ASPASIA KONTOU
MOLECULAR BIOLOGIST
PASTEUR INSTITUTE
ATHENS, GREECE
Introduction
Diagnostic testing is one
of the key strategies for the control of the spreading of the global pandemic
of COVID-19 that began in late 2019, due to the lack of an effective treatment
and vaccine. Figure 1 summarizes the different needs that a diagnostic test may
serve. Different countries have implemented different approaches. The preferred
strategy was prioritized testing for specific groups of persons, mainly
involving hospitalized patients with presentations compatible with COVID-19, as
well as symptomatic persons at risk for poor outcomes. The global shortage of
laboratory supplies leading to limited testing capacity, has also played an
important role in this strategical approach. Nevertheless, there are a few
cases (like South Korea and Singapore) where diagnostic testing on a massive
scale has been a cornerstone for the successful containment of the disease1. Each strategic approach needs a different
testing pipeline. The parameters that need to be taken into account are the
turnaround time, the throughput (ability to perform many test at the same
time), the batching (thee need to have a specific number of specimens before testing),
the ability to perform the test in different settings and infrastructures1,2.
Figure 1.
Basic uses of diagnostic testing of clinical specimens for the detection of
SARs-CoV-2. SARS–CoV-2 = severe acute respiratory syndrome–related
coronavirus-2.1.
Currently, 2 types of
diagnostic tests are available for the detection of SARS-CoV-2: (1) molecular
and (2) serological tests. Molecular tests are real-time
reverse transcription polymerase chain reaction–based assays, which is the
current reference standard for SARS-CoV-2 detection. These tests are performed
mainly on respiratory specimens and at the moment serve the majority of the
needs illustrated in figure 1. PCR test are mainly used for viral detection in
the first month after symptom onset. However, serologic immunoassays and rapid
point-of-care assays are also rapidly emerging1. Serologic assays are expected to serve the
surveillance, epidemic forecasting, and determination of SARS–CoV-2 immunity1.
Figure 2. The phases after the infection of a patient with
SARS-CoV 2 and the diagnostic test that is used at each time point3. SARS-CoV-2:
severe acute respiratory syndrome coronavirus 2, PCR: polymerase chain
reaction.
Laboratory-Based Molecular Testing
The cornerstone for
nucleic acid amplification testing is real-time reverse transcription polymerase
chain reaction-based assays as mentioned before. Samples mainly from the
respiratory tract are taken to assess for the presence of 1 or several nucleic
acid targets specific to SARS–CoV-2. More specifically, four important steps
are involved in the molecular SARS-VoV-2 virus detection (Figure 1).
a)
The first comprises of sample selection and collection.
The SARS-CoV-2 RNA has already been detected in a variety of clinical samples
ranging from the respiratory tract (nasopharyngeal and
oropharyngeal swabs, nasal wash, sputum, saliva, tracheal aspirates,
bronchoalveolar lavage fluid), to samples from the gastrointestinal tract
(feces, anal swabs), blood, as well as endothelial cells of various organs from
patients1. Nevertheless, the preferred initial specimens have
been oropharyngeal and nasopharyngeal swabs due to their easy and
direct sampling[A1] , without requirement of
specialized skills and/or equipment. Samples from the low respiratory tract are
taken mainly from hospitalized patients and may have greater sensitivity than
upper respiratory tract specimens. Samples should be sent directly to the
microbiology laboratory for processing or can be stored at 2-8ºC for 72 hours,
or -70ºC for a greater period of time. Insufficient sampling and poor sample
storage or transport may lead to false negative results1,2.
b)
The sample is processed for nucleic acid extraction.
This involves cell lysis and RNA isolation. The extraction process is performed
either manually or in automated extractors, based on the laboratory supplies of
each diagnostic centre. Methods used in this step are mainly silica based (using
a spin column or magnetic beads). The steps involved are cell lysis, RNA
binding to silica, removal of all other cellular products and RNA elution in an
appropriate buffer2. Rare is the use of phenol-chloroform based assays
for the RNA extraction in diagnostic laboratories.
c)
The isolated viral RNA is reverse transcribed into
cDNA,
d)
which is then used as a template for real-time pcr.
The last 2 steps can be performed in a one-step or a two-step reaction. The genome of the SARS-CoV2 is an unstable RNA molecule, which needs to be transformed into cDNA, through reverse transcription, in order to serve as a template for the final real-time pcr reaction. At this point specific sequence(s) (usually one or two) of the SARS-CoV-2 genome are amplified with the use of oligonucleotide sequence-specific primers and probes and are detected with a fluorescently labelled probe(s). A commonly used strategy is the Taqman-probe detection method. On the other hand, there is always the choice of using non-specific fluorescent dyes, such as SYBR Green which binds to double-stranded DNA regardless of the sequence of the template that is being amplificated. Such protocols may lead to false positive results.
The
real-time pcr reaction also provides a semi-quantitative measure of patient’s
viral copy number in real-time. This is expressed through a meter called the
threshold cycle (Ct) or, according to the MIQE guidelines, quantification cycle
(Cq). More specifically, the
Ct are the number of cycles at which the fluorescence exceeds a determined
threshold. The lower the Ct the higher the viral load of the sample (figure 3).
Figure 3.
Diagram depicting real-time PCR phases (https://help.medicinalgenomics.com/qpcr-vs-end-point-pcr).
The real-time reverse
transcription polymerase chain reaction is mainly performed by
several commercial kits that have been designed for the SARS-CoV 2 detection. CDC (Centres for
Disease Control and Prevention) and the World Health Organization (WHO) have
developed 2 different assays, which have high analytic sensitivity and
specificity for SARS–CoV-2 (have minimal cross-reactivity with other
coronaviruses strains). The first kit contains PCR primer–probe sets for 2
regions of the viral nucleocapsid gene (N1 and N2) and a set for the human
RNase P gene as an internal quality control for the successful RNA extraction.
The second kit targets the SARS–CoV-2 RNA-dependent RNA polymerase (RdRP) and
envelope (E) genes1,2. The SARS-CoV 2 genome contains 6 open reading frames
(ORFs). These include ORF1a/b, spanning 16 non-structural proteins (nsp)
relating to the replication-transcription complex, 4 structural proteins, spike
(S), envelope (E), membrane (M), and nucleocapsid (N), along with several other
non-structural, special structural, and/ or accessory ORFs (ORF3a/b, 6, 7a, 7b,
8, and 10).The primer-probe sets for most diagnostic tests target a combination
of non-structural (ORF1ab region) and structural (S, N, and/ or E) SARS-CoV-2
genes, together with a positive and negative control. The reason for that is
that genome sequence alignment between different coronaviruses has shown more
conservation among the non-structural proteins, compared to the structural ones2.
Figure 4.
The basic steps for the molecular diagnosis of SARS-CoV-2 infection include a)
sample collection, b) extraction of the viral RNA, c) reverse transcription of
the viral RNA into cDNA which is used (https://www.globalbiotechinsights.com/articles/20247/the-worldwide-test-for-covid-19).
Comparison of different clinical samples for SARS-CoV 2 detection
As it has already been mentioned
the SARS-CoV 2 RNA has already been detected in many different specimens
(respiratory tract, feces, blood, endothelial cells of various organs), proving
the presence of the viral genome in extended parts of the human body4. Nevertheless, not all the different specimens have
the same diagnostic value. A study of the detection of SARS-CoV-2 in different
types of clinical specimens proved that samples from the lower respiratory
tract tested most often positive (bronchoalveolar lavage (BAL) fluid specimens
showed a 93% positive rate, followed by sputum with 72%, nasal swabs with 63%
and pharyngeal swabs with 32%)54. This higher diagnostic yield of specimens from the
lower respiratory tract can be explained by the mechanism of the viral infection.
It is supported by the basic science that the target functional receptor of
SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2). The ACE2 protein is
located in various human organs (oral and nasal mucosa, nasopharynx, lung,
stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow,
spleen, liver, kidney, and brain). Lung type I and II alveolar epithelial cells
and enterocytes of the small intestine show abundant surface expression of
ACE2, which is minimal on bronchial epithelial cells and negative on oral,
nasal and nasopharynx. Furthermore, ACE2 is present in vascular endothelium6,7.
Moreover, it was shown that the
detection rate of SARS-CoV-2 was slightly higher when both nasal and oral swabs
were used, compared to solo nasopharyngeal swabs, while the use of only
pharyngeal swabs could lead to false negative results8. On the other hand, bronchoalveolar lavage (BAL)
fluid specimens are the appropriate specimens for viral RNA detection in severe
cases9. Lastly, the time stage of the disease should also be taken into
consideration as higher viral loads have been detected soon after symptom onset10.
SARS-CoV-2 RNA as well as
infectious viral particles have also been detected in faecal samples, implying
that the virus may also be transmitted by the faecal route10,11. Available data suggest that the viral load in faecal
samples is lower than respiratory specimens and that the viral nucleic acid
shedding pattern of patients infected with SARS-CoV-2 resembles that of
patients infected with influenza, rather than that of patients infected with
SARS-CoV10,11. In the same studies a small percentage of blood
samples tested positive, suggesting the systemic nature of the disease in some
cases4.
All the results mentioned before indicate that testing
of specimens from multiple sites may reduce false-negative results and improve
the sensitivity.
Pitfalls of PCR-based assays
Real-time
reverse transcription polymerase chain reaction shows the presence or absence
of the SARS-CoV 2 viral genome in the human host but does not give any further
information for the infectivity of the virus, unless there is proof that the
virus can be isolated and cultured from the particular samples. This explains
why prolonged positive test results that have been detected in some patients
are not in concordance with clinical symptoms of COVID-19. On the other hand,
such results indicate the prolonged shedding of the viral RNA either from the
upper respiratory tract or from the gastrointestinal system1,4.
Moreover,
the RT-PCR tests used for diagnosis can lead to a false negative result for a
variety of reason. For example, the concentration of the viral RNA in the
clinical sample may be below the limit of detection of the method. The limit of
detection is the lowest concentration of SARS-COV-2 that can be detected by the
PCR test and is determined by the detection of the viral RNA in at least 95% of
the cases. If the limit of detection of a method is too high, then patients
infected with SARS-Cov 2 may not test positive. On the other hand, a really low
limit of detection may lead to false positive results, due to the highest risk
for contamination3,4.
As mentioned before the
sampling method combined with the storage and transport conditions play an
important role in the sample quality. RNA is a quite unstable nucleic acid and
can be easily degraded if not properly stored and handled. Therefore, a quality
control test should be performed in every sample (for example the test for the
human RNase P gene). Evidence also shows that the time of sampling together
with the type of sample also influence the PCR result. As mentioned before
samples of the respiratory tract are adequate for COVID-19 diagnosis, as they
have some of the highest viral loads. More specifically, specimens from the
lower respiratory tract show higher sensitivity than nasopharyngeal or
oropharyngeal swabs. Apart from that several studies also corroborate that the
later a specimen is collected after the day symptoms began, the higher the
chance of having a false-negative test result. It is also quite
clear that urine and serum samples are not appropriate specimens for COVID-19 testing2,3,4.
Persistently
positive results, reinfection and coinfections
it is unlikely that patients are becoming reinfected
shortly after their initial infection, however a few cases have been reported.
Lingering positive results are possibly explained if viral RNA remains in
tissues for a considerable amount of time, even when the viral particles
capable of causing infection have been cleared. In most cases, patients that
have tested positive for SARS-CoV-2 after their original infection are asymptomatic or not clinically worse
at the time of retesting. If a patient is positive for the disease, then
has a negative test followed by a positive test within a short period of time,
this is most likely due to a false-negative test occurring between the positive
tests (perhaps due to one of the factors described above)12.
Among patients diagnosed with COVID-19, the occurrence
of concomitant viral infections has been reported to range from below 6% to
greater than 60%. As a result, it is not possible to rule out SARS–CoV-2
infection merely by detecting another respiratory pathogen13.
The lack of an established reference standard, use of
differing sample collection and preparation methods, and an incomplete
understanding of viral dynamics across the time course of infection hamper
rigorous assessment of the diagnostic accuracy of the many newly introduced
SARS–CoV-2 assays.
REFERENCES
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L., Chang, S. P. & Nerurkar, V. R. COVID-19 Special Column: Principles
Behind the Technology for Detecting SARS-CoV-2, the Cause of COVID-19. Hawai’i
J. Heal. Soc. Welf. 79, 136–142 (2020).
3. Sethuraman,
N., Jeremiah, S. S. & Ryo, A. Interpreting Diagnostic Tests for SARS-CoV-2.
JAMA (2020) doi:10.1001/jama.2020.8259.
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W. et al. Detection of SARS-CoV-2 in Different Types of Clinical
Specimens. JAMA (2020) doi:10.1001/jama.2020.3786.
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implications and open questions. J. Cell Sci. 132, jcs227611
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I. et al. Tissue distribution of ACE2 protein, the functional receptor
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R. et al. Virological assessment of hospitalized patients with
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et al. SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected
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12. Iwasaki A. What reinfections mean
for COVID-19. Lancet infect Dis 2020.
October 12, 2020 https://doi.org/10.1016/ S1473-3099(20)30783-0.
13. Garcia-Vidal C,
Sanjuan G, Moreno-Garcia E et al: Incidence of co-infections
and superinfections
in hospitalized patients with COVID-19: a retrospective cohort
study. Clin Microbiol
Inf https://doi.org/10.1016/j.cmi.2020.07.041
[A1]The WHO documents the
summary of the optimum sample collection procedures, which is similar to those
for influenza. Dacron or polyester flocked swabs are recommended for OP/NP
swabs and sterile sample collection containers for washes or bronchoalveolar
lavage fluid (BAL) specimens, urine or stool. For whole blood, EDTA tubes
should be used, and for serum, separator tubes should be used [A1]
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