DIAGNOSTIC MICROBIOLOGY VIRUSES & PARASITES Dr Nazia khan

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.

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

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

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

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

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

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

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

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

MOVIE ELISA

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

MOVIE WESTERN BLOT

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

MOVIE PCR

MOVIE DNA PROBES