Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?

Abstract

Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.

Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia

“…Payne et al wrote that over 60% of errors occur in the pre-analytical phase of any diagnostic process [ 43 ]. In the SARS-CoV-2 context, this phase includes the type of sample, the method of specimen collection, transport medium composition, storage conditions and time from sample collection to the RNA isolation [ 43 , 44 ]. The RNA extraction method and the RT-qPCR assay has also significant impact on Ct values, as demonstrated by this study [ 39 , 40 , 45 , 46 ].…”

“…The Ct value of a more sensitive kit represents a lower viral load compared to the same Ct of a less sensitive kit which needs thousands of more virus copies to reach the same Ct. Additionally, as discussed above, the identical Ct detected by the same assay has distinct infectivity potential if an early or late phase is detected. Without clinical data, the RT-PCR test cannot tell the disease phase [ 44 ]. Discrepancies between Ct values from different assays could be solved by normalisation by standard curve allowing Ct translation into copies/mL [ 52 , 53 , 54 ].…”

Environmental testing for SARS-CoV-2 in three tertiary-care hospitals during the peak of the third COVID-19 wave

“…A cutoff value of 34 cycles using an assay targeting the RdRp gene has been suggested for SARS-CoV-2 infectivity. 10 However, Ct values may well vary across sampling conditions and utilized assays. Furthermore, almost all positive samples fell within the highly transmissible B.1.1.7 lineage.…”

Optimizing testing regimes for the detection of COVID-19 in children and older adults

“…A serious concern in COVID-19 detection is the transportation of samples, and therefore pre-analytical parameters need to be optimized in each laboratory. To follow the protocol, clinical laboratories in the United State need to perform ‘bridging studies’ on FDA-approved 2019-nCoV diagnostic kits [ 116 ]. In this scenario, the effect of different sample types stored in variable transport/collection media on the performance of SARS-CoV-2 RT-PCR kits was evaluated [ 117 ] and concluded that transport media, 0.9% NaCl or amies media are compatible for nasopharyngeal, E-Swab or 3D-printed swabs.…”