Laboratory Diagnosis of Viral Infection

Presented by

Methee Sriprapun, PhD

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

Rapid test (Strip test)

• Antibody or antigen detection

• Immunochromatography technique

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

Embryonated egg

http://www.virology.ws/2009/12/10/influenza-virus-growth-in-eggs/

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

1 HA unit

• หลมไวรสเจอจางมากทสดทใหผลบวก

• อานคา 1HA unit ตอหนวยปรมาตรไวรสททดสอบ (ก l)

References

• พไลพนธ พธวฒนะ. ไวรสวทยา. ส านกพมพอกษรสมย 2540

• ภาวพนธ ภทรโกศล. หลกไวรสวทยาทางการแพทย. ส านกพมพ โนวเลจดเพรส 2550

Thank you for your attention