Applied Microbiology cases

Corynebacterium diphtheriae: Diphtheria
Place the mouse cursor on the links to view the pictures. It may take time to load the images.

 

A 10-year old male child was brought to the hospital emergency with high fever and chills, malaise, sore throat, headache, dysphagia and dyspnea. On examination cervical lymphadenopathy was noted along with grayish white membrane in the throat.

What is your diagnosis?
Clinically, this appears to be a case of pharyngeal or faucial diphtheria. Differential diagnoses include peritonsillar abscess, pharyngitis, epiglottitis, infectious mononucleosis, vincent's angina, oropharyngeal candidiasis and herpes simplex virus infection.

What is pseudomembrane?
A thick, gray, leathery pseudomembrane composed of a mixture of dead cells, fibrin, RBCs, WBCs, and organisms forms locally (tonsillar, pharyngeal) or widely covering the entire tracheobronchial tree. The membrane may cover the tonsils, soft palate, oropharynx, nasopharynx, and uvula. Attempts at scraping the pseudomembrane causes bleeding of the underlying mucosa.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
Nasopharyngeal or pharyngeal swabs must be carefully collected using sterile cotton swabs. At least two sample must be collected, one for microscopy and the other for smear. Smear may also be taken from nose and beneath the membrane but care must be taken not to bleed. Microscopic examination includes a Gram stained smear. If diphtheria is suspected and gram positive bacilli are seen, a special staining technique such as Albert's should be performed. Other way of demonstrating diphtheria bacilli include Immunofluorescent staining. Since microscopy does not provide confirmatory results, culture must be performed. The specimen should be inoculated on Blood agar. If diphtheria is suspected, Loeffler's serum slope and Potassium tellurite agar, Blood tellurite agar (Hoyle's agar/McLeod's agar) or Tinsdale agar should be used. The culture plates should be inoculated at 37oC for 24-48 hours. Selective media may be incubated for 48 hours. If Corynebacterium diphtheriae is identified by biochemical tests, its biotype (gravis, mitis, or intermedius) is determined and the toxigenicity test must be performed using in-vivo or in-vitro methods.

What is your observation?
Gram stained smear reveals few pus cells and gram positive club-shaped bacilli in Chinese letter pattern. Albert stained smear reveals green coloured bacilli in L, V or chinese letter arrangement with few bluish black metachromatic granules throughout the bacilli and especially more so at the bacilli. Blood agar revealed small, greyish, smooth, non-hemolytic colonies. Upon further incubation colonies have dull granular center and glistening periphery and a lighter ring at the edge, resembling frog's egg. Tellurite containing medium showed small black colonies with a black halo. White-grayish colonies are obtained in six hours on Loeffler's serum slope. It is catalase positive, ferments glucose and maltose but not sucrose in Hiss serum water, reduces nitrate but does not hydrolyze urea. Neither glycogen nor starch is fermented.

What is your identification?
The organism isolated is Corynebacterium diphtheriae of the intermedius biotype.

What is the pathogenesis of diphtheria?
Asymptomatic carriers or patients with active infections are chief source of infection. Fomites used by children also serves to transmit infection. Infections occur via respiratory droplets or contact with nasopharyngeal secretions. Overcrowding, poor health, and substandard living conditions facilitate the spread of the disease. C. diphtheriae is not an invasive organism. It infects the superficial layers of the respiratory tract causing local tissue inflammatory reaction followed by tissue necrosis. The occurrence of systemic disease depends on the production of a potent exotoxin by tox+ strains. Only the strains lysogenised by beta phage can produce the toxin. Expression of the gene is regulated by the bacterial host and is iron dependent. Increased toxin production occurs in the presence of low concentrations of iron. The diphtheria toxin is a 62,000-dalton polypeptide composed of two fragments (A and B). The B fragment binds to a receptor on a susceptible cell and undergoes proteolytic cleavage, thus facilitating the entry of segment A, which inhibits peptide chain elongation by inactivating EF-2. The process ultimately functions to inhibit protein synthesis in mammalian cells. Paralysis of the palate and hypopharynx is an early local effect of the toxin. The locally produced toxin is then carried via lymphatics and blood vessels to susceptible tissues. It exhibits a predilection for the myocardium and the cells of the nervous system. Toxin absorption can lead to necrosis of kidney tubules, thrombocytopenia, cardiomyopathy, and demyelination of nerves. Rarely, non-toxigenic strains have caused invasive disease.

Which are the various types of diphtheria?
The site of infection can be faucial, laryngeal, nasal, otitic, conjunctival, genital or cutaneous. Faucial type is the commonest type and may vary from mild catarrhal inflammation to widespread involvement. In malignant or hypertoxic diphtheria, there is severe toxemia with marked submandibular cervical lymphadenopathy (bull neck). Death may occur due to circulatory failure and paralytic sequelae may occur in those who recover. Septic diphtheria manifests as ulceration, cellulitis and even gangrene around pseudomembrane. Hemorrhagic diphtheria is characterized by bleeding from the edge of the membrane, epistaxis, conjunctival hemorrhage, purpura and generalized bleeding.

Which are the various complications of diphtheria?
Asphyxia can occur due to mechanical obstruction by the pseudomembrane and its aspiration can suffocate the patient to death. Infection with the gravis strain is associated with higher mortality rates. Myocarditis can lead to acute circulatory collapse. Other complications include cardiac dilatation and failure, mycotic aneurysm, endocarditis, rhythm disturbances , secondary bacterial pneumonia, cranial nerve dysfunction and peripheral neuropathy, total paralysis, septicemia, septic arthritis, osteomyelitis and death.

Which are the toxigenicity tests?
After isolation of C.diphtheriae, it should be tested for toxin production. It can be detected by in-vitro or in-vivo methods. In vitro-methods include Elek's gel precipitation and tissue culture toxicity test. In-vivo methods involve neutralization of toxin in animal model (guinea pig) challenge test. Polymerase chain reaction (PCR) can detect DNA sequence encoding the A subunit of toxigenic strain.

How is this condition treated?
All cases are promptly isolated and universal precautions are used to limit the number of possible contacts. Immediate airway management is followed by the removal of membrane. Horse-derived diphtheria antitoxin (20,000-1,00,000 units depending on the severity) is administered to neutralize the toxin in circulation. Antibiotics such as penicillin or erythromycin is used for the eradication of organisms, which serves to limit the amount of toxin production. Erythromycin has been shown to be slightly superior in the eradication of the carrier state and throat cultures should be repeated in two weeks as relapses are known to occur.

How is diphtheria prevented?
Diphtheria prophylaxis can be active, passive or combined. Active immunization using diphtheria toxoid is given to the unvaccinated (as DTP or DTaP in children) or as DT or Td (tetanus toxoid and a low dose of diphtheria) to the traveler. It is a part of the national immunization schedule in India to immunize infants, where it is given in three doses of DPT are given at intervals of 4-6 weeks and followed by a fourth dose a year later. A booster dose may be given every five years. Schick test was used in the past to determine if a subject needed to be immunized. Passive immunization involves subcutaneous administration of 500-1000 units of anti-diphtheritic serum as an emergency measure to the exposed unvaccinated susceptible individuals. Such people should be followed up with active immunization. Combined immunization consists of administration of the first dose of diphtheria toxoid on one arm and anti-diphtheritic serum on the other arm, which is followed up by full course of active immunization.

Previous    Applied home    Next

Home           Disclaimer           Contact me           Guestbook          About Me           Sitemap