lab diagnosis og COVID-19

1. LABORATORY DIAGNOSIS
OF COVID-19
Dr.Jageshwar mandal choupal
DNB Resident(Pathology)

2. Corona virus -From common cold to
GLOBAL contagion
• 1.HCoV-NL63-mild symptoms
• 2.HCoV-NL229E-mild symptoms
• 3.HCoV-OC43-mild symptoms
• 4.HCoV-HKU1-mild symptoms

3. • 5.SARS CoV(2003)-8098 cases (10%CFR)
• 6.MERS CoV(2012)-2519 cases(31%CFR)
• 7SARS CoV-2(2019-20 till march) -5406282(3.7%CFR)
• It is assume that covid -19 spread by
BAT→PANGOLIN→HUMAN.

5. ROUTE OF TRANSMISSION
• Air droplets-upto distance of 3 feet,
• may remain active on surface for 24 hrs
• Feco-oral route
• Air borne- 3hrs.

6. carrier
• Asymptomatic carrier
• Symptomatic patient.

7. symptoms
• High fever
• Cough and sneezing
• Breathlessness
• Increase heart rate
• Increase respiration
• Septic shock
• Hypotension

8. Mech. To act in lungs
• Virus have spike on envelope which bind with ACE-2 receptos in
alveolar surface of lung.
• With the help of Rna dependent RNA POLYMERSAE virus form
more copies of ss-rna in lungs.
• By the process of translation virus form protein product.
• These protein products attract macrophages, neutrophilsn
lymphocytes which releases tons of cytokins mainly
IL-1,IL-6,1L-10,TNF-⍺ forming cytokins storm.
• Now these cytokins storn destroy pneumocytes -2 on lung surface.
and increase vasodilation ,capillary permeability .

9. • Now fluid passes to alveolus forming alveolar edema,due to
which suface tension inside alveolus increases very much.
• So to much increase in surface tension alveolus collapse.
• Also proteozyme secreated by macrophages in alveolus
destroys alveolar surface cells leadind to decrease
production of pneumocytes.
• So it causes decrease gas exchange causing
hypoxemia ,thus to maintain oxygen level patient increase
rate of breathing.
• This leads to acute respiratory distress syndrome.

10. WHOM TO TEST ?
• 1.All symptomatic indiviuals with influenza like illness(ILI)
with history of international travelin last 14 days.
• 2. All symptomatic indiviuals with influenza like illness(ILI)
contacts of laboratory confirmed cases.
• 3.All symptomatic influenza like illness(ILI) health care
workers involved in containment and mitigation of
Covid-19.
• 4.All patients of SARI( Severe Acute Respiratory
Infection).

11. • 5.Aymptomatic 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.

12. SPECIMEN TO BE COLLECTED
• 1.Upper respiratory tract specimen-Nasopharyngeal swab
• Oropharyngaeal swab
• 2.Lower Respiratory tract specimen-sputum
• Endotreacheal aspirate
• Bronchoalveolar lavage
• 3.Other specimens-stool
• Blood
• Autopsy material including lung tissue.
• Paired serum- serum contains only specific antibody.

13. Specimen type
Collection
mateials
Storage
temperature
Recommended temp. For
transport to lab.
Nasopharyngeal /
oropharyngeal
swa
Dacron/polyster
flocked swab
2-8℃
2-8℃ <5 days
-70℃>5days
Bronchoalveolar
lavage
Sterile container 2-8℃
2-8℃<2days
-70℃>2days
Encotreacheal
aspirate
Sterile container 2-8℃
2-8℃<2days
-70℃>2days
Sputum Sterile container 2-8℃
2-8℃<2days
-70℃>2days
Tissue from
autopsy/biopsy
Sterile container
with saline
2-8℃
2-8℃<24hours
-70℃>24hours
Serum
Seperate serum
tubes
2-8℃
2-8℃<5 days
-70℃>5days
Whole blood Collection tubes 2-8℃
2-8℃<5 days
-70℃>5days
Stool Stool container 2-8℃
2-8℃<5 days
-70℃>5days

14. Laboratory test for covid -19
• 1.Reverse transcriptase PCR (Nucleic acid amplification
test)
• 2.Serological test
• 3.Viral sequencing
• 4.Viral culture

15. Reverse transcriptase PCR
• DNA amplification test have targeted a combination of the
following genes.
• 1.Envelop
• 2.Rna dependent RNA polymeras
• 3.Nucleocapsid
• 4.Open reading frame 1ab(ORF 1ab)

16. TEST MOLECULAR TARGETS SCOPE
WHO
E gene
RdRp gene
N gene
1st line screening
Confirmatory testing
Additional confirmatory
testing
CDC
N 1/2 gene
RNase P gene
Combined assay
Control assay

18. Used in hybridisation and amplification of cDna.

19. TEST INTERPRETATION(as per WHO)
• 1.Screening test (+) and confirmatory test (+)→, (+) for
covid-19, (SARS -CoV-2 Detected).
• 2.Screening test (+) and confirmatory test (-)→, (-) for
covid-19, (SARS -CoV-2 Not Detected).
• 3.Screening test (-) and confirmatory test (-)→, (-) for
covid-19, (SARS -CoV-2 Not Detected).
• 4.Screening test (-) and confirmatory test (+)→, (-) Retest or
Refer to a reference laboratory for additional testing.
•

20. Factors showing False (-) covid -19 test
• 1.Lack of identification/Misidentification.
• 2.Inadequate procedures for specimen ( e.g swab)
collection,handling,transport and storage.
• 3.Collection of inappropriate material for quality or volume.
• 4.Presence of interfering substances
• 5.Manual errors.
• 6. Testing in patients receiving antiretroviral therapy.

21. ANALYTICAL FACTORS
• 1.Testing carried out outside of the diagnostic window
• 2.Active viral recombination.
• 3.Use of harmonization of primers and probes
• 4.Instrument malfunctioning
• 5.Insufficient or inadequate material
• 6.Non-specific PCR annealing
• Misinterpretation of expression profiles of amplied DNA copies.

23. Serological testing
• 1.Based on the detection of IgM/IgG antibodies.
• 2.Antibody test results are especially important for
detecting previous infections in people who has few or no
symptoms.
• 3.Most Important cross reactivity to other corona viruses
is more challenging.
• It is used as screening test but it is not recommended for
diagnosis by WHO and CDC.

24. Antigen kit testing procedure.

28. Viral sequencing
• It can be useful to monitor for viral genome mutations that
might affect the patients diagnosis and treatment .
• It can done by study of genomic sequence present in
virus genetic mateial.

29. Viral culture
• It can done for the purpose of colony assisted pcr
• In routine patient, viral culture is not done because it is
time taking procedure.

30. Hematological parameters
• 1. RBC- Normocytic Normochromic
• 2.WBC- Leucocytosis
• Neutophile count- Neutrophilia
• Lymphocytes count-lymphocytopenia
• Monocytes count- monocytopenia
• 3.Platelets-Thrombocytopenia.( Giant platelets may be
present)

31. Peripheral smear-Neutrophils
• 1.Heavily clumped chromatin with toxic
granules and cytoplasmic vacuoles.
• 2.Nuclear detachment with elongated
nucleoplasmic and ring shaped nuclei with
platelet attatchment at surface.
• 3.Fetus like nuclei noted with aberrent
nuclear projections named as COVID nuclei.
• 4.Black arrow-fetus like covid nuclei.
• 5.Blue arrow-abberent nuclear projections.
• 6.Yellow arrow-Toxic granulations and
vacuolations
• 7.Red arrow-ring nuclei.
• 8.Green arrow-Elongated nucleoplasm.
Neutrophils

32. LYMPHOCYTES
• 1.Large granular lymphocye with
round to indented nuclei,condensed
chromatin, few prominent nucleoli
also known as covicytes.
• 2.Abundant pale blue cytoplasm
with distinct variable size
azurophilic granules.
• 3.Black arrow-Abundant pale blue
cytoplasm with distinct variable size
azurophilic granules.
• 4.Green arrow-cytoplasmic pod
formation.
• 5.Red arrow-apoptotic lymphocytes
LYMPHOCYTE

33. Monocytes
monocytes.

34. • 1.Activated monocytes seen with shows marked
anisocytosis with prominent cytoplasmic vacuolisation
and few granules.
• 2.Nuclei large,fine chromatin with nuclear
blebbing,nuclear overlapping by vacuoles may be
present.
• 3. Red arrow-activated monocytes with prominent
vacuolisation n few granules.
• 4.Green arrow-Nuclear blebbing.

35. Biochemical parameters
• 1.Albumin-decreased
• 2.ALT-increased
• 3.AST-increased
• 4.Total bilirubin-increased
• 5.LDH-increased
• 6.Urea-increased
• 7.Creatinine-increased
• 8.-Cardiac troponin I-increased
• 9.Na-decreased
• 10.K-decreased
• 11.Ca-decreased

36. •COAGULATION PROFILE
•D-DIMER-Increased
•PROTHROMBIN TIME-Increased
•INFLAMMATORY MARKERS
• 1.Procalcotonin-Increased
• 2.serum ferritin-Increased
• 3.IL-6-Increased
• 4.IL-1-Increased
• IL-10-Increased
• TNF-⍺-Increased

37. THANK YOU