Transmitted and Acquired HIV-1 Drug Resistance from a Family: A Case Study.
ABSTRACT: Antiretroviral drug resistance has become a major threat to the adequate management of human immunodeficiency virus (HIV) infection, but little attention has been paid to the spread and evolution of drug-resistant strains in the family. Here, we described a case of transmitted as well as acquired HIV drug resistance among a father, mother and infant. Epidemiological data were obtained retrospectively. Drug resistance mutations (DRMs) of three patients were tested using a validated In-house Sanger-based sequencing (SBS) method and the Vela next-generation sequencing (NGS) platform. Gene evolution analysis was also performed. According to the epidemiological history and phylogenetic data, in late pregnancy of the mother, the infant's father transmitted HIV-1 to her, and then the mother to the baby, leading to the transmission of V106I as a common mutation of three persons. The mutant frequency was 99.57% (father), 95.38% (mother) and 99.73% (infant), respectively. Mother also acquired K101E (41.03%), K103N (27.56%) and minor mutation of V106M (4.30%) after improperly discontinuing antiretroviral regimen of lamivudine (3TC), tenofovir (TDF) and efavirenz (EFV). Such acquired mutations increased the drug resistance scores on non-nucleoside reverse transcriptase inhibitors (NNRTIs) doravirine, EFV, etravirine, nevirapine and rilpivirine from 10, 0, 10, 10 and 10 to 65, 135, 25, 150 and 55, respectively. Therefore, sexually transmitted diseases, especially DRMs of HIV-1 in families, are of concern and draw attention to the need for enhanced drug-resistance prevention efforts, and accurate surveillance by more sensitive methods in complicated cases.
Project description:The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.
Project description:Rilpivirine (RPV) and Etravirine (ETR) are approved second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment. There is a cross-resistance HIV mutation profile between first- and second-generation NNRTI drugs. We determined the prevalence of HIV-1 drug resistance mutations (DRMs) to RPV and ETR in Botswana. A total of 168 HIV-1 polymerase gene sequences from participants failing nevirapine (NVP)- or efavirenz (EFV)-containing regimens were analyzed for DRMs using the Stanford University HIV drug resistance database. Forty-one sequences were from an adult antiretroviral therapy (ART) study, the Tshepo study, and 127 from a prevention of mother-to-child transmission (PMTCT) study, the Mashi study, all conducted in Botswana. Prevalence of RPV and ETR highest DRM in the adult ART study (n?=?41) were K101E (26.2%), E138A (23.8%), and Y181C (26.2%). The PMTCT cohort's (n?=?127) high prevalence mutations were Y181C (15.7%), E138A (15%), and K101E (11%). A total of 42.9% and 3.2% of patients in the adult ART study and PMTCT study, respectively, had three or more NNRTI mutations at virologic failure. We identified HIV-1 mutations conferring resistance to RPV and ETR even though they have not been used in Botswana. Of concern was the high proportion of sequences from the adult ART study that displayed multiple DRMs; as the number of NNRTI mutations increases, the level of cross-resistance increases. It is plausible that patients displaying such profiles maybe at increased risk of failing second-generation NNRTI drugs, hence, calls for genotyping in patients with prior NVP or efavirenz exposure before prescription of RPV- or ETR-containing cART.
Project description:BACKGROUND:The impacts of genetic polymorphisms on drug resistance mutations (DRMs) among various HIV-1 subtypes have long been debated. In this study, we aimed to analyze the natural polymorphisms and acquired DRM profile in HIV-1 CRF01_AE-infected patients in a large first-line antiretroviral therapy (ART) cohort in northeastern China. METHODS:The natural polymorphisms of CRF01_AE were analyzed in 2034 patients from a long-term ART cohort in northeastern China. The polymorphisms in 105 treatment failure (TF) patients were compared with those in 1148 treatment success (TS) patients. The acquired DRM profile of 42 patients who experienced TF with tenofovir/lamivudine/efavirenz (TDF/3TC/EFV) treatment was analyzed by comparing the mutations at TF time point to those at baseline. The Stanford HIVdb algorithm was used to interpret the DRMs. Binomial distribution, McNemar test, Wilcoxon test and CorMut package were used to analyze the mutation rates and co-variation. Deep sequencing was used to analyze the evolutionary dynamics of co-variation. RESULTS:Before ART, there were significantly more natural polymorphisms of 31 sites on reverse transcriptase (RT) in CRF01_AE than subtype B HIV-1 (|Z value|???3), including five known drug resistance-associated sites (238, 118, 179, 103, and 40). However, only the polymorphism at site 75 was associated with TF (|Z value|???3). The mutation rate at 14 sites increased significantly at TF time point compared to baseline, with the most common DRMs comprising G190S/C, K65R, K101E/N/Q, M184?V/I, and V179D/I/A/T/E, ranging from 66.7 to 45.2%. Moreover, two unknown mutations (V75?L and L228R) increased by 19.0 and 11.9% respectively, and they were under positive selection (Ka/Ks?>?1, log odds ratio [LOD]?>?2) and were associated with several other DRMs (cKa/Ks?>?1, LOD?>?2). Deep sequencing of longitudinal plasma samples showed that L228R occurred simultaneously or followed the appearance of Y181C. CONCLUSION:The high levels of natural polymorphisms in CRF01_AE had little impact on treatment outcomes. The findings regarding potential new CRF01_AE-specific minor DRMs indicate the need for more studies on the drug resistance phenotype of CRF01_AE.
Project description:In HIV-1 subtype-B, specific mutations in Gag cleavage sites (CS) are associated with treatment failure, with limited knowledge among non-B subtypes. We analyzed non-B HIV-1 gag and pol (protease/reverse-transcriptase) sequences from Cameroonians for drug resistance mutations (DRMs) in the gag P2/NC CS, and pol major DRMs. Phylogeny of the 141 sequences revealed a high genetic diversity (12 subtypes): 67.37% CRF02_AG versus 32.6% non-CRF02_AG. Overall, 7.3% transmitted and 34.3% acquired DRMs were found, including M184V, thymidine analogue mutations (T215F, D67N, K70R, K219Q), NNRTIs (L100I, Y181C, K103N, V108I, Y188L), and PIs (V82L). Twelve subjects [10 with HIV-1 CRF02_AG, 8 treatment-naïve and 4 on 3TC-AZT-NVP] showed 3 to 4 mutations in the Gag P2/NC CS: S373Q/T/A, A374T/S/G/N, T375S/A/N/G, I376V, G381S, and R380K. Subjects with or without Gag P2/NC CS mutations showed no significant difference in viral loads. Treatment-naïve subjects harboring NRTI-DRMs had significantly lower CD4 cells than those with NRTI-DRMs on ART (p = 0.042). Interestingly, two subjects had major DRMs to NRTIs, NNRTIs, and 4 mutations in the Gag P2/NC CS. In this prevailing CRF02_AG population with little exposure to PIs (~3%), mutations in the Gag P2/NC CS could increase the risk of treatment failure if there is increased use of PIs-based therapy.
Project description:Introduction:The present study investigated the HIV-1 subtype classification in addition to prevalence of drug resistance mutations (DRMs) in antiretroviral therapy (ART)-experienced and ART-naïve residents of Pontianak, West Kalimantan, Indonesia. Methods:Whole blood samples collected from 30 HIV-1-infected individuals, comprising 19 ART-experienced and 11 ART-naïve individuals, were subjected to RNA and DNA extraction, followed by HIV-1 genes amplification and sequencing analysis. HIV-1 subtyping was classified on viral pol genes encoding reverse transcriptase (RT gene) and protease (PR gene) accompanied by the env and gag genes. DRMs in the RT and PR genes were also analyzed. Results:CRF01_AE was identified as the predominant circulating recombinant form (CRF) of HIV-1 in both ART-experienced and ART-naïve individuals. In addition, CRF02_AG, subtype B, recombinant virus expressing CRF01_AE and subtype B viral genomic fragments, also recombinant virus containing CRF01_AE and CRF02_AG genomic fragments were also identified. Acquired drug resistance (ADR) was identified in 28.5% of ART-experienced individuals, while no transmitted drug resistance was identified in ART-naïve individuals. Conclusions:This study identified CRF01_AE as the most predominant HIV-1 CRF distributing in Pontianak, Indonesia. The prevalence of ADR is considered to be high; thus, further surveillance is needed in this region.
Project description:Increasing incidence of drug resistance is ascertained to be the main obstacles in limiting the virus among the human immunodeficiency virus (HIV) infected individuals. This study investigates the drug resistance mutations (DRMs), genetic variants and origin of transmitted drug resistance of HIV-1 among the HIV-1 infected wives of intravenous drug users (IDUs) in Manipur. 44 HIV pol gene sequences were generated from 56 blood samples by viral gene amplification and sequencing. Sequences were then analysed for drug resistance, genetic variants and origin. The result revealed that among the treatment naive cases, 35.7% had Transmitted Drug Resistance Mutations (TDRMs) while among treatment experienced cases, 50% had Acquired Drug Resistant Mutations (ADRMs). These TDRMs and ADRMs conferred resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and/or protease inhibitors (PIs). Majority of the isolated HIV-1 sequences (77.3%) were subtype C while 9.1% was discordant subtype, 6.8% was subtype B, 4.5% was CRF_01AE and 2.3% was URF_BC. TDRM strains were found to be introduced from Myanmar, Vietnam and mainland India. This study also reveals the appearance of CRF_01AE for the first time in Manipur. The finding of this study indicates high prevalence of drug resistant mutations and complex molecular epidemiology in Manipur.
Project description:Transmission of HIV strains with drug-resistance mutations (DRMs) causes public health problems in resource-rich countries. We use a stochastic model, with data from viral competition experiments, to analyze the effect of fitness costs (FCs) and genetic bottlenecks on limiting transmission of 10 clinically significant DRMs. Transmission of DRMs with low FCs (?0.2%) is similar to wild-type; transmission chains last ?8 generations causing clusters of ?60 infected individuals. Genetic bottlenecks substantially limit transmission of DRMs with moderately high FCs (?0.6%); chains last ?1-3 generations with transmission clusters of 2-7. Transmission of DRMs with extremely high FCs (>6%) only occurs from ?5% of index cases. DRMs can revert to wild-type and remain as minority strains, within treatment-naïve individuals, undetectable by current resistance assays. We calculate, based on assay sensitivity, the length of time each DRM is detectable within individuals. Taken together, our results imply a hidden epidemic of transmitted resistance may exist.
Project description:BACKGROUND: We and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients. METHODS: 27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from naïve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS. RESULTS: Analysis of Sanger sequences of subtype C HIV-1 isolates from naïve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from naïve patients. CONCLUSIONS: While Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger.
Project description:Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resource-limited settings recommend the initiation of lifelong antiretroviral combination therapy (cART) for all pregnant HIV-1 positive women independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of drug resistance are lacking until now. Therefore, we analysed the emergence of drug resistance mutations (DRMs) in women initiating Option B+ in Fort Portal, Uganda, at 12 and 18 months postpartum (ppm).124 HIV-1 positive pregnant women were enrolled within antenatal care services in Fort Portal, Uganda. Blood samples were collected at the first visit prior starting Option B+ and postpartum at week six, month six, 12 and 18. Viral load was determined by real-time RT-PCR. An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic region was performed. PCR-positive samples at 12/18 ppm and respective baseline samples were analysed by next generation sequencing regarding HIV-1 drug resistant variants including low-frequency variants. Furthermore, vertical transmission of HIV-1 was analysed. 49/124 (39.5%) women were included into the DRM analysis. Virological failure, defined as >1000 copies HIV-1 RNA/ml, was observed in three and seven women at 12 and 18 ppm, respectively. Sequences were obtained for three and six of these. In total, DRMs were detected in 3/49 (6.1%) women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Of 49 mother-infant-pairs no infant was HIV-1 positive at 12 or 18 ppm.Our findings suggest that the WHO-recommended Option B+ for PMTCT is effective in a cohort of Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs and vertical transmission. Therefore, these results are encouraging for other countries considering the implementation of lifelong cART for all pregnant HIV-1 positive women.
Project description:BACKGROUND:HIV Prevention Trials Network (HPTN) 061 enrolled black men who have sex with men in the United States. Some men with low/undetectable HIV RNA had unusual patterns of antiretroviral (ARV) drug use or had drugs detected in the absence of viral suppression. This report includes a comprehensive analysis of ARV drug use and drug resistance among men in HPTN 061 who were not virally suppressed. METHODS:The analysis included 169 men who had viral loads >400 copies per milliliter at enrollment, including 3 with acute infection and 13 with recent infection. By self-report, 88 were previously diagnosed, including 31 in care; 137 men reported no ARV drug use. Samples from these 169 men and 23 seroconverters were analyzed with HIV genotyping and ARV drug assays. RESULTS:Forty-eight (28%) of the 169 men had ? 1 drug resistance mutation (DRM); 19 (11%) had multiclass resistance. Sixty men (36%) had ? 1 ARV drug detected, 42 (70%) of whom reported no ARV drug use. Nine (23%) of 39 newly infected men had ? 1 DRM; 10 had ? 1 ARV drug detected. Unusual patterns of ARV drugs were detected more frequently in newly diagnosed men than previously diagnosed men. The rate of transmitted drug resistance was 23% based on HIV genotyping and self-reported ARV drug use but was 12% after adjusting for ARV drug detection. CONCLUSIONS:Many men in HPTN 061 had drug-resistant HIV, and many were at risk of acquiring additional DRMs. ARV drug testing revealed unusual patterns of ARV drug use and provided a more accurate estimate of transmitted drug resistance.