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 Table of Contents  
REVIEW ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 5  |  Page : 24-30

Laboratory testing in human coronaviruses


Department of Medicine, MGM Medical College and LSK Hospital, Kishanganj, Bihar, India

Date of Submission23-Apr-2020
Date of Decision30-Apr-2020
Date of Acceptance02-May-2020
Date of Web Publication04-Jun-2020

Correspondence Address:
Dr. Ashis Kumar Saha
MGM Medical College and LSK Hospital, Kishanganj, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_57_20

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  Abstract 


After 2002–2003 severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, 2012–2013 Middle East respiratory syndrome coronavirus epidemic, ultimately COVID-19 pandemic started in December 2019 and still is going on steadily affecting millions and millions of people taking the precious life of more than one and half millions of people throughout the world, the highest affected country being the United States followed by Italy, France, and so on. Remembering the methods of spreading of infection by droplets and aerosol, high infectivity of this organism, at the same time near absence of biosafety in highly populated areas in the world and the presence of large number of asymptomatic or very minimally symptomatic patients various methods of detection approved by the Food and Drug Administration or different national health organizations, such as rapid antigen test and reverse transcriptase polymerase chain reaction methods, should be started for detection of an infected person for treatment.

Keywords: Coronavirus laboratory rests, different methods, review


How to cite this article:
Saha AK. Laboratory testing in human coronaviruses. Int J Health Allied Sci 2020;9, Suppl S1:24-30

How to cite this URL:
Saha AK. Laboratory testing in human coronaviruses. Int J Health Allied Sci [serial online] 2020 [cited 2020 Nov 26];9, Suppl S1:24-30. Available from: https://www.ijhas.in/text.asp?2020/9/5/24/285961




  Coronavirus and Epidemiology Top


In the order Nidovirales, there are three groups, namely Coronaviridae, Arteriviridae, and Roniviridae. Coronaviridae is subdivided into two subfamilies, such as Coronavirinae and Torovirinae. Coronavirinae is again subdivided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In 1960, a virus was found from tracheal organ culture from a human being admitted in the Common Cold Unit in Salisbury in Wiltshire due to respiratory tract infection by Tyrrell and Bynoe, and it was named as B814. This was published in the British Medical Journal in 1965. The sample was sent to a virologist, June Almeida, for examination. She found the particle like influenza virus but not exactly the same, but it was similar that she found in mouse hepatitis. She sent an article on this particle before the previously published paper but was rejected in peer review. But after watching the electron microscopic picture of B814 and matching it with that of Mrs. Almeida, they convinced with the sent article of Mrs. Almeida and published the paper of Mrs. June Almeida. Name of that virus was given as “coronavirus” as it looked like “crown.” Later on, the strain of these coronaviruses was isolated from upper respiratory tract infection of human beings and named as HCoV-229E and HCoV-OC43.[1],[2] In 2002–2003 epidemic outbreak of severe upper respiratory tract infection in Guangdong Province in China, two new strain of viruses emerged out – these are HCoV-NL63 and HCoV-HKU1.[3],[4] It was originated in bats but later on from civets, raccoon, and dogs to human beings.[5] HCoV-229E and HCoV-NL63 are alphacoronaviruses and HCoV-OC43 and HCoV-HKU1 are lineage A betacoronaviruses (subgenus Embecovirus). In 2012–2013, two (subgenus Merbecovirus) betacoronaviruses, HCoV-HKU4 and HCoV-HKU5, were responsible for Middle East respiratory syndrome (MERS) epidemic outbreak spread from camel, as in some studies, it was shown that viruses isolated from camels and human being were the same.[6],[7],[8] In 2019, the last part of December, severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic started in Wuhan from persons involved in wet animal market, so based on genetic relation with other coronaviruses, it can be assumed that this virus also started from zoonotic reservoir, but real source from where it has creeped into human being is yet to be confirmed. However, due to the similarity of ACE2 of the animals and pets with that in human being, it can be assumed that they may play some role, but we have to wait for that day when all the truth will be open secret.[9] Endemic human coronaviruses, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, usually like the winter season, are distributed throughout the world and usually spread from human being to human being, but HCoV-229E is found sporadically throughout the year.[10] SARS-CoV became pandemic in 2003, but it ended in less than a year after the onset of source case. On the other hand, MERS-HCoV is still being found for the last 7 years after the onset of source case in Saudi Arabia, and animals are the reservoir of this virus. Both the epidemics of SARS and MERS were due to super-spreading event where human beings directly spread to a large number of contacts by nosocomial route. SARS-CoV-2 (COVID-19) spreads through:

  1. Nosocomial route through respiratory droplet
  2. Aerosol droplet
  3. Person-to-person close contact
  4. Fecal-oral route: As SERS-CoV and MERS-CoV produce diarrhea and SERS-CoV RNA was found in stool. Two percent of patients infected with SARS-CoV-2 suffered from abdominal pain, loose stool, as well as vomiting.[11] Again, as per the report of the USA, one patient was found positive for the virus in stool.[12]


The incubation period of COVID-19 is 5.2 days, with the 95th percentile of distribution of incubation period at 12.5 days (95% confidence interval, 4.1–7 days).[13] According to another study, it is up to 14 days (2–14 days).[14]


  Clinical Features Top


At the onset of illness, the symptoms are fever, headache, cough, after a week patient may start with respiratory distress, features of atypical pneumonia, and positive chest X-ray.[9],[15],[16]

Laboratory diagnosis

Methods

Data have been collected searching from the literature in the Internet and different updating materials and news given in the Internet.

Collection of specimens

The main aim is to collect the correct specimen at the right time.

Sites of collection:[10],[17],[18]

  1. Upper respiratory tract:


    • Nasopharyngeal swab
    • Oropharyngeal swab
    • Nasopharyngeal aspirate.


    In China, oropharyngeal swab was taken more frequently than nasopharyngeal swab, but positivity was more in case of later, but positivity was much more in case of the former method (63%) as compared to later method (32%).[19] The United States also recommended the nasopharyngeal swab than oropharyngeal swab. Hence, if oropharyngeal swab is collected, it should be always with nasopharyngeal swab.[20] In both the above cases, flocked nasopharyngeal or oropharyngeal swab is used. Both the specimens should be collected within few days of the onset of illness.

  2. Lower respiratory tract:


    • Sputum: Since in viral pneumonia sputum will not be purulent, so patients should be instructed to cough forcefully till liquid from trachea will come
    • Bronchoalveolar lavage fluid: It should be collected with proper way using personal protective equipment (PPE) by technically sound staff.


    Dynamicity of detection of RNA may confirm the severity of illness. In MERS-CoV illness in case of nonventilated patients, virus was at its peak in the 1st week of the onset of illness, whereas, in case ventilated critically ill patients, it was at the 2nd and 3rd weeks of illness.[21] A similar pattern was observed in case of SARS-CoV infection. In case of SARS-CoV-positive upper respiratory tract infection, viral shedding peaked in the 1st week, whereas in case of lower respiratory tract infection, viral shedding persisted up to the 3rd week from the onset of illness.[22] In comorbid illness, like diabetes, the shedding of virus persisted in MERS-CoV-positive patients for long time.[21]

  3. Blood: If there is difficulty in collection of specimens from the respiratory tract, mainly in case of children, blood should be collected within 3–4 days up to 1 week of the onset of illness in acute phase for reverse transcriptase polymerase chain reaction (RT-PCR) and in convalescent phase for seroconversion. If the test will be delayed, the serum can be stored at 4°C, and if the test will be delayed by >72 h, the serum should be kept frozen at −70°C and transported in an ice pack.[20] However, in case of COVID-19 infection, only 15% of hospitalized patients demonstrated detectable RNA in serum[15]


  4. Rectal swab: In COVID-19 cases, 2% of patients demonstrated a positive reaction in RT-PCR in the US.[12] However, in few exceptional cases, RNA can be detected in stool for about 2 weeks after the onset of illness


  5. Self-collected saliva: In one study, 11 out of 12 patients of SARS-CoV-2 demonstrated positive for viral RT-PCR – it suggested a promising specimen collection method for diagnosis and monitoring this disease.[23]


During collection of respiratory specimens, the following precautions should be undertaken:

  1. It should be taken with minimum health-care staff
  2. Every health-care staff should wear the following:
    • N95 mask
    • Gloves
    • Long-sleeved gown
    • Facial shield.
  3. At the end of the procedure, PPE should be removed followed by hand hygiene
  4. This procedure should be done in the following area:
    • In case of aerosol-producing procedure negative pressure single room is essential.
    • Or, in isolated ventilation room with changing of air in 6–8 cycles/hour during the procedure
    • In some cases, forced ventilation duct system is must.
  5. After completion of the procedure, room must be disinfected followed by vacation of that room for sufficient period for ventilation
  6. During the procedure, nobody must enter or exit the operating room.


During collection of nonrespiratory specimen, the following precautions should be undertaken:

  1. N95 mask
  2. Gloves
  3. Face shield
  4. Long-sleeved gown
  5. Goggles.


Biosafety level in laboratory diagnosis

  1. Biosafety level 3 should be undertaken in the following conditions


    • History travel
    • Exposure to symptomatic patient suggestive of novel coronavirus infection.


    This level should be undertaken till the lysis of specimens.

  2. Biosafety level 2 should be undertaken in the following conditions:


    • In case of routine diagnostic testing of the specimens from the patients with suspected or confirmed SARS-CoV-infected patients.[24]


Cell culture

Only in case of confirmed cases, cell culture for SARS-CoV, MERS-CoV, and SARS-CoV-2 viruses can be performed in the following cell lines:[3],[25],[26],[27]

  1. Primary monkey cell
  2. Vero cell lines
  3. LLc-MK2 cells.


It should not be done in routine laboratories:

  1. For biosafety reasons
  2. Lack of permissive cell lines
  3. Lack of expertise
  4. Long time to get result
  5. Lack of commercial antisera.


Rapid antigen detection test

  1. This test though rapid and of low cost but less sensitive that experienced previously from the test against influenza.[22],[28] Another method is fluorescence immunochromatographic assay which is also a rapid and simple method of detecting nucleocapsid (N) protein of COVID-19[29]
  2. Sensitivity can be increased by incorporation of colloidal gold-labeled immunoglobulin G for detection of antigen of COVID-19[30]
  3. Monoclonal antibody is under preparation in detection of antigen test, but here, antigen should be concentrated and detection phase should be amplified
  4. Canada is preparing one quick response lateral flow test to detect COVID-19 rapidly in 15 min.[31]


However, it should be remembered that sensitivity of test will be positive when viral load will be highest.[32]

Serology

Serology is important for the following reasons:

  1. Detection of antibody in the diagnosis of COVID-19
  2. Understanding the epidemiology of COVID-19
  3. Role of asymptomatic infection in the community.


In early phase of infection, this test will not be positive as antibodies will appear after 5–7 days after the onset of infection. According to one study, antibodies will appear after 5 days of infection. Hence, it is best to use this test after 5 days when swab cannot be collected properly. From China, National Medical Products Administration-approved six serology devices will be sent to different parts of the world in the 1st week of April 2020.

Polymerase chain reaction by reverse transcriptase method

This method has a critical role in the diagnosis of SARS-CoV, MERS-CoV, and COVID-19. SARS-CoV is genetically heterogeneous, so the diagnosis requires pan-CoV assay. Hence, pan-CoV contains different sets, such as:

  1. Multiple primer sets[33]
  2. Single set of nondegenerate primers[34]
  3. Degenerate primers.[35]


Respiratory panels included nucleotides of the following endemic HCoV, such as:[10],[36],[37]

  1. HCoV-E229
  2. HCoV-NL63
  3. HCoV-OC43
  4. HCoV-HKU1
  5. Recently, SARS-CoV-2 has been added to this respiratory panel.[38]


Recently, China has received approval of eleven molecular devices from NMPA. These are given in the [Table 1].
Table 1: Different methods of laboratory diagnosis of SARS-CoV-2 infections

Click here to view


In the certificate, it has been mentioned clearly that this certificate has been given for supplemental diagnosis of pneumonia due to COVID-19. Hence, additional multicentered trial are very urgent if the company requires extension more than a year. Here, nine devices use real-time PCR method with the hydrolysis probe: one uses NGS technique which can detect all the pathogens and the other one uses isothermal amplification followed by detection of chip. In RT-PCR method, nucleic acid will be extracted by hydrolysis probe, the extracted material will be transferred to PCR thermocycler where nucleic acid will be amplified and detected. Now, several protocols have been posted by the WHO at its technical guidance/laboratory guidance site.[39] The United States Centers for Disease Control has developed some protocol (which received emergency use authorization on February 4) for:

  1. Universal diagnosis of SARS-CoV
  2. Specific detection of SARS-CoV1 or COVID-19.


Three separate sets of reactions on RT-PCR targeting nucleocapsid (N) protein antigen:

  1. One for detection of all coronaviruses
  2. Two sets for specific detection of COVID-19.


Specimens are as follows: nasopharyngeal swab, oropharyngeal swab, sputum, aspirates from the lower respiratory tract, and bronchoalveolar lavage fluid.

It also received grant from the US Food and Drug Administration (US FDA) for 13in vitro assay.[40] These are the following:

  1. Above CDC assay
  2. New York SARS-CoV-2 Real-Time PCR Diagnostic Panel (WadsWorth Center, New York State Department of Health)
  3. TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Inc.)
  4. Cobas SARS-CoV-2 (Roche Molecular Systems, Inc.)
  5. Panther Fusion SARS-CoV-2 (Hologic Inc.)
  6. COVID-19 RT-PCR test (Laboratory Corporation of America)
  7. Lyra SARS-CoV-2 assay (Quidel Corporation)
  8. Quest SARS-CoV-2 RT-PCR (Quest Diagnostics Infectious Disease Inc.)
  9. Abbott real-time SARS-CoV-2 assay (Abbott Molecular)
  10. Simplexa COVID-19 Direct (DiaSorin Molecular LLC)
  11. ePlex SARS-CoV-2 test (GenMark Diagnostics Inc.)
  12. Primerdesign Ltd COVID-19 genesig Real-Time PCR assay (Primerdesign Ltd.)
  13. Xpert Xpress SARS-CoV-2 test (Cepheid).


Two RT-PCR has been started by Charite algorithm (Berlin, Germany) in the first step which can detect two genes of subgenus Sarbecovirus, i.e., SARS-CoV and SARS-CoV-2. The genes are as follows:

  1. Envelope € protein
  2. RNA-dependent RNA polymerase.


Both the above tests must be positive to enter into the next step of algorithm.

In the second step, gene target is RNA-dependent RNA polymerase of SARS-CoV-2.

In this test, the following viruses cannot be detected:

  1. Alphacoronaviruses: HCoV-229E and HCoV-NL63
  2. Betacoronaviruses: HCoV-OC43 and HCoVHKU1
  3. MERS-CoV: In this virus, the following genes are the target by RT-PCR:[41]


  • upF
  • ORF1a
  • ORF1b
  • Nucleocapsid (N) protein.


Two assays are followed by Li Ka Shing Faculty of Medicine, University of Hong Kong, to detect the SARS-CoV as SARS-CoV is not in the circulation of human being currently, so automatically SARS-CoV positivity means the patient is SARS-CoV-2.[22],[42] The genes are as follows:

  1. Nucleocapsid (N) gene for screening
  2. ORF1b gene for confirmation of SARS-CoV-2.[43]


In case of MERS-CoV, the following should be the criteria:

  1. A positive result for at least two specific genes in RT-PCR
  2. At least one specific gene positive in RT-PCR which should be followed by confirmation by neutralization test.



  What Is the Future? Top


Since coronavirus is very contagious, so, in the absence of very strict biosafety facility, very simple-to-use diagnostic devices should be used at the point of care.[44] Manufacturers generated few devices for use.

  1. In 2016, for detection of influenza virus, the US FDA accepted the use nasopharyngeal swab as first Chemiluminescence immunoassay-waived nucleic acid-based test[45],[46]
  2. Integrated nucleic acid extraction-independent devices by Xpert* Xpress Flu/RSV and cobas* Liat* Flu A/B and RSV (Roche Molecular Systems, Pleasanton, CA, USA) recently have been approved by FDA and CLIA waiver which can detect and identify influenza A and B and respiratory syncytial virus in nasopharyngeal swab[47]
  3. Recently, FilmArray* respiratory EZ containing 14 respiratory viruses including classical coronaviruses and bacterial pathogens testing by CLIA has been approved.[48]



  Conclusion Top


SARS-CoV, MERS-CoV, and SARS-CoV-2 or COVID-19 are responsible for huge epidemic and the last one is pandemic involving the whole world. Considering its epidemiology, methods of spreading its infectivity through air droplet, aerosols, stool, and at the same time the density of population in different parts of the world including India, fast and accurate diagnosis is urgently needed at point-of-care area in spite of minimal amount of biosafety. For that reason, there should be an arrangement of lysis buffer to inactivate the material before the chance of spreading.

Recommendation

Point-of-care technology helps in rapid detection of the patient, so that they should be sent to quarantine area first to prevent spread to the community and second monitoring the patients to watch any feature of the disease or not.

  1. Most important of any infective communicable disease is the percentage of asymptomatic or negligible symptomatic patients because they are mostly responsible for spreading of the disease in the community. Hence, in COVID-19, most important is to do RT-PCR test the pooled nasopharyngeal swab from at least 10 persons. If it is positive, then every person in that pooled group must be tested individually by RT-PCR method. Before testing, the whole zone must be blocked so that no new person can enter that area and no one can go out. The next day, area beside the tested zone must be selected
  2. The next alternative method may be testing the households of a positive patient and all the residences of that area and office colleagues by the method of pooled RT-PCR test. In that case, travel history should be important
  3. In this pandemic of COVID-19, China sent the rapid test kit, but it must be validated before use so that the result should be accurate because positive result will mark the person as positive and all the households and office colleagues have to enter quarantine area for isolation.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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