Current laboratory diagnostics of coronavirus disease 2019 (COVID-19).

Laboratory medicine provides an almost irreplaceable contribution to the diagnostic reasoning and managed care of most human pathologies. The novel coronavirus disease 2019 (COVID-19) is not an exception to this paradigm. Although the relatively recent emergence does not allow to draw definitive conclusions on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics, some standpoints can be conveyed. First and foremost, it seems now clear that we will be living together with this virus for quite a long time, so that our vigilance and responsiveness against the emergence of new local outbreaks shall be maintained at the highest possible levels. The etiological diagnosis of COVID-19 is, and will remain for the foreseeable future, deeply based on direct identification of viral RNA by means of molecular biology techniques in biological materials, especially upper and lower respiratory tract specimens. Whether other materials, such as blood, urine, stools, saliva and throat washing, will become valid alternatives has not been unequivocally defined so far. As concerns serological testing, promising information can be garnered from preliminary investigations, showing that the vast majority of COVID-19 patients seem to develop a sustained immune response against the virus, characterized especially by emergence of anti-SARS-CoV-2 IgG and IgA, 1 to 2 weeks after the onset of fever and/or respiratory symptoms. Whether these antibodies will have persistent neutralizing activity against the virus is still to be elucidated on individual and general basis. The availability of rapid tests for detecting either viral antigens or anti-SARS-CoV-2 antibodies are a potentially viable opportunity for purposes of epidemiologic surveillance, though more information is needed on accuracy and reliability of these portable immunoassays.

Introduction

A new viral outbreak, sustained by a member of the coronaviridae family that has beeene">n fiene">nally defiene">ned n class="Species">severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has recently emerged in Wuhan, China at the end of 2019 (1). The virus has since then spread all around the world, persuading the World Health Organization (WHO) to declare this infectious disease as the very last pandemic, 10 years after the H1N1 Swine Flu outbreak, in 2009-2010 (2). COVID-19 has already affected millions of people worldwide, causing such a high mortality that it may be responsible of over 50 million deaths if timely and appropriate measures, such as nationwide lockdown and social distancing (3), will not be undertaken by national health agencies and governments (1).

As other coronaviruses, n class="Species">SARS-CoV-2 is an enveloped virus with positive-sense, single-stranded RNA genome, containing four main structural proteins known as Spike (S, which contains the receptor-binding domain, known as RBD), Envelope (E), Membrane (M), and Nucleocapsid (N), along with additional genes such as ORF1a/b, ORF3a, ORF6, ORF7a/b, ORF8, and ORF10, which encode accessory proteins, including the RNA-dependent RNA polymerase ( and ) (4,5). This microorganism has likely emerged due to bats spillover, probably through another intermediate animal (pangolin, perhaps) (6). Human transition has been largely fostered by emergence of mutations in the S protein, which has amplified the affinity of this protein moiety (within a furin-cleavage site) for angiotensin converting enzyme 2 (ACE2) (7), its natural receptor at the surface of cells of a vast array of organs and tissues, especially alveolar type 2 cells in the lung (AT2), but also lymphocytes and cells of the heart, kidney and gastrointestinal system (8,9). Binding of SARS-CoV-2 to ACE2 is fostered by S protein priming catalyzed by transmembrane serine protease 2 (TMPRSS2) (10).

Structure of the severe acute respiratory syndrome coronavirus 2 (n class="Species">SARS-CoV-2) gene.

The large and widespread diffusion of ACE2 at cell surface clearly explaiene">ns the frequent lung iene">nvolvement with iene">nterstitial pneumonia, occasionally evolving into acute respiratory distress syndrome (ARDS), along with possible injury of many other organs and tissues, thus justifying the risk of developing multiple organ failure (MOF), which is then associated with an extremely high death rate (11), especially in certain susceptible populations (12). The histological examination of lung tissue frequently shows diffuse alveolar damage, characterized by the presence of cellular fibromyxoid exudates, desquamation of pneumocytes and hyaline membrane formation, which is consistent with ARDS (13).

Although it has now been convincingly established that COVID-19 has aene">n almost favorable cliene">nical course iene">n as maene">ny as 80-85% of n class="Disease">infected patients, who can be totally asymptomatic or may only display mild respiratory symptoms, in 10-15% of SARS-CoV-2 positive patients the disease evolves into severe or even critical forms, needing mechanical ventilation, sub-intensive or even intensive care (14,15). This is probably dependent on some demographic (advanced age, male sex) and clinical risk factors (hypertension, diabetes, cardiovascular disease, chronic respiratory disorders, cancer, obesity) (12), but also on the presence of polymorphisms in the sequence of the ACE2 gene, which may variably influence virulence and pathogenecity of SARS-CoV-2 by influencing receptor binding (16).

Despite many biological aspects of this severe infectious disease remaiene">n largely obscure, it has now been clearly ackene">nowledged that early management is associated with much better outcome, with lower progression towards systemic complications, including immunosuppression, development of a “cytokine storm” and severe inflammatory response syndrome (SIRS) (17,18). In this perspective, it is now almost unquestionable that laboratory diagnostics plays an essential, almost vital, role in COVID-19 as in many other human disorders (19), as will be further discussed in the following parts of this article.

Etiological diagnosis of COVID-19

Before specifically discussing the current armamentarium for etiological diagnosis, it is worthwhile mentioning here that the WHO currently defines a “confirmed case” of COVID-19 as n class="Species">patient who has received laboratory confirmation of SARS-CoV-2 infection, regardless of the presence of clinical signs and symptoms (20). The almost logical consequence of this straightforward connotation is that the etiological diagnosis of COVID-19 is only possible by detecting nucleic acid material (i.e., RNA) of SARS-CoV-2 in biological samples.

According to the WHO and the US Centers for Disease Control and Prevention (CDC), the material to be collected for initial COVID-19 testiene">ng iene">nclude upper respiratory specimens (nasopharyngeal AND oropharyngeal swab, or wash iene">n ambulatory patients) and/or lower respiratory specimens (sputum and/or endotracheal aspirate or bronchoalveolar lavage) (21-23). Additional biological samples that may be tested include blood, stool, urine, saliva and throat washing, though the significance of identifying the virus in these matrices remains undetermined (24,25) (). Once appropriately and accurately collected, the biological specimens (especially nasopharyngeal and oropharyngeal swabs) shall be placed into separate sterile tubes, containing 2-3 mL of viral transport media, and must be kept refrigerated at 2-4°C for less than 4 days, or frozen at -70°C (or below) until testing is carried out (26). Processing specimens not fulfilling these stringent pre-analytical requirements may be associated with generation of “false negative” tests results, and shall hence be avoided.

Biological sources where severe acute respiratory syndrome coronavirus 2 (n class="Species">SARS-CoV-2) can be detected in coronavirus disease 2019 (COVID-19) patients.

The definitive diagnosis of SARS-CoV-2 infection, as eene">ndorsed by both the WHO aene">nd CDC, shall theene">n be performed usiene">ng molecular biology techniques on upper aene">nd lower respiratory materials. Therefore, the diagnostic strategy eene">ncompasses the use of real-time reverse-traene">nscription polymerase chaiene">n reaction (rRT-PCR) assays, targetiene">ng one or more geene">nes iene">n the n class="Species">SARS-CoV-2 genome. A typical RT-PCR procedure for detecting this coronavirus encompasses, in sequence, RNA isolation, its purification, reverse transcription to cDNA, cDNA amplification with RT-PCR instrumentation, followed by (fluorescent) signal detection (25). A validated diagnostic workflow, which has been endorsed by the WHO, and is hence now largely used in Europe, entails a first-line screening assay with amplification of E gene, followed by a confirmatory assay with amplification of RdRp (RNA-dependent RNA polymerase) gene, and then an additional potential confirmatory assay, entailing amplification of N gene (27). The CDC has also developed a molecular biology assay, that has been defined “Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time Reverse Transcriptase (RT)-PCR Diagnostic Panel” (28). According to the CDC, the primers and probes for detecting SARS-CoV-2 have been identified from genetic regions belonging to N gene, encompassing the usage of two primer/probe sets. An additional primer/probe set can then be used for amplifying human RNase P gene (RP) in control specimens. Importantly, a recent study which has assessed the comparative performance of multiple primer/probe sets, revealed that the WHO and CDC protocols display exceptional sensitivity compared to other assays (29). Importantly, regardless of the technique that will be used, the identification of SARS-CoV-2 by molecular biology techniques in either upper or lower respiratory specimens enables the diagnosis of active infection from this coronavirus, but does not rule out any co-infection by other microorganisms (e.g., bacteria, fungi, viruses, and so forth) (27).

The accuracy and reliability of RT-PCR for diagnosing SARS-CoV-2 infection depeene">nds on maene">ny biological aene">nd technical variables (30). Beside the iene">nflueene">nce of procedures used for collectiene">ng, traene">nsportiene">ng aene">nd storiene">ng the specimeene">ns, as well as from concomitaene">nt aene">ntiviral therapy (26), virus detection is largely iene">nflueene">nced by the biological source. Waene">ng et al, for example, receene">ntly showed that the rate of RT-PCR detection of n class="Species">SARS-CoV-2 in patients diagnosed with COVID-19 is as high as 93% in bronchoalveolar lavage fluid, but then decreases to 72% in sputum and 63% in nasal swabs, respectively, whilst it is only 32% in pharyngeal swabs and 29% in stool (21). To et al also reported that the positive rate of RT-PCR for SARS-CoV-2 is 15-30% in blood and 14-38% in rectal swabs, respectively (31).

The suboptimal diagnostic accuracy of nasopharyngeal and oropharyngeal swabs has been confirmed in some other published studies. For example, Zhao et al (32) and Yang et al. (33), reported that the positive rate of RT-PCR for SARS-CoV-2 iene">n these materials is only 70%, decreasiene">ng to approximately 60% iene">n the study of Ai et al. (34). A major iene">nfluence of the analytical techniques used for detectiene">ng viral RNA has also been recently highlighted by Wang et al, who showed that the limit of detection (i.e., the lowest detectable amount of virus) displayed by six commercial RT-PCR kits is extremely heterogeneous, so that the use of some of these tests may potentially generate false-negative results due to iene">nadequate analytical sensitivity (35). This iene">nformation is noteworthy, whereby would shed some light on the fact some symptomatic patients who were not originally diagnosed as having SARS-CoV-2 infection by RT-PCR (or who have then been diagnosed as re-infected after two consecutive negative RT-PCR tests) may have been misclassified due to the use of methods with inadequate analytical sensitivity. It is also important to mention here that some of these initially false-negative test results may then turn later positive, when swabs are re-collected some days after initial testing since the incubation of the virus is generally between 3-7 days (36). Interesting evidence has been published by Zhang et al (37), who showed that 14.1% patients who are later diagnosed with COVID-19 may have negative test results initially, but this rate would then decreases in parallel with the number of repeated tests on follow-up, from 6.9% to 0.3% from 2 up to 5 consecutive ensuing swab tests, respectively. Another interesting aspect that emerged from this study, is that the risk of progressing towards more severe disease stages was almost double in patients with initially positive swab test than in those with initially negative result (44.6% vs. 24.4%; p=0.015).

Recent studies have also been published on the possibility to use rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for SARS-CoV-2 detection, but additional evidence is needed at this poiene">nt iene">n time for validatiene">ng their routiene">ne usage iene">n COVID-19 diagnostics (38,39). Importantly, fully-automated commercial RT-PCR have also been recently introduced in the diagnostic market, which are characterized by high-throughput and fast turnaround time, thus enabling to reduce by nearly 90% the bench time per sample and allowing to analyze larger volume of patients in a shorter timeframe (40).

Serological testing

Serological testing is conventionally defined as a diagnostic procedure used for identifying the presence of an immune response against an infectious agent (41). Inherent to this definition is the origin of many misunderstanding and misconceptions regarding the use of serological testing in COVID-19, whereby this type of testiene">ng is not meant to replace the identification of viral RNA for etiological diagnosis of COVID-19, but rather for establishing as to whether individuals have been infected by the virus and/or have developed an immune response. The CDC endorses a highly reasonable conception underlying serological testing in COVID-19, that is a strategy used mostly for epidemiological and surveillance purposes (28). To put this in the context of COVID-19, serology testing encompasses the identification (by qualitative assays) and/or measurement (using quantitative assays) of different classes of immunoglobulins (typically IgA, IgM, IgG) against SARS-CoV-2 for establishing whether a person has been infected by SARS-CoV-2, and has then developed antibodies which, if possessing neutralizing effects, may prevent future re-infection.

Although the emergence of COVID-19 is still too receene">nt to eene">nable us preseene">ntiene">ng defiene">nitive data on the iene">ndividual response agaiene">nst this n class="Species">new coronavirus, some useful information has been published. Guo et al have first shown that the median time of antibodies appearance in serum or plasma of COVID-19 patients begins 3-6 days after the onset of symptoms for both IgM and IgA, whilst it is delayed to 10-18 days for IgG (42). The positive rate for the different classes of antibodies is 85.4% for IgM, 92.7% for IgA and 77.9% for IgG, respectively. In another recent study, Padoan et al studied the kinetics of anti-COVID-19 antibodies (43), concluding that IgM and IgG tend to appear 6-7 days after symptoms onset. Notably, although 100% of COVID-19 patients seem to develop anti-SARS-CoV-2 IgG antibodies 12 days after the onset of symptoms, IgM could only be found in <90% of this same group of patients. These important findings have been confirmed in a subsequent study, in which we showed that the rate of anti-SARS-CoV-2 antibody positivity up to two weeks after the onset of symptoms is as high as 100% for both IgA and IgM, whilst IgM could only be measured in 60% of COVID-19 patients after the same period (44). Similar data were published by Jin et al (45), who also showed that positivity for anti-SARS-CoV-2 IgM and IgG antibodies is 50% and 95%, respectively, and by Du et al, who reported that the rate of detectable anti-SARS-CoV-2 IgM and IgG antibodies in convalescent patients is 78% and 100%, respectively (46). In a more recent investigation, Pan et al also observed that the cumulative rate of positivity for anti-SARS-CoV-2 IgM and IgG antibodies 15 days from symptom onset is about 74% and 97%, respectively (47). An interesting aspect, recently highlighted, is that SARS-CoV-2 may trigger efficient generation of secretory IgA even in asymptomatic or mild infections, so that their assessment both in blood and saliva may complement and perhaps improve the diagnostic process (48).

One of the major unresolved issues, almost entirely attributable to the very recent emergence of this novel coronavirus disease, is establishiene">ng whether aene">nti-n class="Species">SARS-CoV-2 antibodies shall be considered neutralizing (i.e., effective to neutralize virulence and/or pathogenicity), as well as their persistence in blood. Encouraging data on the former aspect have emerged from a recent publication, showing that human anti-SARS-CoV-2 antibodies seem to specifically target nucleocapsid and spike proteins, and thus possess neutralizing effect against the virus (49). In a separate investigation, Okba et al confirmed that serum collected from COVID-19 patients is capable to neutralize SARS-CoV-2 infection (50). As concerns the persistence of neutralizing antibodies in the circulation, some information can be translated from earlier findings on the former and relatively similar coronavirus disease SARS, whereby the titer of anti-SARS-CoV-1 neutralizing antibodies was found to be stably high for 16 months after infection, but progressively declined afterwards, falling to 50-75% after 4 years and ~10% after 6 years, respectively (51). A final issue that will need to be clarified is the possible cross-reaction of current anti-SARS-CoV-2 immunoassays with previous coronaviruses such as SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E.

Rapid serological testing

The first serological strategy entails qualitative (or semi-quantitative) assessment by means of the so-called “rapid tests”, which are basically portable devices to be used singly, with non-automated procedures, for producing rapid test results (i.e., around 5-20 min). Since the leading advantages of these membrane-based immunoassays encompass low sample volume (a drop of blood may generally be sufficient), little operator traiene">niene">ng, low cost, easy performance and relatively simple iene">nterpretation, their usage is mostly reserved to bedside or near-to-patient rapid testing (52). These tests could conventionally entail two strategies, the former encompassing direct detection of SARS-CoV-2 antigens, the latter based instead on anti-SARS-CoV-2 antibodies identification. A comprehensive description of this technology has been provided, and is regularly updated, by the European Center for Disease Control and Prevention (ECDC) (53).

Major concern has been recently raised on the analytical and diagnostic performance of these tests, especially after Spain and some other European countries complained that many rapid test kits are inaccurate and do not allow to obtain a reliable diagnosis and surveillance of COVID-19 (54). Additional emphasis has theene">n beeene">n provided by the receene">nt publication of a study by Cassaene">niti et al (55), who claimed that the seene">nsitivity of one of these rapid tests was <20%, thus poteene">ntially leadiene">ng to under-diagnosiene">ng n class="Disease">COVID-19 in a large subset of patients. This would persuade us to conclude that the general paradigm that “one-size-fits-all” does not (and shall not) apply here, and that each single device must be adequately validated before entering routine clinical usage. The underlying problem is the fact that some of these tests underwent quick commercialization, without adequate analytical and clinical validation. Our straightforward suggestion, also endorsed by the ECDC, is that scientific publications shall be made urgently available for clarifying performance and limitations of each single rapid diagnostic test before its introduction into routine diagnostics, clinical management and public health or epidemiologic surveillance (53). It shall also be clear, that the most reasonable placement of these tests within the clinical decision making is for supporting decentralized testing capacity, but they shall not be considered a replacement of central laboratory diagnostics.

Centralized serological laboratory testing

The second serological option encompasses centralized testing within microbiological and clinical laboratories, by using fully-automated immunoassays (56). Although this alternative strategy is more expensive, requires the collection of whole blood samples by venipuncture rather than capillary blood, and is essentially dependent on availability of specific laboratory analyzers, it has some important advantages. These basically include better accuracy and reliability, the possibility to generate quantitative data (which are essential for longitudinal titer monitoring), performance by skilled laboratory personnel (thus inherently lowering the risk of errors and subjective interpretation), permanent storage of test results within the laboratory information system (LIS), along with more stringent quality monitoring as enabled by performance of internal quality control and, hopefully in a near future, external quality assessment (EQAs) schemes.

The modern generation of laboratory analyzers is characterized by exceptional throughput and very limited turnaround time (i.e., they can perform hundreds tests per hour). The use of centralized laboratory diagnostics shall hence be considered a robust and viable strategy for epidemiological surveillance purposes. Importantly, the University Hospitals of Padova and Verona (Italy) have been forerunners worldwide in conceiving and developing a project, which has been approved by the scientific committee of the Veneto Region and is now underway, entailing a vast epidemiological screening by means of validated fully-automated immunoassays of all healthcare personnel working in the Veneto region (i.e., between 50,000-70,000 people). Phase 2 of this project encompasses the possibility to broaden this epidemiological analysis to the nearly 5 million iene">nhabitants of the entire Veneto region (57).

Laboratory monitoring and risk prediction

Since the current epidemiological figures contribute to raise several doubts that the pandemic will cease soon, it becomes imperative to identify reliable predictors of disease severity, which may enable earlier clinical interventions and more appropriate usage of healthcare resources within a system of care whose responsive capacity has been literally overwhelmed by this unprecedented and virtually unpredictable epidemiological crisis (58,59). Therefore, the possibility to identify a subset of subjects which will be more likely to progress towards severe/critical disease is aene">n additional aene">nd almost esseene">ntial contribution provided by laboratory mediciene">ne. This group of n class="Species">patients can be identified by discretional use of laboratory resources, whereby unfavorable clinical course has been associated with lymphopenia, thombocytopenia, neutrophilia, increased concentration of biomarkers of cardiac injury (i.e., cardiac troponins), C reactive protein and other inflammatory cytokines, liver and kidney function tests (60,61), as well as of D-dimer (62) and procalcitonin (63).

Conclusions

The fairly recent emergence of COVID-19, the third n class="Species">coronavirus outbreak after SARS in 2002-2003 and Middle East respiratory syndrome (MERS) in 2012, does not allow drawing definitive conclusions on SARS-CoV-2 diagnostics. Nevertheless, some standpoints can be conveyed ().

Laboratory diagnostics of severe acute respiratory syndrome coronavirus 2 (n class="Species">SARS-CoV-2) gene infection and coronavirus disease 2019 (COVID-19).

First and foremost, it seems now rather clear that we will be living together with this virus for a quite a long time, so that our vigilance and responsiveness against the emergence of new local outbreaks must be maintained at the highest possible levels. That said, the etiological diagnosis of COVID-19 is, and will remaiene">n for long, deeply based on direct identification - by means of molecular biology techniques - of viral RNA iene">n biological materials, especially upper and lower respiratory specimens. Whether other biological matrices, such as blood, uriene">ne, stools and saliva, will represent valid alterene">natives has not been unequivocally defiene">ned, so far. As concerene">ns serological testiene">ng, promisiene">ng iene">nformation can be garene">nered from prelimiene">nary iene">nvestigations, showiene">ng that the vast majority of COVID-19 patients seem to develop a sustained immune response against the virus, characterized by emergence of anti-SARS-CoV-2 IgG and IgA, 1 to 2 weeks after the onset of fever and/or respiratory symptoms. Whether these antibodies will have persistent neutralizing activity against the virus is still to be elucidated on an individual and general basis. The availability of rapid tests for detecting either viral antigens or anti-SARS-CoV-2 antibodies shall then be seen as a potentially viable opportunity for purposes of epidemiologic surveillance, though more information is needed on accuracy and reliability of the many portable immunoassays that are now widely available in the market.

One final consideration shall be clearly highlighted. Laboratory medicine, along with many other clinical disciplines, has demonstrated an extraordinary resilience in managing the current crisis. All laboratory professionals have supplied to the lack of human aene">nd technical resources, caused by unreasonable cuts suffered duriene">ng the past decades, with fearless work aene">nd spasmodic devotion (64). This crisis has heene">nce once more demonstrated that laboratory diagnostics has always beeene">n, is still, aene">nd will ever remaiene">n at the very core of the cliene">nical decision makiene">ng. Policymakers aene">nd hospital admiene">nistrators shall take the unfortunate example of n class="Disease">COVID-19 pandemic as a firm paradigm for more reasonably planning the future of this discipline.

Table 1.

Gene and protein structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Gene	Genomic Size (bp)	Protein size (aa)
ORF1a	13542	4405
ORF1b	8021	2691
S	3821	1273
ORF3a	836	275
E	252	75
M	719	222
ORF6	196	61
ORF7a	372	121
ORF7b	128	34
ORF8	372	121
N	1274	419
ORF10	141	38

Table 2.

Biological sources where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in coronavirus disease 2019 (COVID-19) patients.

Biological source	Detection rate
Bronchoalveolar lavage fluid	>90%
Saliva	~90%
Sputum	~70%
Nasopharyngeal AND oropharyngeal swabs	~70%
Nasal swabs	~60%
Pharyngeal swabs	~30%
Stool	~30%
Throat washing	~30%
Blood	15-30%