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Volume: 24S3 - July 1998 Guidelines for the Control of Diphtheria in Canada APPENDIX BProtocol for the Laboratory Diagnosis of Corynebacterium diphtheriae The role of the laboratory in the diagnosis of diphtheria is to assist clinicians in confirming their clinical diagnosis. However, diphtheria has become rare in industrialized countries since the advent of immunization; experience with the disease is not common, and recognizing it is difficult. Diphtheria may be confused with severe streptococcal tonsillitis, infectious mononucleosis, or Vincent's angina. Therefore, the laboratory must provide accurate and rapid information to help confirm or eliminate a suspected case of diphtheria. Experience with the laboratory identification and confirmation of toxigenicity in isolates of C. diphtheriae is limited; therefore, it is recommended that specific laboratories (to be determined) be considered "reference" laboratories for this purpose. A Specimen collection and transport Respiratory diphtheria Diphtheria is primarily an upper respiratory tract infection. Material for culture should be obtained by collecting throat, nose and/or nasopharyngeal swabs and placed in Amies transport medium. Swabs should be taken from the inflamed areas of the throat and nasopharynx in symptomatic patients. If present, membranous material may also be submitted for examination. For the detection of asymptomatic carriers, nasopharyngeal and throat swabs are useful (swab the tonsillar fossae, posterior pharynx, and uvula). If a delay in transport is anticipated, the swab may be held at either room temperature or 4o C prior to laboratory submission, but it should be transported as soon as possible. Skin diphtheria Another common form is skin diphtheria, which can be clinically indistinguishable from pyodermas. Membranous material may also be seen in skin lesions. Material should be taken from the affected area of the skin, either by aspirate or swab. Remove any crusted material and swab the base of the lesion(s). Note that in cases of impetigo, there may also be Staphylococcus aureus and S. pyogenes present in addition to C. diphtheriae. Use a swab to make smears on clean slides for Gram and Loeffler methylene blue staining to include with the swabs/material taken for culture. Note that a final identification of C. diphtheriae cannot be made on the basis of the smear alone, but direct microscopy of the smear may be used to supplement the culture. Transport Specimens should be transported to the laboratory immediately. The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria. If the specimen cannot be transported immediately to the laboratory, a transport medium such as Amies may be used. If more than 24 hours will elapse before receipt in the laboratory, specimens should be shipped at 4o C by courier to the receiving laboratory. In suspected outbreaks, the receiving laboratory must be notified in advance to have the appropriate amount of specialized media available. B Primary isolation Note: Diagnosis of diphtheria based exclusively upon direct microscopy of a smear is unreliable. Culture specimens should be initially plated on blood agar and Hoyles tellurite agar and incubated at 35o C aerobically. Plates are incubated for 18 to 48 hours. Screen plates for typical dull black colonies by subculturing each morphotype to modified Tinsdale agar and blood agar. Use Gram stain to determine morphology (gram-positive rods). Incubate subcultured plates at 35o C aerobically for 24 hours. Any colonies showing brownish-black halos are positive. Gram stain these colonies, and from the blood agar purity plate, do a catalase test (should be positive), and note colonial morphology (including hemolysis). Any gram-positive bacilli seen on gram stain that exhibit typical colonial morphology should be submitted to a reference facility for further identification and confirmation. C Detection of toxigenicity All isolates identified as C. diphtheriae should be tested for toxigenicity at a reference laboratory. The current method used is the Elek test. Molecular methods for the determination of toxigenicity are being developed. There are primers available for polymerase chain reaction (PCR) detection of the toxin gene (the biologically active fragment A portion). However, some isolates have been found that contain the gene but do not express the protein biologically; therefore, at this point PCR would be recommended as an adjunct to the Elek test. D Antimicrobial susceptibility testing Although most isolates are susceptible to penicillin, susceptibility testing may be undertaken at a reference laboratory. There are no current recommended standards for susceptibility testing of C. diphtheriae, however the following antimicrobials may be tested: penicillin, erythromycin and clindamycin. E Other studies Molecular typing methods are currently being investigated. The use of pulsed-field gel electrophoresis and restriction fragment length polymorphism of rRNA genes has been found to show diversity among the biotypes and among toxigenic and non-toxigenic strains, and were more discriminating than conventional methods. These techniques would be available at a reference laboratory. Recommended reading
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