Corynebacterium diphtheriae (Diphtheria)

History

The earliest written records of diphtheria, some of which are attributed to Hippocrates, can be traced back to the Hellenic period during the 5th or 4th century bc . Vivid depictions of diphtheria were also documented during the Renaissance: Bartholin described the disease as “angina puerorum” and “morbus strangulatorius,” evoking its propensity to cause disproportionate morbidity in young children. Outbreaks occurred sporadically in Spain during the 1600s, and a major epidemic broke out in New England from 1735–40, decimating the population of children in several towns. Belief that divine retribution was the cause of the outbreak may have contributed to the Great Awakening of the mid-1700s. During an 1818–20 epidemic in Tours, Bretonneau described diphtheria's salient clinical findings and differentiated it from other causes of “throat distemper.” He named the disease “diphtheria” (from the Greek “diphthera,” meaning “leather hide”), aptly depicting its characteristic pseudomembrane. He may have also been the first clinician to perform a tracheostomy successfully for pharyngeal diphtheria in 1825.

Understanding of the etiology, pathogenesis, and treatment of diphtheria advanced significantly as discoveries were made in the late 1800s and early 1900s. Klebs isolated C. diphtheriae from a pseudomembrane in 1884, and Loeffler proved it to be the etiologic agent of diphtheria. In 1888 Roux and Yersin discovered the exotoxin and described its clinical effects. The treatment of diphtheria advanced significantly in 1890 when von Behring and Kitasato developed antitoxin in guinea pigs, demonstrating the concept of passive immunity. For this seminal discovery von Behring was awarded the inaugural Nobel Prize for Physiology and Medicine in 1901. A combination toxin-antitoxin vaccine was initially used for prevention of diphtheria, until Ramon developed a safe and immunogenic heat- and formalin-inactivated toxoid vaccine in 1923. Aluminum salt adjuvants were incorporated into the toxoid vaccine to increase its immunogenicity, and by the 1940s an effective vaccine had been developed. Cases of diphtheria in the United States decreased by over 99% from approximately 206,000 cases in 1921 to 5 reported cases since 2000 ( Fig. 204.1 ). In the 1990s, however, there was a resurgence of diphtheria in the former Soviet Union republics, as their health care system was disrupted. At the peak of the epidemic there were more than 140,000 cases and approximately 4000 deaths. Targeted vaccination campaigns and a coordinated international effort halted the outbreak.

The Pathogen

The genus Corynebacterium is grouped within the order Actinomycetales and consists of more than 80 species, several of which are medically important. C. diphtheriae (from “korune” and “diphthera,” Greek for “club” and “leather,” respectively) is named for its characteristic clubbed-shape appearance on Gram stain and its propensity to form a leather-like pseudomembrane. The organism is a gram-positive, unencapsulated, nonmotile, nonsporulating, aerobic rod. On Gram stain the bacilli display a characteristic “Chinese characters” arrangement and may even appear gram variable, due to thinning of the cell wall leading to decolorization of stain. Metachromatic granules containing polyphosphate are found in the polar regions of the bacterium and appear bluish-purple to red when stained with methylene blue. Loeffler developed his eponymous culture medium containing dextrose, horse serum, and beef heart infusion for isolation of C. diphtheriae . Due to the tendency for overgrowth of commensals on Loeffler medium, more selective media containing telluric acid (e.g., Mueller-Miller, Tinsdale) were later developed. On Tinsdale medium potassium tellurite inhibits gram-negative organisms and most upper respiratory flora; C. diphtheriae and C. ulcerans, another medically important Corynebacterium sp., appear as grayish-black colonies with a surrounding brown halo. Urease testing can distinguish between the two organisms, as C. diphtheriae is urease negative, whereas C. ulcerans is urease positive.

C. diphtheriae is subdivided into four biovars (biotypes): belfanti , gravis , intermedius , and mitis . An individual may harbor more than one biovar concurrently. Although clinically similar, biovars may be distinguished on the basis of colony morphology, hemolysis, biochemical reactions (e.g., API Coryne:; bioMérieux, La Balme Les Grottes, France), and, more recently, 16S ribosomal RNA sequencing. Ribotyping and pulsed-field gel electrophoresis have been used to type C. diphtheriae strains during outbreaks and for surveillance purposes; these methods have been supplanted by multilocus sequence typing due to its improved reproducibility.

In the late 19th century Loeffler discovered the presence of avirulent, nontoxigenic strains of C. diphtheriae in healthy carriers and noted that these strains are morphologically indistinguishable from toxigenic strains. Corynebacteriophages carry tox , the gene for exotoxin production, and convert strains of C. diphtheriae from nontoxigenic to toxigenic via a lysogenic cycle. Expression of tox is regulated by DtxR, an iron-activated repressor that is derepressed in low iron states. The potent diphtheria toxin is composed of three domains: a cell receptor binding domain, a transmembrane domain, and a catalytic N-terminal adenosine diphosphate (ADP)-ribosyltransferase domain. C. pseudotuberculosis and C. ulcerans also elaborate the diphtheria toxin; both species may be differentiated from C. diphtheriae by means of biochemical testing. Laboratory methods for detection of toxin include polymerase chain reaction (PCR), enzyme immunoassay (EIA), and immunochromatography.

Epidemiology

The role of the asymptomatic carrier as a reservoir for infection was first recognized in the late 1800s. Humans were originally thought to be the only reservoir, but C. diphtheriae has now been isolated from horses, cattle, and domestic cats. Although diphtheria has been classically understood as an upper respiratory disease acquired via inhalation of infected droplets, cutaneous lesions may provide a more efficient means of transmission, resulting in both respiratory and cutaneous diphtheria. Cutaneous lesions are probably the major reservoir for infection in resource-limited environments, serving as a source of natural immunity in these settings. Transmission of infection between skin lesions and between the respiratory tract and skin lesions (bidirectional) has been documented. Cases of reinfection, probably due to transmission via contaminated fomites, have also been described.

Young children suffered disproportionately from diphtheria during the prevaccine era; up to 70% of cases occurred in children younger than 15 years. In the New England epidemic of 1735–40, 40% of all children below 10 years of age died in a single year in Hampton Falls, New Hampshire. In 1881 greater than 1% of deaths in children younger than 10 years in New York were caused by diphtheria. Children ages 5 to 14 experienced high attack rates of up to 683 per 100,000 population from 1921–24 in Baltimore; the case fatality rate in that city ranged from 5% to 8%. Epidemics tended to peak in the fall at the beginning of the school year and affected those at the age of school entry. In England in the late 1930s diphtheria was the second most common cause of mortality in children, after pneumonia, causing 32 deaths per 100,000 population in those younger than 15 years.

Although introduction of antitoxin treatment in the early 1900s had a beneficial effect on mortality, incidence rates of disease remained elevated. In the immediate prevaccine era the incidence rate in the United States was 237 per 100,000 population per year. Incidence rates decreased worldwide from the 1930s onward, coincident with the introduction of routine toxoid vaccination in children. During World War II, however, outbreaks occurred in European countries that had experienced decreasing rates of infection in the previous years. German soldiers infected with C. diphtheriae biovar gravis reintroduced the disease in several occupied territories. Unlike earlier epidemics in which younger children were disproportionately affected, a relatively high percentage of older children and adults contracted disease, likely due to wartime displacement of large populations and improved living conditions over the preceding decades, leading to decreased exposure of younger children to infection. After World War II incidence rates in industrialized countries declined precipitously as childhood immunization programs were strengthened. By 1965 the attack rate in the United States had declined to 0.08 per 100,000 population. Isolated outbreaks did occur among minority and indigent groups; unimmunized individuals constituted the majority of these cases. One such example was the 1970s outbreak in the Skid Road neighborhood of Seattle, which was concentrated in Native American men with high rates of alcohol dependence and was characterized by a predominance of cutaneous diphtheria.

A major epidemic of diphtheria occurred in the former Union of Soviet Socialist Republics (USSR) in the early 1990s. The incidence rate in the USSR was as low as 0.04 per 100,000 population in the mid 1970s. By the 1980s, however, a combination of factors contributed to the 1990s epidemic: decreased rates of immunization in children due to vaccine shortages and antivaccine propaganda, vaccination of children with the reduced-dose adult formulation of diphtheria toxoid, waning adult immunity, and transmission of infection from unvaccinated members of the military returning from Afghanistan. After the breakup of the USSR in 1991 the health care system collapsed, and the incidence of diphtheria spiked to as much as 50,000 cases in 1995. The predominant strain in Russia, Ukraine, Belarus, the Baltic republics, and northern Kazakhstan was gravis , whereas mitis was predominant in southern Kazakhstan, Tajikistan, Uzbekistan, and Kyrgyzstan. In Russia, where the majority of cases occurred, incidence rates in adolescents and adults were more than 20 per 100,000 population. Mobilization of an intensive vaccination campaigns in coordination with World Health Organization (WHO) and the United Nations Children's Fund brought about an end to the epidemic in the late 1990s.

Global incidence rates of diphtheria have declined steadily as a result of widespread implementation of the WHO's Expanded Programme on Immunization (EPI). From 1980 to 2000 there was a greater than 90% decrease in global incidence rates. Vaccination has prevented an estimated 40 million cases of diphtheria. Despite these impressive figures, outbreaks continue to occur in resource-limited settings, highlighting gaps in vaccination coverage. India is currently the country with the greatest number of cases worldwide. In northern Kerala there were 533 cases in 2016, the majority of which occurred in individuals older than 10 years.

In the prevaccine era placental passage of maternal antibodies afforded passive immunity to neonates. As passive immunity waned at approximately 6 months of age, active immunity developed naturally by means of disease or through clinically silent infection; the majority of children had developed immunity to diphtheria by age 15 years. After the introduction of childhood vaccination, however, the burden of disease shifted to susceptible older children and adults. Examples were clearly seen in Jordan and Sudan, where younger children were mostly affected during epidemics before the implementation of effective childhood vaccination programs, but adults and older children were disproportionately affected during outbreaks occurring after the institution of these programs. Waning immunity to diphtheria underpins the rationale for booster vaccination of older children and adults.

With the decline of disease due to toxigenic C. diphtheriae , the pathogenicity of nontoxigenic strains is becoming increasingly recognized. The tox gene is not necessary for the life cycle or metabolism of C. diphtheriae ; immunized individuals are more likely to harbor nontoxigenic strains, which are consequently more likely to circulate in the community. Nontoxigenic C. diphtheriae has been associated with blood stream infections and endocarditis in individuals with comorbid conditions, such as alcoholism, dental disease, and intravenous (IV) drug use. The majority of nontoxigenic strains do not carry the gene tox ; however, nontoxigenic strains that harbor the gene without expressing the protein exotoxin have recently been detected.

Pathogenesis

Although the diphtheria exotoxin is responsible for much of the disease manifestations, other virulence factors also play a role in pathogenesis. Neuraminidase and trans -sialidase facilitate binding to the host cell and allow the organism to scavenge sialic acid for nutrition and metabolism. SpaA, SpaD, and SpaH pili mediate adherence to pharyngeal, laryngeal, and respiratory epithelial cells. DIP0733 and DIP2093 adhesins facilitate binding to the extracellular matrix, adhesion and invasion of epithelial cells, biofilm formation, and survival in macrophages. Both toxigenic and nontoxigenic strains are capable of converting fibrinogen to fibrin, a finding that may explain the rare occurrence of pseudomembranes in the setting of nontoxigenic C. diphtheriae infection.

The diphtheria exotoxin, containing 535 amino acids, is composed of two covalently bonded fragments, fragment A and fragment B. Fragment B contains the receptor binding and transmembrane domains, which allow cell surface binding and transport into the cytosol, respectively. Fragment A, containing the catalytic domain, facilitates ADP-ribosylation and consequent irreversible inhibition of elongation factor 2, a eukaryotic protein necessary for the coordinated movement of transfer RNA, messenger RNA, and the ribosome during the elongation cycle of protein synthesis. One molecule of the exotoxin is sufficient to kill a cell; the lethal dose in humans may be as little as 100 ng/kg.

C. diphtheriae infection leads to mucosal edema with subsequent necrosis and ulceration. A fibrinous exudate overlying the desquamated mucosae forms the adherent pseudomembrane. On histopathology the pseudomembrane consists of fibrin and denuded epithelial cells with an associated neutrophilic infiltrate and clusters of C. diphtheriae organisms ( Fig. 204.2 ). The pseudomembrane may extend to form a cast of the upper airways. Forcible removal of the pseudomembrane may cause bleeding; dislodgement may lead to aspiration and asphyxiation. Edematous cervical, parabronchial and mediastinal lymph nodes often hemorrhage or necrose.

Clinical Manifestations

C. diphtheriae usually causes upper respiratory tract or cutaneous disease. Cardiac and neurologic complications are the most frequent toxin-mediated manifestations. Both toxigenic and nontoxigenic strains may rarely disseminate to distant sites.