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Chapter 35
Clinical Microbiology
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Specimens
• clinical microbiologist– major function is to isolate and identify
microbes from clinical specimens rapidly
• clinical specimen– portion or quantity of human material that
is tested, examined, or studied to determine the presence or absence of specific microbes
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Figure 35.1
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Working with specimens• safety concerns
– Standard Microbiological Practices have been established by the Centers for Disease Control and Prevention (CDC)
• specimen should:– represent diseased area and other appropriate sites– be large enough for carrying out a variety of
diagnostic tests– be collected in a manner that avoids contamination– be forwarded promptly to clinical lab– be obtained prior to administration of antimicrobial
agents, if possible
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Collection
• numerous methods used
• choice of method depends on specimen
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Skin and mucous membranes• sterile swab
– greater risk of contamination
– limited volume can be collected
Figure 35.2 (a)
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Body fluids, etc.• needle aspiration
– blood and cerebrospinal fluid
• intubation– insertion of tube into body canal or hollow
organ– stomach specimens
• catheter– tubular instrument used to withdraw or
introduce fluids into body cavity– urine
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Figure 35.2 (d)
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Body fluids…
• clean-catch method– collection of midstream portion of
urine
• sputum cup– sputum
• mucous secretion expectorated from lungs, bronchi, and/or trachea
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Figure 35.2 (e)
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Handling
• proper labeling– patient information
– possible diagnosis
– current antimicrobial therapy
– physician information
– type of specimen
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Transport
• should be timely
• may involve use of special media that preserve microbes in specimen
• special treatment may be needed if anaerobe is to be identified in specimen
• temperature control may be needed
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Transport Media
• may require supplementation to support microbial survival or inhibit normal flora– e.g., use of antibacterial antibiotics
such as penicillin to ensure recovery of fungi
– e.g., use of polyvinyl alcohol-based preservatives for fixation of ova and parasites in clinical specimens
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Figure 35.3
used tocollectspecimen
specimen transferredfrom syringe to vial
medium invial retardsoxygendiffusion
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Select Agents Legislation
• governs policy for possession, use and transport (beyond collection point) of potential biothreat agents
• requires specific packaging and approvals for transport of specimens containing these organisms
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Standard Microbiological Practices
• are minimum guidelines that should be supplemented with other precautions based on exposure risks and lab biosafety level regulations
• goal is to protect workers from contact with agents by their taking precautions and by their working in a safe laboratory environment
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More on Standard Microbiological Practices
• e.g., workers can limit their contact with microbes by not eating or smoking in lab and by preventing injuries caused by sharp objects
• e.g., coverings such as lab coats and bandages should be used
• e.g., workers should know how to use emergency eye wash and shower stations
• e.g., work space should be disinfected• e.g., hands should be washed throughly afer
any exposure and before leaving lab
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Biosaftety Levels
• recommended guidelines for additional precautions reflect the laboratory’s biosafety level (BSL)– BSL 1 – not known to cause disease in
healthy adults– BSL 2 – associated with human disease– BSL 3 – disease may have serious or lethal
consequences– BSL 4 – agent poses high risk of life-
threatening disease
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Identification of Microorganisms from
Specimens• preliminary or definitive identification of
microbe based on numerous types of diagnostic procedures– microscopy– growth and biochemical characteristics– immunologic tests– bacteriophage typing– molecular methods
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Microscopy• wet-mount, heat-fixed, or chemically
fixed specimens can be examined• choice of microscopy depends on possible
pathogen– e.g., dark-field microscopy
• detection of spirochetes in skin lesions associated with syphilis
– e.g., fluorescence microscopy• direct microscopic of specimens to detect fungi
• stains often used– Gram stain and acid fast stain
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Monoclonal Antibodies (MAB)
• produced by hybridoma cells
• recognize a single epitope– fluorescently-labeled MABs used
diagnostically • technique has replaced use of polyclonal
antisera for culture confirmation
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Immunofluorescence
• process in which fluorescent dyes are exposed to UV, violet, or blue light to make them fluoresce
• dyes can be coupled to antibody molecules with changing antibody’s ability to bind a specific antigen
• can be used as direct fluorescent-antibody (FA) technique or indirect fluorescent-antibody (IFA) technique assay
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Figure 35.4 (a)
FA technique
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Figure 35.4 (b)
IFA technique
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Growth and Biochemical Characteristics
• techniques used depend on nature of pathogen
• for some pathogens, culture-based techniques have limited use
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Viruses• identified by:
– isolation in living cells
– immunodiagnostic tests
– molecular methods
• replication in culture detected by:– cytopathic effects
• morphological changes in host cells
– hemadsorption• binding of red
blood cells to surface of infected cells
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Fungi
• cultures used to recover fungus from patient specimens
• identification– direct microscopic (fluorescence)
examination– immunofluorescence– serological tests (for some)– rapid identification methods (most yeasts)
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Parasites
• culture-based techniques not commonly used• identification by microscopic examination of
clinical specimens– definitive diagnosis obtained by identification and
characterization of ova, trophozoites and cysts in the specimen
• histological staining of blood, negative staining of other body fluids and immunofluorescence staining are routinely used in identification of parasites
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Bacteria• most bacteria:
– culturing involves use of numerous kinds of growth media
• can provide preliminary information about biochemical nature of bacterium
– additional biochemical tests used following isolation
• some bacteria are not routinely cultured– rickettsias, chlamydiae, and mycoplasmas– identified with special stains, immunologic
tests, or molecular methods such as PCR
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Table 35.1
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Table 35.2
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Figure 35.5 (a)
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Figure 35.5 (b)
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Rapid Methods of Identification
• manual biochemical systems– e.g., API 20 E system
• mechanized/automated systems
• immunologic systems
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Figure 35.6
API 20E system
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Figure 35.7
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Biosensors
• based on the linkage of traditional antibody-based detectrion systems to sophisticated reporting systems
• can be based on– microfluidic antigen sensors– real time PCR– highly sensitive spectroscopy systems– liquid crystal amplification of microbial
immune complexes
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Monoclonal Antibodies (MaBs)
• numerous applications – e.g., tissue typing– e.g., identification and study of microbes,
tumors and surface antigens– e.g., identification of functional populations
of different T cell types– e.g. can be conjugated with molecules to
provide colorimetric, fluorometric or enzymatic activity to report MAB binding to specific antigens
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Table 35.3
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Bacteriophage (phage) Typing
• only done by CDC and certain labs
• based on specificity of phage surface molecules for host cell surface molecules
• phagovar– collection of strains sensitivity to
certain collection of phage types
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Molecular Methods and Analysis of Metabolic
Products
• comparison of proteins
• nucleic acid-based detection methods
• gas-liquid chromatography
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Molecular Methods and Analysis of Metabolic
Products
• molecular methods being wide used– nucleic acid probes
– gas-liquid chromatography
– plasmid fingerprinting
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Nucleic acid-based detection methods
• the use of cloned DNA as a probe– based on ability of probe to hybridize with
complementary sequences in test specimens
• hybridization reaction can be used with a variety of preparations– e.g., bacterial colonies– e.g., purified DNA preparations– e.g., clinical specimens such as pus
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Figure 35.8
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Ribotyping
• used to identify bacterial genera • based on high level of 16S rRNA
conservation among bacteria• rRNA encoding genes or fragments are
amplified by PCR • the nucleotide sequence of the amplified
DNA is determined and compared with those in the National Center for Biotechnology (NCBI)
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Gas-liquid chromatography
• used to identify:– specific microbial metabolites– cellular fatty acids– products of pyrolysis of whole bacterial cells
• involves extracting compounds from cells or growth medium and injecting extract into gas-liquid chromatograph system
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Plasmid fingerprinting
• characterizes bacteria based on number of plasmids and their molecular weight
Figure 35.9
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Immunological Techniques
• detection of antigens or antibodies in specimens– especially useful when cultural methods are
unavailable or impractical or antimicrobial therapy has been started
• use of immunological systems has many advantages– e.g., each to use– e.g., give relatively rapid reaction endpoints– e.g., are sensitive and specific
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Clinical Immunology
• many antibody-antigen interactions that occur in vivo can also be used under controlled laboratory conditions for (in vitro) diagnostic testing
• test selection and timing of specimen collection are essential to the proper interpretation of immunologic tests
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Serotyping
• use of serum antibodies to detect and identify other molecules
• can be used to differentiate serovars or serotypes of microbes that differ in antigenic composition of a structure or product– e.g., since virulence factor genes often occur
in same clone with genes for antigenic cell wall material, it is possible to serologically identify pathogen by testing for cell wall antigens
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More on Serotyping
• e.g., there are ~90 different strains of Streptococcus pneumoniae, each differing in its capsular material
• Quellung reaction– swelling of capsular material following
addition of antisera specific to a capsular type
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Figure 35.10
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Agglutination
• agglutinates– visible clumps or aggregates of cells or
particles
• e.g., Widal test– diagnostic for typhoid fever
• e.g., latex agglutination tests– pregnancy test
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Viral hemagglutination
Figure 35.11
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Figure 35.12
Agglutination tests
titer = reciprocal of highestdilution positive for agglutination
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Complement Fixation
• binding of complement to an antigen-antibody complex
• basis of diagnostic tests that determine if antibodies to an antigen are present in patient’s serum
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Figure 35.13
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Enzyme-Linked Immunosorbent Assay
• ELISA
• can be used to detect antigens in a sample
• can be used to detect antibodies in a sample
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Figure 35.14
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Immunoblotting (Western Blot)
• procedure– proteins separated by electrophoresis– proteins transferred to nitrocellulose sheets– protein bands visualized with enzyme-tagged
antibodies
• sample uses– distinguish microbes– diagnostic tests– determine prognosis for infectious disease
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Figure 35.15
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Immunoprecipitation
• used to detect soluble antigens• binding of bivalent or multivalent
antibodies to antigen forms lattice that precipitates
• lattice formation occurs only when there is an optimal ratio of antigen to antibody
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Figure 35.16
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Immunodiffusion
• precipitation reaction that occurs in agar gel medium
• two commonly used techniques– single radial immunodiffusion (RID)
assay
– double diffusion agar assay (Ouchterlony technique)
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Figure 35.17 (a)
used to quantifyantigen
RID
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Figure 35.17 (b)
precipitatedimmunecomplexes
used todetermineidentityof antigen
Double diffusion agar assay
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Immunoelectrophoresis
• antigens first separated by electrophoresis according to charge
• antigens visualized by precipitation reaction
• has greater resolution than immunodiffusion assays
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Figure 35.18
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Flow Cytometry
• flow cytometry– detects organisms in clinical samples– detection based on cytometric parameters or by use
of fluorochromes• fluorochromes often bound to antibodies or
oligonucleotides
• flow cytometer– forces suspension of cells through laser beam and
measures amount of light scattering of fluorescence– can detect heterogeneous microbial populations with
different responses to antimicrobial treatments
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Radioimmunoassay (RIA)
• purified antigen labeled with radioisotope competes with unlabeled standard for antibody binding
• amount of radioactivity associated with antibody is measured
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Susceptibility Testing• testing for susceptibility to antimicrobial
agents• can be used to identify microbe• particularly useful for determining
proper therapy• determined by:
– dilution susceptibility tests– disk-diffusion tests (Kirby-Bauer method)– E test
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Computers in Clinical Microbiology
• improve efficiency and increase speed and clarity with which results can be reported to physicians
• major uses– test ordering– result entry– analysis of results– report preparation
• also useful for lab management