M icrobiology, V irology and I mmunology Department

1. Microbiology, Virology and Immunology Department Classification and modern methods of diagnostics of infectious diseases

2. METHODS OF EXAMINATION IN MICROBIOLOGY • BACTERIOSCOPIC • BACTERIOLOGICAL • SEROLOGICAL • BIOLOGICAL • ALLERGIC • EXPRESS-DIAGNOSIS

3. BACTERISCOPIC METHOD Staphylococcus aureus Escherichia coli M. tuberculosis M. avium

4. BACTERISCOPIC METHOD Borrelia Yersinia pestis Neisseria gonorrhoeae Corynebacterium diphtheriae

5. BACTERISCOPIC METHOD Bacillus anthracis Clostridium tetani Clostridium botulinum

6. BACTERISCOPIC METHOD

7. BACTERIOLOGICAL METHOD Isolation of pure culture Examples of varous colony morphologies. The appearance of colonies on a plate is species specific and can be very helpful in identifying.

8. Pure culture of conditionally-pathogenic microbes may be causative agents of disease according to the such signs: • microbes are present in tested material from pathologic focus in the amount of 104-105colony-forming units (CFU) in 1 mlor 1 g; • repeated isolation from the same material the same culture; • increasing in the patient’s serum antibodies to the autostrains or microbial culture, which can be causative agent.

9. Necessary rules before collection of tested material: • to take material before antibacterial therapy beginning or after some time after antibiotic inoculation which is necessary for its excretion from the organism (as a rule 8-10 hours); • to take material from infectious focus or examine proper discharges; • hold on to the strict aseptic for the purpose to prevent contamination of the specimen by microflora of environment;

10. Necessary rules before collection of tested material: • material is taken into the sterile boxes; clinical specimen with anaerobic bacteria must be protected from atmosphere oxygen action; • the collection of an adequate specimen is useless if the time between collection and culturing allows the disease-producing organism to die (in another cases it’s necessary to use the refrigerator or special transport media);

11. Necessary rules before collection of tested material: • isolation of viruses, Rickettsia, Chlamidia is made in specialize laboratories; • to clinical specimen a proper document is added, which has data , which has data for correct microbiological examination.

12. BACTERIOLOGICAL METHOD Most specimens received in a clinical microbiology lab are plated onto Blood Agar

13. Gold’s streak

14. Bacillus Proteus BACTERIOLOGICAL METHOD

15. Staphylococcus Streptococcus BACTERIOLOGICAL METHOD

16. BACTERIOLOGICAL METHOD Klebsiella pneumoniae Mannitol SaltMedium Most non-pathogenic staphylococci will not ferment mannitol

17. BACTERIOLOGICAL METHODisolation of pure culture

18. BACTERIOLOGICAL METHODbiochemical identification Biochemical Reactions on API Strip Proteus mirabilis

19. Escherichia coli Lactose Positive Colonies MacConkey Agar Positive Negative Indole Reactions

20. BACTERIOLOGICAL METHOD Serological method Agglutination test

21. BACTERIOLOGICAL METHOD Serological method Agglutination test

22. BACTERIOLOGICAL METHOD Serological method IHT

23. BACTERIOLOGICAL METHOD Serological method Ring precipitation test

24. BACTERIOLOGICAL METHOD Serological method Doublel Immune Diffusion (Ouchterlony’) Test

25. BACTERIOLOGICAL METHOD Serological method Radial Immune Diffusion (Mancini’s) Test

26. BACTERIOLOGICAL METHOD Serological method CFT

27. BACTERIOLOGICAL METHOD Serological method ELISA

28. SEROLOGICAL METHODS Bordetella pertussis Express-diagnosis Treponema pallidum IFA

29. Molecular Diagnostics • Fluorochromes • Fluorescein (490→517nm) • Rhodamine (515→546nm) • Phycoerythrin Immunological Diagnostics Methods Immunofluorescence Protein A has the ability to bind to IgG mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

30. BIOLOGICAL METHOD tetanus botulism

31. BIOLOGICAL METHOD TBC in rabbit TBC in guinea pig

32. ALLERGIC METHOD Mantoux’s test

33. Applications of Molecular Based Testing in Clinical Microbiology • Rapid or high-throughput identification of microorganisms • Detection and analysis of resistance genes • Genotyping • Classification • Discovery of new microorganisms

34. Specimen Collection • Preserve viability/nucleic acid integrity of target microorganisms • Avoid contamination • Appropriate time and site of collection (blood, urine, other) • Use proper equipment (coagulant, wood, or plastic swab shafts) • Commercial collection kits are available • The Clinical and Laboratory Standards Institute (CLSI) has guidelines for proper specimen handling

35. Sample Preparation • Consider the specimen type (stool, plasma, CSF) • More rigorous lysis procedures are required to penetrate cell walls • Consider the number of organisms in the sample • Inactivate inhibitors (acidic polysaccharides in sputum or polymerase inhibitors in CSF) • Inactivate RNases

36. Molecular Diagnostics Immunological Diagnostics Methods ELISA • Addition of a specific antibody (primary antibody) which will bind to the test molecule if it is present. • Washing to remove unbound molecules. • Addition of secondary antibody which will bind to the primary antibody. • The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. • Wash to remove unbound antibody. • Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result. • -> can be used for quasi High-throughput!!!

37. Molecular Diagnostics Immunological Diagnostics Methods ELISA -Variants • Detection based on enzyme catalyzed reactions: • alkaline ⓟ • horseradish peroxidase • β-galactosidase • Detection based on fluorescent labeled secondary antibody

38. Molecular Diagnostics Immunological Diagnostics Methods Western blot SDS-Page: separates the components according to their molecular weight. Blot: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current. Immunoreaction: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab. Detection: a position is visualized by means of an ELISA reaction.

39. Molecular Diagnostics DNA Diagnostic Systems Example: Using PCR to Detect for HIV PCR based methods -> The presence of the appropriate amplified size fragment confirms the presence of the target. -> Specific primers are now available for the detection of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi. • RT-PCR (reverse transcriptase PCR). • HIV has a ssRNA genome. • Specific primers are used to amplify a 156 bp portion of the HIVgag gene. • Using standards the amount of PCR product can be used to determine the viral load. • PCR can also be used as a prognostic tool to determine viral load. • This method can also be used to determine the effectiveness antiviral therapy. Other examples: -> Using PCR to Detect DMD deletions (60% of mutations are deletions)

40. Polymerase Chain Reaction • Capable of amplifying tiny quantities of nucleic acid. • Cells separated and lysed. • Double stranded DNA separated into single strands. • Primers, small segments of DNA no more than 20-30 nucleotides long added. • Primers are complementary to segments of opposite strands of that flank the target sequence. • Only the segments of target DNA between the primers will be replicated. • Each cycle of PCR consists of three cycles: • denaturation of target DNA to separate 2 strands. • annealing step in which the reaction mix is cooled to allow primers to anneal to target sequence • Extension reaction in which primers initiate DNA synthesis using a DNA polymerase. • These three steps constitute a thermal cycle • Each PCR cycle results in a doubling of target sequences and typically allowed to run through 30 cycles, one cylce takes approximately 60-90 seconds.

41. (95°C) (55°C-65°C). (72°C) The final product can be checked on an agarsoe gel to make sure it has correct size and can be sequenced. The final product is available in high conc. and can be used for cloning (gene that has a product), diagnosis (gene of a virus) or fingerprinting (forensic investigation in crime scenes).

42. PCR Polymerase Chain Reaction Continue 30 cycles 230 Copies Denature 21 Copies 22 Copies 23 Copies

43. PCR tubes Thermocycler Sequencing Agarose gel to detect the products

44. Gel Electrophoresis apparatus Loading the gel

45. After reaction is complete, the DNA is loaded into an agarose gel, in order to visualize the bands. The DNA is mixed with Glycerol and specific reagents when loading into the gel. An electric current is applied so the DNA moves towards the + ve electrode according to it s size Small fragments move faster and reach the end of the gel, larger fragments move slower and are at the beginning the gel. Gel is stained to view the DNA bands.

46. Larger bands up Smaller bands down Direction of migration Agarose gel stained with Ethedium bromide and visualized under UV

47. Applications of PCR In forensic medicine: For instance, trace amounts of DNA, in fluids such as blood or semen or in tissue such as hair, can be amplified by PCR and analyzed to see whether the DNA is identical to that of a person suspected of committing a crime. In clinical diagnosis: The technique enabled clinicians to detect infection by the AIDS virus when other methods have failed, in addition to other diseases. Diagnosis genetic diseases, such as sickle cell anemia in fetus still in its mother’s uterus, by amplifying the genetic information provided by just a few fetal cells, which canbe obtained without harming the fetus.

48. Target Microorganisms for Molecular-Based Testing • Those that are difficult or time-consuming to isolate • e.g.,Mycobacteria • Hazardous organisms • e.g.,Histoplasma, Coccidiodes • Those without reliable testing methods • e.g.,HIV, HCV • High-volume tests • e.g.,S. pyogenes, N.gonorrhoeae, C. trachomatis