With the improved availability of generic HAART in India (Kumarasamy et al., 2005
), the demand for accurate but affordable monitoring of patients under treatment is becoming a priority (Kent et al., 2003
). Indeed, increasing access to antiretroviral drugs without adequate monitoring may well result in transmission of drug resistant virus which has been observed in North America and Europe (Bennett et al., 2006
; Geretti, 2007
). The transmission of drug-resistant HIV strains in developing countries may be mitigated by measures that provide guidance for the prescription of highly effective drug combinations, promote adherence and, support the uninterrupted supply of the drugs. The availability of laboratory methods to monitor side effects and the success of treatment and clear guidelines for actions in case of therapeutic failure are also critical. WHO guidelines (WHO, 2008
) suggest that monitoring the success or failure of therapy should include CD4 count at minimum and viral load determinations where possible. When viral load monitoring is performed, genotyping of virologic failures can guide further treatment decisions and provide a direct measure of drug resistance. However, access to commercial assays for viral load monitoring and drug resistance in India is limited primarily by cost.
The present study demonstrated that there was a high concordance between ViroSeq™ and in-house genotyping systems. Mutations associated with the patient’s current failing therapy were more consistently identified by each assay and could be reproduced when repeated independently. Discordant mutations were identified to be less than 0.5% and mainly corresponded to secondary mutations and polymorphisms. ViroSeq™ has detected more mixtures (78%) of WT and mutant virus than by the in-house genotyping (22%), suggesting possibly increased sensitivity of commercial assay in terms of detecting minority variants. In addition to the design of the amplification and sequencing primers there are also differences in the labour and analytic time between the genotyping systems. One advantage of the ViroSeq™ is that it comes with the software that is designed specifically for editing and resitance interpretation, while the in-house assay requires a separate software for assembling sequenced fragments, manual editing and exporting consensus sequence, which needs to be considered. However, this software (SeqScape v2.5), is relatively user friendly and allows simple compilation of all sequence fragments from one sample into one contig, inspection of the sequence traces, and translation and check for frame shifts. The high concordance of mutations (99%) detected by the in-house assay (using SeqScape v2.5 package and Stanford HIV DB) and the ViroSeq™ system showed that the two systems had similar performance on interpreting all the clinically important resistance mutations. There was no significant difference in the time scales required for the assay process to be completed by both the systems. The turn around time for the in-house assay versus the standard assay was 16 and 15 h, respectively.
On the other hand, when genotyping assays are performed, it is important to minimize contamination of reaction mixtures with previously amplified products. Sample cross-contamination is minimized in the ViroSeq™ system by using a single non-nested PCR for amplification, and by using a dUTP/UNG contamination control system. However, the in-house assay, which uses nested PCR amplification and which does not have UNG based contamination control system, requires more vigilance to avoid cross-contamination and strict adherence to good clinical laboratory practices (GCLP) is crucial.
To conclude, the in-house assay performed well in comparison to a gold-standard, US-FDA approved ViroSeq™ system. All the clinically relevant mutations were concordant by both assays and reproducible. Despite very minimal discrepancies, the in-house assay, which costs about 50% ($ 100) of the ViroSeq™ ($ 230), demonstrated a similar capacity to identify clinically relevant mutations compared to the ViroSeq™. Studies spanning a wider spectrum of viral loads and known quantified resistant mutants are required to compare the analytical sensitivity for detection of HIV-1 drug resistance mutations. In addition to this, comparison between the two assays using a wider panel of the different HIV-1 subtypes and CRFs would be useful. Additional issues include the development of new and emerging assays for minority variants (Church et al., 2008
; Johnson et al., 2007
; Villahermosa et al., 2001
; Beck et al., 2002
; Vega et al., 2005
) and assays for genotyping in integrase and envelope, particularly as new drugs against these targets become available (Grant and Zolopa, 2008
; Paar et al., 2008
; He et al., 2008
). In-house approaches to implementing molecular diagnostics can build capacity and provide training in resource-limited settings at a cost substantially lower than commercial assays ($ 100 vs. $ 230). The successful validation and implementation of the in-house assay will also serve to lower the prices and increase competition among commercial suppliers. Wide applications of low-cost genotyping will facilitate surveillance studies and improve clinical decision making for HAART, and thus reduce the risk of further drug resistance-related mutations.