The TRUGENE HIV-1 Genotyping Kit proved to have a consistently high degree of accuracy and reproducibility during an extensive blinded evaluation in several laboratory sites using several technicians over several days and three kit lots. In all laboratories, the base-to-base accuracy was higher than 99% and mutation recognition rates were greater than 82%. Of the 270 assays performed in this study, 259 (96%) assays yielded drug resistance interpretations that exactly matched interpretations derived from the gold standard sequences, and the other 11 (4%) assays provided partially matching interpretations.
Several factors are likely to have contributed to the accuracy and reproducibility of this assay. First, the establishment of explicit training procedures and proficiency evaluation is important, especially for clinical laboratories where medical technologists may not have training or prior experience in molecular biology. Second, the detailed and explicit protocol for the assay contributes to consistent performance, which probably accounts for the improved performance of kit-based assays versus home brew genotyping assays in a recent international proficiency evaluation (W. Keulen, D. Brambilla, M. Buimer, J. Tijnagel, J. Bremer, S. Land, L. de Graaff, H. Versteeg, C. Boucher, and R. Schuurman, 5th Int. Workshop Drug Resist. Treat. Strat., abstr. 166, 2001; L. Shaker-Irwin, A. Scarsella, E. Rogolsky, J. Stryker, and S. Day, 5th Int. Workshop Drug Resist. Treat. Strat., abstr. 160, 2001). Third, the use of dye-labeled primers has been associated with more consistent detection of mixtures of sequences compared with dye-labeled terminators, which may have biased base incorporation rates depending on adjacent sequences. Fourth, the automated data acquisition, analysis, and reporting helps eliminate transcription errors which have accounted for a large portion of sequencing discrepancies in other studies (M. Hoover, D. Wentworth, J. Neaton, et al., 4th Int. Workshop HIV Drug Resist. Treat. Strat., abstr. 78, 2000). Fifth, strict adherence to good manufacturing practices during production of the of the kit, as well as hardware associated with the assay, may explain the lack of variation in accuracy from lot to lot. Finally, the TRUGENE HIV-1 Genotyping Kit has been simplified by elimination of DNA purification steps after the RT-PCR and the CLIP sequencing reactions, by use of the novel CLIP reaction and by use of preformed gel cassettes. These simplifications serve to make the assay more robust through minimization of assay steps.
The results of two assays of infectious molecular clones of HIV-1 were invalidated due to presence of a contaminating HIV-1 sequence. Following completion of data analysis, sequence contamination was investigated using the Genetic Fingerprint function of the OpenGene software. This software function compares the pattern of polymorphisms and mutations of a sequence to all other sequences in the software library's database. Sequences showing identity with another sequence in the database (other than those from the same patient) suggest the possibility of contamination and should be investigated prior to reporting the result. For this study of coded specimens, participating laboratories were instructed not to use the Fingerprint function, which could have biased accuracy results by revealing virus identities in the panel, thereby compromising blinding procedures. Use of the Fingerprint function is, however, recommended during routine laboratory use and was found to detect the contamination events that occurred in this study. Stringent precautions against contamination when performing PCR-based tests are needed (16
), including architectural separation of laboratory areas used for PCR setup and DNA analysis, use of negative template controls, and sequence comparison with sequences determined previously in the same laboratory, as with fingerprinting. Uracil N
-glycosylase is also used to decrease carry-over contamination in PCR-based assays, but was not included in the formulation of the TRUGENE assay which minimizes and detects contamination events by other methods. Further, uracil N
-glycosylase treatment would not eliminate the risk of carryover contamination during the setup of CLIP reactions utilized in the TRUGENE assay.
Our study posed several real-life challenges to a drug resistance genotyping assay, including use of plasma from HIV-1-infected humans. Virus populations in vivo are diverse, which poses significant challenges to molecular diagnostic assays that involve oligonucleotide primers for amplification and sequence detection. These challenges are addressed in the TRUGENE HIV-1 Genotyping Kit through use of degenerate primers and ambiguity codes that allow information about sequence mixtures to be retained in the report. Nevertheless, all of the codon identification errors analyzed in Table (other than those due to mislabeling of aliquots prior to distribution to testing sites) involved incomplete, but partially correct, identification of mixtures of codons present in either the gold standard consensus or the assay result. Even analysis of multiple viral sequence clones from two highly sensitive PCRs, as performed here to establish the gold standard sequence consensus, does not guarantee full representation of the diversity of clinically occurring HIV-1 populations. For example, the consistent detection of the RT L210W mutation (as a mixture with wild type) in 11 of 12 independent assays of specimen VA-MH-005 suggests that the mutation was present in the virus population at low frequency, although it was not detected in any of the 20 clones used to establish the gold standard. Failure to identify the mutation in the clones used for the gold standard could occur due to random sampling error of diverse populations. Ambiguity inherent in establishing gold standard measurements of naturally occurring virus populations is also indicated by the improvement in assay accuracy (Table ) observed when using the 5% cutoff to define the gold standard, which allowed detection of codons present in less than 30% of the virus population to be considered correct. Mutation detection in homogeneous virus stocks derived from infectious molecular clones was also found to be highly accurate and reproducible, representing important measures of the assay performance that is not confounded by virus population diversity.
The challenges for training and quality control were also increased in our study because of the involvement of multiple laboratories (both academic and clinical), multiple testing personnel, and multiple kit lots. Use of large panels of specimens, unique labels for all aliquots, and an external data management and analysis agency eliminated any opportunities for partial unblinding of analysis or reporting in our study. Hence, we believe that the assay performance described here will reflect assay performance in a variety of clinical and research laboratories. Our estimates of assay performance cannot be directly compared with estimates based on less challenging evaluations that involve analysis of molecular clones of HIV-1, extensive use of reference laboratories, or proficiency panels that involve limited numbers of specimens and labeling that may allow laboratories to cross-validate results prior to reporting.
After the study was completed, the TRUGENE assay that is available to clinical laboratories was modified to include a centrifugation step to concentrate the virus from 1 ml of plasma prior to RNA extraction and provision of alternative PCR primers (version 1.5). The centrifugation step was found to decrease the minimum viral load required for genotypic evaluation to less than 100 copies/ml (R. Lloyd, R. Schuuman, H. Stang, DeGroot, L. Hough, D. Burns, R. Mathis, and P. Feorino, 3rd Int. Workshop HIV Drug Resist. Treat. Strat., abstr. 52, 1999). The alternative primers are designed to bind to diverse HIV-1 subtypes to permit testing of non-B HIV-1 subtypes using the TRUGENE system (L. Jagodzinksi, J. Cooley, S. Kelly, and N. Michael, 9th Conf. Retrovir. Opportun. Infect., abstr. 594-T, 2002). The version 1.5 primers are one of the primer sets used in this study to amplify the gold standard sequences. Both assay modifications aim to improve the assay success rate and did not affect the accuracy of sequence determinations in the clinical laboratory that participated in this study (data not shown).
The clinical utility of drug resistance genotyping assays has been consistently demonstrated in clinical trials where laboratory testing is performed in reference laboratories with extensive experience with molecular biology assays (2
). Translation of this experience from clinical trials to clinical practice will require the availability of assay technology that can be mastered and consistently controlled in clinical diagnostic laboratories. The results of this study indicate that the TRUGENE HIV-1
Genotyping Kit is an accurate and robust system suitable for clinical laboratory testing.