Case Report: Bloodstream Infection with Toxigenic Corynebacterium diphtheriae and Gram-Negative Sepsis in a Child with Burns

ABSTRACT.

Corynebacterium (C.) diphtheriae is the agent for a contagious infection, diphtheria. It may manifest as pharyngitis with pseudomembrane formation and cervical lymphadenopathy, cutaneous infection, or as an asymptomatic carrier. Corynebacterium (C.) diphtheriae is not an invasive organism and it remains in the superficial layers of skin lesions and respiratory mucosa. Systemic complications, such as bacteremia, are rare. We report a case of toxigenic C. diphtheriae detected from blood culture of a 1-year-old male patient with burns, who succumbed to the infection after 8 days of stay in the hospital. Patient did not have specific clinical features suggestive of diphtheria. Initial identification of C. diphtheriae was done based on culture, Albert stain findings, biochemical tests and subsequently toxigenicity testing was done by polymerase chain reaction. Although diphtheria vaccination in infancy is universally recommended since the creation of the Expanded Program on Immunization in the 1970s, there have been reports of toxigenic strains of C. diphtheriae in a considerable number of cases. Rapid and accurate identification of C. diphtheriae infection is crucial to prevent mortality. Continued surveillance for diphtheria is needed to reduce transmission and mortality rates.

INTRODUCTION

Corynebacteria are Gram-positive, nonmotile, non-capsulated, non-sporing rods, often club-shaped with metachromatic (volutin) granules. The most important human pathogen is Corynebacterium (C.) diphtheriae, which produces a potent exotoxin causing diphtheria, which is usually a toxemia rather than bacteremia. Respiratory diphtheria is the most common presentation. Bacteremia caused by C. diphtheriae is rare, although invasive infection such as endocarditis, septic arthritis, and skin and soft tissue infections are increasingly recognized to be caused by non-toxigenic C. diphtheriae. The possible route for penetration into bloodstream is invasion from the throat or through skin lesions.^1,2 Nonlytic infection by bacteriophages containing a gene sequence that encodes the diphtheria toxin (DT) allows C. diphtheriae strains to manufacture toxin. The tox gene becomes a component of the bacterial gene operon and is controlled by the dtxR repressor gene, which binds to the tox gene and suppresses its expression in the presence of iron. There is increase in toxin production and absorption in larger membranes. Hence, complication risk is related to the severity of local disease.²

Laboratory identification of C. diphtheriae used to be mainly by conventional methods such as demonstration of metachromatic granules on Albert stain, growth characteristics on potassium tellurite agar (PTA), and biochemical results. With the advent of automated identification systems like matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has provided rapid and accurate identification of C. diphtheriae. Accuracy of the MALDI-TOF MS system for identification of C. diphtheriae, C. ulcerans, and C. pseudotuberculosis is very high (97–100%).³ However, detection of toxin using Elek’s gel precipitation test or tox gene by polymerase chain reaction (PCR) is important to differentiate toxigenic from non-toxigenic isolates for patient management and surveillance purposes.

Superadded bacterial infection is common in burns wound due to loss of protective covering and C. diphtheriae has been very rarely reported to colonize and cause disease in burns patients.⁴ Here, we present a case of bloodstream infection caused by toxigenic C. diphtheriae along with Gram-negative sepsis in a patient with burns.

CASE REPORT

A 1-year-old male child presented with scald burns over his trunk and back, which was sustained at home due to accidental fall into a hot water-bath, 5 hours before coming to hospital. Burns were classified as second degree with 33% involvement. On admission, the child was hemodynamically stable, conscious, and afebrile. After 24 hours in casualty, he was shifted to Pediatric Intensive Care Unit (PICU) for monitoring. On the second day in PICU, he developed high-grade fever and was found to be hypotensive and tachypneic. On that day, Serum procalcitonin was 84.72 ng/mL and total leukocyte count (TLC) was 29,300/μL. Blood culture (BC) was sent to microbiology laboratory on second day of stay in PICU in view of suspected septic shock. Subsequently, he was started empirically on meropenem and cloxacillin.

Paired BCs collected in BacT/ALERT pediatric BC bottles was loaded in the BacT/Alert Virtuo system (BioMérieux, France) as soon as it was received in the microbiology laboratory and incubated aerobically at 37°C. Both the BC bottles flagged positive after 10 hours of incubation. Subculture was performed on 5% sheep blood agar (SBA), MacConkey agar, and chocolate agar (CA) and aerobically incubated at 37°C. Gram stain performed directly from BC bottle revealed Gram-negative bacilli and a few short, Gram-positive bacilli. After 12 to 14 hours of incubation, two types of colony morphology were seen on SBA and CA.

The big, moist and nonhemolytic colony (colony-1) on SBA was Gram-negative bacilli on Gram stain. The small, gray and beta-hemolytic colony (colony-2) on SBA was club-shaped Gram-positive bacilli mostly in cuneiform arrangement. Albert’s stain from colony showed green bacilli with bluish-black metachromatic granules at poles (Figure 1A). Automated identification was performed from colony growth using MALDI-TOF MS (VITEK2, BioMérieux). Colony-1 was identified as Klebsiella pneumoniae and colony-2 as C. diphtheriae with a confidence interval of 99.9%. Subculture of C. diphtheriae was performed on SBA and PTA. Colonies on SBA (after 12–14 hours) were small circular, hemolytic, grayish white, and low-convex (Figure 1B). Growth on PTA showed circular, 1 to 2 mm, black-colored colonies (Figure 1C), and urea was not hydrolyzed. The isolate fermented glucose, maltose, and starch, but not sucrose on Hiss’s serum sugar (Figure 1D). The isolate was identified as “gravis biotype.” Exudate culture from burn wound yielded Acinetobacter baumannii, but C. diphtheriae was not isolated.

Figure 1.

(A) Sheep blood agar showing small circular, hemolytic, grayish white colonies. (B) Albert’s stain showing green-colored bacilli with bluish-black metachromatic granules at ends of bacilli. (C) Potassium tellurite agar showing black colonies. (D) Hiss’s serum sugar showing fermentation of glucose, maltose, and starch, but not sucrose. This figure appears in color at www.ajtmh.org.

Conventional, in-house multiplex PCR was performed from culture isolate for detection of tox gene (tox A and tox B) and dtxR gene to establish the toxigenic potential of the isolate. The PCR has been standardized earlier using reference strains C. diphtheriae NCTC 10648 (strong tox positive), NCTC 3984 (weakly tox positive), and NCTC 10356 (tox negative) and routinely performed in the laboratory. Both the tox and dtxR gene were found to be positive. Although important, in this patient Elek immunodiffusion test was not performed nor was it sent to a regional reference laboratory for the same.

Antimicrobial susceptibility testing (AST) was performed to determine the minimum inhibitory concentration by VITEK-2 automated AST system using AST-P628 card (for Gram-positive cocci). Antimicrobial susceptibility testing results were interpreted using clinical breakpoints available in Clinical and Laboratory Standards Institute (CLSI) M-45 document.⁵ The isolate was susceptible to benzylpenicillin, tetracycline, vancomycin, erythromycin, gentamicin, clindamycin, linezolid, rifampicin and resistant to ciprofloxacin, and cotrimoxazole. Klebsiella pneumoniae was susceptible to cefoperazone–sulbactam, piperacillin–tazobactam, meropenem, and amikacin, but resistant to third-generation cephalosporins, ciprofloxacin, and gentamicin.

During the stay in the hospital, in view of Gram-negative sepsis due to K. pneumoniae and C. diphtheriae, empirical meropenem, and cloxacillin was stopped after 2 days and cefoperazone–sulbactam along with erythromycin was started. Unfortunately, anti-diphtheria antitoxin/serum was not administered to the patient, as there was no finding suggestive of upper respiratory tract involvement and the patient was fully vaccinated for age. Throat swab was not obtained due to lack of signs indicating respiratory involvement. Corynebacterium (C.) diphtheriae was also not isolated from wound culture of the burn site. No repeat BC samples were sent.

The child developed seizure-like episodes, acute renal failure presenting as hyperkalemia & oligoanuria managed on peritoneal dialysis, and subsequently sepsis induced pancytopenia (Hb—7.7 g/dL, platelet count of 10,000/μL, TLC—3,100/μL). Cerebrospinal fluid analysis was not done to rule out meningitis as patient was hemodynamically unstable. During the hospital stay, echocardiogram was done twice with no abnormal finding. Features suggestive of complications of diphtheria, such as myocarditis or neuropathy were absent. Over the period of PICU stay, patient’s condition worsened and succumbed to death after 8 days of hospital stay. Contact tracing of family members was not done and chemoprophylaxis was not given to hospital or household contacts.

DISCUSSION

Corynebacterium (C.) diphtheriae is a Gram-positive bacillus causing respiratory as well as cutaneous diphtheria. The primary route of spread is via respiratory droplets or penetration through skin breach. Skin carriage of C. diphtheriae can act as a silent reservoir for organism and person-to-person spread has been found to be more efficient than from respiratory tract.^6,7 Dissemination through the hematogenous route is rare.¹

Most cases of bacteremia described in the literature are due to non-toxigenic strains of C. diphtheriae.^3,6,8,9 A few cases of septicemia and subacute infective endocarditis due to toxigenic C. diphtheriae has been reported.^10,11 Endocarditis has been associated with cases having history of underlying structural heart disease,¹² however risk factors for other invasive infections are not clearly known. Many patients give a history of intravenous drug abuse, alcohol addiction, or belong to low socioeconomic group of people.⁶ Breach in skin integrity may be an important risk factor for colonization and subsequent infection with C. diphtheriae in patients with burns. The case fatality in invasive infections due to C. diphtheriae is 36% to 47%, mostly associated with non-toxigenic strains.¹ Poor survival rate (∼27%) has been reported in few studies among pediatric age group with blood stream infections (BSIs) caused by toxigenic C. diphtheriae, similar to our case.¹²

There are rare reports of BSIs due to toxigenic C. diphtheriae in pediatric patients with history of previous immunization against DT without clinical evidence of nasopharyngeal or cutaneous diphtheria, often associated with endocarditis.¹² Our patient had received three diphtheria doses in infancy, as documented in his immunization card, despite which he got infected with toxigenic C. diphtheriae. Immunized patients are still prone to C. diphtheriae bacteremia and endocarditis even without the presence of typical toxin-mediated necrotizing lesions because the toxigenic and invasive properties are independent of each other.¹²

The child also had concomitant Gram-negative sepsis that contributed to the fatal outcome. Concomitant Gram-negative sepsis along with lack of evidence of upper respiratory tract symptoms and vaccinated-for-age status had shifted the focus away from considering toxigenic C. diphtheriae as a significant and potential pathogen in our case, leading to non-administration of antidiphtheritic serum, as the toxigenic assay take time to conclude too.¹² In contrast to the West, where non-toxigenic strains predominate due to better immunization coverage, India accounts for over half of all global recorded cases of diphtheria, with toxigenic strains accounting for the majority of cases.¹³ Any BC isolate should be regarded as a possible pathogen and should be evaluated carefully based on the clinical and other investigational parameters. In the resource-limited setting, one should not wait for results of tox PCR tests that are in themselves not confirmatory of toxin production, to make a decision on administration of antitoxin.¹²

Rapid microbiological diagnostics such as use of MALDI-TOF MS has accelerated and improved routine diagnosis and it needs to be integrated into all laboratory workflow, with phenotypic confirmatory testing kept as supplemental test but not as a clinical diagnostic test to make clinical decisions.

CONCLUSION

To conclude, BSIs due to C. diphtheriae, both toxigenic and non-toxigenic strains are rare, but still sporadically reported. Specific management like antidiphtheritic serum may be instituted at the earliest, if there is slightest clinical or laboratory evidence of invasive or localized C. diphtheriae infection. Concomitant Gram-negative bacteremia may cause confusion in clinical decisions, but the importance of BSIs caused by C. diphtheriae should not be taken lightly, and must be addressed along with Gram-negative sepsis.

A limitation of the case study is that toxigenicity testing was not done to rule out non-toxigenic but tox gene-bearing (NTTB) strain infections that may not be effectively prevented by the diphtheria vaccine, which is a partially pure toxoid.