Diagnostic Medical Microbiology

1. Diagnostic Medical Microbiology 29 Safar 1430 25 Febuary 2009

2. Objectives and Learning Outcomes • To acquire basic skills in the identification and isolation of microbial pathogens. • Demonstrate understanding of the principles of diagnostic medical microbiology and the clinical correlations. • Make observations, understand the fundamental elements of experimental design, generate and analyse data on pathogenesis and control of diseases. • Demonstrate proficiency in collection, interpretation, and presentation of scientific data in medical microbiology when conducting a research. • Communicate about case studies using appropriate oral and written means.

3. Lecture Outline: • Normal Bacterial & Fungal flora • Laboratory Aids in the selection of Antimicrobial Therapy • Diagnosis of Chlamydial infections • Diagnosis of Viral Infections • Diagnosis of HIV and AIDS

4. Normal Bacterial & Fungal Flora • Some organisms are considered pathogens whenever they are found in patients. Eg. • Many infections are caused by organisms that are permanent members of normal flora. • Gram-negative rods are strongly suspect as the cause of pneumonia eg. Klebsiella pneumoniae • In some cases, identification of normal flora is more warranted, eg. in abdominal abscesses. • Yeast in small numbers are common flora, not others. • Viruses are usually not part of the normal flora, but some latent viruses eg. _____________ or live vaccine viruses _______________ occasionally appear in viral cultures.

5. Laboratory Aids in the Selection of Antimicrobial Therapy • Drug testing is essential in those bacteria commonly showing resistance, primarily Staphylococcus sp., Neisseria gonorrhoea etc. Testing on fungal and protozoan infections is difficult and often unnecessary. Identification of infectious agents should be attempted as soon as possible, secured before the antimicrobic drug is given. • Choice of drug is based on “informed best guess”. • Disk diffusion susceptibility (or Kirby-Bauer) test – common test, that measures the ability of drugs to inhibit the growth of bacteria. The size of zones of inhibition vary with each different drug. • Tube dilution tests give more sensitive and quantitative results.

7. Laboratory Aids in the Selection of Antimicrobial Therapy • Minimum inhibitory concentration (MIC) = measures the concentration of an antibiotic necessary to inhibit growth of a standardised inoculum under defined conditions. The end point, or minimum inhibitory concentration is the last lowest concentration of drug that gives clear broth, ie. free from microbial growth. This is very useful in gauging the dosage regimen necessary for patients. • In addition, bactericidal effects can be estimated by subculturing the clear broth onto antibiotic-free solid media. The result, which is a reduction of colony-forming units by 99.9% below that of the control, is called the minimal bactericidal concentration (MBC). • The results of antimicrobic sensitivity tests guide the physician’s choice of a suitable drug. Continuous observation of patient’s clinical response is imperative once antimicrobial therapy has begun.

8. Laboratory Aids in the Selection of Antimicrobial Therapy • Drug toxicity – choose one with high/low selective toxicity for the infectious agent and high/low human toxicity. • Therapeutic index (TI) = the ratio of the dose of the drug that is toxic to humans as compared to its minimum effective (therapeutic) dose: • TI = toxic dose (µg/ml) effective dose (µg/ml) Hence, TI=10 is ______________than TI=1.2.

9. Laboratory Aids in the Selection of Antimicrobial Therapy • When antimicrobic treatment fails, this may be due to: • i) the inability of the drug to diffuse into body compartment (brain, joints) • ii) a few resistant cells that did not appear in the sensitivity test in vitro • iii) infection caused by more than one pathogen • Other considerations before administrating drugs: allergy, pregnancy, underlying liver or kidney diseases, age, any genetic or metabolic abnormalities, drugs incompatibility, cost.

10. Diagnosis of Chlamydia • Chlamydia : Structure • Outer cell wall resembles Gram-negative; with relatively high lipid content. • Cell wall is rigid but no peptidoglycan, hence no effect on lysozyme. Perhaps it contains tetrapeptide-linked matrix. • Penicillin-binding proteins occur in chlamydiae. • DNA and RNA are present in elementary and reticulate bodies.

11. Diagnosis of Chlamydial Infections • Although Chlamydia sp. are bacteria, they are obligate intracellular parasites. Hence, procedures are much likely resembled those of diagnosing viruses. • Specimens are collective from respective sites of infections, and carefully placed in transport medium:

12. Chlamydiae • Staining properties: • Giemsa stains: Elementary bodies – purple; larger, noninfective reticulate bodies stain – blue and host cytoplasm - blue. • Fully formed, mature intracellular inclusions of C trachomatis are compact masses near nucleus (dark purple) in Giemsa stain. • Gram staining – negative

13. Chlamydia • Microscopy: • Micro-organisms can sometimes be found in the cytoplasm of squamous cells from conjunctival scrapings due to Chlamydiapsittaci infection. This organism forms compact, oval-shaped grape-like cluster in the cells’ cytoplasm (yellow arrow). Sometimes dispersed elementary bodies can be seen.

14. Diagnosis of Chlamydia • Cell culture techniques for isolation of chlamydia species involves inoculation of the C trachomatis and C psittaci onto cycloheximide-treated McCoy cells (antimetabolites that inhibit host cell replication but allows chlamydiae to use available cell nutrient for growth), and C pneumoniae requires pretreated HEp-2 cells. • Immunoassay –EIAs are used to detect chlamydial antigens from genital swab. • Nucleic acid Hybridization – commercial kits with nonradioisotopic probes are available for C trachomatis 16S RNA sequences. PCR and ligase chain reaction (LCR) are much more sensitive than culture. • Serology: Complement fixation test is widely used to diagnose psittacosis. The microimmunofluorescence method is more sensitive than CF for measuring antichlamydial antibodies. Detection of IgM against C trachomatis is helpful in infant pneumonitis.

15. Diagnosis of Viral Infections • Isolation of a virus may not establish the cause of a given disease. Some viruses persist in human hosts for long periods. • Care for specimens: refrigerated for up to 24hr (except RSV) or freeze -60°C or colder. • Virus is present in pharyngeal secretions; fluid and scrapings of vesicular rashes; conjunctival swabs and tears; brain tissue; and CSF; faeces. • Direct fluorescent antibody is the tests of choice for diagnosis because they provide answers within a few hours after collection cf. virus culture.

16. Detection of Viral Growth • Lytic or cytopathic viruses replicate in cells and produce alterations in cellular morphology (or cell death) and the effect can be seen directly by light microscopy. • For example, enteroviruses often produce cell rounding, pleomorphism and eventual cell death; and measles and RSV cause fusion of cells to produce ____________. • Other viruses are detectable by their production of haemagglutinins; and by a method called interference whereby the virus which produces no cytopathic effect in susceptible cell culture, but can be detected by “challenging” the cell culture with a different virus that normally produces a characteristic cytopathic cell. The second virus fails to infect the cell culture because of the interference by the first virus, which is thus detected. Eg. Rubella virus.

17. Normal monkey kidney cell culture monolayer. B.Enterovirus cytopathic effect in a monkey kidney cell monolayer. Note cell lysis and monolayer destruction. (X 40)

18. C. Normal human diploid fibroblast cell monolayer. D. Cytomegalovirus cytopathic effect in human diploid cell monolayer. Note rounded, swollen cells in a focal area. (Χ40)

19. In vivo Isolation Methods • Embryonated hen’s egg is still used for the initial isolation and propagation of influenza A virus. The egg is incubated to permit viral replication and recognition. • Animal host, commonly the mouse; suckling mice in the first 48h of life are especially susceptible to many viruses. Viral replication is based on development of illness, with signs such as paralysis, convulsions, poor feeding or death. • Further test to elucidate the nature of infecting virus – by histologic and immunofluorescent examinations of tissues or by antibody detection.

20. Virus Identification • On isolation, virus can usually be identified by its cultural characteristics. Further identification may require adequate quantities for testing. • Several diagnostic methods for viral identification are: • 1. Antigen detection • 2. nucleic acid amplification and detection • 3. nucleic acid hybridisation • 4. measuring immune response to virus infection • 5. immune electron microscopy.

21. Diagnostic Virology • 1. Antigen detection: this neutralisation method works by neutralise virus infectivity by mixing it with specific antibody. • Immunofluorescence, enzyme immunoassay (EIA) and latex agglutination are common methods. • Commercial kits are available to detect many viruses inc HSV1 and HSV2, influenza A and B, RSV, adenovirus, parainfluenza virus, rotavirus, cytomegalovirus. • Advantage: allow detection of viruses that do not readily grow in the cell culture (rotavirus, Hep A), or that grow very slowly (CMV)

22. Diagnostic Virology • 2. Nucleic Acid Amplification & Detection: commercial assays are available which inc PCR, reverse-transcriptase PCR. • Detects enteroviruses; and quantifies HIV-1, CMV, EBV. • 3. Nucleic Acid Hybridisation: is a highly sensitive and specific method. • 4. Measuring immune response: cellular immunity may be assessed by dermal hypersensitivity, lymphocyte transformation, cytotoxicity tests. Humoral immune responses- IgM appear initially and are followed by IgG. IgM disappear in several weeks whereas IgG persist for many years. The methods used are neutralisation test, complement fixation, haemagglutination inhibition test, and the immunofluorescence test.

23. Diagnostic Virology • 5. Immune Electron Microscopy (EIA): for viruses not detectable by conventional techniques. • Antigen-antibody complexes or aggregates formed between virus particles in suspension are caused by the presence of antibodies in added antiserum and are detected more readily and with greater assurance than individual virus particles. • IEM detects viruses that cause enteritis and diarrhoea.

24. HIV and AIDS • HIV-1 occurs worldwide, while HIV-2 occurs primarily in West Africa and other geographic areas. • Blood banks use very sensitive tests to detect HIV-1 in donated blood. • Characteristics of HIV: • Family , RNA, • Possess viral enzyme reverse transcriptase. • Commonly assayed viral protein is p24. antibody responses to several HIV gene product: env, gp160, gp120, gp41, gag and pol.

25. Assays for Anti-HIV Antibodies • A. ELISA Enzyme-Linked Immunosorbent Assay: • Primary screening test for HIV-1 infection. • HIV-1 antigens immobilised on a solid surface (plastic wells, or beads). Add patient’s serum. HIV-1 antibodies bound to immobilised antigens are then detected with an enzyme labeled anti-human IgG and a colorimetric reaction. • Results interpretation: the amount of colour is proportionately higher with higher HIV-1 antibodies. • ELISA for HIV-1 is extremely sensitive and specific. • What about infants born with HIV-infected mothers? • ELISA test at 2 months old: • ELISA test at 2 years age:

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

27. Assays for HIV-1 Antibodies • B. Western Blot: • To measure specific HIV-1 antibodies to confirm a positive ELISA result.

28. Western Blot • 1. HIV-1 proteins are separated by electrophoresis. • 2. HIV-1 proteins are transferred onto nitrocellulose strip. • 3. the strip is incubated with patient’s serum. The specific HIV-1 antibodies are subsequently detected using an enzyme-linked antihuman IgG. • 4. positive colorimetric reaction forms bands on the nitrocellulose paper corresponding to p24, gp41 and pg120/160.

29. B. Western Blot: Results • i) Positive test : any 2 bands corresponding to the proteins. • ii) no bands : negative result • iii) bands that do not meet the criterion for a positive test is an indeterminate results. • False-positive and false-negative results are relatively uncommon. • Repeat positive ELISAs and indeterminate Western blots. • Infant born to an HIV-1-infected mother?

30. Assays to Detect HIV Infection • A. Detection of p24 Antigen: • ELISA to detect p24 antigen. p24 antigen is detectable during the acute viraemic stage and in the late stages of AIDS. • A very small proportion of asymptomatic HIV-1 infected persons are p24 antigen +. • B. Detection of HIV-1 RNA: commercial assays inc PCR. NASBA, bDNA. • C. HIV-1 Proviral DNA: DNA extracted from mononuclear cells obtained from anticoagulated peripheral blood. Perform PCR. Useful for infants. • D. HIV-1 Culture: peripheral blood mononuclear cells from a potentially infected patient are cocultured with uninfected cells stimulated with phyto haemagglutinin and interleukin-2. Cultures are looked for: multinucleated giant cells, HIV-1 rev transcrip, p24 Ag. Time-consuming and expensive.

31. Objectives and Learning Outcomes • To acquire basic skills in the identification and isolation of microbial pathogens. • Demonstrate understanding of the principles of diagnostic medical microbiology and the clinical correlations. • Make observations, understand the fundamental elements of experimental design, generate and analyse data on pathogenesis and control of diseases. • Demonstrate proficiency in collection, interpretation, and presentation of scientific data in medical microbiology when conducting a research. • Communicate about case studies using appropriate oral and written means.