Diagnosing Infections And Skin Infections

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Specimen Collection

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Aseptic technique must be used.
-sterile sample containers and tools.
-decontaminate or use antiseptics on tissue surrounding sampling site.
-minimize contamination by the normal microbiota.

Specimen storage
-specimen placed in non-nutritive maintenance media.
-stored in low oxygen-anaerobic conditions.
-refrigeration
-transport quickly to diagnostic lab.

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Phenotypic methods of identifying pathogen in a specimen

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-direct microscopic observation
-bicochemical testing
-phage-typing

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Direct Microscopic observation: Gram stain

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-sesitivity: low- not even to genus level
-advantages: fast/cheap, doesn't require cultivation.
-disadvantage: doesn't tell you much. used only as first stage of identification.

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Direct Microscopic observation: Acid-fast stain

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stain binds to bacteria with mycolic acid in their cell wall. (mycobacteria)
-mycobacteria stain red, other bacteria stain blue
-often used to test for presence of mycobacterium tuberculosis
-sensitivity: genus
-advantages/disadvantages: same as gram stain

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Direct Microscopic observation: Direct Fluorescence antibody test (DFA)

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-Also an immunologic method that uses a specific antibody to detect the pathogen of interest.
-sensitivity - genus, species (depends on antibody chosen)
-advantages- fast and sensitive, doesn't require cultivation
-disadvantages- few. more expensive than non-fluorescence stains.

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How to conduct a DFA

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1. apply specimen to slide.
2. add fluorescent labeled antibodies specific for pathogen.
3. wait 30-60 min for antibodies to bind. Wash off unbound antibodies.
4. Use fluorescence microscope to look for pathogens that fluoresce.

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Biochelmical Testing: Idenitifcation based on nutrient utilization

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-microbe culture in medium with special substrate and then tested for a particular end product (which can be seen with dye and tells whether microb uses that substrate or not)
-most test for use of multiple substrates at a time.
*Enterotube-used for enterics.
*API tests- different tests for many types of pathogens.
-sensitivity- genus, sometimes species
-advantages- easy and cheap.
-disadvantage- time consuming- a pure culture must be obtained first, then the test itsel takes 12-48+ hours.

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Biochemical Testing: Identification based on production of exoenzymes

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-can test for exoenzymes that serve as virulence factors
-Dnase agar: tests for Dnases
-Blood agar: tests for hemolysins
-sensitivity- genus, sometimes species
-advantage- easy/cheap
-disadvantage- time-consuming. must use pure culture and the test takes 24+ hours.

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What are additional bicochemical tests?

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-Catalase test
-Oxidase test- tests for microbes that posses cytochome oxidase

sensitivity- genus, sometimes species
advantages- easy
disadvantages- time consuming because microbes must be cultured first.

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Page-Typing

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-uses bacteriophages to identify microbes in specimens.
-based on the fact that each bateriophage infects a specific species (and sometimes strain) of bacteria
-often used for identifying species and strains of salmonella during foodborne illness outbreaks.
-grow lawn of bacteria on gridded plate
-infect each grid with a different phage and look to see which grids become infected.
-based on lysis of bacterial cells within the grid
-sensitivity- species, strain
-advantage-
-disadvantage- time consuming. you have to grow the bacteria tan wait for the phage to infect.

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Genotypic methods for identifying pathogens in a specimen

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-Fluorescent in-situ hybridizationg (FISH)
-Nucleic acid sequencing
-Polymerase chain reaction (PCR)

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Fluorescent In-situ Hybridization

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-uses a labeled DNA probe to detect a specific pathogen gene.
probe- small piece of DNA complementary to pathogen gene. Labeled with fluorescent dye
-sensitivity- species, strain *depending on specificity of probe
-advantage- sensitive, speed- no culturing required.
-disadvantage- few )enough microbial cells must be present to visualize.

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Nucleic acid sequencing

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-usually sequence the 165 ribosomal RNA gene of microbes within a specimen
-all microbs have the gene, but it's sequence is unique for each species/strain
-sensitivity- species, strain
-advantage- sensitivity, speed- no culturing required.
-disadvantage- few) need enough microbial cells and nucleic acid to sequence.

WIDELY USED METHOD

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What is the method for Nucleic acid sequencing?

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-Method:
1.Obtain specimen and lyse cells
2. Use primer (a probe without the label) to "extract" the 165 gene
3. Sequence the gene and use the sequnce to identify the microbe(s) present in the specimen.

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Polymerase Chain Reaction (PCR)

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-detects presence of a specific pathogen gene
-sensitivity- species, strain*depending on primer you choose
-advantage- sensitivity, detects pathogens when only a few cells are present, speed- no culturing required.
-disadvantage-few

WIDELY USED METHOD

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What is the method for PCR?

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Method:
1. obtain specimen and lyse cells
2.use a primer to "extract" a specific pathogen gene
3.amplify the gene thousands-millions of times and then use various methods to detect presence of the gene.
4.presence of the gene tells you that the pathogen is present in the specimen.

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Immunologic methods for identifying pathogens in a species:

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Serological testing( aka Antigen-antibody reactions)
-Direct antigen tests
-DFA
-Agglutination reactions
-ELISA

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Direct antigen tests

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-usually tests for presence of microbial antigen in specimen
-sensitivity- species, strain
-advantages- QUICK- takes a few minutes, no culturing required. test preformed in-house.
-disadvantages-some tests have high rate of false positives, so this usually requires another test for confirmation.

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What is the method for Direct antigen tests?

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Method:
1. sepciemen(with microbe) added to sample pad.
2. sample pad contains labeled antibodies which form colors when they are bound to antigen(microb)

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What is the Direct antigen tests widely used for?

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USED WIDELY FOR STREPTOCCOCUS, HERPES VIRUS AND CHLAMYDIA.

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Agglutination reactions

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-tests for antibodies in patient's serum.
-based on fact that antibodies bind microbes and cause agglutination.
-can be used to determine if antibody is present and to estimate antibody titer.
-sensitivity- genus, species
-advantages- quick, no culturing required.
-disadvantages- need enough antibody for detection.

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What are the two forms of ELISA (Enzyme-linked immunosorbant assay) test?

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Indirect
Direct

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What does the indirect ELISA test detect?

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Used to detect antibodies in patient's serum

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What does the Direct ELISA test detect?

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used to detect microbe in specimen.

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What's the specificity, advantages and disadvantages to the ELISA tests?

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specificity- species, sometimes strain.
advantages-relatively quick, no culturing required.
disadvantages- laborious, requires trained people or expensive robots.

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What is the method for the indirect ELISA test?

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1. antigen immobilized in well and the serum is added with antibodies.
2. antibodies specific for Ag bind, nonspecific antibodies wash off.
3. a secondary antibody with enzyme label is added. this antibody binds to first antibody.
4. add substrate for the enzyme and look for reaction to occur (Usually a color appears).

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What is the method for the direct ELISA test?

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1.antibody is immobilized in well, specimen with microbe is added.
2. a secondary antibody with enzyme label is added. this antibody also binds to microbe.
3.add substrate for enzyme. Watch for reactions to occur- usually produces a colored product.

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Macule

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flat lesion with color change

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Papule

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small, elevated, solid bump.

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Vesicle

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elevated lesion filled with clear fluid.

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Pustule

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elevated lesion with pus (dead WBC, bacteria, skin cells)

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Scale

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flake portions of skin that peel off