MIIM30011 2024 L5- Detection of pathogens.pdf

 MIIM30011 – Medical Microbiology: Bacteriology Lecture 5: Detecting/identifying pathogens Dr Sacha Pidot Learning objectives Describe and explain the different culture- dependent methods used to diagnose bacterial infections Describe and explain the different...

 MIIM30011 – Medical Microbiology: Bacteriology Lecture 5: Detecting/identifying pathogens Dr Sacha Pidot Learning objectives Describe and explain the different culture- dependent methods used to diagnose bacterial infections Describe and explain the different culture- independent methods used to diagnose bacterial infections  Diagnostic microbiology Use a range of tests to identify disease causing agent Clinical specimen type and handling is important – why? – Where is the disease? – Where is the pathogen, and is it fastidious? – Samples could include: Sputum, Urine, Blood, Feces, Wounds, CSF, etc  When is a pathogen not a pathogen? Normal flora at many sampled body sites Some of these normal flora can also be pathogens, some will appear as contaminants in specimens – Can avoid by disinfection – not possible for all sites (e.g. intestines) Often impossible to collect an uncontaminated sample How do we tell the difference? – Knowledge of disease also important  Diagnostic microbiology Test depends on specimen type and presumptive illness Sensitivity, specificity, speed, cost, also important Culture independent + genomics Culture dependent Tests defined by sensitivity, specificity and speed Sensitivity – the proportion of people WITH Disease X that have a POSITIVE blood test. A test that is 100% sensitive means all diseased individuals are correctly identified as diseased i.e. there are no false negatives. Specificity – the proportion of people WITHOUT Disease X that have a NEGATIVE blood test. A test that is 100% specific means all healthy individuals are correctly identified as healthy, i.e. there are no false positives. Speed – relative and arbitrary. Gram staining CSF from a ? Meningitis patient is a rapid test. PCR may be a rapid test, culture is usually not. Tests are generally becoming more rapid, and ideally at Point of Care (POC). Culture dependent detection methods Bacterial culture Biochemical tests MALDI TOF-MS (depends on sample…)  Bacterial culture Culture-dependent method Involves growth of bacteria on physical media Liquid or agar plates May require cell culture for some bacteria (Chlamydia) Growth can depend on: Temp O2 = aerobes vs anaerobes Time = S. aureus (overnight) vs M. tuberculosis (4-6 weeks) Media type  Selective/differential media Use to “select” or “differentiate” from other organisms within a sample – Selective = inhibit growth of normal flora at same site – Differential = allows growth of both, but EMB agar indicates organism of interest – inhibits G+ve, differentiates b/w lactose fermenters (E. coli – Baird-Parker agar dark centres); non-lactose – Identify S. aureus fermenters (Salmonella, Shigella - inhibits G-ve, S. aureus appear as black – clear colonies) colonies (reduction of tellurite) with halo around them (proteolysis of egg yolk)  Biochemical tests Biochemical tests – Usually based on differential features of closely related organisms or those from similar sites – Most based on enzyme production or activity – catalase, oxidase, urease, etc – Or utilization of carbohydrates, tolerance to particular conditions – 6.5% salt, etc Now rolled into kits for specific organisms – API test strips – 4 – 48 hrs for ID Automated culture systems BACTEC or BACT/ALERT – For blood, sterile fluids, platelets – Grow and scan bottles for bacterial growth (colormetric change) – Alerts when growth detected – bottle removed for further analysis VITEK 2 – For ID and AST in one – All in one machine – Cards inoculated and regularly scanned for growth Full lab automation – WASP system Fully automates plating, biochemical tests, etc https://www.youtube.com/watch?v=ENfS29OeJtA&feature=emb_logo Culture independent detection methods Microscopy of specimen – Staining – Direct and indirect immunofluorescence Immune reactions – ELISA, etc Molecular methods – PCR – DNA sequencing MALDI (depends on sample…)  Microscopic detection Methods for microscopy – Direct examination of smears by staining Which stains? TB commonly diagnosed by staining – 36.8% sensitivity, needs >10,000 bacteria/mL Immunofluorescence – Fluorescent antibody binds directly to pathogen – Viewed under microscope – Difficult to grow pathogens Molecular detection methods PCR/real time PCR – Nucleic acid amplification – Often used where organisms are difficult to culture or speed of diagnosis is essential – Automated systems available eg – GeneXpert for TB diagnosis (other as well) TB usually diagnosed from stain of sputum GeneXpert automates DNA extraction and DNA amplification from sputum samples Cartridge based system – just add sputum, press go Detection for M. tuberculosis in 200 cases → 29 died ?Flu For 5 months, couldn’t grow the organism and symptoms didn’t really fit with known bacterial infections Infected guinea pigs with lung tissue from infected patients – Guinea pigs became sick, but tissue specimens showed mainly scattered rods → ? contaminants Infected eggs with guinea pig tissue – Found masses of rod-shaped bacteria in the eggs Used serum from patients to test for antibodies against these rod-shaped bacteria – Found that patients had antibodies against these bacteria Later found earlier disease “Pontiac fever” = Legionaires disease – Same causative agent – Legionella pneumophila Dr Joseph McDade  Summary Diagnostic tests can be classified into: – Culture dependent Need bacterial growth for test to work – Culture independent Can directly use patient samples Most importantly, good diagnosis relies on combined understanding of the disease and correct interpretation of results!! Finally… Take 2 minutes and write down: – What was the most important thing you learned today? – What questions remain in your mind?