The reproducibility of HIV-1 RT and protease sequencing in clinical settings has attracted much attention because genotypic HIV-1 resistance testing is one of the first applications of sequencing for clinical purposes [1–5]. The evaluation of tests for sequencing HIV-1 is complicated by the fact that HIV-1 exists in vivo as a quasispecies: a mixture of genetically distinct viral variants that evolve from the initial virus inoculum. Suboptimal reproducibility therefore can result from either technical artifacts or variability in the detection of minor viral variants.

The April issue of the Journal of Clinical Microbiology contains a paper describing a collaboration between Stanford University Hospital (SUH) and Virco which provides new insights into the reproducibility of HIV-1 sequencing [6]. The following paragraphs will summarize this study, as well as two previous related studies.

The European Network for Virologic Evaluation (ENVA) has conducted three multicenter blinded sequence comparison studies. The first of these (ENVA-1), which included mixtures of plasmid clones, has been published [7]. ENVA-1 indicated two distinct problems: (1) Three of the 23 participating laboratories were grossly incompetent and reported many inaccurate results; and (2) the remaining laboratories, although they reliably detected mutations that were present in 100% and 50% of the clones, were inconsistent at detecting mutations that were present in 25% of the clones. The first problem indicated the need for ongoing quality control programs for laboratories that are doing sequencing for patients (most of the laboratories in this first study were small research laboratories who just began HIV-1 sequencing). The second problem indicated the need for improved methods for detecting minor mutant virus populations.

In an earlier multicenter study, 13 research laboratories were shipped cell pellets from cultured HIV-1 isolates [3]. The sequence concordance among laboratories was 99.7% at all nucleotide positions. Sequencing cultured cell pellets is simpler than sequencing plasma because RNA extraction and reverse transcription are not necessary and because cultured virus is more homogeneous than uncultured virus. Nonetheless, the high inter-laboratory concordance in this early study attests to the intrinsic reliability of the dideoxynucleoside sequencing of HIV-1.

In the recent study published in the Journal Clin Microbiology, the SUH and Virco clinical laboratories assessed the reproducibility of HIV-1 RT and protease sequencing using plasma aliquots obtained from 46 heavily treated HIV-1 infected individuals. Although both laboratories used sequencing reagents from Applied Biosystems (Foster City, CA), each used a different in-house, non-kit-based ("home brew") protocol for plasma HIV-1 RNA extraction, reverse transcription, PCR, and sequencing.

Overall sequence concordance between the two laboratories was 99.0%. However, 90% of the discordances were partial, defined as one laboratory detecting a mixture while the second laboratory detected only one of the mixture's components. Discordance was more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at about 1% of the nucleotide positions, and, in every case in which one laboratory detected a mixture, the second laboratory detected either the same mixture or one of the mixture's components. The high concordance in detecting mixtures and the fact that most discordance between the two laboratories was partial suggested that most discordances were due to variation in sampling the HIV-1 quasispecies rather than to technical errors.

Taken together, these studies contain a number of take-home messages about sequencing reliability and the significance of nucleotide mixtures: (1) In experienced laboratories, HIV-1 sequencing is highly reproducible at positions that do not contain mixtures; (2) mixtures occur at about 1% of nucleotide positions, although they tend to occur more commonly at codons associated with drug resistance; (3) the presence of mixtures on an electropherogram (two nucleotide peaks at the same position) probably represents mixtures inside a patient rather than laboratory artifact; (4) because mixtures are probably "real" and not artifacts, laboratories should try to improve their sensitivity in detecting mixtures; (5) the difficulty of detecting minor mutant populations is not a consequence of technical inability; rather, it is an unfortunate consequence of HIV-1 biology; and (6) minor mutant populations are more difficult to detect in patients with lower plasma HIV-1 RNA levels.

The inability to detect minor drug-resistant HIV-1 variants is a recognized limitation of clinical HIV-1 drug susceptibility testing using either genotypic or phenotypic susceptibility testing. The possibility of undetected mutant virus should be considered when interpreting the results of drug susceptibility tests, particularly, in patients with complicated antiretroviral treatment histories or in patients who have discontinued one or more antiretroviral drugs. Further studies are also needed to identify patients who are at greater risk of having minor mutant drug-resistant viral populations and to develop sequencing methods with increased sensitivity for these variants.

See also: http://cdli.asm.org/cgi/content/abstract/8/3/560

References