Diagnostic Medical Microbiology & Clinical Correlation

1. Diagnostic Medical Microbiology & Clinical Correlation 22 Safar 1428H 18 Febuary 2009

2. Principles of Diagnostic Medical Microbiology • Physicians who deal with infectious process must know when and how to take specimens, what laboratory examinations to request for. • Laboratory procedures: • Morphologic identification of agent • Culture isolation and identification • Detection of Ag from the agent • DNA-DNA or DNA-RNA hybridization • Ab or cell-mediated immune responses

3. Bacterial & Fungal Infections • Specimens : direct tissue, or fluid samples ( are collected from normally sterile tissue eg. and body fluids eg. and indirect samples (of inflammatory exudates eg. that have passed through sites known to be colonised with normal flora. • Specimen collection  isolation of organism • Recovery of bacteria and fungi vs. normal microbial flora. • A few general rules: i) adequate quantity; ii) sample representative of the infectious process; iii) avoid contamination; iv) prompt examination and v) meaningful specimens must be secured before the administration of antimicrobial drugs.

4. Microscopy & Stains • A relatively simple and inexpensive method, but much less sensitive than culture. • Gram-staining : note on Gram reaction, morphology. The appearance of bacteria on Gram-stained smears provide suggestion of organism, but not definitive. Some non-viable G+ can stain gram negatively.

5. Morphology & Stains • Bacterial morphology has been defined to bacteria grown on agar, however in body fluids and tissue, morphology of bacteria are highly variable. • Mycobacteria: acid-fast organisms (Ziehl-Neelsen stain). Other stains used is auramine-rhodamine but requires fluorescence microscopy. • Immunofluorescent (IF) antibody staining: more specific; fluorescein-labelled Abs are made from antisera produced by injecting animals with whole organisms or complex Ag mixtures  results in polyclonal Abs. Control to minimise nonspecific IF, or use monoclonal Abs. • IF is more useful for specific organisms, isolated on culture media, than for organisms directly isolated from patients.

6. Morphology & Stains • For fungi or other parasites, • Specimens to examine fungi is possible after treatment with 10% _____, which breaks down

7. Gram-staining • 1. Fix smear by heat. • 2. Cover with crystal violet. • 3. Wash with water. Do not blot. • 4. Cover with Gram’s iodine. • 5. Wash with water. Do not blot. • 6. Decolorise for 10-30s with gentle agitation in acetone (30mL) and alcohol (70mL). • 7. Wash with water. Do not blot. • 8. Cover for 10-30s with safranin. • 9. Wash with water and let dry.

8. Mycobacteria staining • Kinyoun carbolfuchsin • Formula: 4g basic fuchsin, 8g phenol, 20mL 95% alcohol, 100mL distilled water. • Stain fixed smear for 3min (no heat necs) cont with Z-N stain. • Ziehl-Neelsen acid-fast stain • 1. fix smear by heat. • 2. Cover with carbolfuchsin, steam gently for 5 min over direct flame (or 20min over a water bath). • 3. Wash with water. • 4. Decolourise in acid-alcohol until only a faint pink colour remains. • 5. Wash with water. • 6. Counterstain for 10-30s with Loeffler’s methlene blue. • 7. Wash with water and let dry.

9. Ziehl-Neelsen staining • Identification of acid-fast bacilli

10. Culture Systems • Standard medium is blood agar, 5% sheep blood for most aerobic and facultatively anaerobic organisms. • Chocolate agar, medium with heated blood. • Selective medium (MacConkey or eosin-methylene blue [EBM] agar) for enteric Gram- rods. • Obligate anaerobes must be plated on at least 2 types of media: i) highly supplemented agar such as brucella agar with hemin, vit K; and ii) selective medium containing substances that inhibit the growth of other non-obligates. • Specialised media - depend on clinical diagnosis and the organism under consideration. Eg. freshly made Bordet-Gengou or charcoal-containing medium to culture for B. pertussis.

11. MacConkey’s Agar

12. Culture Systems • Broth cultures is highly enriched media, important for back-up cultures of biopsy tissues and body fluids. • Many yeasts will grow on blood agar. • Biphasic and mycelial phase fungi grow better on media designed specially for fungi eg. Brain-heart infusion agar (+/- antibiotics) and inhibitory mold agar. • Fungi also grow in media made with plants and vegetable materials.

13. Antigen Detection • Immunologic systems to detect antigens of microorganisms. IF test is one example. • Enzyme immunoassays (EIA) inc enzyme-linked immunosorbent assays (ELISA) and agglutination tests. • In latex agglutination, an antigen-specific antibody (either polyclonal or monoclonal) is fixed to latex beads. Most useful to detect carbohydrate Ags of encapsulated microorganisms. • Immunoblotting (“Western blot”) whereby defined antigens are placed on strips of nitrocellulose paper. Following incubation, the strip is treated with an enzyme-labeled Ab. Addition of the substrate for the enzyme allows detection of Ag-specific bound Ab by colorimetric reaction. Useful for Abs in HIV infection and Lyme disease.

14. Principle of Enzyme Immunoassays First Ab Add antigen Second antibody for the Ag, labelled with enzyme

15. Molecular Diagnostics • Hybridisation of a characterised nucleic acid probe (primer, oligonucleotides) to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. • The nucleic acid probe typically is labeled with enzymes, antigenic substrates, chemiluminescent molecules or radioisotopes to facilitate detection of the hybridisation product.

16. Molecular Diagnosis • A. Identifying bacteria using 16S rRNA • The 16S rRNA has conserved portions of the sequence. • Labeled probe specific for the 16S rRNA of a species are added and then measured. This allows the identification of Mycobacterium sp., Coccidioides immitis, Histoplasma capsulatum. • Portions of the 16S rRNA are conserved across many species and its amplification using primers allows isolation and sequencing of the variable regions of the molecules. These genus- or species-specific allows the identification of pathogens that are impossible or difficult to culture. Eg. Tropheryma whipplei the cause of Whipple’s disease.

17. Molecular Diagnosis • B. Target amplification systems • The polymerase chain reaction (PCR) is used to amplify extremely small amounts of specific DNA. • This technique uses DNA polymerases through alternate changes in temperature to initiate replication in either the 3’ or 5’ direction. The specificity is provided by primers that recognise a pair of unique sites on the chromosome so that the DNA between them can be replicated. • PCR can also be performed on RNA targets, which is called reverse transcriptase PCR. The rev transcriptase is use to transcribe RNA into complimentary DNA for amplification. • PCR assays available commercially for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, cytomegalovirus and enteroviruses.