Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch collaborative study

Abstract

The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism laboratories before embarking on clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/NCT01384682?term=NCT01384682&rank=1].).

Fig 1

Summary of the EQA program for the MARCH HIV coreceptor tropism assay. TAT, turnaround time.

Fig 2

Interlaboratory variation in minimum FPR. There was significant variation across the laboratories for each sample, even when the lowest geno2pheno false-positive rates (FPRs) were examined. Samples were sorted by consensus tropism and then by decreasing interlaboratory agreement. The minimum FPRs obtained from all laboratories from each of the 20 specimens in phase 2 were used to generate box-and-whisker plots for each specimen. Horizontal lines represent the median FPR, boxes represent the interquartile range of the values, and whiskers represent the minimum and maximum values obtained across the laboratories. One laboratory (laboratory B) gave outlier results for 4 samples (with outlier defined as an FPR outside the range mean ± 1.96 standard deviations). These were samples 1, 3, 8, and 15. The other laboratories contributed 0 to 2 outlier results.

Fig 3

Variation by repeated amplifications. The same specimens shown in Fig. 2 were repeatedly sequenced up to 32 times. For comparison, they are displayed in the same order as in Fig. 2. The intensive sequencing revealed marked variation within the samples. Horizontal lines represent the median FPR obtained across all repeated sequences, boxes represent the interquartile ranges of the values, and whiskers represent the minimum and maximum values obtained by repeated sequencing. Samples for which all laboratories gave the same tropism determination often had lower variability according to repeated sequencing analysis (see specimens with asterisks). This approach shows the importance of triplicate sequencing, since many samples had large spreads of FPR values (e.g., sample 8). The method of taking the minimum FPR detected is also key to a sensitive detection of the X4 virus, since higher FPRs might obscure X4 detection (e.g., sample 19). *, all laboratories called X4; **, all laboratories called R5.

Fig 4

Phylogenetic tree of EQA phase 2 sequences for the 14 laboratories. Each sample is represented by a different color. In most cases, the sequences generated from the same sample by different laboratories clustered near each other. There was no evidence of sample mix-ups according to phylogenetic analysis. There was evidence of contamination (i.e., a sequence from one sample clustering with another) for 7 laboratories (laboratories A, B, D, F, H, I, and N), with most cases being restricted to a maximum of 1 sequence in 1 sample. There were 10 possible contamination events identified out of 685 sequences obtained (1.5%).