Clinical Microbiology and Immunology

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Chapter 24

• Clinical Microbiology and Immunology

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Growth-Dep. Clinical Diagnostic Methods

• Isolation of pathogens from clinical specimens
  infected tissues or fluids are collected, ex.
  blood, urine, feces, sputum, cerebrospinal fluid,
  pus.
• What steps should be taken to properly obtain
  clinical specimens?
• General purpose media ex. blood agar, supports
  the growth of most aerobic and facultatively
  aerobic organisms.
• Enrichment media the addition of growth factors
  allows metabolically fastidious to grow, ex.
  Neisseria gonorrhoeae.
• Selective media enhance the growth of certain
  organisms while retarding the growth of others.
• Differential media specialized media that allow
  ID of organisms based on their appearance on the
  media.

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Blood Cultures

• Bacteremia presence of bacteria in the blood,
  uncommon in healthy individuals.
• Septicemia blood infection resulting from the
  growth of a virulent organism entering the blood
  from a focus of infection, results in fever and
  chills and possibly septic shock including organ
  failure.

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Urine Cultures

• Common, causal agents often identical to normal
  flora, ex. E. coli. Enteric bacteria account for
  90 of UTIs.
• Significant UTI 105 organisms per ml of
  clean-catch specimen.
• Dipstick tests test for nitrate production, etc.
  related to large s of organisms in the urinary
  tract.

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Fecal Cultures

• Samples must be collected and preserved properly
  and processed ASAP.
• Processing includes inoculation into a variety
  of selective media for isolation of individual
  bacteria.
• Intestinal parasites are IDed microscopically
  rather than by culture.

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Wounds and Abscesses

• Types of wounds include animal or human bites,
  burns, cuts, penetration of foreign objects.
• Wounds must be carefully sampled and results
  carefully processed since normal flora are likely
  to be present.

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Other Specimens

• Genital specimens (ex. for gonorrhea) and
  anaerobic specimens must be specially processed
  in order to preserve the causal agents for proper
  diagnosis.

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Growth on Selective and Differential Media

• Unknown pathogens are first cultured on primary
  isolation media and then subcultured onto
  selective and differential media for further
  diagnosis.
• Eosin-methylene blue (EMB) selective (for gram
  neg. bacteria) and differential (for diff. genera
  of gram neg. bacteria).
• Biochemical tests measure the presence or absence
  of enzymes involved in the catabolism of
  substrate(s) in the differential medium.
• 100s of tests have been developed for clinical
  use, though only 20 are used routinely. (see
  Table 24.3) Some commercial kits contain a
  battery of tests that ID and individual species,
  ex. S. aureus, M. tuberculosis.

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Testing Cultures for Antimicrobial Drug
Sensitivity

• Minimum Inhibitory Conc. (MIC) broth (or
  antibiotic) dilution assay ? highest dilution
  (lowest conc.) of antibiotic that completely
  inhibits growth.
• Kirby-Bauer disk diffusion method (see Fig. 24.8).

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Safety in the Clinical Lab

• 1. Access to lab restricted.
• 2. Decontamination procedures.
• 3. Personnel must be adequately vaccinated.
• 4. All clinical specimens should be considered
  infectious.
• 5. No mouth pipetting.
• 6. Animals handled only by trained lab
  personnel.
• 7. Lab personnel must wear appropriate safety
  equipment lab coat, sealed shoes, rubber
  gloves, masks, eye protection, etc.
• 8. All clinical specimens should be treated as
  if they contain HIV.

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Immunoassays for Infectious Disease

• Immunoassays detect and measure specific immune
  responses to unique molecular portions of
  pathogens, and are used especially on those
  pathogens (ex. viruses) that are difficult to
  grow.
• New pathogen first infects individual ?
  phagocytized ? phagocytes present Ag from
  pathogen to T helper cells ? T helper cells
  recruit and stimulate other phagocytes and B
  cells ? B cells produce Ab (primary Ab response),
  occurs within 5 days, but Ab do not reach peak
  quantities for several weeks.
• After 1st Ag exposure specific immunity via T and
  B cells develops. After 2nd Ag exposure,
  stimulation of these specific immune cells causes
  a rapid and strong immune response, which quickly
  destroys the pathogen. Thus, the immune system
  has memory, which is characterized by and
  increase in Ab titer (used to track infections).

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Ab Titers, Skin Tests, Diagnosis of Infectious
Disease

• Serological tests are set up by making dilutions
  of patient serum and determining the highest
  dilution at which the Ag-Ab rxn. occurs.
• 2 situations 1. Must see a rise in Ab titer to
  conclude active infection (having Ab present
  could mean prior infection or immunization and
  not current infection). 2. The mere presence of
  Ab is sufficient to indicate infection, ex. HIV.
• Not all infections result in production of
  systemic Ab, ex. Neisseria gonorrhoeae.
• Skin testing ex. tuberculin text intradermal
  injection of a soluble extract of Mycobacterium
  tuberculosis ? pos. inflammatory rxn. at site of
  injection current infection or previous
  exposure to M. tuberculosis.

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Polyclonal and Monoclonal Ab

• Antiserum (serum containing Ab) contains a
  mixture of different Ab directed at the numerous
  determinants on an Ag. This mixture of serum Ab
  is called polyclonal Ab and, though they protect
  the host, they do not provide reproducible
  results for use in clinical diagnostic testing.
• Only a few Ab are directed toward each Ag
  determinant. Ab produced by a single B cell (or
  in vitro clones of a single B cell) monoclonal
  Ab. Monoclonal Ab do provide reproducible
  results for clinical testing ex. for
  immunological typing of bacteria, pregnancy
  testing and blood typing.