19: Diagnosing Infections

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Koch's Postulates
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if disease is of biological origin, you must identify putative biological agent in all patients w the disease. If you inoculate them w putative causative biological agent, volunteers must contract the disease. If they get ill, you should be able to reisolate the agent from all volunteers. *requires heavy use of Five I's
Genotypic (genetic) Techniqueswe read the genome of a microorganism & compare it to othersCollecting a Sampleproper collection, handling & storage of samples is one of most imp considerations in identification & treatment of microbially associated diseases. Use sterile collecting devices and sample containersWays to take a sampleswabs-oral/nasal. Sputum (mucous secretions from lower resp tract) discharged by coughing, or collect it directly using catheterizationClean Catchcollecting urine midstream after cleaning exterior portion of urogenital tractDirty Urine Catchurine that's fist voidedLabelextremely imp to properly labeled samplesImmediately start examining samplewe can take samples directly from patient, stain, & examine under microscopeIncrease Sensitivity of Microscopyby using fluorescent antibodies (glow) that react specifically w a single species of bacteria. If that species is present in sample, it'll glow when viewed under fluorescent microscope (spirochete causes syphilis)Rapid Antigen Testsfor # of pathogens. Along w clinical signs/symptoms these can help rapidly diagnose & aid in treatment. (Rapid Strep Test)Problem w testing directly from samplethere may be few microorganisms to observe. If we suspect a microbial pathogen is causing disease, we culture samples so we can have more to work wSheep's Blood Agarif we suspect a sample contains few microbes or a microorganism that's fastidious or might be easily overgrown by other microorganisms, we may want to 1st inoculate an enriched mediumMannitol Salt Agar or MacConkey Agarif sample is suspected to have high bacterial counts (fecal) we inoculate on selective mediaClues to identify microorganismshemolysis on blood agar & fermentation of sugars in mannitol salt & MacConkeyBlood for Culturecollected directly into a growth medium. Reduces risk of chance of contamination & minimizes shock to fragile microbes of drastic changes in environmental conditions. Growth in these cultures can be directly observed but samples can be periodically taken from these cultures for closer examinationPoint of initial inoculationisolate ind species of microorganisms to get pure cultures that we can perform more tests onWhat can stains tell us?size, shape, arrangementWhat can differential stains tell us?gram state of bacteria, presence of flagella, endospores, & capsulesElectron Microscopyprovides more detail ab bacteria & is only option when looking at virusesExamine physiology of microorganismsability of microorganism to digest or metabolize diff macromolecules (sugars, lipids & proteins)Test Stripsea test strip is inoculated & then incubated. Have an "indicator" (color change, bubble, physical state change). Easy to develop a bacterial "fingerprint". Then use results to identify bacteriaDetermining if a pathogen part of our normal microbiota is causative agentE. Coli. Normal biota. A few hundred to thousands indicates active infection, so it's the disease causing agentDetermining if a pathogen not part of our normal biota is causative agenteasier. If we see pathogens NOT part of our normal flora (Mycobacterium tuberculosis) it's good indication they have a role in disease in questionPolyacrylamide gelthrough which proteins from diff bacterial species has been electrophoresed. Ea black band represents diff kind of protein, & the pattern is the fingerprint specific to an ind species. 1 way we look at chem composition of a microbeGenetic Informationallow us to identify microorganisms, but gives us clues how to attack them if they cause problems for humans. Sequence itself can be used to identify an organism or species or if we isolate NEW species, genetic info allows us to see what other species it's most closely relatedSpecific Nucleic Acid Probeonce we know specific DNA sequence of a specific bacterial species, we can develop this for that bacterial species. They can be labeled w a fluorescent molecule and then added to a solution that's used to bathe a sample (patient or from culture)HybridizeNucleic acid probe will only hybridize (bind to) the nucleic acid sequence it was designed to, and if that sequence is there, the cells will light upPolymerase Chain Reaction (PCR)uses nucleic acid sequences. POWERFUL. Rapidly amplify specific bacterial species (we can make so many copies we can actually see DNA). Can detect specific gene in sample! Aids clinical professionals & epidemiologists. Can determine # of a specific microorganism in a sample (determining pathogenic load) or look for changes in genetic makeup of microorganismsagarose gel electrophoresistechnique used to separate dna fragments by sizeGranulicatella adiacensemerging disease that's associated w bacteremia as well as infections of CNS, eyes, bones, joints, & UT in newbornsSerologybranch of immunology that deals w in vitro diagnostic testing of a persons serumSerological Methodstake advantage of vertebrate immune response, & specifically ability of B cells to produce millions of antibodies to specific antigensSerological Methods allow us to look atimmunological history of patient, & easiest way is to take a patient's serum & mix it w purified antigen of known microbe on microscopic slide. If mixture clumps, the patient has antibodies to the antigen and has been previously exposed or is currently infectedSerological Techniques to determine the actual microorganism in patient or environmental samplesstart w a microbe or sample thought to contain a specific microbe, then mix sample w purified antibody that recognizes a specific microbe. If sample clumps, the antibodies are binding their antigen so the specific microbe the antibodies recognize are present in sampleSpecificityability of an assay to recognize particular antigen & not other antigen [imp characteristic of these assays]Sensitivityability to detect small amts of their target [imp characteristic of these assays. The more sensitive, the more likely we catch infections early]False Positiveswhen we think we detect something but it really isn't there [more specific an assay is, the less this happens]Serological Assays depend onability of antibodies to agglutinate (antibody sticks to more than one particle, clumping a lot of particles together). When whole cells are used we can see clumps easilyPrecipitation Reactionswhere the antigens are in solution and too small to be seen, but we can stick antibodies onto inert particles (small plastic beads) so when they clump we can see reaction w naked eyeWe can use these agglutination reactions to determinetiter (#) of antibodies w/n person's serum. Conducted in tubes or multi well plates, serum in dilated w sterile water and ea dilution is mixed w antigen. The more serum can be diluted and still clump up the antigen, the more antibody is present in undiluted sample. Great way to test efficacy of vaccines in peopleLysinsspecific antibodies that can interact w complement to lyse a target cellComplement Fixationuses this ability to lyse cells, along w sensitized sheep rbcs to detect antibodies to specific antigens. 2 stagesComplement Fixation: Stage 1mix patient's serum w purified antigen. If the patient has antibodies to the antigen, it'll bind that antigen; we then add a known amt of guinea pig complement which will bind to the antibody antigen complex. If patient doesn't have antibodies to the antigen, no antibody antigen complement complex formsComplement Fixation: Stage 2.involves sheep rbcs that already have lysins attached to them and are ready to lyse when exposed to complement. Involves mixing sheep RBCs w stage 1 mixture & if patient has antibodies to the antigen, they bind up the comp which then can't interact w the lysins on the RBCs so the RBCs don't lyse. But if patient does have antibodies to the antigen, the comp isn't bound up and can interact w the lysins on the rbcs so they lyseWhat are Complement Fixation tests used to diagnose?fungal diseases (histoplasmosis) & other diseases which aren't easily detected by other meansInjecting animalsw specific antigens so they produce immune response to it, then harvest antibodies that recognize from the animalsDirect Immunofluorescent Testingtake labeled antibody that recognizes part of a microbe, mix it w a sample we suspect contains that microbe. If sample contains the antigen, the labeled antibody binds to it & when we look w a fluorescent microscope, it glows where the antibodies are bound to the sampleIndirect Immunofluoresent Testingmix serum from patient w known antigen. If it contains antibodies to the antigen, immune complexes will be formed. Then add labeled antibodies that were developed using antibodies as the initial antigen. These anti antibody antibodies will attach to the antibodies attached to the antibodies and we can see the complexes under a fluorescent microscope. No complex, no glowing!Enzyme-Linked Immunosorbent Assay (ELISA)use Indirect ELISA to determine antibody titer by coating ea well w/n multiwell plate w the specific antigen we wish to study. Then put serum in the wells. Once time's been given for antibody antigen complexes to form, wash out well to remove anything that hasn't bound & add anti antibody antibodies which are attracted to an enzyme that can turn colorless substrate into a colored oneIf the antibodies to the antigen were present in the serumcolor will develop in the wells once substrate's added. More antibodies present, stronger the color change. No antibodies, no color changeCapture or Antibody Sandwich ELISAdetermines how much of an antigen's present in a sample. Antibody instead of antigen's used to coat wells of a multiwelled plate. Add samples & controls to ind wells, & if the antigen recognized by the antibody coating the well is present then complexes are formed. We then add another antibody that binds the antigen except this one's also attached to a color changing enzyme. More antigen present the more color develops