A total of 32 pairs of vRNA and proviral DNA protease (PR) and reverse transcriptase (RT) sequences from 25 patients were analyzed. Nineteen patients provided a single samples, 5 patients two sequential yearly samples and one patient was sampled three times at one-year intervals. Median age of the patients was 37.5 (range 16–61) years, 12 males and 13 females with a CD4 count median of 148 (range 3–459) and viral load log median of 4.95 (range 2.58–5.50) ().
Of the 25 subjects enrolled, 21 reported taking prescribed ART regimens within the previous 3 months and 4 patients reported they had not used ART. There were 6 samples obtained from these 4 subjects, with no evidence of drug resistance. The ART regimens of the 21 patients included protease inhibitors (PI) in 19, non-nucleoside reverse transcriptase inhibitors (NNRTI) for 13 and all 21 treated individuals had received NRTIs (see ).
Initially, vRNA and proviral DNA pol sequences were compared at the amino acid level. Sixteen patients had a record of taking protease inhibitors (PI) (19 samples). Ten samples had mutations in the protease gene (). Of these samples, 5 had discrepancies between RNA and DNA mutations. Only one of these 5 (TC049) had a mutation in the DNA pol sequence that was not identified in the matching RNA sequence. However, this sample had two mutations identified in RNA sequence that were not identified in DNA. The remaining 4 sequences (TC045, TC002, TC060, and TC216) had at least one mutation in RNA that was not identified in the matching DNA sequence.
PI, NNRTI and NRTI drug-resistant mutations.
Thirteen patients had received non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs. Overall, 15 patients (16 samples) had NNRTI mutations (). Seven of these 16 pairs had mutations in proviral DNA pol gene sequence not found in RNA sequence. Only 3 of these 16 had mutations in RNA that were absent in DNA (TC060, TC109 and TC111). Sample TC060 had a V108VI mutation in RNA not found in DNA as well as an Y181C mutation in DNA that was not observed in RNA.
Similar to the situation for NNRTIs, analysis of nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations revealed genotypes that differed greatly but with identical phenotypes as measured by resistance scores (). For example, pair TC041 had completely different NRTI resistance mutations identified in RNA and DNA sequences. The RNA sequence identified mutations A62AV, K65R, and K219KQ while the DNA sequence contained L74LV, V75AV, Y115FY and M184MV. Accordingly, while different NRTI resistance mutations were found in DNA versus RNA, the estimated resistances to most NRTIs were similar.
The difference in genotypes between plasma vRNA and PBMC DNA samples were often due to mixtures of wild type and mutant amino acids for a specific resistance position. Of the mutations found only in RNA, 54% were mixtures (14 of 26 mutations) and of those found only in DNA, 47% were mixtures (9 of 19 mutations). In contrast, only 34% of mutations found both in RNA and DNA were mixtures (33 of 96 mutations).
Most samples with different genotypes were similar in predicted phenotype as assessed by the estimates of drug resistance. For example, pair TC012 differed between RNA and DNA in genotype sequence (the mixture of mutation K238KQ at a resistance associated codon was found in DNA but not in RNA). However, once analyzed using the algorithm, both sequences indicated susceptibility to all four NNRTIs.
Numerical resistance score
In examining drug resistance mutations to PIs, 4 pairs (TC060, TC106, TC110, and TC216) demonstrated differences in resistance scores for at least one drug. Only two pairs (TC106 and TC216) had differences in resistance scores greater than 1. Overall, 28 out of 32 pairs had the exact same resistance scores for each of the protease inhibitors for both RNA based and DNA based sequence.
Similarly, when examining resistance mutations to NNRTI drugs, a total of 8 pairs contained a difference in resistance scores for at least one drug of the four analyzed in the algorithm. Two samples (TC070 and TC111) had a difference in resistance score of 2 or greater between RNA- and DNA-based sequences for at least one drug. In general, 24 pairs had the exact same resistance scores for all four drugs.
However, 17 pairs had at least one NRTI drug that had a different resistance score in RNA compared to DNA sequence. Of these 17 pairs, 10 had a difference of 2 or greater for at least one drug analyzed by the algorithm. Fifteen pairs had the exact same numerical resistance score for all 7 NRTIs.
Collapsed resistance score
When the scores were condensed to indicate whether or not the sequence suggested susceptibility or resistance to a specific drug, 3 of 32 pairs demonstrated disagreement between the RNA and DNA sequence for at least one PI (TC060, TC106, and TC216). In all cases, RNA sequence was resistant and DNA sequence susceptible.
Similarly, 5 of the 32 pairs were discordant between RNA and DNA sequence for at least one NNRTI drug included in the algorithm (TC056, TC060, TC070, TC111 and TC118). All 5 samples differed with respect to resistance scores for only one drug and 4 of them were different because of resistance in DNA sequence and not in RNA.
With respect to resistance to NRTI drugs, 11 of the 32 pairs had differences in the collapsed resistance scores of at least one drug. Of these 11, 5 were a result of ABC, TDF and DDI resistance identified in RNA but not in DNA sequence. However, there were 3 pairs in which resistance to at least one NRTI drug was found in DNA and not in RNA. Resistance mutations to 3TC and FTC were generally found together. Similarly, ABC, TDF, DDI and sometimes D4T resistances were also detected together.
Furthermore, differences in resistance scores between plasma vRNA and PBMC DNA for a specific drug class did not correlate with differences in drug resistance mutations in another drug class. Only 4 out of the 32 samples had discrepancies in more than one drug class.
Treatment-specific resistance score
The treatment-specific analysis of resistance, in which the efficacy of current treatment was evaluated, indicated the most similarity between RNA and DNA sequence. Through this analysis, only pair TC106 differed in RNA and DNA sequence, where for current treatment the RNA sequence for this sample scored 0.67 out of 1 for resistance, while DNA scored 0.33.
Phylogenetic evolutionary analysis
Phylogenetic analysis was used to see how closely the sequences in the study were related to one another. Both genetic distances and maximum likelihood trees were generated. The maximum likelihood phylogenic tree is seen in the figure. All of the RNA and DNA sequence pairs were located close to one another on the same branch in the maximum likelihood tree.