Introduction to diagnostic microbiology

1. Introduction to diagnostic microbiology Dr.Ayham Abu Laila

2. Diagnosis of Bacterial Infection Non-microbiological investigations Patient Clinical diagnosis Radiology Haematology Biochemistry Sample Take the correct specimen Take the specimen correctly Label & package the specimen up correctly Appropriate transport & storage of specimen Dr.Ayham Abu Laila

3. A proper clinical assessment is essential for optimal use of laboratory services! Dr.Ayham Abu Laila

4. Dr.Ayham Abu Laila

5. Getting the specimen to the lab • Problems in delay or inappropriate storage• delay in diagnosis & treatment • pathogens die • contaminants overgrow • Blood cultures directly into incubator • not refigerator! • CSF straight to lab • Don't put an entire surgical specimen into formalin! • Send a portion to microbiology in a sterile container Dr.Ayham Abu Laila

6. Collecting the specimen correctly • Take an mid-stream urine • avoids contamination with perineal flora • CSF • Avoid contamination • Avoid bloody tap • Throat swab • Make the patient gag! • Blood cultures • Avoid contamination with skin organisms Dr.Ayham Abu Laila

7. Specimens & 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!!! Dr.Ayham Abu Laila

8. Factors limiting usefulness of bacteriological investigations • 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 Dr.Ayham Abu Laila

9. Diagnosis of Bacterial Infection microscopy unstained or stained with e.g. Gram stain Stain Decolorise Counterstain identification by biochemical or serological tests on pure growth from single colony culture on plates or in broth sensitivities by disc diffusion methods, breakpoints or MICs DNA technologies Serodiagnosis Dr.Ayham Abu Laila

10. MicroscopyUnstained preparations • “Wet prep” • Dark-ground illumination for syphilis Dr.Ayham Abu Laila

11. MicroscopyStained preparations • Gram-stain • Acid-fast stain • Ziehl-Neelsen • Fluorescence • Direct, e.g. auramine • Immunofluorescence Dr.Ayham Abu Laila

12. Culture of Bacteria • 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 Dr.Ayham Abu Laila

13. Advantages of Solid Media • isolation of single clonal colonies • get bacterium in pure culture • identify by colonial morphology • quantification by colony-forming units Dr.Ayham Abu Laila

14. Identification of Bacteria • Morphology • Growth requirements • Biochemistry • Enzymes • Antigens Dr.Ayham Abu Laila

15. Non-cultural diagnostic methods • Antigen detection • e.g. latex agglutination • Antibody detection • e. g. agglutination tests, complement fixation tests, indirect immunofluorescence • Molecular methods • Polymerase Chain Reaction Dr.Ayham Abu Laila

16. Sensitivity tests • on solid media • disc diffusion technique • in liquid media • minimum inhibitory concentration (MIC) test • Breakpoint methods • E-test Dr.Ayham Abu Laila

17. Diagnosis of Viral Infection • Electron microscopy • Antigen detection • Antibody detection • Virus culture • Detect cytopathic effect or antigen • Molecular methods • Polymerase Chain Reaction • Sequencing (e.g. for sensitivities) Dr.Ayham Abu Laila

18. Microbes and humans Very few microbes are always pathogenic Many microbes are potentially pathogenic Most microbes are never pathogenic Dr.Ayham Abu Laila

19. Microbes and humans Disease can come about in several overlapping ways 1. Some bacteria are entirely adapted to the pathogenic way of life in humans. They are never part of the normal flora but may cause subclinical infection, e.g. M . tuberculosis 2. Some bacteria which are part of the normal flora acquire extra virulence factors making them pathogenic, e.g. E. coli 3. Some bacteria which are part of the normal flora can cause disease if they gain access to deep tissues by trauma, surgery, lines, e.g. S. epidermidis 4. In immunocompromised patients many free-living bacteria and components of the normal flora can cause disease, especially if introduced into deep tissues, e.g. Acinetobacter Dr.Ayham Abu Laila

20. How do we know that a given pathogen causes a specific disease? • Koch's postulates • the pathogen must be present in every case of the disease • the pathogen must be isolated from the diseased host & grown in pure culture • the specific disease must be reproduced when a pure culture of the pathogen is inoculated into a healthy susceptible host • the pathogen must be recoverable from the experimentally infected host Dr.Ayham Abu Laila

21. The iceberg concept of infectiousdisease poliomyelitis in a child classical 0.1-1% of infections are clinical disease clinically apparent less severe disease rubella 50% of infections are clinically apparent asymptomaticinfection Spectrum ofvirulence rabies 100% of infections Dr.Ayham Abu Laila are clinically apparent

22. How do we know that a given pathogen causes a specific disease? 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 Recognised syndromes e.g. septicaemia, endocarditis, osteomyelitis meningitis, UTI, pneumonia pharyngitis patient's clinical condition potential pathogen isolated from or detected in clinical samples Dr.Ayham Abu Laila

23. Microbes and humans • Evidence for a potential pathogen being clinical significant (particularly for bacteria) • Isolated in abundance • Isolated in pure culture • Isolated on more than one occasion • Isolated from deep tissues • Evidence of local inflammation • Evidence of immune response to pathogen • Fits with clinical picture Dr.Ayham Abu Laila

24. Normal flora • All body surfaces possess a rich normal bacterial flora, especially the mouth, nose, gingival crevice, large bowel, skin • This can be a nuisance in that • it can contaminate specimens • it can cause disease • This is beneficial in that • it can protect against infection by preventing pathogens colonising epithelial surfaces (colonisation resistance) • removal of the normal flora with antibiotics can cause superinfection, usually with resistant microbes Dr.Ayham Abu Laila

25. How to identify pathogen • Culture characteristics • Morphology and staining • biochemical tests • Serological characteristics • Other characteristics Dr.Ayham Abu Laila

26. Culture characteristics • Shape • Margin • Optical properties • Elevation • Texture • Color • Odor Animation Dr.Ayham Abu Laila

27. Morphology and staining • Cell shape • Arrangement • Staining reaction • Gram stain • Negative stain • End spore stain • Acid fast stain Dr.Ayham Abu Laila

28. Specimen processing • Receiving • Recording • Culturing • Staining • Isolation • Identification • Sensitivity test Dr.Ayham Abu Laila

29. Patient details • Name, Age • Hospital No • Sex, For female: wither pregnant or lactating • Address • Suspected diagnosis • Travel history • immunization Dr.Ayham Abu Laila

30. Identification of specimens • Patient details • Type of specimen • Collection date and time • Laboratory No • Test requested • Name of ordering physician Dr.Ayham Abu Laila

31. Specimen rejection criteria • Mismatch information • Improper container or temperature • Insufficient specimen • Leaking specimen • Formalin specimen • Dried out swap • Late specimen Physician must be informed about rejection Dr.Ayham Abu Laila

32. Report of bacteriology result • CSF, body fluid, blood, and wound: • positive gram stain • Culture and isolation • Identification • Ear: • Potential pathogens; S. aureus, G –ve • Eye: • report identification of any organism Dr.Ayham Abu Laila

33. >Report of bacteriology result • Gastrointestinal: • Routine screen for Salmonella, shigella, campylobacter, Vibrio, and E. coli O157:H7 • Negative culture will be reported as “No enteric pathogens isolated” • Lower respiratory and sputum: • report identification of any organism Dr.Ayham Abu Laila

34. >Report of bacteriology result • Nasal / nasopharyngeal: • report identification of any Gram –ve rod, S. aureus, S. pneumonia, H. influenza, N. meningitides, group A streptococci. • Skin: • Predominant organism • Throat: • group A streptococci, Sensitivity test Dr.Ayham Abu Laila

35. > Report of bacteriology result • Mouth: • you must identify interest to be screened i.e. C. albicans, C. diptheriae • Urine: • report identification and antimicrobial sensitivity on colony count greater than 10.000 CFU • Plates with 3 or more will be reported • Mixed flora of less than 10.000 will be reported as normal skin flora Dr.Ayham Abu Laila

36. > Report of bacteriology result • Vaginal / cervical: • Report predominant organisms • Mixed culture of lactobacillus, diptheroids, staphylococcus, alpha streptococcus, and yeast will be reported as normal vaginal flora. Dr.Ayham Abu Laila

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