Lesson N10: LABORATORY DIAGNOSIS of tuberculosis 1.Scientifically methodical ground of theme After a long decline in incidence that began before the availability of chemotherapy, tuberculosis is making comeback. The hope for eradication tuberculosis is becaming an unlikely possibility. It still remans the most common infectious cause of death in the world. About one-third of the worldâ€™s population is infected with the Mycobacterium tuberculosis. Aroximately 8 million cases of tuberculosis and three million deaths occur every year. Over 20, 000 cases of tuberculosis were reported each year in the USA from 1980 to1993. The disease is seen most often in areas of poverty, overcrowding, and poor hygiene. For the first time in decades, the incidence of tuberculosis is increasing in developing countries. The major factors contributing to this recent increase are: (1) associated of tuberculosis with HIV epidemic;(2) immigration from countries where tuberculosis is common; (3) the transmission of tuberculosis in congregate settings (e.g. healthcare facilities, correctional facilities, homeless shelters); (4) a deterioration a healthcare infrastructure. Near 14% of Mycobacterium tuberculosis isolates were resistent to one or more drugs and about 2% were multiply resistent (MRTB) meaning that they are not sensetive to two or more of the major TB drugs. Patients were actually dying from TB even while undergoing multildrug therapy. Actually, tuberculosis, like a slepping giant is waking. 2.Educational purpose STUDENTS MUST KNOW: 1. Structure, staning properties and cultivation of Mycobacteria. 2. Fermentative properties and toxin production of Mycobacteria. 3. Differential signs of Mycobacteria. 4. Ecology of M. tuberculosis. Resistance. Pathogenicity for animals. 5. Epidemiology and pathogenesis of tuberculosis. 6. Methods of laboratory diagnosis of tuberculosis, value of allergic tests, main methods of prophylaxis and treatment. STUDENTS SHOULD BE ABLE TO:• stain the smears by Ziehl-Nelseenâ€™s technique;• examine the smears under the microscope;• value the growth of Mycobacteria on Loewenstein-Jensen nutrient media;• create the scheme of laboratory diagnosis of tuberculosis.• select preparation for diagnosis and prophylaxis of tuberculosis. 3.Chart of topic content. Causative organisms of tuberculosis in humans and animals are Mycobacterium tuberculosis, Mycobacterium bovis, and M. africanum.
Laboratory diagnosis of tuberculosis consists of bacterioscopic, bacteriological, biological,serological, and allergological examinations. The material to be examined includes, depending on the localization of the process: sputum,pus, cerebrospinal fluid, faeces, and lavage waters from the stomach and bronchi. The obtainedsamples are collected in sterile vessels (sputum into jars, cerebrospinal fluid and other materialinto test tubes). Bacterioscopic examination. Smears are stained with the Ziehl-Neelsen method. M.tuberculosis stained bright red (ruby) appear as either thin, long, slightly curved or short straightrods; occasionally, they may be characterized by granularity. M. tuberculosis are arranged singlyor in irregular groups. In staining the urine sediment destaining should be , made not only withsulphuric acid but also with alcohol since the urine may harbour non-pathogenic acid-fastmycobacteria of smegma (Mycobacterium smegmatis) which, unlike M. tuberculosis, are de-stained by alcohol. If mycobacteria evade detection because of their small numbers present inordinary smears, this difficulty is obviated with the employment of such enriched methods ashomogenization and flotation. homogenization (an equal volume of 1 per cent NaOH solution is added to the sputum, theflask is tightly stoppered and shaken for 5-15minutes until the sputum is dissolved completely;after centrifugation, the precipitate is neutralized by one or two drops of a 10 per centhydrochloric acid solution and smears are prepared); flotation (the homogenized sputum is transferred into a flask which has a rubber stopper andheated in a water bath at 55Â°C for 30minutes, after which it is diluted with distilled water, and 1or 2 ml of xylol, benzine or gasoline are added; the mixture is shaken for 10minutes and after ithas been left to settle for 30 minutes, smears are made from the resulting cream-like layer). There are other methods of sputum preparation which facilitate the demonstration ofmycobacteria. Good results are obtained by employing luminescent microscopy with auramine and examiningthe specimens under the phase-contrast microscope. M. tuberculosis fluoresce with a brightgolden-green light against a dark background. Bacteriological examination is more effective than bacterioscopic one and makes it possibleto reveal in the examined material 20-100 and over mycobacteria per ml and also to determinetheir resistance to drugs, their virulence, type, etc. Add a double volume of 6 per cent sulphuric acid killing acid-sensitive microorganisms to theexamined material in a sterile test tube and shake the tube for 10 min. Then, centrifuge theresultant mixture, pour off the fluid, neutralize the pellet by adding 1-2 drops of 3 per cent sodiumhydroxide or by washing it off several times with isotonic sodium chloride solution, and streak onthe appropriate medium. Faeces are treated with 4 per cent solution of sodium hydroxide, the
mixture is placed in an incubator for 3 hrs, centrifuged, and the residue is neutralized by 8 percent hydrochloric acid, after which inoculation on special media is carried out. International Loewenstein-Jensen medium is recommended by WHO as the-standard mediumfor the primary growth of M. tuberculosis and for determining their resistance to antibacterialdrugs. Dissolve 3.6 g of asparagin, 2.4 g of potassium hydrophosphate, 0.24 g of magnesiumsulphate, O.B g of magnesium citrate, and 0.4 g of potato starch and malachite green in 600 ml ofdistilled water containing 12 ml of glycerol. Sterilize the mixture obtained for 15 min at 120 Â°C.Then pour it into 100 ml of homogeneous suspension from fresh eggs, mix thoroughly, filterthrough a cotton-gauze filter, decant into test tubes, and obtain a slant medium by coagulation at85 Â°C for 45 min. Petragnanis medium. To 150 ml of whole milk, add (with constant stirring) 6 g of potato starch,1 g of peptone and one finely-chopped egg-sized potato. Heat tile mixture until paste is formed,cool to 50 "C, and add four chicken eggs. and one yolk. Mix all the components, pour in 12 ml ofglycerol, and 10 ml of 2 per cent of malachite green solution, filter the mixture through a gauzefilter dispense into test tubes, and coagulate in a slant position at 85 Â°C for 2.5 hrs. Glycerol potato as proposed by Pavlovsky. Peel a potato and immerse it in 1 percent solutionof mercuric chloride for 30 min, wash for 12 hrs in running water, and cut out cylinders by makingdiagonal cuts. Slanted potato is placed into a Roux test tube- Pour 1 ml of 5 per cent glycerolsolution onto the bottom and sterilize the test tube. Sautons synthetic medium. In 200 ml of distilled water dissolve (while constantly heating) 4 gof asparagin, 2 g of citric acid, 0.5 g of potassium dihydrophosphate, and 60 g of glycerol. Filterthe obtained mixture, supplement it with 800 ml of distilled water, add ammonium to bring pH to7.2, decant into flasks, and sterilize for 20 min at 115 Â°C. To protect the mixture from drying, theplugs. of test tubes with nutrient media are sealed with paraffin. The composition of the Finn-2 medium is similar to that of Loewenstein-Jensens medium, butasparagin is replaced in it with sodium glutamate. Samples of the cerebrospinal fluid, exudate, pus, and blood are pipetted onto a nutrientmedium without any preliminary treatment and thoroughly rubbed into it with the aid of a loop,spreading them over the entire surface of the-medium. Cotton plugs are sealed with paraffin (toprevent drying), the inoculated cultures are placed into a 37 Â°C incubator, and kept there for 6-8weeks. An intensive growth of M. tuberculosis is observed on the 15th-25th day on theLoewenstein-Jensen medium and on the 21th-35th day on Petragnanis medium. Colonies of M.tuberculosis are wrinkled, dry, irregular, and protrude above the surface. If no growth is observedwithin 6 weeks, make a scraping from the medium surface and examine it microscopically for thepresence of acid-fast bacteria. To improve growth of M. tuberculosis, it is recommended that the-material examined be treatedwith detergents possessing a bactericidal action (sodium laurilsulphate, rodolan, teapol,laurosept, cetavlon, etc.) or their combination with sodium hydroxide. These-methods make it
possible to achieve a better homogenization of the material, to reduce the time during whichcolonies form on nutrient media, and to do away with the stages of centrifugation, resuspension,and neutralization. If the results are negative, the study is repeated several times (at least 5), and the period ofculture inoculation is lengthened. Rapid methods of the bacterial diagnosis of tuberculosis. The method of microcultures (Pricesmethod). Samples of sputum, pus. urine residue, and lavage waters are spread in a thick layer onseveral sterile glass slides. Take a dried preparation with a sterile forceps and immerse it for 5min in 6 per cent sulphuric acid, and then in a sterile isotonic sodium chloride solution to removeacid. After that place the preparations into vials with citrate blood (add 2 ml of 5 per cent sodiumcitrate to 10 ml of rabbit or sheep blood, dilute the contents in a 1:4 ratio "with distilled water, andpour the mixture into test tubes). Put the inoculated cultures into an incubator. In 48-72 hrs thepreparation is retrieved, fixed, and then stained with the ZiehI-Neelsen method. Microcolonies inthe preparation appear as plaits which form under the impact of the lipid fraction of mycobacteria(the cord factor); the maximal growth is observed on the 7th-10th day. In-depth growth in haemolysed blood (Shkolnikovas method). Into tubes with citrate bloodintroduce material treated with sulphuric acid and washed with isotonic sodium chloride solution.After 6-8 days of incubation, centrifuge the medium and make smears from the pellet. Resistance of the M. tuberculosis to drugs is determined by a serial dilution technique. Forinoculation, one may use both initial material containing no less than 5 mycobacteria per amicroscopic field (direct method) and the culture isolated from it (indirect method). WHOrecommends that the resistance of mycobacteria on Loewenstein-Jensens medium should bedetermined by adding into it, prior to coagulation, various doses of drugs. Resistance of mycobacteria can also be determined in liquid media (with an addition of drugsin corresponding concentrations) in which M. tuberculosis grow in a way similar to that describedby Price and Shkolnikova. At the present time the biological examination fails to find wideemployment in laboratory diagnosis since experimental animals are insensitive to the strains ofmycobacteria resistant to tubazid, phthivazid, isoniazid, and other anti-tuberculosis drugs. Biological tests are utilized for determining the virulence of isolated M. tuberculosis which areinoculated subcutaneously into guinea pigs with negative Mantouxs test. Two-three weeks afterinoculation one should weigh the infected guinea pig, measure its regional lymph nodes, andmake Mantouxs test which is then repeated in 6 weeks. If the results are negative, sacrifice theanimal 4 months after inoculation, examine histologically the internal organs (liver, spleen, lungs,lymph nodes), and inoculate nutrient media. The virulence of the strain is determined by thenumber of specific changes in organs (development of tubercles), changes in the expected lifespan of the animal, weight loss, etc.
The allergy cutaneous test (Mantouxs intracutaneous test with tuberculin) is largely employedfor the determination of contamination of individuals with M. tuberculosis. The results are read in24-48-72 hrs. If the diameter of the infiltrate at the site of tuberculin administration does not exceed 1 mm,the test is considered negative. If the diameter of the infiltrate is 2-4 mm, the test is doubtful, ifover 5 mm, it is positive. The tuberculin reaction may be attended with the development oflymphangitis, regional lymphadenitis, and the appearance of vesicles or necrosis. A positiveallergic response to tuberculin administration indicates the presence of M. tuberculosis in thebody. A negative reaction in adults points to the absence of immunity to tuberculosis. As adiagnostic test, this technique is helpful in recognizing tuberculosis in children, identifyingpopulations requiring revaccination against tuberculosis, and assessing the prevalence oftuberculosis as an epidemiological indicator. Pirquets cutaneous test that was extensively usedin the past has become outdated and is no longer utilized. Serological diagnosis. The complement-fixation reaction is rarely employed in the diagnosis oftuberculosis. The IHA reaction, as proposed by Middlebrook and Dubos is used more extensively.Sensitized red blood cells (tannin-treated sheep or human 0-group erythrocytes) are utilized asan antigen. They are mixed with an extract of M. tuberculosis or purified tuberculin (0.5 ml oferythrocyte sediment and 10 ml of the extract), incubated for 2 hrs at 37 Â°C, and washed off withcentrifugation to remove excessive antigen. To run the test, the patients serum is depleted by asuspension of erythrocytes that have not been treated with the antigen, which excludes thepossibility of a non-specific reaction. The serum to be assayed is diluted, beginning with 1:2, 1:4,1:8, etc. A positive reaction in a 1:8 dilution is definitely diagnostic. Positive results are recordedin 70-90 per cent of tuberculosis patients. To reveal antibodies, the agglutination reaction may be performed. The patients blood serumis diluted with isotonic sodium chloride solution in dilutions varying from 1:40 to 1:640. As anantigen, use non-acid fast cultures of M. tuberculosis obtained as a result of penicillin action andserologically similar to native M. tuberculosis. This reaction is extremely sensitive. It should beremembered that even when the results of bacterioscopic and bacteriological studies arenegative, the diagnosis of tuberculosis may be based on clinical and X-ray findings. FigN1: Classification of Main Mycobacterium SpeciesSpecies Formation of Causes urease nicotine paracin niacin amidase amidase M. + + + + Tuberculosis in humans and othertuberculosis primates, in dogs and other animals that were in contact with asick person
M. africanum + + + â€“ Tuberculosis among inhabitants of tropical Africa (Senegal, and other countries)M. bovis + â€“ + â€“ Tuberculosis in calves, domestic and wild animals, humans and other primatesM. kansasii + + + â€“ Tuberculosis-like disaeses in humans, which is marked by weak activityM. intracellulare â€“ + + â€“ Severe forms of tuberculosis-like in humans, localized lesions in pigsM. xenopi â€“ + + â€“ Lesions of the lungs, urogenital system and granuloma of the skin in humansM. ulcerans + Ulceration of the skin in persons dwelling in Mexico, New Guinea, Malaysia and AfricaM. paratuberculosis Chronic diarrhoea in calves and sheepM. microti + + â€“ + Generalized infection in field miceM. avium â€“ + + â€“ Tuberculosis in birds, some times in calves, pigs and other animals. Infection in humans is rareM. leprae Leprosy in humansM. leprae-murium Endemic affections of rats in different parts of the world Fig N2: Differentiation properties of M. tuberculosis and M. bovis. Sings M. tuberculosis M. bovis Morphology Fine, long rods Short rods Glycerin Stimulate growth Inhibit growth Pathogenicity for animals: Rabbits Chronic Generalized acute tuberculosis, less tuberculosis sensitive then guinea pigs Guinea pigs Generalized Less sensitive then tuberculosis and rabbits death Niacin test Positive Negative Source of infection Humans Animals Mechanism of transmission Air-born Alimentary
4. Studentâ€™s independent study program 1. Classification of Mycobacteria. 2. Morphology, methods of staining, chemical composition, biochemical and antigenic properties of causative agents of tuberculosis. Cultivation. Resistance i surrounding environment. 3. Characteristic of pathogenic substances of M. tuberculosis: cord factor, lipids, endotoxin etc. 4. Pathogenicity for animals. 5. Epidemiology of tuberculosis. Pathogenesis and disease in man. Clinical findings. 6. Non sterile immunity, its features. 7. Allergic state. Allergic test and its practical value. Preparation for use. 8. Main methods of microbiological diagnosis of tuberculosis: bacterioscopic, bacteriological,biological, serological. Enrichment methods â€“ homogenization, flotation. Their values. 9. Nonspecific and specific prophylaxis of tuberculosis. 10. Characteristics of main mycobacteria which are causative agents of different diseases inman. Mycobacteriosis. Laboratory diagnosis of mycobacteriosis.5. Studentsâ€™ practical activities: 1. To study the morphology of causative agents of tuberculosis in sputum smears stained withZiehl-Nelseenâ€™s method. 3. To study the growth of M. tuberculosis on Loevenstein-Jensenâ€™s medium. 4. To familiarize with media for cultivation of M. Tuberculosis (Petragnanis medium, Loevenstein-Jensenâ€™s, Sautons and others) 5. To familiarize with preparation used for the allergic diagnosis, specific prophylaxis andtreatment of tuberculosis.6. Control questions and tests 1. Microscopy of sputum from a patient with acute form of pulmonary tuberculosis is carried outto reveal causative agent of this disease. What method of staining is the most likely to this? A. Gram. B. Ziehl-Neelsen.
C. Burri-Gins D.Romanowsky-Giemsa E. Neisser. 2. Diagnostic test Manteoux was carried out on a 6- year -old child, suspected of activetubercular process. Choose an immunologic preparation, which was injected. A. Vaccine AKDT. B. Vaccine BCG. C. Tuberculin. D.Tularin E. Vaccine Ð ﾐ DT. 3. In the smear from sputum stained by Ziehl-Neelsen method are revealed single or groupingred acid-resistant rods. On nutrient medium the first growth has appeared in 14 days. Whatspecies does the microorganism belong to? A. Yersinia pseudotuberculosis. B. Mycobacterium tuberculosis. C. Histoplasma dubrosii. D. Klebsiella rhinoscleromatis. E. Coxiella burnettii. 4. The patient is 16 years old young man from countryside. The man has negative tuberculinskin test. What should a doctor make?A. To make BCG injection.B. To repeat reaction in 1 month.C. To make serologic diagnostic of tuberculosis.D. Urgently to isolate the young man from collective.E. To make accelerated diagnostics of tuberculosis by a Price method.
5. At a maternity hospital immunization of newborn children against tuberculosis has beenconducted. What vaccine has been used?A. Tetanus toxoid.B. Diphtheritic toxoid.C. BCG.D. Ð¢ABTe.E. DTT. 6.Vaccine against tuberculosis is: A. Toxoid B. Killed corpuscular C. Split vaccine D. Attenuated live E. Associated 7. At school medical inspection of pupils has been conducted. The purpose was to selectchildren for revaccination against tuberculosis. Which one of the following tests has been used? A. Burne test. B. Widal test. C. Tularin skin test.D. Tuberculin skin test. E. Anthraxin skin test. 8. Each of following statements concerning Mycobacterium tuberculosis is correct EXCEPT: A. After being stained with carbolic fuchsin, Mycobacterium tuberculosis resists decolorization with acid alcohol B. Mycobacterium tuberculosis has a large amount of mycolic acid in the cell wall C. Mycobacterium tuberculosis appears as a red rod in Gram-stained specimens D. Mycobacterium tuberculosis appears as a red rod in acid-fast stained specimens9. Each of following statements concerning Mycobacterium tuberculosis is correct EXCEPT: A. Obligate requirement for oxygen
B. Niacin production C. Acid fastness D. Inability to grow on artificial media E. Slow growth 10. According to respiratory type Mycobacterium tuberculosis belong to A. Obligate anaerobes B. Facultative anaerobes C. Obligate aerobes 11. A positive tuberculin test indicate: A. No immunity to Mycobacterium tuberculosis B. Parasitic infectionC. presence of living Mycobacterium tuberculosis bacilli C. Active pulmonary tuberculosis D. Active non-tubercular mycobacterial infection 12.Mycobacterium tuberculosis infections elicit------------ immune response: A. Delayed type of hypersensitivity B. An immediate type of hypersensitivity C. An anaphylactic D. A humoral antibody 13.With the Ziehl-Neelsen stain, acid- fast bacilli appear----------- against a blue background: A. Red B. blue C. Yellow D. Purple E. Pink 14. After BCG inoculation of infants, immunity to tuberculosis lasts until there are live vaccinestrains in the body. Name such kind of immunity? A. Antitoxic B. Passive.
C. Non-sterile (infection). D. Natural E. Sterile 15. Sputum examination on Price method revealed rod- shaped bacteria arranged in helicalcords. Which substance leads to stocking of these bacteria and serpentine- like growth? A. Tuberculin. B. Cord - factor. C. Phosphatides. D. Micolic acid. E. Ð Ð Ð”.7. List of literature:1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,chapter21, p. 123-141.