DETECTION OF PATHOGENS
(DIAGNOSTIC BACTERIOLOGY &
VIROLOGY)
Assist Prof. Dr
Syed Yousaf Kazmi
OBJECTIVES
• Explain the basic concept of diagnostic
microbiology including classical bacteriology
and newer techniques
• Identify the available diagnostic methods
DETECTION OF BACTERIAL INFECTIONS
For diagnosis of various bacterial infections
1. Microscopy & morphology
2. Staining
3. Cultural characteristics
4. Metabolism
5. Resistance
6. Bio chemical properties
MORPHOLOGY
• WET FILM EXAM
– Direct unstained exam
of specimen
– Rapid presumptive
identification e.g. Vibrio
cholerae shooting star
motility
– Suitability of specimen
e.g. Sputum vs saliva
MICROSCOPY
• STAINED SPECIMEN
EXAM
– Gram Stain-Most
bacteria
– ZN Stain-AFB
– Albert Stainmetachromatic granules
of C diphtheriae
– Geimsa Stain-Chlamydia
Gram Stain
Albert Stain
GRAM STAIN
• Most widely used stain
• Color, Shape, Arrangement,
spores and capsules
• Intracellular e.g. Neisseria
• Less sensitive-105 org/ml for
detection
• Differentiation from normal
flora
• Probable identification
Neisseria meningitidis in CSF
Streptococcus pneumoniae
IMMUNOFLUORESCENT ANTIBODY
(IF) STAINING
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•
•
•
•
More specific than other
More cumbersome
More time consuming
Expertise required
Quality control is important
to minimize nonspecific IF
staining
• Bordetella pertussis or
Legionella pneumophila
CULTURE OF SPECIMEN
• Culture of bacteria- Gold
standard for identification
• Usually 24-48 hrs
• 72 hrs for slow growers
• Up to 6 weeks for M
tuberculosis
• Routine media include Blood
Agar, Chocolate agar & Mc
conkeys Agar
• Enriched medium, Differential
medium
CULTURE OF SPECIMEN
• Blood Agar-detects hemolysis
• Other characters e.g. color,
shape and texture of bacterial
colony
• Rate of growth
• Pigment production e.g.
Pseudomonas, Serratia spp.
• Satellitism e.g. Strep
pneumoniae and Staph
aureus, swarming of Proteus
• Allows org to be tested for
antimicrobial sensitivity
Β- hemolysis
Proteus swarming
METABOLISM
• Growth requirements
– Aerobic & Anaerobic
– Micro-aerophilic, Carboxyphilic etc.
– Growth factor requirements e.g.
Haemophilus influenzae factor X, V
FERMENTATION AND BIOCHEMICAL
TESTS
 Sugar fermentation
test
 Indole test
 Methyl red test
 Voges prousker test
 Citrate test
 Catalase test
 Oxidase test
 Urease test
 Nitrate test
OXIDASE TEST
FERMENTATION AND BIOCHEMICAL
TESTS
API 10S
RESISTANCE TO ANTIMICROBIALSANTIBIOGRAM
• Sensitivity
profile of org
• Helps in
identification
• Genetic
resistance or
sensitivity
OTHER TESTS
• Bacteriophage Test
– Mostly for epidemiological
testing
– Not routinely done
– Reference laboratories only
• Animal pathogenicity tests
– Limited value
– Few bacterial infections
– Reference laboratories only
SEROLOGY BASED TESTS
Agglutination Tests
– Latex Agglutination Test e.g. group A streptococcal
pharyngitis, Cryptococcus antigen detection in CSF
– Co-agglutination Test-useful in bacterial identification in
cultures
Enzyme-linked immunosorbent assays (ELISA)
Western Blot Assay
Molecular Diagnosis
– Nucleic acid probe
– PCR
RAPID BACTERIAL IDENTIFICATION
TESTS
• Rapid Serology based
tests
• Rapid presumptive
identification
• Helps in management
• Strep A throat test,
Clostridium difficile rapid
toxin detection test
VIRAL DIAGNOSTIC TESTS
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology
DIRECT TESTS
1.Electron Microscopy
Morphology of virus particles
immune electron microscopy
2. Light Microscopy
Histological appearance of
inclusion bodies e.g. Negri bodies in
rabies
3. Viral Genome Detection
Hybridization With Specific
Nucleic Acid Probes
Polymerase chain reaction (PCR)
INDIRECT TESTS
1. Cell Culture
Cytopathic effect (CPE)
Haemabsorption
Immunofluorescence
2. Animals pathogenicity
Disease or death
ELECTRON MICROSCOPY
Viruses may be detected in the following
specimens.
Faeces
Astrovirus,
Rotavirus,
Vesicle Fluid
HSV
VZV
Skin scrapings
Papillomavirus
Rotavirus
Astroviruses
Cell Cultures
Growing virus may produce
1. Cytopathic Effect (CPE) - such as the ballooning of cells or
syncytia formation, may be specific or non-specific.
2. Haemadsorption - cells acquire the ability to stick to
mammalian red blood cells.
Cytopathic Effect (1)
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.
(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)
Cytopathic Effect (2)
Syncytium formation in cell
culture caused by Resp. Syncytial
Virus (top), and measles virus
(bottom).
(courtesy of Linda Stannard, University of Cape
Town, S.A.)
Problems with cell culture
•
Long period (up to 4 weeks) required for result.
•
Often very poor sensitivity, sensitivity depends on a
large extent on the condition of the specimen.
•
Susceptible to bacterial contamination.
•
Susceptible to toxic substances which may be present in
the specimen.
•
Many viruses will not grow in cell culture e.g. Hepatitis
B, Diarrhoeal viruses, parvovirus, papillomavirus.
SEROLOGY
Classical Techniques
Newer Techniques
• Complement fixation test
• Haemagglutination test
• Immunofourescence tech
• ELISA
• Western Blot
TYPICAL SEROLOGICAL PROFILE AFTER ACUTE
INFECTION
Note that during reinfection, IgM may be absent or present at a low level transiently
COMPLEMENT FIXATION TEST
Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement
fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used)
ELISA for HIV antibody
Microplate ELISA for HIV antibody: colored wells indicate reactivity
WESTERN BLOT
HIV-1 Western Blot
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Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive
USEFULNESS OF SEROLOGICAL RESULTS
• Rapid
• Coincide with clinical conditions
• Cheap
• No contamination effect
PROBLEMS WITH SEROLOGY
• Mild infections e.g. HSV genitalis may not produce
a detectable humoral immune response.
• False positive results-cross-reactivity between
related viruses e.g. HSV and VZV
• Atypical antibodies in SLE etc-false positivity
• Immunocompromised
response.
-↓humoral
immune
MOLECULAR METHODS
• Methods based on the detection of viral genome
• Still small role compared to conventional methods.
• Dot-blot, Southern blot are examples of classical
techniques
• PCR, LCR etc newer techniques
Schematic of PCR
Each cycle doubles the copy number of the target