In the present paper, the performance of the new Roche dual-target CAP/CTM HIV-1 test, version 2.0, is described. We show that the assay is HIV-1 specific and correctly quantifies samples of HIV-1 groups M, N, and O. The viral loads determined with CAP/CTM v2.0 for samples having HIV-1 RNA levels below the threshold of quantification for CAP/CA v1.5 proved to be HIV-1 specific and correlated with therapy failure. In addition, we could show that in patients under therapy, viral load decreased over time until 31 months after initiation of therapy and that levels below 250 cp/ml could regularly be detected with CAP/CTM v2.0 even 100 months after initiation of therapy.
It has previously been reported that CAP/CTM v1.0 identified a significant proportion of samples with levels below the threshold of quantification for CAP/CA v1.5 as HIV-1 positive, with viral loads well over 50 cp/ml. We have previously shown that lack of an UNG system in real-time viral load measurement may pose a significant problem for laboratories that used to measure HCV RNA with CA v1.5 and switch to the use of the Abbott real-time HCV assay, which has no UNG contamination control system built in to its reaction (20
). In the present paper, we demonstrate that the UNG system of CAP/CTM v1.0 and v2.0 is as efficient as the CAP/CA v1.5 UNG system in elimination of dUTP-positive amplicons derived from CAP/CA v1.5. We conclude that low HIV-1 RNA-positive values determined with CAP/CTM v2.0 for samples having HIV-1 RNA levels below the threshold of quantification for CAP/CA v1.5 are true positives. This conclusion was substantiated by the observation that none of the HIV-1-seronegative plasma replicates in the checkerboard analysis was above the lower limit of detection for CAP/CTM v2.0. However, the nature of the low viral titers in treated patients still needs to be resolved. Release of virus particles from latently infected cells or active virus replication could be the cause. We assume that release of virus particles from a decaying pool of latently infected cells is the cause for the long-lasting low viral load in patients under therapy. This assumption is further supported by the observation that in patients whose levels were below the threshold of quantification for CAP/CA v1.5 (<50 cp/ml) but who tested HIV-1 positive with CAP/CTM v2.0, viral load steadily declined during the first 31 months after initiation of therapy and reached a plateau thereafter. The high sensitivity and linearity of CAP/CTM v2.0 at the lower end of quantification (<50 cp/ml) compared to the levels for other HIV-1 viral load assays raises some critical questions for current national and international guidelines for treatment and for clinical trials. Our data show that in each stratum (<12, 12 to 24, 24 to 36, 36 to 48, and >48 months after initiation of therapy), about 20% of patients would be reported as experiencing therapy failure with CAP/CTM v2.0 while being reported as therapy responders with CAP/CA v1.5. In our cross-sectional retrospective data set, a viral load determined by CAP/CTM v2.0 to be above the threshold of quantification for CAP/CA v1.5 was statistically correlated with therapy failure, which suggests that CAP/CTM v2.0 results at the low end have clinical impact. The probability of virological failure in our hospital setting is between 10 and 20%. The therapy failure rate of 11% for viral loads above 50 cp/ml as determined by CAP/CTM v2.0 seems clinically relevant, given that this rate is more than 2-fold higher than that observed for the remaining patients with levels below the threshold of quantification for CAP/CA v1.5. Prospective longitudinal data sets are required, however, for new cutoff values for clinical intervention based on CAP/CTM v2.0 to be defined.
Reliable quantification of subgroup M, including non-B HIV-1 subtypes, is generally considered an important feature of state-of-the-art HIV-1 quantification assays. Non-group-M HIV-1 subgroups N and O are considered to be of minor clinical importance due to the low numbers of infected individuals worldwide. A platform comparison and specificity study using the WHO first reference panel for HIV-1 genotypes was performed with Roche CAP/CA v1.5, CAP/CTM v1.0, and CAP/CTM v2.0 and the Abbott assay. Overall, the platform comparison based on mean titers, Bland-Altman analysis, and the percentage of single strongly deviating measurements shows that there are small quantitation differences between CAP/CTM v2.0 and both CAP/CA v1.5 and CAP/CTM v1.0. These differences are due to (i) titer shifts for individual measurements (e.g., outliers seen in the plots) and (ii) smaller quantitation differences concentrated at the medium and lower assay range seen by the nonzero y intercepts and slopes (Fig. ). This indicates that CAP/CTM v2.0 measures higher target concentrations in samples with medium-to-low viral loads than the two predecessor tests. When the Abbott RealTime test was compared to CAP/CTM v2.0, fewer titer shifts of individual measurements and only negligible differences over the assay range were observed.
Due to the relatively low sequence homology between HIV-1 and HIV-2, development of a viral load assay that is able to reliably quantify all subtypes of both viruses is virtually impossible. However, cross-reactivity may occur, as observed with CAP/CA v1.5 in four out of the six HIV-2 plasma samples, and could lead to misdiagnosis and inappropriate patient management. We were able to show that HIV-2 cross-reactivity of CAP/CA v1.5 is probably solved with CAP/CTM v2.0, but larger numbers of HIV-2-infected individuals need to be tested for this question to be answered.
In conclusion, CAP/CTM v2.0 is, on the basis of the data presented, an accurate and reliable test for HIV-1 viral load measurement relative to the other assays used with respect to specificity, sensitivity, and genotype inclusivity. It can be used for high- and medium-throughput laboratories with low-qualification personnel. The specificity and sensitivity of CAP/CTM v2.0 are good relative to those observed for the other assays used. Although our data suggest that low positive values as determined with CAP/CTM v2.0 for patients under therapy are correlated with therapy failure, additional clinical studies are required for new cutoffs for therapy decisions and patient management to be defined.