1. DIPHTHERIA PRESENTED BY: SHERENE BANAWAN JUNE 23, 2008

2. OBJECTIVES Following the brief PowerPoint Presentation the listener will be able to:  Specify the causative agent of Diphtheria, with understanding of the organism’s virulence factor.  Assess the routes of transmission from person to person.  Describe symptoms of respiratory and cutaneous diphtheria.  Evaluate various media, tests, and procedures carried out to diagnose the disease.  Judge the prognosis of individuals that have acquired the disease and possible treatments.  Deduce possible risk factors, which make individuals susceptible.  Relate methods of prevention to potential risk factors.  Forecast the future of this disease with respect to certain preventative measures in various societies.

3. CAUSATIVE AGENT: Corynebacterium diphtheriae Club-shaped, gram positive rods Production of exotoxin is the distinguishing feature in relation to other corynebacteria. All virulent strains carry a bacteriophage, which carries the diphtheria exotoxin. Figure 1. Microscopic image of C.diphtheriae rods Courtesy of vaccineplace.com

4. Occurrence of Toxigenic Strains Two factors influence the ability of C. diphtheriae to produce the diphtheria toxin: 1.Low extracellular concentrations of iron 2.Presence of a bacteriophage in the bacterial chromosome. Figure 2.The Beta phage that encodes the tox gene for the diphtheria toxin Courtesy of Todar’s Online Textbook of Bacteriology

5. Schematic of Diphtheria Toxin Integration and Expression Figure 3. Diphtheria toxin integration and expression Courtesy of the CDC

6. Routes of Transmission Airborne droplet infection through contact with patient or carrier. Spread depends on closeness and duration of contact. Contact with exudates from skin lesions. Fomites contaminated with organism. Asymptomatic carriers may occur (up to 5% of people in endemic regions). Conditions of poor hygiene and crowding increase risk of transmission.

7. Pathogenicity of Diphtheria Ability to colonize the nasopharyngeal cavity or skin Diphtheria toxin works by causing death of eukaryotic cells and tissues by inhibiting protein synthesis in the cells. Three strains of C. diphtheriae that are differentiated based on the severity of disease caused in humans. The difference in virulence based on relative abilities to produce diphtheria toxin, in both rate and quantity. 1.Gravis 2.Intermedius 3.Mitis

8. Symptoms of Respiratory Diphtheria Incubation period of 1-5 days Onset is relatively slow, moderate fever and mild exudative pharyngitis. Severe cases, a pseudomembrane gradually forms in the throat = asymmetric, grayish-white appearance and strong attachment to the underlying tissue. The pseudomembrane may extend into the nasal cavity and the larynx or suddenly become detached causing obstruction of airways. Local edema causes swelling, which leads to a “bull neck”appearance. Figure 4 and 5. Pseudomembrane(left), Bull neck (right) Courtesy of http://www.vaccineinformation.org

9. Symptoms of Cutaneous Diphtheria Can be caused by both non- toxigenic and toxigenic strains. Often indistinguishable from chronic conditions, such as eczema or impetigo. In a few cases, it produces punched-out ulcers, occasionally with a grayish membrane. Pain, tenderness, erythema, and exudate are typical. If exotoxin is produced, lesions may be numb. Figure 6 and 7. Cutaneous skin lesion Courtesy of http://www.vaccineinformation.org

10. Clinical Manifestations Summary Figure 8. Diagram of Diphtheria Entry and Manifestations Courtesy of gsbs.utmb.edu/microbook/ch032.htm

11. Prognosis and Complications Usually curable in 10 days if treatment is begun promptly, followed by slow recovery for several weeks. A delay in treatment may result in death or long- term heart disease and other complications. Most complications are due to effects of the toxin. Severity of the disease and complications are generally related to the extent of local disease. When the toxin is absorbed, it affects the organs and tissues. Most frequent complications are: myocarditis and neuritis Death: overall fatality rate is 5%-10% with higher rates (up to 20%) in patients 40 years old.

12. Biochemical Reactions of C. diphtheriae Catalase + Urea - Nitrate + Hydrolysis of urea and nitrate production are used to differentiate from other Corynebacterium species.

13. Colony Morphology on Blood Agar Plate Gray Small, granular Irregular edges Small zones of hemolysis. Figure 9. Blood agar culture Figure 10. BAP - intermedius Figure 11. BAP - mitis Courtesy of glowingworks.com

14. Diphtheria Selective Media There are several variations of cultural media to isolate C.diphtheriae. Two common types of media are: Loeffler’s Media Potassium-Tellurite Media- e.g. Cysteine-Tellurite Agar or Modified Tinsdale Agar

15. Loeffler’s Media Primarily used for the promotion of growth and morphological characteristics of Corynebacterium species. Formation of metachromatic granules (Babes-Ernst Bodies) is enhanced within the cells of these organisms. Babes-Ernst bodies must be stained with Albert’s stain (a methylene blue stain). Figure 12. Barred appearance due to Babes-Ernst bodies Courtesy of glowingworks.com

16. Limitations of Loeffler’s Media Although the production of metachromatic granules is characteristic of Corynebacterium sps., other organisms, such as Propionibacterium, Actinomyces and pleomorphic streptococcal strains display stained granules resembling the corynebacteria. Must be used in parallel with other media, such as Cysteine-tellurite agar or Modified Tinsdale agar, for isolation. It is only a tentative identification of C.diphtheriae - must have additional biochemical and toxigenicty tests for differentiation and identification.

17. Cysteine-Tellurite Agar Produces gray to black colonies Varying texture depending on biotypes: 1.Gravis - rough 2.Intermedius - small colonies 3.Mitis - smooth Figure13. Cystine tellurite plate- mitis biotype Figure 14. Cystine tellurite plate-gravis biotype Photos courtesy of glowingworks.com

18. Modified Tinsdale Agar A potassium-tellurite agar C. diphtheriae produces hydrogen sulfide from the cysteine which reacts with the tellurite to produce a smoky brown halo around the black colonies. Figure 15. Modified Tinsdale plate with halo Photos courtesy of glowingworks.com

19. Detection of Toxigenic Strains: ELEK Test Detects the toxigenicity of C. diphtheriae, which is extremely important in the diagnosis of diphtheria. Filter paper impregnated with diphtheria antitoxin is laid crosswise to the organism --> If organism produces the toxin, then a precipitate will form at a 45° angle from the filter paper. If toxin is not produced, then no precipitate line forms. Figure 15 and 16. ELEK Test Controls Courtesy of glowingworks.com

20. Detection of Toxigenic Strains: PCR Most use pure bacterial cultures that focuses on detection of sequences that code for the biologically active subunit of the toxin. PCR assay positive for the presence of diphtheria toxin gene in clinical material suggest that toxigenic C.diphtheriae may be the causative agent. Provides supportive evidence for diagnosis, data are not yet sufficient for PCR to be accepted as a means of confirmation. Advantages: Allows for preliminary presumptive results on toxigenicity within a few hours of specimen collection. Samples held in transport media for prolonged periods of time were PCR positive, while the culture, as expected, was negative.

21. Other Methods of Detection Enzyme immunoassay (EIA) - specific to biologically active subunit of the toxin -takes 3 hours to obtain results -toxigenicity can be detected using bacterial isolates grown on a variety of media -has shown 100% correlation with the Elek test Immunofluorescence - should be used in conjunction with other phenotypic tests – pure cultures are not required Latex immunologic techniques Immunoblotting Passive hemagglutination

22. Treatment 1.Diphtheria antitoxin to neutralize the diphtheria toxin should be given as soon as possible. 2.Antibiotics to eradicate remaining organism, to prevent production of more toxin. The CDC recommends either: Penicillin (intramuscularly) or Erythromycin (orally or injection) for 14 days. The antibiotics do not have an impact on established exotoxic lesions. 3.Prolonged bed rest (3 weeks or until fully recovered), especially if the heart is involved.

23. Prevention = Immunization The nontoxic, immunogenic toxoid is prepared by formalin treatment of the toxin. Children are given monthly injections (3 doses) of this preparation with pertussis and tetanus antigens (DPT vaccine) for three months, after which they receive regular booster injections. Active immunization with diphtheria toxin during childhood and booster doses given every 10 years throughout life.

24. Schick Test: Determination of Immunity Intradermal injection of diphtheria toxin No skin reaction is observed if neutralizing antibodies are present Localized edema with necrosis occurs if neutralizing antibodies are absent, indicating the patient is susceptible to diphtheria. Figure 17. Schick Test Courtesy of lifespan.org

25. Epidemiology Diphtheria is found worldwide, particularly in poor urban areas with overcrowding and low vaccine-induced immunity. C.diphtheriae is maintained in the population by asymptomatic carriers. Spread person to person by respiratory droplets or skin contact Less common in the United States. Figure 18. Woldwide Distribution Courtesy of http://en.wikipedia.org/wiki/Diphtheria

26. Worldwide Reported Cases Worldwide, WHO reported 3,978 cases in 2006. In the US, between 1980 to 2004 there were 57 reported cases of diphtheria. Between 1980-2000, total number of reported cases has decreased by >90%, according to WHO. There is still a need to achieve global vaccination coverage. Figure 19. Woldwide Incidence and Vaccination Courtesy of Todar’s Online Textbook of Bacteriology

27. Who is at risk? Vaccine-induced immunity wanes over time unless periodic booster given or exposure to toxigenic C. diphtheriae occurs. Immunity gaps in adults coupled with large numbers of susceptible children creates potential for new extensive epidemics. 20->50% of adolescents and adults lack immunity to diphtheria toxin in some areas of US with particularly low levels among the elderly. Countries experiencing rapid industrialization or undergoing sociopolitical instability. Endemic in several areas including Africa, India, Bangladesh, Vietnam, tropics of South America. Another reason for reemergence in countries with immunization programs may be due to the introduction of toxigenic C.diphtheriae strains of a new biotype into the general population.

28. REFERENCES “Diphtheria and diphtheria toxoid”. Center of Disease Control and Prevention. www.cdc.gov/vaccines/ed/epivac07/downloads/05-Diphtheria10.ppt. “Diphtheria remains a threat to health in the developing world - an overview”. Scientific Electronic Library Online. 2003. http://www.scielo.br. ESBSCOhost. “Diphtheria Vaccine”. Weekly Epidemiological Record, 2006 Jan 20; 81 (3): 26- 27. www.library.ncahec.net. Gilligan, P., et al. Cases in Medical Microbiology and Infections Diseases. Third Edition. ASM Press, 2003. 134-136. “Loeffler Blood Serum - Loeffler Medium”. 314-315. http://www.bd.com/ds/technicalCenter/inserts/Loeffler_Blood_Serum.pdf. Loh, Gerard. “Corynebacterium”. www.glowingworks.com. Mahon, C., et al. Textbook of Diagnostic Microbiology. Third Edition. Saunders, 2007. 412- 413. Murray, P., et al. Medical Microbiology. Fourth Edition. Mosby, 2002. 252-253. Todar, Kenneth. “Diphtheria”. Todar’s Online Textbook of Bacteriology. University of Wisconsin-Madison. http://www.textbookofbacteriology.net/diphtheria.html. Vaccine Information : Diphtheria Photos. http://www.vaccineinformation.org/diphther/photos.asp.