Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium

Abstract

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Converting to an international unit system improves harmonization of results for SARS-CoV-2 quantification: results from multiple external quality assessments

“…We and others have observed through EQAs that there exists relatively high variability when reporting Ct values, and we presume this is also true outside of ring trials, for example, when measuring virus concentration in patient samples [5,8,9,11,12,19,21]. Our data suggested that converting Ct values to copies per mL (and reporting them as IU/mL) statistically improved precision for all samples.…”

“…We hypothesize that variations in sample preparation (e.g., efficiency of nucleic acid extraction, total volume used as template) contribute to variations within and between laboratories and/or within and between test systems (Figure 3), however this remains to be tested. If these are truly the underlying reasons for the strong within-laboratory within-test system effect, improving the accuracy across all laboratories could come from implementing strict "universal" standard operating procedures or improving external quality assessments to providing laboratories the opportunity to adjustment their protocols to achieve higher accuracy [7,12,19,20].…”

“…There been only tentative efforts to harmonize SARS-CoV-2 RT-qPCR assays and the reporting of quantitative results [12,19].…”

A Hierarchical Framework for Assessing Transmission Causality of Respiratory Viruses

“…A decreasing Ct meaning an increasing genome copy number point to the high likelihood of the donor’s infectiousness without recourse to viral cultures. Such a relationship is reinforced if PCR methods are validated and harmonised on a national of subnational basis to increase consistency [ 30 , 31 ].…”

A Hierarchical Framework for Assessing Transmission Causality of Respiratory Viruses

“…Therefore, coinfection or contamination cannot be fully ruled out. For example, Lednicky et al [33] and harmonised on a national of subnational basis to increase consistency [34,35].…”