This document summarizes laboratory diagnosis of COVID-19. It discusses that molecular (rRT-PCR) tests target genes like E, RdRp, N, and ORF 1ab. Specimens collected include nasopharyngeal swabs, oropharyngeal swabs, sputum, and stool. Interpretation of rRT-PCR tests follows WHO and CDC guidelines. Serological tests detect IgM and IgG antibodies but are not recommended for diagnosis. Viral sequencing and culture are also discussed. Abnormal lab findings in COVID-19 patients include decreased lymphocytes and albumin and increased LDH, D-dimer and inflammatory markers.

1. Dr. Roshan Kumar Mahat
(PhD Medical Biochemistry)
Assistant Professor
Department of Biochemistry
LABORATORY
DIAGNOSIS
OF COVID-19

2. Coronaviruses: From the Common Cold to
Global Contagion
1. HCoV-NL63
2. HCoV-229E
3. HCoV-OC43
4. HCoV-HKU1
Mild symptoms

3. Coronaviruses associated with the severe disease:
1. SARS CoV (2003)
 ≈8098 cases (≈10% CFR)
2. MERS CoV (2012)
 ≈2519 cases (≈35% CFR)
3. SARS CoV-2 (2019-20)
 ≈5406282 (≈7% CFR)
Coronaviruses: From the Common Cold to
Global Contagion

4. Dec 8, 2019
• Reported first known
case of pneumonia of
unknown etiology in
Wuhan
Dec 31, 2019
• China reports a cluster
of cases of pneumonia
with unknown etiology
in Wuhan to WHO
Jan 7, 2020
• A novel coronavirus
2019 was identified
Jan 10, 2020
• Viral genome
sequence was released
Jan 12, 2020
• Four other genome
sequences deposited
in the viral sequence
database
Jan 31, 2020
• WHO declares a public
health emergency of
international concern
Feb 11, 2020
• WHO named the
disease as COVID-19
Feb 11, 2020
• Virus is renamed as
SARS-CoV 2
Mar 11, 2020
• WHO declared COVID-
19 as global pandemic
The timeline of the facts of COVID-19

5. Whom to test?
1. All symptomatic (ILI symptoms) individuals
with history of international travel in the last
14 days.
2. All symptomatic (ILI symptoms) contacts of
laboratory confirmed cases.
3. All symptomatic (ILI symptoms) health care
workers/frontline workers involved in
containment and mitigation of COVID19.
4. All patients of Severe Acute Respiratory
Infection (SARI).

6. 5. Asymptomatic direct and high-risk contacts
of a confirmed case to be tested once
between day 5 and day 10 of coming into
contact.
6. All symptomatic ILI within hotspots/
containment zones.
7. All hospitalized patients who develop ILI
symptoms.
8. All symptomatic ILI among returnees and
migrants within 7 days of illness.
Source: Strategy for COVID19 testing in India (Version 5, dated 18/05/2020, ICMR)

7. Specimens to be collected
• Nasopharyngeal swab
• Oropharyngeal swab
Upper
respiratory
specimens
• Sputum (if produced)
• Endotracheal aspirate
• Bronchoalveolar lavage
Lower
respiratory
specimens
• Stool
• Blood
• Paired serum
• Autopsy material including lung
tissue
Other
specimens

9. Laboratory testing for COVID-19 virus
1. Nucleic acid amplification tests (NAAT)
2. Serological testing
3. Viral sequencing
4. Viral culture

10. Molecular (rRT-PCR) tests have generally
targeted a combination of the following
genes:
1) Envelope (E)
2) RNA dependent RNA polymerase (RdRp)
3) Nucleocapsid (N)
4) Open reading frame 1ab (ORF 1ab)
Nucleic acid amplification test (NAAT): rRT PCR

12. Sampling
Nucleic acid
isolation
RT
Real time
PCR
amplification
Swab RNA cDNA PCR
Figure: General work flow

15. Test interpretation (WHO):
1. Screening test (+) and confirmatory test (+):
positive for COVID-19 (SARS-CoV-2 detected).
2. Screening test (+) and confirmatory test (-):
negative for COVID-19 (SARS-CoV-2 not
detected).
3. Screening test (-) and confirmatory test (-):
negative for COVID-19 (SARS-CoV-2 not
detected).
4. Screening test (-) and confirmatory test (+):
retest or refer to a reference laboratory for
additional testing.

16. Test interpretation (CDC):

17. Potential
vulnerabilities
Preanalytical
General Specific
Analytical
Potential vulnerabilities in the laboratory diagnosis of
coronavirus disease 2019 (COVID-19) using rRT-
PCR:

18. 1. Lack of identification/misidentification
2. Inadequate procedures for specimen (e.g. swab) collection,
handling, transport and storage
3. Collection of inappropriate or inadequate material for quality
or volume
4. Presence of interfering substances
5. Manual (pipetting) errors
General factors

19. 1. Sample contamination
2. Testing in patients receiving antiretroviral therapy
Specific factors

20. 1. Testing carried out outside of the diagnostic window
2. Active viral recombination
3. Use of non-adequately validated assays
4. Lack of harmonization of primers and probes
5. Instrument malfunctioning
6. Insufficient or inadequate material
7. Non-specific PCR annealing
8. Misinterpretation of expression profiles
Analytical factors

21. Source: Lippi et al., 2020

22. • Based on the detection of IgM/IgG antibodies.
• Antibody test results are especially important
for detecting previous infections in people
who had few or no symptoms.
• Cross reactivity to other coronaviruses can be
challenging.
• Not recommended by WHO or CDC for
diagnosis.
Serological testing

24. Figure: Variations in the Levels of SARS-CoV-2 RNA, IgM and IgG after infection.

25. Table: Clinical Significance of an IgM/IgG Serological Test Result

28. Can be useful to monitor for viral genome
mutations that might affect the performance
of medical countermeasures, including
diagnostic tests.
Not recommended as a routine diagnostic
procedure.
Viral sequencing
Viral culture

29. • WBC count- increased
• Neutrophil count- increased
• Thrombocyte count- decreased
• Lymphocyte count- decreased
Hematological
parameters
Main laboratory abnormalities observed in adult
patients with unfavorable COVID-19 progression

30. • Albumin- decreased
• ALT- increased
• AST- increased
• Total bilirubin- increased
• LDH- increased
• Urea- increased
• Creatinine- increased
• Cardiac troponin I- increased
• CKMB- increased
• Sodium- decreased
• Potassium- decreased
• Calcium- decreased
Biochemical
parameters

31. • D-dimer-increased
• Prothrombin time- increased
Coagulation
profile
• Procalcitonin- increased
• Serum ferritin- increased
• IL-6- increased
• CRP- increased
• IL-10- increased
Inflammatory
markers