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Table of Contents
REVIEW ARTICLE
Year : 2020  |  Volume : 17  |  Issue : 3  |  Page : 139-143

Laboratory diagnosis of COVID-19 infection


Department of Microbiology, Apollo Hospitals, jubilee Hills, Hyderabad, Telangana, India

Date of Submission20-Aug-2020
Date of Acceptance29-Aug-2020
Date of Web Publication16-Sep-2020

Correspondence Address:
M S Ratnamani
Apollo Hospitals, Jubilee Hills, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/am.am_105_20

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  Abstract 


Rapid diagnostic tests are required for the early diagnosis of COVID-19 infection. Rapid antigen detection tests are card-based, and the reports are available within 30 min. Sample type is nasopharyngeal swab. Rapid antigen tests help in triaging the patients, early identification, isolation, and contact tracing. Antigen test has high specificity and moderate sensitivity. Symptomatic patients and those with high pretest probability, but are antigen test negative, need to go for real-time polymerase chain reaction testing. Rapid antigen tests aid in the early decision-making and reduce the burden on molecular diagnostics. Serological tests for COVID-19 involve the detection of antibodies in blood. Immunoglobulin G (IgG) antibodies to SARS CoV-2 indicates past infection or past exposure. It should not be used for the diagnostic purpose. IgG, IgM, and total antibodies detection is available on different platforms. IgG antibody testing is useful for seroprevalence, plasma donation, and as epidemiological tool. ICMR has given advisory and algorithm for both antigen and antibody testing.

Keywords: Antibodies, rapid antigen test, specificity and sensitivity


How to cite this article:
Ratnamani M S. Laboratory diagnosis of COVID-19 infection. Apollo Med 2020;17:139-43

How to cite this URL:
Ratnamani M S. Laboratory diagnosis of COVID-19 infection. Apollo Med [serial online] 2020 [cited 2020 Oct 31];17:139-43. Available from: https://www.apollomedicine.org/text.asp?2020/17/3/139/295208




  Introduction Top


COVID-19 pandemic has changed the world of diagnostics. The necessity of rapid diagnostics with good sensitivity and specificity led to the development of new tests for diagnosing this infection.

Real-time polymerase chain reaction (RT-PCR) and nucleic acid amplification testing are molecular-based diagnostic tests. RT-PCR remains the gold standard for COVID-19 diagnosis.

In addition to the molecular techniques, rapid antigen detection tests have recently been introduced for the early diagnosis.


  Rapid Antigen Test for Covid-19 Top


These antigen-based tests detect the presence of viral proteins (antigens) expressed by the SARS CoV-2 virus, mostly spike proteins or nucleoproteins. As they are large enough in size hence, they do not need amplification unlike genetic material in PCR methods. The principle of the antigen test is the use of antibodies that recognize the virus proteins, and if the target antigen is present in sufficient concentrations in the sample, it will bind to specific antibodies fixed to a paper strip enclosed in a plastic casing and generate a visually detectable signal, typically within 30 min.

The antigen detected are expressed only when the virus is actively replicating; therefore, such tests are best used to identify acute or early infection.

The detection depends on the:

  • Concentration or viral loads
  • Stage of infection
  • Quality of sample collected.


In India first kit approved by ICMR is Standard Q COVID-19 Ag from South Korea/India.[1]

Sample type for the STANDARD Q COVID-19 Ag Kit is Nasopharyngeal Swab [Figure 1]{Figure 1}

Recently ICMR also approve two more kits: [Figure 2]{Figure 2}

Advantages with rapid antigen testing methods:

  • Antigen testing is rapid, simpler, and safe. The test results can be interpreted in 15–30 min. Hence, a noticeably short turnaround time. Furthermore, there is no need for biosafety cabinets
  • High specificity-As the tests detects viral proteins present on virus so it has high specificity, i.e., ability to detect true positives
  • The test is quite simple, and no complex steps are involved so expertise for performing the test is not required
  • No complex equipment are needed as it is a visual read
  • Scalability is another major advantage, and it can be done in huge numbers
  • Moreover, the most important advantage of rapid antigen test is that, it is a point of care test (POCT).


Limitations of antigen testing:

  • Detected in the early stage of infection-As all antigen-based tests, it picks up replicating virus proteins; hence, it is detected in the early stage of infection. Best time for antigen detection is within 6 days of developing infection
  • As there is no amplification process, a swab sample may have too little antigen to be detected. Moreover, this may give false-negative result
  • Less sensitive than RT-PCR as a diagnostic tool
  • Depends on the viral loads. A low viral load may not be picked up
  • High specificity (99.3%–100%), moderate sensitivity (50.6% to 84%).


ICMR has given recommendation on whom all to use the antigen detection tests for and the algorithm for its use. The advisory says that symptomatic individuals who test negative for Covid-19 by rapid antigen test should be definitely tested sequentially by RT-PCR to rule out infection, whereas a positive test should be considered as a true positive and does not need reconfirmation by the RT-PCR test [Table 1].{Table 1}

Antigen negative

  1. If the pretest probability for such case is low and antigen test is negative, then we can take it as negative. (True negative). Further, if required such patients can be followed up
  2. If pretest probability is high, then PCR testing needs to be done for such cases.


Antigen positive

It means 100% positive and no further molecular diagnostic testing is required for confirmation.

Best strategy for using it

  1. Implement the advisory from ICMR-Follow the advisory from ICMR for selection of patients on whom the rapid antigen testing is done
  2. Triaging of cases-based on Antigen testing, patients who are likely to have COVID-19 are identified and this reduces the need for molecular confirmatory testing [Figure 3]
  3. Large scale testing: Testing and identifying the infected patients will help in better contact tracing and isolation of infected individuals.


Frequent follow-up testing is also possible with rapid antigen tests. If pretest probability is high and antigen testing is negative, advised to do RT-PCR/follow up retesting.{Figure 3}

In India, antigen testing can be used for the rapid diagnosis of positive cases and hence isolating them and break the chain of infection. Even with moderate sensitivity of antigen tests, we can still identify the positive cases, and this will help in reducing the burden of testing on the molecular diagnosis.

In remote areas or smaller towns trained personnel with appropriate PPE can collect the nasopharyngeal swab and testing can be done in huge numbers.

Antigen testing can be used in health-care sector for triaging covid, suspected and non-COVID patients, and in containment zones for surveillance.


  Serological Test for Covid-19 Top


Serological tests for COVID-19 involve the detection of antibodies in the blood of people who have been infected with COVID-19. Antibodies are produced over days to weeks after infection with the virus. Serologic tests detect resolving or past SARS-CoV-2 virus infection indirectly by measuring the person's humoral immune response to the virus either to nucleocapsid or spike proteins.

It cannot be used for the diagnosis of acute infection but detects past exposure to the virus or past infection.

Timing of seroconversion is a key factor in establishing the appropriate time window to use serology tests.[2]

  • Immunoglobulin M (IgM )– Appears around 7 days, peaks in 14 days and disappears around 21 days
  • Immunoglobulin G (IgG) – appears around 14 days, peaks in 28 days, and persists for about 3 months.


Sample type – whole blood, plasma, or serum.


  Role of Antibody Testing in Covid-19 Infection Top


The significance of IgM antibody is not clear pertaining to this virus. IgM antibody develops in the early phase, and in the early stage, molecular and antigen testing are available. If IgM is detected, further follow up of patients is required. Molecular testing is advised to know the phase of infection [Table 2].{Table 2}

But the IgG Antibody is quite significant, develops late and indicates exposure to SARS CoV-2.

The strength of antibody response depends on the several factors, including age, nutritional status, severity of disease, and certain medications or infections that suppress the immune system.

False negative-As Antibodies start developing after 2–4 weeks after the infection, result will be negative if the test is done during the initial period of infection.

Test result will be negative (false negative) if patient is immunosuppressed or immuno deficient.

False positive: cross reaction with other viruses. To avoid false positives kit selection should be such that they are highly sensitive and highly specific to SARS COVID 2.

IgG antibodies generally start appearing after 2 weeks of onset of infection once the individual is recovering and last for few months only. Therefore, the IgG test is not useful for detecting acute infection.

As per the researchers at the University of Texas MD Anderson Cancer Center published in JCI Insight, Antibodies to the SARS-CoV-2 spike protein receptor-binding domain (S-RBD) are speculated to neutralize virus infection, while the SARS-CoV-2 nucleocapsid protein (N-protein) antibody may often only indicate exposure to the virus, not protections against reinfection.


  Antibody Testing Top


The principle of antibody testing relies on a recombinant antigen produced in the laboratory which is designed to mimic specific structures of the virus, causing any antibodies present in whole blood or serum with a binding affinity to attach to the antigen. They are qualitative tests.


  Two Types Are Available Top


Cassette-based systems which can be read at the point of care through a change in color in an indicator region read visually and laboratory-based immunoassays which require a reader or analyser to detect the reaction.[3]

Rapid tests (immunochromatographic tests)

POCTs detect either only IgG, or combined IgG and IgM in a single cassette in serum, plasma, or whole blood.

These cassette-based tests use the principle of lateral flow immunoassay or immunochromatography.

Few drops of whole blood, serum, or plasma are added to the cassette and a reagent is added. Development of color band indicates the presence of antibodies. The absence of color band indicates no antibodies have been detected.

Advantages of lateral flow immunoassay kits:

  • Rapid tests with results within 10-15 min
  • They do not require expertise. Require low human and facility resource utilization
  • No special equipment, storage, and transportation conditions required
  • Some kits detect both IgM and IgG in a single cassette
  • These tests have high accuracy
  • Can be used for the large number of screenings, produced at low cost and in large quantities.


Limitation of rapid antibody tests:

  • Qualitative
  • Are less sensitive than automated enzyme-linked immunosorbent assay (ELISA).


Automated methods for antibody detection

Automated tests use ELISA or chemiluminescent immunoassay (CLIA) methods for antibody detection. The test principle relies on a recombinant antigen produced in the laboratory which is designed to mimic specific structures of the virus, causing any antibodies present in whole blood or serum with a binding affinity to attach to the antigen which are spike proteins or nucleocapsid proteins.

Based on the reagents, IgG, IgM, can be detected separately or combined as total antibody. These tests demonstrated reliable results with 99.6% specificity and 100% sensitivity for patients tested 14 days or more after symptoms started.

Neutralizing antibody detection: The neutralizing antibody test provides semi-quantitative detection of neutralizing antibodies against the SARS-CoV-2 spike glycoprotein in human serum. These tests are designed to detect potential neutralizing antibodies, often those that prevent interaction of RBD with angiotensin-converting enzyme 2.[4], [5]

It should be noted that lack of scientific data at this time does not allow yet to determine if neutralizing IgG antibodies against SARS-CoV-2 provide long term immunity to the virus or if they protect patients against re-infection.

In India, presently, we have only IgG or total Antibody kit available on CLIA. Some companies are looking at launching IgM kit soon in India.

Advantage of automated test:

  • Simultaneously hundreds of tests can be carries out with better accuracy
  • Their sensitivity and specificity can be higher than cassette-based systems.


Limitation of automated test:

  • Infrastructure costs
  • Additional steps in sample processing, time to obtain results, and practical throughput challenges and substantially increased cost per test
  • It cannot be used at field level and require laboratory set up with skilled professionals.



  Utility of Antibody Testing (Immunoglobulin G) Top


IgG Antibody test for COVID-19 (Only for surveillance and not diagnosis):

  1. Detection of immune response


    1. Serosurveys to understand the proportion of population exposed to infection with SARS-CoV-2 including asymptomatic individuals. Depending upon the level of seroprevalence of infection, appropriate public health interventions can be planned and implemented for prevention and control of the disease
    2. Survey in high risk or vulnerable populations (health care workers, frontline workers, immunocompromised individuals, individuals in containment zones, etc.) to know who has been infected in the past and have now recovered.


  2. Epidemiological tool – Use as epidemiological tool to understand the disease dynamics and help in developing prevention strategies.
  3. Public health department – For planning testing strategy and taking appropriate public health measures. Periodic serosurveillance are useful guide to policy makers
  4. For plasma therapy – Convalescent plasma can be used to treat critical cases. Antibody detection helps in knowing the convalescent phase.
  5. To help develop a vaccine – When a vaccine is developed, it imitates a certain infection, and once injected needs checking for development of immunity. Testing is needed to verify the body's response to a potential vaccine.


ICMR recommendations [Figure 4]{Figure 4}

Newer diagnostic tests

  • CRISPER-SHERLOCK CRISPR SARS-CoV-2 kit uses the SHERLOCK (Specific High-sensitivity Enzymatic Reporter Unlocking) method to program a CRISPR molecule to detect the presence of a specific SARS-CoV-2 genetic signature in specimens collected from patients suspected of COVID-19. Results in about an hour
  • ID NOW COVID 19-POCT RT-PCR test from Abbot which is a fully automated RT-PCR test methodology with rapid turnaround time.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
ADVISORY Newer Additional Strategies for COVID-19 Testing Dated: 23/06/2020, Indian Council of Medical Research Department of Health Research, Ministry of Health and Family Welfare, Government of India; 2020.  Back to cited text no. 1
    
2.
Kumar JS. Laboratory Diagnosis of COVID 19 “The perfect combination” Educational Video.  Back to cited text no. 2
    
3.
Interim Guidelines for COVID-19 Antibody Testing in Clinical and Public Health Settings: CDC; https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines.  Back to cited text no. 3
    
4.
McAndrew KM, Dowlatatshahi DP, Dai J, Becker LM, Hensel J, Snowden LM, et al. Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications on COVID-19 immunity. JCI Insight 2020. https://doi.org/10.1172/jci.insight.  Back to cited text no. 4
    
5.
Jacofsky D, JacofskyEM, Jacofsky M. Understanding Antibody Testing for COVID-19. The journal of Arthroplasty. 2020;35:S74-81.  Back to cited text no. 5
    




 

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  In this article
Abstract
Introduction
Rapid Antigen Te...
Serological Test...
Role of Antibody...
Antibody Testing
Two Types Are Av...
Utility of Antib...
References

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