Identification of drug-related genotypic changes in HIV-1 from serum using the selective polymerase chain reaction.
We attempted to detect drug-related HIV-1 pol gene mutations by selective polymerase chain reaction (PCR) using both proviral DNA and viral RNA isolated from patients (pts) with AIDS or ARC receiving antiretroviral therapy. Peripheral blood mononuclear cell (PBM)-associated proviral DNA and serum-derived viral RNA were obtained from eight patients before and after receiving an alternating regimen of AZT and ddC for 15-41 months or ddI monotherapy for 12-26 months. These specimens were examined for the presence of mutations at positions 70, 74, 215 and 219. We noted that selective PCR results can be ambiguous depending on the quantity of DNA template employed. We, therefore, used the minimal quantity of DNA templates that yielded evaluable PCR products in this study. For all the eight pairs of pre- and post-therapy proviral DNA samples, selective PCR results agreed with independently determined nucleotide sequences. Results of reverse transcription of serum-derived viral RNA followed by selective PCR differed in some cases from those using the proviral DNA. In particular, the use of serum viral RNA appeared to allow earlier detection of changes in drug-related mutations than the use of PBM-associated proviral DNA. We conclude that (i) selective PCR using the minimum and sufficient number of PBM-associated proviral DNA and serum viral RNA copies successfully detects the presence of known pol gene mutations; (ii) drug-related mutations may be distinguished earlier in virions in serum (or plasma) than in proviral DNA in PBM; and (iii) quantification of HIV-1 prior to selective PCR may be an important component in monitoring the therapy of HIV-1 infection.
Anderson
B D
BD
Experimental Retrovirology Section, National Cancer Institute, Bethesda, MD 20892, USA.
Shirasaka
T
T
Kojima
E
E
Yarchoan
R
R
Mitsuya
H
H
D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D016049:Didanosine / Q000494:pharmacology; D004526:Efficiency; D005069:Evaluation Studies as Topic; D015751:Genes, pol / Q000187:drug effects / Q000235:genetics; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014771:Virion / Q000235:genetics; D016047:Zalcitabine / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D015215:Zidovudine; D016047:Zalcitabine; D016049:Didanosine