Research Article: Estimation of measurement error in plasma HIV-1 RNA assays near their limit of quantification

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Viviane D. Lima, Lu Wang, Chanson Brumme, Lang Wu, Julio S. G. Montaner, P. Richard Harrigan, Cecilio López-Galíndez.

http://doi.org/10.1371/journal.pone.0171155

Abstract

Plasma HIV-1 RNA levels (pVLs), routinely used for clinical management, are influenced by measurement error (ME) due to physiologic and assay variation.

To assess the ME of the COBAS HIV-1 Ampliprep AMPLICOR MONITOR ultrasensitive assay version 1.5 and the COBAS Ampliprep Taqman HIV-1 assay versions 1.0 and 2.0 close to their lower limit of detection. Secondly to examine whether there was any evidence that pVL measurements closest to the lower limit of quantification, where clinical decisions are made, were susceptible to a higher degree of random noise than the remaining range.

We analysed longitudinal pVL of treatment-naïve patients from British Columbia, Canada, during their first six months on treatment, for time periods when each assay was uniquely available: Period 1 (Amplicor): 08/03/2000–01/02/2008; Period 2 (Taqman v1.0): 07/01/2010–07/03/2012; Period 3 (Taqman v2.0): 08/03/2012–30/06/2014. ME was estimated via generalized additive mixed effects models, adjusting for several clinical and demographic variables and follow-up time.

The ME associated with each assay was approximately 0.5 log10 copies/mL. The number of pVL measurements, at a given pVL value, was not randomly distributed; values ≤250 copies/mL were strongly systematically overrepresented in all assays, with the prevalence decreasing monotonically as the pVL increased. Model residuals for pVL ≤250 copies/mL were approximately three times higher than that for the higher range, and pVL measurements in this range could not be modelled effectively due to considerable random noise of the data.

Although the ME was stable across assays, there is substantial increase in random noise in measuring pVL close to the lower level of detection. These findings have important clinical significance, especially in the range where key clinical decisions are made. Thus, pVL values ≤250 copies/mL should not be taken as the “truth” and repeat pVL measurement is encouraged to confirm viral suppression.

Partial Text

Long-term suppression of plasma HIV-1 RNA levels (pVLs) below the quantification limit of clinically available assays is the critical goal for patients starting combination antiretroviral therapy (cART) [1]. Maintaining pVLs below this threshold has been shown to promote immune restoration, decrease morbidity and mortality associated with HIV disease, and prevent ongoing viral evolution and HIV transmission [1]. In most resource-rich settings, patients’ pVLs are monitored every 3 to 4 months for early diagnostic of treatment failure, and if failure is confirmed, treatment switch is often recommended. Frequency of monitoring varies in resource-limited settings depending on the availability of the test, however, this issue is rapidly evolving as a result of new guidelines and emerging technologies [2, 3].

To assess the ME of the Amplicor v1.5 and the Taqman v1.0 and v2.0 assays. Additionally, we examined whether there was any evidence that pVL measurements closest to the lower limit of quantification, where clinical decisions are made, were susceptible to a higher degree of random noise than the remaining range.

We longitudinally followed 1933 patients in Period 1, 979 in Period 2, and 429 in Period 3. Overall, in all periods of observation, patients were more likely to be male, to have no history of injection drug use, to have started treatment on a bPI-based regimen, and to have adherence ≥95% during the first year on therapy (Table 1). In terms of CD4 cell count at cART initiation, in Period 1, 60% of patients had CD4 cell count <200 cells/mm3, while in the other periods, the distribution of baseline CD4 cell count was quite similar across all categories. Additionally, in all periods, at the start of cART, the median age was just over 40 years, median pVL was approximately 5.0 log10 copies/mL, and the median number of pVL measurements per patient ranged from 2 to 3 (Table 1). The trajectories of the mean pVL (and associated 95% confidence interval) for these periods from start of cART up to six months were very similar and they are presented in Fig 1. Based on this study, the estimated ME associated with each assay was approximately 0.50 log10 copies/mL, which is consistent the literature [20]. Thus, patients and physicians should be aware that a pVL of 50 copies/mL really means that it is likely that the “true” pVL is between 16 and 158 copies/mL, or a pVL of 250 copies/mL really means that it is likely that the “true” pVL is between 79 and 791 copies/mL. We also observed that there is substantial increase in random noise in measuring pVLs <250 copies/mL, especially close to the lower level of detection of each assay. Interestingly, we also detected a pattern such that pVLs near each assay’s lower limit of quantification were over-represented than values ≥500 copies/mL. There are different possible explanations for this finding. First, each of these repeated measurements may be a genuine representation of underlying distribution of pVLs. Second, repeated pVL testing improves the likelihood of testing below the limit of quantification. Thus, due to ME, a patient whose “true” pVL remains above the limit of quantification can test below this cut-off by chance. Consequently, the more tests are performed in a given patient with a low detectable pVL, the higher the likelihood that at least one measurement will be below the limit of quantification. Finally, low detectable values near the limit of quantification could represent assay’s “false positive” results [12, 13]. As important clinical decisions (e.g., change in drug regimen due to virologic failure) are made based on this range, this study highlights the fact that single pVL results are not reliable given the ME and random noise pertaining to currently used assays, and the likelihood of a pVL being equal to 51 copies/mL, for example, is very small. Instead, physicians should rely on confirmatory retesting to ascertain pVL-based outcomes in patients. The British Columbia Centre for Excellence in HIV/AIDS received approval for this study from the University of British Columbia ethics review committee at the St Paul’s Hospital, Providence Health Care site (H05-50123). The study complies with the BC’s Freedom of Information and Protection of Privacy Act. The study was conducted primarily using anonymized administrative databases, and therefore specific informed consent was not required.   Source: http://doi.org/10.1371/journal.pone.0171155

 

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