scale HIV genotype drug resistance study has not been conducted in Chongqing.MethodsA retrospective study was conducted on people living with HIV(PLWH) who received HIV-1 genotype resistance testing at Chongqing Public Health Medical Center from May 2016 to June 2023. The HIV-1pol gene was amplified through RT-PCR and analyzed in terms of genotypic drug resistance.ResultsOf the 3015 PLWH tested for HIV-1 drug resistance, 1405 (46.6%) were resistant to at least one antiviral drug. Among non-nucleoside reverse transcriptase inhibitors (NNRTIs), 43.8% were resistant, compared to 29.5% for nucleoside reverse transcriptase inhibitors (NRTIs) and 3.4% for protease inhibitors (PIs). V179D/E and K103N/S were identified as the common mutation sites in the NNRTIs class of drugs, M184V/I and K65R/N were reported as the most common mutation sites in NRTIs, while thymidine analogue mutation (TAM) group was identified in 373 samples. L10FIV was the most common mutation in PIs. The dominant HIV-1 subtype was CRF07_BC.ConclusionsThe high prevalence of HIV-1 drug resistance in Chongqing underscores the imperative for rigorous surveillance of the local HIV epidemic. Furthermore, TAMs are associated with HIV-1 multidrug resistance, and timely detection of drug resistance is helpful to reduce the emergence and spread of such drug-resistant strains.

Global and regional genetic diversity of HIV-1 in 2010–21: systematic review and analysis of prevalence

The extensive global genetic diversity of HIV-1 poses a major challenge to HIV vaccine development. We aimed to determine recent estimates of and changes in the global and regional distributions of HIV-1 genetic variants. We conducted a systematic literature review by searching PubMed, Embase, Global Health, and CINAHL for studies containing country-specific HIV-1 subtyping data, published between Jan 1, 2010and Sep 16, 2022. The proportions of HIV-1 subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) in each country were weighted by UNAIDS estimates of the numbers of people living with HIV (PLHIV) in each country to obtain regional and global prevalence estimates of HIV-1 subtypes, CRFs, and URFs with 95% CIs for the time periods 2010-15and 2016-21. The protocol is registered with PROSPERO, CRD42017067164. We obtained 1044datasets, containing HIV-1 subtyping data from 653 013PLHIV from 122countries in 2010-2021. In 2016-2021, subtype Caccounted for 50·4% (95% CI 50·2-50·7; n=18 570 462of 36 823 798) of global HIV infections, subtype Afor 12·4% (12·2-12·6; n=4 571 250), subtype Bfor 11·3% (11·1-11·5; n=4 157 686), subtype Gfor 2·9% (2·9-3·0; n=1 083 568), subtype Dfor 2·6% (2·5-2·7; n=945 815), subtype Ffor 0·9% (0·8-0·9; n=316 724), CRFs for 15·1% (14·9-15·3; n=5 564 566), and URFs for 2·0% (1·9-2·1; n=733 374). Subtypes H, J, and K each accounted for 0·1% or less of infections. Compared with 2010-15, we observed significant (p<0·0001) increases in global proportions of subtype A (0·9%, 95% CI 0·7to 1·1) and subtype C (3·4%, 3·0to 3·7) and decreases in subtype D (-0·5%, -0·6to -0·4), subtype G (-0·8%, -1·0to -0·7), CRFs (-1·0%, -1·3to -0·8), and URFs (-1·8%, -1·9to -1·7), with no changes for subtypes Band F. The global proportion of infections attributed torecombinants decreased from 21·6% (95% CI 21·4 to 21·7; n=7 099 252of 32 622 808) in 2010-15to 19·3% (19·1to 19·5; n=7 094 694of 36 823 798) in 2016-21 (-2·3%, 95% CI -2·6to -2·0; p<0·0001). Regional distributions of HIV-1 variants were complex and evolving, with global trends in the prevalence of HIV-1 variants supported by trends across the regions. Global and regional HIV-1 genetic diversity are complex and continue to evolve. Continued and improved surveillance of HIV-1 variants remains vital for HIV vaccine development and implementation. None.

Role of HLA-B*58:01-restricted CD8+ T cells in HIV-1 subtype AE infection

Abstract HLA-B*58:01 and HLA-B*57 are protective alleles against HIV-1 subtype B or C infection whereas these HLA alleles have not been reported as protective in HIV-1 subtype AE infection. Although HLA-B*58:01-restricted and HLA-B*57-restricted HIV-1-specific CD8+ T cells have been thoroughly analyzed in subtype B or C infection, they have only been partially analyzed in subtype AE infection. We identified six HLA-B*58:01-restricted subtype AE epitopes in Vietnamese individuals infected with subtype AE. HLA-B*58:01-restricted T-cell responses to Gag epitopes, which may control disease progression in HLA-B*58:01+ and HLA-B*57+ individuals infected with subtype B or C, were not protective in subtype AE infection. These findings suggest that the loss of HLA-B*58:01-restricted T cells specific for some Gag epitopes and/or their reduced ability may account for the lack of protective effects conferred by HLA-B*58:01 in subtype AE infection.

Molecular genetic monitoring of HIV-1 variants circulating in St. Petersburg

The aim of the study: to assess the genetic diversity of HIV-1 variants circulating in St. Petersburg.Materials and methods. The study included 289 patients with virological ART failure in 2022 in the St. Petersburg AIDS Center. Fragments of the pol gene encoding integrase, reverse transcriptase and protease were analyzed by polymerase chain reaction and Sanger sequencing. Phylogenetic tree created by the Neighbor-joining method with 1000 repeats of nucleotide sequences, bootstrap values &gt;70. To assess the circulation of HIV genovariants in dynamics in St. Petersburg, sequences and clinical and laboratory parameters obtained from 544 patients since 2018. The total sample included 833 samples (289 were collected in 2022), compared with the results of a study from 1104 HIV-infected patients in 2006–2011.Results and discussion. Monovariants of HIV dominated in the examined patients (95.1%, 275 people), subtype A6 — 88.2% (255 people); subtype B — 5.9% (17 people); C — 0.3% (1 person); G — 0.7% (2 people), the proportion of recombinant forms — 4.9% (14 people). In the sample collection, the proportion of new HIV cases of non-A subtype was 13.3% in 2006–2011, and 11.1% in 2018–2022. A significant increase in the incidence of recombinant forms of HIV-1 was revealed from 1.6% to 3.5% (c2=6.111; p=0.014). In the group (2018–2022), the recombinant form of CRF63_02A6 was more common (15/29 people).Conclusion. Phylogenetic analyses makes it possible to determine HIV subtypes, but also to establish the potential geographical origin of the virus, to identify transmission clusters taking into account the socio-demographic indicators of HIV-infected patients. Molecular epidemiological monitoring can be used to develop and implement programs to counter the spread of HIV among the population. The dominant genetic variant of HIV circulating in St. Petersburg is sub-subtype A6, as it was 10 years ago. The proportion of new cases of infection with non-A subtype of HIV remains stable, with a downward trend. The increase in the frequency of detection of recombinant forms of HIV-1 is probably related to the migration processes of the population.

Comparison of Different HIV-1 Resistance Interpretation Tools for Next-Generation Sequencing in Italy.

Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools. This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

A multiplexed real‐time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low‐and‐middle‐ income countries

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and 82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

Patterns of Transmitted Drug Resistance Mutations and HIV-1 Subtype Dynamics in ART-Naïve Individuals in Veneto, Italy, from 2017 to 2024.

This study investigates the prevalence and patterns of transmitted drug resistance mutations (TDRMs) and HIV-1 subtypes among antiretroviral therapy (ART) naïve individuals in Veneto, Italy, from 2017 to 2024. This research aims to understand the dynamic landscape of TDRMs and HIV-1 genetic diversity to inform treatment strategies effectively. We included all adult ART-naïve people with HIV (PWH) from seven infectious disease units in Veneto, Italy. We collected the genotypic resistance testing conducted to predict drug susceptibility and subtype distribution using the Stanford HIVdb algorithm. We included 762 PWH, showing a slight but statistically significant decline in the B subtype among Italian PWH (p = 0.045) and an increase in non-B subtypes among foreigners, though it was not statistically significant (p = 0.333). The most frequent mutations were in Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs), especially in non-B subtypes, with a notable rise from 10.7% in 2017-2019 to 15.5% in 2020-2024. Notably, TDRMs were consistently detected, highlighting an ongoing challenge despite the stable prevalence observed over the years. In addition, the data revealed a concerning rise in mutations against newer drug classes, such as integrase inhibitors. Conclusively, the study underscores the necessity of continuous surveillance of HIV subtypes and resistance patterns to adapt ART regimens optimally. Despite the stable levels of drug resistance, the emergence of resistance against newer drugs necessitates ongoing vigilance and possible adjustment in treatment protocols to enhance clinical outcomes and manage HIV drug resistance effectively.

Contrasting the effect of hinge region insertions and non-active site mutations on HIV protease-inhibitor interactions: Insights from altered flap dynamics

HIV-1 protease (PR) enzyme is a viable antiretroviral drug target due to its crucial role in HIV maturation. Over many decades, the HIV-1 PR enzyme has exhibited mutations brought on by drug pressure and error-prone nature of HIV-1 reverse transcriptase. Non-active site mutations have played a pivotal role in drug resistance; however, their mechanism of action has not been fully elucidated. We investigated how non-active site mutations affect the conformational stability and drug binding ability of HIV-1 PR. In light of this, we studied a novel HIV-1 subtype C protease variant containing an insertion of valine (↑V) in the hinge region. We analysed the mutations in the presence and absence of ten background mutations. Molecular dynamics simulations revealed that both with and without the background mutations, the PR exhibited increased flexibility of hinge, flaps and fulcrum regions. This allowed the PR to adopt a wider flap conformation when in complex with several inhibitors. Additionally, the simulations revealed that the protease inhibitors (PIs) could not bring the mutated variant proteases into a stable, closed conformation, resulting in increased solvent exposure of the inhibitors. Together, these results suggest that the mutations decrease the favourability of binding by altering the dynamics of the flap regions. Notably, the insertion mutation increased PR hinge flexibility and the introduction of background mutations compensated for this by stabilising the cantilever and hinge regions. Together, these findings provide insight into how non-active site mutations affect PR conformational dynamics in critical areas of the PR thus impacting on drug binding capacity and potentially contributing to drug resistance.

Reverse vaccinology approaches to design a potent multiepitope vaccine against the HIV whole genome: immunoinformatic, bioinformatics, and molecular dynamics approaches

Substantial advances have been made in the development of promising HIV vaccines to eliminate HIV-1 infection. For the first time, one hundred of the most submitted HIV subtypes and CRFs were retrieved from the LANL database, and the consensus sequences of the eleven HIV proteins were obtained to design vaccines for human and mouse hosts. By using various servers and filters, highly qualified B-cell epitopes, as well as HTL and CD8 + epitopes that were common between mouse and human alleles and were also located in the conserved domains of HIV proteins, were considered in the vaccine constructs. With 90% coverage worldwide, the human vaccine model covers a diverse allelic population, making it widely available. Codon optimization and in silico cloning in prokaryotic and eukaryotic vectors guarantee high expression of the vaccine models in human and E. coli hosts. Molecular dynamics confirmed the stable interaction of the vaccine constructs with TLR3, TLR4, and TLR9, leading to a substantial immunogenic response to the designed vaccine. Vaccine models effectively target the humoral and cellular immune systems in humans and mice; however, experimental validation is needed to confirm these findings in silico.

Molecular transmission network analysis reveals the challenge of HIV-1 in ageing patients in China: elderly people play a crucial role in the transmission of subtypes and high pretreatment drug resistance in developed Eastern China, 2019–2023

BackgroundThe number and proportion of HIV/AIDS patients among older people are continuously and rapidly increasing in China. We conducted a detailed molecular epidemiological analysis of HIV-1 epidemic strains in a developed city in eastern China and found that elderly people play a crucial role in the transmission of subtypes and high pretreatment drug resistance (PDR).MethodsA total of 1048 samples were obtained from 1129 (92.8%) newly confirmed HIV-1-positive and treatment-naive patients between 2019 and 2023. The 1316 bp target fragment of the pol gene was amplified by reverse transcription polymerase chain reaction (RT‒PCR) and nested PCR, and Maximum-likelihood (ML) phylogenetic trees and molecular transmission network were constructed to analyse the subtypes and transmission clusters. Molecular transmission network was visualized using Cytoscape with the distance threshold of 0.0075. PDR-associated mutations were determined according to the Stanford University HIV Drug Resistance Database.ResultsA total of 933 pol sequences (89.0%, 933/1048) were successfully obtained, and twelve HIV-1 subtypes were detected. CRF07_BC was the predominant subtype, accounting for 48.1% (449/933) of sequences, followed by CRF01_AE (29.4%, 274/933). A total of 398 individuals (42.7%, 398/933) formed 89 clusters in the network. Multivariable logistic regression analysis revealed that age, nationality, subtype, and PDR were the most significant factors associated with clustering in the transmission network. The prevalence of PDR was 14.6% (136/933).PDR associated with non-nucleoside reverse transcriptase inhibitors (10.0%, 93/933) was much more common than that associated with nucleoside reverse transcriptase inhibitors (1.8%, 17/933) and protease inhibitors (3.2%, 30/933) (χ2 = 77.961, p < 0.001). The most frequent NNRTI mutations were K103N/S/KN/NS (52.2%, 71/136), which led to the highest proportion of high-level resistance to nevirapine and efavirenz (52.2%).ConclusionsOur study revealed the important influence of elderly people on CRF07_BC transmission and the high prevalence of PDR. The clustering of drug-resistant cases was significant, which suggested the potential for localized widespread transmission of drug-resistant strains. HIV screening and the determination of PDR are recommended for older patients to improve early detection and reduce treatment failure and second-generation transmission.