CLINICAL DIAGNOSIS
Laboratory tests can confirm the presence of the toxigenic C. diphtheriae in the throat, nose and skin (Torres et al., 2013). Dacron swabs are used to collect specimens from the pharyngeal tonsils, whereas calcium alginate swabs are inserted through the nares to collect nasopharyngeal swabs (Funke et al. 2012). As mentioned in ‘Pathogenicity’, toxigenic strains increase the number of diphtheria exotoxinin skin lesions. These lesions are often covered with a pseudomembrane and may have to be exposed before swabbing (Natividad et al., 2015). The specimens are then placed into a semisolid transport medium such as Tinsdale tellurite agar for primary isolation and are incubated at 37°C in 5% CO2. This is the best form of isolation as the medium has a short shelf life (Dias et al., 2011), and therefore the nutrients needed to survive would be at optimal quality.
The tellurite bacterium inhibits the growth of other upper respiratory tract bacteria (Schubert JH, et al., 2010) and can indicate whether C.diphtheriae is completely non-haemolytic. The diphtheria bacilli reduces the tellurite to metallic tellurium, producing grey or black colonies on the agar and further degradation of cysteine by the bacterium, produces a brown halo (Burkhardt, 2015), as shown in Figure 7.
As mentioned in, ‘Infectious agents’, C.diphtheriae can be characterized into different groups based on their carbohydrate fermentation patterns, such as mitis, intermedius, or gravis, as shown in Figure 8. They all produce an immunologically identical toxin. The variations in virulence between the strains are due to the rate they produce the toxin. Mitis, intermedius and gravis have a generation time (in vitro) of 180,100 and 60 minutes respectively (Efstratiou A, et.al, 2014). A larger colony is typically observed with the gravis strain, and a faster growth rate can lead to high usage of iron supply in the tissues by the bacterium, paving way for greater production of the diphtheria toxin. Therefore, categorizing the bacterium, allows us to see the severity of the bacterium species. Mitis, intermedius and gravis are correspondingly responsible for the mild, intermediate and acute form of the disease.
To assess the toxicity of these strains, the Elek culture precipitation test is routinely used to test exotoxin production. It is a vital test for diagnosis and should be carried out immediately on any suspected isolate (Burkhardt, 2015).. A filter paper strip is covered with diphtheria antitoxin and placed underneath the agar, whilst it is still liquid. Strains that are to be tested, are streaked on the surface of the agar in a line and perpendicular to the anti-toxin paper strip. After incubation at 37°C for 24 hours, the presence of fine lines at a 45-degree angle to the streaks indicates that toxins from the strains were produced and these reacted with the anti-toxin (J.Clin et.al, 2013).
As an alternative approach for detecting the exotoxin gene, PCR can also be used. A positive culture result confirms a positive PCR assay. A negative culture result after antibiotic therapy along with a positive PCR assay result suggests that the patient is likely to have diphtheria. Although this test is rapid and precise, a positive signal results may be provided in strains in which the toxic gene is not expressed (Spier SJ et.al, 2011).
To observe under a microscope, C. diphtheriae is placed on a glass side and the smears are covered in Albert’s stain for roughly 7 minutes, followed by Albert’s iodine for 2 minutes. After rinsing the glass side, it is placed under a microscope to be observed under oil immersion lens, as shown in Figure 9 (Corynebacterium diphtheriae and Diphtheria, 2020).
REFERENCES
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Efstratiou A, Engler KH, De Zoysa A. Diagnosis and epidemiology of diphtheria. In: Woodford N, Johnson AP, editors. Methods in molecular medicine. Molecular bacteriology, protocols and clinical applications. Vol. 15. Totowa, NJ: Humana Press; 2014. p. 191-212.
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Schubert JH, Bickham ST, Wiggins GL (November 1968). "Tissue culture method for toxigenicity testing of Corynebacterium diphtheriae". Appl Microbiol. 16 (11): 1748–52. PMC 547753. PMID 4973065.
Spier SJ, Leutenegger CM, Carroll SP, Loye JE, Pusterla JB, Carpenter TE, Mihalyi JE, Madigan JE. Use of a real-time polymerase chain reaction-based fluorogenic 5' nuclease assay to evaluate insect vectors of Corynebacterium pseudotuberculosis infections in horses. Am J VetRes. 2004;65:829–834.
Textbookofbacteriology.net. 2020. Corynebacterium Diphtheriae And Diphtheria. [online] Available at: <http://textbookofbacteriology.net/diphtheria.html> [Accessed 1 November 2020].
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