DETECTION OF PATHOGENS (DIAGNOSTIC BACTERIOLOGY

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DETECTION OF PATHOGENS(DIAGNOSTIC BACTERIOLOGY
VIROLOGY)

• Assist Prof. Dr
• Syed Yousaf Kazmi

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OBJECTIVES

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

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DETECTION OF BACTERIAL INFECTIONS

• For diagnosis of various bacterial infections
• Microscopy morphology
• Staining
• Cultural characteristics
• Metabolism
• Resistance
• Bio chemical properties

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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

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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
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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
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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

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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

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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

?- hemolysis
Proteus swarming
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METABOLISM

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

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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
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FERMENTATION AND BIOCHEMICAL TESTS
API 10S
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RESISTANCE TO ANTIMICROBIALS-ANTIBIOGRAM

• Sensitivity profile of org
• Helps in identification
• Genetic resistance or sensitivity

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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

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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

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RAPID BACTERIAL IDENTIFICATION TESTS

• Rapid Serology based tests
• Rapid presumptive identification
• Helps in management
• Strep A throat test, Clostridium difficile rapid
  toxin detection test

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VIRAL DIAGNOSTIC TESTS

• 1. Direct Examination
• 2. Indirect Examination (Virus Isolation)
• 3. Serology

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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)

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INDIRECT TESTS

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

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ELECTRON MICROSCOPY

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

Rotavirus
Astroviruses
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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.

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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)
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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.)
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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.

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SEROLOGY

• Classical Techniques
• Complement fixation test
• Haemagglutination test
• Immunofourescence tech

• Newer Techniques
• ELISA
• Western Blot

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TYPICAL SEROLOGICAL PROFILE AFTER ACUTE INFECTION

• Note that during reinfection, IgM may be absent
  or present at a low level transiently

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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)
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ELISA for HIV antibody

• Microplate ELISA for HIV antibody colored wells
  indicate reactivity

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WESTERN BLOT

• HIV-1 Western Blot
• Lane1 Positive Control
• Lane 2 Negative Control
• Sample A Negative
• Sample B Indeterminate
• Sample C Positive

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USEFULNESS OF SEROLOGICAL RESULTS

• Rapid
• Coincide with clinical conditions
• Cheap
• No contamination effect

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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.

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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

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