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Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots
Published on: February 11, 2022
Operational harmonisation of SARS-CoV-2 RT-qPCR quantification across heterogeneous laboratory systems using WHO
Rie Moriuchi1, Miyuki Nagano2, So Fujiyoshi3
1Microbial Genomics and Ecology Laboratory, The IDEC Institute, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan; Quality Control Section, Tokyo Metropolitan Institute of Public Health, 3-24-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan.
Background:
Quantitative reverse transcription PCR (RT-qPCR) is widely used for SARS-CoV-2 testing; however, quantification cycle (Cq) values are not standardised and cannot be directly compared across platforms. The World Health Organization (WHO) International Standard (IS) allows results to be expressed in IU/mL, although implementation in routine laboratory networks remains insufficiently evaluated. We evaluated whether WHO IS-traceable calibration can operationally harmonise RT-qPCR results across heterogeneous routine laboratory workflows within an external quality assessment (EQA) framework.
Methods:
The study comprised a reference-system evaluation and a real-world EQA implementation. Eight representative RT-qPCR systems were evaluated at reference laboratories. The EQA included 33 datasets from 22 participating laboratories. Cq values were converted to copies/mL using system-specific calibration curves and then to IU/mL based on the dPCR-measured copy concentration and its WHO-assigned IU/mL value. Inter-system comparisons were performed using non-parametric methods.
Results:
Across the eight reference systems, 13 of 28 pairwise comparisons showed significant Cq differences for each sample (p < 0.01). After conversion to IU/mL, no significant pairwise differences remained for Samples 1 and 2, and only one comparison remained significant for Sample 3. In the EQA, reported Cq values spanned 21.4-32.6, whereas corresponding IS values spanned 6.10-6.71 log10 IU/mL and clustered around the target concentration.
Conclusions:
WHO IS-traceable calibration was operationally feasible within the evaluated EQA framework and markedly reduced statistically significant pairwise differences among reference RT-qPCR systems. In the real-world EQA, converted IS-traceable units provided a common quantitative scale across heterogeneous testing workflows.
