Lecture 2: Basic Micro

  • Created by: chandanee
  • Created on: 02-01-19 17:58
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  • Basic Micro
    • Role of medical microbiologist
      • ID OG
      • Provide advice
      • Manage outbreaks
      • Advise on new healthcare
      • Trends in infection
      • Generate local epidemiological data
      • Monitor theatre
    • Samples/Specimen
      • Enrichment culture
        • 1) Direct examination/microscopy
          • Define - reveals pathology which is indicative of a condition
          • Staining - Gram Stain += thicker layer, purple, e.g. Bacillus  Gram - = thinner layer, pink e.g. E. coli. Fixation then crystal violet then iodine then decolourise then safranin. Problem = intracellular, phagocytes may clump, some bacteria stain poorly, rods appear as cocci, mycobacteria difficult to stain
          • Acid fast:
        • 2) Culture - differential/selective
          • Biochemical Tests
            • API ***** = ID enteric bacteria, 550 species, ONPG, CIT, URE, BGal
            • Rapid Staph Plus = ID Staph Plus. Cultures: blood, urine, sputum, gastric, stool.
            • Non-Selective = permits growth of many MO's. Diifferential = discriminates between MO's e.g. blood agar. Selective = contains inhibitors e.g. CCFA. Adv = definite ID, can then isolate. Disadv= 16-24 hours, skilled microbiologist
            • Cytotoxicity = e.g. C difficile  = difficult to diagnose, toxins detected by stool samples, Adv = sens and spec Disadv = slow turn around, variability due to cell lines, experience, variability in protocols and interpretation.
        • 3) Ab detection/ serology
          • 2 approaches: recognise the Ag or detect the Ab. Common tests = slide agglutination e.g. meningitis, Rapid tests
          • ELISA detects viruses and cell surface antigens or toxins. Capture Ab, directed against the protein, linked to a solid support, specimen added if viral Ag present, captured by bound Ab, bound Ag then detected by 2AB linked to an enzyme
          • GLC: anaerobes HPLC: all mycobacteria Adv: rapid, isolation not needed, minimal training for rapid tests. Disadv: false positives from cross reacting Ag, false negs from evolving Ag.
        • 4) Genetic
          • Hybridisation: Virochip gDNA > labelled DNA> clean up >pipette sample >hybridise wash and scan. PCR/RTPCR Adv= rapid, lab culture not needed, sens and specific. Disadv = quant? OG viable?
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    • New Methods
      • xTAG Gi pathogen panel (GPP) Chips = detect and ID bacterial, viral and parasitic all in one test >90% pathogens. Signs of gastroenteriditis in patients, 5 hours.
      • LLMDA: Rapid ID of multiple species in same samples, within 24 hours, contains probes fitted onto a slide. 2000 viruses and 900 bacteria. Fluoresce and read by scanner.
      • Isothermal NA amplification/LAMPPCR = rapid, sens, amplification is extensive. Constant temp unlike most PCRs. 60-65C so no thermocyclers needed. "loop" primers accelerate reaction. Detection by photometry by increasing quant of Mg pyrophosphate. Useful for low income countries. Less sensitive than PCR - useful for diagnostic/detection
      • Mass Spec Matrix (MALDITOF) used for early ID of bacteria in blood, UTIs, cerebrospinal fluid, respiratory tract infections      Peptide mass fingerprints also
      • NGS = faster sequencing at lower cost, template > amp> sequencing - may involve SNPs, new genes, mutations, degree of expression
      • Bacteria or virus - new tool? Use GENES to ID between viruses and bacteria in respiratory infections, blood tests, helps cut back on antibiotic usage, 87% accuracy, long time for results  (10-12 hours) and could become a home based kit.
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    • Synthetic Biology
      • Toehold switch synthetic riboregulator: sample collection > RNA extraction > RNA amp >toehold dertection >visual readout
      • Application: designer probiotics, urogenital healthcare, anti-tumour, inhibition of angiogenesis, sensing disease, managing obesity, diagnosis, antimicrobials
      • Factors to consider: Sens =no of MOs + result Spec= tend to discrimate between MOs. Rapid = C. diff = toxin test Also, reproducibility, differentiation, ease, cost, platform
      • Beyond Diagnosis: 1. Antimicrobial susceptibility test - which drugs? dose? Disc diffusion - zone of inhibition 16-24hr incubation  2. Epidemiology - different cases attributed to same strain. Study of incidence, distribution, disease control, outbreaks, trends, evolution

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