LABORATORY DIAGNOSIS OF VIRAL INFECTION.pptx

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1. LABORATORY DIAGNOSIS OF VIRAL DISEASES Dr Abhishek Kumar Jain Assistant Professor Department of Microbiology RVRS Govt Medical College Bhilwara 1 Saturday, 08 April 2023

2. LABORATORY DIAGNOSIS OF VIRAL DISEASES Laboratory diagnosis of viral infections is useful for the following purposes: To start antiviral drugs Screening of blood donors for HIV, hepatitis B and hepatitis C. Surveillance purpose For outbreak or epidemic investigation To start post-exposure prophylaxis. 2

3. LABORATORY DIAGNOSIS Key steps Specimen selection and collection Specimen transport and storage Laboratory examination Interpretation and reporting

4. Specimen Collection Guideline Avoid causing harm or discomfort to patient. Collect from appropriate site . Obtain specimen at correct time Use appropriate devices Obtain sufficient quantity of specimen Obtain specimen prior to the start of antimicrobial therapy Label correctly . 4

5. SAMPLE SELECTION AND COLLECTION General principle Selection depends upon the specific disease syndrome , viral etiology suspected, Each specimen for virus etiology should be accompanied by a requisition form Requisition form Patient’s Name.............. Age/Sex........... OPD/IPD NO....................... Occupation…………..And Address…… Type Of Specimen.......For...........Test. Clinical History............................ Date And Time Of Collection................ Sign of Clinician

6. Types of Specimen Blood for viral culture Transported in a sterile tube containing anticoagulant Kept at refrigeration temperature (4 ⁰C) until processing Blood for serology Serum should be separated from the clot as soon as possible. Transported in the sterile tube. Serum can be stored for hours or days at 4⁰C. For weeks or months at -20 ⁰C or below before testing.

7. Other common specimens Urine Sputum Stool Pus CSF Swabs Throat Swab, Nasal swab, Oropharyngeal (OP) swab, Nasopharyngeal (NP), Ear swab, Eye swab High Vaginal Swab (HVS), Cervical swab etc. 7

8. LABORATORY EXAMINATION 8 Direct Demonstration of Virus Electron microscopy Immunoelectron microscopy Fluorescent microscopy Light microscopy Detection of Viral Antigens By various formats such as ELISA, direct IF, ICT, flow through assays.

9. LABORATORY DIAGNOSIS OF VIRAL DISEASES 9 Detection of Specific Antibodies Conventional techniques such as HAI, neutralization test and CFT Newer diagnostic formats such as ELISA, ICT, flow through assays. Molecular Methods to Detect Viral Genes Nucleic acid probe—for detection of DNA or RNA by hybridization PCR—for DNA detection by amplification Reverse transcriptase-PCR—for RNA detection Real time PCR—for DNA quantification Real time RT-PCR—for RNA quantification.

10. LABORATORY DIAGNOSIS OF VIRAL DISEASES 10 Isolation of Virus by Animal inoculation Embryonated egg inoculation Tissue cultures: Organ culture, explant culture, cell line culture (primary, secondary and continuous cell lines).

11. Direct detection from specimens For viruses that are difficult to cultivate, EM can be used as primary tool for diagnosis. As an alternative to tissue culture Drawbacks of tissue culture: Highly expensive Has low sensitivity with a detection limit of at least 10 7 virions /ml. The specificity is also low. 11

12. Electron Microscope 12

13. Electron Microscopy Specimens are negatively stained by potassium phosphotungstate and scanned under EM. Shape- Viruses can be identified based on their distinct appearances: Rabies- bullet shaped Rotavirus- wheel shaped Corona virus- petal shaped peplomers Adenovirus- space vehicle shaped 13

14. Immuno-electron Microscopy Sensitivity and specificity of EM can be improved by adding specific antibody to the specimen to aggregate the virus particles which can be centrifuged. Sediment is negatively stained and viewed under EM. 14

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16. Fluorescent Microscopy Direct immunofluorescence technique is employed to detect viral particles in the clinical samples. 16

17. Clinical applications - Clinical applications - Diagnosis of rabies in skin biopsies, corneal smear of infected patients. Syndromic approach- Rapid diagnosis of respiratory infections caused by influenza, rhinoviruses, respiratory syncytial virus, adenoviruses and herpesviruses can be carried out by adding specific antibodies to each of these viruses Detection of adenovirus from conjunctival smears . 17

18. Light Microscopy Inclusion bodies : Histopathological staining of tissue sections may be useful for detection of inclusion bodies which helps in the diagnosis of certain viral infections. 18 Basophilic viral inclusion body

19. Light Microscopy Immunoperoxidase staining: Tissue sections or cells coated with viral antigens are stained using antibodies tagged with horse radish peroxidase (HRP) following which hydrogen peroxide (H 2 O 2 ) and a coloring agent (Benzidine derivative) are added. The color complex formed can be viewed under light microscope. 19

20. Detection of Viral Antigens Various formats are available for detection of viral antigens in serum and other samples Some important antigen detection tests include: HBsAg and HBeAg antigen detection for hepatitis B infection from serum. NS1 antigen detection for dengue virus infection from serum p24 antigen detection for HIV from serum Rotavirus antigen detection from diarrheic stool CMV specific pp65 antigen detection (serum) 20

21. Detection of Viral Antibodies Antibody detection from serum is one of the most commonly used method in diagnostic virology. Appearance of IgM antibody or a four-fold rise of titer of IgG antibody indicates recent infection. Presence of IgG antibody (without a recent rise) indicates chronic or past infection. 21

22. Detection of Viral Antibodies (Cont..) Newer diagnostic formats such as ELISA, ICT, flow through assays are widely used for antibody detection against most of the viral infections such as: Anti- HBc , Anti-HBs and Anti- HBe antibodies for Hepatitis B infection Anti-Hepatitis C antibodies Antibodies against HIV-1 and HIV-2 antigens from serum Anti-Dengue IgM/IgG antibodies from serum. 22

23. Molecular Methods More sensitive, specific and yield quicker results than culture. Polymerase chain reaction (PCR) Reverse transcriptase-PCR (RT-PCR) Multiplex PCR formats BioFire FilmArray Real time-PCR ( rt -PCR) 23

24. Isolation of Virus Viruses cannot be grown on artificial culture media. Cultivated by animal inoculation, egg inoculation or tissue cultures. Being labor intensive, technically demanding and time consuming, virus isolation is not routinely used in diagnostic virology. The specimen should be collected properly and immediately transported to the laboratory. Refrigeration is essential during transportation as most viruses are heat labile. 24

25. Isolation of Virus Because of the ethical issues related to use of animals, animal inoculation is largely restricted only for research use. Research use- To study viral pathogenesis or viral oncogenesis or for viral vaccine trials Diagnostic use- Primary isolation of certain viruses which are difficult to cultivate otherwise; such as arboviruses and coxsackie viruses. 25

26. Egg inoculation Embryonated eggs - first used for viral cultivation by Good pasture in 1931. Embryonated hen’s egg has four sites which are specific for the growth of certain viruses. 26

27. Egg inoculation Yolk sac inoculation Amniotic sac Allantoic sac Chorioallantoic Membrane (CAM) 27

28. Tissue Culture 28 Types Explanation Examples Organ culture (obsolete ) Used for certain fastidious viruses that have affinity to specific organs. Tracheal ring culture for isolation of corona virus Explant culture (obsolete ) Fragments of minced tissue can be grown as 'explants’ Adenoid explants used for adenoviruses. Cell line culture This is the only isolation method which is in use now.

29. Preparation of the Cell Lines Tissues digested – by treatment with proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking. Viral growth medium: Cells are then washed, counted, and suspended in viral growth medium containing balanced salt solution added with essential amino acids and vitamins, salts and glucose supplemented by 5-10% of fetal calf serum and antibiotics. 29

30. Preparation of the Cell Lines (Cont..) Tissue culture flasks 30

31. Preparation of the Cell Lines (Cont..) Incubation: Tissue culture flasks are incubated horizontally in presence of CO 2 , either as a stationery culture or as a roller drum culture. Rolling of the culture bottle in roller drums provides better aeration which is useful for isolation of fastidious viruses (e.g. rotavirus) 31

32. Types of Cell Lines 32 Type Explanation Examples Primary cell line Derived from normal cells. Freshly taken from the organs and cultured Capable of very limited growth in culture, maximum up to 5-10 divisions. Maintain a diploid karyosome Useful for both primary isolation as well as growth of the viruses for vaccine production. Monkey kidney cell line- useful for isolation of myxoviruses , enteroviruses & adenoviruses Human amnion cell line Chick embryo cell line

33. Types of Cell Lines 33 Type Explanation Examples Secondary or diploid cell lines Can divide maximum up to 10-50 divisions before they undergo senescence (death). Also derived from the normal host cells. Maintain the diploid karyosome . Human fibroblast cell line- excellent for the recovery of cytomegalovirus. MRC-5 &WI-38 (human embryonic lung cell strain)

34. 34 Type Explanation Examples Continuous cell lines Derived from cancerous cell lines, hence are immortal. They also possess altered haploid chromosome. Easy to maintain HeLa cell line (Human carcinoma of cervix cell line) Hep-2 cell line (Human epithelioma of larynx cell line)- widely used for respiratory syncytial viruses, adenoviruses and HSV KB cell line (Human carcinoma of nasopharynx cell line) McCoy cell line (Human synovial carcinoma cell line)- useful for isolation of viruses as well as Chlamydia Vero cell line ( Vervet monkey kidney cell line)-used for rabies vaccine production.

35. Types of Cell Lines (Cont..) 35 Human lung fibroblast cell line (Normal) HeLa cell line (normal, uninfected) Vero cell line (normal, uninfected) HEp-2 cell line (normal, uninfected)

36. Detection of Viral Growth in Cell Cultures Two methods are used to detect the growth of the virus in cell cultures. Cytopathic Effect (CPE) Shell Vial Technique 36

37. Cytopathic Effect (CPE) Defined as the morphological change produced by the virus in the cell line detected by light microscope. 37 Type of Cytopathic effect (CPE) Virus Rapid crenation and degeneration of the entire cell sheet Enteroviruses Syncytium or multinucleated giant cellformation Measles, RSV, HSV Diffuse roundening and ballooning of the cell line HSV Cytoplasmic vacuolations SV 40 (Simian vacuolating virus-40) Large granular clumps resembling bunches of grapes Adenovirus

38. Shell Vial Technique Involves centrifugation of cell culture (mixed with the specimen) to enhance the cell contact and viral replication, followed by Detection of early viral antigen in the infected cells by direct fluorescence technique. 38

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