Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

High correlation between the Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1, v2.0 and the Abbott m2000 RealTime HIV-1 assays for quantification of viral load in HIV-1 B and non-B subtypes.

BACKGROUND: HIV-1 viral load assays are critical tools to monitor antiretroviral therapy efficacy in HIV-infected patients. Two assays based on real-time PCR are available, the Abbott Real-Time HIV-1 assay (Abbott assay) and the new Roche COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 test, v. 2.0 (TaqMan(®) test v2.0). OBJECTIVES: We have compared the performance of the two assays in 546 clinical plasma specimens of group M strains from Luxembourg and Rwanda. STUDY DESIGN: Our analyses focused on subtype inclusivity and platforms accuracy for 328 low level viremia samples. RESULTS: Strong agreement and linear correlation were observed between the two assays (R(2) = 0.95) over a wide dynamic range. Bland-Altman analysis showed a mean difference of 0.04 log 10 indicating minimal overall viral load quantification differences between both platforms. One subtype C was severely underquantified by TaqMan(®) test v2.0 for which sequence analysis revealed multiple mismatches between the viral sequence and the primer/probe regions. A non significant lower quantification of the Abbott assay was shown for subtype A1 with a mean log 10 difference of 0.24. For specimens under 200 cp/mL, the overall agreement was 90% at the cut-off of 50 cp/mL and 67% at assay's lower limit of detection of 20 and 40 cp/mL. 309 samples were retested by the COBAS(®) AMPLICOR(®) HIV-1 MONITOR Test, v. 1.5 and a lack of agreement between the three assays around their lower limit of quantification was revealed. CONCLUSIONS: Both real-time tests were closely comparable in the quantification of viral load specimens of ten HIV-1 subtypes and recombinant forms.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Viraemia and HIV-1 drug resistance mutations among patients receiving antiretroviral treatment in Mozambique.

This study was conducted among individuals taking first-line antiretroviral treatment (ART) for at least 12 months under programme conditions in Maputo, Mozambique in order to report on the level of detectable viraemia and the proportion and types of drug resistance mutations among those with detectable viral loads. HIV-1 RNA viral load levels (lower detection limit <50 copies/ml) were measured, and resistance mutations were sequenced. One hundred and forty-nine consecutive patients (69% females, median age 36 years) were included after a mean follow-up time of 23 months. One hundred and seven (72%; 95% CI 64-79) had undetectable viral load, while in 42 (28%, 95% CI 21-36) viral load was detectable (range 50-58884 copies/ml). From 15 patients with viral load >1000 copies/ml, 12 viruses were sequenced: eight were C subtypes and four were circulating recombinant forms (CRF08). Eight (5%; 95% CI 2-9) patients with detectable viral load had one or more major resistance mutations. Nucleoside reverse transcriptase inhibitor (NRTI) and non-NRTI mutations were observed. There were no major mutations for resistance to protease inhibitors. In Maputo, the level of detectable viraemia is reassuringly low. While embarking on ART scale-up, wider surveillance is warranted to monitor programme quality and limit the development of drug resistance, which remains a major potential challenge for the future of ART in Africa.

Differences of nine drug resistance interpretation systems in predicting short-term therapy outcomes of treatment-experienced HIV-1 infected patients: a retrospective observational cohort study.

OBJECTIVE: Drug resistance interpretation systems are used to select the optimal antiretroviral therapy in HIV-infected patients. It is unclear how the systems perform in predicting therapy success and failure and in how far the interpretations are affected by insufficient drug levels. METHODS: The accuracy of nine different interpretation systems in predicting therapy outcomes was evaluated using virological, immunological, pharmacological, and clinical data of 130 patients treated at 13 outpatient centers. Individual susceptibility scores of the interpretation systems were converted into active drug scores (ADS) and correlated with therapy success and failure, defined as viral load reduction of equal to or more (n=66) and less than 1 log10 copies/ml (n=64) at three months after drug resistance testing. RESULTS: Three interpretation systems considered the respective therapies as more active compared to the other interpretation systems (p<0.01). These systems predicted therapy success better than the other systems, while the others performed better in predicting therapy failure. Thus, the overall rate of correctly predicted treatment outcomes was comparable between the different systems (73.1-80.0 %). Univariate and multivariate regression analysis revealed significant correlations between the ADS of all interpretation systems and virological therapy outcomes (p<0.0001). In contrast, only three interpretation systems were significantly correlated with immunological therapy outcomes in univariate and just one in multivariate models (p<0.05). Among 128 determinations of drug levels in 64 patient samples, 19.4 % revealed no detectable drug levels. The consideration of insufficient drug levels significantly improved the prediction accuracy of all interpretation systems (p<0.005). CONCLUSION: Differences between interpretation systems in predicting therapy failures and success need to be considered for future consensus algorithms. The prediction accuracy of interpretation systems can be improved by consideration of plasma drug levels.

Antiviral efficacy and resistance in patients on antiretroviral therapy in Kigali, Rwanda: the real-life situation in 2002.

Our study aimed to complete the published data on ARV therapy in Africa by describing the baseline situation in Rwanda before the launch of a large ARV programme (ESTHER). Prescription habits, frequency and reasons for treatment interruptions but also antiviral efficay, resistance to ARVs and genotypic variability of the viruses present in Rwanda were analysed. Among the 233 patients included in the study, it appeared that a vast majority (91%) were under triple therapy and that half of them had experienced at least one treatment interruption caused mainly by drug shortage or financial difficulties. Among 60 blood samples analysed, 26 were in virological failure with a viral load above 1000 RNA copies/ml and 11 presented major drug resistance mutations. Finally, virological failure could mainly be explained by the high frequency of treatment interruptions but also by the emergence of drug resistance mutations. Consequently the major objective for the ESTHER programme to improve the situation in Rwanda will be to reduce the drug shortage and facilitate the financial accessibility of the treatments.

Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in Rwanda.

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the pol region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.

Updated European recommendations for the clinical use of HIV drug resistance testing.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Initiation of HAART in drug-naive HIV type 1 patients prevents viral breakthrough for a median period of 35.5 months in 60% of the patients.

The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated mutations and polymorphisms in NNRTI-naïve HIV-infected patients.

BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naïve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naïve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.

HIV-associated hematologic disorders are correlated with plasma viral load and improve under highly active antiretroviral therapy.

The relationship between HIV-1 replication and hematologic parameters was examined in two separate studies. The first study was a cross-sectional evaluation of 207 untreated patients. In this study, the proportion of patients with hematologic disorders increased with disease progression. There was a significant inverse correlation between HIV-1 plasma viral load and all hematologic values (r = -0.266 to -0.331). The second study was a longitudinal evaluation of patients on combination antiretroviral therapy (HAART) with hematologic alterations before treatment ( N = 27 with platelets <150,000/microl, 24 with hemoglobin <12 g/dl, 36 with neutrophils <2000/microl and 29 with leukocytes <3000/microl). Samples were analyzed every 3 months for 2 years. At 2 years, >50% of patients experienced a sustained virologic response, with viral loads <500 RNA copies/ml. Hematologic reconstitution occurred progressively for all blood cell lineages and became statistically significant after the sixth month of therapy ( p <.001). Mean values increased from 110 to 180 x 10(3)/microl for platelets, from 10.7 to 12.3 g/dl for hemoglobin (stabilizing finally at 11.4 g/dl), from 1,260 to 2,240/microl for neutrophils, and from 2,260 to 3,600/microl for leukocytes. In conclusion, hematologic disorders are corrected by combination antiretroviral therapy. This suggests a causative role of HIV-1 in hematologic disorders.

Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype.

OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.

Phase I/II dose escalation and randomized withdrawal study with add-on azodicarbonamide in patients failing on current antiretroviral therapy.

BACKGROUND: Azodicarbonamide (ADA), a HIV-1 zinc finger inhibitor, targets a new step in viral replication and cell infectivity. OBJECTIVE: A first phase I/II clinical study of ADA. METHODS: ADA was administered at escalating doses concomitantly with current antiviral therapy during a 3-month open-label period in patients with advanced AIDS and documented virological failure. After 3 months, patients were randomized in a double-blind placebo-controlled withdrawal, ADA being given at the highest tolerated dosage. RESULTS: Fifteen patients with advanced disease failing on combined antiretroviral therapy, 75% of them with proven phenotypic resistance, had a median baseline CD4 cell count of 85 x 10(6) cells/l, CD4/CD8 cell ratio of 0.09 and median plasma RNA viral load of 4.2 log10 copies/ml. Tolerance to ADA was dose dependent and some patients developed nephrolithiasis, glucose intolerance or showed an ADA-related cytotoxicity towards CD4 cells at higher dosages. No patient died during the study period. ADA increased CD4 cell percentage, increased the CD4/CD8 cell ratio and decreased plasma RNA viral load from baseline. At the end of the double-blind period, the ADA group, but not the placebo group, showed a significant response (P < 0.05). No phenotypic resistance to ADA was observed. Overall, 3/11 patients (27%) had consistent viral load reductions > 0.5 log10 copies/ml compared with baseline and 5/ 11 (45%) showed a CD4 cell recovery from baseline > 33%. In responders, ADA induced a median peak increase in CD4 cell percentage change from baseline of 65% (range 47-243%), and viral load decrease of 1.04 log10 copies/ml (range 0.52-1.23). CONCLUSIONS: The maximal tolerated dosage of ADA appears to be 2 g (three times daily). This study provides safety results that will allow larger clinical trials to confirm the preliminary efficacy data.

Variant human immunodeficiency virus type 1 proteases and response to combination therapy including a protease inhibitor.

The objective of this observational study was to assess the genetic variability in the human immunodeficiency virus (HIV) protease gene from HIV type 1 (HIV-1)-positive (clade B), protease inhibitor-naïve patients and to evaluate its association with the subsequent effectiveness of a protease inhibitor-containing triple-drug regimen. The protease gene was sequenced from plasma-derived virus from 116 protease inhibitor-naïve patients. The virological response to a triple-drug regimen containing indinavir, ritonavir, or saquinavir was evaluated every 3 months for as long as 2 years (n = 40). A total of 36 different amino acid substitutions compared to the reference sequence (HIV-1 HXB2) were detected. No substitutions at the active site similar to the primary resistance mutations were found. The most frequent substitutions (prevalence, >10%) at baseline were located at codons 15, 13, 12, 62, 36, 64, 41, 35, 3, 93, 77, 63, and 37 (in ascending order of frequency). The mean number of polymorphisms was 4.2. A relatively poorer response to therapy was associated with a high number of baseline polymorphisms and, to a lesser extent, with the presence of I93L at baseline in comparison with the wild-type virus. A71V/T was slightly associated with a poorer response to first-line ritonavir-based therapy. In summary, within clade B viruses, protease gene natural polymorphisms are common. There is evidence suggesting that treatment response is associated with this genetic background, but most of the specific contributors could not be firmly identified. I93L, occurring in about 30% of untreated patients, may play a role, as A71V/T possibly does in ritonavir-treated patients.

Comparison of DNA sequencing and a line probe assay for detection of human immunodeficiency virus type 1 drug resistance mutations in patients failing highly active antiretroviral therapy.

The resistance of human immunodeficiency virus type 1 (HIV-1) to drugs is a major cause of antiretroviral treatment failure. We have compared direct sequencing to a line probe assay (LiPA) for the detection of drug resistance-related mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. For 26 patients with virological failure despite the use of two nucleoside analogues and one protease inhibitor (indinavir [n = 6], ritonavir [n = 10], and saquinavir [n = 10]), genotypic resistance assays were carried out retrospectively every 3 months for up to 2 years by using direct sequencing (TruGene; Visible Genetics) and a LiPA for detection of mutations in the reverse transcriptase (INNO-LiPA HIV-1 RT; Innogenetics) and the protease (INNO-LiPA HIV Protease, prototype version; Innogenetics) genes. Comparison of the results from both assays found rare major discrepancies (<1% of codons analyzed). INNO-LiPA detected more wild-type-mutant mixtures than sequencing but suffered from a high rate of codon hybridization failures for the reverse transcriptase. LiPA detected earlier and more frequently than sequencing the transient mixed virus population that contained I84V, which appears before V82A in the protease sequence. Mutations M461, G48V, and L90M were often transient and drug pressure related. In conclusion, direct sequencing and LiPAs give concordant results for most clinical isolates. LiPAs are more sensitive for the detection of mixed virus populations. Mutation I84V appears in minor populations in the early steps of the pathways of resistance to indinavir and ritonavir. The fact that some mutations can be found only transiently and in minor virus populations highlights the importance of a low detection limit for resistance assays.