title: Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.
abstract: Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.
  
  
  CONCLUSIONS
  The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
authors: Division of Global HIV/AIDS, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors: Zhiyong
authors: Z
authors: Zhou
authors: Nick
authors: N
authors: Wagar
authors: Joshua R
authors: JR
authors: DeVos
authors: Erin
authors: E
authors: Rottinghaus
authors: Karidia
authors: K
authors: Diallo
authors: Duc B
authors: DB
authors: Nguyen
authors: Orji
authors: O
authors: Bassey
authors: Richard
authors: R
authors: Ugbena
authors: Nellie
authors: N
authors: Wadonda-Kabondo
authors: Michelle S
authors: MS
authors: McConnell
authors: Isaac
authors: I
authors: Zulu
authors: Benson
authors: B
authors: Chilima
authors: John
authors: J
authors: Nkengasong
authors: Chunfu
authors: C
authors: Yang
mesh_terms: D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay / Q000191:economics; D003365:Costs and Cost Analysis; D003906:Developing Countries / Q000191:economics*; D059788:Dried Blood Spot Testing; D024882:Drug Resistance, Viral / Q000235:genetics*; D060005:Genotyping Techniques / Q000191:economics* / Q000379:methods*; D015658:HIV Infections / Q000097:blood / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D006295:Health Resources / Q000191:economics*; D006801:Humans; D007202:Indicators and Reagents / Q000191:economics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011159:Population Surveillance; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000596:Amino Acids; D007202:Indicators and Reagents