Lesson N9: LABORATORY DIAGNOSIS of diphtheria 1.Scientifically methodical ground of theme Diphtheria was formally an important pediatric disease all over the world but following the development of effective prophylactics and mass immunization, the disease has been virtually eradicated from most advanced countries. In those developing countries, where childhood immunization programs have been implemented effectively diphtheria has become rare but in others it still continues to be a serious problem. 2.Educational purpose STUDENTS MUST KNOW: 1. Structure, staining properties and cultivation of Corynebacterium diphtheriaÐµ 2. Fermentative properties and toxin production of Corynebacterium diphtheriaÐµ 3. Epidemiology and pathogenesis of diphtheria. 4. Methods of laboratory diagnosis of disease, the main methods of prevention and treatment. STUDENTS SHOULD BE ABLE TO: â€“ prepare the smears from tested material;stain the smears by Gramâ€™s and Loeffler technique; make microscopical examination of the smears and make up conclusion• value the growth of Corynebacterium diphtheriaÐµ on different media; â€“ choose biological preparation used for laboratory diagnosis and specific prophylaxis of diphtheria ; create scheme of diphtheria diagnosis. 3.Chart of topic content. Fig. N 1: Usual biochemical reaction of C.diphtheriae Catalase Nitrat reduction Urease Helatin Glucose Maltose Sucrose hydrolisis fermentation fermentation fermentation + + - - + + - FigN2:Differential-diagnostic signs of corynebacteria diphtheria and non-pathogenic corynebacteria
Type of Fermenta-tion Toxigenic Additional signscorynebacteri sucrouseeeeeeeeeeeeeeeeeeee glucos starc ity cystina ureas Agglutinatia eeeeee e h se test e test on with antiserumDiphtheriacorynebacteriagravis â€“ + + + + â€“ +mitis â€“ + â€“ â€“ + â€“ +Diphtheroids + + â€“ â€“ + + â€“Pseudodiphth + â€“ â€“ â€“ â€“ â€“ â€“eria bacteriaFig. N3: Mode action of diphtheria toxinC.diphtheriaeDiphtheria toxinCell membraneABABCell deathAPrevents proteinsynthesisInactivates by ribosomeelongationfactor-2ribosomeFig. N 4:Main effects of C.diphtheriae exotoxin
Local SystemicTarget: pharynx Targets: cardiac, nervous tissues and othersInflammatory response withpseudomembraneFig. N 5: Microscopic examinationtakingspecimenswabThroat, nose Prepared smears stain by Gramâ€™s and examine Loeffler techniqueunder microscopeClub-shaping, non-sporing, Gram positive rods, that showmethachromatic granules. Organisms arranged inâ€œChinese letterâ€u patterns, or side- by- side in â€œpalisadesâ€Fig. N 6: Prevention of diphtheriaPrevention of diphtheriaNon specificSpecific(immunization)Isolation of a patient, detection andActive immunization
Passive immunisationisolation of carriersDPT-vaccine ADS-Antitoxin(antidiphtheritic serum)4. Studentâ€™s independent study program 1. Structure and staining properties of diphtheria causative agent 2. Cultivation of Corynebacterium diphtheriaÐµ. Main nutrient media. 3. Biochemical properties of Corynebacterium diphtheriaÐµ. 4. classification of Corynebacterium diphtheriaÐµ. 5. Toxin formation of Corynebacterium diphtheriaÐµ. 6. Epidemiology of diphtheria : a â€“ source of infectious agents; b â€“ mechanism of transmission; c â€“ factors of transmission. 8. Pathogenesis and clinical findings of diphtheria. 9. Laboratory diagnosis of diphtheria. 10. Treatment and prophylaxis of diphtheria. aâ€“ specific prophylaxis of diphtheria. c â€“ urgent prophylaxis of diphtheria. d â€“ treatment 5. Studentsâ€™ practical activities: 1. Stain ready smears of Corynebacterium diphtheriaÐµ by Loeffler technique. Examine under microscope; make up conclusion. Draw and record you observation. (See Fig.N5)
On fixed smear pour alkaline methylene blue to act for 3-5 minutes, wash with water, dry withfilter paper, and examine under the microscope. The cytoplasm of Corynebacterium diphtheriaÐµis stained light-blue, while granules are dark-blue. 2. To familiarize with biological preparation used for laboratory diagnosis and specific prophylaxis of diphtheria.DPT-vaccine, that consists of diphtheria (D) and tetanus (T) toxoid, and pertussis(P) vaccine, allthree generally given together. toxoid is prepared by 0,4% formaline solution treatment fromdiphtheria toxin at 400 C during 4 weeks. It is used for planned immunization against toxoidadministration results in the production of antibodies that specifically neutralize the diphtheriatoxin. Since the disease the results primarily from toxin adsorption rather than microbial invasion,its control can be accomplished most effectively by immunization with toxoid. Unfortunately, theseimmunizations have often been neglected, particularly among socioeconomically disadvantagesgroups, and serious epidemics of the diseases have occurred periodically. Since the 1980s, therehas been an active campaign in the most of the United states, to ensure that children who areentering school are immunized against diphtheria. As a result, only a few cases of diphtheria areannually reported in the USA, as compared to the 30,000 cases reported in 1936.ADS-Antitoxin ( antidiphtheritic serum) is used for treatment and prophylaxis.In1886 Beringfound antibodies in the blood of porpoises, immunized with sublethal doses of diphtherin. In 1896Bering used sheep serum for diphtheria treatment. He was awarded Nobel Prize for creating ofnew treatment method and for the successes of diphtheria treatment. Passive immunization is anemergency measure to be employed when susceptibles are exposed to infection, as a case ofdiphtheria is admitted to general pediatric ward.It is consist of the sucutaneous administration of500-1000 unit of antitoxin As this is a horse serum, precaution against hypersensetivity should beobserve.Antibiotics (erythromycin, penicillin) The bacteria are sensitive to these antibiotics, but suchtreatmen stops only transmission of the disease; it has no effect on the toxin that has beenadsorbed already.Effective treatment of diphtheria depends on giving antiserum against diphtheria toxin to thepatient as soon as possible. Administration of antiserum must be given if the disease issuspected without waiting for proof of the diagnosis, since a delay of the several days need toobtain confirmation from the culture results can be fatal. 3. Start diagnosis of diphtheria carrying. Take specimen from throat by cotton swab. Inoculate it onto Loeffler medium.Throat Thermostat, at370 CCotton swab during 24-48 hours
Inoculate by swab onto Loeffler medium6. Control questions and tests 1. It was necessary to make preventive inoculation of students group because a case ofdiphtheria. What preparation should be used to develop artificial active immunity? A. Diphtheria toxoid B. Antidiphtheritic serum. C. A specific antibody. D. Vaccine DPT (Triple vaccine.) 2. To determine the toxigenicity of causative agents of diphtheria obtained from patients,cultures are inoculated into wells cooped round the central well with diphtheria serum intonutritious agar of Petri dishes. After cultures incubation between central well and some wells withcultures bands (lines of precipitation) are observed. Which serologic test is carried out? A. Test precipitation in gel. B. Tube precipitation test C. Agglutination. D. Floculation E. Coombs test 3. Pure culture of corynebacteria diphtheria is obtained from a patient. What serologic testshould be used for determining toxigenity of culture? A. Agglutinations. B.Test. precipitation in agar C. Complement fixation test D. Hemagglutination inhibition. E. Passive hemaglutination
4. Throat culture isolated from a 5- year- old child was identified as corynebacterium diphtheriaby morphological and fermentative properties, but exotoxin free. What process can make thisstrain toxigenetic?A. Chromosomal mutation.B.Cultivation on telurite mediumC. Passing culture through organisms of sensitive animals.D. Cultivation on medium with antitoxic serum.E. Phage conversion. 5. Corynebacterium diphtheriae was obtained from a sick child with suspicion of diphtheria.What research is necessary to use to be convinced, that this microbe causes diphtheria at thechild?A. Agglutination test.B. Staining a material using Giemaâ€™s method .C. Culturing microbes on a blood agar.D. To infect the rabbit.E. To check up a toxin producing by microbes. 6. Among children there are cases of respiratory infection. The tonsil swab was stained byNeisser technique. There were yellow rods with brown terminal granules, the rods were situatedlike letters V, W, X. What infection can be suspected in this case? A. Infectious mononucleosis. B. Diphtheria. C. Listeriasis. D. Tonsillitis. E. Scarlet fever. 7. Doctor noticed grayish patches â€œpseudomembraneâ€e on pharyngeal tonsils of 6- year-old child at the examination. Bleeding was occurred when there was attempt of remove patches.What symptoms can appear within the next few days without specific treatment?
A. Very strong attack-like cough.B. Lungs edema.C. Toxic lesion of a cardiac muscle, liver, kidneys.D. Papular rash on skin.E. Intermittent fever. 8. The two-year-old boy was not immunizated for diphtheria toxoid. On his pharyngeal tonsilsthere were revealed microorganisms, which on morphological and biochemical properties areidentical to Corynebacterium diphtheriaÐµ. But precipitation test in agar with antitoxic serum hasbeen negative. What form of infectious process can this agent cause in the child withouttreatment?A. Asymptomatic carriage of bacteria.B. The easy nontoxic form of disease.C. The toxic complicated form of disease.D. Chronic disease with damage of the internal organs.E. Persistent infection. 9. The girl, 7 years, with a high body temperature, complaints of sore throat, weakness has beenhospitalized to infectious clinic. The doctor has suspected diphtheria and has given the instructionto take a material from pharynx for obtaining pure culture of the pathogen. Which one of thefollowing points is the most important for confirmation of the diagnosis "diphtheria"?A. Revealing polymetaphosphate granules in agent.B. Demonstrating toxin production.C. Urease test.D. Hemolytic activity of agent.E. Bacteriophage typing. 10. In pediatric department of infectious clinic there was diagnosed diphtheria in the boy. Whichone of the following preparations should be injected first of all?A. Diphtheritic antitoxic serum.
B. Diphtheritic toxoid.C. BCG.D. DPT.E. Ð¢ABTe. 11. In smear from a tonsil of the patient with suspicion for diphtheria there have been revealedblue rods with dark blue beads at the ends. What method of staining has been used? A. Loefflerâ€™s. B. Giemsa. C. Ginsâ€™. D. Gramâ€™s. E. Neisserâ€™s. 12. It is necessary to conduct specific prophylaxis of diphtheria in children at school. Whatpreparation should be used for this purpose? A. Toxoid. B. Antibiotics. C. Probiotics. D. A corpuscular vaccine. E. Immune serum. 13. Each of following statements concerning Corynebacterium diphtheriaÐµ is correct EXCEPT: A. Corynebacterium diphtheriaÐµ is gram positive rod that does not form spores B. toxin production is dependent on the organismâ€™s being lysogenized by bacteriophage C. diphtheria toxoid should not be given to children under the age of 3 years because the incidents of complications is too high D. Antitoxin should be used to treat patients with diphtheria 14. diphtheria toxin is correctly described as:A. Produced by Corynebacterium diphtheriaÐµ both in vitro and in vivo
B. ImmunogenicC. The active agent in Schick testD. Used to vaccinate infants15. The primary virulence factor for Corynebacterium diphtheriaÐµ is: A. Enterotoxin B. Capsule C. Exotoxin D. Endotoxin 16. For practical use of curative antitoxic serums, the patient is always given precisely defineddoses. What units is activity of diphtheria antitoxin measured in? A. International. B. Flocculationâ€™s. C. Antigenic. D. Agglutinationâ€™s E. Neutralizationâ€™s7. List of literature: 1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology,Lugansk, 2004, chapterN17, p. 29-43.