Mycobacterium

Important mycobacteria causing human disease are : mycobacterim tuberculosis , m.bovis, m.leprae and atypical mycobacteria . Mycobacterium tuberculosis Morphology : Tubercle bacilli are slender , straight or slightly curved rods with rounded ends . they measure 1-4 x 0.2-0.8 (average 3 x 0.3 ) in size .
In sputum and other clinical specimens they may occur singly or in small clumps .true branching is occasionally seen in old cultures and in smears from caseous lymph nodes .they are non-motile , non-sporing , noncapsulated and acid-fast stain is useful in staining organisms from cultures or from clinical material .with this staing , the tubercle bacilli staing bright red , while the tissue cells and other organisms are stained blue . Organisms in tissue and sputum smears often staing irregularly and have a beaded or barred appearance , presumably because of their vacuoles and polyphosphate content . tubercle bacilli may alson be stained with auramine o and smears examined by fluorescence microscopy under low magnification .tubercle bacilli appear yellow luminou rods in a dark field. When they hape been detected under low power , the morphology of the bacilli is confirmend by observation with and oil immersion objective . tubercle bacilli are gram-positive but it is difficult to staing them with the gram stain . this is because of the failure of the dye to penetrate the cell wall . Much (1907) demonstrated the presence of gram positive granules in cold abscess pus where there was no evidence of acid fast bacilli but which could produce tuberculosis when injected into susceptible animals . much suggested that these granules ( muchs granules ) were non-acid-fast form of tubercle bacilli . M. bovis appear straighter , stouter and shorter with uniform staining. CULTURAL CHARACTERISTICS M. tuberculosis is an obligate. Optimum temperature for growth is 37C (range 30-40C) and optimum pH is 7.0 (range 6.0-7.6) . Tubercle bacilli can grow on a wide range of enriched culture media but Lowenstein-Jensen (LJ) medium is most widely used. This medium consists of whole egg, asparagines, some mineral salts , malachite green and glycerol or sodium pyruvate and solidified by heating (inspissation).

Malachite green inhibits the growth of organisms other than mycobacteria and provides a colour con-trast against which colonies of mycobacteria can be easily seen. Lowenstein-Jensen glycerol medium is recommended for the isolation of human tubercle bacillus whose growth is enhanced by 0.75% glycerol , but glycerol at this concentration is inhibitory to M. bovis and so it may fail to grow on this medium . however , when the concentration of glycerol is reduced ti 0.5% , it still improves the growth of M. tuberculosis but generally does not inhibit the growth of M. bovis. The average generation time of tubercle bacilli is about 14-15 hours , prolonged incubation is , therefore , necessary . M. tuberculosis grows well on LJ medium (eugenic growth) .it produces visible growth on LJ glycerol medium , incubated at 37C , in about 2 weeks , although on primary isolation from clinical material , colonies may take up to 8 weeks to appear. Colonies are of an off-white or buff colour and are often heaped up or have breadcrumb like appearance . they are tenacious and not easily emulsified. On the other hand , M.bovis grows poorly on LJ glycerol medium ( dysgonic growth) of M. bovis is much better in LJ pyruvate medium. SENSITIVITY TO PHYSICAL AND CHEMICAL AGENTS . Tubercle bacilli are highly resistant to drying. when exposed to direct sunlight , organisms from culture are killed in 2 hours , but bacilli contained in sputum require an exposure of 20-30 hours. In dried sputum , protected from sunlight , they survive for as long as 6 months .the tubercle bacillus is generally more resistant to chemical disinfection than other non-sporeformers, especially when present in sputum. Pathogenesis Humans become infected with M . tuberculosis most frequently by inhaling infective droplets coughed or sneezed into air by a patient with tuberculosis. Bovine tuberculosis spread from animal to animal , and sometimes to human attendants , in moist cough spray . About 1% of infected cows develop lesions in the udder and bacilli are excreted in the milk which can then infect people who drink it raw . Thus, primary human tuberculosis due M. bovis usually involves cervical or mesenteric lymph nodes. Human tuberculosis is divisible into primary and post-primary (secondary) forms. Primary tuberculosis This begins with inhalation of the mycobacteria and ends with a T cellmediated immune response that induces hypersensitivity to the organisms and controls 95% of infections. Inhaled tubercle bacilli are engulfed by alveolar macrophages in with they replicate to form the initial lesion or Ghon focus . it consists of a parenchymal subpleural lesion , often just above or just below the interlobar fissure between the upper and the lower lobes . some bacilli are transported by macrophages to the hilar lymph

nodes . the ghon focus together with the enlarged hilar lymph nodes forms the primary complex . in addition , the bacilli may be seeded by futher lymphatic and haematogenous dissemination in many organs and tissues , including other parts of the lung . in case of M. bovis witch enters the mouth , as in milk borne bovine tuberculosis , the primary complexes involve the tonsil and cervical lymph nodes or the intestine , often the ileocaecal region , and the mesenteric lymph nodes . Post-primary(secondary) tuberculosis It is caused by reactivation of the primary lesion (endogenous) , or by reinfection . reactivation tuberculosis is particularly likely to accur in inmunocompromised individuals including the elderly , transplant recipients and those who are infected with human immunodeficiency virus (HIV). Granulomas of secondary tuberculosis most often occur in the apex of the lungs but may be widely disseminated in the lungs, kidneys , meninges , bones and other organs. The same process of granuloma formation occurs but the necrotic element of the reaction causes tissue destruction and the formation of large areas of caseation termed tuberculomas . proteases liberated by activated macrophages cause softening and liquefaction of de caseous material .two special features of secondary tuberculosis are the presence of caseous necrosis and of cavities , which may rupture into blood vessels , spreading mycobacteria throughout the body , and break in to airways , releasing infectious mycobacteria in aerosols and sputum ( open tuberculosis) . IMMUNITY AND HIPERSENSITIVITY Two immunology responses , antituberculous immunity and tuberculin hypersensitivity , develop simultaneously in naturally infected host .both these are mediated by T-cells sensitized to bacterial antigen . humoral immunity appears to be of no relevance in tuberculosis and antibodies do not influence the course of disease . in the nonimmune host , the bacilli are able to multiply incide phagocytes and lyse the host cells , while in immune host CD4+ helper T cells and CD8+ suppressor Tcells are produced. The former secrete interferon gamma which activates macrophages to kill intracellular mycobacteria and the latter kill the macrophages that are infected with mycobacteria . The tuberculin reactivity was first demon striated in guinea pigs by Robert Koch (1890,1891) . the experiment that led to the demon striation of this reactivity is known as kochs phenomenon . when a healthy guinea pig is inoculated subcutaneously with a pure culture of tubercle bacilli , the puncture site heals quickly and there is no immediate visible reaction . but after 10-14 days a hard nodule appears which soon breaks down to for and ulcer that persists till the animal dies of progressive tuberculosis . the

regional lymph nodes are enlarged and caseous . but something totally different develops when a guinea pig already ill with tuberculosis is inoculated . it is bets to use animals that have been successfully inoculated 4-6 weeks previously .an indurated lesion appears at the site of second inoculation in a day or two which undergoes necrosis in another day to form a shallow ulcer that heals rapidly without involvement of the regional lymph nodes or other tissues . kochs phenomenon is acombination of hypersensitivity and immunity and has three components : 1.- a local reaction of induration and necrosis 2.- a focal response in which there occurs acute congestion and even haemorrhage around tuberculous foci tissue 3.- a systemic response of fever which may sometimes be fatal . TUBERCULIN TEST Principle :delayed , type IV or cell-mediated hypersensitivity . Method : 1:- mantoux test : 0.1ml of purified protein derivative (PPD) containing 5IU is injected intracutaneously into the skin of the volar aspect of the forearem. Those suspected of having tuberculosis , and therefore likely to react strongly , may firs be tested with 1IU.the site of inoculation is palpated 72 hours later. The development of and area of palpable , firm induration 10mm or more in diameter is recorded as positive and those between 5 and 9mm in diameter as doubtful .the extent of the accompanying erythema is irrelevant and should the ignored . if the reaction is completely negative , the test may be repeated by giving and injection of 100IU . 2:-heaf test : this test is done with a multiple puncture apparatus with 6 needles that prick 1-2mm deep into the skin. A drop of undited PPD is spread on the area of skin selected for inoculation. The multiple puncture apparatus is pressed against this area of skin and needles are released .the test is read after 72 hours . erythema and induration around at least 4 of the punctures is regarded as positive . the equipment must be adequately sterilized between each use to prevent transmission of hepatitis and AIDS. Uses of tuberculin test : 1.-to diagnose active infection in infants and young children. 2.-to measure prevalence of infection in the community . 3.-to select susceptible for BCG vaccination . False negative (tuberculin anergy ):

1. Early tuberculosis 2. Advanced tuberculosis 3. Miliary tuberculosis 4. In patients with measles and other exanthematous reactions 5. Occasionally after chemotherapy and removal of lung lesion 6. Advanced age 7. Immunosuppressive therapy and defective CMI 8. Lymphoreticular malignancy 9. Sarcoidosis 10.Severe malnutrition . False positive: false positive reactions may be seen in patients with infection by related mycobacteria ( atypical mycobacteria ). These are usually low grade reaction and can be differentiated by testing with tuberculin prepared from these mycobacteria . Laboratory diagnosis: it can be carried out by direct and indirect methods . bacteriological diagnosis be established by demonstration of tubercle bacilli in clinical specimens by microscopy and cultural techniques . only up to 50% of pulmonary and 25% of extrapulmonary tuberculosis can be diagnosed by smears examination . guinea pig inoculation with clinical specimens once widely used for diagnosis of tuberculosis is now obsolete as in vitro culture methods are equally effective . demonstration of hypersensitivity to tuberculoprotein has some diagnostic significance. The specimen most commonly collected is sputum which consists of pus and mucus secretions coughed up from the lung . patient is instructed to cough up the sputum into a clean widemouthed container . disposable waxed cardboard containers are ideal .a morning specimen may be collected . if sputum is scanty , a 24 hours specimen may be collected . if no sputum is produced , laryngeal swab or bronchial washings are examined . in children , stomach washing may be examined as they tend to swallow sputum . depending upon the site of involvement , cerebrospinal fluid urine and aspirated fluid from bone and joint are exmainen . tissue biopsies are homogenized and examined by microscopy and culture . in pulmonary and renal tuberculosis , three consecutive specimen of sputum and orine should by examined respectively . SMEARS EXAMINATION : Pour the sputum in a petri dish and examine. For preparation of smear, select blood-tinged part of the sputum , if not there then purulent part should be selected. The smears is stained by Ziehl-Neelsen technique and examined under oil immersion lens. Acid-fast bacilli stain bright red against a blue background.

Concentration of specimens Mycobacteria in a specimen can be decontaminated and concentrated into a small volume without inactivation. Such concentrate can be used for microscopy , culture and animal inoculation. Several methods are in use : Petroff`s method : it is a simple and widely used technique. Equal volumes of sputum and 4%sodium hydroxide are mixed and incubated at 37C with frequent shacking