Conceived and designed the experiments: ML M. Kozal. Performed the experiments: JC BS M. Kozal. Analyzed the data: ML JC RY SS VW BS M. Kozal. Contributed reagents/materials/analysis tools: SS JU DS MK MM DM BS ME MK. Wrote the paper: ML RY SS VW JU DS MK MM DM MK.
MJK is the local primary investigator on HIV therapy studies sponsored by Merck, Pfizer, Gilead, Abbott and Bristol-Myers Squibb. MJK receives laboratory research support from Virco and Bristol-Myers Squibb. MJK receives royalties from a patent owned by Stanford University for some HIV diagnostic tests. BS and ME are employees of 454 Life Sciences/Roche. ML, RY, SS, VW, JU, DS, MK, MM, and DM are employees of Bristol-Myers Squibb. The funding of this study by Bristol Myers Squibb does not alter adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
CASTLE compared the efficacy of atazanavir/ritonavir with lopinavir/ritonavir, each in combination with tenofovir-emtricitabine in ARV-naïve subjects from 5 continents.
Determine the baseline rate and clinical significance of TDR mutations using ultra-deep sequencing (UDS) in ARV-naïve subjects in CASTLE.
A case control study was performed on baseline samples for all 53 subjects with virologic failures (VF) at Week 48 and 95 subjects with virologic successes (VS) randomly selected and matched by CD4 count and viral load. UDS was performed using 454 Life Sciences/Roche technology.
Of 148 samples, 141 had successful UDS (86 subtype B, 55 non-B subtypes). Overall, 30.5% of subjects had a TDR mutation at baseline; 15.6% only had TDR(s) at <20% of the viral population. There was no difference in the rate of TDRs by B (30.2%) or non-B subtypes (30.9%). VF (51) and VS (90) had similar rates of any TDRs (25.5% vs. 33.3%), NNRTI TDRs (11.1% vs.11.8%) and NRTI TDRs (24.4% vs. 25.5%). Of 9 (6.4%) subjects with M184V/I (7 at <20% levels), 6 experienced VF. 16 (11.3%) subjects had multiple TAMs, and 7 experienced VF. 3 (2.1%) subjects had both multiple TAMs+M184V, and all experienced VF. Of 14 (9.9%) subjects with PI TDRs (11 at <20% levels): only 1 experienced virologic failure. The majority of PI TDRs were found in isolation (e.g. 46I) at <20% levels, and had low resistance algorithm scores.
Among a representative sample of ARV-naïve subjects in CASTLE, TDR mutations were common (30.5%); B and non-B subtypes had similar rates of TDRs. Subjects with multiple PI TDRs were infrequent. Overall, TDRs did not affect virologic response for subjects on a boosted PI by week 48; however, a small subset of subjects with extensive NRTI backbone TDR patterns experienced virologic failure.
Low abundance drug resistant HIV variants at levels as low as 1% of the circulating viral quasispecies can be detected in antiretroviral (ARV)-naïve individuals by sensitive and quantitative genotyping technologies
Although low abundance resistant variants have been shown to be important in some HIV-infected populations, there are only limited data available on the rate of low abundance transmitted drug resistance (TDRs) mutations in diverse ARV-naïve populations infected with B and non-B HIV subtypes
The CASTLE study was a randomized open label study for treatment naïve persons that compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with tenofovir-emtricitabine (TDF/FTC) in ARV-naïve subjects from 5 continents
In this study, we report the baseline rate of low abundance drug resistant HIV variants detected by ultra-deep sequencing in persons infected with B and non-B subtypes from 5 continents (Africa, Asia, Europe, North and South America). The association of baseline drug resistant variants detected by ultra-deep sequencing on virologic responses at Week 48 in the CASTLE study is explored.
A total of 148 samples were recovered from the CASTLE study sample archives for UDS. All 53 VF specimens and 95 VS specimens were sequenced. The baseline characteristics of these 148 did not differ from the parent study population: mean age 35 years, 34% female, median CD4 cell count 191 cells/mm3. Baseline viral loads ranged from 3030 to >750,000 copies/mL with a median viral load of 132,500 copies/mL.
Of the 148 baseline samples, 90 were subtype B, and 58 were non-B subtype (A, AE, C, BF and F1). UDS results were obtained on 141 of the samples (51 VF and 90 VS, 86 B and 55 non-B subtypes) (
148 samples recovered and sent for UDS: Subtype B (n = 90), Non-B (n = 58); 53 were VF and 95 were VS. 141 samples had UDS data: Subtype B (n = 86), Non-B (n = 55) 51 VF and 90 VS. Of the 7 samples without UDS data, 2 had partial UDS data and were not included; 3 samples were exhausted; and 2 had low viral loads and unable to amplify. VF: Virologic Failure, VS: Virologic Success.
Overall, for subjects with UDS results, 30.5% had a TDR mutation at baseline detected by UDS, and 15.6% had only TDR(s) at <20% rate in the viral population (
WHO TDR Mutation Class | <20% of viral Population |
≥20% of viral Population |
Subjects with any TDR |
Any TDR | 22 (15.6%) | 21 (14.9%) | 43 (30.5%) |
N( |
19 (13.5%) | 16 (11.3%) | 35 (24.8%) |
NNRTI | 12 (8.5%) | 4 (2.8%) | 16 (11.3%) |
PI | 11 (7.8%) | 3 (2.1%) | 14 (9.9%) |
N(
Only TDRs occurring at <20% of the viral population.
At least 1 TDR occurring at ≥20% of the viral population.
WHO TDR ARV class | % of Subjects with a TDR by UDS (n = 141) | % of Subjects with a TDR by SG (n = 147) |
Any TDR | 30.5% | 12.2% |
N( |
24.8% | 9.5% |
NNRTI | 11.3% | 2.0% |
PI | 9.9% | 2.0% |
N(
Of the 141 subjects with a UDS result, 35 (24.8%) had a nucleoside(tide) reverse transcriptase inhibitor N(t)RTI(s) TDR; 26 of these had a thymidine analog mutation (TAM)
Of the 14 subjects with a PI TDRs, 11 (78.6%) had PI TDRs at a <20% levels. The majority of PI TDRs (10/14) were present as solitary PI TDRs (e.g. 24I, 32I, 46I or 58E). Only 4 subjects had >1 PI TDRs: 3 clade B samples with 85V+90M; 46I+84V; and 46I+ 58E+84V+85V+90M; and one clade C sample with 32I+46I. The PI TDR patterns were interpreted for all subjects with the Stanford HIVdb algorithm
Among the 141 samples with a UDS result, there was no difference in the rates of TDRs among those with VS 33.3% (30/90) and VF 25.5% (13/51) by Week 48 (P = 0.35). Rates of TDRs by ARV class in those with VF and VS are shown in
Transmitted Drug Resistance by ARV Class and Virologic Outcome: Note: TDRs are not mutually exclusive (i.e. a subject could have a TDR from more than one ARV class). VS: Virologic Success, VF: Virologic Failure N(t)RTI(s)- nucleoside(tide) reverse transcriptase inhibitor; NNRTI – non-nucleoside reverse transcriptase inhibitor, PI – protease inhibitor. Any: Any transmitted drug resistance mutations. Any TDR: p-value = 0.35, NRTI: p-value = 1, NNRTI: p-value = 1, PI: p-value = 0.02.
Considering specific TDRs that could impact virologic response to the N(t)RTI backbone, TDF/FTC, used in the study, 9 (6.4%) of the subjects had M184V/I identified by UDS at baseline. Seven of 9 M184V/I were at <20% levels and were not detected by standard genotyping. Six of the 9 experienced VF; 4 of these also had TAMs, and 1 had K65R+TAMs (
Specific TDR Mutations | VS (n = 90) | VF (n = 51) | Total # of Subjects with Specific TDR (n = 141) |
M184V/I +/− TAMs +/− K65R | 3 | 6 | 9 |
>1 TAMs | 9 | 7 | 16 |
K65R | 1 | 1 | 2 |
Thymidine Analog Mutations (TAMs); Virologic Success at week 48 (VS), Virologic Failure at week 48 (VF). Transmitted Drug Resistance Mutations (TDRs); 9 subjects with M184V/I +/− TAMs and/or +/− K65R at baseline. 16 subjects with >1 TAMs at baseline. 2 subjects with K65R at baseline.
ID | Clade | 48 w | VL | TAMs | TDRs at baseline with UDS |
1 - ATV | B | VF | 334,000 | 1 | |
2 - ATV | B | VS | 545,000 | ||
3 - ATV | B | VF | 193,000 | 5 | |
4 - ATV | B | VS | 168,000 | ||
5 - ATV | B | VF | 158,000 | 2 | |
6-LPV# | C | VF | 750,000 | 3 | |
7 - LPV | B | VF | 35,000 | ||
8 -LPV | B | VF | 37,000 | ||
9 - LPV | C | VS | 38,100 | 1 | |
10 - ATV | BF | VS | 8840 | 6 |
VF: Virologic Failure VS: Virologic Success NRTI: Nucleoside Reverse Transcriptase Inhibitor NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitor PI: Protease Inhibitor. TAMs: Thymidine Analogue Mutations. HIV VL: HIV Viral Load. TDRs: Transmitted Drug Resistance Mutations. 6-LPV# has both a M184V and a K65R TDR.
Among a representative sample of ARV-naïve subjects from the CASTLE study, HIV variants possessing TDR mutations were commonly detected by UDS. Similar rates of TDRs were identified in B and non-B subtypes. Although the study was not designed to evaluate TDRs by continent nor specific subtype, at least one sample from ARV-naïve subjects from 5 continents (Africa, Asia, Europe, North and South America) had a TDR by UDS. These data suggest that among subjects entering large international clinical trials, TDRs are common in ARV-naive persons from diverse populations and infected with B and non B subtypes. Many of these subjects harboring HIV variants with TDRs were not detected by standard genotyping. The overall TDR rate of 30.5% by UDS among ARV-naïve subjects in our study is similar to other investigations that reported rates between 28 to 33% depending on the populations studied and the sensitive genotyping methods used to identify low abundance resistant variants
When considering TDRs both overall and by specific ARV class, TDRs were not associated with a lower virologic response for subjects initiating a boosted PI-based regimen. However, a small subset of subjects with specific N(t)RTI TDR patterns that could impact the activity of the TDF/FTC backbone used in the study did experience a higher rate of virologic failure. Interestingly, subjects with a PI TDRs identified by UDS at baseline did well on a boosted PI regimen. Possible reasons for this apparently non-intuitive finding include: 1) most PI TDRs were found in isolation (few subjects had multiple PI TDRs), 2) the PI TDRs identified had low Stanford HIVdb scores
There was no difference in the rate of NNRTI TDRs in subjects with VS or VF with approximately 11% having a TDR NNRTI mutation. This data suggests that if a person harbors NNRTI resistant variants, a boosted PI regimen may be successful at suppressing the resistant variants. It has been previously shown that many of the low abundance resistant variants identified by UDS may not affect virologic outcomes if the resistant variants identified are only resistant to ARVs that are not part of the antiretroviral regimen being employed
Although there was no difference in the rate of N(t)RTI TDRs in those subjects with VS or VF, there were subjects with extensive N(t)RTI TDR mutations which are known to impact responses to TDF/FTC. There was a high rate of VF in the small number of subjects with a M184V, even when the TDR was present at <20% levels of the quasispecies and missed by standard genotyping methods. Subjects with multiple TAMs also had disproportionate rates of VF. These results suggest that M184V and specific patterns of N(t)RTI mutation patterns may contribute to predicting response to a boosted PI regimen. However, the number of subjects in this study is small, and more studies should be done to investigate the impact of specific N(t)RTI TDR patterns present in low abundance on virologic responses in subjects initiating boosted PI regimens.
A strength of this study was that the patient population was recently sampled (enrolled between 2005–2006), well characterized, and represented multiple diverse ARV-naïve groups and HIV subtypes. A limitation of this study was that it was a retrospective case control study with the inherent limitations of such a design. The study design allowed us to explore the association of TDRs with clinical responses with a boosted PI regimen and the rate of TDRs in B and non B subtypes. However, investigations into the prevalence of TDRs across continents or patterns of specific TDRs by HIV subtype could not be definitely defined given that the numbers in each category were too few. Future studies should be performed to determine the prevalence of TDRs by UDS in larger retrospective and prospective studies.
Also, recent reports have suggested that the absolute number of viral variants with a TDR mutation, or “mutational load,” may impact the time to virologic failure in a person on therapy
Further studies should be performed to determine how the viral factors of mutational load, mutation linkage (mutations within the same viral genome), and specific mutational patterns interact and impact treatment responses to the many different antiretroviral regimens now used in the clinic. These important virologic parameters will need to be better defined, and the resistance interpretation algorithms accordingly adapted before sensitive genotyping technologies can be incorporated into routine HIV clinical care.
In summary, among a representative sample of ARV-naïve subjects in the CASTLE study, transmitted drug resistance mutations identified by ultra-deep sequencing were common, and B and non-B HIV-1 subtypes had a similar rate of TDRs. TDRs identified by UDS did not affect virologic response for subjects on a boosted PI. However, a small subset of subjects with extensive N(t)RTI backbone TDR patterns were likely to experience virologic failure. Further investigations should be performed to determine the prevalence of TDRs in diverse populations of HIV infected persons across the different regions of the world. Studies should also be done to determine which ARV regimens are best at suppressing HIV variants possessing TDRs at different variant levels and mutation patterns.
The CASTLE study was a 96 week study comparing the antiviral efficacy and safety of ATV/r with LPV/r, each in combination with TDF/FTC in HIV-infected treatment-naïve subjects. The primary objective of the study was to compare the proportion of subjects with HIV RNA levels <50 c/mL at Week 48 between the ATV/r+TDF/FTC and LPV/r+TDF/FTC regimens. The primary analysis of the study confirmed the hypothesis that ATV/r/TDF/FTC was non inferior to LPV/r/TDF/FTC with 78% and 76% of subjects respectively, having HIV RNA <50 c/mL at Week 48. Subjects were enrolled from 5 continents (Africa, Asia, Europe, North and South America) representing 28 countries and had given informed consent
Based on prior studies
Subjects were selected from all 5 continents, and 6 different HIV subtypes were represented. Baseline demography, CD4 cell count and viral load values, and baseline HIV genotype by standard genotyping (Monogram Biosciences, CA) were obtained from the clinical trial database.
Coded baseline plasma samples were sent to Yale for ultra-deep sequencing (UDS) using 454 Life Sciences sequencing center (Branford, CT). The method for UDS has been described elsewhere
Standard genotypic resistance mutation results were determined using Monogram Biosciences GeneSeq technology (South San Francisco CA)
The 2009 World Health Organization reference list of transmitted drug resistance mutations (TDRs) was used to evaluate for the rate of mutations detected by standard and ultra-deep sequencing
Proportions of subjects with TDRs detected by UDS were presented overall and by HIV subtypes, regions, and outcomes at Week 48 (VF or VS). Specific TDR mutation classes (NRTI, NNRTI and PI) were presented similarly. The proportions of subjects with TDRs (all mutations and by specific ARV class) at <20% of the viral population were further calculated. Proportions of subjects with TDRs by B vs. non-B subtypes were compared using Fisher's exact test
The parent CASTLE study is registered with ClinicalTrials.gov, number NCT00272779.