Laboratory Diagnosis of Emerging Human Coronavirus Infections - The State of the Art.

The three unprecedented outbreaks of emerging human coronavirus (HCoV) infections at the beginning of 21st century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. The laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. Newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. This presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (COVID-19) outbreak going on in Wuhan. Viral pneumonias typically do not result in the production of purulent sputum. Thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. Nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. Alternatively, repeated testing can be used because over time, the likelihood of the SARS-CoV-2 being present in the nasal-pharynx increases. Several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of SARS-CoV-2 infections. These assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients.

CoV, MERS-CoV and SARS-CoV-2). In temperate regions endemic HCoVs usually display a winter seasonality, although HCoV-229E has been detected sporadically throughout the year [12]. Endemic HCoVs are globally distributed and are maintained in the human population. The SARS-CoV pandemic came to an end in 2003 (https://www.who.int/csr/resources/ publications/CDS_CSR_ARO_2004_2.pdf?ua=1. Accessed 3 February 2020), less than a year after the first reported case. In contrast, human cases caused by MERS-CoV continue to be reported at the time of writing, more than seven years after the first reported case. Most laboratory-confirmed MERS cases have occurred in the Eastern Mediterranean Region, and the majority of those in Saudi Arabia. Unlike the endemic HCoVs, SARS-CoV and MERS-CoV are maintained in zoonotic reservoirs. The SARS and MERS outbreaks were driven in part by super-spreading events in which individuals directly infected a disproportionally large number of contacts [13]. The SARS-CoV-2-caused coronavirus disease 2019 (COVID- 19) epidemic originated in a Wuhan, China market that sold exotic animals for consumption. Based on genetic relatedness to other betacoronaviruses, SARS-CoV-2 likely has a zoonotic reservoir. However, the precise source of SARS-CoV-2 that initially infected humans remains to be confirmed. The SARS-CoV-2 appears to be substantially more contagious than SARS-CoV ( Table 1). The distribution of SARS-CoV-2 in different mammalian species is unknown. An interesting question is the susceptibility of farm animals and pets, and their role in the epidemiologic cycle as their angiotensin-converting enzyme 2 (ACE2) receptor shares similarity with human ACE2 [14].

Symptoms
Infections caused by endemic HCoVs have an incubation period of 2-5 days and are associated with mild upper respiratory symptoms (the "common cold"). Endemic HCoVs are among the most frequent cause of upper respiratory tract infections. Lower respiratory tract infections (bronchiolitis, pneumonia) are rare. Following an incubation period of usually 4-5 days, patients infected with SARS-CoV often present with symptoms of fever, headache, and myalgias. Respiratory symptoms including cough and dyspnoea usually develop from several days to a week after illness onset. Atypical pneumonia and respiratory deterioration occur in 20-30% of cases. The incubation period and clinical course of MERS are similar to that of SARS, the exception being a higher proportion of cases progressing to respiratory deterioration and distress. The incubation period and clinical course of SARS-CoV-2 infection are probably similar to that of SARS. Li et al. first reported a mean incubation period of 5.2 days [15]. Fever and cough are frequently reported early in the course of illness [16,17]. Infections are also characterized by dyspnoea, respiratory distress and positive chest X-ray [10]. Lower respiratory symptoms often develop about 1 week from the onset of initial symptoms [16].

Morbidity and mortality
Globally over 8000 cases and over 900 deaths due to SARS-CoV were reported, with a case-fatality ratio of approximately 11% (https://www.who.int/csr/sars/en/ WHOconsensus.pdf. Accessed 3 February 2020). Between September 2012 and November 2019, there were 2494 laboratory-confirmed cases of MERS, with 858 deaths (https://www.who.int/emergencies/merscov/en/. Accessed 4 February 2020). The MERS casefatality rate of 34.4% is about triple that of SARS, and persons in the 50-59 year age group are at highest risk for primary cases. In the short time from its emergence in December 2019 to 15 March 2020, the SARS-CoV-2 has been reported in 134 countries. At the time of writing, the situation was evolving rapidly, with over 142,000 confirmed cases reported globally (over 81,000 in China) and 3194 deaths in China (3.9% case-fatality rate) and over 2100 deaths outside of China. Of countries and continents outside of China, South Korea, Iran, and Europe (particularly Italy) have experienced a high number of COVID-19 cases (https://www.who.int/emergencies/diseases/novel-coro navirus-2019/situation-reports/. Accessed 15 March 2020). Mortality rates vary widely, and depend on the age of patients, underlying risk factors, and the denominator definitionhospitalized cases, all symptomatic cases, only moderate to severe cases, etc. In a study of adult patients (mean age 59.7 y; 40% with chronic illnesses) with SARS-CoV-2 pneumonia admitted to the intensive care unit (ICU), 61.5% died within 28 days [18]. In contrast, a study of hospitalized patients (median age 47.5 years) across Beijing showed  [8,20,22,59] 18% of cases to be severe and 73% mild, with a fatality rate of 0.9% [19]. Mortality is highest in older persons, with a median age of 59-75 years [15,17]. Treatment for all severe HCoV infections is supportive although a randomized, double-blinded, control clinical trial has been conducted on a Gilead drug Remdesivir [20] Based on one study focused on children, a total of 28 children aged from 1 month to 17 years have been reported in China. All paediatric cases with laboratory-confirmed SARS-CoV-2 infection were mild cases with no deaths reported [21]. During the first 2 months of the current outbreak, COVID-19 spread rapidly throughout China and caused varying degrees of illness with a death rate of 1.3%. Patients often presented without fever, and many did not have abnormal radiologic findings [22]. The Chinese Centers for Disease Control and Prevention team analysed more than 72,000 patient records, of which 44,672 were laboratory-confirmed cases, 16,186 suspected cases, 10,567 clinically diagnosed cases, and 889 asymptomatic cases. Of the confirmed cases, about 14% of the illnesses were severe, which included pneumonia and shortness of breath, and about 5% have the critical disease, marked by respiratory failure, septic shock, and multi-organ failure. The overall case-fatality rate was 2.3%, and of 1023 deaths included in the study, the majority were in people age 60 and older or those with underlying medical conditions http://www.cidrap.umn.edu/news-perspective/ 2020/02/more-outbreak-details-emerge-covid-19-casestop-70000 (Accessed 18 February 2020).

Specimen collection
It must be appreciated that no matter how accurate and fast laboratory testing methods are, the diagnosis of viral pneumonias such as caused by SARS-CoV-2 involves collecting the correct specimen from the patient at the right time. The endemic HCoVs have been detected from a variety of upper and lower respiratory sources including throat, nasal nasopharyngeal (NP), sputum, and bronchial fluid [12,23,24]. Wang et al have just reported that oropharyngeal (OP) swabs (n = 398) were used much more frequently than NP swabs (n = 8) in China during the COVID-19 outbreak; however, the SARS-CoV-2 RNA was detected only in 32% of OP swabs, which was significantly lower than that in NP swabs (63%) [25]. The US centers for disease control and prevention (CDC) recommends collecting the upper respiratory NP swab. Collection of an OP specimen is a lower priority, and, if collected, should be combined in the same tube as the NP swab (https://www.cdc.gov/coronavirus/ 2019-nCoV/lab/guidelines-clinical-specimens.html. Accessed 16 March 2020). Swab specimens should be placed in a universal or viral transport medium. Nasopharyngeal aspirates are also suitable specimens for the detection of HCoVs.
For the most sensitive detection of SARS-CoV, MERS-CoV, and SARS-CoV-2, the collection and testing of both upper and lower respiratory samples [sputum, bronchoalveolar lavage fluid (BAL)] is recommended [26]. However, the collection of sputum and particularly BAL via bronchoscopy increases biosafety risk to healthcare workers through the creation of aerosol droplets. Proper use of personal protective equipment (PPE) by healthcare workers is important. Bronchoscopy is a highly technical procedure requiring well-trained staff and may not be available in many parts of the world. Upper respiratory specimens are easy to collect, thereby increasing access to testing for patients with mild symptoms, and in the resource limited settings. SARS-CoV and MERS-CoV RNA are also detected from stool, urine and blood specimens, although generally less reliably than from respiratory specimens [26][27][28]]. An exception is SARS-CoV RNA which is consistently detected in feces at about two weeks after symptom onset [26,29]. For the most sensitive detection of endemic HCoVs, upper respiratory specimens should be collected within the first few days of symptom onset. The dynamics of RNA shedding in MERS and SARS patients may reflect the specimen source, severity of illness, as well as underlying risk factors. Among hospitalized patients who did not require ventilator support, MERS-CoV RNA levels in the upper respiratory tract usually peaked in the first week after symptom onset. Among eventual fatal cases requiring ventilation, RNA levels in lower respiratory tract specimens peaked between weeks 2 and 3 [27]. Similar shedding patterns were seen for SARS-CoV: RNA positive rates peaked in upper respiratory tract specimens at 7-10 days after symptom onset and then steadily declined after that, while RNA positive rates in lower respiratory tract specimens remained higher throughout 3 weeks after onset of illness [26]. In one study, diabetes was associated with prolonged MERS-CoV RNA shedding in the respiratory tract [27]. Viral pneumonias typically do not result in the production of purulent sputum. Thus, a nasopharyngeal swab/wash is usually the collection method used to obtain a specimen for testing. Nasopharyngeal specimens may miss early infection; a deeper specimen may need to be obtained by bronchoscopy. Alternatively, repeated testing can be used because over time, the likelihood of the SARS-CoV-2 being present in the nasopharynx increases. Self-collected saliva specimens were tested positive in 11 of 12 COVID-19 patients, suggesting it is a promising non-invasive specimen for diagnosis, monitoring, and infection control in SARS-CoV-2 infections [30]. At the time of writing there was little data on the performance of upper vs. lower respiratory tract specimens for the detection of SARS-CoV-2 [16]. Serum is another source for the detection of SARS-CoV-2. However, only 15% of patients hospitalized with pneumonia had detectable RNA in serum [16]. Specimens collected for laboratory testing of HCoVs should be maintained at refrigerated temperature for up to 72 h, or frozen at −70°C or below (https://www.cdc.gov/coronavirus/ 2019-nCoV/lab/guidelines-clinical-specimens.html. Accessed 15 March 2020). Rectal specimens have been reported positive in patients infected with SARS-CoV-2 [20].

Biosafety considerations
If the patient's travel or exposure history or symptoms suggest possible infection with a high-risk, novel agent, SARS-CoV, or MERS-CoV, then the initial handling of the specimen should be performed under biosafety level 3 (BSL-3) conditions until the specimen or an aliquot is rendered noninfectious by lysis or another method. Virus isolation should not be routinely performed in this situation (https://www.asm.org/ Articles/Policy/Laboratory-Response-Network-LRN-Sentinel-Level-C. Accessed 4 February 2020). The U.S. CDC biosafety guidelines state that routine diagnostic testing of specimens from suspected or confirmed SARS-CoV-2 patients, can be handled in a BSL-2 laboratory using standard precautions (https://www.cdc. gov/coronavirus/2019-nCoV/lab/lab-biosafetyguidelines.html. Accessed 21 March 2020).

Cell culture
Isolation of HCoVs in cell culture is not routinely performed for diagnostic purposes due to the lack of permissive cell lines, time to results, labour and expertise requirements, and the lack of commercial antisera for culture confirmation (Table 2). SARS-CoV and MERS-CoV and SARS-CoV-2 will grow in primary monkey cells and cell lines such as Vero and LLC-MK2, but cell culture should not be performed for suspect cases in routine diagnostic laboratories for biosafety reasons [2,6,31,32]. However, virus isolation in cell cultures is critical to obtain isolates for characterization and to support the development of vaccines and therapeutic agents.

Rapid antigen tests
Rapid antigen tests would theoretically provide the advantage of fast time to results and low-cost detection of HCoVs but are likely to suffer from poor sensitivity based on the experience with this method for influenza (Flu) viruses [33][34][35][36][37] (Table 2). In a pre-peer reviewed article, Diao et al. reported that a fluorescence immunochromatographic assay is an accurate, rapid, early and simple method for detecting nucleocapsid protein of SARS-CoV-2 in NP swab for diagnosis of COVID-19 (https://www.medrxiv.org/content/10.1101/2020.03. 07.20032524v2. Accessed 15 March 2020). The incorporation of colloidal gold-labeled immunoglobulin G (IgG) as the detection reagent is an approach that may increase the sensitivity of rapid antigen tests for respiratory viruses [38]. Monoclonal antibodies specifically against SARS-CoV-2 have been under preparation. Novel approaches to concentrate antigen, or to amplify the detection phase are needed if these methods are to have clinical utility. Sona Nanotech (Halifax, Canada) is developing a quick-response lateral-flow test to screen COVID-19 patients targeting to produce results in 5-15 min (https://sonanano.com/sona-develops-rapidscreening-test-for-coronavirus/. Accessed 15 February 2020). Timing of specimen collection, when viral titres are highest, may improve the diagnostic sensitivity of rapid antigen tests for HCoVs [39].

Serology
Serological assays are not routinely used for diagnosis of HCoV infections due to the lack of commercial reagents, let alone commercial reagents that have been vetted by clinical trials and the regulatory review process [40,41] (Table 2). Serological assays, on the other hand, are important for understanding the epidemiology of emerging HCoVs, including the burden and role of asymptomatic infections.
It has been particularly important for antibody detection in the diagnosis of cases of novel and emerging HCoVs, such as SARS-CoV and MERS-CoV [2,3]. In these situations, affected patients may not test positive for viral RNA, particularly in the early phase of the disease, but retrospectively can be shown to have developed an immune response. When SARS-CoV-2 was identified, especially when rapid antigen testing and/or molecular assays are neither available nor stable, serology can be used as a supplementary diagnostic tool. A recent study demonstrated that both IgM and IgG antibodies were detected 5 days after onset in all 39 patients infected with SARS-CoV-2 infection. The authors recommended to use serology to facilitate the diagnosis of SARS-CoV-2 infections when an NP swab specimen was collected inappropriately and the molecular assays were performed unsatisfactorily [42]. In China, six serology devices have just received urgent approval from the National Medical Products Administration (NMPA) by 12 March 2020 (Table 3). Proper specimen handling and storage are important to maintain the integrity of specimens and the performance of serologic tests.
In China, at the time of revising, eleven molecular devices from Shanghai ZJ Bio-Tech, Shanghai GeneoDx Biotech, BGI Biotech (Wuhan), MGI Tech, Da An Gene, Sansure Biotech, Shanghai BioGerm Medical Biotech Capitalbio (Chengdu), Beijing Applied Biological Technologies, Maccura Biotechnology, and Wuhan Easydiagnosis Biomedicine have received urgent approval from NMPA and their characteristics are contrasted in Table 3. Variable performance has been reported on these devices [47,58]. In their registration certificates, it was clearly indicated that the certificate was for urgent and supplemental diagnosis of pneumonia caused by SARS-CoV-2. Additional multi-centre clinical trial data are needed for extension after one year. Among them, one (MGI Tech) uses its NGS technique to detect all pathogens in a given specimen including SARS-CoV-2 and the other one (Innovita) uses its isothermal amplification followed by chip detection. The other nine devices incorporated real-time PCR technique with hydrolysis probes. After nucleic acids get extracted (separated reagents and systems), the extracts are transferred to a real-time PCR thermocycler (e.g. ABI 7500 Fast Dx Real-Time PCR Instrument) for nucleic acid amplification and detection.
Several RT-PCR protocols for detection of SARS-CoV-2 RNA have been posted by the World Health Organization at https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/technical-guidance/ laboratory-guidance. (Accessed 15 March 2020). Three of these protocols are listed below.
The US CDC developed developed a RT-PCR Diagnostic Panel for universal detection of SARS-like betacoronaviruses and specific detection of SARS-CoV-2 [20]. Three separate RT-PCR reactions target the N gene. One primer/probe set detects all betacoronaviruses, while two sets are specific for SARS-CoV-2. All 3 assays must be positive to report presumptive positive for SARS-CoV-2 (https://www.fda.gov/ media/134922/download. Accessed 15 March 2020). Specimen types included upper and lower respiratory specimens (such as NP or OP swabs, sputum, lower respiratory tract aspirates, BAL, and nasopharyngeal wash/aspirate or nasal aspirate). It The Charité algorithm (Berlin, Germany) begins with two RT-PCR assays that detect E and RdRp genes of subgenus Sarbecovirus (SARS-CoV, SARS-CoV-2, and bat-associated betacoronaviruses). Both assays must be positive to advance to the next step in the testing algorithm. The second step consists of a  [52][53][54] NAAT, monoplex, specific-HCoV High sensitivity and specificity for special species, potential quantification 1-8 h Diagnosis (detection, differentiation, and limited typing) and research [69,70] NAAT, multiplex High sensitivity and specificity, covering other pathogens, FilmArray RP EZ is CLIA-waived 1-8 h Diagnosis (detection, differentiation, and limited typing) and research [12,[55][56][57] NAAT, POCT Rapid and safe, good sensitivity and specificity, some are CLIA-waived

Future direction
All three novel coronaviruses are highly contagious. Fast, safe, simple to use diagnostic devices performed at or near the point of care (POC) (Figure 1) which have been shown to impact patient management and control of infectious disease epidemics [62], are extremely desirable in POC when biosafety facility is limited (Table 3). Several manufactures have been spending efforts to generate devices for POC testing (POCT) [63]. The ID NOW™ (previously Alere i) Influenza A & B assay (Abbott, San Diego, CA) was cleared by the US FDA for direct use on NP swabs as the first-ever Clinical Laboratory Improvement Amendments (CLIA)-waived nucleic acid-based test in January 2016 [64,65]. Similarly, the Xpert® Xpress Flu/RSV (Cepheid, Sunnyvale, CA) and cobas® Liat® Flu A/B & RSV (Roche Molecular Systems, Pleasanton, CA) assays are integrated nucleic acid extraction-independent devices that have recently received FDA clearance and CLIA-waiver for simultaneous detection and identification of FluA, FluB, and RSV in nasopharyngeal swabs [66]. The FilmArray® Respiratory EZ Panel (BioFire, Salt Lake City, UT) so far so far is the only CLIA-waived syndromic panel that covers a set of 14 respiratory viral and bacterial pathogens including classical coronavirus species [67].
Considering the increased levels of mortality and infectivity associated with three novel-coronavirus outbreaks, these random-access, safe and simple tests, which offer fast and accurate detection and identification, are likely to have an immediate impact on prompt clinical and epidemiological decisions [7,63]. Lysis buffer can be used to inactivate the infectivity of specimens so the testing can be run at POC when a biosafety cabinet is not available. Fast near-patient and POCT could help more efficiently triage of suspected cases of novel coronavirus, helping to focus limited resources on enabling appropriate use of quarantine. A handful of diagnostics developers are now striving to bring fast SARS-CoV-2 tests to market as soon as possible, with hopes of ultimately assisting with the ongoing outbreak in China. Molecular diagnostic tests for use at the are in development from Cepheid and HiberGene (Dublin, Ireland). Cepheid has some advantages in the molecular POCT space because it already has instruments placed in China. Mobidiag, meanwhile, may offer additional benefits with a multiplex test for coronavirus and Flu viruses (https://www. genomeweb.com/pcr/diagnostics-firms-rush-developrapid-point-care-tests-novel-coronavirus#.XkeA3SgzY 2x. Accessed 15 February 2020).

Disclosure statement
MJL and Y-WT are employees of Cepheid, the commercial manufacturer of the Xpert Xpress SARS-CoV-2 test. Figure 1. Evolutions in molecular testing procedures. The point-of-care test (POCT) devices incorporate nucleic acid extraction, amplification and detection together into an integrated and sealed cartridge making it simple, rapid and safe. During end-point PCR, DNA is detected or measured at the completion of PCR amplification, requiring post-PCR processing. Real-time PCR is a closed-tube system in which DNA is detected or measured during the exponential phase of amplification.