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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 necessar y Required for proper prognosis

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Clinical microbiology

Clinical Microbiology

Lecture 13

Bio 3124


Identification of pathogens is critical
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

  • Required for proper prognosis

    • Streptococcal pharyngitis might appear like a mild infection

      • Could cause serious heart, kidney complications

  • Track spread of disease

    • Allows faster treatment of others infected

    • Allows identification of cause of infection


Clinical microbiology specimens
Clinical Microbiology, Specimens

  • Clinical microbiology

    • isolate and identify microbes from clinical specimens rapidly

  • Clinical specimen

    • human material tested to determine the presence or absence of specific microbes

  • specimen should:

    • represent diseased area

    • Sufficient quantity to do a variety of diagnostic tests

    • collected aseptically to avoid contamination

    • obtained prior to administration of antimicrobial

    • forwarded promptly and properly to a clinical lab


Clinical techniques
Clinical Techniques

  • Definitive identification relies on:

    • Microscopy: Morphological assessment, fluorescence microscopy for specific detection

    • Biochemical techniques, require growing pathogen

    • Immunologic tests: use of antibodies, Elisa, Agglutination test, complement fixation, immunoprecipitation based tests eg. radial immunodiffusion, double diffusion

    • Molecular techniques: PCR, QPCR, Ribotyping, RFLP also phage typing


Microscopy
Microscopy

  • wet-mount, heat-fixed, or chemically fixed specimens can be examined

    • Morphological, Gram reaction, spore bearing

  • choice of microscopy depends on pathogen

    • e.g., dark-field microscopy

      • detection of spirochetes in skin lesions associated with syphilis

    • e.g., fluorescence microscopy

  • stains often used

    • Simple stains, Gram stain and acid fast stain (Zeil-Neelsen)for mycobacteria


Immunofluorescence microscopy
Immunofluorescence microscopy

  • 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

  • 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

Sample used to infect cells in tissue culture

identified by:

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 (hemagglutinin producing viruses)

Hemadsorption

Growth and Biochemical Characteristics

Syncytium

Polio induced CPE

hPIV3 induced cell fusion


Biochemical identification
Biochemical Identification

Bacteria

  • most bacteria:

    • culturing in 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


Biochemical tests
Biochemical tests

Examples of biochemical tests

  • Biochemical properties represent genetic relatedness

  • Database of biochemical capabilities

    • Can be used to identify bacteria

  • Growing on different substrates as sole carbon source

  • Biochemical signature of test organism

  • Compare with database to find the best match

  • See Flowcharts (algorithms) for ID’ing schemes

Lac- = dark blue

Lac+ = yellow

Hemin

NAD

NAD+Hemin

Oxidase test

H. Influenza requires NAD and hemin

N. meningitidis is has cyt C




Rapid methods of identification
Rapid Methods of Identification

  • manual biochemical systems

    • e.g., API 20 E system

  • mechanized/automated systems:

    • 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
API 20E system

Checks for 20 metabolic markers and generates codes to match

known bacteria


Biolog phenotypic identification array

Each well contains one carbon source

growth results in color change

Biolog phenotypic identification array

  • More than 2500 bacterial, fungi and yeast species

  • Based on colorimetric detection of growth

    • Use of a redox dye coupled to ETC

  • 95 metabolic markers

  • Rapid 4-16 hours

  • Computer based database match

  • Accuracy

Biolog Inc website


Bacteriophage typing
Bacteriophage Typing

  • based on specificity of phage surface molecules for host cell receptors

  • Narrow host range for a collection of phages can be used to typify the hosts

  • Phagovars

    • collection of strains sensitive to certain collection of phage types


Molecular methods
Molecular Methods

  • Nucleic acid-based detection methods

    • Ribotyping

    • Diagnostic PCR

    • Probe hybridization (RFLP analysis)

  • Analysis of proteins: PAGE and Western


Ribotyping
Ribotyping

  • To identify bacterial genera

  • based on high level of 16S rRNA conservation among bacteria

  • PCR amplification of rRNA genes or fragments

  • Sequence of amplified DNA compared with those in the National Center for Biotechnology (NCBI)

  • Strain is determined on the basis of sequence homology


Diagnostic pcr

Clostridium botulinum toxin genes

Type F

Type A

Type B

Type E

Samples

Diagnostic PCR

  • Amplifies small fragment of DNA

  • Allows detection of tiny numbers of bacteria

  • Size of fragment can indicate species, strain

  • Restriction analysis can further indicate strain

  • Single nucleotide differences affect ability to be cut by restriction enzymes


Real time quantitative pcr
Real-Time Quantitative PCR

  • Detection of slow growing viruses, latent infections

  • RT-PCR

    • Reverse Transcriptase makes cDNA from RNA followed by PCR

  • qRT-PCR: quantitative “real-time” PCR

    • Quenched fluorescent probe to amplified DNA

      • Probe is degraded as amplification occurs

      • Separates quencher from fluorophore

    • 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
Immunologic Techniques

  • detection of antigens or antibodies in specimens

    • especially useful when culture methods are unavailable or impractical

  • use of immunological techniques has many advantages

    • easy to use

    • rapid reaction endpoints

    • sensitive and specific


Agglutination
Agglutination

  • 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

  • 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



Enzyme linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay

  • Done in two ways:

    • Direct

    • Indirect

  • Direct:directly detecting antigens in a sample

    • also called antigen capture ELISA

    • Pathogen specific Ab immobilized

    • Patient serum passed

    • Secondary antibody detects binds to captured Ag


Enzyme linked immunosorbent assay1
Enzyme-Linked Immunosorbent Assay

  • Indirect: detecting antibodies in a patients serum

  • interpreted as indirect evidence for exposure to a pathogen

    • Known antigen immobilized

    • Primary Ab detects Ag

    • Enz-linked secondary Ab detects primary Ab

    • Can be quantified



Immunoblotting western blot
Immunoblotting (Western Blot)

  • procedure

    • proteins separated by SDS-PAGE

    • proteins transferred to nitrocellulose sheets

    • protein bands visualized with enzyme-tagged antibodies

  • Examples:

    • distinguish microbes

    • diagnostic tests



Monoclonal antibodies mab and fluorescence microscopy
Monoclonal Antibodies (MAB) and fluorescence microscopy

  • produced by hybridoma cells

  • recognize a single epitope

    • fluorescently-labeled mABs used diagnostically

      • technique has replaced use of polyclonal antisera for culture confirmation

Click to read more about hybridoma technology