Bio303 laboratory diagnosis of infection


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In this Bio303 module talk, I provide an overview of how infections are diagnosed in the clinical microbiology lab, focusing on technologies, old and new, and also on practical issues and workflows crucial to optimal use of the lab.

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  • 95 & 63 strains
  • 95 & 63 strains
  • Change shading Ultimately, we detected three SNP loci which allowed us to distinguish between isolates in the outbreak. These are presented with reference to an unrelated Acinetobacter reference, AB57 which we consider has the “ancestral” state at these loci.
  • Referring back to the outbreak diagram we can plot these consequent genotypes onto each isolate. So when considering C1 it can be seen that it has a unique genotype compared with the others thus making it hard to make a compelling case for transmission from any of the military patients. But when we consider the case of C2 it can be seen that it shares the same genotype as M2 and M4. Given that M2 and C2 were in neighboring beds around week 4 but M4 did not come into contact with C2 at any point, we believe we can make a strong case for transmission from M2 and C2.
  • You can read the full story of these study in the Journal of Hospital Infection where it is available as an online pre-print. Our analyses support transmission of MDR-Aci from the wound of a military patient M2 to the respiratory tract of a civilian patient C2. As MDR-Aci was not isolated from C2 until several weeks after M2 left the adjacent bed, however, we cannot determine when and how transmission occurred. One possibility is that C2 became colonised when the two patients were nursed together, but that colonisation did not reach detectable levels in the sputum until much later. Another possibility is that M2 contaminated the local environment and C2 acquired the organism from the environment only after M2 had left the ward. This latter option would be consistent with a significant role of the environment.
  • Bio303 laboratory diagnosis of infection

    1. 1. Laboratory Diagnosis of Infectious Disease: From Gram-stain to Genomes Professor Mark Pallen
    2. 2. Outline <ul><li>Laboratory Diagnosis of Infection </li></ul><ul><ul><li>Appropriate use of the lab </li></ul></ul><ul><ul><li>M icroscopy </li></ul></ul><ul><ul><li>Culture </li></ul></ul><ul><ul><li>Sensitivities </li></ul></ul><ul><ul><li>Immunoassays </li></ul></ul><ul><li>Rapid Methods </li></ul><ul><li>High-throughput sequencing </li></ul>
    3. 3. <ul><li>Clinical Diagnosis </li></ul><ul><li>Non-microbiology investigations </li></ul><ul><ul><li>Radiology </li></ul></ul><ul><ul><li>Haematology </li></ul></ul><ul><ul><li>Biochemistry </li></ul></ul>Diagnosis of Infection
    4. 4. Laboratory Diagnosis of Infection <ul><li>If physician suspects infection, samples of tissue or fluid collected for </li></ul><ul><ul><ul><li>Microbiological analyses </li></ul></ul></ul><ul><ul><ul><li>Immunological analyses </li></ul></ul></ul><ul><ul><ul><li>Molecular-biological analyses </li></ul></ul></ul><ul><li>Samples include </li></ul><ul><ul><ul><li>blood </li></ul></ul></ul><ul><ul><ul><li>urine </li></ul></ul></ul><ul><ul><ul><li>faeces </li></ul></ul></ul><ul><ul><ul><li>sputum cerebrospinal fluid </li></ul></ul></ul><ul><ul><ul><li>pus </li></ul></ul></ul>
    5. 5. Diagnostic workflow <ul><li>Specimen collection </li></ul><ul><li>Specimen receipt </li></ul><ul><li>Specimen processing </li></ul><ul><li>Testing </li></ul><ul><li>Interpretation </li></ul><ul><li>Reporting </li></ul>
    6. 6. A proper clinical assessment is essential for optimal use of laboratory services!
    7. 7. Factors limiting usefulness of mirobiology investigations <ul><li>Specimens must be obtained and handled properly </li></ul><ul><ul><li>Specimen should be obtained from site of infection </li></ul></ul><ul><ul><li>Sample must be taken aseptically </li></ul></ul><ul><ul><li>Sample size must be large enough </li></ul></ul><ul><ul><li>Metabolic requirements for the organism must be maintained during sampling, storage, and transport </li></ul></ul><ul><li>Wrong sample </li></ul><ul><ul><li>e.g. saliva instead of sputum </li></ul></ul><ul><li>Delay in transport / inappropriate storage </li></ul><ul><ul><li>e.g. CSF </li></ul></ul><ul><li>Overgrowth by contaminants </li></ul><ul><ul><li>e.g. blood cultures </li></ul></ul><ul><li>Insufficient sample / sampling error </li></ul><ul><ul><li>e.g. in mycobacterial disease </li></ul></ul><ul><li>Patient has received antibiotics </li></ul>
    8. 8. Safety in the Microbiology Laboratory <ul><li>Clinical microbiology labs present significant biological hazards for workers </li></ul><ul><li>Standard lab practices for handling clinical samples are in place to protect workers </li></ul><ul><li>Laboratories are classified according to their containment potential, or biosafety level (BSL), and are designated as BSL-1 through BSL-4 </li></ul>
    9. 9. Laboratory Diagnosis of Infection culture on plates or in broth identification by biochemical or serological tests on pure growth from single colony microscopy Decolorise Counterstain Stain unstained or stained with e.g. Gram stain sensitivities Serodiagnosis DNA technologies by disc diffusion methods, breakpoints or MICs
    10. 10. Microscopy <ul><li>Unstained preparations </li></ul><ul><ul><li>“ Wet prep” </li></ul></ul><ul><ul><li>Dark-ground illumination for syphilis </li></ul></ul><ul><li>Stained preparations </li></ul><ul><ul><li>Gram-stain </li></ul></ul><ul><ul><li>Acid-fast stain </li></ul></ul><ul><ul><ul><li>Ziehl-Neelsen </li></ul></ul></ul><ul><ul><li>Fluorescence </li></ul></ul><ul><ul><ul><li>Direct, e.g. auramine </li></ul></ul></ul><ul><ul><ul><li>Immunofluorescence </li></ul></ul></ul>
    11. 11. The Gram Stain Crystal violet Gram's iodine Decolorise with acetone Counterstain with e.g. methyl red Gram-positives appear purple Gram-negatives appear pink
    12. 12. The Gram Stain Gram-positive rods Gram-negative rods Gram-positive cocci Gram-negative cocci
    13. 13. The Gram Stain <ul><li>The Gram stain can be applied to pure cultures of bacteria or to clinical specimens </li></ul><ul><li>Gram-stain not useful for all bacteria </li></ul><ul><ul><li>Mycobacteria have very thick walls and are best seen using an acid-fast staining procedure </li></ul></ul><ul><ul><li>Spirochaetes are long spiral bacteria that are too thin to be seen by Gram-stain </li></ul></ul><ul><ul><li>Specialised intra-cellular bacteria such as chlamydias and rickettsias cannot be seen by Gram stain </li></ul></ul>Pure culture of E. coli (Gram-negative rods) Neisseria gonorrhoeae in a smear of urethral pus ( Gram-negative cocci, with pus cells )
    14. 14. Culture of Pathogenic Microbes <ul><li>Solid media </li></ul><ul><ul><li>Agar plates </li></ul></ul><ul><ul><ul><li>For Identification </li></ul></ul></ul><ul><ul><ul><li>For Enumeration </li></ul></ul></ul><ul><ul><li>Slopes </li></ul></ul><ul><ul><ul><li>For safe long-term culture, e.g. Lowenstein-Jensen media for TB </li></ul></ul></ul><ul><li>Liquid media (broth) </li></ul><ul><ul><li>For enrichment or maximum sensitivity </li></ul></ul><ul><ul><li>E.g. blood cultures </li></ul></ul>
    15. 15. Culture of Pathogenic Microbes <ul><li>Although most pathogenic microbes can be grown after overnight culture in vitro, there are some important exceptions </li></ul><ul><ul><li>Anaerobes or fastidious bacteria may take several days/weks </li></ul></ul><ul><ul><li>Mycobacteria grow very slowly, if at all ( M. leprae uncultivable) </li></ul></ul><ul><ul><li>Treponema pallidum cannot be cultured in vitro </li></ul></ul><ul><ul><li>Obligate intracellular bacteria (e.g. Chlamydia, Rickettsia ) need to be grown in cell culture </li></ul></ul><ul><li>Diagnosis of infection with slow-growing or non-culturable bacteria tends to rely on molecular methods (PCR) or serodiagnosis (antibody detection) </li></ul>
    16. 16. Culture of Pathogenic Microbes <ul><li>Most microbes of clinical importance can be grown, isolated, and identified with specialised growth media </li></ul><ul><ul><li>General Purpose Media </li></ul></ul><ul><ul><ul><li>Support growth of most aerobic and facultatively aerobic organisms (e.g., blood agar) </li></ul></ul></ul><ul><ul><li>Enriched Media </li></ul></ul><ul><ul><ul><li>Contain specific growth factors that enhance growth of certain fastidious pathogens </li></ul></ul></ul><ul><ul><li>Selective Media </li></ul></ul><ul><ul><ul><li>Allow some organisms to grow while inhibiting others </li></ul></ul></ul><ul><ul><li>Differential Media </li></ul></ul><ul><ul><ul><li>Allow identification of organisms based on their growth and appearance on the medium </li></ul></ul></ul>
    17. 17. Advantages of Solid Media <ul><li>tentative identification of an isolate by colonial chararacteristics </li></ul><ul><ul><li>E.g. lactose-fermenter on MacConkey </li></ul></ul><ul><li>isolation of single clonal colonies </li></ul><ul><ul><li>get bacterium in pure culture </li></ul></ul><ul><ul><li>allows detailed tests for definitive identification </li></ul></ul><ul><li>quantification by colony-forming units </li></ul>LF NLF
    18. 18. Identification of Bacteria <ul><li>Morphology </li></ul><ul><li>Growth requirements </li></ul><ul><li>Biochemistry </li></ul><ul><li>Enzymes </li></ul><ul><li>Antigens </li></ul>
    19. 19. Sensitivity tests <ul><li>on solid medium </li></ul><ul><ul><li>disc diffusion technique </li></ul></ul><ul><ul><li>E-test </li></ul></ul><ul><li>in liquid medium </li></ul><ul><ul><li>minimum inhibitory concentration (MIC) test </li></ul></ul>
    20. 20. Antimicrobial Susceptibility Testing <ul><li>Disk Diffusion Test </li></ul><ul><ul><li>Standard procedure for assessing antimicrobial activity </li></ul></ul><ul><li>Inhibition Zones </li></ul><ul><ul><li>Used to determine an organism’s susceptibility to an antimicrobial agent </li></ul></ul>
    21. 21. The E Test
    22. 22. Antimicrobial Susceptibility Testing <ul><li>The MIC (minimum inhibitory concentration) procedure is used to assess antibiotic susceptibility with regard to various concentrations </li></ul>8mg/L 4mg/L 2mg/L 1mg/L 0.5mg/L 0.25mg/L Antibiotic concentration Cloudiness represents growth after overnight incubation means bacteria can grow at that concentration of antibiotic MIC=2mg/L
    23. 23. Diagnosis of Viral Infection <ul><li>Electron microscopy </li></ul><ul><li>Antigen detection </li></ul><ul><li>Antibody detection </li></ul><ul><li>Virus culture </li></ul><ul><ul><li>Detect cytopathic effect or antigen </li></ul></ul><ul><li>Molecular methods </li></ul><ul><ul><li>Polymerase Chain Reaction </li></ul></ul>
    24. 24. Immunoassays for Infectious Disease <ul><li>Identify infection by measuring antibody titre against antigen produced by pathogen </li></ul><ul><ul><li>Agglutination </li></ul></ul><ul><ul><li>ELISA </li></ul></ul><ul><ul><li>Radioimmunoassay </li></ul></ul><ul><li>T Cell based tests </li></ul><ul><ul><li>Skin tests </li></ul></ul><ul><ul><li>Interferon-gamma assays </li></ul></ul>
    25. 25. Agglutination <ul><li>Passive Agglutination </li></ul><ul><ul><li>The agglutination of soluble antigens or antibodies that have been adsorbed or chemically coupled to cells or insoluble particles (e.g., latex beads, charcoal) </li></ul></ul><ul><ul><li>Reactions can be up to five times more sensitive than direct agglutination tests </li></ul></ul>Latex Bead Agglutination Test for Staphylococcus aureus
    26. 26. Rapid Microbiological Methods <ul><li>Growth-Based Technologies </li></ul><ul><li>measurement of biochemical or physiological parameters that reflect growth of microorganisms </li></ul><ul><li>include: </li></ul><ul><ul><li>ATP bioluminescence: AKuScreen to screen for microbial contamination in pharmaceuticals </li></ul></ul><ul><ul><li>colorimetric detection of CO 2 production: Bactec; BacT/Alert </li></ul></ul><ul><li>Cellular Component-Based Technologies </li></ul><ul><li>detection of a specific cellular component </li></ul><ul><li>include: </li></ul><ul><ul><li>Fatty acid profiles </li></ul></ul><ul><ul><li>mass spectrometry </li></ul></ul><ul><ul><li>ELISA </li></ul></ul><ul><ul><li>fluorescent probe detection </li></ul></ul>
    27. 27. Rapid Microbiological Methods <ul><li>Nucleic Acid-Based Technologies </li></ul><ul><li>DNA probes: Gene-Trak; Gene-Probe </li></ul><ul><li>molecular typing </li></ul><ul><li>polymerase chain reaction (PCR) and other nucleic acid amplification technologies (NAATs) </li></ul><ul><li>sequencing </li></ul><ul><li>Automated Methods </li></ul><ul><li>Simplest use classical method for processing sample, then detect colorimetric change to spot growth earlier than visual detection </li></ul><ul><li>Replace human detection methods with machine detection; human judgment with machine intelligence </li></ul><ul><li>include: BacT/ALERT, VITEK </li></ul>
    28. 28. VITEK 2 <ul><li>fully automated system for bacterial/fungal identification and antibiotic susceptibility testing  </li></ul><ul><li>reduces set-up time and minimizes manual steps </li></ul><ul><li>Compact sealed ID/AST cards </li></ul><ul><li>Rapid microorganism identification </li></ul><ul><li>Rapid, same-day antimicrobial susceptibility testing </li></ul><ul><li>Advanced Expert System validates IDs </li></ul><ul><li>Data management software allows for generation of epidemiology reports and antibiograms </li></ul>
    29. 29. Xpert MTB/Rif <ul><li>Sealed cartridge </li></ul><ul><li>Robust sonication/mechanical DNA extraction procedure </li></ul><ul><li>Hemi-nested PCR targets rpoB gene associated with rifampicin resistance </li></ul><ul><li>2 hour result </li></ul>
    30. 30. ‘ Next-generation’ High-throughput sequencing <ul><li>~100x faster, ~100x cheaper than conventional approaches </li></ul><ul><li>Clonal template populations obtained by new methods: </li></ul><ul><ul><li>amplification on solid phase to grow a ‘molecular colony’ </li></ul></ul><ul><ul><li>Massive increase in number of ‘clones’ but shorter read length </li></ul></ul><ul><li>New chemistries for sequence reading </li></ul><ul><ul><li>Pyrophosphate detection (PPi release upon base addition): 454 </li></ul></ul><ul><ul><li>Single (reversibly 3’-blocked) fluorescent base (quenchable) added per step: Solexa </li></ul></ul><ul><ul><li>Sequencing by Ligation (ABI SOLiD) </li></ul></ul>
    31. 32. High-throughput Sequencing in Clinical Microbiology: Applications <ul><li>Pathogen detection and discovery </li></ul><ul><ul><li>Clinical metagenomics </li></ul></ul><ul><li>Polymorphism detection and discovery </li></ul><ul><ul><li>Genomic epidemiology </li></ul></ul><ul><ul><li>Adaptive changes </li></ul></ul><ul><li>Pathogen biology </li></ul><ul><ul><li>Gene detection and discovery </li></ul></ul>
    32. 33. Sequencing Methodologies <ul><li>Culture-dependent </li></ul><ul><li>Culture-independent </li></ul><ul><li>Shotgun library of purified genomic DNA </li></ul><ul><ul><li>Delivers whole-genome sequence </li></ul></ul><ul><li>Phylogenetic profiling: PCR with 16S primers </li></ul><ul><li>Metagenomics: shotgun sequence community DNA </li></ul>
    33. 34. Culture-independent Pathogen Discovery
    34. 36. The Birth of Genomic Epidemiology for Bacteria
    35. 37. The Birth of Genomic Epidemiology for Bacteria
    36. 38. Case Study Acinetobacter baumannii <ul><li>Gram-negative bacillus </li></ul><ul><li>Multi-drug resistant </li></ul><ul><ul><li>colistin and tigecycline as reserve agents </li></ul></ul><ul><ul><li>moving towards pan-resistance </li></ul></ul><ul><li>Associated with </li></ul><ul><ul><li>wound infections and ventilator-associated pneumonia </li></ul></ul><ul><ul><li>bloodstream infections </li></ul></ul><ul><ul><li>returning military personnel from Iraq and Afghanistan </li></ul></ul><ul><ul><li>transmission from military to civilian patients </li></ul></ul>
    37. 39. Acinetobacter Genomic Epidemiology <ul><li>Outbreak in Birmingham Hospital in 2008 </li></ul><ul><li>Isolates indistinguishable by current typing methods </li></ul>
    38. 40. Acinetobacter Genomic Epidemiology <ul><li>454 whole-genome sequencing of 6 isolates </li></ul><ul><li>SNP detection by mapping reads against draft reference assembly </li></ul><ul><li>SNP filtering for false positives </li></ul><ul><li>SNP validation with Sanger sequencing of PCR amplicons </li></ul>
    39. 41. Results: Outbreak Isolates Are Distinguishable At Only Three Loci   SNP 1 SNP 2 SNP 3 AB0057 C A G M1 C A G M2 T A G M3 T A T M4 T A G C1 T T G C2 T A G
    40. 44. Take-away messages <ul><li>Genome sequencing brings the advantages of </li></ul><ul><ul><li>open-endedness (revealing the “unknown unknowns”), </li></ul></ul><ul><ul><li>universal applicability </li></ul></ul><ul><ul><li>ultimate in resolution </li></ul></ul><ul><li>Bench-top sequencing platforms now generate data sufficiently quickly and cheaply to have an impact on real-world clinical and epidemiological problems </li></ul>
    41. 45.
    42. 46. 19 th Century 21 st Century 20 th Century