life

Review
Analytical Performance of COVID-19 Detection Methods
(RT-PCR): Scientific and Societal Concerns †
Roberto Verna 1,2,3,‡ , Walter Alallon 4,‡ , Masami Murakami 5,‡ , Catherine P. M. Hayward 6,‡ ,
Abdel Halim Harrath 7 , Saleh H. Alwasel 7 , Nairo M. Sumita 8,‡ , Ozkan Alatas 9,‡ , Valeria Fedeli 10 ,
Praveen Sharma 11,‡ , Andrea Fuso 10 , Daniela Maria Capuano 2,3 , Maria Capalbo 12 , Antonio Angeloni 10 and
Mariano Bizzarri 10, *,‡

1 In Unam Sapientiam, 00185 Rome, Italy; [email protected]

2 WASPaLM, CT Corporation, P.O. Box 4349, Carol Stream, IL 60197-4349, USA; [email protected]
3 Academy for Health and Clinical Research, 00185 Rome, Italy
4 Department of Clinical Laboratory, Hospital de Clínicas, Facultad de Medicina, Universidad de la República,
Montevideo 11000, Uruguay; [email protected]
5 Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine,
3-39-15 Showa-Machi, Maebashi 371-8511, Japan; [email protected]
6 Health Science Center, Departments of Pathology and Molecular Medicine, and Medicine, Room 2N29A,
McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada; [email protected]
7 Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
[email protected] (A.H.H.); [email protected] (S.H.A.)
8 Grupo Fleury, Central Laboratory Division, Hospital das Clínicas, Faculdade de Medicina, Universidade de
São Paulo, São Paulo 05508, Brazil; [email protected]



9 Department of Medical Biochemistry, Eskisehir Osmangazi University Medical School,


Eskisehir 33400, Turkey; [email protected]
10 Department of Experimental Medicine, Sapienza University, 00160 Rome, Italy;
Citation: Verna, R.; Alallon, W.;
Murakami, M.; Hayward, C.P.M.; [email protected] (V.F.); [email protected] (A.F.); [email protected] (A.A.)
11 Department of Biochemistry, All India Institute of Medical Sciences, Jodhpur 342005, India;
Harrath, A.H.; Alwasel, S.H.; Sumita,
[email protected]
N.M.; Alatas, O.; Fedeli, V.; Sharma, 12 Azienda Ospedaliera Ospedali Riuniti Marche Nord (DG), 61121 Pesaro, Italy;
P.; et al. Analytical Performance of
[email protected]
COVID-19 Detection Methods
* Correspondence: [email protected]
(RT-PCR): Scientific and Societal † The present manuscript has been prepared after the discussion followed by the free WASPaLM webinar held
Concerns. Life 2021, 11, 660. https:// on 17 December 2020. The manuscript has been endorsed by the WASPaLM Board and should be considered
doi.org/10.3390/life11070660 as an official Position Paper of WASPaLM.
‡ Members of the Board of the World Association of Societies of Pathology and Laboratory Medicine
Academic Editor: Patrick Mercié (WASPaLM).

Received: 16 June 2021 Abstract: Background. Health and social management of the SARS-CoV-2 epidemic, responsible for
Accepted: 2 July 2021 the COVID-19 disease, requires both screening tools and diagnostic procedures. Reliable screening
Published: 6 July 2021 tests aim at identifying (truely) infectious individuals that can spread the viral infection and therefore
are essential for tracing and harnessing the epidemic diffusion. Instead, diagnostic tests should
Publisher’s Note: MDPI stays neutral supplement clinical and radiological findings, thus helping in establishing the diagnosis. Several
with regard to jurisdictional claims in analytical assays, mostly using RT-PCR-based technologies, have become commercially available for
published maps and institutional affil-
healthcare workers and clinical laboratories. However, such tests showed some critical limitations,
iations.
given that a relevant number of both false-positive and false-negative cases have been so far reported.
Moreover, those analytical techniques demonstrated to be significantly influenced by pre-analytical
biases, while the sensitivity showed a dramatic time dependency. Aim. Herein, we critically inves-
tigate limits and perspectives of currently available RT-PCR techniques, especially when referring
Copyright: © 2021 by the authors.
to the required performances in providing reliable epidemiological and clinical information. Key
Licensee MDPI, Basel, Switzerland.
Concepts. Current data cast doubt on the use of RT-PCR swabs as a screening procedure for tracing
This article is an open access article
the evolution of the current SARS-COV-2 pandemic. Indeed, the huge number of both false-positive
distributed under the terms and
conditions of the Creative Commons
and false-negative results deprives the trustworthiness of decision making based on those data.
Attribution (CC BY) license (https:// Therefore, we should refine current available analytical tests to quickly identify individuals able to
creativecommons.org/licenses/by/ really transmit the virus, with the aim to control and prevent large outbreaks.
4.0/).

Life 2021, 11, 660. https://doi.org/10.3390/life11070660 https://www.mdpi.com/journal/life

Life 2021, 11, 660 2 of 16

Keywords: COVID-19; SARS-CoV-2; RT-PCR; false positive; false negative; pandemic; clinical pathology

1. The Challenge: Scientific and Political Problems

The spread of the SARS-CoV-2 pandemic around the globe has created several critical
problems that should be addressed concomitantly, with some problems requiring higher
prioritization. The pathophysiology of the disease (that affects presentation and complica-
tions), early detection of infectious individuals, and the need to provide adequate treatment
are among the topics that require very timely and appropriate management. With any
pandemic, recognizing the etiological factor (the virus) is an essential starting point. A key
second step is to establish a reliable and specific method to detect viral infection (especially
the RNA messenger or viral antigens) and/or the related immunological consequences
(specific antibodies, belonging to the IgG, IgM, and IgA clusters) in suspected cases. Un-
doubtedly, the COVID-19 epidemic has promoted the repositioning of laboratory medicine
to a more visible and strategic place in healthcare systems to deal with the current disease
and to prepare for and prevent future pandemics [1].
Awareness of the need for timely development of testing capabilities has led several
regulatory agencies—including FDA—to find acceptable levels of compromise between test
reliability and quick turnaround by readily available and useful diagnostic tools. This has
shortened timelines for assessing mandatory requirements considerably, in some cases, to
just a few weeks. This approach to making rapid approval decisions has led to the approval
of several diagnostic tools without long-term information on reliability, performance, and
diagnostic accuracy, which affect test validity [2].

2. Diagnosis of SARS-CoV-2 Infection

The current diagnostic procedures for the identification of SARS-CoV-2-infected indi-
viduals are primarily based on two distinct approaches [3]. The first is the direct detection
of the virus or its components. This can be carried out by a culture of the virus, detection
of one or more of its proteins or other components, or via amplification of viral nucleic
acids through RT-PCR techniques (usually known as “molecular tests”). The last two are
the most frequently used techniques during the present pandemic, and the main aim of the
present paper is to address some critical considerations regarding the molecular tests.
The second approach is the use of immunological tests to detect the host’s immune
response triggered by the virus through the detection of specific antibodies (IgM, IgG, but
also IgA). Some specialized laboratories are also able to test the host’s cellular immune
response to the virus. In addition, whole-genome sequencing has been applied to determine
the sequence of the SARS-CoV-2 virus in a sample, with the goal to identify changes in
sequence, or so-called variants [4] to further understand the epidemiology and viral factors
that predict disease transmission and severity. However, this is clearly an extremely
expensive approach that cannot be pursued in the frameshift of a rapid, population-based,
screening.
The three most used immunological assays are enzyme-linked immunosorbent assays
(ELISAs), chemiluminescence assays (CLIAs), and lateral flow assays (LFAs) [5]. Virus
neutralization tests are an additional type of specialized immunoassay to specifically detect
neutralizing antibodies, but this type of method is primarily used for assay validation
and research purposes. Preliminary reports on ELISA assays to detect viral infection
have indicated a fairly good correlation between antibody titers and virus-neutralizing
antibodies [6]. In addition to the assays described, many other assays have been developed,
as summarized elsewhere [7].

3. Preanalytical Issues Affecting the Diagnosis of COVID-19

It is widely acknowledged that pre-analytical factors represent a major source of error
in laboratory testing [8]. Such errors include the inappropriate collection of biological
Life 2021, 11, 660 3 of 16

material; inadequate sample storage/transportation (including the presence of additive or

cellular components that may interfere with the assay, in some cases, due to whole-blood
freezing); pipetting errors; contamination; or sample mismatch. Sample contamination is a
key issue to address to ensure the quality of reverse transcription–polymerase chain reaction
(RT-PCR) assays, given that even trace amounts of foreign nucleic acids can jeopardize test
findings. A second critical source of pre-analytic error is the use of improper procedures
for the collection of nasopharyngeal specimens. Indeed, the recommended procedure is
not so straightforward: a nasopharyngeal sample should be obtained by inserting the swab
far into the nostril parallel to the palate to reach the deepest target area, maintaining the
swab in place for few seconds for the absorption of secretions, then immediately putting
the swab into a sterile tube [9]. Failure to comply with the recommended practices can
cause several diagnostic errors [10].

4. Limits of Current RT-PCR Tests

According to several reports, the diagnostic accuracy of many of the currently avail-
able RT-PCR tests for SARS-CoV-2 may be lower than optimal, as false-positive, and
false-negative results are seen in a small but significant proportion of individuals. These
shortcomings have been ascribed to several factors, including lack of harmonization of
primers and probes; technical and analytical errors; and the absence of validation by
independent third parties [11]. The US Food and Drug Administration Emergency Use Au-
thorization (EUA) and Instructions for Use (IFU) documents outline the currently approved
virology tests for SARS-CoV-2, which are largely unstandardized [12]. For example, FDA
data on EUA SARS-CoV-2 virology tests show a wide range of limits of detection (LoD),
spanning >5 orders of log10 differences across different assays. Undoubtedly, assays with
higher LoDs will likely miss more infected patients. These metrics are of critical importance
because each 10-fold increase in the LoD of a COVID-19 viral diagnostic test is expected to
increase the false-negative rate by 13% [13]. Beyond “this variable performance” reported
in IFUs for EUA tests, key attributes of many tests, such as primer sequences, protocol
steps, or viral gene targets, are either unclear or missing [12].
Several different primer-probe sets for use in SARS-CoV-2 detection assays have
been developed and are currently in use. Overall, these tests show high specificity when
tested against a panel of samples positive for several other respiratory viruses, while
their sensitivity is highly variable [14]. It is noteworthy that assays that use the CDC
N2 primer–probe, developed by the Division of Viral Diseases at the Centers for Disease
Control and Prevention (CDC) [15]—and the Corman E-gene primer–probe [16], display
high sensitivity, even in presence of low genomic equivalents of viral RNA. According to
the Corman protocol (further endorsed by World Health Organization (WHO)) [17], the
E-gene assay is used as a first-line screening tool, which is then followed by a confirmatory
assay, based on an RNA-dependent RNA polymerase gene or an N gene test, respectively.
On the contrary, the test supported by the CDC involves three N gene primer/probe sets,
and as such, it is designed for detecting SARS-like coronaviruses (one primer/probe set),
as well the specific detection of SARS-CoV-2 (two primer–probe sets) (Figure 1). The panel
may eventually include a primer/probe for recognizing the human RNase P gene (RP) in
control samples and clinical specimens.
Differences in primers concentrations and/or in DNA probe lengths used by different
protocols have raised some concern, given that it has been argued that those settings do
not conform with the FDA protocols, and this may be one of the factors that lead to false-
positive results [18]. Furthermore, a nearly 100-fold difference in LoD has been recorded
among different assays that use different genes and probes, which cannot be considered
equivalent [19].
Usually, when RT-PCR analyses are designed for the first time, amplification is fol-
lowed by a second step where a separation technique (i.e., electrophoresis) or a sequencing
run is used to confirm that the amplified sequence was correct. Of course, this cannot be
 Life 2021, 11, 660 4 of 16

Life 2021, 11, x FOR PEER REVIEW 4 of 16

routinely performed in population-based screening, but more accurate characterization of
the PCR reactions used to reveal SARS-CoV2 infection is advisable.

Figure 1. Structure
Figure of SARS-CoV-2
1. Structure of SARS-CoV-2 virus. RNA
virus. RNAof the viral
of the genome
viral is constituted
genome by two
is constituted genes—ORF1a
by two genes—ORF1a andand
ORF1b—
ORF1b—
encoding
encoding forfor
thethe
nonstructural
nonstructuralproteins NSP,
proteins NSP,while
whilesmall
smallgenomic
genomicregion
region includes
includes S (spike protein),
protein), M
M (membrane
(membraneprotein),
pro-
tein), E (envelope
E (envelope protein),
protein), andand N (nucleocapsid
N (nucleocapsid protein)
protein) genes.
genes. The The S protein
S protein interacts
interacts with with the angiotensin-converting
the angiotensin-converting enzyme
enzyme 2 (ACE2)
2 (ACE2) receptors
receptors of theofhost
the cell
hostmembrane
cell membrane through
through a receptor-binding
a receptor-binding domain
domain (RBD)(RBD) positioned
positioned onspike
on the the spike
protein.
protein. Once the ACE2/RBD interaction has been established, the virus can be endocytosed into the cell.
Once the ACE2/RBD interaction has been established, the virus can be endocytosed into the cell.

Usually, when RT-PCR

Furthermore, a nearlyanalyses
100-foldare designed
difference for the
in LoD hasfirst
beentime, amplification
recorded among the is fol-
differ-
lowed by a second
ent assays that usestep where agenes
different separation technique
and probes; (i.e., electrophoresis)
therefore, these tests cannot or be
a sequenc-
considered
ingequivalent
run is used to confirm
[19]. Usually,that the amplified
RT-PCR analyses aresequence
followed wasbycorrect.
a secondOfstepcourse,
where this cannot
a separation
be technique
routinely performed in population-based screening, but more accurate
(i.e., mobility of the amplified fragment on a gel) is used to confirm that the characteriza-
tionamplified
of the PCR reactions
substance wasused to reveal
correct. SARS-CoV2
Unfortunately, infection is
in screening advisable.
settings for ascertaining SARS-
Furthermore,
CoV-2 positivity,aRT-PCR
nearly 100-fold
is generallydifference in LoD
not followed byhas been recorded
a second, confirmatoryamongstep.the dif- in
When
ferent assays that
diagnostic use different
settings, with a high genes andprobability
pretest probes; therefore, these tests
of SARS-CoV-2, cannot
RT-PCR be consid-
assays may pro-
ered equivalent
vide important[19]. Usually, RT-PCR
confirmation analysesdiagnosis,
of the suspected are followed basedbyon a second stepradiological
clinical and where a
separation technique
data. However, (i.e.,
when mobility
used of to
as a tool theperform
amplified fragment
screening on a gel)
surveys, is usedRT-PCR
extensive to confirm
testing
thatcannot
the amplified
suffice insubstance
providingwas correct.
reliable Unfortunately,
results. For other endemicin screening
viral settings for ascer-
diseases—including
taining
HCV,SARS-CoV-2
HIV, and chronic positivity, RT-PCR isresults
HBV—positive generally not followed
for specific by aoccur
viral tests second,
afterconfirma-
suggestive
tory step. When
symptoms in diagnostic
appear, and RT-PCR settings,
testswith a high pretest
are usually preceded probability of SARS-CoV-2,
by serological analyses that
greatlyassays
RT-PCR reducemay the provide
risk of false-positive results from of
important confirmation molecular tests. diagnosis, based
the suspected
It isand
on clinical embarrassing
radiological to data.
the field that very
However, few used
when studiesas have
a tooladdressed
to perform thescreening
prevalence
of false-negative
surveys, results for
extensive RT-PCR SAR-CoV-2
testing molecular
cannot suffice assays. Yet,
in providing the critical
reliable results.validation
For other of
diagnostic
endemic viraltests given emergency HCV,
diseases—including approvalHIV,byandregulatory
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HBV—positive indefinitely
results for
specific viral tests occur after suggestive symptoms appear, and RT-PCR tests are usual-
ly preceded by serological analyses that greatly reduce the risk of false-positive results
from molecular tests.
Life 2021, 11, 660 5 of 16

postponed [20]. For SARS-CoV-2, it is unclear if the sensitivity of any authorized commer-
cial test has been assessed in the proper way. Current rules permit indirect demonstration
of the test performance by applying the RT-PCR test to known positive material from
symptomatic people or contrived specimens: such assessments likely overestimate test
sensitivity as swabs may lack viral particle [21]. A reference standard for determining
the sensitivity of SARS-CoV-2 tests in asymptomatic people is still lacking. At the very
beginning of the epidemic in China, at least two studies reported on the impact of missing
truly infected patients due to overestimation of RT-PCR tests sensitivity, resulting in a
false-negative rate ranging from 2 to 30% [22,23]. A recent meta-analysis carried out on
12,057 COVID-19 confirmed cases reports that there is substantial and largely unexplained
heterogeneity in the proportion of false-negative RT-PCR results for SARS-CoV-2, which
cannot be explained by differences in the statistical approach used in different studies [24].
Differences in reliability rates might result from differences in the type of specimen col-
lected, the time to onset of symptoms, the surmised viral load, at the time when the test is
performed, to product-specific factors related to the RT-PCR kit used. All this information
is either partially or unreported in most studies. Noticeably, one report [25] demonstrated
that up to 54% of COVID-19 patients may have, during the first phase of their illness, an
initial false-negative RT-PCR. Accordingly, the field is faced with very low certainty of evi-
dence. Additionally, some published studies have several biases (protocol violation to rule
in/rule out the presence of SARS-CoV-2) and/or fail to report key index test characteristics.
Site of collection of the biological specimen. The analytical specificity of an analytical test
is its capability to recognize solely the molecule that is to be identified—both qualitatively
and quantitatively—in presence of interfering substances or antigenically overlapping
compounds. For molecular tests, the relevant abundance of RNA is essential for bestowing
sensitivity to the assay. In turn, RNA abundance is strongly dependent on the type
and site of collection of the biological sample. Indeed, the sensitivity of RT-PCR for
SARS-CoV-2 is higher for lower respiratory samples than for upper respiratory tract
samples [25,26]. Surprisingly, as reported in a preliminary study of 213 COVID-19 cases,
and 866 respiratory samples obtained onset from different sites of collection during the first
week after symptom, the highest positive rate (>80%) was recorded in salivary samples,
followed by nasopharyngeal (72–73%) and throat swabs (60–61%) [27,28].
A systematic review, performed on pooled data for 8136 clinical specimens tested
for SARS-CoV-2 by RT-PCR, substantially confirmed results for different types of spec-
imens: [29] specimens from the lower respiratory tract showed a positive rate of 71.3%;
bronchoalveolar lavage fluid and saliva samples had the highest positivity rates of 91.8%
and 87.8%, respectively; oropharyngeal swab, feces, and blood showed the lowest posi-
tivity rates (7.6%, 32.8%, and 1%, respectively), whereas no SARS-CoV-2 was detected in
urine samples. Another study showed that the rate of RT-PCR detection of SARS-CoV-2
in patients is as high as 93% in bronchoalveolar lavage fluid, 72% in sputum, and 63% in
nasopharyngeal swabs, while it is only 32% in pharyngeal swabs and 29% in stool [28].
Yet, till now, there has not been an independent assessment of the overall trustworthiness
of different RT-PCR tests, a global comparative analysis of these tests applied to different
settings, or consensus on the most reliable samples across different tests (nasopharyngeal
versus saliva). A few studies found that saliva samples yielded greater detection sensitivity
and more consistent findings throughout the course of infection when compared with
samples taken via nasopharyngeal swabs [30,31]. The very preliminary investigations,
using saliva specimens, displayed huge variability in sensitivity estimates, ranging from 70
to 100%, when compared to the throat and nasopharyngeal swabs tested by RT-PCR [32,33].
As for analyses carried out on nasopharyngeal samples, differences in reported sensitivities
may reflect differences in the timing of sampling, relative to the onset of infection. Indeed,
the highest sensitivity was observed among hospitalized and critically ill patients [34],
while the lowest sensitivity has been recorded when saliva samples were collected during
the late phase from symptom onset or exposure [35]. A recent meta-analysis suggests that
the diagnostic accuracy of saliva-based nucleic acid amplification testing is similar to that
Life 2021, 11, 660 6 of 16

of nasopharyngeal swabs, especially in the ambulatory setting [36]. Overall, the current
information suggests that saliva, when obtained in the early phases of COVID-19 infection
(10 days after the symptom onset), is reliable, and it may be more practical to collect saliva
than nasopharyngeal swabs for the screening and diagnosing of the virus [37].
Viral variants. An important issue of concern that has been increasing during recent
months is the increasing recognition of different viral haplotypes, due to the occurrence of
recombination and/or mutation [38]. High variability in genomic pattern and nucleotide
sequences is in keeping with the rapid spread of the SARS-CoV-2 pandemic [39]. Fur-
thermore, as observed by an extensive survey carried out on 220 SARS-CoV-2 genomic
sequences, different strains of SARS-CoV-2 might coexist [40], thus conferring further
complexity to the diagnostic assessment of the infection. Moreover, another study revealed
that ~79% of primer-binding sequences in at least one gene used in RT-PCR were mutated,
compared to the original SARS-CoV-2 genome (Wuhan-Hu-1, NC_045512) [41]. A bewil-
dering viral diversity has been found even in single patients, with a median number of
four/five intra-individual variants [38,42]. It should be stressed that such heterogeneity in
viral RNAs not only could help explaining differences in intra-individual immunological
response and clinical pathophenotype [43] but can also undermine the accuracy of RT-PCR
detection [44]. Consequently, it is currently assumed that about 10–15% of SARS-CoV-2
variants were not detectable by at least one of the commercialized primers [45].

5. Time Dependency and False-Negative Results

The kinetics of viral load is another factor that varies between individuals and is
influenced by the patients’ epidemiological history, immune response, and treatment or
medication effects [46]. It is another factor that can contribute to false-negative results.
A false-negative case of SARS-CoV-2 infection is defined as an individual with sus-
pected infection and an initial negative result, as determined by the RT-PCR test, with a
positive result on a subsequent test. False-negative results among hospitalized patients
have received considerable attention at the beginning of the COVID-19 pandemic, espe-
cially in those patients in which clinical and radiographic findings were at odds with
negative test results. Most of these false-negative data can be explained by considering
the evolution over time of the viral load. Viral RNA is usually undetectable during the
first two weeks after the infection (when the patient is generally asymptomatic) as well
as two-four weeks later after the onset of clinical symptoms. Viral RNA load declines
quickly two and three weeks after the appearance of symptoms. This indicates that there is
a narrow window for detecting viruses, and there is a potential for false-negative results if
tests are performed outside the period of detectable viral load [47].
In a literature review and pooled analysis, Kucirka et al. [48] analyzed the rate of
false-negative RT-PCR performed on nasopharyngeal swabs of symptomatic patients, with
respect to the timing of symptom onset. The probability of a false-negative result decreased
from 100% on day 1 to 67% (CI, 27–94%) on day 4. On the day with onset of symptoms
the probability of a false-negative rate was 38% and then decreased to 20% 3 days after
symptom onset), while, during the asymptomatic stage (1–4 days), the false-negative rate
ranged from 100 to 94%.
Overall, the sensitivity of RT-PCR testing is therefore severely limited. The sensitiv-
ity in identifying SARS-CoV-2 bearing individuals by RT-PCR test ranges from 44% to
80% [49], as being significantly influenced by viral shedding and by the time of sample
collection when compared to the onset of the infection, as already observed for other
coronaviruses [50]. As a result, it is probable that large numbers of mild, asymptomatic, or
pre-symptomatic COVID-19 cases are not detected by current testing efforts [51] (Figure 2).
 Life 2021, 11, x FOR PEER REVIEW 7 of 16

Life 2021, 11, 660 7 of 16

or pre-symptomatic COVID-19 cases are not detected by current testing efforts [51] (Fig-
ure 2).

Figure2.2.Estimated
Figure Estimatedoverovertime
time likelihood
likelihood of of SARS-CoV-2
SARS-CoV-2 virus
virus detection
detection withwith RT-PCR
RT-PCR teststests and serological
and serological assays assays in re-
in respect
spect to symptom onset. The figure has been reconstructed on the basis of several reports
to symptom onset. The figure has been reconstructed on the basis of several reports and the estimated values are onlyand the estimated values are
only approximate averages. Viral load shows an asymmetric distribution, with an extended tail. The maximum probabil-
approximate averages. Viral load shows an asymmetric distribution, with an extended tail. The maximum probability
ity to detect infectious individuals happens after the first week since the infection and lasts for almost three weeks. Since
tothen,
detect
it isinfectious individuals
quite unlikely happens
to identify afterthat
subjects the could
first week
trulysince the infection
transmit andAs
the disease. lasts for almost three
a consequence, weeks.
no virus Since
develop-
then,
mentitoccurs
is quiteinunlikely
culture to identify
from subjects
biological that could
samples trulyRT-PCR
in which transmitpositivity
the disease.hasAs a consequence,
been ascertained no virus
when thedevelopment
cycle thresh-
occurs
old isinvery
culture
highfrom
(a Ctbiological
< 30 is samples in which
suggested, but itRT-PCR
may vary positivity has been
depending ascertained
on the when the accuracy).
swab procedure cycle threshold is very
Unlike low-
high (a Ct < methods
sensitivity 30 is suggested,
(dashed but it may
line), thevary
highdepending
sensitivityonofthetheswab procedure
RT-PCR method accuracy). Unlike low-sensitivity
could therefore methods
lead to disclosing more
positives
(dashed that,
line), onhigh
the the sensitivity
other hand,ofare thenot contagious
RT-PCR method (solid
could line). Conversely,
therefore lead to false-negative
disclosing more results maythat,
positives occuronduring
the otherthe
first week,
hand, are notwhen active viral
contagious (solidreplication does notfalse-negative
line). Conversely, reach the sensitivity leveloccur
results may required by the
during theRT-PCR test.
first week, For comparison,
when active viral
serologicaldoes
replication analysis shows
not reach thea steady
sensitivityincrease
level in both IgM
required andRT-PCR
by the IgG values
test.only after 4–5 weeks.
For comparison, Thereafter,
serological IgMshows
analysis progres-a
sively decreases after 7–8 weeks, whereas IgG still persists for several months.
steady increase in both IgM and IgG values only after 4–5 weeks. Thereafter, IgM progressively decreases after 7–8 weeks,
whereas IgG still persists for several months.
6. Critical Objectives and False-Positive Results
In theObjectives
6. Critical public health
andsetting, when we
False-Positive are faced with a highly contagious epidemic, a
Results
critical objective is to optimize the early identification of infected individuals that can
In the public health setting, when we are faced with a highly contagious epidemic,
(potentially) transmit the microbe. This is especially relevant in detecting SARS-CoV-2-
a critical objective is to optimize the early identification of infected individuals that can
positive people, who, in many cases, are unaware of their infection. Epidemiologic data
(potentially) transmit the microbe. This is especially relevant in detecting SARS-CoV-2-
have shown that a huge reservoir of asymptomatic/paucisymptomatic subjects has driv-
positive people, who, in many cases, are unaware of their infection. Epidemiologic data
en the diffusion of the current pandemic of COVID-19 [52]. Clinical and epidemiological
have shown that a huge reservoir of asymptomatic/paucisymptomatic subjects has driven
studies
the showof
diffusion that
thethe infectious
current periodof
pandemic begins about 2[52].
COVID-19 daysClinical
after exposure and continues
and epidemiological
to about 12 days after symptom emergence [53]. Overall, the infectious period
studies show that the infectious period begins about 2 days after exposure and continues lasts, on
average, for about two weeks, and during this time, the viral load (RNA
to about 12 days after symptom emergence [53]. Overall, the infectious period lasts,copies per on
ml)
is likely for
average, to increase
about twoprogressively.
weeks, and Therefore,
during thistimely identification
time, the viral load of persons
(RNA that
copies can
per mL)ac-
is likely to increase progressively. Therefore, timely identification of persons that can
actually transmit the virus is mandatory to put in place confinement measures and identify
probable contacts.
Life 2021, 11, 660 8 of 16

However, positive individuals do not overlap automatically with those that can
effectively transmit the virus, although some variants are emerging to have higher rates of
infectivity. Accordingly, the ideal test is not necessarily the one that determines whether
a person has any evidence of SARS-CoV-2—as determined by current PCR-RT-based
swabs—but the one that quickly and accurately identifies those that are truly capable of
transmitting the infection in order to prevent large outbreaks. In other words, we need a
reliable test that could identify the “infecting” individuals. The canonical rules, established
by Koch’s postulates [54], stating that the microorganism must be isolated from the infected
individual and should show growth in culture, have been confirmed in animal models
of SARS-CoV-2 [55]. Indirect evidence of virus replication capability is provided by tests
that identify viral subgenomic mRNA, that can be transcribed in infected cells or detected
in infected cells by fluorescent in situ hybridization techniques that recognize the virus
associated with cells [56]. Of course, this kind of testing is neither rapid nor cheap, and
it is therefore not applicable, so far, for large population screening. However, even if the
antigenic and the genetic “rapid” testing still remain the most useful for tracing the spread
of the virus, it is desirable that more diagnostic tests are settled up in view of a second
phase of the “SARS-CoV2 affair” when the urge for rapid population screening will be less
mandatory and more accurate diagnosis will be relevant.
Overall, all authorized molecular tests (that amplify viral RNA) have higher sensitivity
than antigen tests. Yet, new rapid diagnostic tools, based on lateral flow assays for COVID-
19 antigens, promise to be a reliable alternative for identifying infectious patients [57]. Such
tests capture viral proteins in a lateral flow format that can give results in few minutes and
thus greatly help to rapidly identify those individuals at the highest risk for transmitting
disease [58]. Although less sensitive than reverse transcriptase–polymerase chain reaction
tests, early data suggest that antigen tests can be used to diagnose individuals that are
infectious with COVID-19 [59]. In this study, positivity for viral antigens matched with
virus isolation using an optimal SARS-CoV-2 isolation procedure, i.e., antigen detection
strictly correlates with the presence of virus and can therefore represent a reliable tool
in monitoring the evolution of viral infectivity within the population (epidemiological
surveillance).
Data misinterpretation. Diagnostics can be used in various manners, the so-called use
cases: triage of symptomatic individuals in an epidemic or endemic setting, triage of at-
risk presymptomatic and symptomatic individuals in endemic settings, and confirmatory
testing. The use determines the way in which diagnostic tests are optimally used, namely,
the critical issue is to correctly distinguish between diagnostic and surveillance (screening)
tests, the latter being conceived for ascertaining the epidemiological course of the epidemic,
whereas the former is more critical for the management of individual patients.
For epidemiological testing, the key question is not how well molecules can be detected
in a single sample but how effectively infections can be detected in a population by the
repeated use of a given test as part of an overall testing strategy—the sensitivity of the
testing regimen. Clinical tests are designed for assessing the reliability of diagnosis in
symptomatic individuals and require high sensitivity. On the other hand, tests used in
surveillance regimens should be tailored to detect those individuals that can really diffuse
the virus (true “infecting” persons). RT-PCR detects RNA, not an infectious virus; thus, its
ability to determine the duration of infectivity of patients is very limited, while infectivity
is a critical determinant in informing public health guidelines/interventions. As already
observed in other instances, a critical drawback of RT-PCR tests (but that can be extended
to serological tests too) is that they cannot determine virus infectivity: overall, RT-PCR
sensitivity is good but its specificity for detecting the replicative virus is poor [60].
A recent paper has highlighted that infectivity—recognized by the capability to re-
trieve the virus growing in culture according to Koch’s postulate—is dramatically impaired
when cycle threshold (Ct) values are >24 [61]. A direct relationship has been observed
between Ct values of RT-PCR and the viral load, even if the relationship is less than linear
for low viral loads [62]. Noteworthy, for every one-unit increase in Ct, the odds ratio for
Life 2021, 11, 660 9 of 16

infectivity decreased by 32%. Another report shows that for Ct = 25, the virus can be
identified in up to 70% of samples, while at Ct = 30, this value drops to 20%; for Ct = 35,
only in 3% of cultures the virus can retrieved [63]. Therefore, the straightforward conse-
quence is that almost 50% of newly diagnosed “positive” patients are in fact noninfectious
individuals, given that in these cases, the PCR Ct averages >30, indicating low or even no
viral counts [64]. Indeed, besides the fact that these low levels of detectable RNAs could be
explained by an early or late-stage infection, the prolonged duration of the RNA-positive
tail—during which individuals continue to “release” viral RNA or its fragments—indicates
that a relevant proportion of infected persons are recognized once the infectious period
has already passed [65]. It is generally agreed that replication-competent virus cannot
be successfully cultured more than 9 days after the onset of illness, with a statistically
estimated likelihood of recovering replication-competent virus that approaches zero by
10 days [66,67]. Conclusively, “after about 8 days of onset of symptoms, infectious virus
is no longer present in respiratory tract specimens, despite high amounts of SARS-CoV-2
RNA that are measurable by RT-PCR” [68].
Overall, it has been estimated, in a different cohort of patients, that 88% and 95%
of their specimens no longer yielded replication-competent virus after 10 and 15 days,
respectively, following symptom onset [69,70]. This means the current COVID-19 test
standards for the PCR test would pick up an excessive number of false positives. However,
despite these warnings, an update to the CDC instructions for PCR testing from 1 December
2020, still uses a Ct of 40 [71].
Nonetheless, the implications are relevant, as hundreds of thousands of people
presently being confined based on positive RT-PCR tests are likely to have already passed
the transmissible phase of their infection. These results were anticipated by several investi-
gations [56,72] and prompted different scholars to propose a cutoff Ct value at Ct ≤ 30, with
a duration of eviction of approximately 10 days [73]. Interestingly, prolonged (i.e., with a
duration > 20 days) persistence of positive RT-PCR test, with low Ct values, may indicate
subgroups of infected patients with immunocompromised state or more severe disease,
as suggested by van Kampen [74]. Additionally, it may be surmised that tests with high
Ct thresholds may detect not just live viruses but genetic fragments, leftovers from other
infections (i.e., other Coronavirus for which a cross-reactivity has been documented) [75,76]
that pose no particular risk. Again, these considerations—albeit correct—hardly overcome
the wall of the “field practice.” As a matter of fact, to be able at ascertaining a Ct cutoff to
discern between infecting and noninfective individuals, one should be sure the starting
specimen are homogeneous. Actually, the pre-analytic bias plays a non-neglectable role in
the execution of the swab since different operators introduce a great variability in the sam-
pling. It is clearly arguable that two different specimens taken from the same individuals
can result in very different Cts depending on how accurately the swab has been executed.
The New York Times recently raised the alarm that the number of amplification cycles
needed to find the virus “is never included in the results sent to doctors and coronavirus
patients, although it could tell them how infectious the patients are. In three sets of testing
data that include cycle thresholds, compiled by officials in Massachusetts, New York, and
Nevada, up to 90 percent of people testing positive carried barely any virus” [77].
It is therefore mandatory to use tests that could capture infected people while they
are still infectious. Identification of such persons is a critical objective deemed to reduce the
population prevalence of a respiratory virus. In this respect, as stressed by a recent paper,
“the benchmark standard clinical polymerase chain reaction (PCR) test fails when used in
a surveillance regimen” [67]. Indeed, the distribution over time of RT-PCR positivity in
infected people shows that the diagnostic test still remains positive for longer times after
the stage at which an individual is no longer infectious [78].
Unfortunately, regulatory agencies have not established distinct and clear rules for
validating, as separate tests, those to be used as analytical diagnostic tools versus tests for
ascertaining infectivity and hence public health efforts to reduce community transmission.
Moreover, a reliable assessment of the course of the epidemic worldwide needs different
Life 2021, 11, 660 10 of 16

countries to adopt a common approach for validating performances of both diagnostic tests
and tests used to screen for infectious persons. This is especially urgent when epidemiolog-
ical data (i.e., the number of true infectious people) serve as a base for political decision
making. Conversely, differences in RT-PCR tests, LoDs, or Ct cutoff may contribute to
explaining controversial epidemiological findings, given that some differences among coun-
tries with similar structural sociological traits (population mean age and density, quality
of health services, geographical position) could be ascribed to variances in RT-PCR-based
data acquisition.
As already proposed [79], to improve its reliability, the RT-PCR test should be continu-
ously calibrated against a reference culture in Vero E6 cells in which cytopathic effect has
been observed. The cycle threshold values on each platform for patients who have positive
and negative viral cultures should be correlated with RT-PCR results and the resulting
calibration curve could then be performed to estimate virus viability from the cycle thresh-
old with some certainty [80]. Moreover, evidence shows that viral loads are subject to a
different evolution in symptomatic and asymptomatic people [81]. Conclusively, the gener-
ation of false-positive or false-negative test results jeopardizes the health of the individual
patient and may also derange and disrupt the efficacy of public health policies, emergency
plans, and restrictive measures established by national and international authorities for
containing the outbreak.
Secondly, an alternative to RT-PCR tests could be represented by rapid lateral-flow
antigen tests, and rapid lateral-flow tests based on CRISPR gene-editing technology [82].
These tools provide a sensitive and robust means for high-throughput COVID-19 diagnosis
suitable for use in screening settings and display the potential to enable reliable large-scale
diagnosis [83]. Notably, lateral-flow antigen tests do not have an amplification step, and
their analytic limit of detection is usually 100/1000 times less than RT-PCR tests; to reliably
identify the people that can really “transmit” the virus, such tests need to be adequately
sensitive and would benefit from a clear cutoff value that is established to distinguish
the “infectious” individuals among “positive” individuals. Undoubtedly, the adoption of
such a strategy will have huge consequences for harnessing the epidemic outbreak and
successfully manage the disease spreading.
Altogether, these considerations further outline that clinical symptoms and molecular
analytical tools should be conjugated to perform a correct diagnostic assessment. Some
diseases can be diagnosed based on a test alone; most diseases, however, are defined by the
cluster of symptoms and signs, in addition to test results. In no case can a medical diagnosis
be replaced by simple molecular tests. Indeed, interpreting the results of RT-PCR requires
consideration of patient characteristics, such as symptoms and their severity, contacts
history, presence of preexisting morbidities and drug history, the cycle threshold value,
the number of days from symptom onset to test, and the specimen donor’s age [84]. As
markedly recommended by a recent Cochrane report, a single symptom or sign could not
accurately diagnose COVID-19 [85]. Furthermore, the Oxford Centre for Evidence-Based
Medicine suggests that “the PCR test positivity counts should include a standardized
threshold level of detection, and at a minimum, the recording of the presence or absence
of symptoms. As a disease, the COVID-19 case definition should constitute a disorder
that produces a specific set of symptoms and signs. The in-hospital case definition should,
therefore, also record the CT lung findings and associated blood tests” [86].

7. Conclusions
Reliability of analytical tools in identifying infected individuals, i.e., persons that can
transmit the virus, is mandatory to assess the evolution of the SARS-Cov-2 pandemic and to
provide solid foundations for policy decision making. The life and the liberty of hundreds
of millions of people, as well as the economy of countries around the globe, ultimately
depend on such appropriate assessments. Unfortunately, several scientific studies and
administrative official reports indicate that epidemiological data related to the prevalence
of COVID-19 infection among people are likely to be significantly biased by the relevant
Life 2021, 11, 660 11 of 16

incidence of both negative- and false-positive results. As a matter of fact, optimization of

testing capability, extensive clinical and epidemiological validation, and proper approval
from regulatory agencies are still needed.
To sum up, the following observations can be made:
(1) False-negative results may be ascribed to erroneous/flawed analytical perfor-
mances as well pre-analytical issues, including inappropriate choice of timing of testing
(i.e., pick-up of the biological sample could have been obtained at the wrong timing, too
early or too late with respect to the onset of the infection).
(2) False-positive results may instead arise chiefly as a consequence of extended Ct,
far beyond the recommended threshold of 30–33. Other causes of false-positive results
include wrong timing, cross-reactivity among SARS-CoV-2, and other Coronaviruses.
(3) Last, but not least, false-positive results should be ascribed to administrative errors
and malpractices, as demonstrated recently in Italy. A survey reported by Il Corriere della
Sera [87] shows that positive patients, albeit symptomless, have been persistently added to
the pool of positive even though they may have fully recovered from the time of the initial
test, taken several weeks before.
Even in the ideal case in which a test displays both high sensitivity and specificity, the
number of false-negative and false-positive would still compromise efficient control of the
epidemic. It has been calculated that, with sensitivity and specificity in the range of 98% and
99%, respectively—a goal that currently is far from being achieved—a total of 1000 cases
will be missed when 5 million people are tested, and another 49,500 individuals will receive
false-positive results [88]. Recent data demonstrated that the true picture is even worse,
with a much greater proportion of false-positive ascertained in many countries, including
Italy and the USA. Consequently, many people may be unnecessarily quarantined, and the
public health system unnecessarily burdened with managing contact tracing, confinement,
and other measures. Therefore, it should be mandatory to “interpret” RT-PCR by jointly
examining clinical and radiological data, in the context of the pretest probability of disease.
For COVID-19, the pretest probability assessment includes symptoms, previous medical
history of COVID-19, presence of antibodies, any potential exposure to COVID-19, and
the likelihood of an alternative diagnosis [89], namely, when low pretest probability exists,
positive results should be interpreted with caution and a second specimen tested for
confirmation given that low levels of viral RNA (identified by RT-PCT tests that require
high Ct values) do not equate with infectivity unless the presence of virus particles has
been documented, as clearly recommended by the European Centre for Disease Prevention
and Control [90].
These conclusions cast on doubt the use of RT-PCR swabs as a screening procedure for
tracing the evolution of the current SARS-COV-2 pandemic. The huge number of both
false-positive and false-negative results deprives the trustworthiness of decision making,
which is also influenced by political factors. Health management during such a pandemic
is severely compromised when tests for the infection provide unreliable results [91,92].
Therefore, we should search for a test that can quickly and reliably identify those
individuals—including symptomless people—that are truly infectious and represent a
potential vehicle of virus diffusion. Reliable assessment of such a parameter is indeed
what we need to control and prevent large outbreaks, especially when we are using a
more fitted epidemiological index to track the epidemic advancement [93]. Unfortunately,
current widespread use of RT-PCR swabs appear to be a waste of resources, while the
public health threat warrants large-scale testing—“it would be more effective to authorize
a small number of well-designed, well-developed, and validated tests run on common
high-throughput platforms, followed by a few point-of-care tests, all of which are man-
ufactured in large quantities, than to simultaneously develop and authorize scores of
diagnostics” [91]. Our review has broader consequences on how screening procedures
should be performed. Specifically, it is urgent to set a threshold value for RT-PCR analy-
sis, given that for Ct > 30–35 the incidence of false-positive results would account for the
majority of positive tests. Furthermore, testing in the absence of other proven prevention
Life 2021, 11, 660 12 of 16

strategies is unable to prevent outbreaks [94]. Health authorities should be asked to ur-
gently address such issues in order to establish a proper strategy for managing the current
pandemic, as well as prepare for future threats of a similar kind.
Given that such a problem has been addressed only marginally by few studies, an ex-
tended recognition aimed at assessing the incidence of both false-positive and false-negative
results is urgently warranted. The present review attempts to provide a compelling survey
of available published data, some studies may have been lost notwithstanding. However,
some questions have been left aside and deserve to be studied by future inquiries. For
instance, specific epidemiological investigations should be planned to evaluate how false
negative/positive results arise in correlation with different levels of infection prevalence.
In addition, it must be investigated the relationship between the disappearance of the
virus (analyzed by culturing biological samples to find replicating viral particles) and the
concomitant values of RT-PCR: how long RT-PCR stands positive since the virus has been
removed from biological fluids? Moreover, this information needs to be complemented
with data of seroprevalence, obtained by testing people about the presence of immunologi-
cal markers (IgG, IgM, and IgA) that are the only trustworthy parameters that can provide
a proper estimate of the virus diffusion into the general population. Even more important,
appraisal of immunological status would allow appreciating the extent of the acquired
immunization, a critical health indicator that, after more than 18 months of the pandemic,
still needs to be acquired. Future studies should timely investigate those challenging
questions to plan a better-tailored health strategy aimed at grasping the overall complexity
of the current pandemic, as highlighted by the current review.

Author Contributions: Conceptualization, R.V., W.A., M.M., C.P.M.H., A.H.H., S.H.A., N.M.S., O.A.,
V.F., P.S., A.F., D.M.C., M.C., A.A., M.B.; methodology, A.F., M.C.; validation, M.M., C.P.M.H.; formal
analysis, P.S., O.A.; investigation, C.P.M.H., D.M.C., A.A.; resources, W.A., M.M.; writing—original
draft preparation, M.B.; writing—review and editing, R.V., M.B., V.F., A.F.; visualization, A.H.H.,
S.H.A., N.M.S., O.A.; supervision, R.V., M.B., A.F., A.A.; project administration, R.V., M.B., A.A. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data were obtained from an extended survey based on published
article reported.
Acknowledgments: Abdel Halim Harrath, Saleh H. Alwasel, and Mariano Bizzarri extend their
appreciation to the International Scientific Partnership Program ISPP at King Saud University (KSU)
for supporting this research work through ISPP-122.
Conflicts of Interest: The author declares that he has no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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