Clinical presentation

The time between C. diphtheriae infection and symptom development may range from 1 to 10 days (typically 2–5 days)5. People infected with C. diphtheriae, even if they are asymptomatic, are infective for up to 4 weeks150. Transmission of the disease occurs through direct contact with skin lesions or direct contact or inhalation of airborne oral or respiratory discharges. Infection can also be dispersed by contact with contaminated objects. Lack of vaccination, a compromised immune system, a history of atopic dermatitis (eczema), congested and/or unsanitary living conditions and travel to areas where the disease is endemic are the pragmatic risk factors for diphtheria151. Early diagnosis of diphtheria is based on the typical clinical symptoms (Fig. 4) as this helps to initiate the presumptive treatment quickly. Clinical diagnosis of diphtheria usually relies on the presence of pseudo-membranous pharyngitis. The other typical symptoms of acute diphtheria include enlarged lymph node glands in the neck (bull neck), myocarditis and inflammation of the nerves.

Fig. 4: Clinical presentations of diphtheria. a | Characteristic bull neck caused by enlarged lymph nodes. b | Thick pseudomembrane in the posterior pharynx. The pseudomembrane is a layer of bacteria and debris from necrosis of the surrounding tissues due to diphtheria toxin. c | Cutaneous lesion caused by Corynebacterium diphtheriae. Full size image

Diphtheria is confirmed by isolation of Corynebacterium spp. followed by toxigenicity testing. If the cultures of samples from a patient with suspected diphtheria are negative because antibiotic therapy had been started before the samples were collected, a presumptive diagnosis of diphtheria can be made if C. diphtheriae is isolated from close contacts of the patient, the patient has a minimal anti-DT antibody titre (<0.1 IU) in serum samples obtained before the administration of DAT (although this parameter is not a key diagnostic indicator) and a direct PCR test of clinical swab samples is positive for diphtheria tox genes. Differential diagnoses include acute epiglottitis, oral syphilis, viral pharyngitis, Borellia vincentii infection (also known as Vincent angina or trench mouth), oral candidiasis, infectious mononucleosis and streptococcal pharyngitis. Concurrent diphtheria and infectious mononucleosis with exudative pharyngitis are difficult to distinguish, so accurate diagnosis is essential152, either by culture test for Corynebacterium spp. from throat and/or nasal swabs or by reliable molecular methods. Indirect laryngoscopy is recommended in cases with membrane formation. In patients with pharyngitis and a pharyngeal membrane, diphtheria should be suspected. DT penetrates into Schwann cells and inhibits the synthesis of myelin proteolipid and basic proteins153, leading to diphtheritic polyneuropathy154,155. The period between diphtheria and the development of diphtheritic polyneuropathy varies from 10 days to 3 months155. The initial symptoms of diphtheritic polyneuropathy are paresis of the soft palate and paraesthesia in the distal parts of the extremities, including respiratory muscle pareses. Common symptoms are hyporeflexia or areflexia and hypotonia, sensory symptoms (paraesthesia, hypaesthesia and hyperaesthesia), facial palsy, nerve palsy, diaphragmatic palsy and loss of vasomotor tone. In a typical diphtheritic polyneuropathy, dysfunction of the cranial nerves might present faster than muscular dystrophy (also known as the cranial stage)156. In most cases, diphtheritic polyneuropathy has to be differentiated from Guillain–Barré syndrome, which is common in children. The typical features of diphtheritic polyneuropathy are a high prevalence of bulbar palsy, gradual development of the neuropathy (>4 weeks) and synchronized participation of other organ systems157.

Microbiological diagnosis

The clinical diagnosis of diphtheria must be confirmed by the isolation and identification of one of the three causative Corynebacterium spp. (as diphtheria is a notifiable disease) (Fig. 5). Members of the genus Corynebacterium are Gram-positive, non-motile rods, often with a clubbed end, are aerobic or facultatively anaerobic and convert carbohydrates to lactic acid. Of the >100 species in this genus, only a few, C. diphtheriae, C. pseudotuberculosis and C. ulcerans, are toxigenic and clinically important158. Even though all biotypes of toxigenic C. diphtheriae are virulent, in some findings, strains belonging to the gravis biotype were found to produce larger amounts of DT than strains of the mitis biotype159.

Fig. 5: Diagnostic algorithm. Flow chart showing the methods used in the isolation and identification of Corynebacterium spp. MALDI-TOF, matrix-assisted laser desorption/ionization time of flight mass spectrometry. Full size image

Culture and species identification

Bacteria culture of clinical samples is the gold standard for the isolation and identification of Corynebacterium spp. Swab samples should be collected from the suspected sites of infection, such as the nasopharyngeal cavity, throat, wounds or skin lesions. If a pseudomembrane is present, swabs should be taken from beneath the membrane, or a piece of the membrane can be collected instead. It is essential to collect the samples regardless of whether antibiotic therapy has been started. If delays in the processing of the clinical samples are expected, specimens should be maintained in Amies transportation medium and can be supplemented with charcoal to preserve the viability of the bacteria.

Sheep or horse blood agar or a medium containing potassium tellurite, such as Hoyle's tellurite agar, is used for primary isolation. This medium is not highly selective for C. diphtheriae, as the other bacterial species may also grow. Typical C. diphtheriae colonies are grey to black, whereas Streptococcus spp. grow as tiny black or brownish colonies. On blood agar, corynebacteria grow as convex, greyish, translucent colonies with a granular appearance, mostly with opaque centres. C. ulcerans and C. pseudotuberculosis colonies may exhibit β-haemolysis. Bacteria grown in Löffler’s medium, which contains coagulated serum with phosphate, accumulate volutin granules (a form of intracellular polyphosphate storage). When stained with polychrome methylene blue (Albert stain), the granules appear violet (metachromatic stain), whereas the rest of the bacterial cell appears blue. Colonies of Corynebacterium spp. on tellurite medium appear dark grey or black owing to the intracellular reduction of tellurite to tellurium after 48 h of growth at 37 °C. Using smears made from corynebacterial colonies grown in tellurite medium for immunofluorescence-based toxigenicity tests is not recommended, owing to morphological changes caused by potassium tellurite160.

Colonies isolated from primary culture plates are identified by enzymatic tests and tested for toxin production (see following section). Enzymatic tests include nitrate, urease, catalase, cystinase and pyrazinamidase tests (to detect the presence of nitrate reductase and the other enzymes), which permit the presumptive identification of the potentially toxigenic Corynebacterium spp. within 4 h (ref.161). Kits including combinations of such enzyme assays are commercially available.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) can be used to identify the specific Corynebacterium sp. directly from a colony isolated from the blood agar plates in about 30 min. The accuracy of the MALDI-TOF system for the identification of C. diphtheriae, C. pseudotuberculosis and C. ulcerans is very high (97–100%)162. After MALDI-TOF confirmation of the isolation of a potentially toxigenic colony, bacterial colonies may be used for PCR to identify tox and/or for toxin assays. However, in diphtheria-endemic countries, conventional biochemical tests are still widely used.

Toxigenicity tests

The Elek test works on the principle of antigen and antibody immunoprecipitation. In this assay, a known toxigenic strain (positive control), a non-toxigenic strain (negative control) and the sample strains are inoculated onto Elek agar medium with a paper strip containing DAT (500 IU/ml) placed onto the agar surface163. In the modified Elek test, the test and control strains are inoculated with a disc containing DAT (10 IU/disc) placed in the centre164. After 24–48 h at 37 °C, a clear precipitin line develops at the junction where the toxin produced by the strain and the antibody from the strip or disc meet (Fig. 6). In vitro Vero cell assays and an in vivo rabbit skin test have also been used in the detection or neutralization of DT, but these tests are not recommended for routine use165.

Fig. 6: Elek tests for in vitro detection of diphtheria toxin. The Elek test, also known as the immunodiffusion (immunoprecipitation) technique, is an in vitro virulence test for the detection of toxigenic strains of Corynebacterium spp. a | In a conventional Elek test, the bacterial colonies grown from culture of clinical samples are spread on a dish perpendicular to a filter paper strip containing diphtheria antitoxin (DAT). If the bacteria produce diphtheria toxin (DT), diffused DAT from the filter paper develops a precipitin line at the zone of equivalence. Appearance of the precipitin line indicates that the tested sample (test) produced DT that reacted with the DAT. b | Modified Elek test of the same samples shown in panel a, in which the bacteria are grown around a DAT disc (10 IU/disc) that was placed at the centre of the plate. For clear results, the optimum distance between the inoculum and the DAT disc should be 9 mm. National Collection of Type Cultures (NCTC) 3984 (weak positive, ±) and NCTC 10648 (strong positive, ++) are the positive control strains, and NCTC 10356 (negative, –) is the negative control. Full size image

PCR has been considered a sensitive and specific method for the identification of a specific Corynebacterium sp. or to test the clinical samples from suspected diphtheria cases for the presence of tox. Although the 16S-ribosomal RNA gene-based identification is widely in use, design of species-specific PCR primers can be difficult, especially when the homologous genes have high similarity. Compared with the 16S-ribosomal RNA gene, sequencing the gene encoding the RNA polymerase β-subunit (rpoB) was found to be useful in identifying the Corynebacterium sp.166. The rpoB sequence has a higher degree of polymorphism than the 16S rDNA sequence166. A real-time PCR assay testing for a combination of tox and rpoB genes was reported for the rapid identification of toxigenic and non-toxigenic strains as well as to differentiate C. diphtheriae, C. ulcerans and C. pseudotuberculosis167,168. Of note, whereas a tox-negative result is final and additional toxigenicity assays are not required, the presence of tox does not specify the expression of DT. Hence, the Elek test must be performed on all tox-positive isolates from patients with suspected diphtheria169; however, patients with tox-positive results can be considered for further preventive treatment action, without waiting for the Elek test results. If the clinical laboratories are not equipped for further biochemical or toxigenicity tests, the pure cultures should be submitted to the regional referral centres, in slanted Dorset egg medium or other common agars or on plates at ambient temperature.

The co-agglutination test, passive haemagglutination test, reversed passive latex agglutination assay and bead-based serology assays detecting the expression of glutathione S-transferase fusion proteins are useful for the detection of DT in serum samples and/or pure cultures of toxigenic C. diphtheriae170,171,172,173. Of note, serum samples must always be collected prior to the administration of DAT. However, these techniques have been replaced in many laboratories by a rapid enzyme immunoassay that can detect DT directly from the suspected colonies of corynebacteria174. In this enzyme immunoassay, equine polyclonal antitoxin is the capture antibody and an alkaline phosphatase-labelled monoclonal antibody is the detection antibody. The assay is rapid (within 3 h), sensitive (0.1 ng DT/ml) and specific for the detection of fragment A of the DT molecule174. Several other diagnostic methods were also developed for serological surveillance studies, including enzyme-linked immunosorbent assay-based detection and quantification of anti-DT antibodies175, counterimmunoelectrophoresis176,177,178 and immunofluorescence assay179. Serum anti-DT antibody levels <0.01 IU/ml indicate that an individual is susceptible to diphtheria, levels between 0.01 and 0.09 IU/ml indicate the recommended minimum protective level (basic immunity) and levels ≥0.10 IU/ml are above the protective threshold observed in individuals who have been vaccinated5.

Histology and imaging

Histopathological imaging analysis of the sites of infection might be required for disease detection, diagnosis and prognosis prediction. This analysis is also important to understand the causal reasons for a specific diagnosis and to provide a more comprehensive view of the disease to complement other clinical investigations. In the case of diphtheria, histological examinations are not performed routinely. Pseudomembrane formation can also be caused by Streptococcus spp. and Staphylococcus spp. infections, and, therefore, histology might be required to identify the causative pathogen. In a typical disease state, haematoxylin and eosin stain of the pharyngeal pseudomembrane might display necrosis of the epithelium with fibrinosuppurative exudate. Gram-positive C. diphtheriae cells can also be detected after Brown–Hopps staining114.

Myocardium and peripheral nerves are the most susceptible parts during the acute stage of diphtheria180. In the histology, the affected myocardium might show extensive areas of hyaline degeneration and necrosis with inflammation in the interstitial spaces. In these areas, infiltrates of mononuclear cells with eosinophilic cytoplasm can be seen. Fluorescent staining of tissue sections with anti-DT antibody demonstrates the presence of DT within myocardial fibres, but not in the areas of advanced necrosis. Ultrastructural variations within the affected myofibres (such as enlarged mitochondria with excess lipid droplets, loss of matrix and disorganization of the cristae, with or without dense osmophilic material) can be identified by electron microscopy114. The damaged myofibrils are seen as dislocated scattered foci, with empty, structureless, pale spaces. Clumped chromatin granules can be found near the nuclear membranes120. Echocardiography is advantageous in assessing the ejection fraction and identifying indications of ventricular systolic dysfunction, aortic incompetence and acute mitral valve regurgitation122,181. Electrocardiography is important to monitor patients with diphtheria-associated myocarditis and detect alterations in the ST-segment (elevation or depression) and T wave, which could be a sign of myocardial infarction, sinus tachycardia, multiple atrial ectopic beats, or prolonged PR or QT intervals, which could lead to arrhythmias, among others182,183. CT scans may reveal aortic annular and interventricular septal abscesses and thickening of the pericardium184,185. On X-ray examination, patients with diphtheria may present cardiomegaly and bronchopneumonia with thickened pulmonary markings and/or inflammatory infiltrates186.

Diphtheritic polyneuropathy is due to toxic myelopathy with paranodal demyelination, especially in large myelinated neural fibres. The affected nerves display degeneration of myelin sheaths and axon cylinders. Ranvier nodes appear widened during the early stages of infection, as the paranodal myelin is affected first, followed by demyelination at the later stages187. In the most severe cases, axonal degeneration is common, caused by compression made by the folded myelin and invagination of Schwann cell cytoplasm188. Muscle biopsies during the acute stages of diphtheritic polyneuropathy show scattered, angulated fibres, predominately of type 2B fibres or cores of type 1 fibres. Histology of intramuscular vessels reveals vasculitis (inflammation of blood vessels) with lymphoid cells189. Multiple emboli and diphtheritic neuropathy can be detected using MRI of the brain157, and CT scans can reveal gyriform enhancement of cerebral lesions190.

Biomarkers

In acute diphtheria cases, cardiac complications are common, which develop in 10–25% of patients191 and can result in fatality. In patients with diphtheritic myocarditis, the total leukocyte count and serum glutamic oxalo-acetic transaminase levels are useful biomarkers of prognosis and fatal outcome, as high counts or levels are found to be associated with increased risk191,192. Creatine phosphokinase in muscle/brain (CPK-MB) and cardiac troponins levels might be useful outcome predictors, as they are strongly associated with cardiac mortality183. Increased systemic levels of IL-6 and tumour necrosis factor (TNF) were noticed during diphtheritic endocarditis91.

Vaccination

Diphtheria control is mainly based on immunization of the population through vaccination and prevention of the disease in close contacts of patients with confirmed diphtheria by prompt initiation of antibiotic treatment (chemoprophylaxis) followed by vaccination (the three-dose protocol for individuals who had never received the vaccine or a single booster dose for previously immunized contacts). DTP (also known as DPT in some countries) is a combination vaccine against diphtheria, tetanus and pertussis. The vaccine components include diphtheria and tetanus toxoids (inactivated toxins adsorbed onto an adjuvant (aluminium hydroxide or aluminium phosphate)) and killed whole cells of Bordetella pertussis. In the DTaP vaccine, the pertussis component is acellular. DTP is also combined with other vaccine antigens, such as hepatitis B virus surface antigen and Haemophilus influenzae type b (Hib) conjugates in pentavalent vaccines, and also with inactivated polio vaccine in hexavalent vaccines. A cycle of three doses (via intramuscular injection) of this vaccine is recommended (DTP-3): the first dose should be administered within 6 weeks of age, followed by the other doses at least 4 weeks apart. The third dose has to be completed by 6 months of age. To reduce the number of injections, the DTP vaccine is administered along with other vaccines, such as the Hib vaccine and the hepatitis B vaccine (HepB), scheduled at the same time. Several WHO prequalified vaccines are available in many combinations. Vaccines containing diphtheria toxoid should be stored under refrigerated conditions (2–8 °C) and frozen vaccine should not be used193. In 2012, the CDC (Centers for Disease Control and Prevention) recommended DTP vaccination for individuals of ≥65 years of age194. The vaccine adverse event reporting system did not find any unfavourable events against tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine in this population195. DTP is safe for pregnant women, and especially useful in protecting the young infant against tetanus and pertussis. DTP vaccination during pregnancy also increases immunity and the duration of protection in mothers who had not received the recommended booster doses5.

Based on the WHO and UNICEF estimates, global DTP-3 coverage increased during 2007–2010 (from 79 to 84%) and remained stable from 2010 to 2017 (84–85%)196. The 2018 global DTP-3 coverage was ~86%, and >80% of the countries reached >80% coverage (Fig. 2). This level of coverage seems adequate to maintain the herd immunity, which for diphtheria has a coverage threshold of 85% (ref.197), and lower the risk of outbreaks198; however, it is still not fully protective. Generally, there is a long delay in DTP vaccination, especially among newborn babies in low-income and middle-income countries199. Societal and cultural issues, poverty, false perceptions about the safety and credibility of vaccines and difficulty for parents to comply with the vaccination schedule are possible reasons for this delay200. Completion of vaccination has been significantly correlated with knowledge of mothers on immunization. Parents’ forgetfulness about their child’s immunization status, many siblings in the family, mother’s unemployment and premature birth were significantly associated with a delay in receiving the vaccine15. Children whose mothers had poor school-level education are most likely not to receive the DTP vaccination or to discontinue it than children whose mothers had many years of schooling201,202. With support from the Global Alliance for Vaccines and Immunization, a combined DTwP-HepB-Hib (pentavalent) vaccine was introduced in Kenya in 2001, and this vaccine has now been used by 72 other countries203, targeting about 80 million children for immunization204. Anti-DT antibody levels decrease with ageing owing to changes within the immune system and/or insufficient vaccination earlier in life205. Hence, booster vaccinations during adulthood are recommended to maintain the herd immunity. Reduced antigen-content tetanus, diphtheria and acellular pertussis vaccine is recommended in many countries for boosting immunity in adolescents and adults206. A factor that correlates well with the low rate of serological immunity among adults is the time delay in vaccination during their childhood, that is, a delay up to 3 years207. Several studies on susceptibility to diphtheria in adults also showed that deficiency of seroprotection was more common in women than in men207,208,209,210. This difference might be due to gender-specific immune responses subsequent to vaccination210. Diphtheria vaccination prevents toxin-related symptoms, but does not prevent colonization of invasive NTCD and other non-toxigenic Corynebacterium spp. in the host that could cause substantial health risks to unvaccinated individuals and does not provide protection against asymptomatic carriage of C. diphtheriae211.