Introduction to Medical Microbiology

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Introduction to Medical Microbiology

• Infectious disease
• Interaction between host and microbe
• Commensal close relationship
• Microbes benefit, host unharmed (usually)
• Normal flora, indigenous microbiota, etc.
• Human Microbiome Project (HMP)
• Colonization vs. disease
• Transient colonization
• Permanent colonization
• Disease

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Introduction

• Pathogenic causes disease
• opportunistic pathogen
• strict pathogen
• virulence
• virulence factor (VF)
• exogenous vs. endogenous infection
• normal flora

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Normal Flora of Humans
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Laboratory Dx of Infectious Diseases

• Microscopy chapter 14
• In vitro culture chapter 15
• Molecular diagnosis chapter 16
• Serologic diagnosis chapter 17

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Microscopy

• Microscopic methods used for
• Detection of microorganisms in clinical specimens
• Identification of microorganisms
• Preliminary / presumptive ID
• Definitive ID

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Microscopic Methods

• Brightfield microscopy
• Oil immersion used for best resolution (0.2 µm)
• Best for stained organisms
• Darkfield microscopy
• Improved resolving power (0.02 µm)
• See skinny bacteria (Treponema pallidum)

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Microscopic Methods

• Phase contrast microscopy
• 3D effect useful for living cells
• Cell cultures for virus isolation
• Fluorescence microscopy
• Fluorochrome dyes emit colored light in UV
• Fluorescent antibodies identify microbes
• Electron microscopy
• Not used in clinical labs
• See viruses

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Direct Examination

• wet mount of fresh specimen
• Trichomonas vaginalis, Entamoeba histolytica
• KOH prep of skin, hair, nails
• Dx ringworm (fungi)

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Direct Examination

• India ink
• Detect Cryptococcus in spinal fluid
• Iodine
• Identify parasites in fecal specimens

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Differential Stains

• Gram stain most important for bacteria
• Acid-fast stain Mycobacteria
• Kinyoun, Ziehl-Nielson, auramine methods
• Modified acid-fast stain
• Nocardia, Cryptococcus, etc.
• Wright Giemsa stain blood smears
• protozoa, worms

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Differential Stains

• Silver stain (Gomori methenamine silver GMS)
• Stains fungi in tissue
• Trichrome, iron hematoxylin
• Used for protozoa

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Fluorescence Stains

• calcofluor white
• Stains fungi
• Auramine-rhodamine
• stains acid-fast bacteria
• Fluorescent antibody stain
• Many applications - Legionella, Chlamydia, etc.

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Importance of Microscopy

• definitive Dx by direct microscopy
• worm infections, most protozoa, some fungi
• presumptive identification
• gonorrhea, meningitis, pneumonia gram stain
• acid-fast stain for TB, leprosy
• fluorescent Ab tests

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Dx by In Vitro Culture

• Often the definitive method for Dx of disease
• Especially bacterial diseases
• Types of media
• Enriched nonselective (BAP, CHOC, etc.)
• Selective and / or differential
• MAC, MSA, Hektoen, etc.
• Specialized for specific organisms
• Thayer-Martin, BCYE, etc.
• Cell culture used to isolate viruses,
  intracellular bacteria (e.g., Chlamydia)

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Molecular Dx Methods

• Detection of microbial genes (RNA or DNA)
• RFLP digest DNA with restriction enzymes ?
  electrophoresis ? pattern of fragments
• gene probe based on DNA hybridization
• in situ hybridization performed on clinical
  specimen
• PCR amplifies DNA
• Real-time PCR can quantify HIV

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Using a Gene Probe to Detect Pathogens
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Dx by Serological Methods

• Antibodies (Abs) bind to microbial antigens (Ag)
• Many applications / methods for visualizing Ab-Ab
  reactions
• agglutination (clumping) of cells by Abs
• identify Salmonella, E. coli O157H7, etc.

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Serological Methods - Immunoprecipitation
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Serological Methods - Immunoassays

• solid phase immunoassays
• ELISA uses enzyme detection method
• Immunofluorescence uses fluorescent Ab
• Western blot identifies individual proteins

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ELISA
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Immunofluorescence Assay
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Western Blot

• separate proteins by electrophoresis
• transfer (blot) to filter paper
• incubate with Ab
• incubate with enzyme- conjugated anti-human Ab
• add enzyme substrate, look for color change

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Serology / serodiagnosis

• detect Abs vs. pathogen in patient serum (vs.
  organism itself)
• dilute patient serum ? mix with test Ag
• Ab titer greatest dilution with test
• Abs in serum not proof of disease
• titer Abs twice (acute convalescent samples)
• 4X rise in titer suggests active infection
• many diseases are Dx by serology
• HIV / AIDS, syphilis, viral diseases, etc.

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Sensitivity Specificity

• Sensitivity how few mics. can be detected
• 100 sensitive test is positive in every case of
  disease (true positives)
• insensitive test has lots of false negatives
• determine in lab by using test on dilutions of
  pathogen (sens. limit of detection)

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Sensitivity

• determine sensitivity in clinical practice by
  testing patient samples using two or more methods

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Sensitivity Specificity

• Specificity ability to differentiate the
  pathogen from other similar organisms
• 100 specific test always negative in absence of
  the pathogen (true negatives)
• poor specificity means false positives

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Sensitivity Specificity
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