“Informative Use of Cycle-Threshold Values to Account for Sampling Variability in Pathogen Detection”

Nucleic acid amplification tests, like real-time polymerase chain reaction, are widely used for pathogen detection; however, their interpretation rarely accounts for sampling variability. Instead, cycle threshold values are often categorized reducing precision. We describe how pathogen cycle threshold values can be normalized to endogenous host gene expression to correct for sampling variability and compare the validity of this approach to standardization with a standard curve. Normalization serves as a valid alternative to standardization, does not require making a standard curve, increases precision, accounts for sampling variability, and can be easily applied to large clinical or surveillance datasets for informative interpretation. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the Canadian Institutes of Health Research (#434951) and Genome British Columbia (COV-55). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for the study was obtained from the University of British Columbia human ethics board (H20-01110). Written informed consent was not required as per ethics board approval. Participant data was de-identified prior to analysis; the results of this non-interventional observational study were not linked back to any identifying patient records. The study was deemed to be of minimal risk. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The de-identified participant data that support the findings of this study are available from the British Columbia Ministry of Health. Restrictions apply to the availability of these data, which were used under agreement at the British Columbia Center for Disease Control for this study. * RT-qPCR : real-time quantitative polymerase chain reaction Ct : cycle threshold RNaseP : Ribonuclease P E : Envelope PCC : Pearson’s correlation coefficient SLR. : simple linear regression