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6 laboratory diagnosis of bacterial infection

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6 laboratory diagnosis of bacterial infection

  1. 1. 1 Laboratory Diagnosis of Bacterial InfectionLaboratory Diagnosis of Bacterial Infection
  2. 2. 2 Types of specimen
  3. 3. Basic Principles for Specimen CollectionBasic Principles for Specimen Collection Collecting the correct specimenCollecting the correct specimen Endocervical swabs for GC Pernasal swabs for pertussis whole EMU for TB Sputum, not saliva Blood culture bottles, not clotted blood
  4. 4. Getting the specimen to the lab Problems in delay or inappropriate storage delay in dignosis and treatment ------pathogens die ------contaminants overgrow Blood culture directly into incubator ------not refrigerator CSF straight to lab Don't put an entire surgical specimen into formalin ------send a portion to microbiology in a sterile container
  5. 5. Collecting the specimen correctly Take an mid-stream urine ------avoids contamination with perineal flora CSF ------Avoid contamination ------Avoid bloody tap Blood cultures ------Avoid contamination with skin organisms
  6. 6. Infection Control Please be considerate to lab staff ------Label hazardous specimens Don't send specimens to the lab without proper packing ------Leaking or blood-stained specimens are not acceptable
  7. 7. Factors limiting usefulness of bacteriological investigations wrong sample ------saliva instead of sputum delay in transport/ inappropriate storage ------CSF overgrowth by contaminants ------blood cultures insufficient sample/sampling error ------in mycobacterial disease patient has received antibiotics
  8. 8. Specimen Etiologic diagnosis (blood,urine,stool, cerebrospinal fluid,pus,secreta) Examination of immune responese of the body to an infectious agent Direct examination of the specimen (blood,urine,stool) Isolation, culture and identification of the agent. susceptibility to antimicrobial drugs Tissue cells serum Histochemical stain Antibody detection (ELISA,WB,RIA) Microscopic examination Light microscopy, EM,IEM Component of microbes Antigens(immunofluores cenece,solid-phase radioimmunoassay and ELISA Poison and toxicity test Experimental animal Nucleic acid (nucleic acid electrophoresis, nucleic acid hybridization,PCR ,gene sequencing and gene chips Examination of metabolite (biochemical characteristics) Procedures for detection of pathogens
  9. 9. 9 The main examination methods in diagnosis of bacterial infectionThe main examination methods in diagnosis of bacterial infection ※ Morphological examination ※ Isolation, culture and identification ※ Biochemical Reactions ※Antibiotic Susceptibility Test ※ Antibody detection ※ Antigens or Nucleic acids assay
  10. 10. 10 Morphological examinationMorphological examination ※※ Non-stained microscopic observationNon-stained microscopic observation ▲▲ Dark-field microscopyDark-field microscopy ▲▲ Observe the movement of live bacteriaObserve the movement of live bacteria
  11. 11. 11 ※※ Stained microscopic observationsStained microscopic observations ▲▲ Gram stainGram stain ▲▲ Acid-fast stainAcid-fast stain ▲▲ Fluorescence stain(suchFluorescence stain(such as Auramine stainas Auramine stain)) ▲▲ methylene bluemethylene blue stain metachromatic granule
  12. 12. 12 Isolation & CultureIsolation & Culture 1.Size 2. Shape 3. Color 4.Surface features1.Size 2. Shape 3. Color 4.Surface features 5.Transparency 6. Hemolysis5.Transparency 6. Hemolysis How to describe the feature of bacterial colonies on an agar plate?
  13. 13. 13 Biochemical ReactionsBiochemical Reactions EVERYTHING that a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICAL FINGERPRINT. That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism The theoretic basis of biochemical reaction
  14. 14. 14 Common Tests To identify Bacterial isolates △ Indole assay △ Methyl Red/Voges Proskauer test △ Citrate utilization △ H2S production ( hydrogen sulfide ) △ Urea hydrolysis △ Motility △ Lactose fermentation △ Sucrose fermentation △ Glucose fermentation & gas production △ Oxidase test △ ……
  15. 15. 15 Citrate utilizationCitrate utilizationSugar FermentationSugar Fermentation HH22S TestS Test Examples of Common Biochemical ReactionsExamples of Common Biochemical Reactions
  16. 16. 16 Antibiotic Susceptibility TestAntibiotic Susceptibility Test The wide variation in susceptibility and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy Antimicrobial Susceptibility testing can be down by : Minimum Inhibitory Concentration (MIC) Disk Diffusion Method Minimum Bactericidal Concentration (MBC)
  17. 17. Principle: The tube dilution test is the standard method for determining levels of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).
  18. 18.  Different concentrations of Tetracycline in Nutrient broth: Conc. in mcg/ml ( microgramme ) 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml 1.Minimum Inhibitory Concentration (MIC) :
  19. 19. 2. Disk-diffusion Method (Kirby-Bauer Method):  The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a clinical laboratory where a large number of isolates are tested for susceptibility to numerous antibiotics.  An agar plate is uniformly inoculated with the test organism  A paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface.  Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations.  The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.
  20. 20.  This test must be rigorously standardized since zone size is also dependent on:  inoculum size,  medium composition,  temperature of incubation,  excess moisture and  thickness of the agar.  Zone diameter can be correlated with susceptibility as measured by the dilution method.  Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy.
  21. 21. Staphylococcus aureus (MRSA)  Note the yellowish pigmentation of the bacterial lawn, and the lack of inhibition by the Oxacillin disk
  22. 22. Streptococcus pneumoniae (Pneumococcus):  The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-haemolysis.
  23. 23. Pseudomonas aeruginosa:  The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.
  24. 24. 24 Serological AssaysSerological Assays ※※ Detection antibody in the patient’s serumDetection antibody in the patient’s serum ※※ A current infection should beA current infection should be △△ IgM positiveIgM positive △△ A 4-fold or greater rise on antibody titerA 4-fold or greater rise on antibody titer ■■ the convalescent sample is usually taken 10-14the convalescent sample is usually taken 10-14 days after the acute sample.days after the acute sample. ※※ Major drawbacksMajor drawbacks ■■ A single IgG antibody titer is difficult to interpret,A single IgG antibody titer is difficult to interpret, of course, In certain diseases, a single titer ofof course, In certain diseases, a single titer of sufficient magnitude can be used as presumptivesufficient magnitude can be used as presumptive evidenceevidence ■■ Some exceptionsSome exceptions
  25. 25. Molecular Biology Techniques A- Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labeled probe Probe: consists of specific short sequence of labeled single- stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA Then It is detected by a labeled probe C- Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis
  26. 26. Microbes and humans Very few microbes are always pathogenic Many microbes are potentially pathogenic Most microbes are never pathogenic
  27. 27. Microbes and humans
  28. 28. How do we know that a given pathogen causes a specific disease?
  29. 29. Diagnosis and effective treatment of infection depends not just on isolating an organism, but in establishing a plausible link between the laboratory findings, recognised syndromes and the patient's clinical condition potential pathogen isolated from or detected in clinical samples recognised syndromes e.g. meningitis pneumonia patient's clinic condition
  30. 30. Exercises: 1. What are the basic principles for specimen collectionbasic principles for specimen collection? 2. What’s antibiotic susceptibility test and its useantibiotic susceptibility test and its use? 3.What are the main examination methods in diagnosis ofmain examination methods in diagnosis of bacterial infectionbacterial infection?

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