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1.  Molecular Mechanisms of Resistance to Human Immunodeficiency Virus Type 1 with Reverse Transcriptase Mutations K65R and K65R+M184V and Their Effects on Enzyme Function and Viral Replication Capacity 
Antimicrobial Agents and Chemotherapy  2002;46(11):3437-3446.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutations K65R and M184V result in changes in susceptibility to several nucleoside and nucleotide RT inhibitors. K65R-containing viruses showed decreases in susceptibility to tenofovir, didanosine (ddI), abacavir, and (−)-β-d-dioxolane guanosine (DXG; the active metabolite of amdoxovir) but appeared to be fully susceptible to zidovudine and stavudine in vitro. Viruses containing the K65R and M184V mutations showed further decreases in susceptibility to ddI and abacavir but increased susceptibility to tenofovir compared to the susceptibilities of viruses with the K65R mutation. Enzymatic and viral replication analyses were undertaken to elucidate the mechanisms of altered drug susceptibilities and potential fitness defects for the K65R and K65R+M184V mutants. The relative inhibitory capacities (Ki/Km) of the active metabolites of tenofovir, ddI, and DXG were increased for the RT containing the K65R mutation compared to that for the wild-type RT, but the relative inhibitory capacity of abacavir was only minimally increased. For the mutant viruses with the K65R and M184V mutations, the increase in tenofovir susceptibility compared to that of the mutants with K65R correlated with a decrease in the tenofovir inhibitory capacity that was mediated primarily by an increased Km of dATP. The decrease in susceptibility to ddI by mutants with the K65R and M184V mutations correlated with an increase in the inhibitory capacity mediated by an increased Ki. ATP-mediated removal of carbovir as well as small increases in the inhibitory capacity of carbovir appear to contribute to the resistance of mutants with the K65R mutation and the mutants with the K65R and M184V mutations to abacavir. Finally, both the HIV-1 K65R mutant and, more notably, the HIV-1 K65R+M184V double mutant showed reduced replication capacities and reduced RT processivities in vitro, consistent with a potential fitness defect in vivo and the low prevalence of the K65R mutation among isolates from antiretroviral agent-experienced patients.
doi:10.1128/AAC.46.11.3437-3446.2002
PMCID: PMC128721  PMID: 12384348
2.  N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance 
PLoS Medicine  2007;4(12):e335.
Background
The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.
Methods and Findings
The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 × 10−12). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.
Conclusions
This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.
Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes.
Editors' Summary
Background.
In the 1980s, infection with the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), was a death sentence. Although the first antiretroviral drugs (compounds that block HIV's life cycle) were developed quickly, single antiretrovirals only transiently suppress HIV infection. HIV rapidly accumulates random changes (mutations) in its genetic material, some of which make it drug resistant. Nowadays, there are many different antiretrovirals. Some inhibit the viral protease, an enzyme used to assemble new viruses. Others block reverse transcriptase (RT), which makes replicates of the genes of the virus. Nucleoside/nucleotide RT inhibitors (NRTIs; for example, zidovudine—also called AZT—and lamivudine) and non-nucleoside RT inhibitors (NNRTIs; for example, nevirapine and efavirenz) interfere with the activity of RT by binding to different sites in its so-called “DNA polymerase domain,” the part of the enzyme that constructs copies of the viral genes. Highly active antiretroviral therapy (HAART), which was introduced in the mid 1990s, combines several antiretrovirals (usually a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) so that the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. When treated with HAART, HIV infection is usually a chronic, stable condition rather than a fatal disease.
Why Was This Study Done?
Unfortunately, HIV that is resistant to drugs still develops in some patients. To improve the prevention and management of drug resistance, a better understanding of the mutations that cause resistance is needed. Resistance to RT inhibitors usually involves mutations in the DNA polymerase domain that reduce the efficacy of NRTIs (including thymidine analogue mutations—also known as TAMs—and lamivudine-resistance mutations) and NNRTIs. Blood tests that detect these resistance mutations (genotype tests) have been used for several years to guide individualized selection of HIV drugs. Recently, however, mutations outside the DNA polymerase domain have also been implicated in resistance to RT inhibitors. In this study, the researchers have used data and samples collected since the mid 1990s by Canada's British Columbia Centre for Excellence in HIV/AIDS to investigate the clinical relevance of a mutation called N348I. This mutation changes an asparagine (a type of amino acid) to an isoleucine in a region of RT known as the connection domain. The researchers have also investigated how this mutation causes resistance to RT inhibitors in laboratory tests.
What Did the Researchers Do and Find?
The researchers analyzed the first two-thirds of the RT gene in viruses isolated from a large number of the Centre's patients. Virus carrying the N348I mutation was present in less than one in 100 patients whose HIV infection had never been treated, but in more than one in 10 treatment-experienced patients. The mutation appeared early in therapy, often in viruses that had TAMs, a lamivudine-resistance mutation called M184V/I, and/or NNRTI resistance mutations. Patients treated with zidovudine and nevirapine were 2.6 times more likely to have the N348I mutation than patients not treated with these drugs. Furthermore, the appearance of the N348I mutation often coincided with an increase in viral load, although other mutations that appeared at a similar time could have contributed to this increase. When the researchers introduced the N348I mutation into HIV growing in the laboratory, they found that it decreased the susceptibility of the virus to zidovudine and to NNRTIs.
What Do These Findings Mean?
These findings show that the treatment of patients with RT inhibitors can select a drug-resistant HIV variant that has a mutation outside the enzyme's DNA polymerase domain. Because this N348I mutation, which is commonly selected in vivo and has also been seen in other studies, confers resistance to two classes of RT inhibitors and can emerge early during therapy, it could have a large impact on patient responses to antiviral regimens that contain zidovudine and nevirapine. Although these findings do not show that the N348I mutation alone causes treatment failure, they may have implications for genotypic and phenotypic resistance testing, which is often used to guide treatment decisions. At present, genotype tests for resistance to RT inhibitors look for mutations only in the DNA polymerase domain of RT. This study is the first to demonstrate that it might be worth looking for the N348I mutation (and for other mutations outside the DNA polymerase domain) to improve the ability of genotypic and phenotypic resistance tests to predict treatment outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040335.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including links to fact sheets (in English, French, and Spanish) about antiretrovirals, and chapters explaining antiretroviral resistance testing
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including fact sheets on types of HIV drugs, drug resistance, and resistance tests (in English, Spanish, French, Portuguese, and Russian)
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on treatment (in English and Spanish)
AIDSinfo, a service of the US Department of Health and Human Services provides information for patients on HIV and its treatment
doi:10.1371/journal.pmed.0040335
PMCID: PMC2100143  PMID: 18052601
3.  The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy 
AIDS (London, England)  2009;23(9):1127-1134.
Background
Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi.
Methods
Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed.
Results
Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine.
Conclusion
When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.
doi:10.1097/QAD.0b013e32832ac34e
PMCID: PMC2896488  PMID: 19417582
Africa; antiretroviral failure; public health approach; resistance; resource-limited setting
4.  Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase 
Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.
doi:10.1128/AAC.00127-06
PMCID: PMC1538665  PMID: 16870771
5.  ATP-Dependent Removal of Nucleoside Reverse Transcriptase Inhibitors by Human Immunodeficiency Virus Type 1 Reverse Transcriptase 
Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) ≫ zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.
doi:10.1128/AAC.46.7.2179-2184.2002
PMCID: PMC127313  PMID: 12069972
6.  Major HIV resistance mutations in untreated Romanian patients  
Journal of Medicine and Life  2011;4(2):151-157.
Drug resistance mutations are frequently detected in antiretroviral–naive HIV positive patients, however the data on transmitted resistance in non-B subtypes are limited. As HIV1 subtype F is prevalent in Romania, our goal is to analyze resistance mutations in the pol gene of HIV–1 isolates from drug–naive Romanian patients. HIV–1 pol gene from 12 untreated patients, newly diagnosed (n = 6) and chronically infected (n=6), with detectable HIV RNA viral load was genotyped and the viral subtype was determined by using the Stanford database algorithm. 8/12 strains belonged to the F subtype, 1/12 to the G subtype, and the rest of the studied strains appeared to be K/F, A/F and J/F inter–subtype recombinant forms. The prevalence of HIV–1 strains with at least one major drug resistance mutation in the studied group was unexpectedly high. Major mutations associated with NRTI, NNRTI and PI resistance were detected in 6/12 patients, 2/12 patients and 3/12 patients, respectively; in addition all viral strains had minor mutations in the protease gene. Newly diagnosed patients harbored resistant variants more often than chronically infected ones (4/6 vs. 2/6) did. These data support the use of genotypic resistance testing in treatment–naive HIV positive patients, in order to guide the selection of the first line of antiretrovirals, due to the fact that persons with transmitted drug resistance have a higher risk for both virologic failure and development of resistance at treatment initiation.
HIV–Human immunodeficiency virus; TDR–transmitted drug resistance; HAART–highly active antiretroviral therapy ; SDRM–surveillance list of drug resistance mutations ; NRTIs–nucleos(t)idic reverse–transcriptase inhibitors; NNRTIs– non–nucleosidic reverse transcriptase inhibitors; PIs–protease inhibitors; TAMs–thy–midine analogue mutations; 3TC –lamivudine ; FTC–emtricitabine ; ddI –didanosine ; ABC–abacavir ; ZDV–zidovudine ; d4T–stavudine ; TDF –tenofovir
PMCID: PMC3124271  PMID: 21776297
HIV; subtype F; naive patients; transmitted drug resistance
7.  International Cohort Analysis of the Antiviral Activities of Zidovudine and Tenofovir in the Presence of the K65R Mutation in Reverse Transcriptase▿  
A K65R mutation in HIV-1 reverse transcriptase can occur with the failure of tenofovir-, didanosine-, abacavir-, and, in some cases, stavudine-containing regimens and leads to reduced phenotypic susceptibility to these drugs and hypersusceptibility to zidovudine, but its clinical impact is poorly described. We identified isolates with the K65R mutation within the Stanford Resistance Database and a French cohort for which subsequent treatment and virological response data were available. The partial genotypic susceptibility score (pGSS) was defined as the genotypic susceptibility score (GSS) excluding the salvage regimen's nucleoside reverse transcriptase inhibitor (NRTI) component. A three-part virologic response variable was defined (e.g., complete virologic response, partial virologic response, and no virologic response). Univariate, multivariate, and bootstrap analyses evaluated factors associated with the virologic response, focusing on the contributions of zidovudine and tenofovir. Seventy-one of 130 patients (55%) achieved a complete virologic response (defined as an HIV RNA level of <200 copies/ml). In univariate analyses, pGSS and zidovudine use in the salvage regimen were predictors of the virologic response. In a multivariate analysis, pGSS and zidovudine and tenofovir use were associated with the virologic response. Bootstrap analyses showed similar reductions in HIV RNA levels with zidovudine or tenofovir use (0.5 to 0.9 log10). In the presence of K65R, zidovudine and tenofovir are associated with similar reductions in HIV RNA levels. Given its tolerability, tenofovir may be the preferred agent over zidovudine even in the presence of the K65R mutation.
doi:10.1128/AAC.01380-09
PMCID: PMC2849386  PMID: 20124005
8.  Resistance Patterns Selected by Nevirapine vs. Efavirenz in HIV-Infected Patients Failing First-Line Antiretroviral Treatment: A Bayesian Analysis 
PLoS ONE  2011;6(11):e27427.
Background
WHO recommends starting therapy with a non-nucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTIs), i.e. nevirapine or efavirenz, with lamivudine or emtricitabine, plus zidovudine or tenofovir. Few studies have compared resistance patterns induced by efavirenz and nevirapine in patients infected with the CRF01_AE Southeast Asian HIV-subtype. We compared patterns of NNRTI- and NRTI-associated mutations in Thai adults failing first-line nevirapine- and efavirenz -based combinations, using Bayesian statistics to optimize use of data.
Methods and Findings
In a treatment cohort of HIV-infected adults on NNRTI-based regimens, 119 experienced virologic failure (>500 copies/mL), with resistance mutations detected by consensus sequencing. Mutations were analyzed in relation to demographic, clinical, and laboratory variables at time of genotyping. The Geno2Pheno system was used to evaluate second-line drug options. Eighty-nine subjects were on nevirapine and 30 on efavirenz. The NRTI backbone consisted of lamivudine or emtricitabine plus either zidovudine (37), stavudine (65), or tenofovir (19). The K103N mutation was detected in 83% of patients on efavirenz vs. 28% on nevirapine, whereas Y181C was detected in 56% on nevirapine vs. 20% efavirenz. M184V was more common with nevirapine (87%) than efavirenz (63%). Nevirapine favored TAM-2 resistance pathways whereas efavirenz selected both TAM-2 and TAM-1 pathways. Emergence of TAM-2 mutations increased with the duration of virologic replication (OR 1.25–1.87 per month increment). In zidovudine-containing regimens, the overall risk of resistance across all drugs was lower with nevirapine than with efavirenz, whereas in tenofovir-containing regimen the opposite was true.
Conclusions
TAM-2 was the major NRTI resistance pathway for CRF01_AE, particularly with nevirapine; it appeared late after virological failure. In patients who failed, there appeared to be more second-line drug options when zidovudine was combined with nevirapine or tenofovir with efavirenz than with alternative combinations.
doi:10.1371/journal.pone.0027427
PMCID: PMC3223170  PMID: 22132100
9.  Extensive Drug Resistance in HIV-Infected Cambodian Children Who Are Undetected as Failing First-Line Antiretroviral Therapy by WHO 2010 Guidelines 
Abstract
Antiretroviral therapy in resource-limited settings is monitored clinically and immunologically according to WHO guidelines. Frequent misclassification of virologic failure is reported, mostly in adults, leading to early therapy switch or late failure diagnosis. Pediatric treatment monitoring and resistance data upon first-line failure are limited, particularly when the 2010-WHO pediatric guidelines are used without routine viral load monitoring. We previously reported high treatment failure misclassification rates by pediatric 2010 guidelines in Cambodian children on first-line therapy. Here we determine the extent and patterns of resistance, with yearly viral load and 6-monthly CD4. Drug resistance mutations were determined using the IAS-USA 2011 list. Predicted resistance interpretation was determined with Stanford Database tools. Fifty-one children with available genotypes met inclusion criteria. All but one (subtype B) were CRF01_AE. The most common regimen was stavudine, lamivudine, and nevirapine (96%), taken for a median of 2.2 years. Resistance was seen in 98%; 96% to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs); 51% with ≥4 mutations. The most common NRTI mutations were 184V/I and 67N and the most common NNRTI mutations were 181C/Y/I/V and 190A/S. A total of 22% had multiresistant mutations and 18% had predicted high-level resistance to subsequent therapy options didanosine, abacavir, etravirine, and tenofovir. In 98% of Cambodian children misclassified as nonfailing first-line therapy by 2010 guidelines, 51% had extensive drug resistance to current and 18% to subsequent antiretroviral therapy. Affordable routine viral load monitoring allowing for early and more accurate treatment failure diagnosis is desperately needed in resource-limited settings.
doi:10.1089/aid.2013.0025
PMCID: PMC3685690  PMID: 23506238
10.  In Vitro Antiretroviral Activity and In Vitro Toxicity Profile of SPD754, a New Deoxycytidine Nucleoside Reverse Transcriptase Inhibitor for Treatment of Human Immunodeficiency Virus Infection 
SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 μM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.
doi:10.1128/AAC.50.2.625-631.2006
PMCID: PMC1366874  PMID: 16436719
11.  Efficacy of Short-Course AZT Plus 3TC to Reduce Nevirapine Resistance in the Prevention of Mother-to-Child HIV Transmission: A Randomized Clinical Trial 
PLoS Medicine  2009;6(10):e1000172.
Neil Martinson and colleagues report a randomized trial of adding short-course zidovudine+lamivudine to reduce drug resistance from single-dose nevirapine used to prevent mother-to-child transmission of HIV.
Background
Single-dose nevirapine (sdNVP)—which prevents mother-to-child transmission of HIV—selects non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance mutations in the majority of women and HIV-infected infants receiving it. This open-label, randomised trial examined the efficacy of short-course zidovudine (AZT) and lamivudine (3TC) with sdNVP in reducing NNRTI resistance in mothers, and as a secondary objective, in infants, in a setting where sdNVP was standard-of-care.
Methods and Findings
sdNVP alone, administered at the onset of labour and to the infant, was compared to sdNVP with AZT plus 3TC, given as combivir (CBV) for 4 (NVP/CBV4) or 7 (NVP/CBV7) days, initiated simultaneously with sdNVP in labour; their newborns received the same regimens. Women were randomised 1∶1∶1. HIV-1 resistance was assessed by population sequencing at: baseline, 2, and 6 wk after birth. An unplanned interim analysis resulted in early stopping of the sdNVP arm. 406 pregnant women were randomised and took study medication (sdNVP 74, NVP/CBV4 164, and NVP/CBV7 168). HIV-1 resistance mutations emerged in 59.2%, 11.7%, and 7.3% of women in the sdNVP, NVP/CBV4, and NVP/CBV7 arms by 6 wk postpartum; differences between NVP-only and both NVP/CBV arms were significant (p<0.0001), but the difference between NVP/CBV4 and NVP/CBV7 was not (p = 0.27). Estimated efficacy comparing combined CBV arms with sdNVP was 85.6%. Similar resistance reductions were seen in infants who were HIV-infected by their 6-wk visit.
Conclusions
A short course of AZT plus 3TC, supplementing maternal and infant sdNVP, reduces emergent NNRTI resistance mutations in both mothers and their infants. However, this trial was not powered to detect small differences between the CBV arms.
Trial registration
www.ClinicalTrials.gov NCT 00144183
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Currently, about 33 million people are infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV can be treated with combination antiretroviral therapy (ART), commonly three individual antiretroviral drugs that together efficiently suppress the replication of the virus. HIV infection of a child by an HIV-positive mother during pregnancy, labor, delivery, or breastfeeding is called mother-to-child transmission (MTCT). In 2007, an estimated 420,000 children were newly infected with HIV, the majority through MTCT. Most of these mothers and children live in sub-Saharan Africa where child and maternal mortality rates are high and mortality in HIV-infected children is extremely high. MTCT is preventable and there is a global commitment, agreed at the UN General Assembly Session on HIV/AIDS in 2001, to reduce the proportion of infants infected with HIV by 50% by 2010.
Why Was This Study Done?
In many resource-limited settings, MTCT is prevented by giving a single dose of nevirapine (an antiretroviral drug which has a long duration in the body and protects the fetus during labor and delivery only) to HIV-infected women in labor and also to a baby within 72 hours of birth. However, nevirapine, a non-nucleoside reverse-transcriptase inhibitor (NNRTI), which suppresses the replication of the virus, is associated with increased resistance of HIV, in mother and child, to NNRTI. This resistance reduces the effectiveness of future treatments of both mother and child with combination ART that includes an NNRTI; such regimens are the mainstay for long-term treatment of HIV in developing countries. The researchers investigated whether giving other antiretroviral drugs with nevirapine, during labor and delivery, to both mother and her newborn reduced the chances of them developing resistance to NNRTIs.
What Did the Researchers Do and Find?
The researchers selected 406 HIV-positive pregnant women for study across five sites in South Africa between February 2003 and May 2007. The women and their newborn babies were randomly assigned to receive, either (i) a single dose of nevirapine, (ii) a single dose of nevirapine plus combivir (zidovudine combined with lamivudine) for four days, or (iii) a single dose of nevirapine plus combivir for seven days. At two days, two weeks, and six weeks after delivery blood was collected from mothers and babies. HIV virus from blood samples was analyzed for resistance mutations, and mothers and children with resistance mutations were monitored for a further 96 weeks until no resistance was detected or combination ART (also called “HAART”) was started. Enrollment into the single-dose nevirapine arm was stopped early because a very high rate of NNRTI resistance mutations was found and other investigators reported long-term bad consequences of NNRTI-resistance on subsequent ART. The two nevirapine plus combivir arms were continued. The researchers found that selection of resistance mutations by single-dose nevirapine was reduced in mother and child by the addition of zidovudine and lamivudine for a short period; resistance mutations were found in 59.2% of women who got nevirapine only but only 11.7%, and 7.3% of women treated nevirapine plus four days combivir, and nevirapine plus seven days combivir respectively. A reduction was also seen in new NNRTI resistant mutations in the HIV-infected infants that received combivir. The study did not have enough women to show that there was a real difference between the resistance in the four-day and seven-day combivir regimens.
What Do These Findings Mean?
These findings show that a short-course treatment of zidovudine and lamivudine in addition to a single dose of nevirapine during labor and birth reduces the selection of NNRTI resistance mutations in both mother and child. The drug regimens appeared safe, and easy to provide and adhere to. Preliminary results from this study contributed to a change in clinical practice for the care of pregnant women with HIV; in 2004 the World Health Organisation guidelines introduced a short course of combivir with nevirapine for the management of pregnant HIV-infected women. However, the study had some limitations. It used HIV-positive women who were mainly infected with a subtype of HIV called HIV-1 clade C and who had a lot of virus in their blood. NNRTI resistance after treatment with nevirapine is more common in clade C than in others and this study does not address the effect of these combinations for preventing NNRTI resistance in other HIV subtypes. Also, World Health Organization, national, and international guidelines recommend combination ART during pregnancy, as it decreases HIV transmission from mother to child in the uterus to <2% in resource-limited settings. Although long-term combination treatment may not be available in all locations, this study does not tell us how the short-term combinations during and after delivery tested would compare to longer-term combinations given to pregnant women in reducing both HIV transmission and HIV drug resistance.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000172.
This study is further discussed in a PLoS Medicine Perspective by Lehman et al.
The US Centers for Disease Control and Prevention provide information for HIV treatment and prevention
MedlinePlus provides extensive information on symptoms and treatment for HIV/AIDS as well as access to related clinical trials and medical literature
aidsmap, a nonprofit, nongovernmental organization provides information on HIV and supporting those living with HIV
The World Health Organization gives information on the prevention of mother-to-child transmission of HIV
doi:10.1371/journal.pmed.1000172
PMCID: PMC2760761  PMID: 19859531
12.  How much do antiretroviral drugs penetrate into the central nervous system?  
Journal of Medicine and Life  2011;4(4):432-439.
The central nervous system can act as a compartment in which HIV can replicate independently from plasma, and also as a sanctuary in which, under suboptimal drug pressure, HIV antiretroviral genetic variants can occur. Continuous replication of HIV in brain can contribute to neurocognitive impairment. Therefore, reaching adequate concentrations of antiretrovirals in the central nervous system might be essential in providing neuroprotection and improving neurocognition. Antiretrovirals have a restricted entry into the brain, due to several factors: the unique structure of the blood-brain barrier, and the existence of efficient efflux mechanisms. However, there is a high variability of antiretrovirals in reaching therapeutic drug concentrations in cerebrospinal fluid, that depend on the characteristics of the antiretrovirals (molecular weight, lipophilicity, protein binding) and on their capacity to be substrate for efflux transporters. The review aims to discuss the main mechanisms that interfere with antiretroviral penetration into central nervous system, and to summarize the current data concerning the penetrability of different antiretrovirals into the cerebrospinal fluid.
Abbreviations: ART = antiretroviral treatment; ARV = antiretrovirals; NRTI = nucleos(t)idic reverse-transcriptase inhibitors; NNRTI = non-nucleosidic reverse transcriptase inhibitors; INNRT = integrase inhibitors; CNS = central nervous system; BBB = blood-brain barrier; CMT = carrier-mediated transport; AET = active efflux transports; PGP = P-glycoprotein; MRP = multidrug resistance-associated proteins; SLC = solute carriers; OATP = organic anion transporting polypeptide; OAT = organic anion transporters; OCT = organic cation transporters; EFV = Efavirenz; IDV = Indinavir; ZDV = Zidovudine; d4T = Stavudine; ABC = Abacavir; ddI = Didanosine; 3TC = Lamivudine; TDF = Tenofovir; NVP = Nevirapine; PI = Protease inhibitors; APV = Amprenavir; NFV = Nelfinavir; SQV = Saquinavir; ATV = Ataznavir; TPV = Tipranavir; DRV = Darunavir; T20 = Enfuvirtide; RGV = Raltegravir
PMCID: PMC3227164  PMID: 22514580
antiretroviral treatment; central nervous system; penetrability; HIV
13.  The K65R mutation in HIV-1 reverse transcriptase: genetic barriers, resistance profile and clinical implications 
HIV therapy  2009;3(6):583-594.
Resistance to antiviral therapy is the limiting factor in the successful management of HIV. In general, the K65R mutation is rarely selected (1.7–4%) with tenofovir disoproxil fumarate (TDF), abacavir (ABC), didanosine (ddI), and stavudine (d4T), as compared with the high incidence (>40%) of thymidine analog mutations associated with zidovudine and d4T. The high barrier to the development of K65R may reflect a combination of factors, including the high potency of K65R-selecting drugs, including recommended TDF/emtricitabine and ABC/lamivudine (ABC/3TC) combinations; the partial (low–intermediate level) profile of cross-resistance conferred by K65R to TDF, ABC and 3TC; the favorable viral fitness constraint imposed by K65R and the 3TC/emtricitabine-associated M184V mutations; the bidirectional antagonism between the K65R and thymidine analog mutation pathways; and unique RNA structural considerations in the region surrounding codon 65. Nevertheless, surprisingly high levels of treatment failures and K65R resistance may be associated with triple nucleoside analog regimens. The use of TDF + ABC, TDF + ddI and ABC + d4T in combination with 3TC or emtricitabine should be avoided. This selection of K65R may be reduced by the inclusion of zidovudine in two–four nucleoside reverse-transcriptase regimens. Clinical studies have demonstrated an increased frequency of K65R in association with suboptimal d4T and ddI regimens, as well as nevirapine and its resistance mutations Y181C and G190A. The potential for the development of the K65R mutation in subtype C is particularly problematic wherein a signature KKK nucleotide motif, at codons 64, 65 and 66 in reverse transcriptase, appear to lead to template pausing, facilitating the selection of K65R. Optimizing regimens may attenuate the emergence of K65R, leading to better long-term treatment management in different geographic settings. TDF-based regimens are the leading candidates for first- and second-line therapy, microbicides and chemoprophylaxis strategies.
doi:10.2217/hiv.09.40
PMCID: PMC2826981  PMID: 20190870
HIV-1 drug resistance; K65R; nucleoside analogs; subtype C; tenofovir
14.  Impact of Y181C and/or H221Y mutation patterns of HIV-1 reverse transcriptase on phenotypic resistance to available non-nucleoside and nucleoside inhibitors in China 
BMC Infectious Diseases  2014;14:237.
Background
The aim of this study was to investigate the role of K101Q, Y181C and H221Y emerging in HIV-1 reverse transcriptase with different mutations patterns in phenotypic susceptibility to currently available NNRTIs (nevirapine NVP, efavirenz EFV) and NRTIs (zidovudine AZT, lamivudine 3TC, stavudine d4T) in China.
Methods
Phenotype testing of currently available NNRTIs (NVP, EFV) and NRTIs (AZT, 3TC, d4T) was performed on TZM-b1 cells using recombined virus strains. P ≤ 0.05 was defined significant considering the change of 50% inhibitory drug concentration (IC50) compared with the reference, while P ≤ 0.01 was considered to be statistically significant considering multiple comparisons.
Results
Triple-mutation K101Q/Y181C/H221Y and double-mutation K101Q/Y181C resulted in significant increase in NVP resistance (1253.9-fold and 986.4-fold), while only K101Q/Y181C/H221Y brought a 5.00-fold significant increase in EFV resistance. Remarkably, K101Q/H221Y was hypersusceptible to EFV (FC = 0.04), but was significantly resistant to the three NRTIs. Then, the interaction analysis suggested the interaction was not significant to NVP (F = 0.77, P = 0.4061) but significant to EFV and other three NRTIs.
Conclusion
Copresence of mutations reported to be associated with NNRTIs confers significant increase to NVP resistance. Interestingly, some may increase the susceptibility to EFV. Certainly, the double mutation (K101Q/H221Y) also changes the susceptibility of viruses to NRTIs. Interaction between two different sites makes resistance more complex.
doi:10.1186/1471-2334-14-237
PMCID: PMC4024112  PMID: 24885612
Nucleoside reverse transcriptase inhibitors (NRTIs); Non-nucleoside reverse transcriptase inhibitors (NNRTIs); HIV-1 resistance mutation; 50% inhibitory drug concentration
15.  Development and Evaluation of an Oligonucleotide Ligation Assay for Detection of Drug Resistance-Associated Mutations in the Human Immunodeficiency Virus Type 2 pol Gene▿  
Journal of Clinical Microbiology  2007;45(5):1565-1571.
Human immunodeficiency virus type 2 (HIV-2) is naturally resistant to several antiretroviral drugs, including all of the non-nucleoside reverse transcriptase inhibitors and the entry inhibitor T-20, and may have reduced susceptibility to some protease inhibitors. These resistance properties make treatment of HIV-2 patients difficult, with very limited treatment options. Therefore, early detection of resistance mutations is important for understanding treatment failures and guiding subsequent therapy decisions. With the Global Fund Initiative, a substantial number of HIV-2 patients in West Africa will receive antiretroviral therapy. Therefore, development of cheaper and more sustainable resistance assays, such as the oligonucleotide ligation assay (OLA), is a priority. In this study, we designed oligonucleotide probes to detect the Q151M mutation, associated with phenotypic resistance to zidovudine, didanosine, zalcitabine, and stavudine, and the M184V mutation, associated with phenotypic resistance to lamivudine and emtricitabine, in HIV-2. The assay was successfully developed and evaluated with 122 samples from The Gambia, Guinea Bissau, The Netherlands, and Sweden. The overall sensitivity of the assay was 98.8%, with 99.2% for Q151M and 98.4% for M184V. OLA results were compared with sequencing to give high concordances of 98.4% (Q151M) and 97.5% (M184V). OLA demonstrated a higher sensitivity for detection of minor variants as a mixture of wild-type and mutant viruses in cases when sequencing detected only the major population. In conclusion, we have developed a simple, easy-to-use, and economical assay for genotyping of drug resistance in HIV-2 that is more sustainable for use in resource-poor settings than is consensus sequencing.
doi:10.1128/JCM.02220-06
PMCID: PMC1865891  PMID: 17329450
16.  Impaired Rescue of Chain-Terminated DNA Synthesis Associated with the L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase†  
The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.
doi:10.1128/AAC.49.7.2657-2664.2005
PMCID: PMC1168713  PMID: 15980333
17.  HIV-1 Drug Resistance Emergence among Breastfeeding Infants Born to HIV-Infected Mothers during a Single-Arm Trial of Triple-Antiretroviral Prophylaxis for Prevention of Mother-To-Child Transmission: A Secondary Analysis 
PLoS Medicine  2011;8(3):e1000430.
Analysis of a substudy of the Kisumu breastfeeding trial by Clement Zeh and colleagues reveals the emergence of HIV drug resistance in HIV-positive infants born to HIV-infected mothers treated with antiretroviral drugs.
Background
Nevirapine and lamivudine given to mothers are transmitted to infants via breastfeeding in quantities sufficient to have biologic effects on the virus; this may lead to an increased risk of a breastfed infant's development of resistance to maternal antiretrovirals. The Kisumu Breastfeeding Study (KiBS), a single-arm open-label prevention of mother-to-child HIV transmission (PMTCT) trial, assessed the safety and efficacy of zidovudine, lamivudine, and either nevirapine or nelfinavir given to HIV-infected women from 34 wk gestation through 6 mo of breastfeeding. Here, we present findings from a KiBS trial secondary analysis that evaluated the emergence of maternal ARV-associated resistance among 32 HIV-infected breastfed infants.
Methods and Findings
All infants in the cohort were tested for HIV infection using DNA PCR at multiple study visits during the 24 mo of the study, and plasma RNA viral load for all HIV-PCR–positive infants was evaluated retrospectively. Specimens from mothers and infants with viral load >1,000 copies/ml were tested for HIV drug resistance mutations. Overall, 32 infants were HIV infected by 24 mo of age, and of this group, 24 (75%) infants were HIV infected by 6 mo of age. Of the 24 infants infected by 6 mo, nine were born to mothers on a nelfinavir-based regimen, whereas the remaining 15 were born to mothers on a nevirapine-based regimen. All infants were also given single-dose nevirapine within 48 hours of birth. We detected genotypic resistance mutations in none of eight infants who were HIV-PCR positive by 2 wk of age (specimens from six infants were not amplifiable), for 30% (6/20) at 6 wk, 63% (14/22) positive at 14 wk, and 67% (16/24) at 6 mo post partum. Among the 16 infants with resistance mutations by 6 mo post partum, the common mutations were M184V and K103N, conferring resistance to lamivudine and nevirapine, respectively. Genotypic resistance was detected among 9/9 (100%) and 7/15 (47%) infected infants whose mothers were on nelfinavir and nevirapine, respectively. No mutations were detected among the eight infants infected after the breastfeeding period (age 6 mo).
Conclusions
Emergence of HIV drug resistance mutations in HIV-infected infants occurred between 2 wk and 6 mo post partum, most likely because of exposure to maternal ARV drugs through breast milk. Our findings may impact the choice of regimen for ARV treatment of HIV-infected breastfeeding mothers and their infected infants.
Trial Registration
ClinicalTrials.gov NCT00146380
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Globally, more than 2 million children are infected with the human immunodeficiency virus (HIV) that causes acquired immunodeficiency syndrome (AIDS), and half a million children are newly infected every year. These infections are mainly the result of mother-to-child transmission (MTCT) of HIV during pregnancy, labor and delivery, or through breastfeeding. MTCT can be greatly reduced by treating HIV-positive mothers and their babies with antiretroviral drugs (ARVs). Without ARVs, up to half of babies born to HIV-positive mothers become infected with HIV. This rate of transmission falls to below 5% if a combination of three ARVs is given to the mother throughout pregnancy. Unfortunately, this triple-ARV therapy is too expensive for use in the resource-limited countries where most MTCT occurs. Instead, many such countries have introduced simpler, shorter ARV regimens such as a daily dose of zidovudine (a nucleoside reverse transcriptase inhibitor or NRTI) given to HIV-positive women during late pregnancy coupled with single-dose nevirapine (a non-nucleoside reverse transcriptase inhibitor or NNRTI) at the onset of labor, zidovudine and lamivudine (another NRTI) during labor and delivery, and single-dose nevirapine given to the baby at birth.
Why Was This Study Done?
More than 95% of HIV-exposed children are born in resource-limited settings where breastfeeding is the norm and is crucial for child survival even though it poses a risk of HIV transmission. Consequently, several recent studies have investigated whether MTCT can be further reduced by giving the mother ARVs while she is breastfeeding. In the Kisumu Breastfeeding Study (KiBS), for example, researchers assessed the effects of giving zidovudine, lamivudine, and either nevirapine or nelfinavir (a protease inhibitor) to HIV-infected women from 34 weeks of pregnancy through 6 months of breastfeeding. The results of KiBS indicate that this approach might be a safe, feasible way to reduce MTCT (see the accompanying paper by Thomas and colleagues). However, low amounts of nevirapine and lamivudine are transferred from mother to infant in breast milk and this exposure to ARVs could induce the development of resistance to ARVs among HIV-infected infants. In this KiBS substudy, the researchers investigate whether HIV drug resistance emerged in any of the HIV-positive infants in the parent study.
What Did the Researchers Do and Find?
In KiBS, 32 infants were HIV-positive at 24 months old; 24 were HIV-positive at 6 months old when their mothers stopped taking ARVs and when breastfeeding was supposed to stop. The researchers analyzed blood samples taken from these infants at various ages and from their mothers for the presence of HIV drug resistance mutations (DNA changes that make HIV resistant to killing by ARVs). They detected no resistance mutations in samples taken from 2-week old HIV-positive infants or from the infants who became infected after the age of 6 months. However, they found resistance mutations in a third and two-thirds of samples taken from 6-week and 6-month old HIV-positive infants, respectively. The commonest mutations conferred resistance to lamivudine and nevirapine. Moreover, resistance mutations were present in samples taken from all the HIV-positive infants whose mothers who had received nelfinavir but in only half those taken from infants whose mothers who had received nevirapine. Finally, most of the mothers of HIV-positive infants had no HIV drug resistance mutations, and only one mother-infant pair had an overlapping pattern of HIV drug resistance mutations.
What Do These Findings Mean?
These findings indicate that, in this KiBS substudy, the emergence of HIV drug resistance mutations in HIV-infected infants whose mothers were receiving ARVs occurred between 2 weeks and 6 months after birth. The pattern of mutations suggests that drug resistance most likely arose through exposure of the infants to low levels of ARVs in breast milk rather than through MTCT of drug-resistant virus. These findings need confirming but suggest that infants exposed to ARVs through breast milk—a situation that may become increasingly common given the reduction in MTCT seen in KiBS and other similar trials—should be carefully monitored for HIV infection. Providers should consider the mothers' regimen when choosing treatment for infants who are found to be HIV-infected despite maternal triple drug prophylaxis. Infants exposed to a maternal regimen with NNRTI drugs should receive first-line therapy with lopinavir/ritonavir, a protease inhibitor. The significance of the NRTI mutations such as M184V with regard to response to therapy needs further evaluation. The M184V mutation may result in hypersensitization to other NRTI drugs and delay or reverse zidovudine resistance. Given the limited availability of alternative drugs for infants in resource-limited settings, provision of the standard WHO-recommended first-line NRTI backbone, which includes 3TC, with enhanced monitoring of the infant to ensure virologic suppression, could be considered. Such an approach should reduce both illness and morbidity among infants who become HIV positive through breastfeeding.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/ 10.1371/journal.pmed.1000430.
The accompanying PLoS Medicine Research article by Thomas and colleagues describes the primary findings of the Kisumu Breastfeeding Study
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on HIV/AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on children, HIV, and AIDS and on preventing mother-to-child transmission of HIV (in English and Spanish)
UNICEF also has information about children and HIV and AIDS (in several languages)
The World Health organization has information on mother-to-child transmission of HIV (in several languages), and guidance on the use of ARVs for preventing MTCT
doi:10.1371/journal.pmed.1000430
PMCID: PMC3066134  PMID: 21468304
18.  Use of nucleoside reverse transcriptase inhibitors and risk of myocardial infarction in HIV-infected patients enrolled in the D:A:D study: a multi-cohort collaboration 
Lancet  2008;371(9622):1417-1426.
Summary
Background
Whether nucleoside reverse transcriptase inhibitors increase the risk of myocardial infarction in HIV-infected individuals is unclear. Our aim was to explore whether exposure to such drugs was associated with an excess risk of myocardial infarction in a large, prospective observational cohort of HIV-infected patients.
Methods
We used Poisson regression models to quantify the relation between cumulative, recent (currently or within the preceding 6 months), and past use of zidovudine, didanosine, stavudine, lamivudine, and abacavir and development of myocardial infarction in 33 347 patients enrolled in the D:A:D study. We adjusted for cardiovascular risk factors that are unlikely to be affected by antiretroviral therapy, cohort, calendar year, and use of other antiretrovirals.
Findings
Over 157 912 person-years, 517 patients had a myocardial infarction. We found no associations between the rate of myocardial infarction and cumulative or recent use of zidovudine, stavudine, or lamivudine. By contrast, recent—but not cumulative—use of abacavir or didanosine was associated with an increased rate of myocardial infarction (compared with those with no recent use of the drugs, relative rate 1.90, 95% CI 1.47-2.45 [p=0.0001] with abacavir and 1.49, 1.14-1.95 [p=0.003] with didanosine); rates were not significantly increased in those who stopped these drugs more than 6 months previously compared with those who had never received these drugs. After adjustment for predicted 10-year risk of coronary heart disease, recent use of both didanosine and abacavir remained associated with increased rates of myocardial infarction (1.49, 1.14-1.95 [p=0.004] with didanosine; 1.89, 1.47-2.45 [p=0.0001] with abacavir).
Interpretation
There exists an increased risk of myocardial infarction in patients exposed to abacavir and didanosine within the preceding 6 months. The excess risk does not seem to be explained by underlying established cardiovascular risk factors and was not present beyond 6 months after drug cessation.
doi:10.1016/S0140-6736(08)60423-7
PMCID: PMC2688660  PMID: 18387667
19.  Clonal Resistance Analyses of HIV-1 after Failure of Therapy with Didanosine, Lamivudine, and Tenofovir 
Antiviral therapy  2010;15(3):437-441.
Background
The rapid failure of initial therapy with combinations of nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) that exclude zidovudine has not been fully explained by standard virus population analyses of HIV-1 drug resistance. We therefore investigated HIV-1 genotype and phenotype at the single genome level in samples from patients on a failing regimen of tenofovir (TNV), didanosine (ddI), and lamivudine (3TC).
Methods
Single genome sequencing was performed on nine failure samples containing both K65R and M184V mutations by standard genotype, either as wild-type/mutant mixtures (6/9) or as mutant only (3/9). Recombinant clones with different combinations of observed mutations were generated and tested for NRTI susceptibility.
Results
Of the 204 single genome sequences analyzed, 50% were K65R/M184V double-mutants, 38% were M184V single-mutants, 10% were M184I single-mutants, and only 1% (2 sequences) were K65R single-mutants. Phenotypic testing of recombinant clones showed a significant increase in resistance for double-mutants: mean fold-resistance to ABC, ddI, and TNV was 6.5, 4.3, and 1.6 for K65R/M184V double-mutants versus 2.5, 1.9, and 0.6 for M184V single-mutants, respectively (p<0.001).
Conclusions
Mutants with K65R and M184V linked on the same genome were the most common HIV-1 variants in samples analyzed from patients failing TNV, ddI, and 3TC with both mutations detected by standard genotype. The double-mutant exhibited reduced susceptibility to all three NRTI in the regimen. This resistant phenotype, resulting from just two linked point mutations, likely contributes to rapid failure of NRTI combinations that exclude zidovudine.
doi:10.3851/IMP1539
PMCID: PMC2902251  PMID: 20516563
20.  Comparison of the Precision and Sensitivity of the Antivirogram and PhenoSense HIV Drug Susceptibility Assays 
Objective
Although 2 widely used susceptibility assays have been developed, their precision and sensitivity have not been assessed.
Design and Methods
To assess the precision of the Antivirogram and PhenoSense assays, we examined susceptibility results of HIV-1 isolates lacking drug resistance mutations and containing matching patterns of drug resistance mutations. To assess sensitivity, we determined for each assay the proportion of isolates with common patterns of matching drug resistance mutations having reductions in susceptibility greater than those in isolates without drug resistance mutations.
Results
We analyzed protease inhibitor (PI) susceptibility results obtained by the Antivirogram assay for 293 isolates and by the PhenoSense assay for 300 isolates. We analyzed reverse transcriptase (RT) inhibitor susceptibility results obtained by the Antivirogram assay for 202 isolates and by the PhenoSense assay for 126 isolates. For wild-type and mutant isolates, the median absolute deviance of the fold resistance of nucleoside RT inhibitor susceptibility results was significantly lower for the PhenoSense assay than for the Antivirogram assay. The PhenoSense assay was also significantly more likely than the Antivirogram assay to detect resistance to abacavir, didanosine, and stavudine in isolates with the common drug resistance mutations M41L, M184V, and T215Y (±L210W). We found no significant differences between the 2 assays for detecting PI and nonnucleoside RT inhibitor resistance.
Conclusion
The PhenoSense assay is more precise than the Antivirogram assay and superior at detecting resistance to abacavir, didanosine, and stavudine.
PMCID: PMC2547471  PMID: 15764961
antiretroviral therapy; HIV reverse transcriptase; HIV protease; drug resistance; sensitivity; specificity
21.  Amino Acid Mutation N348I in the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Multiclass Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors▿ †  
Journal of Virology  2008;82(7):3261-3270.
We identified clinical isolates with phenotypic resistance to nevirapine (NVP) in the absence of known nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations. This resistance is caused by N348I, a mutation at the connection subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Virologic analysis showed that N348I conferred multiclass resistance to NNRTIs (NVP and delavirdine) and to nucleoside reverse transcriptase inhibitors (zidovudine [AZT] and didanosine [ddI]). N348I impaired HIV-1 replication in a cell-type-dependent manner. Acquisition of N348I was frequently observed in AZT- and/or ddI-containing therapy (12.5%; n = 48; P < 0.0001) and was accompanied with thymidine analogue-associated mutations, e.g., T215Y (n = 5/6) and the lamivudine resistance mutation M184V (n = 1/6) in a Japanese cohort. Molecular modeling analysis shows that residue 348 is proximal to the NNRTI-binding pocket and to a flexible hinge region at the base of the p66 thumb that may be affected by the N348I mutation. Our results further highlight the role of connection subdomain residues in drug resistance.
doi:10.1128/JVI.01154-07
PMCID: PMC2268505  PMID: 18216099
22.  Genotypic and Phenotypic Resistance Patterns of Human Immunodeficiency Virus Type 1 Variants with Insertions or Deletions in the Reverse Transcriptase (RT): Multicenter Study of Patients Treated with RT Inhibitors 
Genomic rearrangements in the 5′ part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI). We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements. Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories. Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay. Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5′ part of the RT, with an extensive molecular variation. The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each. In four cases, codon 67 was deleted. High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively. Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations. Resistance to 3TC could develop in the absence of specific mutations. Low-level resistance to ddI was noticed in 40% of the patients. The deletions of codon 67 seemed to have little effect on NRTI sensitivity. Most of the rearrangements were shown to contribute to cross-resistance to NRTI. The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.
doi:10.1128/AAC.45.6.1836-1842.2001
PMCID: PMC90554  PMID: 11353634
23.  A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus 
Virology Journal  2011;8:33.
Background
The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.
Methods
Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.
Results
Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.
Conclusions
The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.
doi:10.1186/1743-422X-8-33
PMCID: PMC3038945  PMID: 21255423
24.  The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity 
Journal of Virology  2014;88(3):1536-1547.
Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.
doi:10.1128/JVI.02904-13
PMCID: PMC3911599  PMID: 24227862
25.  Prediction of Abacavir Resistance from Genotypic Data: Impact of Zidovudine and Lamivudine Resistance In Vitro and In Vivo 
Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI). Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V. Abacavir resistance has also been observed in NRTI multidrug-resistant samples. Furthermore, abacavir resistance has been described in the context of zidovudine resistance. To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples. Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively. If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible. Therefore, the relative frequencies of other mutations were evaluated. Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples. Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant. Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only. Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase. Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction. In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant. Thus, options for useful sequential combinations of NRTI are limited.
doi:10.1128/AAC.46.1.89-94.2002
PMCID: PMC126991  PMID: 11751116

Results 1-25 (846492)