View source for COVID-19 Testing, Reporting, and Information Management in the Laboratory/Diagnostic testing of COVID-19 and other coronaviruses/Testing conducted on previous coronaviruses

===2.2 Testing conducted on previous coronaviruses===

====2.2.1 Severe acute respiratory syndrome (SARS)====
[[Severe acute respiratory syndrome]], otherwise known as SARS, arose in South China in late 2002. Caused by the SARS coronavirus (SARS-CoV) and believed to have originated from horseshoe bats<ref name="McKieScientists17">{{cite web |url=https://www.theguardian.com/world/2017/dec/10/sars-virus-bats-china-severe-acute-respiratory-syndrome |title=Scientists trace 2002 Sars virus to colony of cave-dwelling bats in China |author=McKie, R. |work=The Guardian |date=09 December 2017 |accessdate=03 April 2020}}</ref>, SARS eventually was contained in the summer of 2003. The last known infection was in April 2004, due to a laboratory accident.<ref name="NormileMounting04">{{cite journal |title=Mounting Lab Accidents Raise SARS Fears |journal=Science |author=Normile, D. |volume304 |issue=5671 |pages=659–61 |year=2004 |doi=10.1126/science.304.5671.659 |pmid=15118129}}</ref> During that time, the following sample collection and test procedures evolved from the related outbreaks (note that this is only a summary; consult the cited literature directly for full details)<ref name="NYSDHLab04">{{cite web |url=https://www.health.ny.gov/diseases/communicable/sars/sars_reporting/attachment_6_dear_doctor_lab.htm |title=Laboratory Testing for SARS |author=New York State Department of Health |publisher=State of New York |date=February 2004 |accessdate=03 April 2020}}</ref><ref name="CDCSevere04">{{cite web |url=https://www.cdc.gov/sars/guidance/f-lab/downloads/F-lab-full.pdf |format=PDF |title=Public Health Guidance for Community-Level Preparedness and Response to Severe Acute Respiratory Syndrome (SARS), Version 2 - Supplement F: Laboratory Guidance |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=21 May 2004 |accessdate=03 April 2020}}</ref><ref name="KnoblerLearning04">{{cite book |chapter=Appendix C: In the absence of SARS-CoV transmission worldwide: Guidance for surveillance, clinical and laboratory evaluation, and reporting |title=Learning from SARS: Preparing for the Next Disease Outbreak |editor=Knobler, S.; Mahmoud, A.; Lemon, S. et al. |publisher=National Academies Press |pages=292–302 |year=2004 |isbn=9780309182157 |doi=10.17226/10915}}</ref><ref name="WHO_SARSInt04">{{cite web |url=http://www.who.int/csr/resources/publications/en/SARSReferenceLab.pdf |title=WHO SARS International Reference and Verification Laboratory Network: Policy and Procedures in the Inter-Epidemic Period |author=World Health Organization |publisher=World Health Organization |date=23 January 2004 |accessdate=03 April 2020}}</ref><ref name="LiangLab04">{{cite journal |title=Laboratory Diagnosis of Four Recent Sporadic Cases of Community-acquired SARS, Guangdong Province, China |journal=Emerging Infectious Diseases |author=Liang, G.; Chen, Q.; Xu, J. et al. |volume=10 |issue=10 |pages=1774–81 |year=2004 |doi=10.3201/eid1010.040445 |pmid=15504263 |pmc=PMC3323270}}</ref>:

* Determine that the patient is indicating clinical and/or epidemiological evidence of SARS (meets case definitions). As Knobler ''et al.'' put it: "SARS-CoV testing should be considered if no alternative diagnosis is identified 72 hours after initiation of the clinical evaluation and the patient is thought to be at high risk for SARS-CoV disease (e.g., is part of a cluster of unexplained pneumonia cases)."<ref name="KnoblerLearning04" />

* Collect multiple specimen types at different time points of the patient's illness. Respiratory and plasma or serum specimens should be collected early into the first week of illness. Respiratory samples should be from [[Nasopharyngeal swab|nasopharyngeal aspirates and swabs]] of the upper respiratory tract or, in some cases, fluids from the lower respiratory tract using [[bronchoalveolar lavage]], tracheal aspiration, or a pleural tap. (Sputum can also be collected.) Whole blood (5 to 10 ml) is collected into either a serum separator tube for blood serum or EDTA tube for blood plasma. Stool samples are also of import early on for virus isolation or detection and are useful in at least the first and second weeks of the illness. Blood serum is useful in weeks two and three for detecting a rising titre. Additionally, the literature also makes reference to methods of collecting specimens ''postmortem''.

* Conduct testing. At the time, the two primary test types used were enzyme immunoassay (EIA; today more commonly known as [[ELISA]]<ref name="LequinEnzyme05">{{cite journal |title=Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA) |journal=Clinical Chemistry |author=Lequin, R.M. |volume=51 |issue=12 |pages=2415–18 |year=2005 |doi=10.1373/clinchem.2005.051532 |pmid=16179424}}</ref>) for detection of serum antibody and reverse transcription polymerase chain reaction (RT-PCR) for detection of the virus' RNA. The U.S. [[Centers for Disease Control and Prevention]] (CDC) had this to say about these tests in May 2004<ref name="CDCSevere04" />: 

<blockquote>Both the EIA and the RT-PCR tests are sensitive and highly specific for SARS-CoV. The ability to diagnose SARS-CoV infection in a patient is often limited, however, by either the low concentration of virus in most clinical specimens (RT-PCR assays) or the time it takes a person to mount a measurable antibody response to SARS-CoV (serologic assays). The likelihood of detecting infection is increased if multiple specimens (e.g., stool, serum, respiratory tract specimens) are collected at several times during the course of illness.</blockquote>

:The literature also makes reference to an [[Immunofluorescence|immunofluorescence assay]] (IFA) for detecting antibody, with the CDC calling its results "essentially identical to those for the EIA for SARS antibody."<ref name="CDCSevere04" /> Tangentially, isolation of SARS-CoV in cell culture from a clinical specimen is also referenced, though such activity is reserved for [[Biosafety level|Biosafety Level 3]] (BSL-3) laboratories.

* Confirm the results. Laboratory confirmation is based on one of 1. initial local lab detection and subsequent national reference lab confirmation of a validated serology-based test detection; 2. isolation of SARS-CoV in cell culture with subsequent confirmation from a validated test; or 3. initial local lab detection and subsequent national reference lab confirmation of SARS-CoV RNA from a validated RT-PCR test which used either two clinical specimens from different sources or two same-source clinical specimens from two different days. 

:Additionally, in the case of serology, one of the following must be true: 
# SARS-CoV serum antibodies are detected in a single serum specimen; or,
# a "four-fold or greater increase in SARS-CoV antibody titer between acute- and convalescent-phase serum specimens tested in parallel"<ref name="CDCSevere04" /> is detected; or,
# a "negative SARS-CoV antibody test result on acute-phase serum and positive SARS-CoV antibody test result on convalescent-phase serum tested in parallel"<ref name="CDCSevere04" /> is detected.

:Of note is the [[World Health Organization]]'s (WHO) January 2004 cautionary message about serological diagnostics in not only SARS-CoV but other types of coronaviruses. At that time, they showed a level of unsurety in regards to how coronaviruses elicited serological cross-reactions and generated antigenic recall. They also preached caution in interpreting serological results in non-epidemic periods and when no viral sequence data are available. Finally, they also mentioned the added difficulties of rate cases when coinfection with a related human coronavirus occurs, "although the use of expressed proteins in Western blots may help to sort this out."<ref name="WHO_SARSInt04" /> More than 15 years later, Loeffelholz and Tang put this concept into clearer terms, indicating that while "serological assays are not routinely used for diagnosis of &#91;human coronavirus&#93; infections due to the lack of commercial reagents," they still have important value "for understanding the epidemiology of emerging &#91;human cornaviruses&#93;, including the burden and role of asymptomatic infections," as well as for antibody detection of novel and emerging coronaviruses.<ref name="LoeffelholzLab20">{{cite journal |title=Laboratory diagnosis of emerging human coronavirus infections – The state of the art |journal=Emerging Microbes & Infections |author=Loeffelholz, M.J.; Tang, T.-W. |volume=9 |issue=1 |pages=747–56 |year=2020 |doi=10.1080/22221751.2020.1745095 |pmid=32196430}}</ref>

* Arrange for confirmatory testing to be performed by an appropriate test site in the case of a positive RT-PCR test.

* Report to state or local health departments details of patients radiographically confirmed with pneumonia with at least one SARS-CoV risk factor for exposure, clusters of healthcare workers with unexplained pneumonia, and any positive SARS-CoV test results. Additional international reporting of SARS by WHO Member States in regards to probable and laboratory-confirmed cases is also requested.

* Send off for an additional verification by an external member of the WHO's SARS Reference and Verification Laboratory Network before internationally announcing results as a laboratory-confirmed case.

====2.2.2 Middle East respiratory syndrome (MERS)====
[[File:MERS-CoV Particles (8414774264).jpg|left|thumb|Colorized transmission electron micrograph showing particles of the Middle East respiratory syndrome coronavirus that emerged in 2012]]Unlike SARS, Middle East respiratory syndrome, or MERS, continues to appear in the human population. Since its appearance in 2012, several thousand laboratory-confirmed cases of MERS have been reported to the WHO.<ref name="Bernard-StoecklinCompar19">{{cite journal |title=Comparative Analysis of Eleven Healthcare-Associated Outbreaks of Middle East Respiratory Syndrome Coronavirus (Mers-Cov) from 2015 to 2017 |journal=Scientific Reports |author=Bernard-Stoecklin, S.; Nikolay, B.; Assiri, A. et al. |volume=9 |at=7385 |year=2019 |doi=10.1038/s41598-019-43586-9 |pmid=31089148 |pmc=PMC6517387}}</ref> The virus MERS-CoV is believed to have originated from bats, which at some unknown point spread to Dromedary camels. Approximately 55 percent of MERS-CoV infections have come from direct contact with such camels, though it's not entirely clear how the rest of known cases have been caused<ref name="BanerjeeBats19">{{cite journal |title=Bats and Coronaviruses |journal=Viruses |author=Banerjee, A.; Kulcsar, K.; Misra, V. et al. |volume=11 |issue=1 |at=E41 |year=2019 |doi=10.3390/v11010041 |pmid=30634396 |pmc=PMC6356540}}</ref> (Alshukairi ''et al.'' suggest asymptomatic or mildly symptomatic camel workers may serve as a possible transmission source<ref name="AlshukairiHigh18">{{cite journal |title=High Prevalence of MERS-CoV Infection in Camel Workers in Saudi Arabia |journal=mBio |author=Alshukairi, A.N.; Zheng, J.; Zhao, J. et al. |volume=9 |issue=5 |pages=e01985-18 |year=2018 |doi=10.1128/mBio.01985-18 |pmid=30377284 |pmc=PMC6212820}}</ref>). The following sample collection and test procedures have evolved from working with the MERS-CoV virus (note that this is only a summary; consult the cited literature directly for full details)<ref name="CDCLabTestMERS19">{{cite web |url=https://www.cdc.gov/coronavirus/mers/lab/lab-testing.html |title=CDC Laboratory Testing for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=02 August 2019 |accessdate=04 April 2020}}</ref><ref name="CDCInterimMERS19">{{cite web |url=https://www.cdc.gov/coronavirus/mers/guidelines-clinical-specimens.html |title=Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons Under Investigation (PUIs) for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) – Version 2.1 |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |date=02 August 2019 |accessdate=04 April 2020}}</ref><ref name="WHOLabMERS18">{{cite web |url=https://www.who.int/csr/disease/coronavirus_infections/mers-laboratory-testing/en/ |title=Laboratory testing for Middle East Respiratory Syndrome Coronavirus: Interim guidance |work=WHO/MERS/LAB/15.1/Rev1/2018 |author=World Health Organization |publisher=World Health Organization |date=January 2018 |accessdate=04 April 2020}}</ref><ref name="Al-AbdelyMiddle19">{{cite journal |title=Middle East Respiratory Syndrome Coronavirus Infection Dynamics and Antibody Responses among Clinically Diverse Patients, Saudi Arabia |journal=Emerging Infectious Diseases |author=Al-Abdely, H.M.; Midgley, C.M.; Alkhamis, A.M. et al. |volume=25 |issue=4 |pages=753-766 |year=2019 |doi=10.3201/eid2504.181595}}</ref><ref name="Kelly-CirinoAnUpdated19">{{cite journal |title=An updated roadmap for MERS-CoV research and product development: focus on diagnostics |journal=BMJ Global Health |author=Kelly-Cirino, C.; Mazzola, L.T.; Chua, A. et al. |volume=4 |issue=Suppl. 2 |at=e001105 |year=2019 |doi=10.1136/bmjgh-2018-001105 |pmid=30815285 |pmc=PMC6361340}}</ref>:

* Determine that the patient is indicating clinical and/or epidemiological evidence of MERS (meets case definitions). "Testing for other respiratory pathogens using routinely available laboratory procedures, as recommended in local management guidelines for community-acquired pneumonia, should also be performed but should not delay testing for MERS-CoV."<ref name="WHOLabMERS18" />

* Collect at a minimum both lower respiratory and upper respiratory tract samples. Lower respiratory tract specimens are typically the most revealing, as they have been shown to contain the highest viral load (due to the expression of the virus's cellular receptor DPP4 in the lower respiratory system). Bronchoalveolar lavage, tracheal aspiration, or a pleural tap can be used to collect specimens from the lower respiratory tract. (Sputum can also be collected.) Upper respiratory tract specimens (in this case, both a nasopharyngeal and an oropharyngeal swab are recommended) are also valuable in diagnosis, though extra care should be taken to ensure nasopharyngeal swabs gather secretions from the [[Pharynx#Nasopharynx|nasopharynx]] and not just the nostril. Nasopharyngeal aspiration is also an acceptable sample collection method for the upper respiratory tract.

:Regarding serum specimens, slight differences in guidance appear between WHO guidance and CDC guidance. The WHO appears to differentiate between symptomatic and asymptomatic patient testing, whereas the U.S. CDC seems to only indirectly differentiate the two. The WHO suggests if testing symptomatic patients, stick with lower and upper respiratory tract specimens, which will be tested using nucleic acid amplification (molecular) testing (NAAT). Serological testing of serum specimens should be used for symptomatic patients "only if NAAT is not available."<ref name="WHOLabMERS18" /> If this is the case, the WHO recommends paired samples, one collected within the first week of illness and the second about three to four weeks later. For asymptomatic patients in high-contact outbreak scenarios, the WHO recommends all three sample types (with respiratory samples taken preferably within 14 days of last documented contact). 

:The current CDC guidance differentiates between molecular testing for active infections and serology for previous infections. The CDC adds that "MERS-CoV serology tests are for surveillance or investigational purposes and not for diagnostic purposes."<ref name="CDCLabTestMERS19" /> Whether or not to collect a serum specimen in MERS diagnostics may depend on the assay used, however. For example, the CDC, in its Version 2.1 guidance, indicates that testing using the CDC MERS rRT-PCR assay requires collection of serum in addition to upper and lower respiratory tract specimens. For that specific assay, the CDC differentiates between patients who've had symptom onset less than 14 days prior and those who've had it 14 days or later: if prior, serology is for the rRT-PCR test, and if later, serology is for antibody testing. In either case, 200 µL of serum is required.

* Conduct testing. NAAT methods like real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays have been the most common tool for diagnosing MERS-CoV infection due to their high sensitivity. According to late 2018 research by Kelly-Cirino ''et al.'', at least 11 commercial single assay and five commercial multiplex assay kits are available (see [https://gh.bmj.com/content/bmjgh/4/Suppl_2/e001105/DC1/embed/inline-supplementary-material-1.pdf Table S1], a PDF file, from their highly relevant paper), perhaps more as of April 2020. Serological antibody detection is performed using ELISA, [[Immunofluorescence#Secondary (indirect)|indirect immunofluorescence]] (IIF), and [[Neutralisation (immunology)|microneutralization]].

* Confirm the results. Laboratory confirmation of MERS-CoV infection is the same for both the WHO and the CDC: one of either a validated NAAT test providing a positive result for at least two different genomic targets, or a validated NAAT test providing a positive result for a specific genomic target along with sequencing confirmation of a separate genomic target. Persons under investigation who receive one negative NAAT result on a recommended specimen are considered to be negative for active MERS-CoV infection. The laboratory should consider testing additional specimens after the first negative. The CDC considers known MERS patients to be negative for active MERS-CoV infection after two consecutive negative NAAT tests on all specimens. The WHO adds: "A patient with a positive NAAT result for a single specific target without further testing but with a history of potential exposure and consistent clinical signs is considered a probable case."<ref name="WHOLabMERS18" /> The WHO also has additional guidance on using serology for confirming MERS-CoV infection for purposes of reporting under the International Health Regulations.

* Report using national reporting requirements. More broadly, state or local health departments should receive details about received specimens to be tested for MERS-CoV, even before testing begins. Regardless of result, the final positive or negative laboratory confirmation should also be reported to national authorities. If the infection becomes widespread, updates for each new confirmed case or suspected positive should also be made.

====2.2.3 The common cold====
Approximately 10 to 15 percent of cases of what we call the "common cold" are associated with an endemic coronavirus, of which are two distinct groups: HCoV-229E and HCoV-OC43.<ref name="WatTheCommon04">{{cite journal |title=The common cold: A review of the literature |journal=European Journal of Internal Medicine |author=Wat, D. |volume=15 |issue=2 |pages=79–88 |year=2004 |doi=10.1016/j.ejim.2004.01.006 |pmid=15172021}}</ref> Disease symptoms associated with these coronaviruses—typically in the form of respiratory infection and the symptoms that come with it—by themselves are typically mild<ref name="LoeffelholzLab20" />, and laboratory testing isn't necessarily indicated for those immunocompetent individuals capable of self-limiting.<ref name="BabadyMulti18">{{cite journal |title=Multicenter Evaluation of the ePlex Respiratory Pathogen Panel for the Detection of Viral and Bacterial Respiratory Tract Pathogens in Nasopharyngeal Swabs |journal=Journal of Clinical Microbiology |author=Babady, N.E.; England, M.R.; Jurcic Smith, K.L. et al. |volume=56 |issue=2 |at=e01658-17 |year=2018 |doi=10.1128/JCM.01658-17 |pmid=29212701 |pmc=PMC5786739}}</ref> However, symptom overlap with pharyngitis and bronchitis, as well as the complication of pharyngitis and sinusitis also potentially having bacterial origin, can complicate clinical diagnosis. Additionally, as more antivirals that target a specific virus are created, and as concerns of unnecessarily using antibiotics to treat viral diseases grows<ref name="JenisonRapid16">{{cite web |url=https://www.statnews.com/2016/11/30/antibiotic-resistance-molecular-diagnostics/ |title=Rapid lab tests can help reduce antibiotic resistance |author=Jenison, R. |work=STAT |date=30 November 2016 |accessdate=03 April 2020}}</ref><ref name="RoyRapid18">{{cite web |url=https://www.healio.com/news/infectious-disease/20180926/rapid-test-for-viral-infections-reduces-unnecessary-antibiotic-prescribing |title=Rapid test for viral infections reduces unnecessary antibiotic prescribing |author=Roy, K. |work=Healio |date=26 September 2018 |accessdate=06 September 2021}}</ref>, laboratory methods of respiratory virus diagnosis—particularly for those who are immunocompromised—have value.<ref name="WatTheCommon04" /><ref name="BabadyMulti18" /> 

RT-PCR, a molecular method, has been used for well over a decade for detecting coronaviruses.<ref name="WatTheCommon04" /><ref name="MahonyDetect08">{{cite journal |title=Detection of Respiratory Viruses by Molecular Methods |journal=Clinical Microbiology Reviews |author=Mahoney, J.B. |volume=21 |issue=4 |pages=716–47 |year=2008 |doi=10.1128/CMR.00037-07 |pmid=18854489 |pmc=PMC2570148}}</ref> However, as molecular methods of analysis have expanded over the years, more rapid solutions for testing have been developed. For example, the GenMark ePlex rapid multiplex molecular diagnostics instrument and the ePlex Respiratory Pathogen Panel were evaluated in a multicenter trial by Babady ''et al.'' in 2017.<ref name="BabadyMulti18" /> The panel is capable of testing for the presence of 15 viral types—including the -229E, -OC43, and two other coronaviruses—and two bacterial types in nasopharyngeal swab specimens, with results in typically less than two hours.<ref name="BabadyMulti18" /> The cost associated with these sorts of tests, compared to their benefits, likely limits ubiquitous use at the first sign of a cold<ref name="BabadyMulti18" />, but as molecular diagnostic technologies become more compact and easy-to-use, testing for infection by endemic human coronaviruses may become slightly more commonplace. However, as the authors point out, with no treatment for these endemic coronaviruses, any additional utility beyond diagnosing an illness as viral rather than bacterial would primarily be found in epidemiological studies of the associated genotyping data.<ref name="BabadyMulti18" />