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Gram positive rods Dr Alex Owusu-Ofori
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Gram positive rods Non spore-forming AEROBIC ANAEROBIC Spore-forming
Corynebacteria C. diphtheriae diphtheroids Listeria monocytogenes Erysipelothrix rusiopathiae ANAEROBIC Lactobacillus spp. Spore-forming AEROBIC Genus: Bacillus B. anthracis B. cereus B. subtilis ANAEROBIC Genus: Clostridium C. tetani C. botulinum C. difficile C. perfringens
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Species of Corynebacteria
Corynebacterium Pathogenic C. diphtheriae Commensal "Diphtheriods" C. hofmannii, C. xerosis, C. acne C. diphtheriae is the only pathogenic members of this genus Normal flora of RT, urethra, vagina, Skin
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Corynebacteria - Overview
Gram positive, non motile bacilli with irregularly stained segments C. diphtheriae : most important member of this genus, causes diphtheria Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva. Corynebacteria is the most successfully studied bacteria – clinical description started by Hippocrates in the 4th Century BC
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Historical overview I. Corynebacterium diphtheriae (Klebs–Loeffler bacillus)
Bretonneau 1826 Clinical characterisation of diphtheria – diphtherite Klebs 1883 First decribed the bacterium Loeffler 1884 First solated/cultivated the bacterium Roux and Yersin 1888 Discovering the diphtheria toxin Behring and Kitasato Discovering the diphtheria antitoxin Antitoxic immunity (therapy and prevention) Roux 1894 Treatment with antitoxin
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Historical overview II.
Emil von Behring 1901 Produced antitoxin - Nobel prize Behring 1913 Active immunisation I. with toxin-antitoxin mix Schick 1913 Skin test Ramon 1923 Active immunisation II. Anatoxin = toxoid Freeman 1951 PHAGE (lysogenia, toxin production)
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Corynebacterium spp Gram positive bacilli, with characteristic morphology (club shaped and beaded) Non motile Non spore forming Non capsulated Facultative anaerobic (A facultative anaerobe is a microorganism that can exist regardless of whether there is molecular oxygen. When there is oxygen, the anaerobe creates ATP using aerobic respiration. If oxygen is not present, then the organism uses anaerobic resp or fermentation) Breakdown glucose by oxidation and fermentation C. diphtheriae is fastidious while diphtheriods are non-fastidious Catalase positive Oxidase negative
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Important features of C. diphtheriae
Slender Gram positive bacilli Pleomorphic, Chinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules Metachromatic Granules: made up of polymetaphosphate Bluish purple color with Loeffler’s Methylene blue Special stains: Albert’s, Neisser’s & Ponder’s Grows aerobically at 37°C
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Virulence factor Exotoxin – Diphtheria toxin: Protein in nature
very powerful toxin Responsible for all pathogenic effects of the bacilli Produced by all the virulent strains Two fragments A & B
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Diphtheria Toxin Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox+ phage) or beta phages Can be toxoided by - Prolonged storage Incubation at 37°C for weeks Treatment with 0.2 – 0.4 % formalin or Acid pH. Strain used for toxin production – ‘Park Williams 8’ strain Antibodies to fragment B - protective
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Epidemiology Habitat – nose, throat, nasopharynx & skin of carriers and patients Spread by respiratory droplets, usually by convalescent or asymptomatic carriers Nasal carriers habour the bacilli for longer time than pharyngeal carriers Local infection of throat - toxemia Incubation period of diphtheria – 3 to 4 days In tropics, cutaneous infection is more common than respiratory infection
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Site of infection Faucial (palatine tonsil) – commonest type Laryngeal
Nasal Ear Conjunctival Genital – vulval, vaginal, prepucial Cutaneous – usually a secondary infection on pre-existing lesion, caused by non toxigenic strains
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Pathogenesis & Clinical Manifestations
Human Disease Usually begins in respiratory tract Virulent diphtheria bacilli lodge in throat of susceptible individual Multiply in superficial layers of mucous membrane Elaborate toxin which causes necrosis of neighboring tissue cells Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria) Usually appears first on tonsils or posterior pharynx and spreads upward or down In laryngeal diphtheria, mechanical obstruction may cause suffocation Regional lymphnodes in neck often enlarged (bull neck)
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Complications of diphtheria
Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin. Asphyxia – due to obstruction of respiratory passage Acute circulatory failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recovery Sepsis – pneumonia & otitis media
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Diphtheria - Clinical Classification
Based on the severity of clinical presentation: Malignant or hypertoxic – severe toxemia with marked adenitis Septic – ulceration, cellulitis, & gangrene around the pseudomembrane Hemorrhagic – bleeding from the edge of membrane, epistaxis, conjunctival hemorrahge, purpura & generalized bleeding tendency.
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Diagnosis of diphtheria
Clinical Diagnosis Laboratory Diagnosis Specific treatment must be never delayed for laboratory results To confirm the clinical manifestation
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Diagnosis of diphtheria
Diagnosis of case Diagnosis of carrier Symptomatic patient Asymptomatic
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Laboratory diagnosis Specimen: A throat swap
Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37°C for 24. On Loeffler's serum medium: Corynebacteria grow much more readily than other respiratory pathogens Used to enhance the characteristic microscopical appearance of corynebacteria The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges
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Loefflers serum medium
Developed by Friedrich Loeffler in 1887 Loefflers medium is used to grow corynebacteria Most recent modification by Buck in 1940 to enhance the growth of C. diphtheria Consists of Beef serum, heart muscle and animal tissue peptone, sodium chloride, dextrose and egg. The primary value of Loeffler Medium is in the growth and morphological characterization of members of the genus Corynebacterium; enhances the formation of metachromatic granules within the cells of the organism.
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Laboratory Diagnosis Culture – isolation of bacilli requires media enriched with blood, serum or egg Blood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrs Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies Hoyle’s media modifications of TBA McLeod’s media BA – to differentiate from staphylococal or streptococcal pharyngitis C.diph colonies are Small, granular, irregular edges and gray with small zones of hemolysis
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Growth of diphtheria bacilli
Blood agar Tellurite blood agar Loeffler’s serum slope
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Laboratory Diagnosis Specimen – swab from the lesions Microscopy
Gram stain: Gram +ve bacilli, chinese letter pattern Immunofluorescence Albert’s stain for metachromatic granules
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Gram stain of C. diphtheriae
C. diphtheriae on BTA
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Cultural characteristics
On blood tellurite agar (Mcloed’s blood agar) It is selective medium for isolation of C. diphtheriae (Potassium tellurite) 3 biotypes of C. diphtheriae are characterized on BTA i.e. Gravis, mitis and intermedius biotypes The most severe disease is associated with the gravis biotype Colony of gravis biotype is large, non-hemolytic & grey. Colonies of mitis biotype are small, hemolytic and black Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.
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Morphology Stain Gram +ve, nonspore forming nonmotile bacilli
Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance) Beaded (metachromatic granules) Stain Gram stain: C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped Polychrome methylene blue stain: C. diphteriae appears beaded due to the presence of intercellular “Metachromatic or volutin" granules By stain, the granules appear red while the rest of organism appears blue.
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Biotypes of Diphtheria bacilli
Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types: Features 1. Gravis 2. Intermedius 3. Mitis Case fatality rate High Low Complications Paralytic, hemorrhagic Hemorrhagic Obstructive Predominance In epidemic areas Epidemic areas Endemic areas Spread Rapid Rapidly than mitis Less rapid Colony on TBA ‘Daisy head” colony ‘Frog’s egg colony ‘Poached egg’ colony Hemolysis Variable Nonhemolytic Usually hemolytic
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Biochemical Reaction All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)
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Carbohydrate Fermentation Test:
Principle: Each species of corynebacteria has its specific carbohydrate fermentation pattern C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only Procedure: Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism Glucose Maltose Sucrose Incubate the tubes at 37oC for 24 hrs.
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Results: Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH Glucose Maltose Sucrose Glucose Maltose Sucrose +ve +ve +ve +ve +ve - ve C. xerosis C. diphtheriae
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Laboratory Diagnosis Virulence tests - Invitro tests
Elek's gel precipitation test filter paper saturated with antitoxin (1000units/ ml) is placed on agar plate with 20% horse serum bacterial culture streaked at right angles to filter paper Dr Ekta, Microbiology
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Treatment specific treatment must not be delayed if clinical picture suggests of diphtheria rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin) early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV
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Prophylaxis Active Immunization (Vaccination)
Formol toxoid (fluid toxoid) incubation of toxin with 0.3% formalin at pH at 37°C for 3 to 4 weeks fluid toxoid is purified and standardized in flocculating units (Lf doses) Adsorbed toxoid (more immunogenic than fluid toxoid) purified toxoid adsorbed onto insoluble aluminium phosphate or aluminium hydroxide given IM (DTP or TD)
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Prophylaxis Adsorbed Toxoid
DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine DaT - contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid. (smaller dose used to diminish likelihood of adverse reactions) Schedule i) Primary immunization - infants and children - 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry ii) Booster immunization - adults -Td toxoids used (travelling adults may need more) SHICK test - to test susceptibility to vaccine, not done now-a-days
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Prophylaxis Passive immunization ADS (Antidiphtheritic serum, antitoxin) - made from horse serum - 500 to1000 units subcutaneously Combined immunization First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation
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Diphtheria Cased by C. diphtheriae
Mycocarditis, neuroitis, palate perforation Acute, Toxin mediated Recovery or complication & death (if more toxin absorbed) Childhood disease affect upper respiratory tract Diphtheria Respiratory obstruction due to extensive membrane formation Transmitted by droplet infection 2-6 days I.P. Sore throat, Pharyngitis 2-3 days, Bluish white adherent pseudo membrane
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