Morphology & IdentificationThe mycobacterium are rod-shaped non sporeforming, aerobic bacteria that do not stain readilybut, once stained, resist de colorization by acidor alcohol and are therefore called “acid-fast”bacilli. Mycobacterium are rich in lipids. It causechronic diseases producing lesions of theinfectious granuloma type. The Ziehl-Neelsentechnique of staining is employed foridentification of acid-fast bacteria. In sputum orsections of tissue, mycobacterium can bedemonstrated by yellow-orange fluorescenceafter staining with fluorochrome stains (eg,auramine, rhodamine).
Pathology: Two principal lesions ofmycobacterium tuberculosis is Exudativetype- this consists of an acute inflammatoryreaction, with edema fluid, and develops,rapidly spreads to the lymphatic and regionallymph nodes. In tissue often heals rapidly.The lymph node undergoes massivecaseation, which usually calcifies. Thetuberculin test becomes positive. AndProductive type- when fully developed, thislesion, a chronic granuloma.
Mycobacterium tuberculosis infects the lung, andis distributed systemically within macrophagesand survives intracellularly. Inhibition ofphagosome-lysosome fusion and resistance tolysosomal enzymes have both been suggestedto play a role. Cell-mediated immunity developswhich causes infiltration of macrophages andlymphocytes with development of granulomas(tubercles). The disease can be diagnosed byskin testing for delayed hypersensitivity withtuberculin (also know as protein purified purifiedfrom Mycobacterium tuberculosis, PPD). Apositive test does not indicate active disease;merely exposure to the organism.
Laboratory diagnosis of tuberculosis The presence of acid fast bacteria in sputum is a rapid presumptive test for tuberculosis (link to method). Subsequently, when cultured, M. tuberculosis will grow very slowly producing distinct non- pigmented colonies after several weeks. M. tuberculosis can be differentiated from most other mycobacterium by the production of niacin. A rapid alternative to culture is polymerase chain amplification (PCR).
Growth Characteristics: Mycobacterium areobligate aerobes. Increased CO2 tensionenhances growth. Ordinarily, mycobacteriumgrow in clumps or masses because of thehydrophobic character of the cell surface.Biochemical activities are not characteristic,and the growth rate is much slower than thatof most bacteria. Incubation of the inoculatedmedia is continued for up to 8 weeks.Isolated bacteria should be tested for drugsusceptibility. Virulent strains of tuberclebacilli form microscopic “cord factor” in whichacid-fast bacilli are arranged in parallelchains.
Tuberculosis is usually treated forextensive time periods (6-9 months orlonger) since the organism growsslowly and may become dormant. Byusing two or more antibiotics (includingrifampicin and isoniazid), the possibilityof resistance developing during thisextended time is minimized.M. tuberculosis causes disease inhealthy individuals and is transmittedman-man in airborne droplets.
Three types of media are employed.1. Simple synthetic media,2. Oleic acid-albumin media,3. Complex organic media(Lowenstein Jensen )
Immunity: In the course of primaryinfection, the host also acquireshypersensitivity to the tuberclebacilli. This is made evident bythe development of a positivetuberculin reaction. Antibodiesform against a variety of thecellular constituents of thetubercle bacilli.
The BCG vaccine (Bacillus deCalmette et Guerin, an attenuatedstrain of M. bovis) has not beeneffective. In the Russia, where theincidence of tuberculosis is low,widespread vaccination is notpracticed. Indeed immunization(resulting in a positive PPD test)is felt to interfere with diagnosis.
Tuberculosis is usually treated forextensive time periods (6-9 months orlonger) since the organism growsslowly and may become dormant. Byusing two or more antibiotics (includingrifampicin and isoniazid), the possibilityof resistance developing during thisextended time is minimized. M.tuberculosis causes disease in healthyindividuals and is transmitted man-man in airborne droplets.
Tuberculin skin test purified protein derivative injected, sensitized T cells react giving delayed hypersensitivity reactionBCG vaccine attenuated strainEfficacy questionable, interferes with skin test
2004 the Tuberculosis ofrespiratory bodiesAverage statistic disease on 100.000population = 90 on Republic Moldova,In Chisinau = 97 on 100.000 populationChildrens disease in city’s 0-16 years= 0,15 cases on 1000 childrenTotal 3238 cases of disease havebeen registered in 2004 years
Leprosies mycobacterium and their morpho-biological characters The organism does not grow in culture media. However, it grows well in the armadillo (which has a low body temperature), allowing production of M. leprae antigens and pathogenesis studies. M. leprae has traditionally been identified on the basis of acid- fast stains of skin biopsies and clinical picture.
M. leprae is the causative agent of leprosy(Hansens Disease), a chronic diseaseoften leading to disfigurement.. It is rarelyseen in the Russia but common in thethird world. The organism infects the skin,because of its growth at low temperature.It also has a strong affinity for nerves. In"tuberculoid" leprosy, there are feworganisms due to control by active cell-mediated immunity. In "lepromatous"leprosy, due to immuno-suppression bythe organism, the opposite is found.Treatment with antibiotics is effective andthe overall disease incidence worldwide isdown. Lepromin is used in skin testing.
Corynebacterium are gram-positive rods,non-motile and non spore-forming, that oftenposses club shaped ends and irregularlystaining granules. They are often incharacteristic arranjaments in forms V or N.Irregularly distributed within the rod (oftennear the poles) are granules straining deeplywith aniline dyes (metacromatic granules)that give the rod a beaded appearance.Several species form part of the normal floraof the human respiratory tract, other mucousmembranes, and skin. Corynebacteriumdiphtheria produces a powerful exotoxin thatcauses diphtheria in humans.
The principal human pathogen of thegroup is C. diphtheria. In nature it occursin the respiratory tract, in wounds, or onthe skin of infected persons or normalcarriers. It is spread by droplets or bycontact to susceptible individuals; virulentbacilli then grow on mucous membranesand start producing toxin. All toxigenic C.diphtheria are capable of elaborating thesame disease-producing exotoxin. Thefactors that control toxin production invivo are not well understood.
Diphtheria toxin is absorbed into the mucousmembranes and causes destruction ofepithelium and a superficial inflammatoryresponse. The necrotic epithelium becomesembedded in exuding fibrin and red and whitecells, so that a grayish “pseudo membrane” isformed – commonly over the tonsils, pharynx,or larynx. Any attempt to remove the pseudomembrane exposes and tears the capillariesand thus results in bleeding. The regionallymph nodes in the neck enlarge, and theremay be marked edema of the entire neck. Thediphtheria bacilli within the membranecontinue to produce toxin actively, resultingoften in paralysis of the soft palate, eyemuscles, or extremities.
Colonization of the upper respiratory tract(pharynx and nose) and less commonly skin withC. diphtheria can lead to diphtheria. Theorganism does not produce a systemic infection.However, in addition to a pseudo membranebeing formed locally (which can cause choking)systemic and fatal injury results primarily fromcirculation of the potent exotoxin (diphtheriatoxin). The latter begins over a period of a week.Thus treatment involves rapid therapy with anti-toxin. The gene for toxin synthesis is encoded ona bacteriophages (the tox gene).Corynebacterium not infected with phage, thusdo not generally cause diphtheria.
Culture: On coagulated serum medium, thecolonies are small, granular, and gray, withirregular edges. On blood agar containingpotassium tellurite, the colonies are gray to blackbecause the tellurite is reduced intracellular.The 3 types of C. diphtheria typically have thefollowing appearance on such media:var gravis – nonhemolytic, large, gray, irregular,striated colonies;var mitis – hemolytic, small, black, glossy,convex colonies;var intermedius– nonhemolytic, small colonieswith characteristics between the 2 extremes.
If is suspect the diphtheria: Swabs from thenose, throat, or other suspected lesions must beobtained before antimicrobial drugs areadministered. Smears stained with alkalinemethylene blue or Gram’s stain show beadedrods in typical arrangement. Inoculate a bloodagar plate, a Loeffler slant, and a tellurite plate,and incubate all 3 at 37oC. Unless the swab canbe inoculated promptly, it should be keptmoistened with sterile horse serum so the bacilliwill remain viable. In 12-18 hours, the Loefflerslant may yield organisms of typical “diphtherialike” morphology. In 36-48 hours, the colonies ontellurite medium are sufficiently definite forrecognition of the type of C. diphtheria.
C. diphtheria are identified by growthon Loeffler is medium followed bystaining for metachromatic bodies(polyphosphate granules, Babes-Ernst bodies). Characteristic blackcolonies are seen on tellurite agarfrom precipitation of tellurium onreduction by the bacteria. Productionof exotoxin can be determined by invivo or in vitro tests.
Such tests are really tests for toxigenicityof an isolated diphtheria like organism.In vivo test – a culture is emulsified and4 ml is injected subcutaneously into eachof 2 guinea pigs, one of which hasreceived 250 units of diphtheria antitoxinintraperitoneally 2 hours previously. Theunprotected animal should die in 2-3days, whereas the protected animalsurvives.
In vitro test – a strip of filter papersaturated with antitoxin is placed on anagar plate containing 20% horse serum.The cultures to be tested for toxigenicityare streaked across the plate at rightangles to the filter paper. After 48 hour isincubation, the antitoxin diffusing from thepaper strip has precipitated the toxindiffusing from the toxigenic cultures andresulted in lines radiating from theintersection of the strip and the bacterialgrowth.
Tissue culture test – thetoxigenicity of C.diphtheriaecan be shown byincorporation of bacteria intoan agar overlay of cell culturemonolayer. Toxin produceddiffuses into cells bellow andkills them.
Immunity in diphtheria determining theantitoxic immunity by the Schick reaction(in vivo) and IHAR (in vitro). Resistanceto the disease depends largely on theavailability of specific neutralizingantitoxin in the bloodstream and tissues.Thus, the treatment of diphtheria restslargely on rapid suppression of toxin-producing bacteria by antimicrobials andthe early administration of specificantitoxin against the toxin formed by theorganisms at their site of entry andmultiplication.
Antitoxic immunity to diphtheria may beactive or passive. The relative amount ofantitoxin that a person posses at a giventime can be estimated in one of 2 ways:Titration of Serum for Antitoxin Content:Serum is mixed with varying amounts oftoxin and the mixture injected intosusceptible animals. The greater theamount of toxin neutralized, the higherthe concentration of antitoxin in theserum.
Schick Test : This test is based on the fact that diphtheria toxin is very irritating and results in a marked local reaction when injected intradermally unless it is neutralized by circulating antitoxin. One Schick test dose (amount of standard toxin that, when mixed with 0.001 unit of the US Standard diphtheria antitoxin and injected intradermally into a guinea pig, Will induce a 10-mm erythematous reaction) is injected into the skin of one forearm and an identical amount of heated toxin is injected into the other forearm as a control. (Heating for 15 minutes at 60oC destroys the effect of the toxin.)
The test should be read at 24 and 48hours and again in 6 days andinterpreted as follows:Positive reaction ( susceptibility todiphtheria toxin, absence of adequateamounts of neutralizing antitoxin; lessthan 0,01 Lf units/ml) – Toxin producesredness and swelling that increase forseveral days and then slowly fade,leaving a brownish pigmented area.The control site shows no reaction.
Negative reaction (adequate amount ofantitoxin present: usually in excess of0,02 Lf units/ml) – Neither injection siteshows any reaction.Pseudo reaction - Schick test reactionsmay be complicated by hypersensitivityto materials other than the toxincontained in the injections. A pseudoreaction shows redness and swelling onboth arms which disappearsimultaneously on the second or thirdday. It constitutes a negative reaction.
Combined reaction – beginslike a pseudo reaction, withredness and swelling at bothinjection sites; the toxin latercontinues to exert its effects,however, whereas the reactionat the control site subsidesrapidly. This denoteshypersensitivity as well asrelative susceptibility to toxin.
Diphtheria is now a disease of almost historicimportance due to effective immunization ofinfants (in conjunction with pertussis andtetanus, DPT) with a toxoid (inactive toxin)which causes production of neutralizingantibodies.However, colonization is not inhibited andthus C. diphtheria is still found in the normalflora. Immunity can be monitored with theSchick skin test. Treatment in non-immuneindividuals primarily involves injection of anti-toxin. Antibiotics are also administered at thistime.
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