1. DIAGNOSTIC MICROBIOLOGY VIRUSES & PARASITES
Dr Nazia khan

2. OBJECTIVES
Discuss various techniques utilized in diagnosis of viral infection with clinical examples
Explain principle of molecular techniques in diagnostic microbiology.
Briefly discuss diagnostic modalities available for parasitic infections with clinical examples.

3. DIAGNOSTIC MICROBIOLOGY
Viruses & parasites not routinely cultured on conventional agar
Diagnosis of viruses and parasites require special procedures
Indirect methods are more suitable then direct methods
Viruses are too small to be seen by light microscopy
Conventional stains are ineffective against them

4. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
MICROSCOPY
Light microscopy useful for some protozoa and parasites
Scotch tape test-Enterobius ova
Not fit for viruses
WET FILM EXAM
Useful for motile Giardia, Entameoba histolytica etc.
Also for ovas, cysts of parasites
Usually urine, stool examination
Direct visualization of worm

5. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
STAINED SPECIMEN
Negri bodies of Rabies virus
Giemsa stain for Plasmodium in blood
ZN stain for Cryptosporidium hominis
Iodine stain for Giardia cyst, Entamoeba cyst
Viruses are not stained
Negri body

6. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
ELECTRON MICROSCOPY
Not routinely used
For certain viruses not grown in cell cultures
Example: Rotavirus in stool
Zaire ebola virus was diagnosed by EM in recent outbreak
Monkeypox outbreak in USA
Immune electron microscopy improve virus detection by reacting with specific antibodies
Rota virus
Ebola virus

7. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
IMMUNOFLUORESCENCE
Direct exam of viral particle
Monoclonal antibodies against viral or parasitic antigens
Fix on slide
Immuno-fluorescent staining
Rapid and sensitive test
Cryptosporidium in stool
RSV in respiratory secretion

8. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
VIRUS CULTURE
On special cell lines e.g. vero cells, monkey kidney cells
Specimens are inoculated on cell lines
Bactericidal chemicals added
Incubated
Observe effects on cell lines
Cytolysis, syncytia formation etc.
Laborious, time consuming
RSV, Measles, Influenza virus

9. DIAGNOSTIC MICROBIOLOGY-DIRECT METHODS
VIRUS CULTURE NEWER TECHNIQUES
Immuno-fluorescent staining of intracellular antigens on cultured cells
Use monoclonal antibodies against virus antigen
Label the antibodies
Put these antibodies on cell lines
Observe the fluorescence
Advantage-diagnosis in 24 hours possible

10. DIAGNOSTIC MICROBIOLOGY-INDIRECT METHODS
ANTIGEN DETECTION
Rapid tests like swabs for Influenza virus
ELISA TEST for HBsAg detection, p24 antigens
Latex agglutination tests
Complement fixation tests

11. MOVIE ELISA

12. DIAGNOSTIC MICROBIOLOGY-INDIRECT METHODS
ANTIBODY DETECTION
Western Blot Immunoassays
Viral antigens are separated on a filter paper
Serum is flooded on the filter paper
Ag-Ab react
Dark bands of Ag-Ab visible
Can identify specific antibodies

13. MOVIE WESTERN BLOT

14. DIAGNOSTIC MICROBIOLOGY
MOLECULAR DIAGNOSIS
PCR TEST
PCR for HSV in CSF-sensitive test for HSV encephalitis
PCR for toxoplasma gondii in amniotic fluid
NUCLEIC ACID PROBES
FLUORESCENCE IN SITU HYBRIDIZATION
EBV in cell cultures
Dengue virus
DNA HYBRIDIZATION
Leishmania tropica in skin smears

15. MOVIE PCR

16. MOVIE DNA PROBES