Technical and Regulatory Shortcomings of the TaqMan Version 1 HIV Viral Load Assay

<div><h3>Background</h3><p>The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility. Sudden increases in the number of patients with detectable pVL have been reported following the introduction of the TaqMan version 1 assay.</p> <h3>Methods</h3><p>Between October 2009 and April 2010 all routine pVL samples from British Columbia, Canada, with 40–250 copies/mL by TaqMan were re-tested by Amplicor (N = 1198). Subsequent short-term virological and resistance outcomes were followed in patients with unchanged therapy (N = 279; median 3.2 months follow-up).</p> <h3>Results</h3><p>TaqMan and Amplicor values correlated poorly at low pVL values. Low-level pVL by TaqMan was not associated with impending short-term virological failure; only 17% of patients with 40–250 copies/mL by TaqMan had detectable pVL by Amplicor at follow-up. During the follow-up period only 20% of patients had an increase in pVL by TaqMan (median [IQR]: 80 [36–283] copies/mL). In addition, in ∼2.4% of samples pVL was dramatically <em>underestimated</em> by TaqMan due to poor binding of the proprietary TaqMan primers.</p> <h3>Conclusions</h3><p>The replacement of Amplicor with the TaqMan assay has altered the previously accepted definition of HIV treatment failure without any evidence to support the clinical relevance of the new definition. Given the systematic differences in measurement in the low pVL range the British Columbia HIV treatment guidelines now use a threshold of >250 copies/mL by TaqMan to define treatment failure.</p> </div

Kaplan Meier estimates for the probability of mortality by the levels of the programmatic compliance score.

<p>Kaplan Meier estimates for the probability of mortality by the levels of the programmatic compliance score.</p

Results from the predictive model for the probability of mortality based on the programmatic compliance score.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047859#s3" target="_blank">Results</a> from the predictive model for the probability of mortality based on the programmatic compliance score.</p

Plasma viral load testing and reporting protocol in British Columbia between October 2009 and April 2010.

<p>All plasma samples were initially tested with the Roche COBAS AmpliPrep/COBAS TaqMan v1 HIV-1 Test (“TaqMan v1”) assay. TaqMan v1 pVL results <40 or ≥250 copies/mL were reported to physicians. Samples with TaqMan v1 pVL ≥40 and <250 copies/mL were re-tested by the Roche COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR UltraSensitive Test, version 1.5 (“Amplicor v1.5”). The Amplicor v1.5 test results were reported to physicians.</p

Systematic underestimation of plasma viral load by TaqMan v1 in a minority of patients.

<p>A minority (2.4%) of samples had viral loads <i>underestimated</i> by >0.5 log₁₀ copies/mL by TaqMan v1 after re-testing with Amplicor v1.5. Depicted here is the viral load history of one representative patient showing systematic underestimation of viral load by TaqMan v1 when compared to results obtained by re-testing samples with the Amplicor v1.5, TaqMan v2 and/or Abbott assays. TaqMan v1 viral load results are shown as solid triangles (▴) joined by a solid line. Overlaid are the corresponding results from the Amplicor v1.5 (solid squares ▪), TaqMan version 2 (unshaded triangles Δ) and Abbott (unshaded squares γ) assays where available. For this patient TaqMan v1 systematically under-reported pVL by an average of 1.3 log₁₀ copies/mL over a period of 18 months.</p

Patient characteristics of 279 HAART-treated patients with low-level viremia followed longitudinally.

<p>IQR: Interquartile Range.</p

Relationship between the programmatic compliance score and mortality.

<p>(A) Bivariable associations between each of programmatic compliance score components and mortality for individuals who started antiretroviral therapy between 2006 and 2009. (B) Results from the multivariable explanatory model for the probability of mortality based on the programmatic compliance score for individuals who started antiretroviral therapy between 2006 and 2009. Area Under the Curve: 0.896.</p

Descriptive statistics by mortality status at the end of follow-up.

<p>Notes: HA: Health Authority, Q1: 25th percentile; Q3: 75th percentile.</p

Distribution of the programmatic compliance score over time.

<p>Distribution of the programmatic compliance score over time.</p

The proportion of samples detectable by Amplicor v1.5 increases as a function of the TaqMan v1 value.

<p>Of 1198 samples with pVL results 40–250 copies/mL by TaqMan v1, only 34% were detectable when re-tested by Amplicor v1.5 (>50 copies/mL). When TaqMan v1 results were grouped into 50 copies/mL strata we observed a stepwise increase in detectability by Amplicor v1.5 with increasing viral load by TaqMan v1.</p