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Clinical Microbiology
Lecture 13
Bio 3124
Identification of pathogens is critical

Use appropriate treatments
• Antibiotics don’t work on all bacteria
• Many bacteria are now drug-resistant
• Proper choice of antibiotics necessary
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Required for proper prognosis
• Streptococcal pharyngitis might appear like a mild
infection
• Could cause serious heart, kidney complications
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Track spread of disease
• Allows faster treatment of others infected
• Allows identification of cause of infection
Clinical Microbiology, Specimens
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Clinical microbiology
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isolate and identify microbes from clinical specimens rapidly
Clinical specimen
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human material tested to determine the presence or absence
of specific microbes
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specimen should:
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represent diseased area
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Sufficient quantity to do a variety of diagnostic tests
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collected aseptically to avoid contamination
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obtained prior to administration of antimicrobial
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forwarded promptly and properly to a clinical lab
Clinical Techniques
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Definitive identification relies on:
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Microscopy: Morphological assessment, fluorescence
microscopy for specific detection
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Biochemical techniques, require growing pathogen
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Immunologic tests: use of antibodies, Elisa, Agglutination
test, complement fixation, immunoprecipitation based
tests eg. radial immunodiffusion, double diffusion
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Molecular techniques: PCR, QPCR, Ribotyping, RFLP
also phage typing
Microscopy
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wet-mount, heat-fixed, or chemically fixed specimens
can be examined
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choice of microscopy depends on pathogen
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Morphological, Gram reaction, spore bearing
e.g., dark-field microscopy
• detection of spirochetes in skin lesions associated
with syphilis
e.g., fluorescence microscopy
stains often used
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Simple stains, Gram stain and
acid fast stain (Zeil-Neelsen)
for mycobacteria
Immunofluorescence microscopy
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fluorophores are exposed to UV, violet, or blue light to
make them fluoresce

coupled to antibody molecules without changing
antibody’s ability to bind a specific antigen
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can be used as direct fluorescent-antibody (FA) assay
or indirect fluorescent-antibody (IFA) assay
Direct and indirect Immunofluorescence
detection
Cytomegalovirs
infected cells
Herpes simplex infected cells
Growth and Biochemical Characteristics
Viruses
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Sample used to infect cells in tissue
culture
identified by:

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immunodiagnostic tests
molecular methods
replication in culture detected by:
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Polio induced CPE
cytopathic effects
• morphological changes in host cells
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Syncytium
hemadsorption
• binding of red blood cells to surface of
infected cells (hemagglutinin producing viruses)
Hemadsorption
hPIV3 induced cell fusion
Biochemical Identification
Bacteria
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most bacteria:
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culturing in growth media
• can provide preliminary information about biochemical nature
of bacterium

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additional biochemical tests used following isolation
some bacteria are not routinely cultured

rickettsias, chlamydiae, and mycoplasmas
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identified with special stains, immunologic tests, or
molecular methods such as PCR
Biochemical tests
Examples of biochemical tests

Biochemical properties
represent genetic relatedness
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Database of biochemical
capabilities

Can be used to identify
bacteria

Growing on different substrates
as sole carbon source
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Biochemical signature of test
organism
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Compare with database to find
the best match

See Flowcharts (algorithms) for
ID’ing schemes
Lac- = dark blue
Hemin
NAD
NAD+Hemin
H. Influenza requires NAD and hemin
Lac+ = yellow
Oxidase test
N. meningitidis is has cyt C
Identification
scheme for
G+ bacteria
Identification
scheme for Gbacteria
Rapid Methods of Identification
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manual biochemical systems
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mechanized/automated systems:
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e.g., API 20 E system
Biolog phenotypic arrays
immunologic systems
Reference book: Bergey's manual of determinative bacteriology / [edited by] John G. Holt et al., Baltimore : Williams & Wilkins,
c1994
API 20E system
Checks for 20 metabolic markers and generates codes to match
known bacteria
Biolog phenotypic identification array

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More than 2500 bacterial, fungi and
yeast species
Based on colorimetric detection of
growth
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Use of a redox dye coupled to ETC
95 metabolic markers
Rapid 4-16 hours
Computer based database match
Accuracy
Biolog Inc website
Each well contains one carbon source
growth results in color change
Bacteriophage Typing
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based on specificity of phage surface
molecules for host cell receptors
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Narrow host range for a collection of
phages can be used to typify the hosts
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Phagovars
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collection of strains sensitive to
certain collection of phage types
Molecular Methods
 Nucleic
acid-based detection methods
• Ribotyping
• Diagnostic PCR
• Probe hybridization (RFLP analysis)
 Analysis
of proteins: PAGE and Western
Ribotyping
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To identify bacterial genera
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based on high level of 16S rRNA conservation among
bacteria
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PCR amplification of rRNA genes or fragments
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Sequence of amplified DNA compared with those in the
National Center for Biotechnology (NCBI)
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Strain is determined on the basis of sequence homology
Diagnostic PCR
Amplifies small fragment of DNA
 Allows detection of tiny numbers of bacteria
 Size of fragment can indicate species, strain
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Clostridium botulinum toxin genes
Samples
 Restriction analysis can further
indicate strain
 Single nucleotide differences
affect ability to be cut by
restriction enzymes
Real-Time Quantitative PCR
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Detection of slow growing viruses,
latent infections
RT-PCR
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Reverse Transcriptase makes cDNA from
RNA followed by PCR
qRT-PCR: quantitative “real-time” PCR
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Quenched fluorescent probe to amplified
DNA
• Probe is degraded as amplification occurs
• Separates quencher from fluorophore
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Measure appearance of fluorescence
• Faster the gain, the more template present
• Indicates more viral RNA or DNA in sample
Animation: Real-time PCR
Genomic fingerprint: RFLP analysis
RFLP (restriction fragment
length polymorphism)
• Genomic DNA restriction
• Electrophoesis
• Chemical denaturation
• Southern transfer: transfer of
ssDNA onto nylon membranes
• Probe hybridization:
short complementary DNA labelled
with 32P or tagged with an enzyme
eg Alkaline phosphatase
• Detection: chromogenic
reaction or by X-ray
autoradiography
- Related strains show similar RFLP patterns
Animation: Southern blot and RFLP analysis
Immunologic Techniques

detection of antigens or antibodies in specimens
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especially useful when culture methods are
unavailable or impractical
use of immunological techniques has many
advantages

easy to use

rapid reaction endpoints
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sensitive and specific
Agglutination
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agglutinates
 visible immune complexes formed by cross-linking cells
with antibodies
 eg., Agglutination of S. thyphi by serum from infected
patient (Widal Test)
 Can be used to titre the serum antibodies for a pathogen
titer = reciprocal of highest
dilution positive for agglutination
Complement Fixation
Complement fixation: binding complement to an
antigen-antibody complex; compl. used up
 basis of diagnostic tests that determine if
antibodies to an antigen are present in patient’s
serum
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Animation: Complement fixation
Enzyme-Linked Immunosorbent Assay
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Done in two ways:
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Direct
Indirect
Direct: directly detecting
antigens in a sample
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also called antigen capture
ELISA
Pathogen specific Ab
immobilized
Patient serum passed
Secondary antibody detects
binds to captured Ag
Enzyme-Linked
Immunosorbent Assay
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Indirect: detecting
antibodies in a patients
serum
interpreted as indirect
evidence for exposure to a
pathogen
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Known antigen immobilized
Primary Ab detects Ag
Enz-linked secondary Ab
detects primary Ab
Can be quantified
Animation: Direct and Indirect ELISA
Immunoblotting (Western Blot)
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procedure
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proteins separated by SDS-PAGE
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proteins transferred to nitrocellulose sheets
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protein bands visualized with enzyme-tagged antibodies
Examples:
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distinguish microbes
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diagnostic tests
Extra slides if you like to seek for more
Monoclonal Antibodies (MAB) and
fluorescence microscopy
produced by hybridoma
cells
 recognize a single
epitope

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fluorescently-labeled
mABs used
diagnostically
• technique has replaced
use of polyclonal antisera
for culture confirmation
Click to read more about hybridoma technology