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.

Figure 1

(a) Genome of SARS-CoV-2 with the most common RT-PCR targets highlighted. (b) Essential steps of the RT-PCR diagnostic workflow including sample collection, storage in a transport medium, lysis and RNA extraction, reverse transcription, amplification, and detection. (c) Example of a real-time RT-PCR amplification curve for SARS-CoV-2 and postrun analysis to interpret results.

Figure 2

Quantification of SARS-CoV-2 following exposure to different inactivation conditions: (a) surface plasmon resonance (SPR) binding curves recorded on a T200 Biacore for the receptor-binding domain (RBD, 200 nM) and RBD for different time intervals. The interface was modified with SARS-CoV-2-specific VHH-72 nanobodies. (b–d) RNA stability after heat inactivation: SARS-CoV-2 RNA-positive nasopharyngeal swab specimens were pooled and divided into equal volumes and heated for 30 min at 60 °C. The aliquots were either kept at room temperature (RT), 4 °C, or −80 °C for the indicated times before RNA extraction and RT-PCR was performed in triplicate. As a control, untreated specimens were included. Ct values are indicated for the SARS-CoV-2 targets IP2 (b), IP4 (c), and the cellular control G6PDH (d) (Figure 2−2d report mean values of three samples).

Figure 3

Clinical significance of SARS-CoV-2 RT-PCR results. (a) Timeline of SARS-CoV-2 infectivity taking into account our own findings and those of others.^, (b) Ct values (target IP2) as a function of time after the symptom onset in nasopharyngeal swab specimens of COVID-19 patients. Ct of specimens with undetectable SARS-CoV-2 RNA were set to 50. (c) Correlation of Ct values with SARS-CoV-2 infectivity. Vero E6 cells were infected with 10-fold dilutions of a SARS-CoV-2 isolate. The plates were incubated for 6 days in 5% CO₂ at 37 °C and examined daily using an inverted microscope (ZEISS Primovert) to evaluate the extent of the virus-induced cytopathic effect in cell culture. The calculation of the estimated virus concentration was carried out by the Spearman and Karber method^, and expressed as TCID₅₀/mL (50% tissue culture infectious dose). TCID₅₀/mL values were transformed to PFU mL^–1 using the formula PFU mL^–1 = TCID₅₀/mL × 0.7. RNA extraction and RT-PCR (target IP4 and target IP2) were performed in duplicate for each dilution. Ct of dilutions with undetectable SARS-CoV-2 RNA were set to 50.