It has been emphasized that fast and accurate diagnostic tests are essential for controlling the ongoing COVID-19 pandemic. Some evaluations have shown that saliva specimens and nasopharyngeal swabs have similar sensitivity in detecting SARS-CoV-2 [7–9]. In the present study, unexpectedly, a high number of samples with Ct values on the border of Ct < 40 and low sensitivity of our results for patients (53.4%) were observed. According to the CDC 2019-nCoV Real-time RT-PCR Diagnostic Panel protocol , we carefully reviewed our internal and external controls, which revealed the following: i) The known positive control consistently yielded a Ct value in the range of 30 to 33, and the negative controls did not yield fluorescence growth curves in any of the batches assayed. ii) Ct values were highly consistent between duplicates, and the external no-template control never yielded fluorescence growth curves. iii) The RP primers and probe set, as extraction RNA controls, yielded fluorescence growth curves; however, on many occasions, they crossed the Ct = 40 threshold, particularly in those patients from whom scarce saliva was obtained. For the mechanically ventilated patients, saliva specimens were obtained using a disposable sterile plastic like-Pasteur pipette, which yielded a low amount of saliva (approximately 500 µl). iv) All assays were performed with the same batch of primers, probes and reagents. The CDC 2019-nCoV Real-time RT-PCR Diagnostic Panel Results Interpretation Guide states that if only one of the two targets (N1 and N2) is positive, the test result is inconclusive, and the sample should be reanalysed, with new nucleic acid re-extraction and RT-PCR steps. In addition, the guide states that negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for treatment decisions or other patient management decisions. In addition, false-negative results may occur if an inadequate number of organisms are present in the specimen .
Ct values may vary due to a variety of factors; however, the algorithm used to calculate them assumes that all amplification efficiencies are equal to 2 or at least equal among all reactions, and possible variation in amplification efficiency among sequences or samples  or among instruments  are not considered. Interestingly, in a retrospective study of nasopharyngeal-oropharyngeal specimens from symptomatic individuals, the proportion of specimens containing a viral load close to the assay limit of detection (LoD) was small; therefore, there was a low risk of false negatives when testing symptomatic patients by qRT-PCR .
The qRT-PCR has high sensitivity and is therefore helpful for the initial diagnosis of COVID-19 . However, according to Tom and Mina , reporting the result as a binary measure, i.e., positive or negative, can confuse physicians by eliminating information useful for clinical decision making. It has been reported that after complete resolution of symptoms, patients infected with SARS-CoV-2 continue to yield positive qRT-PCR results for many weeks . At later time points in such cases, Ct values are often very high, representing the presence of low copy numbers of viral RNA (as low as fewer than 100), but the results are reported to the clinicians simply as positive. This situation leaves the clinician with little choice but to interpret the results similarly to the clearly positive results of samples from patients with a high viral load, in which RNA copies typically reach 100 million or more . Therefore, it is advisable that clinicians be informed of the Ct values obtained during the amplification of the viral markers as well as the Ct values corresponding to the LoD of viral RNA, which vary according to the characteristics of each system and amplification protocol used. As Binnicker  suggests, Ct value criteria must be established by each healthcare institution. In addition, the incorporation of viral load value would be useful to detail the number of copies of viral RNA in a sample.
A common concern is the presence of asymptomatic SARS-CoV-2-infected individuals at work (return-to-work) or hospital settings. A study regarding viral dynamics in 31 asymptomatic COVID-19 patients showed that 22 of them presented symptoms after their hospital admission and that their Ct values (Ct = 39) before hospital admission were significantly higher than those of symptomatic patients (Ct = 34.5). These findings showed that although the asymptomatic patients with COVID-19 exhibited a lower viral load, they undergo a certain period of viral shedding, allowing the possibility of transmission during their asymptomatic period . In addition, some authors have stated that a positive qRT-PCR result may be due to the presence of active, replicating virus or residual viral nucleic acid, i.e., noninfectious virus; a small proportion of recovered patients may yield positive virus detection results after discharge, and this positivity does not necessarily mean that the patient is transmissive [29, 30]. In the present study, we performed reverse transcription using oligo(dT) primers, which is the method most widely used for the conversion of mRNA into cDNA. During reverse transcription, an oligo(dT) primer is first annealed to the poly(A) sequences present at the 3' end of nearly every mRNA by T:A base-pairing. Subsequently, the reverse transcriptase extends from the annealed oligo(dT) primer along the mRNA template, resulting in the copying of the mRNA sequence into the cDNA sequence . Thus, during reverse transcription, we captured all viral mRNA templates, implying that a possible active viral transcription process is underway and that the potential infective SARS-CoV-2 virus may be shedding. Furthermore, differences within the fluorescence growth curves for the three markers (N1, N2, N3) of the same gene can be explained by the reverse transcription by oligo(dT) primers: A high frequency of truncated cDNAs through internal poly(A) priming has been demonstrated ; therefore, during qPCR, different amounts of cDNA template can modify the yield of the fluorescence growth curves. Although most of the AHW in the present study who were found positive by qRT-PCR showed high Ct values with 1 or 2 markers, suggesting a low viral load, the number of positive results increased almost 3 times when we changed the Ct cut-off from 40 to 46. Nevertheless, 8 AHW exhibited 2 or 3 amplified markers with high viral load (Ct < 20), implying a risk of transmission for their contacts. However, caution should be taken when interpreting the results, since wide flexibility in the Ct cut-off criteria could lead to an over-reporting of positive tests that would complicate potential clinical decisions.
Critically ill patients infected with SARS-CoV-2 have been reported to exhibit higher viral loads and more prolonged shedding in the lower respiratory tract than in the upper respiratory tract. Sampling from the lower respiratory tract may be required to assess the true viral clearance in such patients . Therefore, considering the technical difficulties in obtaining a sufficient amount of saliva from patients who required assisted mechanical ventilation, it is not advisable to use saliva as test specimens in critically ill patients.
The Ct value is inversely related to the viral load, and every ~ 3.3 increase in Ct value reflects a 10-fold reduction in starting material . Interestingly, a significant association between Ct < 40 for N2 and mechanical ventilation in patients (p = 0.013) was found, and significant correlations between the Ct values of N1 and N3 and the values of ALT, AST, LDH, CK-MB and ferritin were identified. We do not have a clear virological explanation for this finding. Different expression levels between markers can arise due to the reverse transcription process used in the present protocol; however, we cannot rule out differential expression in certain regions of the structural genes of the virus, especially the nucleocapsid gene, in which multiple copies must be expressed synchronously to assemble the SARS-CoV-2 virions that will be expelled from the infected cell. Nevertheless, our findings are in concordance with other reports. Azzi et al.  found an inverse correlation between the LDH values obtained in haematochemical analyses and Ct value; in addition, a retrospective cohort study of 678 inpatients showed that the risk of intubation was higher in patients with a high viral load (Ct < 25) than in those with a medium (Ct 25–30) or low viral load (Ct > 30) . Furthermore, in a meta-analysis of 60 studies that reported laboratory findings, Nascimento et al.  found differences in patients with COVID-19 for ALT, AST and LDH. Another meta-analysis performed by Shi et al.  showed that elevated levels of CK-MB were associated with the severity of COVID-19 in patients. Furthermore, another study found that the serum levels of ferritin were markedly increased in patients with very severe COVID-19 compared with patients with severe COVID-19 . Ferritin is particularly relevant as it is a mediator of immune dysregulation; it has been proposed that under extreme hyperferritinaemia, ferritin exerts direct immune-suppressive and pro-inflammatory effects, contributing to the cytokine storm observed in patients with COVID-19 . For this reason, it is important to continue carrying out virological studies of this new coronavirus to clearly understand the molecular process of viral replication and find potential markers of disease progression as well as targets of therapeutic drugs that will allow the control of COVID-19.