Laboratory Diagnosis of Infectious Diseases

Laboratory Diagnosis of Infectious Diseases

Learning Outcomes

• Describe different types of microscopy and their uses.
• Explain the use of selected stains in microscopy.
• Describe how bacteria and fungi are cultured.
• Explain why different types of culture conditions are required for some bacteria.
• Describe several different types of culture media and know the specific organisms that grow on each.
• Describe how viruses are cultured.

The Diagnostic Process

• Diagnostic microbiology is part of the diagnostic puzzle.
• Starts with patient seeking help:
  • History is taken:
    • Chief complaint.
    • History of present illness.
    • Past, family, and/or social history.
  • Examination.
  • Order tests if relevant.
• Aim is to determine the cause (pathogen).
• May need to isolate (culture) pathogen or not.
• May be able to identify without isolating.
• Isolation of pathogen.
• Identification of pathogen.

Diagnostic Tests

• Five major categories of tests:
  1. Microscopy
  2. Culture
  3. Biochemical tests
  4. Molecular testing
  5. Rapid tests and immunoassays

Microscopy

• Direct microscopic examination purpose:
  • Determine if organisms are present.
  • May provide a preliminary identification of organisms / diagnosis.
  • Can provide relevant information rapidly.
• Particularly relevant:
  1. For sites that are normally sterile
     • e.g., cerebral spinal fluid (CSF)
  2. Where the pathogen is visually distinct
     • e.g., Fungi: morphology of hyphae or conidia
     • e.g., Protozoa: morphology or ova, trophozoites, cysts
  3. Where staining will show relevant characteristics
     • e.g., Cryptococcus capsule
     • e.g., Streptococcus cells in chains

Microscopy - Specimens

• Specimens can be examined directly or fixed and stained.
  1. Direct examination: e.g., wet mount
     • Examine as is, or add things
       • e.g., KOH to dissolve background materials (for fungi)
       • e.g., lactophenol cotton blue or India ink (for Cryptococcus capsule)
  2. Common stains for fixed smears
     • Gram stain: Gram positive or negative
     • Calcofluor white: stains cellulose & chitin
       • for fungi
     • Acid-fast stains: organism retains dye when acidic decolouriser is used
       • e.g., Kinyoun, Ziehl-Neelsen for Mycobacteria, Nocardia

Microscopy - Types

• Types of microscopy:
  • Bright field microscopy
  • Dark field microscopy
  • Phase contrast
  • Fluorescence microscopy
  • Electron Microscopy

Bright Field Microscopy

• Basic components:
  • Light source
  • Condenser (to focus the light)
  • Objective and ocular lenses (for magnification)
• Specimen illuminated by transillumination:
  • Light passes up through condenser to specimen
  • Image magnified by lenses
• Used to examine
  • Heat- or chemically fixed specimens
  • Or wet mounts
• Specimens are usually stained e.g. Gram stain
  • Provides cell shape, Gram reaction, and arrangement
• Maximum magnification x 1000

Dark Field Microscopy

• Basic components:
  • Same objective and ocular lenses as light microscopy
  • Different condenser:
    • Prevents transmitted light from directly illuminating specimen
• Specimen brightly illuminated against a dark background
• Same resolution as light microscopy but increased contrast
• Historically used for detection of Treponema pallidum (syphilis) in chancre fluid
  • Extremely thin cells, not easily seen by light microscopy
  • Largely superseded now by serology testing

Phase Contrast Microscopy

• Uses parallel beams of light
  • One beam out of phase relative to the other by 1/4 wavelength.
  • Light diffracted differently by the specimen and by background
• Difference amplified by phase plate (in phase ring)
• Light appears brighter
• Used for unstained preparations
• Same resolution as light microscopy but increased contrast
• Often used for fecal samples
  • Looking for parasite ova or cysts: internal morphology important for diagnosis

Fluorescence Microscopy

• Uses fluorochromes (fluorescent dye)
  • Absorb short-wave light and emit energy at a higher wavelength (visible).
• Fluorochromes may be:
  • Non-specific
    • e.g., nucleic acid dyes
    • Acridine orange, DAPI
  • Specific
    • Antibody + fluorophore
    • e.g., fluorescein isothiocyanate (FITC) conjugated to an antibody

Fluorescence Microscopy - Specific Fluorochromes

• Specific fluorochromes may work directly or indirectly:
  • Direct immunofluorescence:
    • Detects antigen
    • Fluorophore conjugated to primary antibody
  • Indirect immunofluorescence:
    • Uses two antibodies
    • Fluorophore conjugated to secondary antibody
    • Multiple secondary antibodies can bind
    • More sensitive
• Organisms appear bright against a dark background

Electron Microscopy

• Magnetic coils (electromagnets) used to focus electrons rather than glass lenses
• Very high resolution
  • e.g., viral particles can be seen
• Two types:
  • Transmission electron microscope:
    • Light passes through the specimen: 2D
  • Scanning electron microscope:
    • Electrons bounce off the surface: 3D effect
• Used more for research than for diagnostics

Types of Laboratory Tests: Culture

• Purpose of culturing specimens:
  1. To determine if pathogens (bacteria, fungi, virus, other) are present
  2. To grow enough organism to use in identification tests
  3. To grow enough organism for antibiotic susceptibility testing
• Culture not always appropriate, feasible, or necessary:
  • Not all pathogens can be cultured
    • e.g., Mycobacterium leprae, until recently Treponema pallidum
  • The process may be too slow for some organisms/diseases
  • Faster, cheaper ID methods may already exist
• Isolation of pathogen.
• Identification of pathogen.

Bacterial Culture

• Routine culture conditions:
  • Aerobic (ambient air), 37°C, 24 h, Blood agar
• Does not suit all pathogens
• Culturing conditions to consider:
  1. Nutritional requirements
  2. Gaseous atmosphere
  3. Incubation temperature
  4. Incubation time

Bacterial Culture Conditions: Nutrition

• General categories of culture media:
  1. Non-selective and enriched
  2. Selective (and/or)
  3. Differential
  4. Specialized
• Non-selective and enriched
  • Supports growth of most organisms
  • Including many fastidious organisms e.g.:
    • Blood agar: basal medium enriched with ~5% blood
    • Chocolate agar: basal medium enriched with hemin and isovitalex supplements
    • Thioglycollate broth: can be enriched with hemin and vitamin K to support the growth of anaerobes

Bacterial Culture Conditions: Nutrition - Selective/Differential

• Selective (and/or) Differential media
  • Selective: inhibits the growth of unwanted organisms
  • Differential: shows characteristic reactions
    • Blood agar is differential: shows different hemolysis patterns
  • Examples:
    • MacConkey agar
      • Differentiates Gram-negative lactose fermenters from non-fermenters
      • Contains bile salts and crystal violet to inhibit Gram positives
    • Xylose-lysine deoxycholate (XLD) agar
      • Selective for Shigella and Salmonella
      • Contains sodium deoxycholate to inhibit many non-pathogenic bacteria
      • Contains fermentable sugars and other compounds

Bacterial Culture Conditions: Nutrition - Specialized Media

• Specialized media
  • Composition designed specifically to suit particular microbes
  • Microbes will not grow without specific supplements/components
    • Examples:
      • Buffered charcoal yeast extract (BCYE) agar
        • For Legionella and Nocardia
        • Legionella not actually fastidious: widespread in the environment
        • Just not a fan of laboratory media
        • Medium contains:
          • Charcoal to neutralize hydrogen peroxide produced by Legionella
          • L-cysteine and ferric pyrophosphate: essential nutrients
      • Regan Lowe agar
        • For Bordetella pertussis
        • Contains: charcoal to neutralize hydrogen peroxide and fatty acids
        • And horse blood for enrichment / nutrients
      • Lowenstein-Jensen agar
        • For Mycobacterium tuberculosis
        • Contains: whole eggs, defined salts, glycerol, potato flour, malachite green (to inhibit other bacteria)
        • Poured in slopes in tubes (not plates)
        • The organism takes weeks to grow
        • Agar would dry out; tubes do not

CHROMID

• Commercial media containing specific chromogenic substrates
  • Microbes produce specific colors after incubation
  • Simultaneous culture and (preliminary) ID
• Different media for specific specimens or suspected pathogens
  • e.g., plates available for (suspected)
    • Vibrio
    • ESBL
    • Candida
    • MRSA
    • Salmonella

Bacterial Culture Conditions: Nutrition - Obligate Intracellular Parasites

• Some bacteria will not grow on agar
  • e.g., obligate intracellular parasites: require living cells
    • e.g., Rickettsia, Bartonella, Coxiella, Chlamydia
  • Options:
    • Cell culture (mammalian cells)
      • Also used for viruses
    • Embryonated hens’ eggs
  • Usually, diagnostic methods other than culture are used for these organisms
    • Culture is slow; impractical

Bacterial Culture Conditions: Atmosphere

• Condition and Composition:
  • Ambient: ~78% N2, 21% O2, 0.04% CO_2
    • Suits many common pathogens
    • Suitable for facultative anaerobes: can grow with or without oxygen
  • Anaerobic: typical 85% N2, 10% H2, 5% CO_2
    • Organisms cannot tolerate oxygen; will die
    • Anaerobic jars use gas paks:
      • Reaction consumes O2, generates CO2
      • e.g., Bacteroides, Clostridium
  • Capnophilic: 5% CO_2
    • For capnophilic organisms
    • e.g., Streptococcus pneumoniae, Haemophilus influenzae, Neisseria spp.
  • Microaerophilic: prefer ~5% O2, some also like ~10% CO2
    • Use candle jar (similar to anaerobic jar)
    • e.g., Helicobacter, Campylobacter

Bacterial Culture Conditions: Temperature

• Most common pathogens are incubated at 35 - 37°C
• Examples of exceptions:
  • Campylobacter species 42°C
  • Yersinia enterocolitica 25°C
  • Mycobacterium marinum 30°C
  • Mycobacterium xenopi 42°C

Bacterial Culture Conditions: Time

• Most bacterial pathogens will form colonies on agar after 18-24 h
• Examples of exceptions:
  • Brucella species: 3-7 days to 3 weeks
  • Legionella pneumophila: 2-3 days to 2 weeks
  • Campylobacter species: up to 96 hours
  • Mycobacterium tuberculosis: 15-25 days

Blood Culture Systems

• Used to detect organisms in the blood
  • e.g., septicemia, bacteremia
  • Organisms may be present in very low numbers:
    • Cannot directly plate as there are too few
• Blood inoculated into bottle at bedside and sent to lab
  • Bottles placed in automated machine
  • Signal positive if/when sufficient growth
• Gram stain and culture from positive bottles
• Different bottles for different organisms
  • Routine is aerobic and anaerobic

Virus Culture

• Viral culture not often used for diagnostic testing
  • Replaced by nucleic acid amplification tests
  • Culture still used for research purposes
• Viruses can replicate in the laboratory in:
  1. Cell culture
  2. Embryonated hens’ eggs
     • Was standard prior to the 1950s (before cell culture was available)
     • Still used to make egg-based flu vaccine
  3. Animals
     • No longer used in diagnostics

Viral Culture – Cell Monolayers

• Viruses cultured in monolayers of (usually mammalian) cells
  • Grown on the walls of flasks
  • Specific viruses grow best in specific cell lines
• Cell lines may be continuous or primary:
  • Continuous: can be maintained indefinitely in the lab; immortalized
  • Primary: will die out after 20-80 passages
• Examples and origins of cell types:
  • Vero African green monkey kidney (fibroblasts)
  • HeLa Human cervical adenocarcinoma (epithelial)
  • MRC-5 Human diploid fetal lung (fibroblasts)
  • A549 Human alveolar adenocarcinoma (basal epithelial)
  • Hep-2 carcinoma of larynx (actually these are now HeLa cells)

Viral Culture – Detection of Virus

• Virus detected by observing any of the following:
  1. Cytopathic effect
     • Detected by light microscopy
     • May be very characteristic between a virus and cell type
     • e.g., rounding, focal degeneration, foamy degeneration
  2. Hemadsorption/hemagglutination
     • Add red blood cells: viral hemagglutinin causes RBCs to clump
  3. Fluorescent monoclonal antibody staining (immunofluorescence)
     • Labelled antibody is added
     • Fluorophore absorbs UV light and emits visible light
     • Can be used for cultured virus or for examining specimens directly (without culture)

Culture of Fungi

• Common culture media:
  • Sabouraud’s dextrose agar
    • Can be made selective to inhibit bacteria: reduced pH + high sugar + antibiotics
  • Corn meal agar
    • Useful for suppressing vegetative growth and inducing chlamydospore formation by Candida albicans
• Culture conditions
  • Most fungi grow slowly (slower than bacteria)
  • Some prefer cooler temperatures (< 37°C)
• Identification of fungi relies heavily on morphological features
  • May be visible by microscopy
  • Culture may also show characteristic features:
    • Colony appearance or color, structures

Fungal culture - features used in identification

1. Yeast on agar
   • Single cells, form bacteria-like colonies
2. Mould on agar
   • Furry or fluffy appearance due to aerial hyphae
3. Colony pigmentation
   • e.g., Trichophyton rubrum shows red on the reverse
4. Dimorphic growth
   • e.g., Sporothrix schenckii
     • 30°C: mould form
     • 35°C: yeast form
5. Microscopic appearance of characteristic features e.g. mycelia or conidia
   • Back to the first technique: microscopy!

Fungal Culture

• Epidermophyton floccosum
  • Club-shaped macroconidia, no microconidia
• Microsporum canis
  • Spindle-shaped macroconidia
• Aspergillus flavus
  • Thick-walled conidiophores
• Mucor sp.
  • Ball-shaped clusters of spores (sporangia)