Due to the rapid spread and increasing number of coronavirus disease 19 (COVID-19)
cases caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), rapid and accurate detection of virus and/or disease is increasingly
vital to control the sources of infection and help patients to prevent the illness
progression. Since December 2019, there has been considerable challenge regarding
the use of nucleic acid test or clinical characteristics of infected patients as the
reference standard to make a definitive diagnose of COVID-19 patients. As the early
diagnosis of COVID-19 is critical for prevention and control of this pandemic, clinical
characteristics cannot alone define the diagnosis of COVID-19, especially for patients
presenting early-onset of symptoms.
Along with the advancement in medical diagnosis, nucleic acid detection-based approaches
have become a rapid and reliable technology for viral detection. Among nucleic acid
tests, the polymerase chain reaction (PCR) method is considered as the ‘gold standard’
for the detection of some viruses and is characterized by rapid detection, high sensitivity,
and specificity. As such, real-time reverse transcriptase-PCR (RT-PCR) is of great
interest today for the detection of SARS-CoV-2 due to its benefits as a specific and
simple qualitative assay [1–3]. Moreover, real-time RT-PCR has adequate sensitivity
to help us much for diagnosing early infection. Therefore, the ‘criterion-referenced’
real-time RT-PCR assay can be considered as a main method to be applied to detect
the causative agent of COVID-19, SARS-CoV-2.
An important issue with the real-time RT-PCR test is the risk of eliciting false-negative
and false-positive results. It is reported that many ‘suspected’ cases with typical
clinical characteristics of COVID-19 and identical specific computed tomography (CT)
images were not diagnosed [4]. Thus, a negative result does not exclude the possibility
of COVID-19 infection and should not be used as the only criterion for treatment or
patient management decisions. It seems that combination of real-time RT-PCR and clinical
features facilitates management of SARS-CoV-2 outbreak. Several factors have been
proposed to be associated with the inconsistency of real-time RT-PCR [5]. In the following,
we attempt to discuss various challenges regarding the detection of SARS-CoV-2 by
real-time RT-PCR. It is expected that this could provide beneficial information for
the comprehension of the limitations of the obtained results and to improve diagnosis
approaches and control of the disease.
It is well known that results from real-time RT-PCR using primers in different genes
can be affected by the variation of viral RNA sequences. Genetic diversity and rapid
evolution of this novel coronavirus have been observed in different studies [6,7].
False-negative results may occur by mutations in the primer and probe target regions
in the SARS-CoV-2 genome. Although it was attempted to design the real-time RT-PCR
assay as precisely as possible based on the conserved regions of the viral genomes,
variability causing mismatches between the primers and probes and the target sequences
can lead to decrease in assay performance and potential false-negative results. In
this regard, multiple target gene amplification could be used to avoid invalid results.
Several types of SARS-CoV-2 real-time RT-PCR kit have been developed and approved
rapidly, but with different quality. Importantly, the sensitivity and specificity
of the real-time RT-PCR test is not 100%. All of them behind the laboratory practice
standard and personnel skill in the relevant technical and safety procedures explain
some of the false-negative results.
According to the natural history of the COVID-19 and viral load kinetics in different
anatomic sites of the patients, sampling procedures largely contribute to the false-negative
results. Optimum sample types and timing for peak viral load during infections caused
by SARS-CoV-2 remain to be fully determined. A study has reported sputum as the most
accurate sample for laboratory diagnosis of COVID-19, followed by nasal swabs, while
throat swabs were not recommended for the diagnosis [8]. They also suggested the detection
of viral RNAs in bronchoalveolar lavage fluid (BALF) for the diagnosis and monitoring
of viruses in severe cases. However, gathering of BALF needs both a suction tool and
an expert operator, in addition to being painful to the patients. While BALF samples
are not practical for the routine laboratory diagnosis and monitoring of the disease,
collection of other samples such as sputum, nasal swab, and throat swab is rapid,
simple, and safe. To avoid inconsistent results, it would be better to use different
specimen types (stool and blood) besides respiratory specimen during different stages.
It is worth noting that samples should be obtained by dacron or polyester flocked
swabs and should reach the laboratory as soon as possible after collection. False-negative
results may occur due to the presence of amplification inhibitors in the sample or
insufficient organisms in the sample rising from inappropriate collection, transportation,
or handling.
Viral load kinetics of SARS-CoV-2 infection have been described in two patients in
Korea, suggesting a different viral load kinetics from that of previously reported
other coronavirus infections [9]. In the first patient, the virus was detected from
upper respiratory tract (URT) and lower respiratory tract (LRT) specimens on days
2 and 3 of symptom onset, respectively. On day 5, the viral load was increased from
day 3 in the LRT specimen. However, the viral loads decreased from around day 7 in
both URT and LRT specimens. Real-time RT-PCR continued to be positive at a low level
until day 13 (LRT specimens) and day 14 (URT specimens). Finally, the assay became
undetectable for two consecutive days from day 14 (LRT specimen) and day 15 (URT specimen),
respectively. In the second patient, SARS-CoV-2 was detected in both URT and LRT specimens
on day 14 of symptom onset. However, the initial viral loads were relatively lower
than those of patient 1 in whom the test was performed on day 2 of symptom onset.
From day 18 (URT specimen) and day 20 (LRT specimen), real-time RT-PCR became undetectable
for two consecutive days, respectively. URT sample of day 25 was again positive for
RdRp and E genes. However, it was interpreted as negative due to high Ct value of
the RdRp gene (Ct value of 36.69). These findings indicate the different viral load
kinetics of SARS-coV-2 in different patients, suggesting that sampling timing and
period of the disease development play an important role in real-time RT-PCR results.
Finally, the Centers for Disease Control and Prevention (CDC) has designed a SARS-CoV-2
Real-Time RT-PCR Diagnostic Panel to minimize the chance of false-positive results
[10]. In accordance, the negative template control (NTC) sample should be negative,
showing no fluorescence growth curves that cross the threshold line. The occurrence
of false positive with one or more of the primer and probe NTC reactions is indicative
of sample contamination. Importantly, the internal control should be included to help
identify the specimens containing substances that may interfere with the extraction
of nucleic acid and PCR amplification. Because of the several risks to patients in
the event of a false-positive result, all clinical laboratories using this test must
follow the standard confirmatory testing and reporting guidelines based on their proper
public health authorities.
1.
Expert opinion
In conclusion, according to the mentioned reasons, the results of real-time RT-PCR
tests must be cautiously interpreted. In the case of real-time RT-PCR negative result
with clinical features suspicion for COVID-19, especially when only upper respiratory
tract samples were tested, multiple sample types in different time points, including
from the lower respiratory tract if possible, should be tested. Importantly, combination
of real-time RT-PCR and clinical features especially CT image could facilitate disease
management. Proper sampling procedures, good laboratory practice standard, and using
high-quality extraction and real-time RT-PCR kit could improve the approach and reduce
inaccurate results.