Topic P08: Laboratory diagnostics of tuberculosis, actinomycetes and nocardiae Task 1: Microscopy of acid-fast and partially acid fast microoorganisms While entirely acid-fast microorganisms (Mycobacterium) cannot be stained using Gram staining. only partialy acid-fast ones (Actinomyces, Nocardia) can be Gram strained, but they stain inconstantly; they also tend to have branched filamentous forms. a) Staining of (negative) clinical material using Ziehl-Neelsen staining method ________________________________________________________________________________ Ziehl-Neelsen staining is used for mycobacteria (M. tuberculosis, M. leprae), but also for some parasites (Cryptosporidium parvum, Cyclospora cayetanensis). The acid-fast organisms are stained only when heated during staining*, but then they are not decororized even by so clled „acid alcohol“ (solution of alcohol with HCl or H[2]SO[4]). Decolorized bacground is then counerstained. Stain the negative sputum according to the Ziehl-Neelsen method (methylene blue variant). It is not likely that acid-fast rods would be present. Observe in microscope (immersion). Draw the results; at least, you will see the bacground, e. g. leucocytes, epithelia and other objects. Do not forget do describe your picture (use lines)! Describe also the staining procedure – fill in the following table with names of used reagents. 1. During the staining the preparation is until 2. This reagent is made of and 3. Instead of this reagent, it is also possible to use b) Microscopy of a mycobacterial culture ________________________________________________________________________________ Examine microscopically (immersion oil, immersion 100× objective) the preparation of mycobacterial culture stained by Ziehl-Neelsen staining method. Evaluate presence of red acid-fast rods. Draw observed structures. Do not forget do describe your picture (use lines)! c) Microscopic examination of actinomycetes and nocardia strains ________________________________________________________________________________ Examine microscopically the slide stained by Gram. Describe and draw observed formations. Observe high polymorphism of the microorganisms (from coccal shape, through rods to fibre/strings, often branched; Gram-positive, but often staining half Gram-negative). Do not forget do describe your picture (use lines)! Task 2: Culture of mycobacteria, Actinomyces and Nocardia. The culture requests of acid fast and partialy bacteria are very different. v For Mycobacterium tuberculosis we use special media: liquid media (Šula, Banič) and solid media (Ogawa, Löwenstein-Jenssen). The solid media are different from majority of other solid media used in medical microbiology; they do not contain agar, they are „solid“ because of coagulated egg proteins. Before culture, the medium should be specially treated. v For Nocardia a current blood agar is sufficient. v For Actinomyces we need VL-agar and culture in anaerostat/anaerobic jar (see P07), as this organism is anaerobic. *Heating may be eventually substituted by use of highly concentrated carbolfuchsin and higly concentrated phenol; this modification of Ziehl Neelsen staining (Kinyoun modification) does not require heating. a) Describe media for mycobacterial cultivation Medium name liquid/solid colour notes b) Describe and draw the growth of Mycobacterium, Actinomyces and Nocardia on (in) given media Bacterium Medium name Presence/absence of growth, eventually growth character (use your own words to characterize the growth) Mycobacterium Actinomyces blood agar VL agar Nocardia blood agar VL agar Task 3: Assessment of antimicrobial drugs susceptibility For treatment of mycobacterial infections, it is necessary to use special drugs, called antituberculotics. The way of testing is different from other bacteria, too: antituberculotics are added directly to the culture media. On the oter hand, Actinomyces and Nocardia are treated by „normal“ antibiotics and also „normal“ diffusion disc test is used for testing. a) Assessment of mycobacterial susceptibility to antituberculotics By comparing with a control test-tube, read the results of antituberculotic susceptibility tests of Mycobacterium tuberculosis strain. Antituberculotic Growth control Growth Y/N Interpretation b) Antibiotic susceptibility of Nocardia and Actinomyces Perform in vitro susceptibility testing of Nocardia and Actinomyces to suitable antibiotics. Into the table, write the abbreviation of the antibiotics according to a card and for all tested strains measure the susceptibility zones. On your card, you have limit zones – according to them, interprete the zones as susceptible (S) resistants (R) and dubious (D). Strain Antibiotic (full name) Zone Æ (mm) Interpretation Zone Æ (mm) Interpretation Task 4: PCR in diagnostics of TB As the culture of mycobacteria is complicated, PCR becomes a very important method in its diagnostics. Read a result of PCR TB diagnostics (from slideshow), write the results and interprete them. Patient No. Sample band Control band Interpretation 1 2 3 4 Task 5: Diagnostics of leprosy Leprosy is a disease that still affects millions of people in underdeveloped countries. Its laboratory diagnostics is difficult because Mycobacterium leprae does not grow on artificial media. Fill in the following table. The name of this animal is It is used to produce and this substance is used for Picture source: http://www.1-costaricalink.com/costa_rica_fauna/nine_banded_armadillo.htm Task 6: Indirect TB detection by means of QUANTIFERON^©-TB Gold test It is a test of induced interferon gamma release checking and by means of this, checking of the cell-mediated immunity. Test principle: It was proven that in TB, including latent TB, tuberculosis antigens activate T-lymphocytes and they produce big amounts of interferon gamma. Similarly those T-lymphocytes may be activated non-specifically by so called mitogenem; that is why mitogene is used as a positive control (MIT). As a negative control we use a test tube containing nothing (NIL). The test tube with proper TB antigen is labeled "TB". Interferon itself is detected by ELISA reaction. Interpret the Quantiferon-TB Gold examination in four patients with use of interpretation table. Anna: MIT = 4.8 TB = 1.2 NIL = 1.1 Your interpretation: _____________________ Berta: MIT = 5.3 TB = 4.8 NIL = 2.1 Your interpretation: _____________________ Cecil: MIT = 0.9 TB = 0.9 NIL = 0.8 Your interpretation: _____________________ Dimos: MIT = 8.4 TB = 8.3 NIL = 8.2 Your interpretation: _____________________ (all values are in IU/ml) Interpretation table (according to test recommendations; simplified!) NIL TB minus NIL MIT minus NIL Final test interpretation Presence of infection M. tuberculosis ≤ 8,0 < 0.35 ≥ 0.5 negative Not likely ≥ 0.35 any value positive Likely < 0.35 < 0.5 unsure Cannot be determined > 8,0 any value any value