1. DETECTION OF PATHOGENS (DIAGNOSTIC BACTERIOLOGY & VIROLOGY)
Assist Prof. Dr
Syed Yousaf Kazmi

2. OBJECTIVES
Explain the basic concept of diagnostic microbiology including classical bacteriology and newer techniques
Identify the available diagnostic methods

3. DETECTION OF BACTERIAL INFECTIONS
For diagnosis of various bacterial infections
Microscopy & morphology
Staining
Cultural characteristics
Metabolism
Resistance
Bio chemical properties

4. 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

5. MICROSCOPY STAINED SPECIMEN EXAM Gram Stain-Most bacteria ZN Stain-AFB
Albert Stain-metachromatic granules of C diphtheriae
Geimsa Stain-Chlamydia
Gram Stain
Albert Stain

6. 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

7. IMMUNOFLUORESCENT ANTIBODY (IF) STAINING
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

8. CULTURE OF SPECIMEN
Culture of bacteria- Gold standard for identification
Usually 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

9. 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

10. METABOLISM Growth requirements Aerobic & Anaerobic
Micro-aerophilic, Carboxyphilic etc.
Growth factor requirements e.g. Haemophilus influenzae factor X, V

11. 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

12. FERMENTATION AND BIOCHEMICAL TESTS
API 10S

13. RESISTANCE TO ANTIMICROBIALS-ANTIBIOGRAM
Sensitivity profile of org
Helps in identification
Genetic resistance or sensitivity

14. OTHER TESTS Bacteriophage Test Animal pathogenicity tests
Mostly for epidemiological testing
Not routinely done
Reference laboratories only
Animal pathogenicity tests
Limited value
Few bacterial infections

15. 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

16. RAPID BACTERIAL IDENTIFICATION TESTS
Rapid Serology based tests
Rapid presumptive identification
Helps in management
Strep A throat test, Clostridium difficile rapid toxin detection test

17. VIRAL DIAGNOSTIC TESTS
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology

18. 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)

19. INDIRECT TESTS 1. Cell Culture Cytopathic effect (CPE) Haemabsorption
Immunofluorescence
2. Animals pathogenicity Disease or death

20. ELECTRON MICROSCOPY
Viruses may be detected in the following specimens.
Faeces Rotavirus, Astrovirus,
Vesicle Fluid HSV
VZV
Skin scrapings Papillomavirus
Rotavirus
Astroviruses

21. 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.

22. 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)

23. 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.)

24. 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.

25. SEROLOGY Classical Techniques Newer Techniques
Complement fixation test
Haemagglutination test
Immunofourescence tech
Newer Techniques
ELISA
Western Blot

26. TYPICAL SEROLOGICAL PROFILE AFTER ACUTE INFECTION
Note that during reinfection, IgM may be absent or present at a low level transiently

27. 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)

28. ELISA for HIV antibody
Microplate ELISA for HIV antibody: colored wells indicate reactivity

29. WESTERN BLOT HIV-1 Western Blot Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive

30. USEFULNESS OF SEROLOGICAL RESULTS
Rapid
Coincide with clinical conditions
Cheap
No contamination effect

31. 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 -↓humoral immune response.

32. 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

33. Schematic of PCR
Each cycle doubles the copy number of the target