title: Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.
abstract: OBJECTIVE
  Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.
  
  
  METHODS
  The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.
  
  
  METHODS
  The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000-1.8 million RNA copies/ml.
  
  
  RESULTS
  Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective.
  
  
  CONCLUSIONS
  The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.
authors.affiliation: Drug Resistance Lab, National AIDS Research Institute, Indian Council of Medical Research, Bhosari, Pune, India.
authors.forename: Devidas N
authors.initials: DN
authors.lastname: Chaturbhuj
authors.affiliation: Department of Epidemiology & Biostatistics, National AIDS Research Institute, Indian Council of Medical Research, Bhosari, Pune, India.
authors.forename: Amit P
authors.initials: AP
authors.lastname: Nirmalkar
authors.affiliation: National AIDS Research Institute, Indian Council of Medical Research, Bhosari, Pune, India.
authors.forename: Ramesh S
authors.initials: RS
authors.lastname: Paranjape
authors.affiliation: National JALMA Institute Of Leprosy And Other Mycobacterial Diseases, Indian Council of Medical Research, Bhosari, Agra, India.
authors.forename: Srikanth P
authors.initials: SP
authors.lastname: Tripathy
mesh_terms: D000465:Algorithms; D019380:Anti-HIV Agents / Q000494:pharmacology; D003362:Cost-Benefit Analysis; D024882:Drug Resistance, Viral; D005838:Genotype; D060005:Genotyping Techniques / Q000191:economics* / Q000379:methods; D015658:HIV Infections / Q000097:blood*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics* / Q000379:methods; D012984:Software; D019562:Viral Load
chemical_list: D019380:Anti-HIV Agents