In this study, we evaluated the yield of HIV genotyping of DBS samples obtained from HIV-infected children enrolled in clinical trials in sub-Saharan Africa (1
). HIV genotypes for reverse transcriptase were successfully obtained for 94 (90%) of 105 samples analyzed, despite collection and long-term storage in countries with differing temperature, humidity, and storage conditions. However, it is important to emphasize that three different primer sets were required to achieve this success rate, with 16.1% (15 of 94 samples) failing to amplify on the first attempt and requiring a second step of testing (one-step PCR on previously synthesized cDNA with a different method that produced shorter, overlapping amplicons). While incorporation of repeat testing with multiple short amplicons enhanced the recovery of genotypes in the samples tested from the first clinical trial (tested using assay #1), switching to assay #3, which included a combination of the outermost primers of the short, overlapping primers that we previously reported for pyrosequencing of subtype C HIV reverse transcriptase (10
) and involved testing of samples from five countries (South Africa, Uganda, Malawi, Zimbabwe, and Tanzania), increased the yield of genotyping to nearly 90% for samples that were stored optimally. The addition of the repeat testing increased the success rate by 3% for optimally stored samples and 13% for those that were suboptimally stored, highlighting the importance of optimal storage conditions for the genotyping of DBS. Importantly, with assay #3, a subset of the samples from the P1060 trial had matched plasma samples that were analyzed previously using an FDA-cleared HIV genotyping kit. For the pretreatment samples that were analyzed with both methods in a blind manner, results from DBS genotyping were 100% concordant with plasma genotyping for major drug-resistant mutations obtained using the FDA-cleared genotyping kit. Together, these findings suggest that the primers used in assay #3 may be suitable for application in sub-Saharan Africa.
There are several limitations to this study. First, all of the samples tested were obtained from infants and children who were not receiving antiretroviral therapy at the time of sample collection. Therefore, HIV viral loads in the samples were high, facilitating HIV nucleic acid recovery and amplification from DBS samples. The proportion of samples successfully genotyped with DBS may be lower when samples with lower viral loads (e.g., those obtained at the time of treatment failure) are tested. Second, most of the samples analyzed did not have drug resistance mutations; further evaluation of the sensitivity for detecting mutations using DBS samples is needed, using samples with a variety of drug resistance mutations present at different levels in the viral population. Third, the methods used in this report provide information for HIV reverse transcriptase only. Methods that include analysis of HIV-1 protease are needed, particularly since the WHO now recommends protease inhibitor-based regimens for infants and children with prior exposure to nevirapine prophylaxis (14
); NVP-based regimens are widely used for prophylaxis in resource-constrained countries. Analysis of both HIV reverse transcriptase and protease from DBS samples is likely to require amplification of multiple amplicons because HIV RNA can become degraded during storage and/or extraction; in contrast, commercially available assays that use plasma for analysis typically obtain information for both HIV protease and HIV reverse transcriptase from a single amplicon. Fourth, the methods used in this report rely on nested PCR for DNA amplification; nested PCR can increase the risk of sample cross-contamination and may bias the proportion of viral variants represented in the amplified DNA product. Finally, we used three different methods for RT-PCR (different primer sets and amplification conditions); different numbers of DBS spots were also used for testing. Therefore, the amounts of blood used for analysis varied among the samples tested.
Nevertheless, the overall success of obtaining genotypes in this study and the high concordance with plasma genotypes coupled with the fact that many laboratories in resource-constrained settings are already familiar with use of DBS samples for infant diagnostic testing (6
) suggest that in-house DBS genotyping assays provide an alternative for antiretroviral drug resistance testing in children in resource-constrained regions but may require region-specific optimization before widespread use.