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Objective: To assess the prevalence of modest (< 10-fold) decreases in baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) susceptibility and their impact on virological response to NNRTI-containing triple therapy in drug-naive individuals., Methods: Baseline HIV resistance phenotype, genotype and response to therapy were examined retrospectively for all antiretroviral-naive individuals initiating therapy with two nucleoside analogues and an NNRTI in British Columbia, Canada, between 05/1997 and 08/1999 (n = 279), followed until July 31 2001. Time to viral suppression (first of at least two consecutive plasma viral loads < 400 copies HIV RNA copies/ml) and viral rebound (to > or = 400 copies/ml after first pVL < 400 copies HIV RNA copies/ml), were estimated by Kaplan-Meier methods. Multivariate analyses were performed using Cox proportional hazards regression., Results: Nevirapine was the most commonly prescribed NNRTI (96%). Four- to 10-fold decreased susceptibility to NNRTIs was observed in > 30% of untreated individuals at baseline, an observation strongly driven by decreased susceptibility to delavirdine (22.4%). A > 10-fold decrease in susceptibility to any NNRTI was observed only rarely (< 2%). There was no association between four- and 10-fold decreased baseline susceptibility to NNRTIs and virological outcome (P > 0.05). In multivariate analyses, the strongest predictors of poor virological response to NNRTI-based therapy were baseline plasma viral load and the proportion of time on therapy in the first year of follow-up. There was no relationship between the presence of previously reported mutations associated with decreased NNRTI susceptibility (at codons 135 and 283 in HIV reverse transcriptase) and virological response., Conclusions: These data suggest that the clinically significant level of resistance to NNRTIs, particularly nevirapine, in drug-naive individuals is likely greater than four- to 10-fold.

The Y318F substitution in the 3' region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been linked to nonnucleoside RT inhibitor (NNRTI) resistance in vitro. A systematic search of a large phenotypic-genotypic database (Virco) linked the Y318F substitution with a >10-fold decrease in NNRTI susceptibility in >85% of clinically derived isolates. There was a significant association between Y318F and use of delavirdine (P = 10(-11)) and nevirapine (P = 10(-6)) but not efavirenz (P = 0.3). Site-directed HIV-1 Y318F mutants in an HXB2 background displayed 42-fold-decreased susceptibility to delavirdine but <3-fold-decreased susceptibility to nevirapine or efavirenz. Combinations of Y318F with K103N, Y181C, or both resulted in decreased efavirenz susceptibility of 43-, 3.3-, and 84-fold, respectively, as well as >100- and >60-fold decreases in delavirdine and nevirapine susceptibility, respectively. These results indicate the importance of the Y318F substitution in HIV-1 drug resistance.

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

Purpose: To relate baseline plasma HIV genotypic and virtual phenotypic antiretroviral drug susceptibility to subsequent virological response in patients receiving saquinavir (SQV)-enhancing therapy. Individuals were randomized to receive stavudine (d4T), SQV, and one of ritonavir, nelfinavir, or delavirdine to enhance SQV blood levels., Method: The protease and reverse transcriptase baseline sequences of 31 treatment-experienced patients were analyzed by genotype and virtual phenotype and were related to viral load at weeks 12 and 24. Genotypic resistance to SQV was defined by the presence of G48V and/or L90M mutations in the protease gene. Potential cross-resistance to d4T in zidovudine (ZDV)-experienced individuals was defined by the presence of thymidine-associated mutations in the reverse transcriptase gene., Results: ZDV-associated mutations did not affect the virological response at 24 weeks. Individuals who were sensitive to SQV at baseline as determined by either genotyping or virtual phenotyping showed a greater decrease in viral load at week 24 than those resistant to SQV, irrespective of treatment arm. By genotyping, SQV-sensitive individuals had a median log decrease of 1.12 compared to 0.32 for those individuals who were SQV resistant. By virtual phenotyping, SQV-sensitive individuals had a median log decrease of 1.0 compared to a rise of 0.08 in resistant individuals., Conclusion: Thymidine analogue-associated mutations at baseline did not influence the response to subsequent therapy involving d4T. Individuals who were sensitive or resistant to SQV by genotyping or virtual phenotyping responded to SQV-enhancing regimens, but the virological response was greater in those who were sensitive.

This study examines the association between presence of drug resistance mutations and phenotypic resistance at baseline to virologic response to salvage therapy in a community setting. The study population consisted of 58 antiretroviral drug-experienced patients with HIV-1 infection who had recently switched therapy because of virologic failure. Drug resistance mutations in the reverse transcriptase- and protease-coding regions and phenotypic susceptibility to 13 antiretroviral drugs were assessed at baseline. Plasma HIV-1 RNA levels were assessed at baseline and at subsequent clinic visits. Results showed that three variables were significant in predicting virologic response: HIV-1 levels at baseline, number of protease mutations, and phenotypic sensitivity score for the regimen at baseline. For four drugs there was a significant association between the presence of specific drug resistance mutations and >10-fold phenotypic resistance to that drug. With phenotypic resistance defined as >4-fold resistance, the association between specific drug resistance mutations and phenotypic resistance was significant for seven drugs. Overall, these data show that phenotypic susceptibility and absence of drug resistance mutations, particularly protease mutations, are significant predictors of virologic response. For several drugs, specific combinations of drug resistance mutations are associated with decreased phenotypic susceptibility and might provide useful clinical guidelines in selecting therapeutic options.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Objectives: To examine the natural phenotypic variability in drug susceptibility among recombinant HIV-1 isolates from a large number of untreated HIV-positive individuals from wide-ranging geographic locations, and to use this information to establish biologically relevant cut-off values for phenotypic antiretroviral susceptibility testing., Methods: Phenotypic susceptibility to 14 antiretroviral agents was determined for HIV-1 samples from > 1000 treatment-naive individuals in seven clinical trials. Samples were from the USA (n = 351), Germany (n = 306), Canada (n = 265), and South Africa (n = 358). Geometric mean fold-resistance and confidence intervals were determined relative to a standard laboratory wild-type virus., Results: Baseline fold-resistance was approximately log-normally distributed for all antiretroviral agents examined. There was no evidence of large geographical differences in average antiviral susceptibility. Geometric mean fold-resistance for each of 14 antiviral agents was similar (+/- 0.5-fold) for samples derived from the USA, Canada, Germany, or South Africa. The non-nucleoside reverse transcriptase inhibitors (NNRTI) exhibited the broadest distribution of susceptibility; approximately 97.5% of all isolates had < 2.5-4.0, < 3.0-4.5, and < 5-10 fold-decrease in susceptibility to five protease inhibitors, six nucleoside analogues, and three NNRTI, respectively. No consistent geographic pattern or clade effect (B versus C) in either the mean or the distribution of baseline antiretroviral susceptibility was observed., Conclusions: Phenotypic drug susceptibility of HIV-1 in untreated individuals varies markedly from drug to drug, with broadly similar patterns world-wide. These results have important implications in defining the 'normal range' of phenotypic susceptibility to antiretroviral agents and establish biologically relevant cut-off values for this phenotypic drug susceptibility test.

Using a large panel of human immunodeficiency virus type 1 site-directed mutants, we have observed a higher correlation than has previously been demonstrated between zidovudine (AZT)-triphosphate resistance data at the reverse transcriptase (RT) level and corresponding viral AZT resistance. This enhanced-resistance effect at the RT level was seen with ATP and to a lesser extent with PP(i) when ATP was added at physiological concentrations. The ATP-dependent mechanism (analogous to pyrophosphorolysis) appears to be dominant in the mutants bearing the D67N and K70R or 69 insertion mutations, whereas the Q151M mutation seems independent of ATP for decreased binding to AZT-triphosphate.

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.

Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.

To describe prevalence of antiretroviral (ARV) drug-resistant HIV-1 strains among patients with a history of earlier treatment with ARV drugs in Abidjan, Côte d'Ivoire, we determined mutations that confer HIV-1 ARV drug resistance by sequencing the viral reverse-transcriptase and protease genes derived from plasma viral RNA of 68 individuals consecutively enrolled in the Joint United Nations Program on AIDS Drug Access Initiative (UNAIDS-DAI) with a history of earlier ARV drug treatment in Abidjan between August 1998 and April 1999. Phenotypic ARV drug resistance was assessed using a recombinant virus assay. Primary mutations associated with ARV drug resistance to at least one of the reverse-transcriptase inhibitors or protease inhibitors were detected in 39 (57.4%) of the 68 patients. The prevalence of mutations associated with resistance to ARV drugs was: 29 (42.6%) to zidovudine, 10 (14.7%) to lamivudine, one (1.5%) to didanosine, one K103N mutation (associated with resistance to delavirdine, nevirapine, and efavirenz), one Y181C mutation (associated with resistance to delavirdine and nevirapine), two to both indinavir (M46I/L and V82A) and saquinavir (G48V and L90M), and one each to ritonavir (V82A) and nelfinavir (D30N). Phenotypic resistance to at least one nucleoside reverse transcriptase inhibitor (RTI) was seen in 25 (39.7%) patients, to nonnucleoside RTIs in 5 (8%) patients, and to protease inhibitors in 4 (6%) patients. The high prevalence we observed in this study may limit in future the effectiveness of ARV programs in the Côte d'Ivoire.

The presence of the lamivudine-associated M184V RT mutation increases tenofovir susceptibility in multiple HIV genotypes. Tenofovir is uniquely active against multinucleoside-resistant HIV expressing the Q151M mutation, but shows reduced susceptibility to the T69S insertion mutations. HIV with common forms of zidovudine and lamivudine resistance are susceptible to tenofovir, corroborating phase II clinical results demonstrating the activity of tenofovir DF in treatment-experienced patients.

A variety of key mutations in HIV reverse transcriptase (RT) have been associated with nucleoside reverse transcriptase inhibitor (NRTI) exposure, which give rise to a diverse range of effects in terms of altered drug susceptibilities, viral replicative capacity and RT biochemistry. There are three basic mechanisms of resistance conferred by specific mutations in the coding region of RT. The first is drug discrimination, whereby a particular drug or drugs are either selectively excluded from uptake or from the RT-primer-template catalytic complex. Drug discrimination is, for the most part, relatively specific for individual drugs. Repositioning of the template-primer to prevent a catalytically competent complex in the presence of a bound drug molecule has also been observed in some instances, and forms a second mechanism. The third, and potentially most significant for long-term efficacy of the NRTIs, is pyrophosphorolysis, the primary mode of resistance to zidovudine. Mutations selected by this drug or stavudine serve to elevate the natural rate of the reverse reaction for RT. Pyrophosphorolysis uncouples the last nucleoside monophosphate added to the proviral transcript, and attaches it to either a free pyrophosphate (regenerating a deoxynucleoside triphosphate) or to a nucleoside di- or triphosphate (usually ATP). Uncoupling a chain-terminating NRTI residue therefore rescues reverse transcription and reduces drug susceptibility across the class, since the process is not specific for the selecting drug. Of all the nucleoside-associated mutations, the best known and most studied are the six associated with thymidine analogue exposure. These six mutations (M41L, D67N, K70R, L210W, T215Y/F, K219Q) enhance RT pyrophosphorolysis to confer high-level viral resistance to zidovudine, and clinically significant loss of response to stavudine and didanosine. They have also been found to confer reduced susceptibility to lamivudine and abacavir, particularly when present alongside other NRTI-induced changes. Other key mutations generally confer more limited resistance to specific agents, although the primary lamivudine- and abacavir-associated M184V substitution generates a broad spectrum of drug-dependent phenotypes, and uncommon mutational complexes conferring resistance across the entire class are well known. In addition to 'classical' multi-nucleoside-resistant genotypes, database-driven 'virtual phenotyping' for accumulations of NRTI-associated mutations around a core of thymidine analogue-induced changes predicts drug susceptibilities below wild-type across the entire NRTI class, even in the absence of key mutations associated with individual agents. When the natural range of drug susceptibilities for treatment-naive isolates is used as the basis for defining resistance, retrospective analysis of clinical isolates in the Virco database shows a significantly increased incidence of reduced susceptibility for the dideoxy NRTIs (didanosine, stavudine and zalcitabine) that was undetected in previous assays. These data imply a cumulative degradation of response to

Objective: To analyse the immunological and virological effects of treatment interruptions in HIV-1-infected patients with treatment failure and multidrug-resistant virus., Methods: Drug susceptibility was assessed using Antivirogram and genotypic analysis was based on population and clonal sequencing for 48 patients who had interrupted treatment (> or = 2 months)., Results: Treatment interruption resulted in viral load increases (mean 0.7 log 10 copies/ ml; P = 0.0001) and CD4 cell count decreases (mean 89 x 10(6) cells/l; P = 0.0001). A complete shift to wild-type virus at the phenotypic, genotypic and clonal level was observed in 28/45 patients. These patients differed from those that did not show a shift to wild type in baseline CD4 cell counts (192 versus 59 x 10(6) cells/l; P= 0.007) and in the relationship between baseline viral load and CD4 cell count (no correlation versus a significant negative correlation; P= 0.008). Response to re-initiation of treatment fell with increasing viral load [relative hazard (RH) 0.33; P= 0.001] and with increasing total number of drugs with reduced susceptibility (RH 0.51; P = 0.0003); it improved with the number of new drugs received (RH 2.12; P = 0.0002) and a shift to wild type (RH 5.22, P = 0.006)., Conclusions: Changes in surrogate markers suggest that treatment provided benefit in spite of virological failure and resistant virus. Although patients with a shift to wildtype virus responded better in the short term to treatment re-initiation, the long-term effects are not known and the risk of immune deterioration needs to be carefully considered.

Development of drug resistance is the inevitable consequence of incomplete suppression of virus plasma levels in HIV-1-infected patients treated with highly active antiretroviral therapy. Resistance mutations previously characterized have been found in B subtype viruses of developed countries. Moreover, mutation profiles for non-B and more divergent B subtype viruses found in developing countries shall be analyzed together with their ex vivo phenotyping in order to establish an exact correlation between the genotyping data and the clinical management counseling for those uncommon virus subtypes. In the present study, we evaluated the mutation profile for individuals infected with B subtype and non-B subtype viruses. Viral DNA fragments corresponding to the RT gene were amplified, sequenced, and subtyped. Phenotyping analysis for reverse transcriptase nucleoside (NRTI) and nonnucleoside inhibitor susceptibility was performed using the recombinant virus assay technology. Brazilian non-B subtypes (subtype F, n = 4, and subtype A, n = 1) isolates showed essentially the same B subtype mutation profile, presenting an NRTI drug resistance with similar MIC50% and MIC90% values for all drugs analyzed regardless of their subtypes. A strong cross-resistance phenotype among AZT, 3TC, and abacavir could be seen in all isolates analyzed. A novel result was that some RT sequences not only revealed the presence of G333D/E mutations but also correlated to the presence of mutation T386I that could abrogate the M184V-surpassing effect of L210W or L210W plus G333D/E. These findings suggest that Brazilian non-B subtype HIV-1 strains use an identical RT drug resistance mutation pattern when compared to B isolates and will contribute to the validation of the genotypic and phenotypic tests in these predominant worldwide-spread viral variants., (Copyright 2000 Academic Press.)

Objective: While transmission of drug-resistant HIV-1 has been reported, estimates of prevalence of resistance in drug-naïve populations are incomplete. We investigated the prevalence of genotypic mutations and phenotypic antiretroviral resistance in a cohort of HIV-1 infected U.S. military personnel prior to the institution of antiretroviral therapy., Design: Cross-sectional cohort study., Methods: Plasma was obtained from 114 recently HIV-1 infected subjects enrolled in an epidemiological study. Genotypic resistance was determined by consensus sequencing of a PCR product from the HIV-1 pol gene. Sequences were interpreted by a phenotypic-genotypic correlative database. Resistance phenotypes were determined by a recombinant virus cell culture assay., Results: Genotypic mutations and phenotypic resistance were found at a higher than expected frequency. Resistance to non-nucleoside reverse transcriptase inhibitors was most common, with a prevalence of 15% of 95 subjects by genotype and 26% of 91 subjects by phenotype. Genotypic and phenotypic resistance respectively were found in 4% and 8% of subjects for nucleoside reverse transcriptase inhibitors and in 10% and 1% for protease inhibitors. One subject harbored virus with resistance to all three drug classes., Conclusions: A substantial frequency of resistance to antiretroviral drugs was identified in a therapy-naïve U.S. cohort. In most cases, the genotypic and phenotypic assays yielded similar results, although the genotypic assay could detect some protease inhibitor resistance-associated mutations in the absence of phenotypic resistance. These data suggest the need for optimization of treatment guidelines based on current estimates of the prevalence of drug resistance in HIV-1 seroconverters.

Although efavirenz-containing regimens effectively suppress plasma levels of human immunodeficiency virus (HIV) RNA, it is now clear that undetectable plasma viremia may not reflect a lack of viral replication. Because lymphoid tissue is an active site of HIV replication, the lymph node virus burden was analyzed in persons who received highly active antiretroviral therapy (HAART) containing either efavirenz or a protease inhibitor (PI). Testing with in situ hybridization revealed no detectable follicular dendritic cell-associated HIV RNA in either group, and only 2 of 8 persons in the efavirenz group and 1 of 4 in the PI group had detectable RNA in lymph node mononuclear cells (LNMC) when tested by use of nucleic acid sequencebased amplification. Low levels of replication-competent HIV were identified in both groups by use of quantitative coculture assays. There was no evidence of development of resistance to either regimen in virus isolated from LNMC. These data support the use of efavirenz as an alternative to a PI in initial HAART regimens.

Abacavir (1592U89) is a nucleoside inhibitor of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT). Resistance to abacavir was studied with abacavir alone and with abacavir in combination with other nucleoside analogues in cell culture, in virus isolates from zidovudine/lamivudine clinical trials, and in the first dose-escalating 12-week clinical trial (CNA2001) to evaluate abacavir clinical potency. Abacavir alone in vitro selected for mutations at HIV RT codons K65R, L74V, Y115F, and M184V. However, abacavir combined with zidovudine selected against virus with the M184V mutation. Abacavir therapy in vivo resulted in large decreases in HIV load (>1 log), even in 1 subject who had the M184V mutation at baseline. A total of 51% of subjects showed new mutations at any of codons K65R, L74V, and M184V after abacavir monotherapy, compared with 11% who received zidovudine/abacavir. Small changes (2- to 4-fold) in abacavir susceptibility were detected. On stopping therapy, reselection of the pretherapy sequence occurred within 4 weeks.

Objective: To assess the relationship between viral susceptibility at baseline and virological response in human immunodeficiency virus (HIV)-infected patients treated with multi-drug salvage regimens after multiple previous treatment failures., Design: Retrospective analysis of 50 patients from the Frankfurt HIV cohort who had received treatment with a minimum of six drugs, and for whom a sample for baseline viral phenotyping was available., Methods: Viral drug susceptibility was measured retrospectively from stored samples using the Antivirogram, a recombinant virus assay based method. Virological response was defined as a viral load of < 400 copies/ml at week 24. For analysis of treatment response, drop-outs were dealt with in two ways, either as failures (DAF) or censored (DAC). Several logistical regression models were applied to identify predictors of response, including baseline virus load, number of new drugs and phenotypic sensitivity scores., Results: At baseline, drug resistance was extensive: 96% of patients had viruses resistant to at least one drug class and 32% had viruses resistant to all three drug classes. In the DAF analysis, 39 patients experienced virological failure. In the DAC analysis, eight were censored and 31 patients experienced virological failure. In multivariate models that adjust for baseline viral load, the number of new drugs and total phenotypic sensitivity scores, the baseline viral load and phenotypic sensitivity score remained significantly associated with virological outcome, whereas in those adjusted for baseline viral load, the number of new drugs, NRTI phenotypic sensitivity score and PI phenotypic sensitivity score, only the latter remained significantly associated with virological outcome. Both the DAF and DAC analyses produced similar results. In all models used, virological failure was shown to be significantly associated with baseline viral load and phenotypic sensitivity score., Conclusions: In this retrospective analysis based on a small number of patients, viral drug susceptibility at baseline was strongly associated with virological outcome at 24 weeks, independent of covariates such as baseline viral load and treatment history. Baseline viral load also maintained a significant, independent association with virological outcome in most models.

We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.

Objective: To determine the rate of nevirapine resistance in patients failing a nevirapine plus protease inhibitor (PI)-based regimen, and whether these isolates remain susceptible to other non-nucleoside reverse transcriptase inhibitors (NNRTI)., Design and Setting: A retrospective cohort study in two tertiary university hospitals., Patients: Eighty-eight HIV-infected, NNRTI-naive patients receiving nevirapine plus PI as a rescue regimen after PI treatment failure., Main Outcome Measures: Genotypic and phenotypic resistance data at inclusion (73 and 60 plasma samples, respectively) and after 24 weeks (53 and 42 samples)., Results: Baseline phenotypic susceptibility to nevirapine was found in 70% of patients, and similar data were observed for efavirenz (91%) and delavirdine (71%). NNRTI resistance-associated mutations were found in 11 patients (12.5%). At 24 weeks, resistant isolates to nevirapine were found in 92% of patients, and correlated with similar resistance to efavirenz (68%) and delavirdine (73%). In the genotypic analysis, the Y181 C mutation was observed in 76% of mutants, and the most common changes were a combination of mutations at positions Y181C/K103N (23%) and the single mutation Y181C (15%). The development of nevirapine resistance was associated with baseline resistance to PI included in the regimen (P= 0.01). For isolates containing the single amino acid substitution Y181C, 29% remained fully susceptible to efavirenz, whereas 14% showed intermediate resistance to efavirenz and delavirdine., Conclusion: The failure of a nevirapine plus PI-containing regimen is associated with nevirapine resistance in most patients, with the most common mutation occurring at amino acid residue 181. Although there is a high degree of cross-resistance among NNRTI, nearly one third of resistant isolates carrying the single Y181C mutation remain susceptible to efavirenz.

Human immunodeficiency virus type 1 (HIV-1) strains dually resistant to zidovudine and lamivudine (3TC) may arise during zidovudine-3TC combination therapy. The objective of this cross-sectional study (n = 43 patients) was to test the association between therapy response (clinical and immunologic) to zidovudine-3TC and the level of phenotypic zidovudine resistance and zidovudine resistance-associated genotype of 3TC-resistant isolates. Other variables included were baseline CD4+ cell count, baseline Centers for Disease Control and Prevention (CDC) classification, virus load, and time receiving zidovudine. Phenotypic resistance was assessed using a recombinant virus assay. Genotypic analysis was based on population sequencing of plasma HIV-1. In a univariate analysis using a logistic regression model, it was found that therapy response was significantly associated with phenotypic and genotypic zidovudine resistance, baseline CD4+ cell count, and virus load. After adjustment for all variables, phenotypic resistance to zidovudine remained the only significantly associated factor, independent of baseline CD4+ cell count, baseline CDC classification, and virus load.

Objective: To investigate the prevalence and magnitude of M184V-mediated changes in susceptibility to zalcitabine, didanosine, stavudine and abacavir (1592U89 succinate) in a cohort of lamivudine-treated patients., Design and Methods: A total of 255 samples from patients treated with lamivudine and zidovudine with or without other nucleoside reverse transcriptase inhibitors (NRTI) were analysed for susceptibility to zidovudine, lamivudine, zalcitabine, didanosine and stavudine using a recombinant virus assay. Seventy-three samples originated from patients exposed to zidovudine and lamivudine only. A subset of 27 samples was investigated for cross-resistance to abacavir. Resistance was defined as a change in median inhibitory concentration more than fivefold compared with wild-type (high-level resistance, > 10-fold). A genotypic analysis of plasma-derived reverse transcriptase coding regions was carried out in samples with cross-resistance., Results: The majority of samples displayed wild-type or greater than wild-type sensitivity to zalcitabine, didanosine and stavudine: resistance was seen in 17.2, 9 and 6.3% of the total sample population, respectively. Of these, 1.2, 2.7 and 2.4%, respectively, showed high-level resistance. The prevalence of resistance to a particular NRTI was lower in samples from patients not pretreated with that NRTI and in samples from patients exposed to zidovudine-lamivudine only. Cross-resistance was more prevalent in samples with high ZDV resistance. There was no obvious correlation between cross-resistance and genotype; all but two samples were mutant at codon 184. There were no consistent changes at positions associated with zidovudine resistance. The majority of samples from a subset (n=27) were four- to eightfold less sensitive to abacavir. There were no other genotypic changes in addition to M184V known to be associated with abacavir resistance., Conclusions: Cross-resistance was not commonly observed in this lamivudine-treated cohort. M184V per se is not expected to compromise subsequent treatment with NRTI such as didanosine-stavudine or combinations containing abacavir.

Replication of mixtures of two or more human immunodeficiency virus type 1 (HIV-1) variants would be expected to result in the eventual selection of the fittest virus due to Darwinian competition among the variants. The relative proportions of known HIV-1 variants (which may differ only by a single nucleotide from a standard "wild-type" virus, HIV-1HXB2) in mixed viral cultures were quantified by analysis of automated sequence signals of reverse transcriptase PCR products. With this method, the relative levels of replicative fitness of several zidovudine (3'-azidothymidine)-resistant HIV-1HXB2 variants were estimated under controlled in vitro conditions by measuring the rate of change in the proportions of viral variants as they replicated in cell cultures both in the presence and in the absence of drug selection pressure. These variants were engineered to contain commonly observed zidovudine resistance mutations in the HIV-1 reverse transcriptase (M41L, K70R, T215Y, and M41L+T215Y). In the absence of zidovudine, all variants tested displayed reduced replicative fitness compared to wild-type HIV-1HXB2. The order of relative fitness was wild type > K70R >> T215Y = M41L+T215Y > M41L. Mixed cultures in the presence of zidovudine showed a dose-dependent selection pressure against the wild-type virus which varied according to the resistance profile of each virus. The information gathered from this approach provides insight into competition among multiple HIV-1 variants, which likely occurs in vivo with drug selection pressure, and may be applicable in more complex mathematical models for predicting the emergence of HIV-1 variants after the initiation of antiretroviral therapy.

Combinations of antiretroviral drugs that prevent or delay the appearance of drug-resistant human immunodeficiency virus-type 1 (HIV-1) mutants are urgently required. Mutants resistant to 3'-azidothymidine (AZT, zidovudine) became phenotypically sensitive in vitro by mutation of residue 184 of viral reverse transcriptase to valine, which also induced resistance to (-)2'-deoxy-3'-thiacytidine (3TC). Furthermore, AZT-3TC coresistance was not observed during extensive in vitro selection with both drugs. In vivo AZT-3TC combination therapy resulted in a markedly greater decreased in serum HIV-1 RNA concentrations than treatment with AZT alone, even though valine-184 mutants rapidly emerged. Most samples assessed from the combination group remained AZT sensitive at 24 weeks of therapy, consistent with in vitro mutation studies.

The reverse transcriptase enzyme of human immunodeficiency virus type 1 (HIV-1) is the target for many inhibitors. Amino-acid substitutions in functional regions of the enzyme that abolish reverse transcriptase activity also prevent HIV-1 replication. But selection pressure by drugs such as AZT (3'-azido-3'deoxythymidine, zidovudine), ddI (2',3'-dideoxyinosine) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) causes outgrowth of resistant variants due to non-lethal mutations in the enzyme. Reports of synergy and lack of cross-resistance between reverse transcriptase inhibitors (refs 7, 9, 10, 12-14, 17, 18, 20, 21), plus the reversal of AZT resistance by mutations induced by ddI and NNRTIs, have indicated that specific drug combinations directed at reverse transcriptase might curtail resistance. Chow et al. extended this concept in a report that specific multiple combinations of resistance mutations in the reverse transcriptase can significantly impair HIV-1 replication. They concluded that evolutionary limitations may exist to prevent the emergence of multidrug resistance to inhibitors of reverse transcriptase. We report here that HIV-1 co-resistant to AZT, ddI and the NNRTI nevirapine can be readily selected in cell culture starting with dual AZT- and ddI-resistant virus. We found no evidence for 'replication incompatible' combinations of resistance mutations, although a mutation (M184-->V) conferring oxathiolane-cytosine nucleoside resistance in reverse transcriptase completely suppressed AZT resistance in a triple-resistant background. These in vitro observations suggest that triple drug combination therapy might ultimately result in co-resistant HIV-1, although they do not preclude assessment of such combinations for treatment of HIV-1 disease.

Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations.

It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations (Asn-67, Arg-70, and Tyr-215). Construction of a molecular clone with this genotype gave rise to only a partially resistant virus, raising the possibility that an additional mutation existed in some clinical isolates. Using an HIV marker rescue system, we have mapped and identified a fifth mutation conferring resistance to zidovudine, namely, methionine to leucine at codon 41 of HIV RT. An infectious molecular clone containing this mutation together with three previously identified mutations in the RT coding sequence yielded highly resistant HIV after transfection of T cells. Direct detection of the fifth mutation in DNA samples from cocultured peripheral blood lymphocytes by the PCR revealed that it occurred relatively early in the development of zidovudine resistance. However, this mutation was only detected after the appearance of the codon 215 change in the RT coding sequence. Identification of this mutation in addition to the other known mutations conferring resistance enables rapid and direct correlation between an RT genotype and sensitivity of the virus.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.