ABSTRACT Zidovudine resistance (ZDV-R) is associated with classic genotypic changes at codons 41, 67, 70, 210, 215, and 219 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene as well as with the multinucleoside resistance (MNR) complexes (Q151M MNR complex; 6-bp insertion/A62V complex). In addition, enhanced resistance to ZDV in the context of the classic ZDV mutations plus the M184V mutation has been associated with additional mutations at positions 208, 211, 214, and 333. In this study we investigated phenotypic ZDV-R determined by a recombinant virus assay (Antivirogram; Virco) in 223 clinical samples in relation to the above genotypic changes. 150 out of 223 clinical samples had the M184V mutation. Phenotypic ZDV-R ranged from 0.3- to 5,338-fold. Sixteen samples (15 with high ZDV-R ranging from 90- to 3,571-fold) contained MNR-associated patterns. Analysis of classic mutational patterns broadly demonstrated increasing ZDV-R with increasing number of ZDV mutations. A comparable correlation was obtained when ZDV-R was analyzed only relative to the T215Y/F mutation. Site-directed mutagenesis experiments investigating the influence of the additional mutations H208Y, R211K, and L214F on ZDV-R resulted in a 7.4- or 21-fold increase in ZDV-R when the R211K/L214F or H208Y/R211K/L214F mutations, respectively, were added to a highly ZDV-R virus. In the clinical sample data set we analyzed, the combination of R211K/L214F appeared most frequently. The H208Y change was detected only in highly ZDV-R viruses, whereas the G333E/D change was distributed equally. All changes were independent of the M184V mutation. A 2.4- or 8-fold increase in ZDV-R was observed in the clinical samples with high ZDV-R containing the R211K/L214F or H208Y/R211K/L214F mutations, respectively. We have shown that the combination of the additional mutations H208Y, R211K, and L214F in HIV-1 RT may influence ZDV-R and should be considered when assessing ZDV-R.

Two large, independent human immunodeficiency virus type 1 resistance databases containing >7700 reverse-transcriptase (RT) sequences were used to analyze the epidemiology of amino acid substitutions at codons 44 and 118, which confer moderate lamivudine resistance in the presence of zidovudine resistance. As expected, E44A/D and V118I mutations were strongly associated with M41L, D67N, L210W, and T215Y but also with other mutations, including K43E/N/Q, T69D, V75M, H208Y, R211K, and K219R. Both E44D and V118I were more frequently associated with stavudine and didanosine than with zidovudine and lamivudine treatment. However, selection of E44A/D and V118I was also detected in association with a switch to other nucleoside RT inhibitors, including zalcitabine and abacavir. Site-directed mutagenesis confirmed that 44D and 118I can decrease phenotypic susceptibility not only to lamivudine but also to most other nucleoside analogues, particularly stavudine and abacavir. Thus, substitutions at RT codons 44 and 118 have broad implications in nucleoside RT inhibitor resistance in the setting of several nucleoside-associated mutations.

The development of decreased viral susceptibility to one or more of the antiretroviral agents used in the combination treatment of HIV-1-infected patients frequently causes failure to achieve or maintain complete suppression of the virus. A decrease in antiretroviral drug susceptibility may develop during the course of therapy through the accumulation of resistance-associated mutations. Alternatively, an individual may become infected with a virus that already harbours mutations conferring decreased susceptibility to nucleoside reverse transcriptase inhibitors (NRTI), non- nucleoside reverse transcriptase inhibitors (NNRTI), or protease inhibitors (PI). Therapeutic options for such newly infected patients may be limited as a consequence. Although transmission of virus with decreased susceptibility to each of the three classes of drugs has been demonstrated, only limited and mainly retrospective data on the frequency of drug resistance in newly infected patients is available [1–12]. Two prospective studies analysed the results of 81 and 57 patients, respectively [13,14]. We report here the detailed results of a prospective study performed on 230 antiretroviral-naive HIV-1-infected patients from the USA. Both the genetic sequence and the phenotypic drug susceptibility profiles of the patient HIV-1 isolates were studied. The eligibility criteria for a subject to be enrolled in the study were as follows: laboratory evidence of acute primary HIV-1 infection (detectable HIV-1 RNA in plasma using sensitive polymerase chain reaction or branched DNA assays together with negative or indeterminate HIV antibody test) or seropositivity for HIV-1 infection (enzyme-linked immunosorbent assay and Western blot positive) first documented within the past three years (i.e. not known to be seropositive for more than 3 years) and no previous antiretroviral therapy of any kind, (including NRTI, NNRTI, PI, and therapeutic HIV vaccines). Patients were enrolled in the study solely with the aim of determining the prevalence of resistance in therapy-naive patients. All eligible patients who gave their informed consent to participate in the study were enrolled until the targeted number of subjects was reached. Plasma samples were obtained from 230 antiretroviral-naive HIV-1-infected individuals between August 1998 and January 1999. The samples originated from nine clinical centres across six states in the USA. The samples were shipped to the laboratory on dry ice and stored at −70°C until analysis. Phenotypic drug susceptibility testing was performed using a recombinant virus assay (Antivirogram®) [15–17]. The results of this analysis are expressed as the fold-increase in mean IC50 (μM) of a particular drug when tested with patient-derived recombinant virus isolates, relative to the mean IC50 (μM) of the same drug obtained when tested with a reference wild-type virus (IIIB/LAI). Drug susceptibility values generated by the phenotypic assay were classified into one of three drug susceptibility categories: sensitive (S), intermediately resistant (I) and resistant (R), i.e. with a substantial decrease in susceptibility, representing fold-changes in the IC50 values of 4 or less, between 4 and 10, and over 10, respectively, compared with the IC50 value for the reference wild-type virus. Genotypic analysis was performed by automated, population-based, full-sequence analysis (Applied Biosystems Inc., ABI, Foster City, CA, USA). Sequencing results are reported as amino acid changes at positions in protease and reverse transcriptase (RT) compared with the wild-type (HXB2) reference sequence. The sequencing methodology allows the detection of the simultaneous presence of different nucleotides at any position of the region sequenced. The level of sensitivity of detecting such mixtures is of the order of 10–20% for the minority population. Whenever such a mixture of sequences was observed in a sample, the clinical isolate was classified as being mutant. Phenotypic drug susceptibility testing and genotypic analyses were performed on 192 and 199 patient samples, respectively. The ages of the patients enrolled varied between 21 and 64 years (median age 34). Ninety per cent were men. The racial distribution was 64% Caucasian, 23% black, 9% Hispanic, and 4% other. The mode of HIV-1 transmission was homosexual, heterosexual, bisexual, transfusion, drug use or other in the following proportions: 74, 15, 4, 3, 3 and 1%, respectively. Eleven per cent of the patient cohort had a viral load of less than 1000 copies HIV-1 RNA/ml, 21% had a viral load of between 1000 and 10 000 copies/ml and 68% had a viral load greater than 10 000 copies/ml. The distribution of patients across the participating states was: California 31%, Florida 25%, Georgia 22%, Massachusetts 12%, Minnesota 7%, and Pennsylvania 3%. Table 1 shows the frequencies of drug resistance-associated mutations [18–22] found in the RT and protease genes of HIV-1 isolated from this antiretroviral drug-naive patient cohort. The list includes primary as well as so-called 'secondary' mutations [18–22]. The percentage of subjects with virus exhibiting decreased phenotypic susceptibility and the percentage of subjects having virus with mutations associated with decreased susceptibility are represented for each drug in Fig. 1. Overall, 35 and 6% of 192 patients tested had virus with a moderate or substantial decrease in phenotypic susceptibility to one or more antiretroviral drugs, respectively. Patients showed decreased phenotypic susceptibility to one, two or to all three classes of antiretroviral agents in 23.4, 10.4 and 1% of cases, respectively, for intermediately resistant virus, and in 4.7, 1 and 0% of cases for resistant virus. Genotypically, 14 and 16% of 199 patients tested had virus with one or more mutations associated with a decrease in susceptibility to NRTI and NNRTI, respectively. For PI, the figure was 54% if mutations at position 77 are included, and 33% if not. Primary mutations in protease were present in six patients (3%) and secondary mutations in 53% (including mutations at position 77 of protease) and 32% of patients (excluding mutations at position 77 of protease). Of the six patients harbouring primary mutations in protease, four also harboured secondary mutations. The primary mutations in protease were found predominantly in the recently infected group, but a greater sample size would be needed to confirm these preliminary observations. All cases of 41L, 215Y, 184V and 103N mutations of RT were found in the recently infected group. The mode of transmission of the virus, sex of the patient, race or city of origin had no effect on the prevalence of resistance in this patient group. It should be emphasized that the primary objective of this virological study was to characterize the drug susceptibility profile of HIV-1 in such patients. The clinical implications of these findings will need to be addressed in further studies.Fig. 1.: Prevalence of (a) phenotypic drug susceptibility and of (b) mutations associated or possibly associated with drug resistance in therapy-naive patients. Abac, Abacavir; Adef, adefovir; ddC, zalcitabine; ddI, didanosine; d4T, stavudine; DLV, delavirdine; EFV, efavirenz; IDV, indinavir; NLV, nelfinavir; NVP, nevirapine; RTV, ritonavir; SQV, saquinavir; 3TC, lamivudine; ZDV, zidovudine.Table 1: Frequency of mutations found in reverse transcriptase and protease at positions in which changes are known to be or may be associated with drug resistance. Mutation K103N in RT, which can develop rapidly during NNRTI monotherapy [23], was detected in three individuals. A total of 2.6 and 3.6% of the study subjects had virus with a greater than 10-fold reduction in phenotypic susceptibility to nevirapine and delavirdine, respectively, and 15.6 and 17.7% had virus with an intermediate decrease in phenotypic susceptibility (Fig. 1). The significance of the size of these 'intermediate' groups should be considered in the light of recent data suggesting that the clinically significant level of resistance to NNRTI may be greater than 10-fold [24]. Patients who have virus with moderate decreases in phenotypic susceptibility to NNRTI may still respond to therapy regimens that include this class of drugs [25]. In the case of the PI mutations, 'primary' mutations at positions 46, 50, 82, 84 and 90 were detected, but not those at positions 30 and 48. These 'primary' mutations occur at low frequencies, whereas 'secondary' mutations such as those at positions 10, 36, 71 and 77 were each found to occur at greater than 10% frequency in this patient group. Similar distributions in the frequencies of these classes of mutations in protease have been observed in previous studies evaluating HIV-1 mutational patterns in therapy-naive patients [7,9,14]. Some studies reported not finding any 'primary' mutations and only a number of 'secondary' mutations in therapy-naive patients [4,13]. Secondary mutations have often been considered as natural polymorphisms, alone contributing little or nothing to drug resistance [4,26]. The high frequency of 'secondary' mutations in protease observed in the viral isolates in our study does not translate into a high incidence of decreased phenotypic susceptibility in those isolates. However, some of these secondary mutations are possibly involved in the development of cross-resistance among almost all PI. It seems that even though a number of primary mutations are necessary for the development of resistance to a single PI, co-resistance to several PI may have a different genetic basis [27]. The administration of PI to patients whose virus already harbors such 'secondary' mutations and the subsequent acquisition of 'primary' mutations may lead to the development of broad PI co-resistant virus. The transmission of such 'secondary' mutations may, therefore, compromise future therapy success in that initial therapy with PI may be successful despite the presence of secondary mutations [13], but once 'primary' mutations appear at first therapy failure in this mutational background, broad PI cross-resistance may rapidly develop.#OIn conclusion, we present a substantial HIV-1 susceptibility data set from therapy-naive individuals. This study confirms the substantial presence of pre-existing drug resistance, presumably caused by transmission. Continued surveys of this nature will be important to gain insight into the significance of the transmission of HIV-1 drug-resistant strains. Werner Verbiesta Stephen Brownb Calvin Cohenc Marcus Conantd Keith Henrye Susan Huntf Michael Sensiong Alan Steinh Richard Strykeri Melanie Thompsonj Patricia Schela Remi Van Den Broeckk Stuart Bloorl Timothy Alcornm Margriet Van Houttea Brendan Larderl Kurt Hertogsa

ABSTRACT We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

A method of determining a virtual phenotype of a human immunodeficiency virus comprising: a) obtaining a genetic sequence of said virus. b) identifying at least one mutation pattern in said genetic sequence wherein said genetic sequence comprises at least one mutation, and wherein said at least one mutation or mutation pattern is associated with resistance to at least one therapy, c) searching a base genotype data for entries genotypes with a pattern similar to at least one of the mutation patterns identified in the genetic sequence in b) d) correlating each input genotype a similar mutation pattern with a phenotype mutation in a base phenotype data, and e) calculating the number of times of the virtual resistance of said virus from all phenotypes of the database identified.

Objective: To characterize HIV-1 phenotypic resistance patterns and genotypic mutations among patients taking antiretroviral medications in Uganda.Methods: We reviewed charts and retrieved archived plasma specimens from patients at an AIDS specialty center in Uganda where antiretroviral therapy has been used since 1996. Phenotypic and genotypic resistance testing was done on specimens associated with a viral load of 1000 copies/ml.Results: Resistance testing of specimens was completed for 16 patients. Among 11 specimens collected before initiation of antiretroviral therapy, no phenotypic resistance or primary genotypic mutations were found. Among 8 patients taking lamivudine, phenotypic resistance was found for 9 (90%) of 10 specimens and was associated with an M184V mutation in all nine cases. Among 12 patients taking zidovudine, no phenotypic resistance and few primary mutations were found. For 6 patients who were receiving protease inhibitors, we observed no phenotypic resistance and only one primary genotypic mutation associated with resistance.Conclusions: The absence of apparent resistance among samples collected before antiretroviral therapy supports the notion that a similar approach to selection of antiretroviral therapy can generally be used against non-B subtypes. A genotypic marker of antiretroviral resistance to lamivudine in HIV-1 subtypes A, C, and D was similar to those in subtype B infections. These results suggest that the methods used for monitoring for the emergence of drug resistance in antiretroviral programs in Africa may be similar to those used in developed settings.

The gastrointestinal mucosa is a major lymphoid tissue reservoir for human immunodeficiency virus (HIV) replication. Genotypic and phenotypic resistance patterns of HIV type 1 (HIV-1) RNA isolated from colonic mucosa were compared with those from the plasma and peripheral blood mononuclear cells (PBMC) of 7 patients. Genotyping was performed using full-sequence analysis, and phenotyping was performed using a recombinant virus assay. Mutations in the reverse-transcriptase (kappa=.84) and protease (kappa=.73) genes were highly concordant among compartments. Similarly, phenotypic resistance patterns were highly concordant among compartments (intraclass correlation coefficient,.91). In 5 instances among 3 patients, a different genotypic result was observed between plasma and the other tissue compartments. Mixtures of wild-type and mutated HIV-1 RNA were present in the mucosa and PBMC but not in the plasma. Despite significant concordance among compartments, mucosal- and PBMC-derived viral RNA showed instances of discordance with plasma-derived virus that may suggest compartmentalization of virus.

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.

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 (greater than or equal to 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/I; 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. (C) 2000 Lippincott WiIliams & Wilkins.

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.

ObjectiveTo evaluate in HIV-1 the extent of phenotypic and genotypic antiretroviral drug resistance and cross-resistance towards the protease inhibitors (PIs) saquinavir, ritonavir, indinavir and nelfinavir among a set of patient samples originating from European and US routine clinical practice and submitted for phenotypic drug resistance testing and/or genotypic analysis. The mutational pattern(s) underlying both resistance and cross-resistance to PIs was investigated. MethodOver 6000 patient isolates with plasma viral load greater than 1000 copies/ml plasma were analysed. Phenotypic resistance was evaluated by a recombinant virus assay. Phenotypic resistance is expressed as the fold-increase of the 50% inhibitory concentration (IC50) value of a compound for a patient-derived recombinant virus isolate compared with that for a wild-type laboratory virus. Genotypic analysis is reported as amino acid changes at positions in the HIV-1 protease compared to a wild-type reference. ResultsPhenotypic resistance to any single PI was observed in 17 to 25% of the clinical isolates investigated. Phenotypic cross-resistance among PIs (> 10-fold increase in IC50 value) was detected in 59 to 80% of the samples resistant (> 10-fold increase in IC50 value) to at least one PI. The prevalent mutations in PI-resistant isolates involved substitutions at codons 10, 36, 46, 54, 71, 77, 82 and 90. The most frequent mutational pattern in samples with PI cross-resistance involved combined substitutions at positions 10 and 90, extended with substitutions at positions 54, 71, 77, 82 or 84. ConclusionsExtensive use of first-generation PIs leads to the emergence of HIV-1 isolates possessing cross-resistance to all members of this class. Identification of particular mutational profiles among these isolates may assist in the design of new generation inhibitors with specific activity against protease-mutant HIV strains.

ABSTRACT 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.

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.

ObjectiveAlthough the use of HIV-1 protease inhibitors (PI) has substantially benefited HIV-1-infected individuals, new PI are urgently needed, as broad PI resistance and therapy failure is common. MethodsThe antiviral activity of tipranavir (TPV), a non-peptidic PI, was assessed in in vitro culture for 134 clinical isolates with a wide range of resistance to currently available peptidomimetic PI. The susceptibility of all 134 variants was then re-tested with the four PI simultaneously with TPV, using the AntivirogramTM assay. ResultsOf 105 viruses with more than tenfold resistance to three or four PI and an average of 6.1 PI mutations per sample, 95 (90%) were susceptible to TPV; eight (8%) had four- to tenfold resistance to TPV and only two (2%) had more than tenfold resistance. ConclusionsThe substantial lack of PI cross-resistance to TPV shown by highly PI-resistant clinical isolates makes TPV an attractive new-generation HIV inhibitor.

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.