Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes.
BACKGROUND
High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes.


OBJECTIVE
Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping.


METHODS
A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs).


RESULTS
All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful.


CONCLUSIONS
A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).
Department of Medical Microbiology, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands. s.c.aitken@umcutrecht.nl
Susan C
SC
Aitken
Aletta
A
Kliphuis
Carole L
CL
Wallis
Mei Ling
ML
Chu
Quirine
Q
Fillekes
Roos
R
Barth
Wendy
W
Stevens
Tobias F
TF
Rinke de Wit
Rob
R
Schuurman
D000293:Adolescent; D000328:Adult; D000349:Africa; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D005260:Female; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D055815:Young Adult
D017931:DNA Primers; D054303:HIV Reverse Transcriptase; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1