CHAPTER 90 Diphtheria 931

SUBSECTION III Gram-Positive Bacilli Diphtheria 90 Barbara W. Stechenberg

Diphtheria is an acute infectious disease caused by Corynebacterium in mortality rates by a therapeutic intervention. In Germany alone, an diphtheriae or, less commonly, C. ulcerans. C. pseudotuberculosis, which estimated 45,000 lives were saved each year.62 primarily causes infections in sheep and goats, is not discussed here because it only rarely causes a diphtheria-like disease in humans. Infection by toxigenic strains of C. diphtheriae causes disease that is mediated by ETIOLOGY the production of an extracellular protein. Nontoxigenic strains also Corynebacteria (Klebs-Löffler bacilli) are irregularly staining, gram- can cause disease, but it is usually less severe. positive, nonmotile, nonsporulating, pleomorphic bacilli.54 The club- Before the discovery of antitoxin at the turn of the 20th century, shaped appearance of the bacillus is not a true morphologic feature, the “strangling angel of children,” as diphtheria once was called, was a but results from attempting to grow the bacillus on media that are significant cause of mortality in children and adults.108 Apparent reference nutritionally inadequate (Löffler media). The organism can be recovered to diphtheria can be traced to the 5th century BC in the works of Hip- most readily on media containing selective inhibitors that retard the pocrates, Epidemics III, case 7.79 In 1821, it was recognized as a specific growth of other microorganisms; a sheep blood agar–based medium entity by Brettoneau, who suggested that the disease was caused by a containing fosfomycin (for selectivity) and Tindale medium (tellurite germ and that it could be transmitted from person to person. Brettoneau medium with cystine) are ideal.43,44 C. diphtheriae and C. ulcerans coined the origin of the modern term diphtheria from the Greek root grow on supplement-free blood agar, chocolate agar, and other standard diphtheria, which means “skin” or “hide.”50 In 1883 the causative agent media.43 was identified by Klebs in stained smears from diphtheritic membranes; Colonies of C. diphtheriae (with the exception of the lipophilic, gray in 1884, Löffler grew the organism on artificial media and showed that C. intermedius) and C. ulcerans appear grayish white on Löffler medium. in guinea pigs it caused a fatal infection closely resembling human On tellurite medium, three diphtheria colony types can be distinguished: disease. mitis, gravis, and intermedius. Mitis colonies are smooth, black, and The toxin was purified in 1889 by Roux and Yersin, who found that convex; they do not ferment starch or glycogen and are hemolytic. toxin alone could cause the disease. Shortly thereafter, Behring and Gravis colonies are gray, radially striate, and semirough; they ferment Kitasato discovered antitoxins when they immunized animals with toxins starch and glycogen and usually are not hemolytic. Intermedius colonies rather than bacteria. The use of antitoxin to treat children with diphtheria are small and smooth and have a black center; they do not ferment at the turn of the 20th century resulted in one of the largest decreases starch or glycogen and are not hemolytic. C. ulcerans colonies resemble 932 SECTION 16 Bacterial Infections gravis on Tindale medium but differ in that they are hemolytic. Similar Diphtheria toxin is lethal to humans in an amount of approximately to gravis, they ferment starch and glycogen. All diphtheria biotypes and 130 µg/kg body weight. Cytoplasmic internalization of 1 molecule of C. ulcerans are characterized by cystinase activity and absence of pyra- toxin has been shown to cause cell death.11 Toxigenic and nontoxigenic zinamidase activity. C. ulcerans may be distinguished from C. diphtheriae strains of C. diphtheriae can cause disease, but only strains that produce by its urease activity and ability to liquefy gelatin. Biotype belfanti, toxin cause disease with symptoms of myocarditis and neuritis. which does not occur in a toxigenic form, may be distinguished from the three potentially toxigenic diphtheria biotypes by its inability to reduce nitrate on Tindale medium and from C. ulcerans by its lack of EPIDEMIOLOGY production of urease.43 Ribotyping and pulsed-field gel electrophoresis, Asymptomatic human carriers serve as the reservoir for C. diphtheriae. both of which involve restriction digestion of genomic bacterial DNA, Infection by C. diphtheriae is acquired by contact with either a carrier followed by gel electrophoresis and Southern blotting, permit more or an individual with active disease. The bacteria may be transmitted specific typing within each diphtheria biotype and aid in the epide- via droplets during coughing, sneezing, or talking. Rarely transmission miologic study of outbreaks.41,95 of C. diphtheriae occurs from skin lesions or fomites. Some reports C. diphtheriae biotypes intermedius, gravis, and mitis and C. ulcerans suggest that skin carriers of C. diphtheriae are more infectious than all have been observed in a toxigenic form. Intermedius was the biotype nose or throat carriers and that skin carriers may serve as potential isolated most commonly in the United States between 1971 and 1981. reservoirs for the initiation of epidemic spread.12,68 In areas in which Of the strains isolated, intermedius was found to be toxigenic more skin infections are endemic, levels of natural immunization may be often than mitis or gravis.36 In the United Kingdom between 1993 and high.20 This phenomenon is illustrated particularly well in a survey of 1998, and similarly in other parts of Europe, the biovar gravis has tetanus and diphtheria immunity in a rural Kenyan community, where represented most nontoxigenic isolates, followed by mitis and belfanti.45 age was not found to be predictive of immunity, and no correlation Of the four nontoxigenic isolates obtained during a surveillance study was found between levels of antibody for tetanus and diphtheria.77 of a U.S. Northern Plains Native American Community in 1996, two Person-to-person transmission of C. ulcerans is not known to occur, were of biotype mitis and two were gravis.59 According to a UK diphtheria although C. ulcerans was isolated from the siblings of two patients reference laboratory, between 1993 and 1998, the toxigenic isolates reported in the United Kingdom between 1995 and 1997.17 Cases of originating from Asia, Africa, and the Middle East were reported to be respiratory diphtheria caused by C. diphtheriae and C. ulcerans have of the biotype mitis or gravis, with the exception of one intermedius been documented in association with contaminated unpasteurized milk isolate.44 Golaz and associates59 reported similar findings when they taken from cows with infected teats.18,60,104 C. diphtheriae has been isolated surveyed the different biotypes in South Dakota. A significant overall from horses, dogs, and other domestic animals. C. ulcerans has been increase in the proportion of nontoxigenic isolates has been observed reported to infect ground squirrels in the United States, but transmission in Europe and Australia in recent years.58 The reason for this increase to humans has not been reported.67,93 C. ulcerans is a commensal in is unclear, but one hypothesis is that increased immunity to toxigenic animals and has been isolated in a wide range of wild and domestic strains secondary to immunization has altered this epidemiology. animals.105 The complete genome sequence of C. diphtheriae biotype gravis has According to World Health Organization (WHO) reports, diphtheria been elucidated.33 The genome is approximately 2.48 Mb with a G+C is distributed worldwide and remains endemic in many developing areas content of 53% and has approximately 2320 predicted coding sequences. of the world, including Asia, Africa, South America, and the Mediterranean Metabolic analysis of the genome has revealed that C. diphtheriae has regions.19 Worldwide, diphtheria epidemics have occurred in a cyclic a complete set of enzymes for glycolysis, gluconeogenesis, pentose- manner since the 16th century. In 2014, 7321 cases of diphtheria were phosphate pathways, anaerobic and aerobic respiration, amino acid reported to the WHO, although many others were likely unreported. biosynthesis, and purine nucleotide biosynthetic pathway. Most of the In the United States, the incidence and mortality rates from diphtheria enzymes for the tricarboxylic acid cycle are present, with the exception in the 1920s were 140 to 150 cases/100,000, with 13,000 to 15,000 deaths of an enzyme succinyl coenzyme A (CoA) synthetase that catalyzes the each year. The number of cases gradually declined to 15 cases/100,000 conversion of succinate to succinyl CoA. An alternative enzyme present population in 1945 with the extensive use of diphtheria toxoid vaccine. in C. diphtheriae may fulfill this conversion, however. The pyrimidine From 1970 through 1979, the average number of cases of diphtheria pathway in C. diphtheriae lacks an enzyme that interferes with the reported annually in the United States was 196.8 From 1980 to 2004, production of cytidine; the pathway to the biosynthesis of thymidine only 57 cases of diphtheria were reported in the United States. Approxi- is complete. The genome has revealed 13 regions that are unique to C. mately 75% of the cases were in patients older than 15 years who were diphtheriae and may serve as pathogenicity islands.33 unimmunized or inadequately immunized.8 Only two cases of diphtheria No functional or significant differences have been detected in the have been reported in the United States between 2004 and 2015. exotoxins elaborated by the three strains of C. diphtheriae or by C. Maintenance of immunity in adults requires a booster vaccination every ulcerans. Only strains that are lysogenic for β-prophage or a closely 10 years. The Centers for Disease Control and Prevention (CDC) related phage carrying the gene for toxin production produce diphtheria estimated in the mid-1990s that fewer than 50% of adults in the United toxin. One or more tox gene sequences may exist in the bacterial genome, States had received their 10-year boosters and that 40% to 50% of and the most highly toxigenic strains contain three or more copies.82 adults were susceptible to diphtheria.52,96 In addition, the toxoid vaccine Phage multiplication is not a prerequisite for the production of toxin. does not provide protection against nontoxigenic strains. The capacity to synthesize toxin depends on genetic and nutritional The incidence peaks during the cooler autumn, winter, and spring factors. Toxin-producing cells apparently are cells in which spontaneous months. Several epidemics, primarily in the southern United States, induction of the prophage to the phage occurs.53 The most important have occurred in late summer and fall and corresponded to a high factor controlling the yield of toxin is the concentration of inorganic prevalence of C. diphtheriae skin infections. Between the years 1971 iron in the culture medium.39 Growth of C. diphtheriae in iron-deficient and 1981, the incidence of diphtheria was highest in the western United media prolongs the duration of induction lysis and is associated with States. A 100-fold greater incidence of diphtheria occurs in Native a high yield of toxin. High concentrations of iron inhibit the production Americans than in the general population.36 This difference may be of toxin. Production of toxin also can be increased by the use of ultraviolet attributable to socioeconomic factors more than to race. In 1996, a radiation. Conversion to a toxigenic strain occurs in nature, as has been surveillance study was conducted in a Northern Plains Native American shown by restriction enzyme studies of carriers of toxigenic and community after C. diphtheriae was isolated from the skin of a resident nontoxigenic strains in Manchester, England. of the community. The woman was a chronic alcoholic admitted to the The ability of a strain of C. diphtheriae to elaborate diphtheria toxin hospital for detoxification and treatment of severe necrotizing leg ulcers, can be shown by using several methods. In vivo studies involving necrosis from which a toxigenic mitis biotype was isolated. In the following 4 of tissue in guinea pigs has been replaced by the widely used Elek46 or months, 11 positive cultures were obtained from the community, modified Elek test.49 Enzyme immunoassay48 and polymerase chain including four positive throat swabs from asymptomatic household reaction (PCR)94 have been used to detect toxigenicity. contacts of the index cases. Ribotyping indicated that the isolates were CHAPTER 90 Diphtheria 933 related closely to each other genetically and to strains obtained from toxins also mediate target protein ribosylation by this mechanism. In past cases from the same area; they were different from organisms addition to the inhibition of cellular protein synthesis, an independent obtained from other parts of the United States and from the former mechanism of cytolysis has been described. In the presence of calcium Soviet Union, where an ongoing epidemic was occurring at the time.25,59 and magnesium, diphtheria toxin has a nuclease-like activity that causes A similar study was conducted in a Koorie (Aborigine) community in DNA fragmentation that results in cytolysis.34,35,70,78 Victoria, Australia, in 1994, after three cases of nontoxigenic C. diphtheriae Marked toxin-mediated tissue necrosis occurs in the vicinity of C. endocarditis were diagnosed in the community. After screening 359 diphtheriae colonization and induces a robust local inflammatory asymptomatic (with the exception of four people who had chronic skin response. The inflammatory response coupled with the necrotic tissue ulcers swabbed) contacts of the index cases, 12 produced positive cultures produces a patchy exudate that initially can be removed. As the infection for nontoxigenic C. diphtheriae. Of them, five were of the same biovar progresses, the increased production of toxin causes a centrifugal gravis clone as the three index cases.69 Evidence that diphtheria is widening of the area of infection, and eventually a fibrinous exudate diagnosed more frequently in chronic alcoholics and the indigent than develops. A tough adherent membrane results from coagulation of the in the general population is significant. In a 1993 to 1994 outbreak in exudate. The color of the pseudomembrane initially is white but, over St. Petersburg, Russia, 69% of a total of 42 deaths occurred in individuals the course of time, becomes dirty gray. Late in the course of the infection, classified as chronic alcoholics.98 Between 1972 and 1982, three outbreaks green or black spots appear on the membrane, representing areas of occurred in the indigent alcoholic population living in Seattle’s Skid necrosis. Histologic analysis of the pseudomembrane differs based on Road.66 Cutaneous infections accounted for 86% of the 1100 total cases. the site of formation and maturation of the membrane. Analysis of the The first outbreak was caused by a single toxigenicintermedius biotype pharyngeal pseudomembrane shows fibrin; inflammatory cells, primarily clone, whereas the other two involved nontoxigenic mitis and gravis composed of neutrophils, red blood cells, and colonies of organisms; strains. The incidence was highest in winter and spring. and superficial epithelial cells. A major epidemic began in 1990 in the new independent states of With severe infections, significant vascular congestion, interstitial the former Soviet Union and spread throughout the area. Between 1990 edema, fibrin exudate, and intense neutrophilic infiltration develop.64 and 1997, approximately 150,000 cases of diphtheria were reported, Profuse bleeding can occur when the membrane is torn off. The with approximately 4000 fatalities.65,95 The epidemic was attributed to edematous tissue and the diphtheritic membrane may encroach on the decreasing immunization rates and immunity in adults and children airway. The membrane sloughs spontaneously during the recovery and to movement of large numbers of people during the collapse of period, although sloughing can occur during the acute phase of the the former Soviet Union.23,106 Apparently multiple foci of infection illness, leading to aspiration. Occasionally secondary bacterial infection existed across the continent. Most of the epidemic isolates from Ukraine (classically caused by Streptococcus pyogenes) develops. Respiratory and Russia were biotype gravis, but further molecular characterization embarrassment or suffocation may occur, with involvement of the larynx of the tox genes from these areas revealed distinct epidemic strains in or tracheobronchial tree. Bronchopneumonia may develop if the exudate each location.70 A mass immunization program was initiated in Russia enters the small airways and alveoli. Infection of these sites is an uncom- in 1993, with a resultant 10% decrease in the number of new cases mon occurrence, however. Infections of the esophagus and stomach, reported between 1994 and 1995 (vs. two- to threefold increases in the with pseudomembranous lesions indistinguishable from lesions found number of cases each year for the preceding 3 years).57 The WHO also in the respiratory tract, have been reported.72 held training workshops and assembled laboratory kits to assist in Toxin produced at the site of infection is distributed throughout establishing the proper diagnosis of diphtheria. Because cases also began the body by the bloodstream and the lymphatics. This distribution to appear in Europe with increasing frequency during this period, the occurs most readily when the pharynx and tonsils are covered by a European Working Group on Diphtheria (ELWGD) and reference diphtheritic membrane. Any organ or tissue can be damaged as a result laboratories were assembled to assist in an effort to increase routine of diphtheria toxin, but lesions of the heart, nervous system, and kidneys screening for diphtheria. The ELWGD since has expanded and currently are particularly prominent. Clinical manifestations appear after a variable includes 20 participating countries, including representatives in Western latent period of 10 to 14 days for myocarditis and 3 to 7 weeks for and Eastern Europe, the United States, Australia, and Southeast Asia.45 manifestations in the nervous system, such as peripheral neuritis. In a Surveillance from Europe from 2000 to 2009 has documented a decrease study of 102 patients who died of diphtheria caused by C. diphtheriae, in incidence of more than 95% across the region, with the Russian the hearts appeared dilated, flabby, and pale, with a characteristic “streaky” Federation and Latvia accounting for 83% of the cases. In Western appearance in the myocardium. The most prominent pathologic findings Europe, toxigenic C. ulcerans has increasingly been identified, often in are necrosis and hyaline degeneration of the myocardium. The myo- association with contact with companion animals.107 More recently there cardium also appears edematous and is infiltrated with mononuclear have been documented cases, particularly of cutaneous diphtheria, in cells with eosinophilic cytoplasm. In a significant proportion of cases, refugees and asylum seekers in Europe. fatty accumulation in muscle fibers and the conducting system may be observed.89 Burch and associates21 showed mitochondrial damage with depletion of glycogen and accumulation of lipid droplets in the damaged PATHOGENESIS AND PATHOLOGY myofibrils. Toxin may be observed within the myocardial cells with Diphtheria is initiated by entry of C. diphtheriae into the nose or mouth, fluorescent antibody staining.64 If the patient survives, muscle regenera- where the bacilli remain localized on the mucosal surfaces of the upper tion and interstitial fibrosis can be seen. respiratory tract. Occasionally the ocular or genital mucous membranes Peripheral neuropathy occurs secondary to C. diphtheriae infections. serve as the site of localization. The bacilli are unable to invade intact Histologic studies have shown that affected nerves have significant skin but may infect preexisting skin lesions. After a 2- to 4-day period degeneration of myelin sheaths and axons. Toxic neuritis with fatty of incubation, lysogenized strains may elaborate toxin. degeneration of paranodal myelin can be noted early in the disease Diphtheria toxin is secreted as a single polypeptide of 535 amino course; segmental demyelination occurs later.9 Axonal damage is second- acids with a molecular weight of 58,342 Da.70 The toxin is composed ary to the application of external pressure from the swollen Schwann of two subunits: a large B subunit that is involved in receptor binding cell cytoplasm and myelin.81 and an A subunit that is the enzymatically active portion of the toxin. C. diphtheriae infections also can lead to necrosis and hyaline The toxin initially is absorbed onto the target cell membrane by binding degeneration of the liver, which can lead to hypoglycemia. Adrenal a receptor on the cell surface and then undergoes receptor-mediated hemorrhage and acute tubular necrosis of the kidney have been known endocytosis. When it is in the endolysosomal complex, it undergoes a to occur secondary to C. diphtheriae infections.64 conformational change with subsequent release of the A subunit into the cytoplasm. The A subunit transfers an adenosine diphosphate (ADP)-ribosyl group from nicotinamide adenine dinucleotide (NAD) CLINICAL MANIFESTATIONS to elongation factor 2. This ADP-ribosylation inactivates elongation The signs and symptoms of diphtheria depend on the site of infection, factor 2 and inhibits protein synthesis in the cell. Cholera and pertussis the immunization status of the host, and whether toxin has been 934 SECTION 16 Bacterial Infections distributed to the systemic circulation. The incubation period is 1 to begin as vesicles or pustules that progress to typical ulcers with sharply 6 days (range, 1 to 10 days). Diphtheria can be classified clinically on defined borders, membranous bases, and surrounding erythema and the basis of the anatomic location of the initial infection and the edema. They may be covered with a dark pseudomembrane. The lesions diphtheritic membrane (nasal, pharyngeal/tonsillar, laryngeal or occur most commonly on the legs, feet, and hands. For the first 1 to 2 laryngotracheal, skin, and others) involved. More than one anatomic weeks, the lesions are painful. Spontaneous healing generally takes 6 site may be involved simultaneously. to 12 weeks, but lesions have been reported to persist for 1 year.68 Nasal diphtheria initially resembles a common cold and is character- Conjunctival, aural, and vulvovaginal diphtheria also may occur. ized by mild rhinorrhea and a paucity of systemic symptoms. Gradually Conjunctival lesions usually are limited to the palpebral conjunctiva, the nasal discharge becomes serosanguineous and then mucopurulent. which appears red, edematous, and membranous. Rarely conjunctival A foul odor may be noticed, and careful inspection reveals a white lesions have been associated with corneal erosion.99 Diphtheria infections membrane on the nasal septum. In severe cases, the infection may of the ear are characterized by the development of otitis externa with excoriate the nares and upper lip. Nasal diphtheria is a mild form of a persistent purulent and frequently foul-smelling discharge. the disease because absorption of toxin usually is slow from this site. Clinical presentations other than typical diphtheria have been Frequently delays in establishing an accurate diagnosis of nasal diphtheria associated with isolation of the organism from patients with meningitis, occur because of the lack of systemic symptoms. The nasal form of the endocarditis, osteomyelitis, and hepatitis. In most cases, these infections disease occurs most often in infants. have occurred in patients with underlying problems, such as structural Pharyngeal and tonsillar diphtheria begin insidiously with anorexia, or valvular heart disease or intravenous drug use, or in individuals malaise, low-grade fever, and pharyngitis. Within 1 or 2 days, a membrane from poor socioeconomic backgrounds.40,104 appears. The extent of membrane formation correlates with the immune Several cases of septic arthritis caused by nontoxigenic C. diphtheriae status of the host; in some partially immune individuals, a membrane have been described.1,63,104 Afghani and Stutman1 reported the case of may not develop. The white or gray adherent membrane may cover the a 27-month-old child who had septic arthritis of the hip and skin tonsils and pharyngeal walls and extend on to the uvula and soft palate lesions on the lower extremities. In this case, the skin lesions were or down on to the larynx and trachea. Attempts to remove the membrane presumed to be the portal of entry for nontoxigenic C. diphtheriae. are followed by bleeding. Cervical lymphadenitis varies. In some cases, Although the child had received four doses of diphtheria and tetanus it is associated with edema of the soft tissues of the neck and may be toxoids and pertussis vaccines, immunization with toxoid does not so severe that it gives the appearance of a “bull neck.” In a 1970 epidemic, provide protection against nontoxigenic strains of C. diphtheriae. In “erasure” edema of the neck was noted in patients with pharyngeal this case, the organism was sensitive to penicillin, cefuroxime, cephalothin, diphtheria.85 Patients with erasure edema did not have a classic bull and clindamycin but was resistant to oxacillin, an antistaphylococcal neck appearance, but the edema was characterized by obliteration of antibiotic often used for the treatment of septic arthritis when the the sternocleidomastoid muscle border, the mandible, and the median causative organism cannot be identified. A similar case was described border of the clavicle. The edema was brawny, pitting, warm to the in an immunocompetent, fully vaccinated 2-year-old child who had touch, and tender to palpation. Erasure edema was noted in 29% of skin lesions from which C. diphtheriae was isolated. The skin lesions immunized patients and 30% of nonimmunized or inadequately also were assumed to be the portal of entry for the organism because immunized patients. It occurred most commonly in children older than pan-sensitive C. diphtheriae were isolated from the skin and the articular 6 years and generally was associated with infection by the gravis or aspirate.63 intermedius strain of C. diphtheriae. Within a 12-month period, in New South Wales, Australia, four The course of pharyngeal diphtheria depends on the degree of cases of septic arthritis complicating endocarditis caused by the non- elaboration of the toxin and the extent of the membrane. In severe toxigenic gravis variety of C. diphtheriae were reported. In addition, cases, respiratory and circulatory collapse may occur. The pulse rate is the same strain caused three cases of endocarditis without the develop- increased disproportionately to body temperature, which generally ment of septic arthritis. Demographic distribution of these seven cases remains normal or slightly elevated. The palate may be paralyzed. This included a 12-year-old boy who died, five patients who were in their paralysis may be unilateral or bilateral and associated with difficulty 20s, and a patient who was 49 years old. Three of the patients had swallowing and nasal regurgitation of swallowed fluids.42 Stupor, coma, underlying cardiac abnormalities, and one had a history of intravenous and death may occur within 7 to 10 days. In less severe cases, recovery drug use.104 This same clone of nontoxigenic C. diphtheriae was isolated may be slow and may be complicated by the development of myocarditis from three patients in Koorie, an aborigine community in Victoria, or neuritis. In mild cases, the membrane sloughs off in 7 to 10 days, Australia, who had endocarditis, and five asymptomatic contacts.69 Two and recovery is uneventful. of the three patients with endocarditis were members of the same family, Laryngeal diphtheria generally reflects a downward extension of the and one of them had a history of alcohol abuse. The third patient had membrane from the pharynx. Rarely laryngeal diphtheria is primary a septic sternoclavicular joint, in addition to endocarditis, with isolation and does not reflect an extension of disease from the pharynx. In these of the same organism. Nontoxigenic C. diphtheriae sepsis can lead to cases, toxicity and signs of toxemia generally are less prominent. Two splenic and hepatic abscesses, as reported in a patient with chronic cases of isolated diphtheritic tracheitis have been reported in the litera- lymphocytic leukemia in British Columbia, Canada.71 ture.13,103 The clinical findings of laryngeal diphtheria are indistinguishable Complications secondary to elaborated diphtheria toxin may affect from those of other types of infectious croup. Noisy breathing, progressive any system, but myocarditis and involvement of the nervous system stridor, hoarseness, and a dry, barking cough may be noted. Suprasternal, are most characteristic. Myocarditis may occur after mild and severe subcostal, and supraclavicular retractions reflect severe laryngeal obstruc- cases of diphtheria. Generally it develops in patients in whom administra- tion, which may be fatal unless alleviated. Occasionally in a mild case, tion of antitoxin is delayed. Myocarditis most commonly appears in an acute and fatal obstruction may occur because of a partially detached the second week of the disease, but it can appear as early as the first or piece of membrane that occludes the airway. In severe cases of laryngeal as late as the sixth week of illness. Tachycardia, a muffled 1S , murmurs, diphtheria, the membrane may extend downward and invade the entire and arrhythmias such as atrioventricular dissociation indicate myocardial tracheobronchial tree. involvement. Echocardiography may show left ventricular dysfunc- Cutaneous disease, in contrast to pharyngeal disease, is more common tion.3,61,80 Although some cases may result in cardiac failure, most in warmer climates and often is caused by nontoxigenic strains. In some myocardial complications are temporary. countries with tropical and subtropical climates, such as Uganda, Neurologic complications appear after a variable latent period. Tanzania, Sri Lanka, and Samoa, C. diphtheriae has been isolated from Approximately 75% of all patients with severe diphtheria develop 60% of skin lesions in children.74 Cutaneous diphtheria is more con- neuropathies. The incidence of neurologic sequelae has been shown to tagious than respiratory diphtheria. Cutaneous diphtheria may be an correlate with the severity of respiratory symptoms; 20% of all patients important source of person-to-person transmission of diphtheritic with respiratory problems develop polyneuritis. Neurologic complications organisms and outbreaks in indigenous populations in which overcrowd- from diphtheria infections predominantly are bilateral, are motor rather ing and poor hygiene are important risk factors.12,20,68 The skin lesions than sensory, and usually resolve completely. Paralysis of the soft palate CHAPTER 90 Diphtheria 935 is the most common occurrence and generally appears in the third assay.7,74,75,91,92,94 The absence of the tox gene by PCR excludes the diagnosis week. It is manifested by a nasal quality in the voice, nasal regurgitation, of diphtheria. PCR may give false-positive results, however, because it and difficulty swallowing. Ocular paralysis usually occurs around the does not differentiate between partial or nonfunctional tox genes and fifth week of illness and is characterized by blurring of vision and functional tox gene products. An increasing number of cases of non- difficulty with accommodation. Internal strabismus also may be noted. toxigenic diphtheria that are positive for the tox gene by PCR have been Paralysis of the diaphragm, peripheral neuropathy involving the limbs, reported from Ukraine and Russia.58,83 and loss of deep tendon reflexes likewise are reported as complications The immune status of patients can be determined by toxin neutraliza- of diphtheria. When they occur, along with an elevated cerebrospinal tion in Vero cells.88 This method is used frequently, although it is difficult fluid protein, the syndrome is clinically indistinguishable from Guillain- to standardize and relies heavily on individual interpretation of results. Barré syndrome. Enzyme-linked immunosorbent assay is a more rapid and quite sensitive Rarely, 2 or 3 weeks after the onset of illness, involvement of the method, but it detects some nonspecific antibodies; when antitoxin vasomotor centers results in hypotension and cardiac failure. Gastritis, levels are in the low range, the assay may generate falsely elevated results.86 hepatitis, nephritis, and hemolytic-uremic syndrome also have been Finally a delayed fluorescence immune assay method was developed reported as complications of diphtheria.102 by Aggerbeck and colleagues2 in 1996 and has been reported to have Information on the effects, if any, of diphtheria on the fetus during good sensitivity, specificity, and reproducibility.16 Levels of diphtheria pregnancy is sparse. El Seed and associates47 reported a case of pharyngeal antitoxin of 0.01 IU/mL or greater generally are accepted as protective. diphtheria in a pregnant woman that occurred during the first trimester A skin-testing method, the Schick test, also has been used to assess of pregnancy. Apart from vaginal bleeding, no complications of pregnancy immunity. were noted. Severe diphtheritic toxemia in the mother was characterized The Schick test was used previously to determine the immune status by quadriparesis, from which she fully recovered. A physically normal of the patient. It is not helpful in establishing an early diagnosis because female infant was delivered at term. In this single case, severe diphtheritic it cannot be read for several days, and currently it is not widely used. toxemia during pregnancy was not associated with any teratogenic effect In the Schick test, a measured amount of purified diphtheria toxin (0.1 in the fetus and did not impair intrauterine fetal growth. mL) is injected subcutaneously. A hypersensitivity reaction indicates an inadequate presence of antitoxin. A toxoid control also is injected, in the opposite arm, to help distinguish between a reaction to toxin DIAGNOSIS and a reaction to other antigens in the toxin preparation. The diagnosis of diphtheria should be based on clinical findings because Other laboratory studies are of little diagnostic value. The white any delay in initiating therapy poses a serious risk to the patient. Isolation blood cell count may be normal or elevated. Rarely anemia develops of the organism is used to confirm the clinical diagnosis. Material as a result of rapid hemolysis of red blood cells. Examination of obtained from beneath the membrane, where organisms are concentrated cerebrospinal fluid may reveal a minimal elevation of protein and, most highly, or a portion of the membrane itself should be obtained rarely, a mild pleocytosis in patients with diphtheritic neuritis. for culture.5,33 Hypoglycemia, glucosuria, or both may occur and reflect hepatic toxicity. C. diphtheriae is relatively resistant to drying. The use of a nonnutri- An elevation in blood urea nitrogen may develop in patients with acute tive, moisture-reducing transport medium helps prevent the overgrowth tubular necrosis. An electrocardiogram should be obtained and may of other microorganisms. The laboratory should be notified about the reveal ST-segment and T-wave changes or arrhythmias indicative of possibility of diphtheria so that appropriate culture media are inoculated. myocarditis. A Löffler slant, a tellurite plate, and a blood agar plate should be inocu- lated. Tellurite-containing media inhibit the growth of normal oral Differential Diagnosis flora, allowingC. diphtheriae to grow into characteristic black colonies. Mild forms of nasal diphtheria in a partially immunized host may Other corynebacteria, staphylococci, and yeast also can reduce tellurite resemble the common cold. When the nasal discharge is more serosan- and grow into black colonies.38 Although examination of direct smears guineous or purulent, nasal diphtheria must be distinguished from a of colonies or diphtheritic lesions remains an important supplement foreign body in the nose, sinusitis, adenoiditis, or the snuffles of congenital to clinical examination, it often is inaccurate. Screening the colonies syphilis. Careful examination of the nose with a nasal speculum, sinus from the tellurite plate for catalase, urea, nitrate, pyrazinamidase, and radiographs, and appropriate serologic tests for syphilis are helpful in cystinase is important. Most biotypes of C. diphtheriae are catalase excluding these disorders. positive, urease negative, nitrate positive (except biotype belfanti), Tonsillar or pharyngeal diphtheria must be differentiated from pyrazinamidase negative, and cystinase positive.43 streptococcal pharyngitis. Generally streptococcal pharyngitis is associated All diphtheria bacilli that are recovered should be tested for toxigenic- with more severe pain on swallowing, higher temperature, and a ity. In 1949, the Elek immunoprecipitation assay replaced in vivo testing nonadherent membrane limited to the tonsils. In some patients, pha- for toxigenicity using guinea pigs or rabbits.46 The Elek test is based on ryngeal diphtheria and streptococcal pharyngitis coexist. gel diffusion and immunoprecipitation of toxin from organisms inocu- Tonsillar and pharyngeal diphtheria also must be differentiated from lated onto agar adjacent to an antitoxin-containing well. A strain that infectious mononucleosis (lymphadenopathy and splenomegaly are is positive for toxin is indicated by the formation of a precipitin band common findings, atypical lymphocytes are generally present, and between the toxin and antitoxin.44,46 The Elek test takes 48 hours to heterophile antibody may be present), nonbacterial membranous yield results on the toxigenic nature of a C. diphtheriae strain. A modified tonsillitis (white blood cell count generally is low, throat cultures reveal Elek test that consists of placement of an antitoxin-impregnated disk normal flora, and the course is unaffected by antibiotics), primary onto an agar plate surrounded by inoculates of clinical specimen and herpetic tonsillitis (presence of gingivitis, stomatitis, and discrete lesions positive control has been described. In contrast to the conventional of the tongue and palate may be helpful), Vincent angina (may be Elek test, the modified test has the advantage of using “spot” inoculations indistinguishable), and thrush (constitutional symptoms are absent, of numerous colonies directly from the primary plate. In addition, the and lesions are present on the buccal mucosa and tongue). Tonsillar modified Elek test has fewer false-positive and false-negative results and pharyngeal diphtheria also must be differentiated from blood and yields results more rapidly (16 to 24 hours).43 dyscrasias such as agranulocytosis and leukemia (complete blood count A rapid enzyme immunoassay is another test for detection of and bone marrow study are helpful); post-tonsillectomy faucial mem- diphtheria toxin. This method uses equine polyclonal antibody to capture branes (membranes are stationary and do not spread); and oropharyngeal the diphtheria toxin and an alkaline phosphatase–labeled monoclonal involvement by toxoplasmosis, Arcanobacterium, cytomegalovirus, antibody to detect fragment A of the toxin. It is a rapid test that takes tularemia, and salmonellosis (associated signs and symptoms and 3 hours and has a limit of detection of 100 pg/mL.48 appropriate cultures and serologic tests may be diagnostic).55 Rapid testing for diphtheria toxin by PCR specific for the A or B Laryngeal diphtheria must be differentiated from spasmodic or portion of the toxin gene, tox, is sensitive and has produced positive nonspasmodic croup; acute epiglottitis; laryngotracheobronchitis; results in specimens stored for 12 months before performing the bacterial tracheitis (particularly staphylococcal); aspirated foreign bodies; 936 SECTION 16 Bacterial Infections peripharyngeal and retropharyngeal abscesses; and laryngeal papillomas, during an epidemic in Texas showed no statistical difference in the risk hemangiomas, or lymphangiomas. A careful history, followed by careful for diphtheria infection developing in individuals with full, lapsed, visualization in the hospital under controlled conditions, aids in making inadequate, or no previous diphtheria immunization; however, a 30-fold a correct diagnosis. increased risk for development of symptomatic diphtheria in individuals with no immunization and an 11.5-fold increase in individuals with inadequate immunization were noted.87 PREVENTION The most important health problem in the United States today is Diphtheria is prevented on a community-wide basis most effectively inadequate immunization of the population. Immunization rates in by active immunization. Diphtheria toxoid is available in combination adults are poorer than those in infants and children because of failure with tetanus toxoid as pediatric DT or adult Td and in combination to maintain adequate immunity through appropriate booster immuniza- with acellular pertussis as DTaP and Tdap. Combination regimens with tion. A 70% to 80% immunization level is thought to be required to DTaP and inactivated poliovirus and hepatitis B (Pediatrix) and prevent epidemic spread.36 inactivated poliovirus and Haemophilus influenzae type B (Pentacel) Prevention of diphtheria also depends on management of the are also available. Pediatric formulations of diphtheria toxoid vaccines contacts of known cases of diphtheria and carriers of the organism and contain three to four times more diphtheria toxoid but the same tetanus on isolation of patients to minimize the spread of disease. Individuals toxoid in contrast to the adult formulations. Children younger than 7 at risk for contracting the disease from the index case include those years should be given the pediatric formulations of vaccine, whereas who have had close respiratory or physical contact or prolonged children older than 7 years should receive the adult Td. Two forms of close proximity with the infected individual, including members of the Tdap are available: Boostrix, which is approved for children beginning index case’s household.17 Specifically, family members who share at age 10 through 65 years or older, and Adacel, which is approved for body towels and cups or eating utensils, share a bed or a bedroom with individuals 11 to 64 years old.22,26–29 more than two people, or take a bath less than once per week have a Primary immunization is carried out conveniently and effectively significantly greater risk for contracting the disease from the infected by giving diphtheria and tetanus toxoids and pertussis vaccine, DTaP, patient. A history of eczema in the contact also has been associated at 2, 4, and 6 months of age, with booster doses given at 15 to 18 months with a significantly increased risk for contracting diphtheria from the and again when the child is 4 to 6 years of age. Booster doses with the index case.97 The patient is infectious until diphtheria bacilli no longer adult type of diphtheria and tetanus toxoids adsorbed (Td) should be can be cultured from the site of infection. Two or three consecutive given at 10-year intervals to all immunized individuals. Current recom- negative cultures at least 24 hours apart are required, and antibiotic mendations are to give Tdap as the first booster dose in patients who therapy must be complete for 24 hours before the patient is released are 11 to 12 years old and to anyone older who has not received a dose from isolation. If obtaining cultures is impossible, isolation may be of Tdap.30 Td and Tdap contain 2 to 2.5 flocculation units (Lf) diphtheria ended after the completion of 14 days of appropriate antibiotic toxoid per dose in contrast to 7 to 25 Lf in the pediatric diphtheria, treatment.6 tetanus toxoid, and pertussis vaccine preparations (DTaP and DT). Cultures should be taken from the nose and throat of all close contacts, Primary immunization of children older than 7 years may be performed who should be kept under surveillance for 7 days (surveillance as an with Td. Two doses are given intramuscularly at least 4 weeks apart, outpatient is acceptable).5 Regardless of their immunization status, with a booster dose provided 1 year later. Children and adults who are contacts should be treated with a single intramuscular dose of benzathine severely immunocompromised or undergoing long-term hemodialysis penicillin G 600,000 U for individuals weighing less than 30 kg and 1.2 should use the standard immunization schedule, although response million U for individuals weighing more than 30 kg or a 7-day course may be suboptimal.51,56,76 of erythromycin 40 to 50 mg/kg per day (maximum 2 g/day) divided Most local and systemic reactions to the diphtheria and tetanus into four doses.5 The immune status of each contact should be deter- toxoids and whole cell pertussis vaccine (DTP), including fever, were mined; individuals for whom immune status is inadequate, including related to the pertussis component.10,37 Administration of tetanus and individuals who have had the primary series but more than 5 years has diphtheria toxoids is not followed by the high incidence of reactions elapsed since they received their last booster dose, should receive an associated with the use of pediatric DTP vaccines. At least one study injection of diphtheria toxoid. In addition, patients with diphtheria showed that 7.5 Lf toxoid can be given safely to adults without a higher should be immunized during convalescence because infection may not risk for reactions occurring.14 Primary immunization against diphtheria confer immunity.6 for infants with progressive neurologic disorders and completion of Asymptomatic carriers who previously were not immunized against the primary immunization series in patients who had experienced an diphtheria should have cultures taken, receive diphtheria toxoid and untoward reaction to an earlier DTP vaccine injection may be performed penicillin or erythromycin (as described earlier), and be seen daily by with diphtheria and tetanus toxoids rather than diphtheria and tetanus a physician. Asymptomatic contacts who are found to carry a toxigenic toxoids and pertussis vaccine.4 strain should be subjected to the same isolation and treatment measures A report of 97 preterm infants who received diphtheria and tetanus as the index case.6 If daily surveillance is impossible, benzathine penicillin toxoids and acellular pertussis vaccine (DTaP) (94 of these infants also is preferred over erythromycin for treatment because failure to adhere received H. influenzae type B vaccine) showed that most infants tolerated to an oral drug regimen is a concern. If a contact is experiencing the vaccination without side effects, although a subgroup of infants symptoms when seen, treatment of diphtheria is indicated. It is important with very low birth weight (mean, 873 g) had either a recurrence or an to initiate prophylactic therapy in contacts who have not been immunized increase in the number of apneic and bradycardic episodes in the 48 before the results of culture are received. Management of carriers is hours after receiving vaccination. The apneic and bradycardic episodes described in the next section. Contacts whose occupations involve were present before immunization in every case.100 close contact with unimmunized children or food handling (especially Booster doses of tetanus and diphtheria toxoids should be given at milk) should refrain from working until cultures are confirmed to be 10-year intervals to all immunized individuals. The CDC Advisory negative.6 Committee on Immunization Practices now recommends that all adults aged 19 years and older who have not received a dose of Tdap should receive a single dose.31,84 A recent update recommends a dose TREATMENT of Tdap during every pregnancy.32 As mentioned earlier, levels of Treatment of diphtheria is predicated on neutralization of free toxin diphtheria antitoxin of 0.01 IU/mL or greater generally are accepted as and eradication of C. diphtheriae or C. ulcerans with antibiotics. Disease protective. caused by C. ulcerans should be treated in the same manner as disease Diphtheria immunization is not always followed by complete protec- caused by C. diphtheriae.3,24 The decision to administer equine antitoxin tion.78 Immunization is directed against the phage-mediated toxin, not should be based on the site and size of the membrane, the degree of against infection. Fully immunized individuals may be carriers or may toxicity, and the duration of illness and should be made clinically as have disease caused by nontoxigenic strains. An investigation conducted soon as possible.15 CHAPTER 90 Diphtheria 937

Antitoxin can neutralize circulating toxin or toxin that is absorbed patient with myocarditis may receive digitalization if congestive heart onto cells, but is ineffective when cells have been penetrated. Early failure develops. Digitalization for arrhythmias caused by diphtheria treatment is essential to limit tissue damage. An adequate dose of antitoxin may be contraindicated, however. In severe disease, prednisone 1 to 1.5 must be administered intravenously as early as possible to neutralize mg/kg per day for 2 weeks has been shown to lessen the incidence of all free toxins. A single dose is used to avoid the risk for developing myocarditis. sensitization from repeated doses of horse serum. Tests for sensitivity Hydration should be maintained, and a high-calorie liquid or soft to horse serum must be performed before antitoxin is administered. diet should be provided. Secretions should be suctioned as needed to Antitoxin and its indications for use as well as instructions for sensitivity prevent aspiration. Palatal and pharyngeal paralysis increases the risk testing and administration are available through the CDC (Emergency for aspiration occurring, so gavage via a nasogastric tube is indicated Operations Center, 770-488-7100; www.cdc.gov/diphtheria/dat.html).5 in these patients. The antitoxin dosage is empirical. Pharyngeal or laryngeal disease of The quality of the voice and the gag reflex should be checked regularly 48 hours’ duration or less should be treated with 20,000 to 40,000 U, for assessment of progression of the disease. Laryngeal diphtheria may nasopharyngeal disease with 40,000 to 60,000 U, and severe pharyngeal require relief of obstruction with a tracheostomy. This procedure should or laryngeal diphtheria with 80,000 to 120,000 U of antitoxin. The last be performed before the patient has become exhausted. dose also should be given to patients with mixed clinical symptoms Adequate immunity does not develop in at least half of patients and to patients with brawny edema or disease of longer than 48 hours’ who recover from diphtheria, and they remain subject to reinfection. duration. The value of antitoxin in the treatment of cutaneous disease Immunization is indicated after the patient recovers. is debated, but some experts recommend 20,000 to 40,000 U because toxic effects have been reported.5 The use of intravenous immunoglobulin (IgIV) for therapy of respiratory or cutaneous diphtheria has not been PROGNOSIS approved or evaluated for efficacy. A human monoclonal antibody to Many factors affect the prognosis in cases of diphtheria, the most replace equine diphtheria antitoxin is under development and holds important being the immunization status of the host. Morbidity and promise as a potential therapeutic agent.101 mortality rates are increased significantly in patients who are unim- Although antibiotics are not a substitute for treatment with antitoxin, munized or inadequately immunized. The rapidity with which medical they should be given when diphtheria is suspected clinically. Penicillin care is sought and the diagnosis of diphtheria is suggested has a great and erythromycin are still effective against most strains of C. diphtheriae. impact on outcome. If specific treatment is provided on the first day Penicillin and erythromycin also are effective in eradicating group A of disease, the mortality rate may be reduced to less than 1 percent; hemolytic streptococci, which may complicate 30% of cases of diphtheria. delay in providing treatment until day 4 may be associated with a 20-fold Treatment consists of a 14-day course of penicillin or erythromycin. increase in the mortality rate. Penicillin may be given as aqueous penicillin G 100,000 to 150,000 U/ The virulence of the infecting organism and the location of infection kg per day in four divided doses intravenously or as procaine penicillin are important prognostic factors. Infection with a nontoxigenic C. 25,000 to 50,000 U/kg per day (maximum of 1.2 million U) in two diphtheriae strain may cause disease but does not lead to myocarditis, divided doses intramuscularly. Patients who are sensitive to penicillin neuritis, and other toxin-related phenomena. Toxigenic disease may should be given erythromycin in a daily dosage of 40 to 50 mg/kg vary from mild to severe. In cases of mild diphtheria, membrane (maximum of 2 g/day) in four divided doses for 14 days. When the sloughing and full recovery generally occur within 7 days. Disease caused patient is able to tolerate oral medications, erythromycin or penicillin by toxigenic gravis strains tends to be more severe and carries a poorer V may be used in place of the intravenous antibiotics.5 prognosis. Although diphtheria may affect the skin, nasopharynx, and Follow-up cultures should be obtained at least 2 weeks after antibiotic other mucous membranes, involvement of the larynx heralds a more therapy is completed; if they are positive, erythromycin should be given complicated course. Laryngeal diphtheria increases the risk for develop- for an additional 10 days.5 Some resistance to erythromycin has been ment of airway obstruction and promotes systemic absorption of the observed, but it is uncommon, and its epidemiologic significance is toxin. These patients require close monitoring of respiratory function unknown.5,6 Penicillin is recommended as first-line treatment in Vietnam, and for involvement of other organ systems. Laryngeal diphtheria is based on sensitivity data.73 Amoxicillin, rifampin, and clindamycin more likely to be fatal in infants. provided in appropriate dosages also may be effective. Lincomycin and Few laboratory parameters indicate the severity of diphtheria. The tetracycline have proved to be less effective, and cephalexin, oxacillin, development of amegakaryocytic thrombocytopenia and leukocytosis and colistin have been shown to be ineffective against C. diphtheriae. with counts of greater than 25,000 cells/mm3 has been associated with The endpoint of therapy is two or three consecutive negative cultures a poor outcome. at least 24 hours apart. In addition to receiving antibiotic therapy, patients The prognosis in a patient with diphtheria remains guarded until with diphtheria should be immunized during convalescence because recovery is complete. At any time during the course of the illness, infection may not confer immunity.5 complications such as laryngeal obstruction, shock, and ventricular The carrier state has been treated effectively with a single intramus- fibrillation may occur suddenly and unexpectedly. In patients with cular dose of benzathine penicillin G 600,000 U for children weighing myocardial involvement, permanent damage to the heart, specifically less than 30 kg or 1.2 million U for individuals weighing 30 kg or greater fibrosis, may occur and lead to later complications. In addition, potentially or oral erythromycin 40 to 50 mg/kg per day for children and 1 g/day severe neurologic manifestations, such as phrenic nerve paralysis, may for adults for 7 to 10 days.5 Carriers should have repeat pharyngeal appear late in the course of the disease. cultures performed a minimum of 2 weeks after antibiotic therapy is Persistence of C. diphtheriae may be noted in the nasopharynx of complete; if the repeat cultures are positive, carriers should receive an 5% to 10% of convalescing patients. Recovery from diphtheria is followed additional course of antibiotics. by immunity that is demonstrable for at least 1 year after illness in 50% Treatment of deep infections caused by nontoxigenic C. diphtheriae of patients. Second attacks are rare; nonetheless, immunization should can be accomplished with a wide variety of susceptible agents. Endo- be performed after the patient recovers. carditis with these organisms treated with either a β-lactam alone or Before the use of antitoxin and the availability of antibiotics, the in combination with an aminoglycoside has been associated with favorable mortality rate from diphtheria was 30% to 50%. Death was most common outcomes.90 in children younger than 4 years old and was the result of suffocation. At present, the worldwide mortality rate is 5% to 10%, with no clear Supportive Treatment association with age. Bed rest is extremely important and should be required for 2 to 3 weeks. Serial electrocardiograms should be obtained two or three times each week for 4 to 6 weeks to detect myocarditis as early as possible. NEW REFERENCES SINCE THE SEVENTH EDITION Absolute bed rest must be enforced if myocarditis is detected, 5. American Academy of Pediatrics. Diphtheria. In: Kimberlin DW, Brady MT, because sudden death has been precipitated by excessive activity. A Jackson MA, et al, eds. Red Book: Report of the Committee on Infectious Diseases. 30th ed. Elk Grove Village, IL: American Academy of Pediatrics; 2015:60-63, 325–9. ADDITIONAL READING 32. Centers for Disease Control and Prevention. Updated recommendation for the Barksdale L. Corynebacterium diphtheriae and its relatives. Bacteriol Rev. use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine 1970;34:378-422. (Tdap) in pregnant women – Advisory Committee on Immunization Practices Pappenheimer AM Jr. Diphtheria toxin. In: Ajl SJ, Kadis S, Montie TC, eds. Microbial (ACIP), 2012. MMWR Morb Mortal Wkly Rep. 2013;62:131-135. Toxins. Vol. 11B. New York: Academic Press; 1973. 101. Sevigny LM, Booth BJ, Rowley KJ, et al. Identification of a human monoclonal Wood WB Jr. From Miasmas to Molecules. New York: Columbia University Press; antibody to replace equine diphtheria antitoxin for the treatment of diphtheria 1961. intoxication. Infect Immun. 2013;81:3992-4000. Zamiri I. Corynebacterium. In: Collee VG, Fraser AG, Marmion BP, Simmons A, eds. Mackie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill The full reference list for this chapter is available at ExpertConsult.com. Livingstone; 1996:299-307. CHAPTER 90 Diphtheria 938.e1

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