TRACING THE CRIMINAL Part eight: Acidoresistant (acid-fast) criminals Institute for microbiology shows Intro: Spitoons in fight with TB In Czechoslovakia between World War I and World War II the a society was formed with personal engagement of president Masaryk and his „League against TB“. It showed a big effort in fight against this disease. The part of this was education for people not to spit to the floor, but to use spittoons. Survey of individual parts Clinical characteristics of acid-fast bacteria Story One n Johny did know allready for many years that he is HIV positive. He knew pretty well that he is more vulnerable than other people and that each infection can get him more quickly than other people n Nevertheless he was surprised that.he started to cough recently. His doctors tried various variants, but after roentgen, PCR examination and culture examination came to conclusion that it is a miliar (granular) form of tuberculosis. The criminal was n Mycobacterium tuberculosis, although TB may be caused by Mycobacterium bovis, too. n Interesting for this microbe: it lives inside cells. This is also related with the fact that antibody response is weak in tuberculosis (so neither antigen nor antibodies are detected) and cell imunity is very important (in vaccination, too). n As in HIV infection just cell immunity is dammaged, TB is one of oportune infections. Tuberculosis Pulmonary form of TB is not the only one TB worldwide Once more TB Brain tuberculoma More special things in TB Cell wall of mycobacteria Story two n Mr Hassan lived in desert part of Sudan, where wars and unrests were very common. n Recently even friends that still did neither run away nor die started to dislike contacts with mr. Hasan. Mutilation of face of mr. Hassan was a clear mark, that mr. Hassan suffers the disease still too common in this part of world. n Good luck that Hassan met members of a non-governement organisation, that where sure about diagnose. Using dapson it was possible to help Mr. Hassan. Causative agent is Leprosy Story Three n Mr. Piranha was a furious aquarist. Last month he had a problem: he had to use his left hand only to do anything inside the fish tank, as he had an ulcus on his right hand. n After examination, his case was closed as so named fish tank granuloma, common in aquarists. A simillar disease in swimmers is called swimming pool granuloma. n Causative agents are… Atypical mycobacteria n Besides M. tuberculosis and M. leprae there exist also plenty of other mycobacteria. Some of them, e. g. Mycobacterium marinum, are so named. atypical mycobacteria, sometimes causing wound infections and other problems. n Some mycobacteria are non-pathogenous and they are normal part of human microflora, e. g. M. smegmatis – see picture Infection of M. marinum Story four n Mrs. Fistula found several months ago some small formations in her neck n Recently one of them opened to skin and dense, yelow, unpleasantly spelling pus appeared in the orifice. So, Mrs. Fistula visited her doctor. n The doctor sent the pus for microbiological examination. Examination durated very long time – mrs. Fistula was already angry that microbiologists are lazy and don‘t want release the results. Finally the criminal was found… …it was actinomycosis Actinomycosis Nocardiosis Nocardial pneumonia with septicaemia Brain nocardiose on CT Special properties of acid-fast bacteria Acidoresistance + alcaliresistance Consequences for clinical doctors Diagnostics of acid-fast bacteria How to search for criminals n Microscopy: We use Ziehl-Neelsen stain and fluorescence stain n Culture: We use special media, and before the culture the specimen should be treated by a hydroxide. The aim is to kill other bacteria, that would be more succesfull as they grow more quickly. Alcaliresistant mycobacteria survive that easily. n Automatic culture: Various types of culture automats are used: they are able to detect culture positivity much sooner than classic culture n Biochemical differenciation is possible in specialized laboratories n Animal experiment: guinea pig is used sometimes n PCR diagnostics is more and more important PCR kit for TB diagnostics Ziehl-Neelsen staining n In step 1 we stain by carbolfuchsine (Gabbet) in hot until steam rises. Without heating mycobacteria could not be stained at all. n In step 2 we decolorize by „acid alcohol“, what is mixture of alcohol with a mineral acid, most commonly HCl n In step 3 we counterstain the background, so everything decolorized in Step 2. We counterstain by malachit green or methylene blue. n Result: red acidoresistant rods on blue or green backround Ziehl-Neelsen stain Ziehl-Neelsen stain Nocardia and Actinomyces microscopy n These bacteria are Gram staining, although they staining poorly and thay are very pleomorphous. n Both of them are typical by their branched filaments, staining Gram-positive, although some parts of the filament may stain Gram-negative or they might remain unstained at all n Sometimes, short (coccoid) forms may also occur in microscopy Culture of mycobacteria n Hydroxide should be used before culture n We use liquid Šula or Banić media and egg Ogawa or Löwenstein-Jenssen media. Egg media are solid because of egg white coagulation, they do not contain agar n Even solid media are in test tube and closed firmly. This is not only because personel would be endangered, but also as media would dry. n Results are read after 3, 6 and for sure after 9 weeks of culture. (Positive results are mostly found after 6 weeks of culture.) Nocardia and Actinomyces culture n Although both genera are similar in many properties, one is different: Nocardia is strictly aerobic, while Actinomyces is strictly anaerobic bacterium To liquid Šula medium n Even positive test tube is clear by first view, as the growth of mycobacteria is visible only at the bottom („blue mess“, as student J. H. called it J) Tests of antituberculotic susceptibility (not antibiotic!) n Antituberculotics are strange chemicals, different from antibiotics (with exceptions) n Allways we combine 2 or 3 of them: resistances appear quickly, and some have only intra- of only extracellullar effect n We cannot use diffusion disk tests n Antituberculotics are added directly into culture media, growth control is added („Kontrola růstu“) n Growth present à mycobacteria resistant n Growth absent à mycobacteria susceptible Survery of commonly used antituberculotics Antibiotic susceptibility of Actinomyces and Nocardia n Unlike mycobacteria, in nocardiae and actinomycetes antibiotic susceptibility can be tested using diffusion disc test. We have to know, that they grow slowly and badly. PCR for TB PCR is a method used in TB diagnostics more than diagnostics of other bacteria. The reason is that it makes the diagnostics much faster and the risk of environmental contamination is not so serious. Lepromin test in leprosy diagnostics n There is an animal. Its name is nine banded armadillo. n It is necessary for production of lepromin. n This substance is used in lepromin test, the equivalent of tuberculin test for TB Pictures of acid-fast bacteria Criminal photos: Mycobacterium tuberculosis Actinomyces israeli Nocardia asteroides Appearance of mycobaterial colonies The End