Genotypic Resistance Assays Research Articles

Antiretroviral Genotypic Resistance Mutations in HIV-1 Infected Korean Patients with Virologic Failure

Resistance assays are useful in guiding decisions for patients experiencing virologic failure (VF) during highly-active antiretroviral therapy (HAART). We investigated antiretroviral resistance mutations in 41 Korean human immunodeficiency virus type 1 (HIV-1) infected patients with VF and observed immunologic/virologic response 6 months after HAART regimen change. Mean HAART duration prior to resistance assay was 45.3±27.5 months and commonly prescribed HAART regimens were zidovudine/lamivudine/nelfinavir (22.0%) and zidovudine/lamivudine/efavirenz (19.5%). Forty patients (97.6%) revealed intermediate to high-level resistance to equal or more than 2 antiretroviral drugs among prescribed HAART regimen. M184V/I mutation was observed in 36 patients (87.7%) followed by T215Y/F (41.5%) and M46I/L (34%). Six months after resistance assay and HAART regimen change, median CD4+ T cell count increased from 168 cells/µL (interquartile range [IQR], 62-253) to 276 cells/µL (IQR, 153-381) and log viral load decreased from 4.65 copies/mL (IQR, 4.18-5.00) to 1.91 copies/mL (IQR, 1.10-3.60) (P<0.001 for both values). The number of patients who accomplished viral load <400 copies/mL was 26 (63.4%) at 6 months follow-up. In conclusion, many Korean HIV-1 infected patients with VF are harboring strains with multiple resistance mutations and immunologic/virologic parameters are improved significantly after genotypic resistance assay and HAART regimen change.

Evaluation of HIV-1 viral load and genotypic resistance assays for resource-limited settings

There is an urgent need for low-cost assays for HIV-1 quantitation to ensure adequate follow-up of HIV-infected patients on antiretroviral therapy (ART) in resource-limited countries. Two low-cost viral load assays are evaluated, a reverse transcriptase activity assay (ExavirLoad v2, Cavidi) and a real-time reverse transcriptase PCR assay (Generic HIV viral load, Biocentric). Both tests were compared with the ultrasensitive HIV Amplicor Monitor assay. Samples were collected in Mombasa, Kenya, from 20 HIV-1 seronegative and 150 HIV-1 seropositive individuals of whom 50 received antiretroviral treatment (ART). The ExavirLoad and the Generic HIV viral load assay were performed in a local laboratory in Mombasa, the Amplicor Monitor assay (version 1.5, Roche Diagnostics) was performed in Ghent, Belgium. ExavirLoad and Generic HIV viral load reached a sensitivity of 98.3% and 100% and a specificity of 80.0% and 90.0%, respectively. Linear regression analyses revealed good correlations between the Amplicor Monitor and the Generic HIV viral load (r= 0.935, p< 0.001) with high accuracy (100.1%), good precision (5.5%) and a low percent similarity coefficient of variation (5.4%). Bland–Altman analysis found 95% of the samples within clinically acceptable limits of agreement (−1.19 to 0.87 log copies/ml). Although, the ExavirLoad also showed a good linear correlation with the Amplicor Monitor (r= 0.901, p< 0.001), a problem with false positive results was more significant. The cost per test remains relatively high (US$ 30 for ExavirLoad and US$ 20 for the Generic HIV viral load). Hence, false positive results and the need for an expensive PCR instrument for the Generic HIV viral load assays still limit the implementation of these tests in less equipped, less experienced laboratories. © 2007 Elsevier B.V. All rights reserved.

GENOTYPIC RESISTANCE PROFILE OF HIV-1 PROTEASE GENE: A PRELIMINARY REPORT FROM VELLORE, SOUTH INDIA

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Evaluation of HIV-1 viral load and genotypic resistance assays for resource-limited settings

Evaluation of HIV-1 viral load and genotypic resistance assays for resource-limited settings

Trends of antiretroviral drug resistance in treatment-naive patients with human immunodeficiency virus type 1 infection in Taiwan

To determine the prevalence and trends of antiretroviral drug resistance among HIV-1-infected Taiwanese patients who have been provided with free-of-charge antiretroviral therapy (ART) since 1990. Blood samples collected from 786 HIV-1-infected patients from 1999 to 2006 were subjected to genotypic resistance assay. Antiretroviral resistance mutations were identified in accordance with the antiretroviral resistance mutation list of the International AIDS Society-USA Consensus Guidelines. Trends of resistance were studied in patients enrolled in two periods: before (period 1, January 1999 to December 2003) and after (period 2, January 2004 to December 2006) the CRF07_BC outbreak among injection drug users (IDUs). The frequency of HIV-1 isolates harbouring one or more primary mutations associated with antiretroviral resistance to reverse transcriptase inhibitors or protease inhibitors increased significantly from 6.6% in period 1 to 12.7% in period 2 (P = 0.003). A significant increase in prevalence of antiretroviral drug resistance was observed among men who have sex with men and patients infected with HIV subtype B. In multivariate analysis, hepatitis C virus (HCV) exposure, which exhibited collinearity with injection drug use and infection with CRF07_BC, represented a lower risk for infection with resistant viruses. Our findings suggest that the prevalence of antiretroviral resistance has increased in Taiwan over the past 8 years after the introduction of combination ART. IDUs who were HCV-seropositive and infected with CRF07_BC were at lower risk for infection with antiretroviral-resistant viruses.

Genotypic resistance profile of HIV-1 protease gene: A preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Reproducibility of a high-throughput HIV-1 genotypic resistance assay over time (2001–2007)

Reproducibility of a high-throughput HIV-1 genotypic resistance assay over time (2001–2007)

Genotypic resistance to lopinavir and fosamprenavir with or without ritonavir of clinical isolates from patients failing protease inhibitors-containing HAART regimens: Prevalence and predictors

The aim of this study was to establish the prevalence and predictors of genotypic resistance of HIV-1 to lopinavir and fosamprenavir from patients failing protease inhibitors (PI)-based regimens. We selected 643 HIV-1-infected patients with available treatment history who underwent genotypic resistance assays for virological failure from a clinical site and from the Stanford database. According to the genotypic resistance interpretation of the Stanford algorithm, proportions of viruses showing full susceptibility to fosamprenavir and lopinavir were 32% and 34%, respectively (p =ns). Proportions of viruses fully susceptible to lopinavir/r and fosamprenavir/r according to the Agence Nationale pour la Recherche sur le SIDA (ANRS) algorithm, were 81% and 81%, respectively. According to the Rega algorithm, proportions of viruses showing full susceptibility to fosamprenavir/r and lopinavir were 80% and 70%, respectively (p<0.001). According to the ANRS and Rega interpretations, the time on therapy predicted susceptibility to lopinavir/r, while susceptibility to fosamprenavir/r according to ANRS was predicted by the number of prior PI regimens experienced. According to the Stanford interpretation, prior indinavir exposure predicted resistance to lopinavir/r and fosamprenavir/r while prior nelfinavir use predicted susceptibility to both drugs. After failing PI-based regimens, the majority of viruses retained a predicted susceptibility to fosamprenavir/r and lopinavir/r. In patients failing PIs, the interpretation of genotypic resistance to fosamprenavir may change considerably according to the different algorithms and in respect to the effect of pharmacokinetic boosting with ritonavir.

Phenotypic resistance of resistant strains of HIV type-1 subtype B in China

This study was aim to explore the characteristics of phenotypic resistance of resistant strains of HIV type-1 (HIV-1) subtype B and to compare the concordance between the phenotypic resistance and genotypic resistance. The genotypic resistance assay for the HIV-1 clinical isolates was performed. One isolate without resistance mutation was chosen as a drug-sensitive reference strain and seven subtype B isolates with resistance mutations were phenotypically tested. Fifty percent inhibitory concentrations (IC50) between resistant and sensitive viruses were compared. The resistance extent was determined by the folds of the increased IC50. The concordance between the phenotypic resistance and genotypic resistance was also analyzed. IC50 of resistant isolates were 0.0006 - 0.1300 micromol/L for zidovudine (AZT), 0.0016 - 0.0390 micromol/L for lamivudine (3TC), 0.0104 - 0.4234 micromol/L for nevirapine (NVP), and 0.0163 - 0.1142 micromol/L for indinavir (IDV), respectively. Genotypic and phenotypic resistance assays indicated that the resistant strains were intermediately and highly resistant to nucleotide analog reverse transcriptase inhibitors and non-nucleotide analog reverse transcriptase inhibitors. The phenotypic assay was consistent with the genotypic assay. For measuring the potential resistance, the genotypic assay was more sensitive than the phenotypic. In evaluating the resistance to protease inhibitors, these two assays were discrepant. Both the phenotypic and genotypic assays indicate that the resistant viruses exist in HIV-infected patients in China who have received treatment. Phenotypic and genotypic assays have high concordance, and the genotypic assay could replace the phenotypic assay to predict the HIV-1 resistance.

HBV virological assessment

Management of hepatitis B virus (HBV) infected patients involves serological diagnosis, quantitation of HBV-DNA and measurement of HBV drug resistance. Different serological markers such as HBsAg, anti-HBs, anti-HBc (total and IgM), HBeAg and anti-HBe are assessed by immunoassays in order to define the infection status. The emergence of surface mutants however is a continuous challenge to design more effective immunoassays. Commercially available quantitative HBV-DNA assays with increased sensitivity and wider linear range give a more accurate estimate of viral replication and contribute decisively in the initiation and the monitoring of the response to HBV therapy. Genotypic drug resistance assays are important diagnostic tools, since the administration of nucleoside/nucleotide analogues to HBV infected patients leads to the development of drug resistance patterns very much dependent on the treatment regimen. Special issues have to be taken into consideration regarding HBV/HIV-1 co-infected patients, since concominant HIV and HBV replication results in higher rates of HBV replication. Current efforts are focused on the standardization of HBV-DNA assays (qualitative and quantitative), of HBV drug resistance assays as well as in the development of new assays and markers that will help in the prognosis and management of HBV infection (quantitative detection of pre-core mutants and HBV ccc-DNA assays).

Effects on Immunological and Virological Outcome of Patients Using One Protease Inhibitor or One Non-Nucleoside Reverse Transcriptase Inhibitor in a Triple Antiretroviral Therapy: Normal Clinical Practice Versus Clinical Trial Findings

We compared the response of two standard 3-drug regimens containing two-nucleoside reverse transcriptase inhibitor (Zidovudine +Lamivudine) plus either a protease inhibitor (PIs) (Indinavir) or a non-nucleoside reverse transcriptase inhibitor (NNRTIs) (Efavirenz) among treatment-naïve or treatment-experienced HIV-infected persons. The obtained results will be compared to clinical trial findings. Through a retrospective study, we compared the virological and immunological response of 119 Tunisian HIV-1 infected patients (North Africa) who started for the first time/ in salvage use a triple antiretroviral treatment containing one NNRTI (group A1/group A2) or one PI (group B1/group B2). Viral load (VL) was analysed with Amplicor HIV-1 Monitor test and drug resistance mutations were examined by using two distinct line probe assays (LiPA). The analysis according to the received treatment showed an average of 0.45 log(10) drop in the mean VL among groups A2 and B2. For naïve patients, 62.5% of group A1 reached an undetectable VL versus 53.5% for group B1 (p < 0.001). With regard to the CD4 cell count change, we observed a mean increase of more than 65% versus baseline within group A1 in comparison with 42% for group B1 (p < 0.001). Genotypic resistance assays showed that patients of group A2 had significantly more resistance mutations than those of group B2 (2.66 vs. 0.75 (p = 0.0039)). Finally, 15 patients who were failing were switched to indinavir or efavirenz. When indinavir was replaced by efavirenz, we observed an increase in the plasma VL. With regard to the effects on immunological and virological outcome of patients using PIs or NNRTIs in a triple antiretroviral therapy, our observations in normal clinical practice supported the reported clinical trial findings but are not in favour of replacing indinavir by efavirenz in failing regimen.

Efficacy of Highly Active Antiretroviral Therapy in HIV-Infected Children Participating in Thailand's National Access to Antiretroviral Program

Programs for access to antiretroviral treatment were only recently implemented in developing countries. This study aimed to describe the effect of highly active antiretroviral therapy (HAART) in treating human immunodeficiency virus (HIV)-infected children in Thailand's National Access to Antiretroviral Program for People Living with HIV/AIDS. From August 2002 to July 2003, a total of 107 children were enrolled in the study. They received HAART consisting of either nevirapine or efavirenz, together with lamivudine and stavudine. Generic drugs and/or adult formulations were used. CD4 lymphocyte count, plasma HIV RNA level, and weight-for-age and height-for-age z scores were measured before, 2 months after, and every 6 months after initiation of HAART. A genotypic resistance assay was performed for patients with poor virological response. The mean age of the patients was 7.7 years (range, 2.1-13.8 years). At baseline, the median CD4 cell percentage was 3%, and the plasma HIV RNA level was 5.4 log10 copies/mL. Four patients died from HIV-related illness. After 72 weeks of HAART, the median CD4 cell percentage was 21%, and 76% of patients had HIV RNA levels of < 50 copies/mL. The mean weight-for-age and height-for-age z scores increased from -1.9 to -1.3 (P < .0001) and from -2.3 to -2.0 (P < .0001), respectively. The percentage of patients who took > or = 95% of prescribed medications during the interval between every follow-up visit was 86% For patients with suboptimal virological response, the most common resistance mutations among HIV isolates were associated with lamivudine and with nonnucleoside reverse-transcriptase inhibitors. In this resource-limited setting, HAART is safe and effective for HIV-infected children despite initiation of treatment during the advanced stage of disease. The use of generic and nonpediatric drug formulations is feasible.

Comparative performance evaluation of the HIV-1 LiPA protease and reverse transcriptase resistance assay on clinical isolates

Background Assays that provide information regarding HIV-1 resistance to antiretroviral drugs are widely used to help manage antiretroviral treatment. The most commonly used HIV genotypic resistance assays are based on DNA sequencing (TRUGENE ®, ViroSeq ™, and home-brew) or reverse hybridization (LiPA). Objectives This study compares the results from clinical specimens using two assay methods: the LiPA HIV-1 protease (PR) and reverse transcriptase (RT) resistance assay and DNA sequencing. Study design Operators at each of three sites tested 10–20 randomly selected clinical specimens using LiPA (three strips total with probes for PR codons 30, 46, 48, 50, 54, 82, 84, and 90, and RT codons 41, 69, 70, 74, 75, 103, 106, 151, 181, 184, and 215) and DNA sequencing (TRUGENE ® HIV-1 Genotyping Assay or home-brew methodology). Results from the two methods were categorized for each codon as follows: (i) concordant (LiPA and sequencing having the same result for wild-type (WT), mutant, and mixture); (ii) partially concordant (mixture by one method and not by the other); (iii) indeterminate (no result by LiPA); and (iv) discordant (LiPA and sequencing detecting different amino acids). Results A total of 50 clinical specimens were tested using the LiPA PR strip; 40 of these were also tested using the LiPA RT strip. For PR, 91.3% of the codon results were concordant, 3.0% were partially concordant, 4.5% were indeterminate by LiPA, and 1.3% were discordant. For RT, 88.0% of the codon results were concordant, 5.9% were partially concordant, 5.2% were indeterminate, and 0.9% were discordant. LiPA detected 3.0% (PR) and 6.4% (RT) WT/mutant mixtures, compared to 0.5% (PR) and 3.2% (RT) mixtures by sequencing. Conclusions More WT/mutant mixtures were detected using LiPA, possibly indicating increased sensitivity. Relatively high concordance and low discordance rates were observed between LiPA and DNA sequencing. The indeterminate rate for LiPA was moderately high and may limit the clinical utility of this assay.

A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene

Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naïve and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naïve samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36–45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.

Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation.

High incidence rates of isoniazid-, rifampin-, and multiple-drug-resistant tuberculosis have been reported in countries of the former Soviet Union (FSU). Genotypic (unlike phenotypic) drug resistance assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For these assays to be clinically useful, they must be able to detect the range of mutations seen in isolates from the population of tuberculosis patients to which they are applied. Two novel macroarrays were applied to detect mutations associated with rifampin (rpoB) and isoniazid (katG and inhA) resistance. In a sample of 233 isolates from patients in Samara, central Russia, 46.5% of isolates possessed mutations in both the rpoB and the katG (or inhA) genes. Combined results from the macroarrays demonstrated concordance in 95.4 and 90.4% of phenotypically defined rifampin- and isoniazid-resistant isolates, respectively. The contribution of different mutations to resistance was comparable to that reported previously for non-FSU countries, with 90% of rifampin-resistant isolates and 93% of isoniazid resistant isolates due to rpoB531 and katG315 mutations, respectively. The percentage of phenotypically resistant rifampin isolates with no mutations in the rpoB codons 509 to 536 was 4.2%, which was similar to previous reports. Novel macroarrays offer a rapid, accurate, and relatively cheap system for the identification of rifampin-, isoniazid-, and multiple-drug-resistant Mycobacterium tuberculosis isolates.

Virological significance, prevalence and genetic basis of hypersusceptibility to nonnucleoside reverse transcriptase inhibitors

Nonnucleoside reverse transcriptase inhibitors (NNRTI) are used to treat HIV-infected individuals in combination with nucleoside analogues (NRTI) and protease inhibitors. Long-term treatment with antiretroviral agents results in the emergence of strains with decreased susceptibility (resistance) to the drugs and is one of the major factors in loss of drug efficacy. Conversely, there have been recent reports of HIV strains with increased susceptibility (hypersusceptibility) to NNRTIs. These isolates emerge in patients on long-term antiretroviral therapy particularly in individuals receiving NRTIs. The prevalence of NNRTI hypersusceptibility ranges between 17.5 and 50% in NRTI-treatment experienced compared to 10% in NRTI-naïve patients. There is an inverse correlation between NNRTI hypersusceptibility and phenotypic NRTI resistance and a direct correlation between the number of NRTI resistance mutations present in the HIV reverse transcriptase. Re-sensitisation of phenotypic NNRTI resistance has been reported by NRTI mutations and is not likely to be detected using genotypic resistance assays. Recent studies demonstrate that NNRTI hypersusceptible virus at baseline is likely to predict better virological outcomes in patients on NNRTI-based salvage regimens compared to patients with NNRTI susceptible virus. These studies have implications for the sequence of antiretroviral drug use where patients may benefit from NRTI therapy before the introduction of NNRTIs, however more studies are needed to examine this treatment rationale.

Genotypic Resistance Assay to Antiretrovirals in HIV-1 Infected Individuals

Genotypic Resistance Assay to Antiretrovirals in HIV-1 Infected Individuals

Resistenz in der antiretroviralen Therapie der HIV-Infektion*

@ Despite impressive success of highly active antiretroviral therapy, a durable control of viral replication cannot be achieved in a substantial proportion of HIV-infected patients. The most important reason is development of resistance, defined as capability of mutant virus to replicate in the presence ofa higher concentration of substance in comparison to wild-type virus. In the treated HIV-positive population resistance is prevalent in almost 50%, whereas in treatment-naives resistance can be found in 10-14%. For diagnosis, genotypic resistance tests (sequencing ofpol gene) and phenotypic resistance assays (susceptibility against antiretrovirals of an isolate in vitro) are available. A special problem is the interpretation of complex mutation patterns. Elaborate databases are implemented for this task. A number of clinical studies have demonstrated the value of resistance testing in the situation of virological failure. Resistance-guided salvage therapy was significantly more effective in these trials in comparison to former standard of care. This led to the recommendation of resistance testing in this clinical situation by German and international expert panels.

A simple and sensitive ‘in-house’ method for determining genotypic drug resistance in HIV-1

Antiretroviral combination therapy is a major advance in the treatment of HIV infection, but development of antiretroviral resistance is still an important cause of treatment failure. Therefore, resistance testing was recommended recently for follow-up of HIV-1 infected individuals. The aim of this study was to develop a new genotypic resistance assay because simple and affordable assays with sufficient sensitivity for different genetic subtypes and low copy number samples are still lacking. Different methods and primers for RNA extraction from plasma, cDNA synthesis, nested PCR and sequencing on an ABI310 automated sequencer were evaluated and optimised. The PCR was designed to amplify a fragment covering the protease and the first half of the reverse transcriptase (RT), which harbour most known resistance mutations to licensed antiretroviral drugs. Resistance mutations were identified using resistance analysis tools available over the Internet. The optimised assay had a sensitivity of approximately 200 RNA copies per ml. The method was evaluated on plasma samples from treated patients infected with different subtypes of HIV-1 and appeared to have similar sensitivity for all subtypes. Samples that had failed previously in routine testing were analysed successfully with the new assay. The new assay is more sensitive and robust than the current routine method.

Extent and importance of cross-resistance to efavirenz after nevirapine failure.

The objective of this study was to evaluate the activity of efavirenz after the failure of a nevirapine-containing regimen. This prospective study included 47 patients with plasma HIV loads >1000 copies/ml, and who had received nevirapine for at least 16 weeks, included in an efavirenz-based salvage regimen. The main outcome measure was virological response, defined as an HIV RNA level decrease of at least 1 log(10) copies/ml after 24 weeks, according to genotypic and phenotypic resistance to efavirenz. Phenotypic resistance was defined as a >10-fold increase in the IC(50). The median CD4(+) cell count was 236 x 10(6)/liter and the median HIV RNA level was 4.5 log(10) copies/ml. Mutations known to decrease susceptibility to nonnucleoside reverse transcriptase inhibitors were observed in 79% of patients, predominantly at residues 181 (49%), 103 (40%), and 106 (19%), but phenotypic resistance to efavirenz was seen in 62% of cases. All the strains with the K103N mutation showed high-level resistance to efavirenz, in contrast with 20% of those carrying exclusively the Y181C mutation. By week 24, 38% of patients had responded and 19% had achieved an undetectable HIV load. Virological failure was observed in patients with phenotypic resistance to efavirenz (67 vs. 11%; relative risk [RR], 4; 95% confidence interval [CI], 1.07-14.89; p = 0.04), or in presence of the K103N mutation (52 vs. 17%; RR, 1.77; 95% CI, 1.12-2.79; p = 0.02), and these results remained unchanged after adjusting for HIV load, or by resistance to the accompanying drugs in the salvage regimen. A previous longer period of nevirapine therapy was significantly associated with the emergence of efavirenz resistance (288 vs. 170 days, p < 0.01). We conclude that genotypic and/or phenotypic resistance assays permit the sequential use of nonnucleoside reverse transcriptase inhibitors in the clinical setting. Our data suggest that an early change after nevirapine failure could avoid the emergence of efavirenz resistance.