DETECTION OF PATHOGENS
IMMUNODIAGNOSTICS
Assist Prof Dr.
Syed Yousaf Kazmi
OBJECTIVES
• Discuss various laboratory techniques for
diagnosis of microorganisms
• Explain the basis of immunodiagnostic
laboratory procedures
• Briefly discuss clinically important
immunodiagnostic tests
DIAGNOSIS OF MICROORGANISMS
• Take detailed history
• Perform meticulous clinical
examination
• Focus on few differential
diagnosis
• Plan to take appropriate
specimens for lab testing
• Start on empiric treatment
• Get the results from lab
• Shift to definitive treatment
DIRECT VISUALIZATION OF
MICROORGANISM
DIRECT MICROSCOPY
CSF, Stool, Urine, Other body
fluids
May observe motile organisms
e.g. Vibrio cholerae, Giardia
lamblia in stool
STAINED SPECIMEN
Giardia lamblia
Gram Stain
ZN Stain
Leishman’s Stain
India Ink preparation
(Movie clip Giardia, Entamoeba)
Bacteria in CSF
DIRECT VISUALIZATION OF
MICROORGANISM
• Capsular stains
• Capsular swelling reaction
for Streptococcus
pneumoniae
• Indirect visualization of
the capsule of several
organisms
• Helps rapid identification
India ink for Cryptococcus
RAPID BEDSIDE DIAGNOSTIC TESTS
• Based on antigen antibody
reaction
• Antigen/ Antibody is tagged
on strip
• Sample is added-Result
• Aim is to save time
• Rapid Streptococcus pyogenes
throat swab test
• Rapid Malaria test
• Rapid Viral diagnostic tests
• (Movie clip Strep, HIV)
CULTURE OF SPECIMEN
• Routine for most bacterial and
fungal infections
• Most bacteria are rapid growers
• Slow grower org (M tuberculosis)
• Appropriate culture media
• Appropriate oxygen and
temperature requirements
• Observe culture characteristicshelps in identification
Hemolysis on Blood Agar
IDENTIFICATION OF BACTERIA
• Enzyme tests e.g. Catalase
test, Coagulase test,
Oxidase test, Urease test
• Biochemical testing of
pathogen
• Antimicrobial sensitivity
testing
• Help clinicians in review of
antimicrobial prescription
ROLE OF SEROLOGY IN ORGANISM
IDENTIFICATION
DETECTION OF MICROBE
ANTIGEN WITH KNOWN
ANTISERA
Quellung Reaction (Strep
pneumoniae)
Detection of pathogens by
slide agglutination tests e.g.
Staph aureus rapid identification
IDENTIFICATION OF SERUM
ANTIBODIES
Complement Fixation test
• Still used for antibodies
against viruses
Direct Hemagglutination
• Agglutinins in EBVagglutination of sheep RBC
• Cold agglutinins in
infection Mycoplasma
pneumoniae-human RBCs
at 4oC
AGGLUTINATION TESTS
Antigen antibody reaction when
antigen is in particulate form e.g.
bacterial cell, antigen coupled with
latex beads
It is simple, inexpensive & sensitive
• Slide Agglutination tests
– Many bacterial isolate identification
– Blood grouping and cross matching
• Tube test
– Serological diagnosis of typhoid,
Brucella, typhus, atypical
pneumonia
(Movie clip Agglutination test)
PRECIPITATION REACTION
IMMUNOELECTROPHORESIS
When electrophoresis is applied
to accelerate gel diffusion
procedure
Utilized for
• Presence/absence of specific
proteins
• Ig classes immunodeficiency
• Immunoproliferative disorder
• (Movie clip Radial ID, Double
Imunoassay)
FLUORESCENT ANTIBODY TEST
DIRECT FAT
• Antigen in question is fixed
on slide
• Fluorescent dye labeled
antibodies put on slide
• Fluorescent microscope to
detect the reaction
• Example: Chlamydia Ag
INDIRECT FAT-detect
antibodies
Used for RSV, Pneumocystis
jirovecii , Syphilis etc.
(Movie clip IF)
RADIOIMMUNOASSAY & ENZYME
LINKED IMMUNOABSORBENT ASSAYS
• Highly sensitive
• Can detect 10-9g of material
• Used for detection of infectious disease
antigens/ antibodies, hormones, drugs,
antibiotics, serum proteins, tumor markers
• Both tests same principle except in RIA
radiolabelled product is detected while in
ELISA an enzyme mediated color change is
perceived
RADIOIMMUNOASSAY & ENZYME LINKED
IMMUNOABSORBENT ASSAYS
• An antigen/ antibody is
coated onto microtitre
plate
• Antibody/ antigen in
question is flooded,
• Enzyme labeled antibody
• Substrate is added- color
change measured
• (Movie clip ELISA
direct/ID)
WESTERN BLOT
• Different antigens are
separated by electrophoresis
and fixed on a membrane
• Patient’s serum containing
antibodies is flooded-AgAb
reaction
• Antihuman Ig conjugated with
enzyme/ radioactive label
added
• Different Ag are visualized
• (Movie clip Western Blotting)