2. Outline
•The objectives of laboratory diagnosis
•Specimen collection
•Principles of laboratory diagnosis of viral infection
3. Objectives
•Diagnosis and confirm viral infection
•Epidemiological study
•Confirm new viral pathogen or emerging viral infection
•Study pathology and pathogenesis of viral infection
•Vaccine study and novel test development for viral diagnosis
4. Specimen collection
•Blood specimen : plasma/serum, PBMCs
•Body fluids: urine, saliva, CSF
•Swab: nasopharyngeal swab
•Feces
•Tissue
Should be collected in transport medium
Sent to laboratory as soon as possible
Keep at suitable temperature
สิ่งส่งตรวจต้องส่งคู่กับ
ใบส่งตรวจเสมอ
โดยจะต้องระบุข้อมูลต่างๆ
ในการตรวจให้ชัดเจน
5. Specimen collection (2)
•Specimen collection & type of viral infection
•Specimen collection & laboratory purpose
Influenza virus nasopharyngeal swab
Rotavirus infection, hepatitis A feces
Hepatitis B virus blood specimen
Rabies virus saliva, brain biopsy
Viral isolation : collecting during onset of illness in transport medium sent to laboratory immediately
Serological assay : pair serum (acute and convalescent periods)
single serum rapid test
6. Principles of Laboratory diagnosis of viral infection
•Direct specimen examination
•Viral isolation and identification
•Antibody detection or serological assay
8. Electron microscope for viral detection and identification
Coronavirus infection: SARS
http://www.redorbit.com/education/reference_library/health_1/viruses/2583898/coronavirus/
9. Immunostaining assay
•Using monoclonal antibody to viral antigen in clinical specimen
•Secondary antibody to develop the signal of positive staining
Secondary antibody tagged with fluorescence dye Immunofluorescence staining assay (IFA)
Secondary antibody tagged with peroxidase + develop signal with substrate Immunoperoxidase staining (IHC, ICC)
11. Cytopathology assay
•Observation of cell change during viral infection in clinical specimens
Multinucleated (giant) cell or syncytial cell from Tzanck smear
http://cai.md.chula.ac.th/chulapatho/chulapatho/lecturenote/infection/Pathology%20of%20infection/inclusion%20bodies/herpesimplex.html
Negri’s body in cytoplasm
of neuron cells
http://cai.md.chula.ac.th/chulapatho/chulapatho/lecturenote/infection/Pathology%20of%20infection/inclusion%20bodies/Rabies.htm
12. Latex agglutination assay
• Either antibody or antigen detection
http://library.tcmedc.org/webpath/microbio/microbe/microbe05.htm
13. Latex agglutination assay
•Either antibody or antigen detection
http://library.tcmedc.org/webpath/microbio/microbe/microbe05.htm
14. Rapid test (Strip test)
•Antibody or antigen detection
•Immunochromatography technique
15. Molecular assay
•DNA virus: PCR or qPCR
•RNA virus: RT-PCR or qRT-PCR
•Nested PCR & Nested RT-PCR
16. Viral isolation and identification
•Gold standard for viral diagnosis
•Confirm direct examination and serological assay
•Require facilities for laboratory setting
•Time consuming
20. Viral isolation using cell culture
•Types of cell culture
1. Primary cell culture
2. Secondary or diploid cell culture
3. Continuous or Established cell line
•ปัจจุบันนิยมใช้ cell line ในการแยกเชื้อไวรัส •จะต้องศึกษาว่าไวรัสแต่ละชนิดใช้ cell line ชื่ออะไรในการเลี้ยงได้ บ้าง เช่น Rabies virus ใช้ Vero cells เป็นต้น
21. Viral identification
•Animal model
- Clinical sign observation
- Specimen collection or sacrifice animal model
for viral detection and identification
•Embryonated egg
- hemeagglutination assay : amniotic/allantoic fluid
- Pock formation : chorioallantoic membrane
- Embryo observation: died
- Immunostaining
22. Viral identification (2)
•Cell culture
- Change of cell formation: cytopathic effect
(CPE), hyperplasia,
inclusion body, etc.
- Immunostaining, molecular assay
- Cell culture medium: viral identification using
immunoassay (HI, HA,
ELISA)
23. Cytopathic effect (CPE)
•Cell formation varies in each virus
http://virology-mania.blogspot.com/2009/02/next-up-cytopathic-effect-cpe.html
24. Antibody detection or serological assay
•Widely used in routine laboratory
•Require pair serum : acute and convalescent
periods (2-3 weeks)
•The change of antibody titer (4-fold rising)
•ต้องระวังในการแปลผล การที่มีแอนติบอดีอาจไม่ได้บ่งบอกว่าติดเชื้อ อยู่ขณะนั้น อาจมาจากการได้รับวัคซีน หรือ เคยติดเชื้อมาแล้ว
25. Tools for antibody detection
•Neutralization test
•ELISA
•Complement fixation test
•Hemagglutination inhibition test (HI or HAI) ใช้ กับไวรัสที่มี hemeagglutinin gene เช่น influenza, rubella, dengue, JE เป็นต้น
•Western blot
•Rapid testต่างๆ
26. 1 HA unit
•หลุมไวรัสเจือจางมากที่สุดที่ให้ผลบวก •อ่านค่า 1HA unit ต่อหน่วยปริมาตรไวรัสที่ทดสอบ (กี่ l)