Routine Drug Resistance Testing Research Articles

Evaluation of the HIV-1 drug resistance to elsulfavirine and the effectiveness of it among Russian treatment-naïve patients

The aim of the study: to analyze the prevalence of resistance mutations to elsulfavirine and to evaluate the effectiveness of it among HIV-infected treatment-naïve patients in real clinical practice.Materials and methods. The study included 578 patients with HIV infection, which divided into 3 groups. The first group is 354 HIV-infected treatment-naïve patients for whom HIV-1 nucleotide sequences were obtained as part of routine drug resistance testing. The second study group included 111 HIV-infected treatment-naïve patients, tested for drug resistance before the antiretroviral therapy containing elsulfavirine. The third study group included 113 HIV-infected treatment-naïve patients, each of whom was assigned a treatment regimen containing elsulfavirine without prior drug resistance testing. The observation period for patients of the second and third groups who received treatment was 24 weeks. To assess the effectiveness of antiretroviral therapy in patients, viral load, CD4+ T-cell counts, and adherence to therapy were assessed. HIV-1 subtypes and drug resistance mutations were determined using the Stanford HIV Resistance Database (v. 8.9-1). To clarify the results of subtyping, phylogenetic analysis of nucleotide sequences was carried out using the MEGA program (v. 6.0).Results. The prevalence of mutations associated with decreased susceptibility to elsulfavirine among HIV-infected treatment-naïve patients was 1.7% and 4.5% for the first and second groups of patients, respectively. All of the patients have only single resistance mutations which, according to the results of preclinical studies, cannot cause drug resistance. The use of elsulfavirine in real clinical practice among treatment-naïve patients has demonstrated good virological and immunological efficacy of the drug. As a result of 24 weeks of therapy in patients of the second group, no treatment ineffectiveness, and the development of drug resistance were observed. Among the patients of the third group, 6 patients (5.3%) have the virological failure of therapy associated with the resistance to the used drugs. All patients with virological failure had a resistance mutation profile associated with a high level of drug resistance to one of the drugs in the treatment regimen, lamivudine. Additionally, 1 patient had a combination of mutations that reduce susceptibility to elsulfavirine, and 4 patients had mutations that can reduce susceptibility to elsulfavirine in combination with other mutations.Conclusion. The low prevalence of mutations associated with a decrease in susceptibility to elsulfavirine and the absence of combinations of mutations make it possible to predict the successful use of this drug for Russian treatment-naïve patients. Reported cases of virological failure of antiretroviral therapy are difficult to interpret in the context of elsulfavirine due to the lack of an exact list of mutations and their combinations, and associations with the degree of resistance to it. This study describes for the first time the mutation profiles in patients with virological failure of therapy containing elsulfavirine and demonstrates the necessity of the further study of drug resistance profile to drug in vitro and in vivo.

Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay

BackgroundHIV-1 DNA genotypic drug resistance testing is increasingly performed to guide treatment switching or simplification in controlled patients. The Sentosa NGS platform is a fully automated system marketed for drug resistance testing on HIV-1 RNA samples. ObjectivesThe aim of this study was to evaluate this automated NGS solution for routine resistance genotypic resistance testing in proviral HIV-1 DNA. Study designSanger sequencing (SS) of the reverse transcriptase (RT), protease (PR) and integrase (IN) genes was performed using the French ANRS protocol. NGS was performed retrospectively on frozen samples, using the Sentosa platform combined with the Sentosa SQ HIV genotyping Assay. ResultsA total of 77 samples were run once using NGS. A successful sequencing of the three HIV-1 genes (RT, PR, IN) was obtained for 45 samples. The number of cumulated RAMs was 179, 185 and 219 with SS, NGS 20% and NGS 10% respectively; however most of them were minor mutations in the PR region. The mutation detection rate was similar between SS and NGS 20%. Several discordances were observed between both methods in the RT and PR regions, mainly due to the use of different DNA extracts, and hypermutation. ConclusionsHIV-1 DNA genotypic resistance testing can be performed with the Sentosa platform. Few technical optimizations are still needed to include the extraction step and to improve the sequencing efficiency.

Routine drug resistance testing in proviral HIV-1 DNA: Prevalence of stop codons and hypermutation, and associated factors.

We investigated the presence of stop codons (SC) and/or hypermutation (HM) in HIV-1 DNA sequences generated for routine drug resistance testing in proviral HIV-1 DNA, and sought for associated factors. At least one SC was identified in 6.2% of HIV-1 DNA sequences, among which 54.8% were hypermutated. The defective virus group (SC w/o HM) was similar to the non-SC group regarding the characteristics of HIV-1 infection, and before drug exposure. In addition, the HIV-1 DNA levels were not different between both groups. Sequences with SC/HM displayed a higher proportion of RAMs. The impact of the SC/HM associated RAMs on clinical responses requires further investigation.

HIV drug resistance among naïve HIV-infected patients in Iran.

Background:Antiretroviral (ARV) therapy extends life for persons living with HIV. Antiretroviral treatment (ART) has been rapidly expanding coverage around the world, including in Iran. However, ART drug resistance also rapidly develops with expanding use and limits effectiveness and treatment options. The aim of this study was to monitor the appearance of new mutations conferring HIV pretreatment drug resistance in the treatment of naïve patients with HIV in Iran.Materials and Methods:Blood samples were obtained from ARV treatment-naïve patients from 8 different provinces in Iran in 2016 for genotyping for drug resistance mutations.Results:Sequences were successfully obtained from 90 specimens. Of these, 2 (2%) mutations conferring resistance to protease inhibitors, 2 (3%) conferring resistance to nucleoside reverse transcriptase inhibitors (NRTIs), and 9 (13%) conferring resistance to non-NRTI (NNRTI) were detected. Any ARV-resistant drug mutation was found in 11 patients (12%).Conclusion:Nearly one in 8 ARV-naïve patients had mutations associated with NNRTI resistance in diverse areas of Iran in 2016. Iranian ARV therapy guideline for HIV could consider non-NNRTI-based first-line therapies and expand routine drug resistance testing before treatment initiation as according to HIV drug resistance recommendations of the World Health Organization.

Prevalence of Darunavir Resistance in the United States from 2010 to 2017.

The emergence and transmission of antiretroviral drug resistance have been and remain a concern among people living with human immunodeficiency virus (HIV)-1 infection. The protease inhibitor (PI) darunavir has been approved for use in the United States for more than 10 years and has demonstrated a high barrier to resistance. Previous analyses identified significant reductions in the prevalence of samples with darunavir resistance-associated mutations (RAMs) and with phenotypic resistance to darunavir and other PIs between 2006 and 2012. This analysis extends those findings by evaluating darunavir and PI resistance among clinical samples submitted for routine drug resistance testing (combined genotyping and phenotyping) in the United States from 2010 to 2017. Frequencies of 11 darunavir and 23 primary PI RAMs, and phenotypic susceptibility, were assessed yearly among all samples and in a subset of samples with distinct phenotypic resistance to one or more PIs. Among all samples (N = 60,760), the proportion with 0 darunavir RAMs was 91.7% in 2010 and 95.8% in 2017. The proportions of all samples with phenotypic susceptibility to darunavir, atazanavir, and lopinavir were, respectively, 97.4%, 94.2%, and 94.7% in 2010 and 98.6%, 97.7%, and 97.5% in 2017. Among the 4,799 samples with phenotypic resistance to one or more PIs, the proportions with phenotypic susceptibility to darunavir, atazanavir, and lopinavir were, respectively, 73.3%, 41.5%, and 46.0% in 2010 and 70.7%, 53.7%, and 48.8% in 2017. The prevalence of darunavir RAMs among commercially tested HIV-1 samples remained low and generally stable from 2010 to 2017, and high proportions showed phenotypic darunavir susceptibility.

Completeness of HIV nucleotide sequence ascertainment and its potential impact on understanding HIV transmission – Maryland, 2011–2013

ABSTRACTHIV nucleotide sequences generated through routine drug resistance testing (DRT) and reported to Maryland's Molecular HIV Surveillance system are most effective for elucidating transmission patterns and identifying outbreaks if DRT is ordered promptly and sequences are reported completely. Among reported cases of HIV infection newly diagnosed during 2011–2013 in Maryland residents aged ≥13 years, we assessed sequence ascertainment completeness. To better understand which populations were most likely to have a sequence, we examined associations between sequence ascertainment and clinical and demographic characteristics. During 2011–2013, 4423 new HIV infection diagnoses were reported; sequences were ascertained for 1282 (29.0%). Among 3267 cases with complete data, odds for having a sequence ascertained were highest for cases in persons living inside Maryland's Central Region with initial CD4 counts ≤500 cells/mm3 (adjusted odds ratio [aOR] 2.4, 95% confidence interval [CI] 1.9–3.1). Sequence ascertainment did not vary significantly by patient age, sex, race/ethnicity or HIV transmission category. Educational interventions, policy changes and improved processes to increase timely DRT and subsequent sequence reporting with a focus on testing at entry to care, particularly for those with higher CD4 counts and those living outside the Central Region, might improve ascertainment completeness.

Time to overcome pretreatment HIV drug resistance

Time to overcome pretreatment HIV drug resistance

HIV Phylogeographic Analyses and Their Application in Prevention and Early Detection Programmes: The Case of the Tijuana–San Diego Border Region

HIV Phylogeographic Analyses and Their Application in Prevention and Early Detection Programmes: The Case of the Tijuana–San Diego Border Region

HIV drug resistance testing by high-multiplex "wide" sequencing on the MiSeq instrument.

Limited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n = 759) and from a Ugandan study cohort (n = 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.

The prevalence of RT 245 codon polymorphisms and its association with duration of infection among HIV-1 patients in Serbia

Amino acid (aa) substitutions at position 245 of HIV-1 reverse transcriptase (RT) have been described to be associated to the presence of human leukocyte antigen (HLA)–B*5701 allele in the host, in particular in subtype B infection. Preliminary data show HLA–B*5701 prevalence of around 6.5% in Serbian population. In this study, we investigated the prevalence of RT codon 245 substitutions among HIV infected patients in Serbia. Furthermore, we analyzed the association between RT 245 aa substitutions with duration of infection, estimated by proportion of ambiguous basecalls per sequence. The study included 184 consenting, subtype B HIV infected patients aged 18 or more. The majority of patients were newly diagnosed, 150/184, while 34/184 patients were on treatment. Pol region sequences, covering protease and minimally 250 RT codons, were obtained within routine drug resistance testing. The fraction of ambiguous nucleotides in each sequence was calculated for samples drawn from naive patients. We used ambiguity percentage of 0.47% as a cut-off value delimiting recent (less than 1 year) vs. chronic infection (longer than 1 year). In total, predominant aa at RT codon 245 was the wild type valin (V) found in 118/184 (64.1%), hence 35.9% (66/184) contained mutation at this position (among naive patients this percentage was 38% (57/150). The most common substitution at RT codon 245 was methionine (M) 29/184 (15.7%), followed by glutamic acid (E) 20/184 (10.9%) and others. Based on the percentage of ambiguous basecalls, a total of 55.3% of naive samples (83/150) were classified as recent infection, while among these, 57.8% (48/83) had V at position 245. A total of 45.3% (68/150) were classified as chronic infection, with the presence of V at RT codon 245 found in 69.1% (47/68). We did not find statistically significant association between polymorphisms at codon 245 and duration of infection (p=0.2080). The frequency of RT 245 substitution found in our study exceeds the estimated prevalence HLA–B*5701 in Serbian population. This may be related to the presence of other, similar HLA alleles, limiting specificity of the correlation between HLA-B*5701 and RT codon 245 variation. Furthermore, no statistically significant difference was found in the prevalence of RT 245 substitutions between recent and chronic infection. This may suggest early fixation of an HLA induced selective imprint, during viral evolution in an infected patient.

A systems analysis of mutational effects in HIV-1 protease and reverse transcriptase

The development of a quantitative understanding of viral evolution and the fitness landscape in HIV-1 drug resistance is a formidable challenge given the large number of available drugs and drug resistance mutations. We analyzed a dataset measuring the in vitro fitness of 70,081 virus samples isolated from HIV-1 subtype B infected individuals undergoing routine drug resistance testing. We assayed virus samples for in vitro replicative capacity in the absence of drugs as well as in the presence of 15 individual drugs. We employed a generalized kernel ridge regression to estimate main fitness effects and epistatic interactions of 1,859 single amino acid variants found within the HIV-1 protease and reverse transcriptase sequences. Models including epistatic interactions predict an average of 54.8% of the variance in replicative capacity across the 16 different environments and substantially outperform models based on main fitness effects only. We find that the fitness landscape of HIV-1 protease and reverse transcriptase is characterized by strong epistasis.

Epidemiological network analysis in HIV-1 B infected patients diagnosed in Italy between 2000 and 2008

This study, through a phylogenetic analysis, is aimed to identify potential epidemiological networks and sequence interrelationships between acute/early and chronic infections in both drug-naïve and drug-experienced individuals within a local, well-defined setting and to investigate the population dynamics of transmitted resistance and the potential contribution of untreated patients to the spread of antiretroviral resistance. A total of 884 HIV-1 B subtype pol gene sequences from 306 drug-naïve (40 recently and 266 chronically infected) and 578 drug-treated HIV-1 infected patients were collected through routine drug-resistance testing between 2000 and 2008 in a single center (Division of Infectious Disease, Bergamo, Northern Italy). Bayesian phylogenetic tree was reconstructed and transmission clusters were recognized using a posterior probability as statistical support of each cluster. Differences among clustered and non-clustered drug-resistance mutations were assessed by Fisher's exact test.In our cohort we identified five clusters including ≥6 sequences with the root posterior probability of 100%. Dated phylogenies reconstructed through Bayesian Markov chain Monte Carlo model was possible for only two main clade (≥10 sequences) originated between 1990 and 2002.Among the 306 drug-naïve individuals, 12% carried a viral strain with at least 1 major mutation associated with transmitted drug resistance and 36% of these strains were involved in significant clusters.We report for the first time that many (34%) of HIV-1 subtype B transmission clusters indentified in Italy were only composed by drug-naïve individuals and that the 14% of transmitted drug resistance was linked to transmission clusters composed only of newly diagnosed individuals. The phylogenetic analysis was performed on a large cohort of drug-naïve recently/chronically infected individuals where drug-experienced patients represent almost all infected individuals in a restricted geographical area.Our findings highlight the role of newly diagnosed individuals, not yet exposed to antiretroviral drugs, in the transmission of drug-resistant HIV-1 strains, providing new insights for the planning and management of treatment programs in developing countries.

Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections

BackgroundThe number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission.MethodsBaseline HIV-1 pol sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics.ResultsMultivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with Chlamydia (p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), Chlamydia infection (p = 0.013) and primary HIV (p = 0.017).ConclusionsCombination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.

A Simple Screening Approach to Reduce B*5701-Associated Abacavir Hypersensitivity on the Basis of Sequence Variation in HIV Reverse Transcriptase

Abacavir hypersensitivity is strongly associated with the human leukocyte antigen (HLA)-B*5701 allele; however, the cost of routine high-resolution HLA typing before initiation of therapy remains prohibitive. We propose a simple approach to reduce B*5701-associated abacavir hypersensitivity based on the screening of human immunodeficiency virus (HIV) reverse transcriptase (RT) for a signature B*5701-associated cytotoxic T lymphocyte escape mutation at RT codon 245. The correlation between HLA-B*5701 and RT codon 245 variation was investigated in 392 HIV-infected, antiretroviral-naive adults who were initiating highly active antiretroviral therapy. The relationship between codon 245 variation and premature abacavir discontinuation was investigated in a larger cohort of treated individuals (n=982). Associations between HLA-B*5701 and codon 245 variants were determined using Fisher's exact test or the chi (2) test. A very strong association between HLA-B*5701 and RT codon 245 variation was observed. Only 1 (4.2%) of 24 subjects with B*5701 harbored virus with the clade B "wild-type" amino acid 245V, compared with 278 (75.5%) of 368 who did not have B*5701 (P<.001). The sensitivity and specificity of codon 245 substitutions for predicting HLA-B*5701 were 96% and 75%, respectively, and the positive and negative predictive values were 20% and 99.6%, respectively. This association remained robust even after antiretroviral treatment was administered (negative predictive value, 100%; n=269). In abacavir-treated individuals (n=982), codon 245 substitutions were predictive of premature abacavir discontinuation (P=.02). As HIV RT sequence is incidentally obtained as a part of routine drug-resistance testing, the examination of sequence variation at RT codon 245 could be adopted as a simple, low-cost screening method to identify individuals who could be safely treated with abacavir and/or who could benefit from HLA characterization.

Short Communication:Low Prevalence of Genotypic Drug Resistance Mutations among Antiretroviral-Naive HIV Type 1 Patients in Malaysia

To assess the prevalence of mutations associated with drug resistance in antiretroviral-naive patients in Kuala Lumpur, Malaysia, genotypic resistance testing was conducted among drug-naive HIV-1 patients attending the University Malaya Medical Center (UMMC) between July 2003 and June 2004. Reverse transcriptase (RT) and protease genes of plasma virions were sequenced from 100 individuals. The majority of the patients were recently diagnosed. Codons 20-255 of the RT and 1-96 of the protease gene were examined for major and minor mutations associated with antiretroviral resistance reported by the International AIDS Society- USA (IAS-USA) Drug Resistance Mutations Group. The prevalence of patients with at least one major mutation conferring drug resistance was 1%, with only one patient having a Y181C amino acid substitution in the RT gene that confers high-level resistance to nevirapine and delavirdine. Minor mutations were detected in high prevalence in the protease gene. Amino acid substitutions I13V, E35D, and M36I were associated with CRF01_AE while L63P, V77I, and I93L were associated with subtype B. Baseline prevalence of major mutations associated with resistance to antiretroviral drugs was low among antiretroviral-naive HIV-1 patients, suggesting that routine drug resistance testing may be unnecessary for all individuals newly diagnosed with HIV or all patients beginning antiretroviral therapy.

Phenotypic susceptibilities to tenofovir in a large panel of clinically derived human immunodeficiency virus type 1 isolates.

Tenofovir is a nucleotide analogue human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor, and its oral prodrug, tenofovir disoproxil fumarate, has recently been approved for the treatment of HIV-1 infection in the United States. The objective of this study was to characterize the in vitro susceptibility profiles of a large panel of clinically derived HIV-1 isolates for tenofovir. The distribution of tenofovir susceptibilities in over 1,000 antiretroviral-naive, HIV-1-infected individuals worldwide was determined using the Virco Antivirogram assay. In addition, phenotypic susceptibilities to tenofovir and other RT inhibitors were determined in a panel of nearly 5,000 recombinant HIV-1 clinical isolates from predominantly treatment-experienced patients analyzed as a part of routine drug resistance testing. Greater than 97.5% of isolates from treatment-naive patients had tenofovir susceptibilities <3-fold above those of the wild-type controls by the Antivirogram. The clinically derived panel of 5,000 samples exhibited a broad range of antiretroviral drug susceptibilities, including 69, 43, and 16% having >10-fold-decreased susceptibilities to at least one, two, and three antiretroviral drug classes, respectively. Greater than 88% of these 5,000 clinical isolates were within the threefold susceptibility range for tenofovir, and >99% exhibited <10-fold-reduced susceptibilities to tenofovir. Decreased susceptibility to tenofovir was not directly associated with resistance to other RT inhibitors; r(2) values of log-log linear regression plots of susceptibility to tenofovir versus susceptibility to other RT inhibitors were <0.4. The results suggest that the majority of treatment-naive and treatment-experienced individuals harbor HIV that remains within the normal range of tenofovir susceptibilities and may be susceptible to tenofovir disoproxil fumarate therapy.