Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing.
OBJECTIVE
To demonstrate the use of HIV-1 reverse transcriptase (RT) recovered directly from plasma for phenotypic drug susceptibility testing.


METHODS
Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RT eluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits.


RESULTS
The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC(50)) of 1.5 +/- 0.93 microM for nevirapine, 0.21 +/- 0.099 microM for efavirenz, 7.1 +/- 3.2 microM for delavirdine, 0.42 +/- 0.15 microM for azidothymidine triphosphate and 0.059 +/- 0.018 microM for didehydrothymidine triphosphate. The increase in IC(50) value for RT with drug resistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs.


CONCLUSIONS
RT derived from virions recovered from the plasma of HIV infected individuals can be used for analysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysing phenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs.
Shao
Xing-Wu
XW
MTC, Karolinska Institute, Stockholm, Sweden.
Malmsten
Anders
A
Lennerstrand
Johan
J
Sönnerborg
Anders
A
Unge
Torsten
T
Gronowitz
J Simon
JS
Källander
Clas F
CF
D019380:Anti-HIV Agents / Q000494:pharmacology; D023241:Antiretroviral Therapy, Highly Active; D054306:Dideoxynucleotides; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy; D054303:HIV Reverse Transcriptase / Q000032:analysis* / Q000302:isolation & purification; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D015203:Reproducibility of Results; D018119:Stavudine / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000494:pharmacology; D014771:Virion / Q000737:chemistry; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000494:pharmacology
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate; D018119:Stavudine; D054303:HIV Reverse Transcriptase