Diagnosis of Infectious Diseases

1. Diagnosis of Infectious Diseases

2. Laboratory Investigation of Microbial infections Examining specimens to detect isolate and identify pathogens: 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4- Serological identification: 5- Molecular biology techniques 6- Bacteriophage typing

3. 1- Microscopy Microorganisms can be examined microscopically for: a- Bacterial motility: Hanging drop method: A drop of bacterial suspension is placed between a cover slip and glass slid b- Morphology and staining reactions of bacteria: Simple stain: methylene blue stain Gram stain: differentiation between Gm+ve and Gm–ve bacteria . Primary stain (Crystal violet) . Mordant (Grams Iodine mixture) . Decolorization (ethyl alcohol) . Secondary stain ( Saffranin) Ziehl-Neelsen stain: staining acid fast bacilli . Apply strong carbol fuchsin with heat . Decolorization (H2SO4 20% and ethyl alcohol . Counter stain (methylen blue)

4. 2- Culture Techniques * Culture media are used for: - Isolation and identification of pathogenic organisms - Antimicrobial sensitivity tests * Types of culture media: a- Liquid media: - Nutrient broth: meat extract and peptone - Peptone water for preparation sugar media - Growth of bacteria detected by turbidity b- Solid media: - Colonial appearance - Hemolytic activity - Pigment production

5. Types of solid media 1- simple media: Nutrient agar 2- Enriched media: media of high nutritive value . Blood agar . Chocolate agar . Loffler’s serum 3- Selective media: allow needed bacteria to grow . Lowenstein–Jensen medium . MacConkeys agar . Mannitol Salt Agar 4- Indicator media: to different. between lact. and non lact. ferment . MacConkeys medium . Eosine Methlyne blue Agar 5- Anaerobic media: for anaerobic cultivation . Deep agar, Robertson’s Cooked Meat Medium

6. Colonial appearance on culture media * Colony morphology: . Shape . Size . Edge of colony . Color * Growth pattern in broth: . Uniform turbidity . Sediment or surface pellicle * Pigment production: . Endopigment production (Staph. aureus) . Exopigment production (Ps. aeruginosa) * Haemolysis on blood agar: . Complete haemolysis (Strept. Pyogenes) . Partial haemolysis (Strept. Viridans) * Growth on MacConkey’s medium: . Rose pink colonies (Lactose fermenters) . Pale yellow colonies (Non lactose fermenters)

7. 3- Biochemical Reaction Use of substrates and sugars to identify pathogens: a- Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar b- Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface e- H2S production: Depends on production H2S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate

8. 3- Biochemical Reaction (cont.) c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator d- Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Color of medium turns pink (positive) e- Action on milk: Fermentation of lactose with acid production Red color if litmus indicator is added

9. 3- Biochemical Reaction (cont.) f- Oxidase test: Some bacteria produce Oxidase enzyme Detection by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive) g- Catalase test: Some bacteria produce catalase enzyme Addition of H2O2 lead to production of gas bubbles (O2 production) h- Coagulase test: Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method i- Urease test: Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH3 Alklinity of mediaand change color of indicator from yellow to pink

10. 4- Animal pathogenicity * Animal pathogenicity test: Animals commonly used are guinea pigs, rabbits, mice * Importance of pathogenicity test: - Differentiate pathogenic and non pathogenic - Isolation organism in pure form - To test ability of toxin production - Evaluation of vaccines and antibiotics

11. Serological identification A- Direct serological tests: - Identification of unknown organism - Detection of microbial antigens by using specific known antibodies - Serogrouping and serotyping of isolated organism B- Indirect serological tests: - Detection of specific and non specific antibodies (IgM & IgG) by using antigens or organisms

12. Molecular Biology Techniques A- Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labeled probe Probe: consists of specific short sequence of labeled single- stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA Then It is detected by a labeled probe C-Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis

13. Bacteriophage

14. Bacteriophage Bacteriophages are viruses that parasitize bacterial cell Replication of Bacteriophage : A- Lytic or vegetative cycle: End by lysis of bacterial cell and release of copies of the phage 1) Adsorption: Adsorption occurs between attachment sites on the phage (tail fibres) and specific receptor sites on bacteria It is specific strep (sensitivity of bacteria to different phages) 2) Penetration: The tail sheath will contract and inject DNA into bacterial cell

15. A- Lytic or Vegetative Cycle 3) Eclipse phase: Viral DNA directs the host cell metabolism to synthesize new enzymes and proteins for phage synthesis 4) Intracellular synthesis: Host cell machinery is directed by genetic information provided by phage nucleic acid to synthesize phage coats and nucleic a. 5) Assembly: Protein subunits of the phage head and tail aggregate Each capsid acquires nucleic acid molecule to become a mature phage particle 6) Release: Accumulation of huge number of phage The cell bursts and phage particles are released

16. II- Temperate Phage cycle “Lysogenic cycle” * Adsorption and penetration take place as in lytic cycle * Virus DNA integrate with host chromosome (Prophage) and replicate as part of host chromosome * The bacterial cell is called a”lysogenic bacterium” * Lysogenic bacterium has certain characters: a- Immune to infection by another phage b- Acquire new properties e.g. production of exotoxin Diphtheria bacilli, Cl. Botulinum, Strpt. Pyogen erthrogenic toxin

17. Outcome of Temperate cycle 1) The cell continue carrying prophage indefinitely, passing it to daughter cells 2) The prophagedetach from the bacterialchromosome and start a lytic cycle 3) As prohage is detached it may carry genetic material of bacterial chromosome As it infects another bacterium , it will transmit to it new characters

18. Practical applications using phages * Phages are important as a research tools * Phages are used as vectors in DNA recombinant technology * Phage typing of bacteria is important in tracingsource of infection for epidemiologic purposes

19. Antimicrobial Susceptibility testing • Introduction: • Identification of a bacterial isolate from a patient provides guidance in the choice of an appropriate antibiotic for treatment • Many bacterial species are not uniformly susceptible to a particular anti-bacterial compound • This is particularly evident among the Enterobacteriaceae, Staphylococcus spp., and Pseudomonas spp. • The wide variation in susceptibility and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy

20. Antimicrobial Susceptibility testing can be down by three ways: • Minimum Inhibitory Concentration (MIC) • Disk Diffusion Method • Minimum Bactericidal Concentration (MBC)

21. 1. Minimum Inhibitory Concentration (MIC) : Principle: • The tube dilution test is the standard method for determining levels of resistance to an antibiotic. • Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. • The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).

22. Different concentrations of Gentamycin in Nutrient broth: Conc. in mcg/ml 0.1 0.2 0.4 0.8 1.6 3.1 Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml

23. Different concentrations of Tetracycline in Nutrient broth: Conc. in mcg/ml 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml

24. 2. Disk-diffusion Method (Kirby-Bauer Method): • The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a clinical laboratory where a large number of isolates are tested for susceptibility to numerous antibiotics. • An agar plate is uniformly inoculated with the test organism • A paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface. • Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations. • The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.

25. This test must be rigorously standardized since zone size is also dependent on: • inoculum size, • medium composition, • temperature of incubation, • excess moisture and • thickness of the agar. • Zone diameter can be correlated with susceptibility as measured by the dilution method. • Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy.

26. Using a dispenser, antibiotic-impregnated disks are placed onto the agar surface. • As the bacteria on the lawn grow, they are inhibited to varying degrees by the antibiotic diffusing from the disk.

27. Staphylococcus aureus (MRSA) • Note the yellowish pigmentation of the bacterial lawn, and the lack of inhibition by the Oxacillin disk

28. Streptococcus pneumoniae (Pneumococcus): • The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-haemolysis.

29. Pseudomonas aeruginosa: • The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.