Quality control trial for human immunodeficiency virus type 1 drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results

Abstract

Between January and March 2000, a quality control panel for human immunodeficiency virus (HIV) drug resistance testing was analyzed by 20 laboratories in five countries. The panel consisted of three clinical samples with different drug resistance genotypes and phenotypes and one HIV-negative plasma. Participants were asked to report the methods used for amplification and sequencing, a list of drug resistance-associated mutations that were detected in the protease and reverse transcriptase of each sample, and an interpretation concerning the susceptibility or resistance to 14 antiretroviral drugs. A total of 22 genotypic data sets were generated, which showed an overall good technical quality except for three participants, who failed to report key mutations for drug resistance. Problems were encountered in three respects: (i). resistant minorities of L90M in the protease, which were determined to about 12% by real-time amplification, were only detected by one-fourth of the participants; (ii). newly described resistance mutations were frequently not reported; and (iii). interpretations of drug resistance-associated mutations varied widely, in particular for protease inhibitors. In some cases, different interpretations were caused by differences in the detection of resistant minorities, but even for the same genotypic profile, interpretations varied considerably. Similar discrepancies were revealed if current Web-based interpretation systems were used to predict drug resistance for samples of the proficiency panel. This indicates that a consensus for the interpretation of drug resistance-associated mutations is urgently needed.

FIG. 1.

Results of the real-time ARMS for 90L and 90M in the protease. (A) Downstream primer sets for the amplification of wild-type 90L (WT) and mutant 90M (Mut) sequences for sample 3 of the quality control trial. The mismatch at the 3′ end is responsible for the differences in threshold cycles (ΔC_t), which result from authentic amplification (e.g., WT template and WT primers) compared to amplification based on mispriming (e.g., WT template and MUT primers). The addition of one (WT1/Mut1) or three (WT3/Mut3) internal destabilizing mutations resulted in a higher discriminatory window, which consequently improved the detection limit for minorities. (B) Results of the real-time amplification of 10⁶ copies of WT sequence per reaction, with WT3 or Mut3 as downstream primers (assay run in duplicate; black arrow indicates ΔC_t). The corresponding upstream primer was identical for WT and Mut amplification. The amplification was monitored through the binding of SybrGreen to double-stranded DNA. ΔRn, difference between the normalized reporter fluorescence and baseline. (C) Mixture of 10⁴ copies of mutant template with increasing amounts of WT template DNA (10³ to 10⁸ copies/reaction). The first column shows quantitation of Mut template alone. Up to a 1,000-fold excess of WT template did not influence the accuracy of quantitation. (D) Real-time amplification of sample 3. The relation between 90L and 90M was calculated as 88% versus 12%, after the exact copy numbers had been determined by generating specific standard curves for wild-type and mutant template (see Results).