Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance

Abstract

As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi ( n = 58), Tanzania ( n = 60), and China ( n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.

Field Study of Dried Blood Spot Specimens for HIV-1 Drug Resistance Genotyping

“…Genotyping of the protease and RT regions of the HIV-1 pol gene was carried out using a broadly sensitive in-house method (15,31). Briefly, a 1,084-base-pair segment of the 5= region of the pol gene was generated by RT-PCR followed by nested PCR using total nucleic acid extracted from a DBS.…”

A Single Early Introduction of HIV-1 Subtype B into Central America Accounts for Most Current Cases

“…Sequence analysis of the protease (PR) and the first part of the reverse transcriptase (RT) of the HIV-1 pol gene (nucleotides 2268 to 3257 of reference strain HXB2, PR nucleotides 16 to 297, and RT nucleotides 1 to 708) was performed on the plasma or serum samples by using a published protocol (21,22,24). Dried-blood-spot samples were sequenced using another published genotyping assay developed by the CDC (25,26).…”

Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

“…This novel assay can simultaneously detect 45 alleles in a single reaction tube and has good concordance with a well-validated in-house sequencing-based method (13). Specimens from both ART-experienced and ARTnaive patients were analyzed successfully by the MAS assay, indicating the potential utility of this assay to detect both acquired and transmitted DRMs.…”

“…The tests conducted for the MAS assay using deidentified specimens were determined to be non-human subject research by the associate director for Science at the Center for Global Health at CDC. The nested PCR products obtained previously by a broadly sensitive genotyping assay (13,14) were analyzed using the MAS assay. Verification of low-abundance alleles detected by the MAS assay.…”

“…Conventional sequencing-based assays, such as the FDA-approved and commercially available genotyping assays, ViroSeq (11) and TruGene (12), and many in-house assays (13)(14)(15), have been widely used for HIVDR detection analyses in clinical settings and surveillance purposes. These assays can provide detailed sequence information and detect all possible mutations that are present in an amount above the detection limit, but they are generally labor-intensive and not sensitive enough to detect low-abundance mutations.…”