Expert Review of Molecular Diagnostics

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Diagnosis of SARS-CoV-2 infection in the setting of

the cytokine release syndrome

Marwan M. Azar, Junghee J. Shin, Insoo Kang & Marie Landry

To cite this article: Marwan M. Azar, Junghee J. Shin, Insoo Kang & Marie Landry (2020)
Diagnosis of SARS-CoV-2 infection in the setting of the cytokine release syndrome, Expert Review
of Molecular Diagnostics, 20:11, 1087-1097, DOI: 10.1080/14737159.2020.1830760

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EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
2020, VOL. 20, NO. 11, 1087–1097
https://doi.org/10.1080/14737159.2020.1830760

SPECIAL REPORT

Diagnosis of SARS-CoV-2 infection in the setting of the cytokine release syndrome

Marwan M. Azara, Junghee J. Shinb, Insoo Kangb and Marie Landrya,c
a
Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA; bDepartment of Internal Medicine,
Section of Rheumatology, Allergy & Immunology, Yale School of Medicine, New Haven, CT, USA; cDepartment of Laboratory Medicine, Yale School
of Medicine, New Haven, CT, USA

ABSTRACT ARTICLE HISTORY

Introduction: Coronavirus disease (COVID-19) can trigger a cytokine response storm (CRS) that is Received 7 August 2020
associated with high mortality but for which the underlying pathophysiology and diagnostics are not Accepted 28 September
yet well characterized. This review provides an overview of the underlying immune profile of COVID-19- 2020
related CRS as well as laboratory markers for acute diagnosis and chronic follow-up of patients with KEYWORDS
SARS-CoV-2 and CRS. SARS-CoV-2; cytokine-
Areas covered: Innate and acquired immune profiles in COVID-19-CRS, RNA-detection methods for SARS- release syndrome; COVID-19;
CoV-2 in the setting of CRS including factors that affect assay performance, serology for SARS-CoV-2 in the cytokines; diagnosis; RNA;
setting of CRS, and other biomarkers for COVID-19 will be discussed. sensitivity
Expert opinion: Studies support the implication of CRS in the pathogenesis, clinical severity and
outcome of COVID-19 through the production of multiple inflammatory cytokines and chemokines
from activated innate and adaptive immune cells. Although these inflammatory molecules, including IL-
6, IL-2 R, IL-10, IP-10 and MCP-1, often correlate with disease severity as possible biomarkers, the
pathogenic contributions of individual molecules and the therapeutic benefits of targeting them are yet
to be demonstrated. Detection of SARS-CoV-2 RNA is the gold standard method for diagnosis of COVID-
19 in the context of CRS but assay performance varies and is susceptible to false-negative results even
as patients clinically deteriorate due to decreased viral shedding in the setting of CRS. Biomarkers
including CRP, ferritin, D-dimer and procalcitonin may provide early clues about progression to CRS and
help identify thrombotic and infectious complications of COVID-19.

1. Introduction severe COVID-19 disease including acute respiratory distress

syndrome and septic shock with multi-organ failure.
The novel Coronavirus, SARS-CoV-2, was first linked to an
Development of CRS is strongly linked to increased morbid­
acute respiratory syndrome in December of 2019 after cases
ity and mortality [3]. Though certain clinical findings includ­
of respiratory failure were reported in Wuhan, China. Since
ing anosmia and ageusia in the context of an interstitial
then, the world had witnessed a global pandemic not seen
pneumonia are suggestive of COVID-19, no signs or symp­
since the Spanish Influenza. As of this writing, more than
toms are pathognomonic of infection. Similarly, epidemiolo­
200,000 people have succumbed to SARS-CoV-2 in the
gic factors such as increased age, clinical signs like rising
United States, compared to an estimated 675,000 from the
oxygen supplementation requirement and radiologic fea­
Spanish Influenza. The predominant clinical manifestations
tures like worsening pulmonary infiltrates are associated
of coronavirus disease (COVID-19) are respiratory including
with a greater risk for severe disease but remain imperfect
upper and lower respiratory tract symptoms but other sys­
markers of progression to CRS in individual patients [4].
tems such as the gastrointestinal tract, coagulation cascade,
A combination of clinical, epidemiologic and laboratory
vascular and central nervous systems are commonly
markers are needed to discriminate between patients who
involved [1]. Clinical and physiologic studies of COVID-19
are at risk of or in the course of progressing to the inflam­
have suggested a biphasic pattern of illness with an initial
matory phase of COVID-19. Determining whether patients
viral response phase lasting 7 to 10 days after symptom-
have progressed to CRS has important therapeutic implica­
onset characterized by rapid viral replication followed by an
tions, since anti-inflammatory agents are likely to be more
inflammatory response phase driven by innate and adaptive
useful than anti-virals in this setting [5,6]. In this paper, we
immune responses to the virus [2]. In a subset of indivi­
review the underlying innate and acquired immune profile
duals, immune dysregulation leads to a catastrophic mala­
of COVID-19-related CRS and discuss laboratory markers for
daptive hyperimmune response. This cytokine-release
acute diagnosis and chronic follow-up of patients with
syndrome (CRS), which is well described in the setting of
SARS-CoV-2 and CRS.
cancer immunotherapy, is associated with progression to

CONTACT Marwan M. Azar marwan.azar@yale.edu Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven,
CT 06510, USA
This article has been republished with minor changes. These changes do not impact the academic content of the article.
© 2020 Informa UK Limited, trading as Taylor & Francis Group
1088 M. M. AZAR ET AL.
Article highlights
● CRS is a systemic inflammatory response with multi-organ failure that
can be triggered by a variety of factors including infections, rheu­
matic diseases, and malignancies, resulting in the overactivation of
immune cells, largely innate immune cells and T cells.
● In CRS, activated immune cells produce high levels of pro-
inflammatory cytokines such as IL-1β, IL-6, TNF-α and IFN-γ, leading
to the activation of endothelial cells with capillary leakage, hypoten­
sion, coagulopathy, and augmented inflammation.
● Patients with COVID-19 can develop CRS with increased levels of
inflammatory cytokines and chemokines, encompassing IL-1β, IL-1
receptor antagonist (IL-1RA), IL-7, IL-8, IL-9, IL-10, G-CSF, GM-CSF,
IFN-γ, IP-10, MCP-1, MIP-1a, and MIP-1b, with some of them correlat­
ing with disease severity.
● CRS is associated with progression to severe COVID-19 disease includ­
ing acute respiratory distress syndrome and septic shock with multi-
organ failure, with increased morbidity and mortality
● The clinical sensitivity of SARS-CoV-2 RNA testing is dependent on
several factors including assay analytical sensitivity, specimen type,
specimen quality, severity of disease and the timing of specimen
collection
● Specimens obtained ‘too early’ prior to the ramping up of viral
replication or ‘too late’ after viral replication had declined below
the testing assay’s limit of detection may lead to false-negative
results.
● RNA testing during CRS may lead to false-negative results since
clinical deterioration due to immune dysregulation can coincide
with a decrease of viral replication
● Persistent low level positive nucleic acid testing for weeks after initial
diagnosis likely do not indicate ongoing active infection
● The analytical sensitivity of commercial NAAT assays for SARS-CoV-2
varies but a more accurate assessment is a comparison of assays’
performance using parallel testing low positive clinical samples.
● Though generally not recommended for diagnosis of acute SARS-CoV
-2 infection, serology may be useful when clinical suspicion for
COVID-19 is high despite negative-repeated RNA testing, a scenario
which may occur in late disease including in the CRS phase.
● Several biomarkers including CRP, ferritin, D-dimer and procalcitonin
may provide early clues about progression to CRS and help identify
thrombotic and infectious complications of COVID-19.
2. Cytokine-release syndrome and COVID-19
CRS is a systemic inflammatory response with multi-organ
failure that can be triggered by a variety of factors such as
infections, rheumatic diseases, malignancies, and certain drugs
[7]. The term ‘cytokine release syndrome’ was first described in
the early ‘90s, when the anti-T-cell antibody muromonab-CD3
(OKT3) was introduced as an immunosuppressive treatment
for solid organ transplantation [8]. Subsequently, CRS has
been reported after infusion of chimeric antigen receptor
(CAR) T cells, antibody-based therapies, and administration of
non-protein-based cancer drugs as well as in association with
rheumatic diseases and severe viral infections, especially those
with pathogenic coronaviruses (CoV) including Middle East
respiratory syndrome coronavirus (MERS-CoV), severe acute
respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV
-2 [9–1617––18–19]. Indeed, the morbidity and mortality of
COVID-19 are due primarily to excessive immune activation,
inflammation, and sepsis [20], with acute respiratory distress
syndrome (ARDS) the proximate cause of death [21]. In CRS,
the overactivated immune cells, largely innate immune cells
and T cells, produce high levels of pro-inflammatory cytokines
such as IL-1β, IL-6, TNF-α and IFN-γ, leading to the activation
of endothelial cells with capillary leakage, hypotension, coa­
gulopathy, and augmented inflammation [22,23]. Patients with
CRS can develop a hemophagocytic lymphohistiocytosis (HLH)
or macrophage activation syndrome (MAS)-like syndrome with
the rise of other cytokines and chemokines including IL-8, IL-
18, IP-10, MCP-1, MIG and MIP-1β [24]. Patients with COVID-19
was found to have increased levels of inflammatory cytokines
and chemokines, encompassing IL-1β, IL-1 receptor antagonist
(IL-1RA), IL-7, IL-8, IL-9, IL-10, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-
1, MIP-1a, and MIP-1b, with some of them correlating with
disease severity (Table 1) [25].
2.1. Innate immunity-related cytokines in COVID-19
Up to 20% of COVID-19 cases present with severe disease,
with ARDS and multiple-organ failure driven largely by CRS
[26]. Both SARS-CoV and SARS-CoV-2 use the angiotensin-
converting enzyme-
related carboxypeptidase (ACE2) to gain entry to cells [27].
This molecule is widely expressed not only in cardiopulmonary
and gastrointestinal tissues but also in some hematopoietic
cells including innate immune monocytes, macrophages, and
dendritic cells. SARS-CoV-2 infection of monocytes, macro­
phages, and dendritic cells infected leads to activation of
pattern recognition receptors (PRR) and its downstream tran­
scription factors such as NF-kB, leading to the production of
an array of proinflammatory cytokines and chemokines [3].
Probably, the most potent pro-inflammatory cytokines of
innate immunity are IL-1β, IL-6 and TNF-α which are produced
mainly by monocytes, macrophages, mast cells, endothelial
Table 1. Changes of cytokines and chemokines in COVID-19.
Cytokines Changes in COVID-19
IL-6 Increased circulatory levels in association with disease severity.
[23,26,27]
Patients with SpO2 < 90% had higher levels of IL-6 than
patients with SpO2 ≥ 90% [29].
TNF-α Increased circulatory levels in association with disease severity.
[23,26,27]
IL-1β Increased circulatory levels in association with disease severity.
[26,27]
IL-10 Increased circulatory levels. [23,48–50]
Patients with SpO2 < 90% had higher levels of IL-6 than
patients with SpO2 ≥ 90% [29].
IFN-γ Increased circulatory levels in association with disease severity
[44] and viral loads [45]
Decreased frequency of IFN-γ-secreting CD4 + T cells in
severe cases than in moderate cases [26]
IL-7 Patients requiring ICU admission displayed higher levels of
circulatory IL-7 compared to patients not requiring ICU
admission [23]
IL-13 Increased circulatory levels [23,48–50]
IL-4 Increased circulatory levels. [23,48–50]
IL-2 Patients requiring ICU admission showed higher IL-2 level
compared to patients not requiring ICU admission [23]
Chemokines
IP-10 Patients requiring ICU admission had higher levels of IP-10
(CXCL10) compared to patients not requiring ICU admission [23] in
association with disease severity [44]
MCP-1 Patients requiring ICU admission had higher levels of MCP-1
(CCL2) compared to patients not requiring ICU admission [23]
MCP-3 Increased in association with disease severity [44]
(CCL7)
MCP-1α Patients requiring ICU admission had higher levels of MCP-1 α
(CCL3) compared to patients not requiring ICU admission [23] in
association with disease severity [44]

and/or epithelial cells [28,29]. CRS likely results from a sudden
increase in pro-inflammatory cytokines with the influx of
immune cells, including macrophages, neutrophils, NK cells
and T cells, from the circulation into the infected site [29]. In
addition to pro-inflammatory cytokines, growth factors and
chemokines such as vascular endothelial growth factor
(VEGF), monocyte chemoattractant protein-1 (MCP-1), and IL-
8 can contribute to the destabilization of endothelial cells in
CRS, leading to the development of vascular barrier impair­
ment and capillary damage with multiorgan failure [29].
Patients with COVID-19 have higher circulatory levels of IL-
1β, IL-6, IL-8, IL-10, TNF-α compared to healthy controls, cor­
relating with disease severity [25,30,31]. For instance, levels of
IL-6 and IL-10 in peripheral blood were higher in patients with
severe COVID-19 than in patients with mild COVID-19 [32]. In
fact, patients with SpO2 < 90% had higher levels of IL-6 and IL-
10 than patients with SpO2 ≥ 90% [33]. Although SARS-CoV-2
viral RNA was detected in only 15% of COVID-19 patients,
these patients had elevated levels of IL-6 with poor clinical
prognosis [34]. A transcriptional and serological analysis of
COVID-19 patients showed increased levels of pro-
inflammatory cytokines, including IL-6, along with low type
I and III IFNs and high chemokine signature [35], suggesting
reduced antiviral defense coupled with dysregulated inflam­
matory responses. The inflammatory cytokines IL-1β, IL-6, and
TNF-α account largely for the clinical manifestations, including
fever, hyperferritinemia, acute phase reactions, and coagulo­
pathies in COVID-19 given the known proinflammatory prop­
erties of these molecules [36–37–38]. These findings suggest
a possible pathologic role of IL-6 in COVID-19-associated CRS,
raising the consideration of IL-6 blockade as in CRS due to
other conditions like CAR-T therapy [39,40]. Indeed, IL-6 inhi­
biting therapy has been employed in COVID-19 using the anti-
IL-6R antibodies tocilizumab and sarilumab as well as the anti-
IL-6 antibody siltuximab, with mixed results. Some early
uncontrolled, open-labeled, and retrospective observational
studies reported the possible clinical benefits of tocilizumab
with the improvement of fevers and inflammatory markers as
well as with a reduced risk of invasive mechanical ventilation
or death in severe COVID-19 [5,41,42]. However, a recent
phase 3 clinical trial of sarilumab in patients with COVID-19
was discontinued after finding no benefit for preventing
deaths or getting off ventilation [43]. IL-6 deficient mice had
impaired immunity to viral and intracellular bacterial infec­
tions [44]. Patients with rheumatoid arthritis treated with toci­
lizumab developed a higher rate of serious infections
compared to those who received alternative biologics
[45,46]. In a pre-printed platform, a study reported an
increased risk of secondary infections with higher mortality
in critically ill COVID-19 patients who received tocilizumab
compared to patients who did not receive this drug [47].
2.2. T cell immunity-related cytokines in COVID-19
In addition to innate immune cells, analysis of immune mole­
cules in blood indicates the activation of T cells in COVID-19.
Activated T cells can further stimulate innate immune
responses by producing cytokines. T helper 1 (Th1) cells that
dominantly produce IFN-γ can activate monocytes,
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 1089
macrophages, and NK cells while Th2 and Th17 cells can
activate eosinophils and neutrophils via producing IL-5 and
IL-17, respectively [48,49]. Regulatory T cells such as FOXP3
+ regulatory CD4 + T cells can regulate early innate immune
responses by suppressing inflammatory cytokine production,
providing checks and balances between host defense and
inflammatory damage. Patients with COVID-19 appear to
have alterations in adaptive immunity which includes reduced
numbers of circulatory T cell numbers but increased circula­
tory levels of T cell cytokines [28,50]. Indeed, patients with
COVID-19 have increased circulatory levels of the T cell cyto­
kines IL-2, IFN-γ, IL-17, and the soluble IL-2 receptor alpha
chain (IL-2 R or CD25), which is cleaved from the surface of
activated T cells [25,30–32]. It is noticeable that innate
immune cytokines IL-1β, IL-6, and IL-12 can promote the
activation, differentiation, and cytokine production of T cells
with the expression of the receptors for these cytokines. IL-1β
can enhance IL-17 production from CD4 + T cells, namely
T helper (Th) 17 cells, while IL-12 induces IFN-γ-producing
Th1 cells [51,52]. IFN-γ can further activate innate immune
cells like monocytes, macrophages, and natural killer cells
with the expression of IFN-γ receptor, resulting in augmented
production of cytokines and chemokines as a feed-forward
mechanism. Such interface of T cells and innate immune
cells can be mediated by chemokines like monocyte chemoat­
tractant protein 1 (MCP1 or CCL2), MCP3 (CCL7) and macro­
phage inflammatory protein 1 alpha (MCP-1α or CCL3)
released from monocytes and macrophages [53]. IFN-γ can
also induce the chemokine IFN-γ–induced protein 10 (IP-10
or CXCL10) from monocytes, endothelial cells and fibroblasts,
which can recruit T cells, NK cells, and monocytes. In COVID-
19, the expression levels of IFN-γ, IP-10, MCP-3, and MIP-1α
were shown to be increased and highly associated with dis­
ease severity during disease progression [25,54]. IFN-γ level
was also found to correlate with viral load and disease severity
in patients with COVID-19 [55], suggesting the relationship of
the viral burden with T cell activation. Th2 cells produce high
levels of IL-4, IL-5 and IL-13 with the capacity to promote
tissue fibrosis [56] whereas IL-10 from monocytes, Th2 cells
and regulatory T cells can inhibit pro-inflammatory cytokines
[57]. Increased levels of IL-4, IL-10 and IL-13 were reported in
patients with COVID-19 compared to healthy controls [25,58–
59–60]. In persistent viral infections, CD4+ and CD8+ T cells
lose their effector function, become unable to control viral
infection, and secrete high levels of TGF-β and IL-10 [61].
Thus, elevated IL-10 and IL-13 in COVID-19 could further dam­
pen the effector function of T cells and promote tissue fibrosis.
IL-7 with the capacity to promote T cell survival increases in
COVID-19 [25] although patients with this disease have
decreased frequencies of lymphocytes, including CD4+ and
CD8 + T cells [32,62]. The increased IL-7 could be driven in
part by lymphopenia in COVID-19 as lymphopenia is known to
increase IL-7 [63].
3. SARS-CoV-2 RNA detection for diagnosis of COVID
in the setting of CRS
The primary diagnostic method for acute COVID-19 infection
is measurement of SARS-CoV-2 RNA via nucleic acid testing,
1090 M. M. AZAR ET AL.
most commonly polymerase chain reaction (PCR). Several
assays are available including from commercial laboratories
and the CDC (Table 2), with varying analytical sensitivities
among other parameters. The clinical sensitivity of RNA detec­
tion is dependent on the amount of viral shedding in clinical
specimens. In COVID-19, viral replication begins 2–5 days
before symptom onset and peaks between 0.6 days to
5 days after symptom onset when measured in upper respira­
tory tract specimens [64,65]. Testing done ‘too early’ i.e.
before viral loads increase beyond the assay’s limit of detec­
tion will lead to false-negative results (Figure 1). However,
viral replication may peak prior to development of symptoms
and high viral loads in the pre-symptomatic stage likely
account for a significant portion of transmissions, up to 44%
in one mathematical model [64]. In one study, the median
time from detection of SARS-CoV-2 to symptom onset in pre-
symptomatic patients was 15 days [66]. After 7 days of illness,
viral loads drop rapidly but may remain above the threshold
of nucleic acid detection for weeks post-symptom onset.
Though viral loads peak early in the course of COVID-19,
they are higher and persist longer in severe disease. In
a study comparing mild and severe cases, the median and
peak viral loads were 1 log10 higher in the latter group [67].
Similarly, patients with severe COVID-19 exhibited higher viral
loads and more prolonged shedding than mild cases (21 vs.
14 days) [68] suggesting that clinical sensitivity of PCR testing
is higher in more severe COVID19. Notably, among patients
with mild disease, the median viral load of patients with
symptomatic and asymptomatic disease are not significantly
different [66]. It is important, however, to understand the viral
kinetics of SARS-CoV-2 infection in the context of the COVID-
19 disease process in which severity of disease, particularly in
the latter stages appears to be driven by a cytokine storm
rather than purely by virus-related cytotoxicity (Figure 1). In
a subset of individuals who clinically deteriorate 1–2 weeks
after initial symptom onset, reflecting inflection from a viral
response phase to a cytokine response phase, nucleic acid
testing is likely to be less sensitive despite clinical decompen­
sation since viral loads are significantly lower at the inflam­
matory stage of disease. Falsely negative PCR tests may
theoretically occur in this period (‘too late’), especially when
sampling upper respiratory tract specimens, which are less
sensitive than lower respiratory tract specimens (such as spu­
tum or endotracheal fluid) at any one point after symptom
onset [67]. Conversely, PCR testing may remain positive for
more than 6 weeks after initial diagnosis, with a median of
24 days in the general population. Prolonged shedding is
more common with advanced age, certain comorbidities like
diabetes and hypertension and immunocompromising condi­
tions [69,70]. Though viral RNA can be detected for weeks
after symptom onset including during the cytokine response
phase of illness, it is unclear whether persistently low viral
loads represent infectious virus. In a studies of patients with
COVID, viral culture was successful only from specimens col­
lected within of symptom onset or with viral loads ≥6 log
copies/mL [71,72]. In another study, virus was successfully
cultured only from upper respiratory specimens when the
PCR cycle threshold was below 34, correlating to a low viral
load [73]. Taken together, these studies suggest that
prolonged low-level viral shedding may not reflect live infec­
tious virus. Regardless of other factors, poor sampling espe­
cially of upper respiratory specimens can lead to false-
negative results.
3.1. Factors that influence SARS-CoV-2 RNA detection
assay performance
3.1.1. Specimen collection, storage and transport
The pre-analytical phase of laboratory testing is the most
common source of laboratory errors and testing for SARS-
CoV-2 is no exception [74,75]. Regardless of the sample type,
adequate specimen collection is fundamental to accurate
nucleic acid testing. Proper specimen collection is critical in
obtaining accurate results and poor specimen collection can
lead false negative tests. The recommended collection techni­
que varies across specimen types. For example, nasopharyn­
geal (NP) specimens require insertion of the swab tip through
the nostril to the pharynx until resistance is encountered, after
which the swab should be left in place for a few seconds to
absorb secretions. Deep nasal or mid-turbinate samples are
collected by inserting a swab one inch into the nose, to the
level of the turbinates, rotating several times, then repeated in
the other nostril using the same swab. For anterior nasal
specimens, the swab should be inserted 0.5 inches inside
each nostril and rotated along the nasal membrane 5 times,
then repeated in the other nostril using the same swab. After
collection, specimens must be stored in appropriate condi­
tions. Instructions vary based on the transport medium
employed by different assays, but CDC has a general recom­
mendation for storage at 2–8°C for up to 72 hours after
collection and at ≤70°C if transport is not possible within
72 hours. Improper storage can accelerate nucleic acid degra­
dation especially for RNA which is significantly less stable than
DNA even in optimal conditions, leading to false-negative
results. In addition, each laboratory must use appropriate pre-
analytical processes to reduce contamination between clinical
specimens. Both mid-turbinate and anterior nasal swabs can
be self-collected with instructions under the observation of
a healthcare worker.
3.1.2. Considerations for home collection
Home collection has several advantages including increased
convenience, decreased risk of exposing other patients and
healthcare workers and decreased need for personal protec­
tive equipment [76]. However, from a testing standpoint, these
approaches present unique challenges including an increased
likelihood of suboptimal specimen collection and an increased
likelihood of specimen degradation during transport. The FDA
recommends against self-collected NP swabs for safety.
Therefore, in addition to standard validation studies, the FDA
requires additional stability and usability studies for assays
that employ home collection. Self-collection kits that facilitate
and optimize collection (such as for saliva), instructional
pamphlets and videos that clarify testing steps for laypersons
and testing of less invasive specimen like nasal swabs can
improve specimen collection and diagnostic accuracy. Home
collection must be approved by the FDA and tests must
Table 2. Characteristics of a selection of commercial assays for detection of SARS-CoV-2 RNA.
Developer/ Reported
Manufacturer Assay Method Gene Targets Ct values LOD* Specimen Sample volume Features Assay time
Abbott Abbott RealTime SARS CoV-2 Real time RT- N, RdRp Yes 100 copies/ NP, OP, Nasal 500 µL Automated 3.5 hr
on m2000 system PCR mL High throughput
Abbott ID NOW COVID-19 Isothermal RdRP No 125 copies/ NP, Nasal, OP Dry nasal swab placed Manual 5–13
nucleic mL directly into the Sample Low Throughput min
acid Receiver (1 sample per run)
amplification Low skill level. Can be used
(NEAR) at Point of Care.
Becton Dickinson BD BioGX SARS-CoV-2 for BD Real time RT- N1, N2 Yes 40 copies/ NP, OP, Nasal, MT 750 μL Automated 3.5 hr
MAX system PCR mL Two duplex reactions
based on CDC assay
24 samples per batch
Centers for Disease CDC 2019-Novel Coronavirus Real time RT- N1, N2 (original Yes 1–3 NP, OP, Nasal, MT, BAL, TA, 140–200 µL into separate Manual 3.5 hr
Control and real-time RT-PCR PCR assay copies/ Sputum extraction Low throughput (designed
Prevention diagnostic panel included N3) µL as 3 singleplex PCR
reactions)
96-well plate
High skill level
Cepheid Xpert Xpress SARS CoV-2 Real time RT- N2, E Yes 250 copies/ NP, OP, Nasal, MT 300 μL Automated 45 min
PCR mL Random access
Inoculate single sample
into cartridge and place on
instrument
Diasorin SimplexaCOVID-19 Direct Real time RT- ORF1ab, S Yes 242 copies/ NP, Nasal, BAL 50 μL Automated 1.5 hr
PCR mL Batched
Inoculate 1–8 wells in
wheel
Hologic SARS CoV-2 Assay (Panther Real time RT- ORF1ab Yes 0.01 TCID50 NP, OP, nasal, MT, BAL 500 µL into lysis tube Automated 2.5 hr
Fusion) PCR regions 1 /mL Random access
and 2
Hologic Aptima SARS CoV-2 Assay Transcription ORF1ab No 0.01 TCID50 NP, OP, nasal, MT 500 μL into lysis tube or Automated 3.5 hr
(Panther System) mediated regions 1 /mL direct testing of Aptima High Throughput
amplification and 2 Multitest swab
(TMA)
Quest SARS-CoV-2 RNA, Qualitative Real time RT- N1, N3 Yes 136 copies/ NP, OP, Nasal, MT, BAL, TA, 200 µL into separate Batched testing 3.5 hr
Real-time RT-PCR PCR µL Sputum, home collected extraction Multiplex RT-PCR based on
nasal swabs CDC assay
96 well plate
Can test unusual samples
types
RP control required for
home collection
Roche Cobas SARS CoV-2 for use on Real time RT- ORF1ab, E Yes 0.009 NP, OP, nasal, MT 600 µL Automated 3.5 hr first
6800/8800 Systems PCR TCID50/ High throughput 96 then
mL 96 tests per run 1.5 hr
Thermofisher TaqPath COVID-19 Combo Real time RT- ORF1ab, N, S Yes but not 10 copies NP, OP, Nasal, MT, BAL 200 or 400 µL into separate Manual or automated 3.5 hr
Kit PCR readily /reaction extraction High Throughput
obtained 96 or 384 well plates
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS

High skill level

*LOD varies per specimen type. Lowest LOD is included here. LOD is an in vitro assessment that may not correlate when tests are compared in parallel using clinical specimens.
RT-PCR, reverse transcription polymerase chain reaction.
Gene targets: N, nucleoprotein; RdRp, RNA dependent RNA polymerase; E, envelope; ORF1ab, open reading frame 1ab; S, spike.
LOD, limit of detection or lowest concentration of analyte that can be detected 95% of the time.
1091

NP, nasopharyngeal; OP: oropharyngeal; MT, mid-turbinate; BAL, bronchoalveolar lavage; TA, tracheal aspirate.
1092 M. M. AZAR ET AL.
ensure human material has been collected, such as including
PCR for human RNase P gene.
3.1.3. Sample type
As a general rule, deeper specimens yield greater sensitivity.
Nucleic acid testing on nasopharyngeal specimens is overall
more sensitive than for other URT specimens with mid-
turbinate, anterior nasal and oropharyngeal specimens being
incrementally less sensitive [77]. The data are mixed, however
with some studies showing mid-turbinate swab specimens to
be equally sensitive to nasopharyngeal specimens [78].
Similarly, saliva is less or equally sensitive to nasopharyngeal
specimens in most studies though saliva collection methods
are inconsistent, making an accurate analysis difficult [79,80].
Lower respiratory track (LRT) specimens such as sputum,
endotracheal fluid and bronchoalveolar lavage fluid are more
sensitive than upper respiratory tract (URT) specimens and
should be collected if initial URT testing is negative but suspi­
cion remains moderate to high [67]. Though certain conclu­
sions can be made from available studies, variability in study
designs, collection and test methodologies, patient popula­
tions including demographics and clinical severity inject sig­
nificant heterogeneity into results.
3.1.4. Sensitivity and specificity
The clinical sensitivity of SARS-CoV-2 nucleic acid testing is
defined as the ability of the test to correctly identify patients
with COVID-19. Clinical sensitivity for SARS-CoV-2 testing is
dependent on several factors including severity of disease,
specimen type, specimen quality and analytical sensitivity.
Clinical sensitivity also encompasses test result interpretation
in a clinical context in order to differentiate acute infection
from colonization (for example, with Pneumocystis jiroveci) or
prolonged shedding (for example, with rhinovirus). In the case
of COVID-19, SARS-CoV-2 RNA detection can be prolonged
with a subset of patients shedding virus beyond 6 weeks
after initial infection [69]. Therefore, positive SARS-CoV-2 test
result may indicate acute recent COVID-19 infection but could
also indicate prolonged shedding weeks or months after acute
infection. Since prolonged positivity may not indicate infec­
tiousness, making this distinction is important clinically and for
infection prevention purposes [71–73].
The analytical sensitivity of a SARS-CoV-2 nucleic acid test is
its ability to detect the target analyte (SARS-CoV-2 RNA in this
case) in a biologic sample with the limit of detection (LOD)
being the concentration SARS-CoV-2 RNA in a specimen that
can be consistently detected ≥ 95% of the time. These mea­
sures vary across various laboratory-developed and commer­
cial SARS-CoV-2 nucleic acid tests with most available assays
reporting an LOD from 150 to 1,000 genome copies/mL (Table
2). However, a more accurate assessment is a comparison of
assays’ performance using parallel testing of low positive clin­
ical samples. Recently, falsely negative NAAT testing has been
reported with mutated strains of SARS-CoV-2 highlighting the
need for epidemiologic monitoring of circulating SARS-CoV-2
viruses [81,82].
It should be noted that supply chain disruptions have
affected all aspects of laboratory testing, including swabs
and transport media, nucleic acid extraction reagents and
buffers, plasticware including pipet tips, test kits and reagents,
and instrumentation. These shortages have led to a constant
struggle to quickly validate alternative materials and methods,
the need to maintain multiple test platforms, and to compro­
mise and sometimes accept less than optimal results.
4. SARS-CoV-2 serology for diagnosis of COVID in
the setting of CRS
After COVID-19, most individuals develop a serologic response
to SARS-CoV-2, with a median time to IgG positivity of
11–14 days depending on several factors [83,84]. Patients
with very mild disease and those with underlying immuno­
compromising conditions may not mount an antibody
response whereas those with critical illness may have higher
and more sustained antibody titers [85]. False-negative testing
may occur with poorly sensitive assays (particularly lateral flow
assays), if testing is done prior to 11 days after symptom onset
or in very mild cases. Serology is not recommended for clinical
diagnosis of SARS-CoV-2 infection as it is less sensitive than
RNA detection for acute infection, and the implications of
positive serology for either immunity or infectivity to others
have not been sufficiently characterized. Though not routinely
recommended, serology may have a role in the clinical diag­
nosis of COVID in certain circumstances. These include diag­
nosis of suspected late COVID-19 complications such as
Multisystem Inflammatory Syndrome in Children without
prior positive PCR/RNA and suspected recent COVID-19 with
negative-repeated RNA testing despite high clinical suspicion,
a scenario that may occur in the setting of CRS (Figure 1) [86].
SARS-CoV-2 IgM antibodies peak within 3 weeks of illness
onset with most patients seroconverting to IgG between
week 3 and week 7. Since development of SARS-CoV-2 IgM
and potentially IgG, coincide with the cytokine response phase
of illness, serology has diagnostic utility in the diagnosis of
COVID when PCR testing is negative but suspicion for COVID
remains high [87] In a study of patients with COVID-19, serol­
ogy was positive in 28–100% of patients with negative upper
respiratory PCR testing suggesting clinical value in this setting.
Moreover, the sensitivity of IgG was substantially higher than
PCR (80% vs.46%) in patients who were in a later phase (15–­
39 days) from symptom onset [84]. If initial testing with serol­
ogy is negative, a convalescent sample should be obtained
2–4 weeks later to exclude delayed seroconversion.
5. Laboratory markers of severe COVID and CRS
Laboratory testing is central to both the initial diagnosis and
follow-up of SARS-CoV-2 infection and can moreover provide
early clues of progression to CRS. Laboratory findings asso­
ciated with COVID-19 include decreased lymphocyte counts,
platelet counts and albumin levels as well as elevated CRP,
ferritin, lactate dehydrogenase (LDH), transaminases,
D-dimer, procalcitonin and interleukin-6 and 10 levels
among others. In retrospective case series of 393 patients
with COVID-19 in New York, lymphopenia was present in
90% of cases, elevated ferritin in 66%, elevated CRP in
43%, elevated D-dimer in 36%, elevated alanine

Figure 1. Viral, serologic and diagnostic correlates of SARS-CoV-2 Infection.
After SARS-CoV-2 acquisition, a subset of individuals (40–45%) will become symptomatic after a median incubation period of 5 days. Viral replication accelerates rapidly in upper respiratory
tract cells (viral replication phase), reaching a peak viral load between 0.6 days to 5 days after symptom onset, before beginning to decrease gradually. Some patients may develop clinical
worsening during viral load decline as a result of an overactive inflammatory response (cytokine response phase). Testing done too early (>~5 days before symptom onset) or too late
(>~21 days after symptom onset) in the course of the infection may yield false-negative PCR results as viral load may be below the threshold of detection of the assay (purple-shaded
areas). SARS-CoV-2-specific serum IgG develops at a median of 11 days after infection.
aminotransferase in 32% and elevated procalcitonin in 17%
[88]. In addition to aiding in the initial diagnosis of COVID-
19, several biomarkers may serve as indicators of greater
disease severity at baseline as well as markers of disease
progression, often a clinical manifestation of developing
CRS. CRP is perhaps the best predictor of COVID-19 severity.
Elevated CRP levels have been associated with more severe
disease at baseline, more extensive lung infiltrates on ima­
ging and increasing CRP levels are a risk factor for COVID-19
progression [89,90]. Decreased lymphocytes counts and
increased LDH levels have also been associated with severe
COVID-19 both as individual biomarkers and when inte­
grated into severity prediction indexes [91,92]. Immune dys­
regulation is also manifested by higher neutrophil-
lymphocyte-ratio, and lower percentages of monocytes,
eosinophils, and basophils. Moreover, decreased T-cells sub­
sets, particularly helper T cells have been observed with
severe disease [31].
Though progressive respiratory failure is the main driver of
morbidity and mortality in COVID-19, superimposed processes
including bacterial superinfection and coagulopathy are
known complications. When significantly elevated procalcito­
nin levels are strongly suggestive of a superimposed bacterial
infection [93]. Indeed, while viral infections trigger production
of interferon-γ which inhibits production of procalcitonin,
bacterial infections stimulate production of IL 1β, IL-6 and
tumor necrosis factor α which enhance the production and
release of procalcitonin from extrathyroidal tissues. However,
mildly elevated procalcitonin levels have been observed in
patients with severe COVID-19 and no evidence of bacterial
infection. In these cases, it is possible that elevated inflamma­
tory cytokines in the context CRS-related immune dysregula­
tion lead to increases procalcitonin synthesis [31]. This is
consistent with findings from severe influenza, in which
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 1093
procalcitonin may not be sufficiently sensitive to rule out
bacterial pneumonia as a stand-alone marker [94].
As with secondary bacterial infection, the development of
a systemic coagulopathy is now a well-recognized complica­
tion of severe COVID-19. Increased concentrations of inflam­
matory cytokines induce a prothrombotic cascade. In
particular, IL-6 induces tissue factor expression on mono­
nuclear cells driving coagulation activation and thrombin
generation [95]. Complications which include deep vein
thrombosis (DVT), pulmonary embolism (PE), ischemic
stroke, myocardial infarction and systemic arterial events
have been reported in up to approximately 30% of patients
with COVID-19 in the intensive care unit [96]. The most
common findings of COVID-19-related coagulopathy are ele­
vated D-dimer, mild thrombocytopenia and prothrombin
time prolongation. D-dimer, a product of fibrin degradation,
is a sensitive marker of venous thromboembolism (VTE).
However, increased D-dimer levels are often driven by
underlying inflammatory processes including sepsis, severe
COVID-19 and CRS. Since elevated D-dimer levels may be
a manifestation of both increased disease severity and an
increased risk of thrombosis, the discriminatory power for
VTE is reduced [97]. Accordingly, rising D-dimer levels, var­
ious institutionally determined D-dimer thresholds and clin­
ical signs of VTE including sharp rises in oxygen
requirements have been employed as indications for institu­
tion of therapeutic anticoagulation in the setting of severe
COVID-19 and CRS.
6. Conclusion
Studies support the implication of CRS in the pathogenesis,
clinical severity and outcome of COVID-19 through the elevated
production of multiple inflammatory cytokines and chemokines
1094 M. M. AZAR ET AL.
from activated innate and adaptive immune cells. Although these
inflammatory molecules, including IL-6, IL-2 R, IL-10, IP-10 and
MCP-1, often correlate with disease severity as possible biomar­
kers, the pathogenic contributions of individual molecules and
the therapeutic benefits of targeting them are yet to be demon­
strated. Detection of SARS-CoV-2 RNA is the gold standard
method for diagnosis of COVID-19 in the context of CRS but
assay performance varies according to analytical and clinical
characteristics. Biomarkers including CRP, ferritin, D-dimer and
procalcitonin may provide early clues about progression to CRS
and help identify thrombotic and infectious complications of
COVID-19.
7. Expert Opinion
Studies clearly indicate the rise of multiple inflammatory cyto­
kines and chemokines in patients with COVID-19, possibly
contributing to the pathogenesis of this disease accompanied
by systemic inflammation, especially in the form of CRS. As
these cytokines and chemokines form a highly interactive
network, often serving as a feed-forward mechanism, it
would be challenging to identify the most significant
upstream driver(s) dictating systemic inflammatory responses
in COVID-19. Such molecules could be produced from the
innate and/or adaptive immune cells in response initially to
SARS-CoV-2 and later to molecules released from damaged
cells as damage-associated molecular patterns. High-dimen­
sional cytokine analysis done in a prospective manner,
together with careful clinical data collection and computa­
tional integration of both biological and clinical data, may be
able to reveal the possible upstream inflammatory cytokine(s)
accountable for CRS with systemic inflammation and ARDS,
the major mortality driving manifestations, in COVID-19. Such
an investigation could be achieved using recently developed
high-dimensional single cell analytic tools like single cell RNA
sequencing and mass cytometry in addition to cytokine multi­
plex. The results of this study can lead to the development of
a new therapeutic approach with an appropriate selection of
cytokine blockade(s) and its introductory time point, hopefully
before the progress to the full-blown CRS and ARDS. The same
cytokine(s) also can serve as a biomarker guiding additional
therapeutic strategies such as timing of initiating certain treat­
ment modalities and predicting prognosis.
Many sensitive NAAT have been developed, yet the labora­
tory diagnosis of SARS CoV-2 remains a challenge due to
escalating demands that outpace testing capacity. Detection
of viral RNA is preferred, but access has been limited by
unreliable supply chains, and inability to obtain collection
swabs, instruments, reagents and supplies. Test backlogs at
reference laboratories lead to delays of a week or more before
results are received. Due to staffing shortages, risk to person­
nel, and need for scarce PPE, sample collection has been
inadequate. These challenges have led to alternative strate­
gies, such as testing saliva, self-collection of nasal swabs, and
home collection. High-throughput genetic testing laboratories
have adapted their methods to SARS CoV-2 screening. Novel
technologies such as CRISPR, as well as pooling of samples to
increase throughput and reduce cost, have been implemented
for the first time for diagnostic testing. Rapid, sensitive, simple,
and affordable molecular tests that can be performed at the
point of care or at home are expected soon. Less sensitive
methods such as rapid antigen tests, with results in 15-30 min
and minimal equipment requirements, may also help to fill the
gap in some settings such as frequent re-testing schemes.
While concerns that the virus may mutate and elude detection
are valid, the use of multiple viral targets in most assays
provide some assurance of continued efficacy. As these multi­
ple strategies to improve access and shorten time to diagnosis
come to fruition, SARS CoV-2 infections should be more
quickly detected and controlled, thus facilitating the safe
reopening of society.
Funding
This paper was not funded.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
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