Clinical MicrobiologyClinical Microbiology

Lecture 13Lecture 13Bio 3124Bio 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 necessaryRequired 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, SpecimensClinical Microbiology, Specimens Clinical microbiologyClinical microbiology

isolate and identify microbes from clinical specimens rapidly

Clinical specimenClinical specimen

human material tested to determine the presence or absence

of specific microbes

specimen should: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

MicroscopyMicroscopy

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

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

Polio induced CPE

hPIV3 induced cell fusion

Syncytium

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

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 Identification

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

Biochemical testsExamples of biochemical testsExamples of biochemical tests

Lac+ = yellowLac- = dark blue

Hemin NAD NAD+Hemin

H. Influenza requires NAD and hemin N. meningitidis is has cyt C

Oxidase test

Identification scheme for G+ bacteria

Identification scheme for G- bacteria

Rapid Methods of Identification

manual biochemical systemse.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

Checks for 20 metabolic markers and generates codes to matchknown bacteria

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

Each well contains one carbon sourcegrowth results in color change

Biolog Inc website

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

Nucleic acid-based detection methods• Ribotyping• Diagnostic PCR• Probe hybridization (RFLP analysis)

Analysis of proteins: PAGE and Western

RibotypingRibotyping

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

Amplifies small fragment of DNAAllows detection of tiny numbers of bacteriaSize of fragment can indicate species, strain

Type A

Type B

Type E

Type F

Samples

Clostridium botulinum toxin genes

Restriction analysis can further indicate strain

Single nucleotide differences affect ability to be cut by restriction enzymes

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

Real-Time Quantitative PCR

Animation: Real-time PCR

Genomic fingerprint: RFLP analysisGenomic 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 TechniquesImmunologic 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

AgglutinationAgglutination

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 highestdilution 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

Animation: Complement fixation

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 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

Animation: Direct and Indirect ELISA

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

Extra slides if you like to seek for moreExtra slides if you like to seek for more

Monoclonal Antibodies (MAB) and fluorescence microscopy

produced by hybridoma cells

recognize a single epitopefluorescently-labeled

mABs used diagnostically • technique has replaced

use of polyclonal antisera for culture confirmation

Click to read more about hybridoma technology