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Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA

1998; Elsevier BV; Volume: 75; Issue: 1 Linguagem: Inglês

10.1016/s0166-0934(98)00100-1

1879-0984

Lisa M. Demeter, Richard T. D’Aquila, O. S. Weislow, Eric Lorenzo, Alejo Erice, Joseph Fitzgibbon, Robert W. Shafer, Douglas D. Richman, Thomas M. Howard, Yuqi Zhao, Eva Fisher, Diana Huang, Douglas L. Mayers, S. Arthur Sylvester, Max Q. Arens, Kim Sannerud, Suraiya Rasheed, Victoria A. Johnson, Daniel R. Kuritzkes, Patricia Reichelderfer, Anthony J. Japour,

HIV/AIDS Research and Interventions

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39 043 nucleotide assignments spanning codons 10–256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among ≥75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.