The goal of TAQAS is to use quality assessment to build capacity in laboratories in the Asian region, by assuring the quality of their HIV genotyping processes and by ensuring parity of test outcome between participating laboratories including a reference laboratory. A further goal is to use TAQAS as a vehicle to develop a collaborative laboratory network, and thereby to encourage regional dissemination of laboratory know-how and expertise, to facilitate troubleshooting of testing problems, and to support laboratories setting up HIV genotyping.
TAQAS is an ongoing program. The present study reports the results of participants testing the first three TAQAS panels. Most (13 of 15) of the samples were plasma derived from HIV-1-infected, ARV-treated or -naive individuals; two samples were normal plasma spiked with co-culture-derived HIV-1. Others offering quality assessment for HIV genotyping have used constructed samples (Neuwald et al., 2002
; Schuurman et al., 1999
). Their samples were characterised so that the DRMs presented and the proportion of nucleotide mixtures were known to the QA provider, and therefore the participants’ detection of DRMs and nucleotide mixtures could be quantified. However, it has been argued that the use of constructed samples does not adequately assess the HIV genotyping process because naturally occurring viral quasi-species are not present in constructed samples (Huang et al., 2003
; Sayer et al., 2003
). Furthermore, intra-laboratory differences have been demonstrated to occur at the RT-PCR level of the process, and therefore DNA was considered an inappropriate starting template for assessing an RT-PCR assay (Galli et al., 2003
). For these reasons, the aim of TAQAS is to use clinical plasma samples and low passage virus isolates as analytes in the program.
Comparison with a TG, generated for each sample using a rules based algorithm from the edited protease and reverse transcriptase nucleotide sequences submitted by all participants, including a reference laboratory, was considered to be an impartial method of assessing inter-laboratory agreement (Sayer et al., 2003
). All participants achieved a high level of agreement at the nucleotide sequence level (>98%) when they tested their second and, in many cases, first TAQAS panel. In an analogous QA program, 97.9% agreement at the nucleotide sequence level was reported between nine laboratories testing four plasma samples (Sayer et al., 2003
). Therefore, TAQAS participants were considered to have achieved a level of inter-laboratory agreement at the level of edited nucleotide sequences comparable with a group of laboratories testing in well-resourced settings.
Consistent with the findings of other studies, only a minority of the nucleotide sequence differences (<0.5%) were complete differences (Demeter et al., 1998
; Sayer et al., 2003
; Shafer et al., 2001
). Differences reported were either a single nucleotide at a position that was defined as a nucleotide mixture in the TG, or a nucleotide mixture different from the mixture in the TG. TAQAS participants used different sequencing methods, and therefore presumably different sequencing primers, to produce their sequences. Furthermore, they were sequencing samples that contained viral quasi-species. Therefore a low level of partial nucleotide sequence differences between participants’ sequences should be expected, but it should be ensured that the level remains low and does not affect the participants’ detection of DRMs. Importantly, participants that reported a lower level of agreement with the TG in their first TAQAS increased their level of agreement in subsequent panels.
Most participants detected at least 80% of the DRMs in each of the three TAQAS panels, and many participants matched the performance of the reference laboratory in detecting >90% of the DRMs. Two participants that detected fewer DRMs also detected few nucleotide mixtures. Substantial inter-laboratory variation in detection of DRMs has been reported in other QA programs. Participants testing constructed samples in a world-wide proficiency testing program were reported to have detected between 0% and 88% of DRMs (Schuurman et al., 1999
). However, participants testing clinical samples, similar to those presented by TAQAS, detected ≥82% of the DRMs (Sayer et al., 2003
). Thereby, the detection of DRMs by TAQAS participants in samples containing multiple DRMs and nucleotide mixtures was comparable to other laboratories’ performances.
Increased sample complexity potentially explained the decreased detection of DRMs by many of the participants in TAQAS 3 compared with previous TAQAS panels. Others have demonstrated that complexity of sequence interpretation is increased by the presence of mixed virus populations, and that sample complexity influences the detection of DRMs (Descamps et al., 2006
; Erali et al., 2001
; Neuwald et al., 2002
). Furthermore, laboratories may fail to detect DRMs presented as viral mixtures when they made up only a portion of the viral population (Schuurman et al., 1999
A relationship was demonstrated between the detection of DRMs and nucleotide mixtures in the participants’ edited nucleotide sequences. The same correlation has been found by another HIV genotyping quality assessment program, supporting the contention that under-detection of nucleotide mixtures could potentially lead to underreporting of DRMs (Erali et al., 2001
; Land et al., 2004
; Sayer et al., 2003
). Therefore, in TAQAS, of nucleotide mixtures was considered an indicator of the quality of the HIV genotyping outcome. The participants’ detection of all nucleotide mixtures in the three TAQAS panels varied. Most participants demonstrated an increased detection of nucleotide mixtures between the first and second TAQAS panels, but in general this increase was not maintained in TAQAS 3. The complexity of TAQAS 3, as previously discussed, may have influenced this outcome. In addition, several participants consistently detected low levels of or no nucleotide mixtures in all the TAQAS panels. No common aspects in the testing methods limited to the participants who under detected nucleotide mixtures were identified however, participants supplied only limited information about their respective HIV genotyping methods. Under-detection of DRMs by two participants (ID: 2 and ID: 12) may be explained by their low detection of nucleotide mixtures. However, suboptimal detection of nucleotide mixtures did not always correlate with low detection of DRMs, which may indicate that some participants reported only the mutant nucleotide base when a mixture was present (ID: 5 and ID: 10). Because of their clinical significance, the importance of the detection of nucleotide mixtures has been a focus of TAQAS. Identifying nucleotide mixtures, which relies in part on visual inspection of electropherograms, has been the subject of training at past TAQAS workshops, and further training in this aspect of the test process is planned.
Not surprisingly, concordance in the interpretation of ARV resistance was higher when all participants used the same, as compared to various, interpretation system. Importantly, the extent of parity between participants when they used various systems, and the extent when they used the same system, were the same as those achieved in a national EQAS conducted in Australia (Land et al., 2004
). Studies have found differences between systems to interpret ARV resistance profiles to combinations of DRMs (de Luca et al., 2003
; Fox et al., 2007
; Huang et al., 2003
; Ravela et al., 2003
; Ross et al., 2005
; Snoeck et al., 2006
). The findings of TAQAS support the previous evidence that different interpretations of ARV resistance will occur when different systems are used, arguing for a single standard.
The limitations of the TAQAS program reported here include the use of predominantly Subtype B virus in a geographical region where non-B subtypes are the norm, a small number of laboratory participants, limited information about the participants’ HIV genotyping methods, and the lack of defined criteria to assess laboratory performance. It is a future objective of TAQAS to address the deficiencies that have been identified in the initial stages of the program.
To date participation in TAQAS by 10 Asian laboratories has shown that these laboratories produce quality output from HIV genotyping that is on par with the performance of a reference laboratory, and with the performances of laboratories in well-resourced settings. Low levels of inter-laboratory differences were demonstrated between edited nucleotide sequences, detection of DRMs, and interpretation of ARV resistance to multiple drugs.
Over the course of testing up to three quality assessment panels, participants in TAQAS have improved or maintained a high standard of quality in HIV genotyping outcome. The impact of the variations that exist as a result of the differences in test methods could be minimised by adopting standardized approaches in, for example, interpretation of edited nucleotide sequence and editing raw sequence. These options will be explored as future TAQAS initiatives.