We have shown, as have others [10
], that viral mutations associated with nevirapine resistance are much more common 6 weeks after sd-NVP than suggested by conventional sequence-based genotyping. Using an allele-specific real-time PCR assay for the most common mutation (K103N), we found almost all women had detectable levels of this mutation at 6 weeks. K103N is important since it is associated with resistance to nevirapine and efavirenz, which are both popular NNRTI for therapeutic regimens and it is also the most persistent [15
]. The nested ‘in-house’ population-sequencing method we used on these same samples detected mutations only if present in > 0.1 of the viral population. This is a slightly greater sensitivity than has been observed with other commercial population-sequencing assays possibly because of the nested PCR reaction and shorter target region. However, the lower limit of detection of the allele-specific assay was 0.002 which is at least 50-fold more sensitive than our ‘in-house’ assay and closer to 100-fold more sensitive than commercial genotyping assays.
Over time both detection of K103N-resistant variants and the proportion of the viral RNA population found to harbor these variants declined rapidly in plasma. By 12 months after sd-NVP, our sensitive allele-specific assay could detect K103N variants in only 11% of RNA samples, and these variants were present at low frequency. It remains to be determined whether resistant variants at such low levels have clinical consequences. A study in Thailand showed that women who received nevirapine for prevention of mother-to-child transmission had a poorer virological response to treatment than unexposed women even in the absence of detectable resistance mutations [17
]. These results imply a clinical role for these low frequency mutants. However, the Thai study also found that among the nevirapine-exposed, those without detectable resistance mutations were more likely to achieve virologic suppression after treatment than women with detectable resistance [17
]. As shown here, women without detectable K103N by conventional genotyping have resistant variants at significantly lower frequency than those detected by conventional genotyping. Thus the frequency of resistant variants may influence treatment response in a dose-dependent manner. Clinical studies should investigate specifically whether sd-NVP has any influence on women’s response to an NNRTI-treatment regimen when started more than 12 months later by which time clinical consequences, if any, may be small.
There were appreciable individual differences in the frequency and persistence of resistant variants. Women who had higher frequencies of mutant viral RNA at 6 weeks (who would have been detected by conventional genotyping) were more likely to have resistance variants detected at later time points. However, the rate of decline was faster among those starting with higher frequencies. These results are consistent with more rapid viral kinetics being associated both with higher peak frequencies of resistant variants and with their more rapid decline. The net result was that for most women resistant variants had declined below detection by one year. Individual variation in drug clearance, viral dynamics or host immune factors may explain the small number of women who displayed an increase in the frequency of K103N variants between 6 weeks and 3 months before declining. Our data also suggest that markers of more advanced disease, including low CD4 cell count and higher viral load, tended to be associated with persistence of resistance mutations. Further clarification of these predictors may have clinical utility. These associations also caution against uncritical comparison across different clinical cohorts since the profile of disease within cohorts may affect the extent of resistant mutations observed.
A limitation of our study is that we examined only the most common resistant variant, K103N. Other mutants may show different dynamics due to differences in viral fitness [18
]. A further limitation is that pre-treatment samples were not available. Although resistance mutations may exist at very low frequencies even in the absence of drug pressure [19
] other studies among drug-naive, subtype C-infected women have not detected K103N mutations [11
]. This suggests that pre-existing mutants, if present, are below the detection threshold of even these kinds of assays. Resistance mutations and other polymorphisms may occur in primer binding sites which could affect the sensitivity of the real-time PCR assay. However, we designed subtype C-specific PCR primers in highly conserved regions and single nucleotide changes would not result in complete loss of primer binding. These limitations seem unlikely to explain the dramatic changes in the detection and frequency of K103N variants as time elapsed after sd-NVP.
Our study documented that even once mutants are no longer detectable in plasma viral RNA, persistence in cellular DNA can occur after sd-NVP. However, this could be detected in less than 5% of women. We only examined persistence in the total leukocyte pool and therefore cannot rule out whether there might be mutants present in other cell reservoirs or in lymph nodes. A limitation is that we were unable to quantify cellular HIV DNA copies for input into our PCR. Furthermore it was not possible to determine whether viral DNA was integrated or extra-chromosomal. These factors may have resulted in an underestimation of K103N in DNA. It will be important to test in clinical studies whether the presence of these variants in DNA identifies women most vulnerable to failing NNRTI-based regimens.
Our data suggest that although readily selected by sd-NVP therapy, resistant viral genotypes do not persist for long periods in peripheral blood viral RNA and, although persistence in the cellular DNA compartment occurs, it can be detected in only a minority. If these markers predict subsequent response to NNRTI treatment, when treatment is started more than 12 months post-delivery, previous exposure to sd-NVP may affect treatment responses among only a minority. This is reassuring since the numbers of women needing treatment in the immediate post-partum period will decrease as HIV care programs are implemented more widely and women who meet criteria for drug treatment (low CD4 cell count and symptomatic disease) are started on treatment before or during pregnancy. Furthermore, as subsequent deliveries will generally occur only at some interval of more than 12 months the consequences of previous exposure to sd-NVP for future transmission may be less than anticipated.