Corynebacterium diphtheria : Corynebacterium diphtheria By- Mr. Gunjal Prasad N. P.D.V.V.P.F’s Medical College Ahmednagar PowerPoint Presentation: Corynebacteria are Gram positive bacilli. Frequently show club shaped swelling; hence the name. Most imp. pathogenic spp. – C. diphtheriae ; The disease was first recognised by Bretonneau (1826) . “Dephtherite” because of “Tough leathery pseudo membrane formed in this disease. (Diphtheros – Leathery). Also known as “Klebs - Loeffler’s bacillus (KLB)”. Theodor A. E. Klebs (1883) first observed & described it. Friedrich Loeffler (1884) first cultivated it. MORPHOLOGY: GPB, 3-6 0.6-0.8 . Tendency of clubbing at one or both ends. Pleomorphic. Klebs Loeffler Non-sporing, non-motile, non-capsulated, non acid –fast. Though small chains of mycolic acids are present in cell wall. MORPHOLOGY PowerPoint Presentation: Arrangement of bacilli Arranged at various angles to each other. Resembling the letters V or L known as – Chinies Letter pattern or Cuneiform arrangement. Due to incomplete separation of daughter cells. Corynebacteria : Corynebacteria Albert’s Stain Slide showing Green colour bacillary body & Brown or Dark purple colour Metachromatic granules / Babe Earnests Granules/ Inclusion bodies etc. PowerPoint Presentation: GRANULES Show granules composed of polymetaphosphates. Metachromatic granules — When stained with Loeffler’s methylene blue; take up a bluish purple color. Other names – Volutin granules. Babes –Ernst granules. Polar bodies. Staining -- Loeffler’s methylene blue Albert’s stain. Neisser’s stain. Ponder’s stain. Corynebacteria : Cultural characteristics Aerobe and facultatively anaerobe. Growth is scanty on ordinary media. Enrichment with Blood, Serum, Eggs is necessary for good growth. Optimum temperature – 37 0 C. pH – 7.2. Enriched media — Loeffler’s serum slope. K-tellurite blood agar. Loeffler’s serum slope Rapid growth; 6-8 hrs. Colonies – Small, circular, white, opaque. On cont. incubation - Acquire distinct yellow tint. Corynebacteria PowerPoint Presentation: Cultural characteristics K-tellurite blood agar K- Tellurite - Selective agent. May take 2 days to grow. Diphtheria bacilli reduce K-tellurite to metallic tellurium—black colonies. Modifications of K-tellurite media – McLeod’s and Hoyle’s. Tellurite (0.04%) inhibits the growth of other organisms acting as selective agent. Corynebacteria : Based on colony morphology & other properties McLeod’s classified C.diphtheriae into three types - - C. diphtheriae Gravis – Causes serious disease. - C. diphtheriae Intermedius – Causes intermediate. - C. diphtheriae Mitis - Causes mild disease. Association is not constant. Gravis and Intermedius - high case fatality rate. Mitis infection is less lethal. Paralytic complications – Gravis. Hemorrhagic – Gravis & Intermedius. Obstructive lesion in air passage – Mitis. Generally Mitis is predominant in endemic area. Gravis & Intermedius are tend to be epidemic. Corynebacteria Corynebacteria : Biochemical Reactions Sugar fermentation tested in Hiss’s serum water . Ferments Glucose, Galctose, Maltose. Some virulent strains of Corynebacteria are found to ferment sucrose with production of acid only no gas. Urease negative ( C.ulcerans – Urease positive ). Corynebacteria Corynebacteria : Toxin Powerful Exotoxin. Patogenicity is due to the potent Exotoxin only. About 95-99% strains of Gravis & Intermedius are toxigenic. Only 80-85% strains of Mitis are toxigenic. Avirulent strains are common among convalescents, contact and carriers. The strain most widely used for production of toxin is “Park Williams 8” strain which belongs to either Mitis or Intermedius group. Corynebacteria Toxin : Toxin It is protein, molecular wt. 62,000 Kd. Labile. Potent & lethal dose for 250gms Guinea pig is 0.0001 mg. Consists of Fragment A & Fragment B. 24,000 & 38,000 respectively. Both are necessary for toxic action. Toxin : Frag A – Active part of toxin. Frag B - Binds toxin to cells. When released by bacterium toxin is inactive. Active site on frag A is masked. Activation is achieved by proteases present in infected tissues or in culture medium. Antibodies to B are protective, as act against binding of B fragment to host cells. Toxin Toxin : Prolonged storage, incubation at 37 0 C.For 4-6 wks., Treatment with 0.2-0.4% formalin or acid pH converts it to TOXOID. Toxoid is toxin – with lost toxicity but has antigenicity. It is capable of producing antitoxin and act specifically on it. Toxigenecity depends upon a bacteriophage called Corynephage (tox+) genetic determinant controlling toxin production. Toxin Corynebacteria : MECHANISM OF ACTION OF TOXIN In the presence of nicotinamide adenine dinucleotide (NAD) fragment A inactivates the elongation factor -2 (EF2). Therefore inhibition of polypeptide chain elongation leading to inhibition of protein synthesis this causes cell death. Toxin has special affinity for – Myocardium Adrenals Nerve endings Corynebacteria Corynebacteria : PATHOGENICITY Incubation period – 3-4 days. May be as short as one day. - Prolonged in carriers. Site of infection –Faucial —Commonest. - Laryngeal - Nasal - Otitic - Conjuntival - Vulval, Vaginal, - Prepucial - Cutaneous Cutaneous infections are usually due to nontoxigenic strains. Corynebacteria Corynebacteria : CLASSIFICATION OF DIPHTHERIA ACCORDING TO CLINICAL SERVERITY Malignant or Hypertoxic – Severe toxemia. Marked adenitis (Bull neck). Death due to circulatory failure. Paralytic complications are common in those recovered. Corynebacteria Corynebacteria : Septic – Ulceration present - a circumscribed inflammatory and often suppurating lesion on the skin. Cellulitis present - an inflammation of body tissue (especially that below the skin) characterized by fever and swelling and redness and pain Gangrene around pseudomembrane . Corynebacteria PowerPoint Presentation: Haemorrhagic – Bleeding from the edge of pseudomembrane. Epistaxis - hemorrhage from the nose. Conjunctival haemorrhage. Purpura - A rash of purple spots on the skin caused by internal bleeding from small blood vessels. Generalized bleeding tendency. COMPLICATIONS : COMPLICATIONS COMPLICATIONS : COMPLICATIONS Corynebacteria : PATHOPHYSIOLOGY Bacilli remain at the site of entry. They multiply and produce toxin. Toxin causes local necrotic changes. Fibrinous exudate together with necrosed cells i.e. epithelial cells WBC, RBC & bacteria. Form pseudomembrane. This is characteristic of diphtheria. Pseudomembrane causes mechanical complications & toxin causes systemic effects. Corynebacteria Corynebacteria : COMPLICATIONS Asphyxia – A condition arising when the body is deprived of oxygen, causing unconsciousness or death; suffocation Due to pseudomembrane which causes mechanical Obstruction of airway. Acute circulatory failure. Pseudodiphtheritic paralysis – Typically occurs in 3 rd or 4 th week of disease. Palatine & ciliary paralysis occurs. No pupillary paralysis. Spontaneous recovery occurs. Septic – Pneumonia & Otitis media. Corynebacteria Corynebacteria : LABORATORY DIAGNOSIS Immediately start the treatment on suspicion of diphtheria. Do not wait for laboratory tests. Any delay may be fatal. Lab confirmation is required only for initiation of control measures & for epidemiological purposes & not for treatment . Laboratory Diagnosis consists of – Isolation of diphtheria bacilli. Demonstration of its toxicity. Corynebacteria Corynebacteria : 1. Isolation of diphtheria bacilli - Specimen – Swabs taken from lesion. Microscopy Gram’s stain – Irregularly stained GPB with Granules. But resembles normal oral flora not confirmatory. Is imp. To differentiate Vincent’s angina. In this, Vincent’s Spirochetes & Fusiform bacilli are seen. Corynebacteria Corynebacteria : Special Stain – Commonly used – Albert’s stain. Green bacilli with brownish granules in Chinese letter pattern are seen. Corynebacteria Corynebacteria : CULTURE – BLOOD AGAR – To differentiate Streptococcal& Staphylococcal pharyngitis. LOEFFLER’S SERUM SLOPE – Quick growth. (6-8 hrs.) Yellowish white colonies. TELLURITE BLOOD AGAR – Important for isolating diphtheria bacilli from convalescents, contacts, and carriers as in these cases other commensals bacteria may over grow them. Tellurite inhibits other bacteria. Wait for 2 days to label it as negative. Black colonies are seen. Corynebacteria Corynebacteria : 2.Demonstration of toxicity i.e. virulence test a. In vivo test Subcutaneous test. Intracutaneous test. b. In vitro test 1. Elek’s gel precipitation test. 2.Tissue culture test. Corynebacteria Corynebacteria : Corynebacteria 1. Subcutaneous Test – Diphtheria antitoxin Emulsified in broth. Small quantity injected S.C. in guinea pig. GP protected with 500 U. GP not protected. 18-24 hrs. Previously. Does not die. Dies in 4 days if strain is virulent. DISADVANTAGE – Wastage of animal. PowerPoint Presentation: 2.Intracutaneous Test Broth emulsion injected intracutaneously in GP or Rabbit. Protected with 500U 50 U antitoxin given IP after 4 hrs. antitoxin previous day Control animal Test animal No change Inflammatory reaction at injection site & progresses to necrosis in 48-72 hrs. ADVANTAGES – - No wastage of animal - As many as 10 strains can be tested at a time on a rabbit. PowerPoint Presentation: 3.Elek’s Gel Precipitation Test Procedure— Rectangular strip of filter paper impregnated with diphtheria antitoxin (1000 u/ml) Incorporated in 20% horse serum agar when it is still fluid Agar sets surface is dried. Narrow streaks of strains are made at right angles to filter paper strip. Positive & negative controls are put. Plate incubated at 37 o c for 24-48 hrs Elek’s Gel Precipitation Test: Elek’s Gel Precipitation Test Test PC NC Filter paper soaked in Antitoxin Observation - Toxin produced by strain diffuses into agar , where it meets antitoxin at optimum conc. Line of precipitation formed - toxigenic strain No ppt. lines are formed - nontoxigenic strain Corynebacteria : Corynebacteria 4. Tissue Culture Test Strain is incorporated in agar overlay of cell culture monolayers. Toxin produced diffuses into the cells. Cells are killed. Immunoprophylaxis: Immunoprophylaxis Diphtheria can be controlled by immunization— Active Passive Combined Active immunization — Provides herd immunity - Leads to eradication of disease. Passive & Comb.Immu. - Provide emergency protection to susceptible exposed to risk 1.Active Immunisation: 1.Active Immunisation A. Formol Toxoid – Toxin is incubated with formalin until the product is devoid of toxicity but retains immunogenicity. B. Adsorbed Toxoid— This is a purified toxoid adsorbed onto aluminium phosphate. Much more immunogenic than FT. Universally preferred agent. Immunization schedule : Immunization schedule Starts at the age of 1&1/2 months. 3 doses given IM at interval of 1 month. 1st Booster – 1&1/2 year of age. 2nd Booster - at school entry. It is given as a trivalent preparation, Containing Tetanus Toxoid and Pertussis Vaccine as DPT Vaccine. 2.Passive Immunisation: 2.Passive Immunisation Emergency measure. SC administration of 500-1000 U of antitoxin i.e. antidiphtheritic serum i.e. ADS. Horse serum precaution against hypersensitivity should be observed. Given to susceptible when exposed to risk. Ex. – When a case of diphtheria admitted to general pediatric ward. 3.Combined Immunisation: 3.Combined Immunisation 1st dose of adsorbed toxoid given on one arm and ADS on other arm. Followed by full course of active immunization. Ideally those who receive ADS prophylactically should receive combine immunization. Treatment: Treatment 1. Antitoxin - Should be given immediately. Dose - 20,000 – 1,00,000 U Half dose IV, half dose IM. Not indicated in cutaneous type as caused by nontoxigenic strains. Treatment: Treatment 2. Antibiotic - Only supplements the antitoxin. Does not replace the antitoxin therapy. Sensitive to Penicillin. Erythromycin – in carriers. Other pathogenic Corynebacteria : Other pathogenic Corynebacteria C.ulcerans, C. jakeium, C. pseudotuberculosis, C. renale. DIPHTHEROIDS : DIPHTHEROIDS Commensals in throat, skin, conjunctiva, and other areas. Sr. No. Diphtheria Bacilli Diphtheroids 1. Generally irregularly stained. Stained more uniformly. 2. Possess metachromatic granules. Possess few/no metachromatic granules. 3. Arranged in cuneiform pattern. Arranged in pallisades. Differentiation is generally done by virulence test and biochemical reactions. EX - 1. C. pseudodiphtheriticum – Throat. 2. C. xerosis – Conjunctival sac. Schick test : Schick test Diphtheria can be controlled by immunization. In the old days immunizing agents were less & not free from risk. Therefore it was customary to test for susceptibility before active immunization was done. Susceptibility testing was done by Schick test. It is not used now a day. Principle – When diphtheria toxin is injected intradermally, it causes a local reaction in susceptible person. No reaction occurs in immune person as antitoxin in circulation neutralizes toxin. Thank you !: Thank you !