Prevalence of codon 215 revertant mutations.
Between 1999 and 2007, plasma samples from 156 (3.4%) of 4,533 KPNC persons undergoing genotypic resistance testing at SUH had one or more sequences with a codon 215 revertant. Seventy-three of these persons were ARV naive at the time their plasma was submitted for sequencing, including 68 whose samples did not also have a Y or F present (in combination with a revertant). The sequences from these 68 persons included 19 with T215D, 15 with T215E, 12 with T215S, 10 with T215C, 4 with T215V, 2 with T215L, 1 with T215N, 1 with T215I, and 4 with mixtures of revertants including SC (2 sequences), DE (1), and DA (1).
Among the 4,533 KPNC persons undergoing genotypic resistance testing between 1999 and 2007, 1,339 persons were ARV naive at the time of their genotyping; 2,120 persons were ARV experienced at the time of their first genotyping; and definitive treatment histories were not available for 1,074 persons although the majority of these persons had some form of poorly documented ARV therapy. Thus, the prevalence of T215 revertants in ARV-naive persons during this 9-year period was 5.1% (68/1,339) among those known to be ARV naive and no lower than 2.8% (68 divided by the sum of 1,330 ARV-naive persons plus the 1,074 persons for whom treatment was not definitively known).
Availability of samples with codon 215 revertant mutations.
Plasma HIV-1 RNA levels were ≥4.5 log copies/ml in 46 of the 68 persons with T215 revertants. From these 46 persons, samples with ≥500 μl of cryopreserved plasma were available for UDPS in 25 cases. From these 25 samples, ≥40 viral templates could be extracted and amplified from 22. The median number of extractable and amplifiable virus templates was lower for the first 8 samples for which PfuUltra was used for amplification (80; range, 40 to 160) than with the subsequent 14 revertant samples for which Expand High-Fidelity PCR was used (160; range, 120 to >480) despite the fact that multiple reverse transcription and PCRs were performed for several viruses in the first set of samples.
Clinical characteristics of persons with codon 215 revertant samples undergoing UDPS.
Table shows the year of sampling, the plasma HIV-1 RNA levels, and the CD4 counts for each of the persons with T215 revertants that were available for UDPS. Four persons had been acutely infected within 1 year prior to genotypic resistance testing, three additional persons had been infected some time within the 3 years prior to testing, seven had been infected 2.5 or more years prior to testing, and no information on the timing of infection could be gleaned from the chart review of eight persons.
Clinical characteristics and UDPS results for 22 antiretroviral-naive persons with T215 revertants by standard direct PCR Sanger sequencing
Direct PCR sequence results of samples with T215 revertants.
Each of the 22 sequences belonged to subtype B. Direct PCR sequencing revealed a median of eight differences from the consensus B sequence (range, 2 to 15) including the following 215 revertants: T215C in six persons, T215S in six persons, T215D in four persons, T215E in four persons, T215DE in one person, and T215DA in one person. Seven of 22 samples also had one or more established RT inhibitor resistance mutations by direct PCR sequencing (Table ) including the nucleoside RT inhibitor (NRTI) mutations M41L (three persons), D67N (one person), L210W (one person), and M41L D67N T69D L210LW (one person) and the non-NRTI (NNRTI) resistance mutation V108I (1 person). In addition, three persons had the nonpolymorphic NRTI-associated mutation H208Y, occurring alone in two persons and with V108I in another person. Protease inhibitor resistance mutations were not identified in any of the 22 T215 revertant samples.
The sequences from samples 7900, 25674, and 27979, which were obtained in 2000, 2005, and 2006, respectively, differed by a mean 1.4% of their nucleotides and formed a clade with 100% bootstrap support in a neighbor-joining tree created from the 22 sequences in this study and 200 randomly selected sequences from untreated persons sequenced at SUH (data not shown). The possibility of an epidemiologic relationship between the persons from whom these samples were obtained was not investigated.
UDPS results of samples with T215 revertants.
UDPS identified all 133 RT mutations (6.0 per sample) detected in pure form by direct PCR sequencing and 22 of the 24 mutations detected as part of an electrophoretic mixture. UDPS detected at a prevalence of ≥2.0% a mean of 3.8 RT mutations that were not detected by direct PCR sequencing (range, 0 to 12). UDPS also detected at a prevalence of ≥2.0% the consensus B amino acid at a mean of 1.0 RT positions for which the direct PCR sequence contained a pure mutation (range, 0 to 4) and a mean of 6.8 silent mutations per sample (range, 0 to 35).
UDPS detected additional RT inhibitor resistance mutations in samples from 10 persons including additional T215 revertants in 4 persons, other NRTI resistance mutations in 4 persons, and the minor NNRTI resistance V108I mutation in 2 persons. The additional T215 revertants (Table ) included T215S in 25% of sequence reads of sample 26420, T215D in 7% of sequence reads of sample 3930, T215C in 1.0% of sequence reads of sample 16412, and T215E and T215S at 1.1% and 1.0%, respectively, of sequence reads of sample 14661. The six additional samples with non-T215-revertant RT mutations included 1816 and 25685, which contained M41L in 11% and 2% of sequence reads, respectively; 23835, which contained M1841 in 1.1% of sequence reads; 30154, which contained K70R, V751, and F77L in 4%, 1.0%, and 1.0%, respectively; and 25674 and 39210, which contained V1081 in 3% and 1.8% of reads, respectively.
T215Y and T215F were not detected at a level above 0.1%, a level far below the reliability cutoff, in any sample. One additional T215 revertant mutation was detected in the sample 14661, T215N at 0.8%. No protease inhibitor resistance mutations were detected at a level ≥1.0% in any of the 22 samples. Of 83 mutations detected only by UDPS, 79 (95%) were present as the dominant variant in ≥0.1% of about 15,000 pooled treated and untreated persons with group M HIV-1 infection in the Stanford HIV Drug Resistance Database (19
Confirmatory limiting dilution and molecular clonal sequencing.
Limiting dilution sequences was determined on the three samples for which additional NRTI mutations were detected by UDPS. For sample 1816 which contained M41L in 11% of pyrosequencing reads, 20 clonal and 4 oligoclonal sequences were performed. M41L was detected in 3 of 20 clones, and M41M/L was detected in one of four oligoclonal sequences. Of the additional RT mutations detected only by UDPS, E36K (2% of pyrosequencing reads), K102R (6%), D123E (6%), I142V (12%), and K173R (15%) were detected in one or more limiting dilution sequences whereas E28Q (2%), E40A (2%), and E42K (2%) were not detected in 20 clonal and 4 oligoclonal sequences.
For sample 3930 which contained T215D in 7% of pyrosequencing reads, 45 clonal and 13 oligoclonal sequences were performed. T215D was detected in 2 of 45 clones, and T215D/C was detected in 2 of 13 oligoclonal sequences. Of the additional RT mutations detected only by UDPS, K20R (21% of pyrosequencing reads) was detected in nine clonal and four oligoclonal sequences.
For sample 14661, which contained T215S (1.0%), T215E (1.1%), and T215N (0.8%), each present in about 1% of pyrosequencing reads, 43 clonal and 8 oligoclonal sequences were performed. None of these mutations was detected in the 43 clonal sequences; T215E was detected in two of eight oligoclonal sequences. Of the additional RT mutations detected only by UDPS, I178M (3%), T200A (4%), R172K (6%), and T200I (4%) were detected by limiting dilution sequencing.
For sample 30154 which contained K70R, V75I, and F77L in 4%, 1%, and 1% of pyrosequencing reads, 146 molecular clones, encompassing RT positions 1 to 134, confirmed the presence of K70R in eight clones and V75I and F77L each in one (but not the same) clone. Of the additional RT mutations between positions 1 to 134, T69A (3%), E79D (8%), and V118I (3%) were present in 1, 15, and 3 clones, respectively.
Direct PCR and UDPS sequence results obtained on the 29 control samples.
UDPS detected all 116 RT mutations detected in pure form by direct PCR sequencing of the 29 control samples (3.9 per sample) and 37 of 41 mutations detected as part of an electrophoretic mixture. UDPS detected at a prevalence of ≥2.0% a mean of 4.8 mutations that were not detected by direct PCR sequencing (range, 0 to 11) (see Table S1 in the supplemental material). UDPS also detected at a prevalence of ≥2.0% the consensus B amino acid at a mean of 1.1 RT positions at which the direct PCR sequence contained a pure mutation (range, 0 to 5) and a mean of 6.4 silent mutations (range, 0 to 54).
In four control samples, UDPS detected additional RT inhibitor resistance mutations including D67N in 5% of reads in sample 16356; D67N, V108I, and H208Y at 3%, 1.1%, and 1.2%, respectively, of reads in sample 9918; K65R at 1.7% in sample 9635; and the NNRTI-resistance mutation G190E in 1.5% of sample 14177. No protease inhibitor resistance mutations were detected at a level above 1.0% in any control sample. T215Y and T215F were not detected at a level above 0.1%, a level far below the reliability cutoff, in any sample. T215S was detected at a level of 0.8% in one sample. Of 138 mutations detected only by UDPS, 128 (93%) were present as the dominant variant in ≥0.1% of about 15,000 pooled treated and untreated persons with group M HIV-1 infection in the Stanford HIV Drug Resistance Database (19
The prevalence of minority RT inhibitor resistance mutations at additional RT positions was not significantly higher among the 22 revertant samples (6/22, or 27%) compared with the 29 control samples (4/29, or 14%; P = 0.4). However, if the additional T215 revertants (found in four revertant samples) were considered RT inhibitor resistance mutations, then the difference between the two populations would be significantly different (10/22, or 45%, versus 4/29, or 14%; P = 0.03).
Response to ARV therapy.
Table summarizes the treatments and virological responses of the 22 persons with T215 revertants. Fifteen of the persons received one or more ARV regimens within the year following their genotyping. Ten treated persons sustained complete virological suppression, defined as multiple persistent plasma HIV-1 RNA levels below 75 copies/ml, the lower limit of detection of the bDNA assay (Versant HIV-1 RNA 3.0 assay; Siemens, Berkeley, CA). Two treated persons had initial virological responses followed by planned treatment interruption and virological rebound. Three persons experienced virological failure with the development of resistance. In sample 9650, virological failure occurred after 44 months of virological suppression. However, in samples 16412 and 30154, virological failure occurred upon initial therapy.
ARV therapy and virologic response in 22 persons with T215 revertant mutations undergoing UDPS
Person 16412, whose sequence contained T215S, the NRTI resistance mutation D67N, and the unusual mutation K70S, developed virological failure after treatment with zidovudine plus lamivudine plus nelfinavir. A phenotypic assay following virological failure showed reduced susceptibility to lamivudine and nelfinavir. However, subsequent therapy with tenofovir plus didanosine plus lopinavir-ritonavir led to complete virologic suppression which has persisted for 48 months. Person 30154, whose sequence had T215E and three clonally confirmed minor variants (K70R, V75I, and F77L), developed successive virological failures with tenofovir plus emtricitabine plus efavirenz followed by tenofovir plus emtricitabine plus atazanavir-ritonavir. Genotyping following the second virological failure demonstrated the NNRTI resistance mutations K103N and P225H and the NRTI resistance mutations M184I. V75L but none of the original minority variants (K70R, V75I, and F77L) was detected in this follow-up sample.