HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.
In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.
Brun-Vézinet
F
F
Laboratories of Virology, Hôpital Bichat-Claude Bernard, Paris, France.
Ingrand
D
D
Deforges
L
L
Gochi
K
K
Ferchal
F
F
Schmitt
M P
MP
Jung
M
M
Masquelier
B
B
Aubert
J
J
Buffet-Janvresse
C
C
D002474:Cell-Free System; D003470:Culture Media; D005838:Genotype; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D007700:Kinetics; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000235:genetics; D014779:Virus Replication; D015215:Zidovudine / Q000494:pharmacology*
D003470:Culture Media; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase