The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing

Abstract

Background Resolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown. Methods Here we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD. Results Ct values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%. Conclusions The assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods.

COVID-19 RT-PCR diagnostic assay sensitivity and SARS-CoV-2 transmission: A missing link?

“…16 Similar studies performed on samples with Ct >30 about 3-25% of the samples retested as negative depending on the kit used. [17][18][19] However, a recent study by Arnaut et al, highlights that the sensitivity of currently approved assay varies over 10,000fold and several low sensitivity (high LoD) assays will miss the majority of infected patients that could be easily identified by assays having high sensitivity (low LoD). 17 Further, a study by Winnet et al, identifies a case of pre-symptomatic household transmission from a healthy young adult to a sibling and a parent, with high Ct (low viral load) results in the early phase (pre-symptomatic) of the infection that could prevent transmission in the family.…”

“…[17][18][19] However, a recent study by Arnaut et al, highlights that the sensitivity of currently approved assay varies over 10,000fold and several low sensitivity (high LoD) assays will miss the majority of infected patients that could be easily identified by assays having high sensitivity (low LoD). 17 Further, a study by Winnet et al, identifies a case of pre-symptomatic household transmission from a healthy young adult to a sibling and a parent, with high Ct (low viral load) results in the early phase (pre-symptomatic) of the infection that could prevent transmission in the family. 10 Furthermore, an analysis of serial RT-PCR assays and chest CT scans has demonstrated that the mean interval between the initial negative to positive RT-PCR results was 5.1 days ± 1.5, and the mean interval between initial positive to subsequent negative RT-PCR results was 6.9 days ± 2.3.…”

“…8 Low Ct has been associated with a higher risk for severe COVID-19, 6,7 whereas, high Ct has been associated with pre-as well as post-symptomatic phases of the disease. 9,10 Appallingly, decision analytical models have assessed that 59% of all transmission are asymptomatic transmission, comprising 35% from pre-symptomatic and 24% from asymptomatic individuals. 11 However, measures to reduce transmission from individuals who do not have COVID-19 symptoms have been politicized as these likely had negative effects on the economy.…”

Nucleic Acid Testing of SARS-CoV-2

“…In general, sputum samples show higher viral loads than throat swab samples, whereas low viral RNA is detected in urine or stool samples [ 75 ]. The two main factors that influence the quantitative measurement of viral roads are Cq values that are repeatable with acceptable uncertainty and a reliable means of converting from the Ct value to viral load [ 76 , 77 , 78 ]. For molecular diagnostic assays, a limit of detection (LoD) and a limit of quantification (LoQ) are also considered the lowest concentrations of target RNA that can be detected by RT-qPCR [ 79 ].…”

Nasal Swab Performance by Collection Timing, Procedure, and Method of Transport for Patients with SARS-CoV-2

“…However, there are conflicting reports as to which of several specimen types bear the highest viral load ( 1 – 3 ), and ultimately, the “preferred-specimen” specification was removed from interim CDC guidance on 29 April 2020 ( 4 ). Sensitivity is a complex issue, however, as detection in the upper airways (nasopharynx and oropharynx) is affected by multiple factors, including duration of illness prior to testing ( 5 ) and the limit of detection (LoD) of the reverse transcription (RT)-PCR assay used ( 6 ).…”