In this study, we report the development of a broadly sensitive RT-PCR-based genotyping assay for surveillance and monitoring of HIV-1 DR in the pol
gene region. By applying this assay, we genotyped DBS from 3 countries for transmitted HIV-1 DR. This study confirms and extends previous reports that DBS are a useful sample collection device for HIV-1 DR genotyping analysis (5
). However, the current study provides additional support for the use of DBS for HIV DR genotyping in several ways. (i) Samples were collected under routine clinical conditions and transported to CDC Atlanta for genotyping. (ii) We genotyped multiple HIV-1 group M subtypes and CRFs in samples from China, Cameroon, Malawi, and Tanzania, reported in this study, and also from many other countries, including Botswana, Honduras, Ethiopia, Kenya, Nigeria, Thailand, and Zambia (data not shown). Together, these results indicated that this genotyping assay is broadly sensitive for all the major HIV-1 subtypes and CRFs. (iii) The assay is one-step RT-PCR-based and covers both the protease (amino acids [aa] 13 to 99) and RT (aa 1 to 251) regions, which results in the reduction of genotyping cost and the possibility of laboratory-related cross-contaminations because fewer hands-on manipulations are needed. Compared to commercially available genotyping assays, our assay reduces the cost for genotyping reagents by almost 50%. (iv) Lastly, the assay can be successful in genotyping at least 90% of samples collected under resource-limited field conditions and with a viral load of ≥400 copies/ml of plasma. However, the current 5′ nested-PCR primer (Prt-F2) for the protease region starting at codon 13 is a few amino acids shorter than those in commonly used genotyping assays, such as the Trugene HIV-1 genotyping system (Siemans Medical Solutions Diagnostics, Tarrytown, NY) and the Viroseq HIV-1 genotyping system (Celera Diagnostics, Alameda, CA), and does not cover all the amino acid positions associated with DR against protease inhibitors. Currently, it will not affect the genotyping results, as many countries are only using the first-line regimens recommended by WHO (34
), which do not include protease inhibitors. We are currently evaluating a new primer set to overcome this issue.
The unusually high viral diversity of HIV-1 poses problems historically for HIV diagnosis using nucleic acid-based tests (1
). The two commercially available genotyping assays, the Viroseq HIV-1 genotyping system (Celera, Alameda CA) and the TruGene HIV-1 genotyping system (Siemans Medical Solutions Diagnostics, Tarrytown, NY), were designed and approved by the FDA for HIV-1 subtype B only. Although studies have indicated that these assays can be used for other HIV-1 group M subtypes, the reported sensitivities vary (2
). The assay we report here and the one reported recently by Buckton et al. (7
) are the only assays that have directly addressed subtype-genotyping-efficiency issues. In the analysis of Buckton et al., using plasmid clones representing major HIV-1 group M subtypes, they found that the genotyping assay was less efficacious for subtypes A2, CRF01_AE, CRF02_AG, F, and H in the RT region and for CRF02_AG, F, and H in the protease region, respectively. We have genotyped DBS and plasma specimens from more countries than reported here using our broadly sensitive genotyping assay. We found that the genotyping efficacies ranged from 90% to 100% for plasma samples (data not shown) and from 82% to 100% for DBS samples representing HIV-1 group M subtypes A1, A2, B, C, D, F1, F2, and G, circulating recombinant forms CRF01_AE, CRF02_AG, CRF07_BC, CRF08_BC, and CRF10_CD, and many unique recombinant forms (Tables , , and ).
In summary, we have developed a broadly sensitive genotyping assay that is restricted to the pol gene region. Using this assay, we were able to genotype diverse HIV-1 group M viral strains from DBS samples collected under field conditions in several countries where multiple subtypes and CRFs are cocirculating. The assay is likely to become a useful tool for HIV DR surveillance in many developing countries.