HIV-1 Subtyping Research Articles

SNAPPy: A snakemake pipeline for scalable HIV-1 subtyping by phylogenetic pairing.

Human immunodeficiency virus 1 (HIV-1) genome sequencing is routinely done for drug resistance monitoring in hospitals worldwide. Subtyping these extensive datasets of HIV-1 sequences is a critical first step in molecular epidemiology and evolution studies. The clinical relevance of HIV-1 subtypes is increasingly recognized. Several studies suggest subtype-related differences in disease progression, transmission route efficiency, immune evasion, and even therapeutic outcomes. HIV-1 subtyping is mainly done using web-servers. These tools have limitations in scalability and potential noncompliance with data protection legislation. Thus, the aim of this work was to develop an efficient method for large-scale local HIV-1 subtyping. We designed SNAPPy: a snakemake pipeline for scalable HIV-1 subtyping by phylogenetic pairing. It contains several tasks of phylogenetic inference and BLAST queries, which can be executed sequentially or in parallel, taking advantage of multiple-core processing units. Although it was built for subtyping, SNAPPy is also useful to perform extensive HIV-1 alignments. This tool facilitates large-scale sequence-based HIV-1 research by providing a local, resource efficient and scalable alternative for HIV-1 subtyping. It is capable of analyzing full-length genomes or partial HIV-1 genomic regions (GAG, POL, and ENV) and recognizes more than ninety circulating recombinant forms. SNAPPy is freely available at: https://github.com/PMMAraujo/snappy/releases.

Increased predominance of HIV-1 CRF01_AE and its recombinants in the Philippines.

The growth rate of new HIV infections in the Philippines was the fastest of any countries in the Asia-Pacific region between 2010 and 2016. To date, HIV-1 subtyping results in the Philippines have been determined by characterizing only partial viral genome sequences. It is not known whether recombination occurs in the majority of unsequenced genome regions. Near-full-length genome (NFLG) sequences were obtained by amplifying two overlapping half genomes from plasma samples collected between 2015 and 2017 from 23 newly diagnosed infected individuals in the Philippines. Phylogenetic analysis showed that the newly characterized sequences were CRF01_AE (14), subtype B (3), CRF01/B recombinants (5) and a CRF01/CRF07/B recombinant (1). All 14 CRF01_AE formed a tight cluster, suggesting that they were derived from a single introduction. The time to the most recent common ancestor (tMRCA) for CRF01_AE in the Philippines was 1995 (1992–1998), about 10–15 years later than that of CRF01_AE in China and Thailand. All five CRF01/B recombinants showed distinct recombination patterns, suggesting ongoing recombination between the two predominant circulating viruses. The identification of partial CRF07_BC sequences in one CRF01/CRF07/B recombinant, not reported previously in the Philippines, indicated that CRF07_BC may have been recently introduced into that country from China, where CRF07_BC is prevalent. Our results show that the major epidemic strains may have shifted to an increased predominance of CRF01_AE and its recombinants, and that other genotypes such as CRF07_BC may have been introduced into the Philippines.

Global and regional molecular epidemiology of HIV-1, 1990–2015: a systematic review, global survey, and trend analysis

Global genetic diversity of HIV-1 is a major challenge to the development of HIV vaccines. We aimed to estimate the regional and global distribution of HIV-1 subtypes and recombinants during 1990-2015. We searched PubMed, EMBASE (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) for HIV-1 subtyping studies published between Jan 1, 1990, and Dec 31, 2015. We collected additional unpublished HIV-1 subtyping data through a global survey. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. We grouped countries into 14 regions and analysed data for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of HIV-1 subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV (PLHIV) in each country to generate regional and global estimates of HIV-1 diversity in each time period. The primary outcome was the number of samples designated as HIV-1 subtypes A, B, C, D, F, G, H, J, K, CRFs, and URFs. The systematic review is registered with PROSPERO, number CRD42017067164. This systematic review and global survey yielded 2203 datasets with 383 519 samples from 116 countries in 1990-2015. Globally, subtype C accounted for 46·6% (16 280 897/34 921 639 of PLHIV) of all HIV-1 infections in 2010-15. Subtype B was responsible for 12·1% (4 235 299/34 921 639) of infections, followed by subtype A (10·3%; 3 587 003/34 921 639), CRF02_AG (7·7%; 2 705 110/34 921 639), CRF01_AE (5·3%; 1 840 982/34 921 639), subtype G (4·6%; 1 591 276/34 921 639), and subtype D (2·7%; 926 255/34 921 639). Subtypes F, H, J, and K combined accounted for 0·9% (311 332/34 921 639) of infections. Other CRFs accounted for 3·7% (1 309 082/34 921 639), bringing the proportion of all CRFs to 16·7% (5 844 113/34 921 639). URFs constituted 6·1% (2 134 405/34 921 639), resulting in recombinants accounting for 22·8% (7 978 517/34 921 639) of all global HIV-1 infections. The distribution of HIV-1 subtypes and recombinants changed over time in countries, regions, and globally. At a global level during 2005-15, subtype B increased, subtypes A and D were stable, and subtypes C and G and CRF02_AG decreased. CRF01_AE, other CRFs, and URFs increased, leading to a consistent increase in the global proportion of recombinants over time. Global and regional HIV diversity is complex and evolving, and is a major challenge to HIV vaccine development. Surveillance of the global molecular epidemiology of HIV-1 remains crucial for the design, testing, and implementation of HIV vaccines. None.

Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay

BackgroundThe widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals.MethodsWe implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George’s University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals.ResultsAlthough subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02_AG, and CRF01_AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient’s antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George’s, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4+ T cell counts in the NHS Lothian patients.ConclusionsThis is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.

Appearance of Drug Resistance Mutations Among the Dominant HIV-1 Subtype, CRF01_AE in Maumere, Indonesia.

Human Immunodeficiency Virus (HIV) is still a major health issue in Indonesia. In recent years, the appearance of drug resistance-associated mutations has reduced the effectiveness of Antiretroviral Therapy (ART). We conducted genotypic studies, including the detection of drug resistance-associated mutations (from first-line regimen drugs), on HIV-1 genes derived from infected individuals in Maumere, West Nusa Tenggara. Maumere, a transit city in West Nusa Tenggara, which has a high HIV-1 transmission rate. We collected 60 peripheral blood samples from 53 ART-experienced and 7 ART-naive individuals at TC Hillers Hospital, Maumere between 2014 and 2015. The amplification and a sequencing analysis of pol genes encoding protease (the PR gene) and reverse transcriptase (the RT gene) as well as the viral env and gag genes were performed. HIV-1 subtyping and the detection of drug resistance-associated mutations were then conducted. Among 60 samples, 46 PR, 31 RT, 30 env, and 20 gag genes were successfully sequenced. The dominant HIV-1 subtype circulating in Maumere was CRF01_AE. Subtype B and recombinant viruses containing gene fragments of CRF01_AE, subtypes A, B, C, and/or G were also identified as minor populations. The major drug resistance-associated mutations, M184V, K103N, Y188L, and M230I, were found in the RT genes. However, no major drug resistance-associated mutations were detected in the PR genes. CRF01_AE was the major HIV-1 subtype prevalent in Maumere. The appearance of drug resistance-associated mutations found in the present study supports the necessity of monitoring the effectiveness of ART in Maumere.

Burmese injecting drug users in Yunnan play a pivotal role in the cross-border transmission of HIV-1 in the China-Myanmar border region

ABSTRACT Injecting drug users (IDUs) are the major risk group for HIV-1 infection in the China-Myanmar border area. There are a large number of Burmese IDUs living in Yunnan (Yunnan-mIDUs) who might be associated with the cross-border transmission of HIV-1. From 2010 to 2013, 617 Yunnan-mIDUs were recruited from three counties of Yunnan, 19.0% of whom were detected to be HIV-1 positive by serological testing. Partial HIV-1 p17, pol, vif-env, and env genes were amplified from the positive samples and were sequenced. Phylogenetic and HIV-1 subtyping analyses revealed that HIV-1 recombinant forms (RFs), including RF_BC (36.4%), RF_01BC (26.1%), RF_01C (9.1%) and RF_01B (1.1%), were predominant among this cohort. Of the identified HIV-1 strains, 14.8%, 9.1% and 3.4% belonged to subtype C, CRF01_AE and subtype B, respectively. Transmission cluster analysis showed that sequences from the Yunnan-mIDUs formed transmission clusters not only with those from Burmese IDUs but also with those from Chinese IDUs, indicating that Yunnan-mIDUs might acquire HIV-1 infection from or spread HIV-1 to both Burmese and Chinese IDUs. Phylogeographic analyses revealed three cross-border transmission patterns associated with Yunnan-mIDUs, in which Yunnan-mIDUs served as the crucial nodes linking the Burmese and Chinese IDUs. These results suggest that Yunnan-mIDUs are a potential viral reservoir for the diffusion of HIV-1 in Yunnan and play a pivotal role in the bidirectional cross-border transmission of HIV-1 in the China-Myanmar border region. More intervention efforts that focus on Yunnan-mIDUs are recommended in Yunnan’s campaign against HIV/AIDS.

The Transmission and Evolution of HIV-1 Quasispecies within One Couple: a Follow-up Study based on Next-Generation Sequencing

Next-generation sequencing (NGS) has been successfully used to trace HIV-1 infection. In this study, we investigated the transmission and evolution of HIV-1 quasispecies in a couple infected through heterosexual behavior. A heterosexual couple in which both partners were infected with HIV-1 was followed up for 54 months. Blood samples including whole-blood and plasma samples, were collected at various time points. After HIV-1 subtyping, NGS (Miseq platform) was used to sequence the env region of the HIV-1 quasispecies. Genetic distances were calculated, and phylogenetic trees were generated. We found both partners were infected with HIV-1 subtype circulating recombinant form (CRF), CRF65_cpx. The quasispecies distribution was relatively tightly clustered in the phylogenetic tree during early infection. Over time, the distribution of HIV-1 quasispecies gradually became more dispersed at 12th months, with a progressive increase in gene diversity. By 37th months, the sequences obtained for both partners formed different clusters in the phylogenetic tree. These results suggest that the HIV-1 contact tracing results generated by the Miseq platform may be more reliable than other conventional sequencing methods, which can provide important information about the transmission and evolution of HIV-1. Our findings may help to better target preventative interventions for promoting public health.

Absence of HIV-1 Drug Resistance Mutations Supports the Use of Dolutegravir in Uganda.

To screen for drug resistance and possible treatment with Dolutegravir (DTG) in treatment-naive patients and those experiencing virologic failure during first-, second-, and third-line combined antiretroviral therapy (cART) in Uganda. Samples from 417 patients in Uganda were analyzed for predicted drug resistance upon failing a first- (N = 158), second- (N = 121), or third-line [all 51 involving Raltegravir (RAL)] treatment regimen. HIV-1 pol gene was amplified and sequenced from plasma samples. Drug susceptibility was interpreted using the Stanford HIV database algorithm and SCUEAL was used for HIV-1 subtyping. Frequency of resistance to nucleoside reverse transcriptase inhibitors (NRTIs) (95%) and non-NRTI (NNRTI, 96%) was high in first-line treatment failures. Despite lack of NNRTI-based treatment for years, NNRTI resistance remained stable in 55% of patients failing second-line or third-line treatment, and was also at 10% in treatment-naive Ugandans. DTG resistance (n = 366) was not observed in treatment-naive individuals or individuals failing first- and second-line cART, and only found in two patients failing third-line cART, while 47% of the latter had RAL- and Elvitegravir-resistant HIV-1. Secondary mutations associated with DTG resistance were found in 2%-10% of patients failing third-line cART. Of 14 drugs currently available for cART in Uganda, resistance was readily observed to all antiretroviral drugs (except for DTG) in Ugandan patients failing first-, second-, or even third-line treatment regimens. The high NNRTI resistance in first-line treatment in Uganda even among treatment-naive patients calls for the use of DTG to reach the UNAIDS 90:90:90 goals.

ACQUIRED DRUG RESISTANCE TO NRTI CLASS IN TREATMENT-EXPERIENCED HIV INFECTED PATIENTS FROM THE CONSTANTA COUNTY: THERAPEUTIC IMPLICATIONS

Objective. To determine the prevalence of acquired drug resistance (ADR) and of resistance patterns in treatment-experienced HIV infected patients from Constanta in order to establish the best therapeutic options in NRTI class. Material and methods. A retrospective study which included 144 treatment-experienced HIV patients with confirmed viral failure. The strains isolated from these patients were analysed in the Molecular Genetic Laboratory of „Matei Bals“ National Institute of Infectious Diseases, Bucharest and the resulting sequences were saved in FASTA format. The HIV-1 subtyping was based on „REGA HIV01&2 Automated subtyping tool version 2.0“ algorithm. „Stanford HIVdb Program version 8.4“ was used in order to determine the therapeutic options. For statistical calculations, the R-Project software was used. Graphic representations were performed using GNUPLOT program. Results. The prevalence of the acquired drug resistance was 92.36%. The most frequent mutation occurred at the level of the codon 184. The TAM-2 path was more frequently selected compared to TAM-1. Association between TAM1 and TAM 2 were also found, mutation K65R being rarely met. Conclusions. The prevalence of the acquired drug resistance in our study was high, The most valuable therapeutic option in the INRT class remains tenofovir, due to the mutational profile, which was selected on account of the extensive use of thymidine analogues.

Profile of HIV subtypes in HIV/HBV- and HIV/HCV-coinfected patients in Southern Brazil.

HIV and viral hepatitis infections are major causes of chronic disease worldwide and have some similarities with regard to routes of transmission, epidemiology, front barriers faced during access of treatment, and strategies for a global public health response. The objective was to describe the HIV-1 subtypes, viral tropism and single-nucleotide polymorphisms (SNPs) of interleukin 28B (IL28B) from a case series of HIV/viral hepatitis coinfected patients from southern Brazil. Clinical and epidemiological data were evaluated by a review of medical records. Periodic blood draws were taken to determine the viral and host characteristics. This study included 38 patients with HIV/HBV or HIV/HCV coinfection; the median age was 49 years. Thirty-seven (97.4%) were on antiretroviral therapy, 32 (84.2%) had an undetectable viral load, a median CD4+ T-cell count of 452 cells/mm3. HIV-1 subtyping showed 47.4 and 31.6% of patients with subtypes C and B, respectively. Analysis of viral co-receptor usage showed a predominance of the R5 variant (64.7%), with no significant difference between the subtypes. Twenty patients with HIV/HCV coinfection were eligible to receive HCV therapy with pegylated-interferon-alpha plus ribavirin, and 10/20 (50%) of them achieved sustained virological response. SNPs of IL28B were evaluated in 93.3% of patients with HIV/HCV coinfection, and 17 (60.7%) presented the CC genotype. In the present case series, a higher frequency of HIV subtype C was found in coinfected patients. However such findings need to be prospectively evaluated with the inclusion of data from regional multicenter analyses.

Comparative Evaluation of Subtyping Tools for Surveillance of Newly Emerging HIV-1 Strains.

HIV-1 non-B subtypes/circulating recombinant forms (CRFs) are increasing worldwide. Since subtype identification can be clinically relevant, we assessed the added value in HIV-1 subtyping using updated molecular phylogeny (Mphy) and the performance of routinely used automated tools. Updated Mphy (2015 updated reference sequences), used as a gold standard, was performed to subtype 13,116 HIV-1 protease/reverse transcriptase sequences and then compared with previous Mphy (reference sequences until 2014) and with COMET, REGA, SCUEAL, and Stanford subtyping tools. Updated Mphy classified subtype B as the most prevalent (73.4%), followed by CRF02_AG (7.9%), C (4.6%), F1 (3.4%), A1 (2.2%), G (1.6%), CRF12_BF (1.2%), and other subtypes (5.7%). A 2.3% proportion of sequences were reassigned as different subtypes or CRFs because of misclassification by previous Mphy. Overall, the tool most concordant with updated Mphy was Stanford-v8.1 (95.4%), followed by COMET (93.8%), REGA-v3 (92.5%), Stanford-old (91.1%), and SCUEAL (85.9%). All the tools had a high sensitivity (≥98.0%) and specificity (≥95.7%) for subtype B. Regarding non-B subtypes, Stanford-v8.1 was the best tool for C, D, and F subtypes and for CRFs 01, 02, 06, 11, and 36 (sensitivity, ≥92.6%; specificity, ≥99.1%). A1 and G subtypes were better classified by COMET (92.3%) and REGA-v3 (98.6%), respectively. Our findings confirm Mphy as the gold standard for accurate HIV-1 subtyping, although Stanford-v8.1, occasionally combined with COMET or REGA-v3, represents an effective subtyping approach in clinical settings. Periodic updating of HIV-1 reference sequences is fundamental to improving subtype characterization in the context of an effective epidemiological surveillance of non-B strains.

Prevalence of HIV-1 transmitted drug resistance in recently infected, treatment-naïve persons in the Southwest of Iran, 2014-2015.

The emergence and transmission of drug resistant HIV mutants is a major concern, especially in resource-limited countries with expanding antiretroviral therapy. Studies have recently reported the prevalence of HIV-1 transmitted drug resistance (TDR) mutations in certain Iranian cities; however, no information is currently available about the level of TDR, as well as the nature of the circulating HIV-1 subtypes, in the Southwestern bordering province of Iran, Khuzestan. Herein, we used a WHO-recommended TDR survey method to classify the prevalence of TDR in indigenous people of Khuzestan province. For this purpose, between March 2014 and February 2015, blood samples were collected from 52 newly diagnosed, antiretroviral treatment-naïve, HIV-1 infected persons aged from 18 to 30 years. TDR mutations were determined by sequencing the protease (PR) and reverse transcriptase (RT) genes and interpreted using the WHO drug resistance mutations surveillance list. HIV-1 subtypes were characterized by sequencing the PR-RT, C2-V5, and p17 regions of the pol, env and gag genes, respectively. Two participants had non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, specifically K103N in one individual and K101EK/K103KN/G190AG in the other. No nucleoside reverse transcriptase inhibitor (NRTI) or major protease inhibitor (PI) mutations were identified. HIV-1 subtyping revealed that all participants were infected with HIV-1 CRF35_AD. According to the WHO sequential sampling method, the prevalence of HIV-1 TDR in the sampling area (Khuzestan province) was classified as moderate for NNRTIs and low for NRTIs and PIs. This is the first HIV-1 drug resistance threshold survey in the Khuzestan province of Iran and shows a predominance of NNRTI TDR mutations in this area.

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil

IntroductionThe widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. MethodsPatients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal–Wallis and Chi-square tests. ResultsForty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1–30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1–24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. ConclusionsThe high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population.

Surveillance of HIV-1 pol transmitted drug resistance in acutely and recently infected antiretroviral drug-naïve persons in rural western Kenya.

HIV-1 transmitted drug resistance (TDR) is of increasing public health concern in sub-Saharan Africa with the rollout of antiretroviral (ARV) therapy. Such data are, however, limited in Kenya, where HIV-1 drug resistance testing is not routinely performed. From a population-based household survey conducted between September and November 2012 in rural western Kenya, we retrospectively assessed HIV-1 TDR baseline rates, its determinants, and genetic diversity among drug-naïve persons aged 15–59 years with acute HIV-1 infections (AHI) and recent HIV-1 infections (RHI) as determined by nucleic acid amplification test and both Limiting Antigen and BioRad avidity immunoassays, respectively. HIV-1 pol sequences were scored for drug resistance mutations using Stanford HIVdb and WHO 2009 mutation guidelines. HIV-1 subtyping was computed in MEGA6. Eighty seven (93.5%) of the eligible samples were successfully sequenced. Of these, 8 had at least one TDR mutation, resulting in a TDR prevalence of 9.2% (95% CI 4.7–17.1). No TDR was observed among persons with AHI (n = 7). TDR prevalence was 4.6% (95% CI 1.8–11.2) for nucleoside reverse transcriptase inhibitors (NRTIs), 6.9% (95% CI 3.2–14.2) for non- nucleoside reverse transcriptase inhibitors (NNRTIs), and 1.2% (95% CI 0.2–6.2) for protease inhibitors. Three (3.4% 95% CI 0.8–10.1) persons had dual-class NRTI/NNRTI resistance. Predominant TDR mutations in the reverse transcriptase included K103N/S (4.6%) and M184V (2.3%); only M46I/L (1.1%) occurred in the protease. All the eight persons were predicted to have different grades of resistance to the ARV regimens, ranging from potential low-level to high-level resistance. HIV-1 subtype distribution was heterogeneous: A (57.5%), C (6.9%), D (21.8%), G (2.3%), and circulating recombinant forms (11.5%). Only low CD4 count was associated with TDR (p = 0.0145). Our findings warrant the need for enhanced HIV-1 TDR monitoring in order to inform on population-based therapeutic guidelines and public health interventions.

English

HIV-1 genetic diversity in sub-Saharan Africa is broad and the AIDS epidemic is driven predominantly by recombinants in Central and West Africa. The classification of HIV-1 strains is therefore necessary to understand diagnostic efficiency, individual treatment responses as well as options for designing vaccines and antiretroviral (ARV) treatment guidelines. More so, accurate subtyping of a partial or full genome would represent the population dynamics of HIV and provide evidence for designing surveillance strategies within a geographic region. Evaluating the performance of rapid subtyping tools with options that incorporate phylogeny could be fast, more user-friendly and of high performance. A total of 570 HIV-1 partial sequences from Cameroon, Angola, Democratic Republic of Congo, Gabon and Senegal were obtained from the Los Alamos National Library (LANL) HIV Sequence Database. Phylogeny was performed using MEGA v6 and the results were used to evaluate the performance of eleven different rapid HIV-1 subtyping tools: REGA v2, REGA v3, NCBI, Stanford HIVDB, SUDI, Geno2Pheno, Euresist, STAR, jpHMM, COMET and SCUEAL. The performance of these subtyping tools differed among HIV-1 clades and across different viral genes. NCBI and SUDI showed the highest performance in subtyping. The discordance observed between the rapid subtyping tools and phylogeny implies that phylogenetic analysis is still the more suitable method for HIV-1 classification. However, the need to update the reference datasets of the subtyping tools, and validate algorithms for rapid subtyping and quality control is imperative as this information is relevant for clinical use and policy-making to the AIDS response.   Key words: HIV, phylogeny, performance, subtyping tools, algorithm.

Genetic Diversity and Transmitted Drug Resistance of HIV-1 Subtypes in Blood Donors Candidates in Northern Mozambique

In order to evaluate the genetic diversity of HIV-1 subtypes and transmitted drug resistance (TDR) mutations in northern Mozambique, we analyzed 120 plasma specimens obtained from drug naive blood donors candidates, who tested positive during routine HIV screening in three blood banks in that region. The genotyping for HIV-1 resistance was performed using Trugene Genotyping SystemTM. HIV-1 genetic subtypes were defined based on entire PR and partial RT gene regions, using REGA HIV-1 Subtyping Tool version 3.0 algorithm and confirmed by phylogenetic inference, using NJ algorithm. The majority of genotyped samples were classified as HIV-1 subtype C (80.0%), followed by subtype A1 (10.5%), subtype D (3.2%) and subtype G (2.1%). The inter-subtype recombinant forms (A1/C, A1/D and C/D) were identified in four participants (4.2%). TDR mutations associated with nucleoside and nonnucleoside reverse transcriptase inhibitors (K219E, G190A, K101E and K103N) were observed in five (5.3%) subjects. Although a large proportion of HIV-1 subtype C was observed, non-C and recombinant forms together correspond to 20% in this study, which is different from what was described in central and southern regions, where subtype C was almost 100%. However, the profile mutations from this study correlate with the ARVs used both for first line schemes and PMTCT in Mozambique. This data reinforces the necessity of continuous surveillance of HIV-1 diversity, TDR and routes of spread, through inner cities of Mozambique to understand better the dynamics of the HIV-1 epidemic and support national public health policies in the country.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

BackgroundAlthough biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs.MethodsPlasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database (http://hivdb.stanford.edu) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates.ResultsAll subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions.ConclusionAlthough the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

HIV-1 transmitted drug resistance mutations among antiretroviral therapy-Naïve individuals in Surabaya, Indonesia.

BackgroundThe emergence of transmitted drug resistance (TDR) compromises the effect of antiretroviral therapy (ART), resulting in treatment failure of human immunodeficiency virus (HIV) disease. Although more than a decade has passed since ART was introduced into Indonesia, information on TDR is limited. Here, a genotypic study of TDR among ART-naïve individuals was conducted in Surabaya, Indonesia.MethodHIV-1 seropositive participants were recruited from the communities of commercial sex workers and intravenous drug users as well as from the university teaching hospital in Surabaya. Protease (PR) and reverse transcriptase (RT) genes were sequenced in order to conduct HIV-1 subtyping and phylogenetic analysis and to detect TDR. TDR was defined as the presence of at least one surveillance drug resistance mutation on the WHO list or major drug resistance mutations in the International AIDS Society-USA panel.ResultFifty two and 47 of the PR and RT genes, respectively, were successfully sequenced in the 58 samples. HIV-1 subtyping revealed that 86.3% (50/58) of the sequenced samples were classified as CRF01_AE, 8.6% as subtype B, 3.4% as B/CRF01_AE, and 1.7% as A/G/CRF01_AE. TDR of PR inhibitors was not detected in this study. In contrast, TDR of RT inhibitors was detected in 4.3% (2/47) of samples. In addition, minor drug resistance mutations were detected in 98.1% (51/52) and 12.8% (6/47) of PR and RT genes, respectively.ConclusionThis study clarified the predominance of the CRF01_AE strain in Surabaya, Indonesia. The prevalence of TDR was below 5%, indicating that the currently available first-line regimen is still effective in Surabaya. However, the prevalence might be underestimated since we detected only major population of HIV-1 in individuals. Therefore, continuous surveillance is required in order to detect the emergence of TDR in the early phase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12981-015-0046-y) contains supplementary material, which is available to authorized users.

Molecular characterization of HIV-1 infection in Northwest Spain (2009–2013): Investigation of the subtype F outbreak

BackgroundHIV-1 subtype B is the predominant one in European regions several, while other subtypes and recombinants are also circulating with high prevalence. A sub-epidemic of subtype F with specific characteristics and low response to treatment has been recently identified in Galicia. In this study we investigated the characteristics of the HIV-1 subtype F sub-epidemic in A Coruña and Santiago de Compostela in Northwest Spain. Methods420 newly HIV-1 diagnosed patients during 2009–2013 were enrolled in this study. HIV-1 subtyping was carried out using automated subtyping tools and phylogenetic analysis. Molecular epidemiology investigation of subtypes B and F was performed by means of phylogenetic analysis using fast maximum likelihood. Phylodynamic analysis was performed using Bayesian method as implemented in BEAST v1.8. ResultsSubtype B found to be the predominant (61.2% and 70.4%) followed by subtype F (25.6% and 12.0%) in both areas (A Coruña and Santiago de Compostela, respectively). The latter found to mainly spread among men having sex with men (MSM). The vast majority of subtype F lineages from both areas clustered monophyletically, while subtype B sequences clustered in several tree branches. The exponential growth of subtype F sub-epidemic dated back in 2008 by means of phylodynamic analysis. Most of new infections during 2009–2013 occurred within the subtype F transmission cluster. ConclusionsSubtype F circulates at high prevalence in A Coruña and Santiago de Compostela in Northwest Spain, suggesting that the HIV-1 epidemic in this region has distinct characteristics to the rest of Spain. Subtype F has being spreading among MSM and is currently the most actively spreading network. The single cluster spread of this local sub-epidemic might provide an explanation for the distinct characteristics and the low response to antiretroviral treatment.

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany.

IntroductionThe aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007.Materials and MethodsPlasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms.ResultsOverall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%.ConclusionsDetailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand the development of resistance in the presence of the drug. Our results will contribute to define the relevance of integrase polymorphisms in HIV-strains resistant to integrase inhibitors and to improve resistance interpretation algorithms.