Virologic failure of protease inhibitor-based second-line antiretroviral therapy without resistance in a large HIV treatment program in South Africa

BACKGROUND: We investigated the prevalence of wild-type virus (no major drug resistance) and drug resistance mutations at second-line antiretroviral treatment (ART) failure in a large HIV treatment program in South Africa. METHODOLOGY/ PRINCIPAL FINDINGS: HIV-infected patients ≥ 15 years of age who had failed protease inhibitor (PI)-based second-line ART (2 consecutive HIV RNA tests >1000 copies/ml on lopinavir/ritonavir, didanosine, and zidovudine) were identified retrospectively. Patients with virologic failure were continued on second-line ART. Genotypic testing for drug resistance was performed on frozen plasma samples obtained closest to and after the date of laboratory confirmed second-line ART failure. Of 322 HIV-infected patients on second-line ART, 43 were adults with confirmed virologic failure, and 33 had available plasma for viral sequencing. HIV-1 RNA subtype C predominated (n = 32, 97%). Mean duration on ART (SD) prior to initiation of second-line ART was 23 (17) months, and time from second-line ART initiation to failure was 10 (9) months. Plasma samples were obtained 7(9) months from confirmed failure. At second-line failure, 22 patients (67%) had wild-type virus. There was no major resistance to PIs found. Eleven of 33 patients had a second plasma sample taken 8 (5.5) months after the first. Median HIV-1 RNA and the genotypic resistance profile were unchanged. CONCLUSIONS/ SIGNIFICANCE: Most patients who failed second-line ART had wild-type virus. We did not observe evolution of resistance despite continuation of PI-based ART after failure. Interventions that successfully improve adherence could allow patients to continue to benefit from second-line ART therapy even after initial failure.     

Sustained Virological Response on Second-Line Antiretroviral Therapy following Virological Failure in HIV-Infected Patients in Rural South Africa

Objective

This study aims to describe the virological, immunological and clinical efficacy of protease inhibitor (PI)-based second-line antiretroviral therapy (ART) in rural South Africa.

Methods

An observational cohort study was performed on 210 patients (including 39 children) who initiated PI-based second-line therapy at least 12 months prior to data collection. Biannual clinical, immunological and virological monitoring was performed. Primary endpoints were adequate virological response (plasma HIV-1 RNA<400 copies/ml), full virological suppression (plasma HIV-1 RNA<50 copies/ml) and treatment failure (virological failure (plasma HIV-1 RNA>1000 after initial virological response) or on-going viremia (plasma HIV-1 RNA never<400 copies/ml for more than six months)). Data were analyzed by an on-treatment (OT) and intention-to-treat (ITT) approach. Analyses were primarily performed on the group of patients who switched following first-line virological failure.

Results

Median duration of follow-up after switch to second-line treatment was 20 months [IQR 11–35]. 191 patients had switched to second-line ART due to first-line virological failure. 139/191 of them (72.8%, ITT) were in care and on treatment at the end of follow-up and 11/191 (5.8%, ITT) had died. After twelve months, an adequate virological response was seen in 92/128 patients (71.9%, OT), of which 78/128 (60.9%, OT) experienced full virological suppression. Virological response remained stable after 24 months. Virological efficacy was similar amongst adult and pediatric patients. As in first-line ART, we observed a lack of correlation between virological failure and WHO-defined immunological failure.

Conclusions

Good virological outcomes following first-line failure can be achieved with PI-based, second-line antiretroviral therapy in both adult and pediatric patients in rural South Africa. Retention rates were high and virological outcomes were sustainable during the two-year follow-up period, although persisting low-level viremia occurred in a subset of patients. The observed viro-immunological dissociation emphasizes the need for virological monitoring.     

Sequencing-based detection of low-frequency human immunodeficiency virus type 1 drug-resistant mutants by an RNA/DNA heteroduplex generator-tracking assay

Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.     

Polymorphism in Gag gene cleavage sites of HIV-1 non-B subtype and virological outcome of a first-line lopinavir/ritonavir single drug regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial.     

Virologic failures on initial boosted-PI regimen infrequently possess low-level variants with major PI resistance mutations by ultra-deep sequencing

Background

It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects.

Objective

To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen.

Methods/Results

Patients experiencing VF on a Tenofovir/Emtricitabine+PI/r regimen were evaluated by ultra deep sequencing (UDS) for mutations classified/weighted by Stanford HIVdb. Samples were evaluated for variants to 0.4% levels. 36 VF subjects were evaluated by UDS; 24 had UDS for PI and RT DRMs. Of these 24, 19 (79.2%) had any DRM by UDS. The most common UDS-detected DRM were NRTI in 18 subjects: M184V/I (11), TAMs(7) & K65R(4); PI DRMs were detected in 9 subjects: M46I/V(5), F53L(2), I50V(1), D30N(1), and N88S(1). The remaining 12 subjects, all with VLs<10,000, had protease gene UDS, and 4 had low-level PI DRMs: F53L(2), L76V(1), I54S(1), G73S(1). Overall, 3/36(8.3%) subjects had DRMs identified with Stanford-HIVdb weights >12 for ATV or LPV: N88S (at 0.43% level-mutational load 1,828) in 1 subject on ATV; I50V (0.44%-mutational load 110) and L76V (0.52%-mutational load 20) in 1 subject each, both on LPV. All VF samples remained phenotypically susceptible to the treatment PI/r.

Conclusion

Among persons experiencing VF without PI DRMs with standard genotyping on an initial PI/r regimen, low-level variants possessing major PI DRMs were present in a minority of cases, occurred in isolation, and did not result in phenotypic resistance. NRTI DRMs were detected in a high proportion of subjects. These data suggest that PIs may remain effective in subjects experiencing VF on a PI/r-based regimen when PI DRMs are not detected by standard or UDS genotyping.     

Medication Possession Ratio Associated with Short-Term Virologic Response in Individuals Initiating Antiretroviral Therapy in Namibia

The visual-analogue scale (VAS), Likert item (rating scale), pills identification test (PIT), and medication possession ratio (MPR) provide estimates of antiretroviral therapy (ART) adherence which correlate with HIV viral suppression. These simple adherence measures are inexpensive and easy to administer; however, require validation and adjustment prior to implementation. The objective of this study was to define the optimal adherence assessment measure in Namibia to identify patients at risk for sub-optimal adherence and poor virologic response 6 months after ART initiation. We conducted a cross-sectional survey in HIV-infected adults receiving ART for 6–12 months prior to the adherence assessment. Adherence measures included 30-day VAS, 30-day Likert item, self-reported treatment interruptions, PIT, and MPR. Association of adherence measures with 6-month HIV-1 RNA level was assessed using two thresholds (1000 copies/mL and 5000 copies/mL). Adherence was assessed in 236 patients, mean age 37.3 years, 54% female. Mean adherence was 98.1% by 30-day VAS, 84.7% by 30-day Likert item, 97.0% by self-reported treatment interruptions, 90.6% by PIT, and 98.8% by MPR. Agreement between adherence measures was poor using kappa statistic. 76% had HIV-1 RNA <1000 copies/ml, and 88% had HIV-1 RNA <5000 copies/ml. MPR (continuous) was associated with viral suppression <5000 copies/ml (p = 0.036). MPR <75% was associated with virologic failure at ≥5000 copies/ml with OR 3.89 (1.24, 12.21), p = 0.013. Adherence was high with all measures. Only MPR, was associated with short-term virologic response, suggesting its cross-culturally utility for early identification of patients at high risk for virologic failure.     

Week 120 Efficacy of Tenofovir,Lamivudine and Lopinavir/r-Based Second-Line Antiretroviral Therapy in Treatment-Experienced HIV Patients

Background

Tenofovir (TDF) and ritonavir-boosted lopinavir (LPV/r) were not introduced to China as second-line medications until 2009. The efficacy and safety of TDF/3TC/LPV/r based second-line regimen have not been evaluated in Chinese HIV patients who failed first-line regimens.

Methods

This was a multicenter cohort study recruiting patients from Beijing, Shanghai, Guangdong, and Henan provinces between November 2008 and January 2010. Eighty HIV infected patients failing first-line regimens with serum creatinine lower than 1.5 times the upper limit of normal received TDF+ lamivudine (3TC)+ LPV/r were followed up for 120 weeks. CD4 cell count, viral load, and estimated glomerular filtration rate (eGFR) were monitored at each visit.

Results

At baseline, 31.2% and 48.8% of patients had moderate/high-level resistance to TDF and 3TC, respectively; while 2.5% of patients had only low-level resistance to LPV/r. During 120 weeks of follow-up, virological suppression rate reached over 70% (<40 copies/ml) and 90% (<400 copies/ml), and median CD4 cell count increased from 157 cells/μL at baseline to 307 cells/μL at week 120. Baseline drug-resistance mutations had no impact on the efficacy of second-line antiretroviral therapy. Median eGFR dropped from 104.7 ml/min/1.73m² at baseline to 95.6 ml/min/1.73m² at week 24 and then recovered after week 96.

Conclusion

This study for the first time demonstrated that TDF+ 3TC+ LPV/r was efficacious as second-line regimen with acceptable nephrotoxicity profiles in patients who failed zidovudine or stavudine based first-line regimens in China.

Trial Registration

ClinicalTrials.gov NCT00872417     

Determinants of sustained viral suppression in HIV-infected patients with self-reported poor adherence to antiretroviral therapy

Background

Good adherence to antiretroviral therapy (ART) is critical for successful HIV treatment. However, some patients remain virologically suppressed despite suboptimal adherence. We hypothesized that this could result from host genetic factors influencing drug levels.

Methods

Eligible individuals were Caucasians treated with efavirenz (EFV) and/or boosted lopinavir (LPV/r) with self-reported poor adherence, defined as missing doses of ART at least weekly for more than 6 months. Participants were genotyped for single nucleotide polymorphisms (SNPs) in candidate genes previously reported to decrease EFV (rs3745274, rs35303484, rs35979566 in CYP2B6) and LPV/r clearance (rs4149056 in SLCO1B1, rs6945984 in CYP3A, rs717620 in ABCC2). Viral suppression was defined as having HIV-1 RNA <400 copies/ml throughout the study period.

Results

From January 2003 until May 2009, 37 individuals on EFV (28 suppressed and 9 not suppressed) and 69 on LPV/r (38 suppressed and 31 not suppressed) were eligible. The poor adherence period was a median of 32 weeks with 18.9% of EFV and 20.3% of LPV/r patients reporting missed doses on a daily basis. The tested SNPs were not determinant for viral suppression. Reporting missing >1 dose/week was associated with a lower probability of viral suppression compared to missing 1 dose/week (EFV: odds ratio (OR) 0.11, 95% confidence interval (CI): 0.01–0.99; LPV/r: OR 0.29, 95% CI: 0.09–0.94). In both groups, the probability of remaining suppressed increased with the duration of continuous suppression prior to the poor adherence period (EFV: OR 3.40, 95% CI: 0.62–18.75; LPV/r: OR 5.65, 95% CI: 1.82–17.56).

Conclusions

The investigated genetic variants did not play a significant role in the sustained viral suppression of individuals with suboptimal adherence. Risk of failure decreased with longer duration of viral suppression in this population.     

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.     

Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection

Objective

To evaluate minority variant drug resistance mutations detected by the oligonucleotide ligation assay (OLA) but not consensus sequencing among subjects with primary HIV-1 infection.

Design/Methods

Observational, longitudinal cohort study. Consensus sequencing and OLA were performed on the first available specimens from 99 subjects enrolled after 1996. Survival analyses, adjusted for HIV-1 RNA levels at the start of antiretroviral (ARV) therapy, evaluated the time to virologic suppression (HIV-1 RNA<50 copies/mL) among subjects with minority variants conferring intermediate or high-level resistance.

Results

Consensus sequencing and OLA detected resistance mutations in 5% and 27% of subjects, respectively, in specimens obtained a median of 30 days after infection. Median time to virologic suppression was 110 (IQR 62–147) days for 63 treated subjects without detectable mutations, 84 (IQR 56–109) days for ten subjects with minority variant mutations treated with ≥3 active ARVs, and 104 (IQR 60–162) days for nine subjects with minority variant mutations treated with <3 active ARVs (p = .9). Compared to subjects without mutations, time to virologic suppression was similar for subjects with minority variant mutations treated with ≥3 active ARVs (aHR 1.2, 95% CI 0.6–2.4, p = .6) and subjects with minority variant mutations treated with <3 active ARVs (aHR 1.0, 95% CI 0.4–2.4, p = .9). Two subjects with drug resistance and two subjects without detectable resistance experienced virologic failure.

Conclusions

Consensus sequencing significantly underestimated the prevalence of drug resistance mutations in ARV-naïve subjects with primary HIV-1 infection. Minority variants were not associated with impaired ARV response, possibly due to the small sample size. It is also possible that, with highly-potent ARVs, minority variant mutations may be relevant only at certain critical codons.     

Analysis of HIV-1 sequences before and after co-infecting syphilis

Increasing syphilis incidence among men who have sex with men (MSM) has been reported. The index case was a human immunodeficiency virus type 1 (HIV-1)-positive MSM who presented coincidentally with the secondary syphilis and a rebound of plasma viral load after complete suppression of HIV-1 (below 50 copies/ml) for 13 months with potent antiretroviral therapy (PART), suggesting a possibility of HIV-1 superinfection. We analyzed HIV-1 sequences before and after syphilis in four HIV-1-positive patients including the index case to explore drug resistance mutations (DRMs) and a possibility of HIV-1 superinfection. There were patients who obtained DRMs around syphilis infection but no evidence of HIV-1 superinfection was obtained. Our results underline the importance of strict adherence to PART.     

An in silico pharmacological approach toward the discovery of potent inhibitors to combat drug resistance HIV-1 protease variants

Protease inhibitors (PIs) are crucial drugs in highly active antiretroviral therapy for human immunodeficiency virus-1 (HIV-1) infections. However, resistance owing to mutations challenge the long-term efficacy in the medication of HIV-1-infected individuals. Lopinavir (LPV) and darunavir (DRV), two second-generation drugs are the most potent among PIs, hustling the drug resistance when mutations occur in the active and nonactive site of the protease (PR). Herein, we strive for compounds that can stifle the function of wild-type (WT) HIV-1 PR along with four major single mutants (I54M, V82T, I84V, and L90M) instigating resistance to the PIs using in silico approach. Six common compounds are retrieved from six databases using combined pharmacophore-based and structure-based virtual screening methodology. LPV and DRV are docked and the binding free energy is calculated to set the cut-off value for selecting compounds. Further, to gain insight into the stability of the complexes the molecular dynamics simulation (MDS) is carried out, which uncovers two lead molecules namely NCI-524545 and ZINC12866729. Both the lead molecules connect with WT and mutant HIV-1 PRs through strong and stable hydrogen bond interactions when compared with LPV and DRV throughout the trajectory analysis. Interestingly, NCI-524545 and ZINC12866729 exhibit direct interactions with I50/50′ by replacing the conserved water molecule as evidenced by MDS, which indicates the credible potency of these compounds. Hence, we concluded that NCI-524545 and ZINC12866729 have great puissant to restrain the role of drug resistance HIV-1 PR variants, which can also show better activity through in vivo and in vitro conditions.