Diagnosing infections
Module 9
© 2021 McGraw Hill. All rights reserved. Authorized only for instructor use in the classroom. Amy P. Rossi, ScM
No reproduction or further distribution permitted without the prior written consent of McGraw Hill.
Images from Microbiology: A Systems Approach, Cowan, 6th Edition
Step 1 : History and physical exam
► History –
• duration (bacterial vs viral)
• discharge/pus (bacterial vs viral)
• location of symptoms (lower vs upper respiratory infection)
• sick contacts
• hobbies and occupation (environmental exposures)
• sexual history
• social history - smoker (non-infectious bronchitis or opportunistic infection),
alcoholism (aspiration pneumonia)
► Physical Exam – signs of infection, purulent discharge (bacterial vs viral),
decreased breath sounds (lower vs upper respiratory), fever, low oxygen
saturation
Step 2 : Specimen Collection
Specimen Collection: Culturette
Cultivation of Specimen
► Use of specialized media that reveal identifying characteristics such as colony
appearance, motility, gas requirements
► Selective media:
• Used to enrich the specimen
• Used to encourage the growth of only the
suspected pathogen
► Differential media:
• Used to identify definitive characteristics and fermentation patterns
Step 3: Identifying Genus and Species
► Phenotypic:
• Observation of microbe’s
microscopic and macroscopic
morphology, physiology, and
biochemical properties
► Immunologic:
• Analysis of microbe using
antibodies, or of patients’
antibodies using prepackaged
antigens
► Genotypic:
• Analysis of microbe’s DNA or RNA
Phenotypic methods
► “quick and dirty” e.g. microscopic urinalysis
► Gram stain and acid-fast stain
• Morphology and arrangement
• Narrow down potential causative organisms
• Presence of multiple organisms can make interpretation difficult
► Culturing
• Not all organisms can be cultured
• Time consuming, e.g. TB takes weeks to grow on culture plates
• Allows for a pure culture to be obtained using selective media
• Differential medial allows identification of definitive characteristics
Biochemical Testing
► Observe reactions from combining unknown bacteria with
nutrients or substrates
► Observe change in color, viscosity, or gas production
► Indirect evidence of enzyme present in bacteria
Dichotomous Keys
Determining Clinical Significance of Cultures
► Important to determine if an isolate is clinically important or merely
a contaminant or normal biota
• Focus on the number of microbes in a specimen
• A few colonies of Escherichia coli can be normal biota
• A few hundred colonies of E. coli can mean an active infection
• The presence of a single colony of a true pathogen, such as Mycobacterium
tuberculosis in a sputum culture or an opportunist in sterile sites is highly
suggestive of its role in disease
Immunological methods – Blood as a window to the body
► Alterations in WBC count
• Eosinophilia – parasite?
• Neutrophilia – bacteria/fungi >> virus
► Serology (includes serum, urine, CSF, saliva, whole tissue)
• Does the patient have antibodies to the suspected organism
• IgM vs IgG –> new vs remote/latent infection
• IgE –> parasites, allergies
► Signs of inflammation
• C-reactive protein (non-specific indication of inflammation)
• Erythrocyte sedimentation rate
Antibody-based tests – agglutination
► Confirm identity of
organism (direct)
► Detect antibodies to
particular organism
(indirect)
► Quantify antibody titers
Antibody-based tests – immunochromatographic test
► Identify unknown organism
• e.g. Rapid strep test
► Identify presence of antibodies
• e.g. Rapid HIV test
► Requires additional confirmatory
testing
Genotypic methods – Polymerase Chain Reaction
► Quickly identify fastidious, slow growing organisms
► Identify viruses without culturing (time and labor intensive)
► Determine drug resistance by detecting known mutations
► Multiple similar assays with the principle of amplifying unique
nucleic acid sequences for a specific organism so we can detect it
in a specimen
Genotypic methods – Polymerase Chain Reaction
► Principle of test
• Isolate DNA and/or RNA from specimen (RNA -> cDNA)
• Use short sequences of DNA that bind to specific, unique regions of the
organism’s genome (i.e. primers)
• Using special enzymes and nucleic acids, the sample goes through cycles
of amplification driven by changes in temperature
• In conventional PCR, the amplified DNA is run on a gel that separates DNA
fragments by size. A positive result will have a band while a negative one
won’t
• In Real Time or quantitative PCR, fluorescence is detected with each
amplification and can be quantified by comparing it to a reference
Mass Spectrometry – MALDI TOF
► Ionizes protein fragments and identified the ions based on their mass-to-charge
ratio
► Results are compared to a database of known organisms, each with a unique
signature
► Multiple unknown species can be identified in a single sample
► Antibiotic susceptibility
► Results within minutes
► Does not require knowing potential organisms vs PCR
Mass Spectrometry – MALDI TOF
Sensitivity vs Specificity
► Sensitivity: ► Specificity:
• The detection of even minute • The property of a test to focus on
quantities of antibodies or antigens only a certain antibody or antigen
in a specimen
• Does not react with unrelated or
• Reflects the degree to which a test distantly related antigens
will detect every positive person
• The degree to which a test does
not falsely detect people who do
not have a condition
▽ false negatives ▽ false positives