laboratory diagnosis of viral infection
TRANSCRIPT
Outline
• The objectives of laboratory diagnosis
• Specimen collection
• Principles of laboratory diagnosis of viral infection
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
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
สงสงตรวจตองสงคกบ ใบสงตรวจเสมอ โดยจะตองระบขอมลตางๆ ในการตรวจใหชดเจน
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
Principles of Laboratory diagnosis of viral infection
• Direct specimen examination
• Viral isolation and identification
• Antibody detection or serological assay
Direct specimen examination
• เปนการตรวจการตดเชอไวรสโดยตรงในสงสงตรวจตางๆ
• รวดเรว ใชเวลานอย
Diagnosis tests
- Electron microscope (EM) - Immunostaining assay (IFA, IHC, ICC) - Cytopathology assay: multinucleated cell (RSV, VZV) : inclusion body (Negri’s body) - Agglutination assay : Latex agglutination - Rapid test - Molecular assay (PCR & RT-PCR & qPCR & qRT-PCR)
Electron microscope for viral detection and identification
Coronavirus infection: SARS
http://www.redorbit.com/education/reference_library/health_1/viruses/2583898/coronavirus/
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)
Immunofluorescence staining
http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_10.pdf
WNV-infected Vero cells
http://www.virologyj.com/content/3/1/71/figure/f3?highres=y
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
Latex agglutination assay
• Either antibody or antigen detection
http://library.tcmedc.org/webpath/microbio/microbe/microbe05.htm
Latex agglutination assay
• Either antibody or antigen detection
http://library.tcmedc.org/webpath/microbio/microbe/microbe05.htm
Molecular assay
• DNA virus: PCR or qPCR
• RNA virus: RT-PCR or qRT-PCR
• Nested PCR & Nested RT-PCR
Viral isolation and identification
• Gold standard for viral diagnosis
• Confirm direct examination and serological assay
• Require facilities for laboratory setting
• Time consuming
Tools for viral isolation
• Experimental animal (ไมนยมในปจจบน เนองจากตองอาศยสถานทจ าเพาะ และมการควบคมทด)
• Embryonated egg
• Cell culture
Viral isolation using embryonated egg
• Influenza virus, Mumps, HSV-1, HSV-2, rabies virus
• Route of infection
- Amniotic sac : influenza virus
- Allantoic sac: New castle virus
- Embryo
- York sac: rabies virus
- Chorioallantois (chorioallantoic membrane): HSV
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
• ปจจบนนยมใช cell line ในการแยกเชอไวรส
• จะตองศกษาวาไวรสแตละชนดใช cell line ชออะไรในการเลยงไดบาง เชน Rabies virus ใช Vero cells เปนตน
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
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)
Cytopathic effect (CPE)
• Cell formation varies in each virus
http://virology-mania.blogspot.com/2009/02/next-up-cytopathic-effect-cpe.html
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)
• ตองระวงในการแปลผล การทมแอนตบอดอาจไมไดบงบอกวาตดเชออยขณะนน อาจมาจากการไดรบวคซน หรอ เคยตดเชอมาแลว
Tools for antibody detection
• Neutralization test
• ELISA
• Complement fixation test
• Hemagglutination inhibition test (HI or HAI) ใชกบไวรสทม hemeagglutinin gene เชน influenza, rubella, dengue, JE เปนตน
• Western blot
• Rapid testตางๆ
References
• พไลพนธ พธวฒนะ. ไวรสวทยา. ส านกพมพอกษรสมย 2540
• ภาวพนธ ภทรโกศล. หลกไวรสวทยาทางการแพทย. ส านกพมพ โนวเลจดเพรส 2550