The document discusses laboratory diagnosis methods for infectious diseases, detailing techniques such as microscopy, culture, immunological tests, and high-throughput sequencing. It emphasizes the importance of proper sample collection and handling to ensure accurate diagnoses while outlining various microbiological and molecular methods for identifying pathogens. Additionally, it highlights the advancements in genomic epidemiology, especially in understanding and tracking multi-drug resistant infections.

1. Laboratory Diagnosis of Infectious Disease: From Gram-stain to Genomes Professor Mark Pallen

2. Outline Laboratory Diagnosis of Infection Appropriate use of the lab M icroscopy Culture Sensitivities Immunoassays Rapid Methods High-throughput sequencing

3. Clinical Diagnosis Non-microbiology investigations Radiology Haematology Biochemistry Diagnosis of Infection

4. Laboratory Diagnosis of Infection If physician suspects infection, samples of tissue or fluid collected for Microbiological analyses Immunological analyses Molecular-biological analyses Samples include blood urine faeces sputum cerebrospinal fluid pus

5. Diagnostic workflow Specimen collection Specimen receipt Specimen processing Testing Interpretation Reporting

6. A proper clinical assessment is essential for optimal use of laboratory services!

7. Factors limiting usefulness of mirobiology investigations Specimens must be obtained and handled properly Specimen should be obtained from site of infection Sample must be taken aseptically Sample size must be large enough Metabolic requirements for the organism must be maintained during sampling, storage, and transport Wrong sample e.g. saliva instead of sputum Delay in transport / inappropriate storage e.g. CSF Overgrowth by contaminants e.g. blood cultures Insufficient sample / sampling error e.g. in mycobacterial disease Patient has received antibiotics

8. Safety in the Microbiology Laboratory Clinical microbiology labs present significant biological hazards for workers Standard lab practices for handling clinical samples are in place to protect workers Laboratories are classified according to their containment potential, or biosafety level (BSL), and are designated as BSL-1 through BSL-4

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. Microscopy Unstained preparations “ Wet prep” Dark-ground illumination for syphilis Stained preparations Gram-stain Acid-fast stain Ziehl-Neelsen Fluorescence Direct, e.g. auramine Immunofluorescence

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. The Gram Stain Gram-positive rods Gram-negative rods Gram-positive cocci Gram-negative cocci

13. The Gram Stain The Gram stain can be applied to pure cultures of bacteria or to clinical specimens Gram-stain not useful for all bacteria Mycobacteria have very thick walls and are best seen using an acid-fast staining procedure Spirochaetes are long spiral bacteria that are too thin to be seen by Gram-stain Specialised intra-cellular bacteria such as chlamydias and rickettsias cannot be seen by Gram stain Pure culture of E. coli (Gram-negative rods) Neisseria gonorrhoeae in a smear of urethral pus ( Gram-negative cocci, with pus cells )

14. Culture of Pathogenic Microbes Solid media Agar plates For Identification For Enumeration Slopes For safe long-term culture, e.g. Lowenstein-Jensen media for TB Liquid media (broth) For enrichment or maximum sensitivity E.g. blood cultures

15. Culture of Pathogenic Microbes Although most pathogenic microbes can be grown after overnight culture in vitro, there are some important exceptions Anaerobes or fastidious bacteria may take several days/weks Mycobacteria grow very slowly, if at all ( M. leprae uncultivable) Treponema pallidum cannot be cultured in vitro Obligate intracellular bacteria (e.g. Chlamydia, Rickettsia ) need to be grown in cell culture Diagnosis of infection with slow-growing or non-culturable bacteria tends to rely on molecular methods (PCR) or serodiagnosis (antibody detection)

16. Culture of Pathogenic Microbes Most microbes of clinical importance can be grown, isolated, and identified with specialised growth media General Purpose Media Support growth of most aerobic and facultatively aerobic organisms (e.g., blood agar) Enriched Media Contain specific growth factors that enhance growth of certain fastidious pathogens Selective Media Allow some organisms to grow while inhibiting others Differential Media Allow identification of organisms based on their growth and appearance on the medium

17. Advantages of Solid Media tentative identification of an isolate by colonial chararacteristics E.g. lactose-fermenter on MacConkey isolation of single clonal colonies get bacterium in pure culture allows detailed tests for definitive identification quantification by colony-forming units LF NLF

18. Identification of Bacteria Morphology Growth requirements Biochemistry Enzymes Antigens

19. Sensitivity tests on solid medium disc diffusion technique E-test in liquid medium minimum inhibitory concentration (MIC) test

20. Antimicrobial Susceptibility Testing Disk Diffusion Test Standard procedure for assessing antimicrobial activity Inhibition Zones Used to determine an organism’s susceptibility to an antimicrobial agent

21. The E Test

22. Antimicrobial Susceptibility Testing The MIC (minimum inhibitory concentration) procedure is used to assess antibiotic susceptibility with regard to various concentrations 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. Diagnosis of Viral Infection Electron microscopy Antigen detection Antibody detection Virus culture Detect cytopathic effect or antigen Molecular methods Polymerase Chain Reaction

24. Immunoassays for Infectious Disease Identify infection by measuring antibody titre against antigen produced by pathogen Agglutination ELISA Radioimmunoassay T Cell based tests Skin tests Interferon-gamma assays

25. Agglutination Passive Agglutination The agglutination of soluble antigens or antibodies that have been adsorbed or chemically coupled to cells or insoluble particles (e.g., latex beads, charcoal) Reactions can be up to five times more sensitive than direct agglutination tests Latex Bead Agglutination Test for Staphylococcus aureus

26. Rapid Microbiological Methods Growth-Based Technologies measurement of biochemical or physiological parameters that reflect growth of microorganisms include: ATP bioluminescence: AKuScreen to screen for microbial contamination in pharmaceuticals colorimetric detection of CO 2 production: Bactec; BacT/Alert Cellular Component-Based Technologies detection of a specific cellular component include: Fatty acid profiles mass spectrometry ELISA fluorescent probe detection

27. Rapid Microbiological Methods Nucleic Acid-Based Technologies DNA probes: Gene-Trak; Gene-Probe molecular typing polymerase chain reaction (PCR) and other nucleic acid amplification technologies (NAATs) sequencing Automated Methods Simplest use classical method for processing sample, then detect colorimetric change to spot growth earlier than visual detection Replace human detection methods with machine detection; human judgment with machine intelligence include: BacT/ALERT, VITEK

28. VITEK 2 fully automated system for bacterial/fungal identification and antibiotic susceptibility testing reduces set-up time and minimizes manual steps Compact sealed ID/AST cards Rapid microorganism identification Rapid, same-day antimicrobial susceptibility testing Advanced Expert System validates IDs Data management software allows for generation of epidemiology reports and antibiograms

29. Xpert MTB/Rif Sealed cartridge Robust sonication/mechanical DNA extraction procedure Hemi-nested PCR targets rpoB gene associated with rifampicin resistance 2 hour result

30. ‘ Next-generation’ High-throughput sequencing ~100x faster, ~100x cheaper than conventional approaches Clonal template populations obtained by new methods: amplification on solid phase to grow a ‘molecular colony’ Massive increase in number of ‘clones’ but shorter read length New chemistries for sequence reading Pyrophosphate detection (PPi release upon base addition): 454 Single (reversibly 3’-blocked) fluorescent base (quenchable) added per step: Solexa Sequencing by Ligation (ABI SOLiD)

32. High-throughput Sequencing in Clinical Microbiology: Applications Pathogen detection and discovery Clinical metagenomics Polymorphism detection and discovery Genomic epidemiology Adaptive changes Pathogen biology Gene detection and discovery

33. Sequencing Methodologies Culture-dependent Culture-independent Shotgun library of purified genomic DNA Delivers whole-genome sequence Phylogenetic profiling: PCR with 16S primers Metagenomics: shotgun sequence community DNA

34. Culture-independent Pathogen Discovery

36. The Birth of Genomic Epidemiology for Bacteria

37. The Birth of Genomic Epidemiology for Bacteria

38. Case Study Acinetobacter baumannii Gram-negative bacillus Multi-drug resistant colistin and tigecycline as reserve agents moving towards pan-resistance Associated with wound infections and ventilator-associated pneumonia bloodstream infections returning military personnel from Iraq and Afghanistan transmission from military to civilian patients

39. Acinetobacter Genomic Epidemiology Outbreak in Birmingham Hospital in 2008 Isolates indistinguishable by current typing methods

40. Acinetobacter Genomic Epidemiology 454 whole-genome sequencing of 6 isolates SNP detection by mapping reads against draft reference assembly SNP filtering for false positives SNP validation with Sanger sequencing of PCR amplicons

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

44. Take-away messages Genome sequencing brings the advantages of open-endedness (revealing the “unknown unknowns”), universal applicability ultimate in resolution Bench-top sequencing platforms now generate data sufficiently quickly and cheaply to have an impact on real-world clinical and epidemiological problems

45. http://pathogenomics.bham.ac.uk/blog/2011/08/are-diagnostic-and-public-health-bacteriology-ready-to-become-branches-of-genomic-medicine/

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