TUBERCULOSIS, TUBERCULOSIS, DIPTHERIA AND DIPTHERIA AND

WHOOPPING COUGHWHOOPPING COUGH

USMF, Microbiology, Virusology &Immunology ( Natalia Florea )USMF, Microbiology, Virusology &Immunology ( Natalia Florea )

Classification of Classification of Mycobacterium:Mycobacterium:Mycobacterium tuberculosisMycobacterium tuberculosis

Mycobacterium bovisMycobacterium bovis

Mycobacterium aviumMycobacterium avium

Mycobacterium ulceransMycobacterium ulcerans

Mycobacterium kansasii …Mycobacterium kansasii …

Mycobacterium lepraeMycobacterium leprae

Morphology & IdentificationMorphology & IdentificationThe mycobacterium are rod-shaped non spore The mycobacterium are rod-shaped non spore forming, aerobic bacteria that do not stain readily forming, aerobic bacteria that do not stain readily but, once stained, resist de colorization by acid but, once stained, resist de colorization by acid or alcohol and are therefore called “acid-fast” or alcohol and are therefore called “acid-fast” bacilli. Mycobacterium are rich in lipids. It cause bacilli. Mycobacterium are rich in lipids. It cause chronic diseases producing lesions of the chronic diseases producing lesions of the infectious granuloma type. The Ziehl-Neelsen infectious granuloma type. The Ziehl-Neelsen technique of staining is employed for technique of staining is employed for identification of acid-fast bacteria. In sputum or identification of acid-fast bacteria. In sputum or sections of tissue, mycobacterium can be sections of tissue, mycobacterium can be demonstrated by yellow-orange fluorescence demonstrated by yellow-orange fluorescence after staining with fluorochrome stains (eg, after staining with fluorochrome stains (eg, auramine, rhodamine). auramine, rhodamine).

PathologyPathology:: Two principal lesions of Two principal lesions of mycobacterium tuberculosis is mycobacterium tuberculosis is Exudative Exudative type-type- this consists of an acute inflammatory this consists of an acute inflammatory reaction, with edema fluid, and develops, reaction, with edema fluid, and develops, rapidly spreads to the lymphatic and regional rapidly spreads to the lymphatic and regional lymph nodes. In tissue often heals rapidly. lymph nodes. In tissue often heals rapidly. The lymph node undergoes massive The lymph node undergoes massive caseation, which usually calcifies. The caseation, which usually calcifies. The tuberculin test becomes positive. And tuberculin test becomes positive. And Productive type-Productive type- when fully developed, this when fully developed, this lesion, a chronic granuloma.lesion, a chronic granuloma.

Mycobacterium tuberculosisMycobacterium tuberculosis infects the lung, and infects the lung, and is distributed systemically within macrophages is distributed systemically within macrophages and survives intracellularly. Inhibition of and survives intracellularly. Inhibition of phagosome-lysosome fusion and resistance to phagosome-lysosome fusion and resistance to lysosomal enzymes have both been suggested lysosomal enzymes have both been suggested to play a role. Cell-mediated immunity develops to play a role. Cell-mediated immunity develops which causes infiltration of macrophages and which causes infiltration of macrophages and lymphocytes with development of lymphocytes with development of granulomasgranulomas (tubercles). The disease can be diagnosed by (tubercles). The disease can be diagnosed by skin testing for delayed hypersensitivity with skin testing for delayed hypersensitivity with tuberculintuberculin (also know as protein purified purified (also know as protein purified purified from from Mycobacterium tuberculosisMycobacterium tuberculosis, , PPDPPD). A ). A positive test does not indicate active disease; positive test does not indicate active disease; merely exposure to the organism.merely exposure to the organism.

Laboratory diagnosis of tuberculosisLaboratory diagnosis of tuberculosis The presence of acid fast bacteria in The presence of acid fast bacteria in sputum is a rapid presumptive test for sputum is a rapid presumptive test for tuberculosis (link to method). Subsequently, tuberculosis (link to method). Subsequently, when cultured, when cultured, M. tuberculosisM. tuberculosis will grow will grow very slowly producing distinct non-very slowly producing distinct non-pigmented colonies after several weeks. pigmented colonies after several weeks. M. M. tuberculosistuberculosis can be differentiated from most can be differentiated from most other mycobacterium by the production of other mycobacterium by the production of niacinniacin. A rapid alternative to culture is . A rapid alternative to culture is polymerase chain amplification (PCR).polymerase chain amplification (PCR).

Growth Characteristics:Growth Characteristics: Mycobacterium are Mycobacterium are obligate aerobes. Increased CO2 tension obligate aerobes. Increased CO2 tension enhances growth. Ordinarily, mycobacterium enhances growth. Ordinarily, mycobacterium grow in clumps or masses because of the grow in clumps or masses because of the hydrophobic character of the cell surface. hydrophobic character of the cell surface. Biochemical activities are not characteristic, Biochemical activities are not characteristic, and the growth rate is much slower than that and the growth rate is much slower than that of most bacteria. Incubation of the inoculated of most bacteria. Incubation of the inoculated media is continued for up to 8 weeks. media is continued for up to 8 weeks. Isolated bacteria should be tested for drug Isolated bacteria should be tested for drug susceptibility. Virulent strains of tubercle susceptibility. Virulent strains of tubercle bacilli form microscopic “cord factor” in which bacilli form microscopic “cord factor” in which acid-fast bacilli are arranged in parallel acid-fast bacilli are arranged in parallel chains. chains.

Tuberculosis is usually treated for Tuberculosis is usually treated for extensive time periods (6-9 months or extensive time periods (6-9 months or longer) since the organism grows longer) since the organism grows slowly and may become dormant. By slowly and may become dormant. By using two or more antibiotics (including using two or more antibiotics (including rifampicin and isoniazid), the possibility rifampicin and isoniazid), the possibility of resistance developing during this of resistance developing during this extended time is minimized. extended time is minimized.

M. tuberculosisM. tuberculosis causes disease in causes disease in healthy individuals and is transmitted healthy individuals and is transmitted man-man in airborne droplets.man-man in airborne droplets.

Three types of media are employed. Three types of media are employed.

1. Simple synthetic media, 1. Simple synthetic media,

2. Oleic acid-albumin media, 2. Oleic acid-albumin media,

3. Complex organic media 3. Complex organic media (Lowenstein Jensen )(Lowenstein Jensen )

Immunity:Immunity: In the course of primary In the course of primary infection, the host also acquires infection, the host also acquires hypersensitivity to the tubercle hypersensitivity to the tubercle bacilli. This is made evident by bacilli. This is made evident by the development of a positive the development of a positive tuberculin reaction. Antibodies tuberculin reaction. Antibodies form against a variety of the form against a variety of the cellular constituents of the cellular constituents of the tubercle bacilli. tubercle bacilli.

The BCG vaccine (The BCG vaccine (Bacillus de Bacillus de Calmette et GuerinCalmette et Guerin, an attenuated , an attenuated strain of strain of M. bovisM. bovis) has not been ) has not been effective. In the Russia, where the effective. In the Russia, where the incidence of tuberculosis is low, incidence of tuberculosis is low, widespread vaccination is not widespread vaccination is not practiced. Indeed immunization practiced. Indeed immunization (resulting in a positive PPD test) (resulting in a positive PPD test) is felt to interfere with diagnosis.is felt to interfere with diagnosis.

Tuberculosis is usually treated for Tuberculosis is usually treated for extensive time periods (6-9 months or extensive time periods (6-9 months or longer) since the organism grows longer) since the organism grows slowly and may become dormant. By slowly and may become dormant. By using two or more antibiotics (including using two or more antibiotics (including rifampicin and isoniazid), the possibility rifampicin and isoniazid), the possibility of resistance developing during this of resistance developing during this extended time is minimized. extended time is minimized. M. M. tuberculosistuberculosis causes disease in healthy causes disease in healthy individuals and is transmitted man-individuals and is transmitted man-man in airborne droplets.man in airborne droplets.

Tuberculin skin test purified protein derivative injected, sensitized T cells react giving delayed hypersensitivity reaction

BCG vaccine attenuated strain

Efficacy questionable, interferes with skin test

2004 the Tuberculosis of 2004 the Tuberculosis of respiratory bodies respiratory bodies

Average statisticAverage statistic disease on 100.000 disease on 100.000 population = 90 on Republic Moldova, population = 90 on Republic Moldova, In Chisinau = 97 on 100.000 In Chisinau = 97 on 100.000 populationpopulation

Children's disease in city’sChildren's disease in city’s 0-16 years 0-16 years = 0,15 cases on 1000 children= 0,15 cases on 1000 children

Total 3238 cases of disease have Total 3238 cases of disease have been registered in 2004 yearsbeen registered in 2004 years

Leprosies mycobacterium and their Leprosies mycobacterium and their morpho-biological characters morpho-biological characters

The organism does not grow in The organism does not grow in culture media. However, it grows well culture media. However, it grows well in the armadillo (which has a low body in the armadillo (which has a low body temperature), allowing production of temperature), allowing production of M. lepraeM. leprae antigens and pathogenesis antigens and pathogenesis studies. studies. M. lepraeM. leprae has traditionally has traditionally been identified on the basis of acid-been identified on the basis of acid-fast stains of skin biopsies and clinical fast stains of skin biopsies and clinical picture.picture.

M. lepraeM. leprae is the causative agent of leprosy is the causative agent of leprosy (Hansen's Disease), a chronic disease (Hansen's Disease), a chronic disease often leading to disfigurement.. It is rarely often leading to disfigurement.. It is rarely seen in the Russia but common in the seen in the Russia but common in the third world. The organism infects the skin, third world. The organism infects the skin, because of its growth at low temperature. because of its growth at low temperature. It also has a strong affinity for nerves. In It also has a strong affinity for nerves. In "tuberculoid" leprosy, there are few "tuberculoid" leprosy, there are few organisms due to control by active cell-organisms due to control by active cell-mediated immunity. In "lepromatous" mediated immunity. In "lepromatous" leprosy, due to immuno-suppression by leprosy, due to immuno-suppression by the organism, the opposite is found. the organism, the opposite is found. Treatment with antibiotics is effective and Treatment with antibiotics is effective and the overall disease incidence worldwide is the overall disease incidence worldwide is down. down. LeprominLepromin is used in skin testing. is used in skin testing.

Classification of Corynebacterium Classification of Corynebacterium diphtheria, biovariants: diphtheria, biovariants:

C. diphtheriaC. diphtheria

C. pseudo diphthericumC. pseudo diphthericum

C. ulcerans C. ulcerans

C. xerosisC. xerosis

C. haemolyticum (pyogenes)C. haemolyticum (pyogenes)

C. hofmanniiC. hofmannii

Corynebacterium are gram-positive rods, Corynebacterium are gram-positive rods, non-motile and non spore-forming, that often non-motile and non spore-forming, that often posses club shaped ends and irregularly posses club shaped ends and irregularly staining granules. They are often in staining granules. They are often in characteristic arranjaments in forms V or N. characteristic arranjaments in forms V or N. Irregularly distributed within the rod (often Irregularly distributed within the rod (often near the poles) are granules straining deeply near the poles) are granules straining deeply with aniline dyes (metacromatic granules) with aniline dyes (metacromatic granules) that give the rod a beaded appearance. that give the rod a beaded appearance. Several species form part of the normal flora Several species form part of the normal flora of the human respiratory tract, other mucous of the human respiratory tract, other mucous membranes, and skin. Corynebacterium membranes, and skin. Corynebacterium diphtheria produces a powerful exotoxin that diphtheria produces a powerful exotoxin that causes diphtheria in humans. causes diphtheria in humans.

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MorfologyMorfology

The principal human pathogen of the The principal human pathogen of the group is C. diphtheria. In nature it occurs group is C. diphtheria. In nature it occurs in the respiratory tract, in wounds, or on in the respiratory tract, in wounds, or on the skin of infected persons or normal the skin of infected persons or normal carriers. It is spread by droplets or by carriers. It is spread by droplets or by contact to susceptible individuals; virulent contact to susceptible individuals; virulent bacilli then grow on mucous membranes bacilli then grow on mucous membranes and start producing toxin. All toxigenic C. and start producing toxin. All toxigenic C. diphtheria are capable of elaborating the diphtheria are capable of elaborating the same disease-producing exotoxin. The same disease-producing exotoxin. The factors that control toxin production in factors that control toxin production in vivo are not well understood. vivo are not well understood.

Diphtheria toxin is absorbed into the mucous Diphtheria toxin is absorbed into the mucous membranes and causes destruction of membranes and causes destruction of epithelium and a superficial inflammatory epithelium and a superficial inflammatory response. The necrotic epithelium becomes response. The necrotic epithelium becomes embedded in exuding fibrin and red and white embedded in exuding fibrin and red and white cells, so that a grayish “pseudo membrane” is cells, so that a grayish “pseudo membrane” is formed – commonly over the formed – commonly over the tonsils, pharynx, tonsils, pharynx, or larynxor larynx. Any attempt to remove the pseudo . Any attempt to remove the pseudo membrane exposes and tears the capillaries membrane exposes and tears the capillaries and thus results in bleeding. The regional and thus results in bleeding. The regional lymph nodes in the neck enlarge, and there lymph nodes in the neck enlarge, and there may be marked edema of the entire neck. The may be marked edema of the entire neck. The diphtheria bacilli within the membrane diphtheria bacilli within the membrane continue to produce toxin actively, resulting continue to produce toxin actively, resulting often in paralysis of the soft palate, eye often in paralysis of the soft palate, eye muscles, or extremities. muscles, or extremities.

Colonization of the upper respiratory tract Colonization of the upper respiratory tract (pharynx and nose) and less commonly skin with (pharynx and nose) and less commonly skin with C. diphtheriaC. diphtheria can lead to diphtheria. The can lead to diphtheria. The organism does not produce a systemic infection. organism does not produce a systemic infection. However, in addition to a pseudo membrane However, in addition to a pseudo membrane being formed locally (which can cause choking) being formed locally (which can cause choking) systemic and fatal injury results primarily from systemic and fatal injury results primarily from circulation of the potent exotoxin (diphtheria circulation of the potent exotoxin (diphtheria toxin). The latter begins over a period of a week. toxin). The latter begins over a period of a week. Thus treatment involves rapid therapy with anti-Thus treatment involves rapid therapy with anti-toxin. The gene for toxin synthesis is encoded on toxin. The gene for toxin synthesis is encoded on a a bbacteriophages (the tox gene).acteriophages (the tox gene). CorynebacteriumCorynebacterium not infected with phage, thus not infected with phage, thus do not generally cause diphtheria. do not generally cause diphtheria.

ColonColonyy on the culture mediumon the culture medium

Culture:Culture: On coagulated serum medium, the On coagulated serum medium, the colonies are small, granular, and gray, with colonies are small, granular, and gray, with irregular edges. On blood agar containing irregular edges. On blood agar containing potassium tellurite, the colonies are gray to black potassium tellurite, the colonies are gray to black because the tellurite is reduced intracellular. because the tellurite is reduced intracellular.

The 3 types of C. diphtheria typically have the The 3 types of C. diphtheria typically have the following appearance on such media: following appearance on such media:

var var gravis gravis – nonhemolytic, large, gray, irregular, – nonhemolytic, large, gray, irregular, striated colonies;striated colonies;

var var mitis mitis – hemolytic, small, black, glossy, – hemolytic, small, black, glossy, convex colonies; convex colonies;

var var intermediusintermedius– nonhemolytic, small colonies – nonhemolytic, small colonies with characteristics between the 2 extremes. with characteristics between the 2 extremes.

If is suspect the diphtheria: Swabs from the If is suspect the diphtheria: Swabs from the nose, throat, or other suspected lesions must be nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are obtained before antimicrobial drugs are administered. Smears stained with alkaline administered. Smears stained with alkaline methylene blue or Gram’s stain show beaded methylene blue or Gram’s stain show beaded rods in typical arrangement. Inoculate a blood rods in typical arrangement. Inoculate a blood agar plate, a Loeffler slant, and a tellurite plate, agar plate, a Loeffler slant, and a tellurite plate, and incubate all 3 at 37oC. Unless the swab can and incubate all 3 at 37oC. Unless the swab can be inoculated promptly, it should be kept be inoculated promptly, it should be kept moistened with sterile horse serum so the bacilli moistened with sterile horse serum so the bacilli will remain viable. In 12-18 hours, the Loeffler will remain viable. In 12-18 hours, the Loeffler slant may yield organisms of typical “diphtheria slant may yield organisms of typical “diphtheria like” morphology. In 36-48 hours, the colonies on like” morphology. In 36-48 hours, the colonies on tellurite medium are sufficiently definite for tellurite medium are sufficiently definite for recognition of the type of C. diphtheria. recognition of the type of C. diphtheria.

C. diphtheriaC. diphtheria are identified by growth are identified by growth on Loeffler is medium followed by on Loeffler is medium followed by staining for metachromatic bodies staining for metachromatic bodies (polyphosphate granules, Babes-(polyphosphate granules, Babes-Ernst bodies). Characteristic black Ernst bodies). Characteristic black colonies are seen on tellurite agar colonies are seen on tellurite agar from precipitation of tellurium on from precipitation of tellurium on reduction by the bacteria. Production reduction by the bacteria. Production of exotoxin can be determined by in of exotoxin can be determined by in vivo or in vitro tests.vivo or in vitro tests.

Such tests are really tests for Such tests are really tests for toxigenicitytoxigenicity of an isolated diphtheria like organism. of an isolated diphtheria like organism.

In vivo testIn vivo test – a culture is emulsified and – a culture is emulsified and 4 ml is injected subcutaneously into each 4 ml is injected subcutaneously into each of 2 guinea pigs, one of which has of 2 guinea pigs, one of which has received 250 units of diphtheria antitoxin received 250 units of diphtheria antitoxin intraperitoneally 2 hours previously. The intraperitoneally 2 hours previously. The unprotected animal should die in 2-3 unprotected animal should die in 2-3 days, whereas the protected animal days, whereas the protected animal survives. survives.

In vitro testIn vitro test – – a strip of filter paper a strip of filter paper saturated with antitoxin is placed on an saturated with antitoxin is placed on an agar plate containing 20% horse serum. agar plate containing 20% horse serum. The cultures to be tested for toxigenicity The cultures to be tested for toxigenicity are streaked across the plate at right are streaked across the plate at right angles to the filter paper. After 48 hour is angles to the filter paper. After 48 hour is incubation, the antitoxin diffusing from the incubation, the antitoxin diffusing from the paper strip has precipitated the toxin paper strip has precipitated the toxin diffusing from the toxigenic cultures and diffusing from the toxigenic cultures and resulted in lines radiating from the resulted in lines radiating from the intersection of the strip and the bacterial intersection of the strip and the bacterial growth. growth.

Tissue culture testTissue culture test – the – the toxigenicity of C.diphtheriae toxigenicity of C.diphtheriae can be shown by can be shown by incorporation of bacteria into incorporation of bacteria into an agar overlay of cell culture an agar overlay of cell culture monolayer. Toxin produced monolayer. Toxin produced diffuses into cells bellow and diffuses into cells bellow and kills them. kills them.

Immunity in diphtheria determining the Immunity in diphtheria determining the antitoxic immunity by the Schick reaction antitoxic immunity by the Schick reaction (in vivo) and IHAR (in vitro). Resistance (in vivo) and IHAR (in vitro). Resistance to the disease depends largely on the to the disease depends largely on the availability of specific neutralizing availability of specific neutralizing antitoxin in the bloodstream and tissues. antitoxin in the bloodstream and tissues. Thus, the treatment of diphtheria rests Thus, the treatment of diphtheria rests largely on rapid suppression of toxin-largely on rapid suppression of toxin-producing bacteria by antimicrobials and producing bacteria by antimicrobials and the early administration of specific the early administration of specific antitoxin against the toxin formed by the antitoxin against the toxin formed by the organisms at their site of entry and organisms at their site of entry and multiplication.multiplication.

Antitoxic immunity to diphtheria may be Antitoxic immunity to diphtheria may be active or passive. The relative amount of active or passive. The relative amount of antitoxin that a person posses at a given antitoxin that a person posses at a given time can be estimated in one of 2 ways:time can be estimated in one of 2 ways:

Titration of Serum for Antitoxin ContentTitration of Serum for Antitoxin Content: : Serum is mixed with varying amounts of Serum is mixed with varying amounts of toxin and the mixture injected into toxin and the mixture injected into susceptible animals. The greater the susceptible animals. The greater the amount of toxin neutralized, the higher amount of toxin neutralized, the higher the concentration of antitoxin in the the concentration of antitoxin in the serum. serum.

Schick Test Schick Test : : This test is based on the fact that This test is based on the fact that diphtheria toxin is very irritating and results in a diphtheria toxin is very irritating and results in a marked local reaction when injected marked local reaction when injected intradermally unless it is neutralized by intradermally unless it is neutralized by circulating antitoxin. One Schick test dose circulating antitoxin. One Schick test dose (amount of standard toxin that, when mixed with (amount of standard toxin that, when mixed with 0.001 unit of the US Standard diphtheria 0.001 unit of the US Standard diphtheria antitoxin and injected intradermally into a guinea antitoxin and injected intradermally into a guinea pig, Will induce a 10-mm erythematous reaction) pig, Will induce a 10-mm erythematous reaction) is injected into the skin of one forearm and an is injected into the skin of one forearm and an identical amount of heated toxin is injected into identical amount of heated toxin is injected into the other forearm as a control. (Heating for 15 the other forearm as a control. (Heating for 15 minutes at 60oC destroys the effect of the toxin.) minutes at 60oC destroys the effect of the toxin.)

The test should be read at 24 and 48 The test should be read at 24 and 48 hours and again in 6 days and hours and again in 6 days and interpreted as follows:interpreted as follows:

Positive reaction Positive reaction ( susceptibility to ( susceptibility to diphtheria toxin, absence of adequate diphtheria toxin, absence of adequate amounts of neutralizing antitoxin; less amounts of neutralizing antitoxin; less than 0,01 Lf units/ml) – Toxin produces than 0,01 Lf units/ml) – Toxin produces redness and swelling that increase for redness and swelling that increase for several days and then slowly fade, several days and then slowly fade, leaving a brownish pigmented area. leaving a brownish pigmented area. The control site shows no reaction. The control site shows no reaction.

Negative reactionNegative reaction (adequate amount of (adequate amount of antitoxin present: usually in excess of antitoxin present: usually in excess of 0,02 Lf units/ml) – Neither injection site 0,02 Lf units/ml) – Neither injection site shows any reaction.shows any reaction.

Pseudo reaction - Pseudo reaction - Schick test reactions Schick test reactions may be complicated by hypersensitivity may be complicated by hypersensitivity to materials other than the toxin to materials other than the toxin contained in the injections. A pseudo contained in the injections. A pseudo reaction shows redness and swelling on reaction shows redness and swelling on both arms which disappear both arms which disappear simultaneously on the second or third simultaneously on the second or third day. It constitutes a negative reaction. day. It constitutes a negative reaction.

Combined reactionCombined reaction – begins – begins like a pseudo reaction, with like a pseudo reaction, with redness and swelling at both redness and swelling at both injection sites; the toxin later injection sites; the toxin later continues to exert its effects, continues to exert its effects, however, whereas the reaction however, whereas the reaction at the control site subsides at the control site subsides rapidly. This denotes rapidly. This denotes hypersensitivity as well as hypersensitivity as well as relative susceptibility to toxin. relative susceptibility to toxin.

Diphtheria is now a disease of almost historic Diphtheria is now a disease of almost historic importance due to effective immunization of importance due to effective immunization of infants (in conjunction with pertussis and infants (in conjunction with pertussis and tetanus, DPT) with a toxoid (inactive toxin) tetanus, DPT) with a toxoid (inactive toxin) which causes production of neutralizing which causes production of neutralizing antibodies. antibodies.

However, colonization is not inhibited and However, colonization is not inhibited and thus thus C. diphtheriaC. diphtheria is still found in the normal is still found in the normal flora. Immunity can be monitored with the flora. Immunity can be monitored with the Schick skin test. Treatment in non-immune Schick skin test. Treatment in non-immune individuals primarily involves injection of anti-individuals primarily involves injection of anti-toxin. Antibiotics are also administered at this toxin. Antibiotics are also administered at this time.time.