Quality Assessment Program for Genotypic Antiretroviral Testing Improves Detection of Drug Resistance Mutations

Abstract

Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.

Model for Assessment of Proficiency of Human Immunodeficiency Virus Type 1 Sequencing-Based Genotypic Antiretroviral Assays

“…This would mean that if a mixed base is represented in the GCS, then data returned by a laboratory should show a mixed base in the same position to be considered homologous. Sequence homology to a group consensus sequence was shown to be slightly lower by Sayer et al (8), but these results may be influenced by the high proportion of in-house assays in the group evaluated (six of nine laboratories). Although some samples in our study displayed Ͻ98% concordance to the GCS, it is possible that the discrepancies were generated in part by the use of RUO kits where less-stringent quality control of kit reagents was in place.…”

“…The ability to accurately report the identity of each nucleotide at each position along the template could be considered in the assessment and evaluation of genotyping proficiency. This issue was also described by Sayer et al (8) in their analysis of, predominantly, in-house assays and by Shafer et al, who examined replicate testing by two laboratories also using inhouse assays (9). In our testing, all laboratories used the same sequencing platform and the group consensus sequence for each sample was generated by alignment using a 60% threshold.…”

“…Another indicator that might contribute to assessing proficiency is the generation of discordant data along the template sequence (8,9). By examining the patterns of editing reflected by the case designation of each base reported, potential difficulty in technical performance may be identified.…”

Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

“…HIV genotyping is routinely performed by Sanger sequencing of plasma HIV RNA; however, this technology is too costly and technically demanding to implement in most low-resource laboratories and clinics. Furthermore, the sensitivity of consensus sequencing is limited for minor variants constituting Ͻ25% of the viral population (7,8), although low-frequency HIV drug resistance mutations, particularly involving resistance to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs), have been associated with increased risks of virological failure with first-line ART (9)(10)(11)(12)(13)(14)(15). Therefore, sensitive and affordable assays are needed to monitor HIV drug resistance as part of global HIV treatment programs.…”

Mapping Protease Inhibitor Resistance to Human Immunodeficiency Virus Type 1 Sequence Polymorphisms within Patients

“…Shafer et al (69) previously showed that most discordances between two laboratories were due to inherent sampling variation in sequencing a heterogeneous population rather than exogenous differences in the sequencing process. However, Sayer et al (66) found that nine laboratories processing the same set of HIV-1 protease and RT samples varied consistently in their rates of reporting mixtures. They were unable to resolve whether differences in reporting mixtures were due to experimental procedures or subsequent sequence editing.…”