Processing of NEQAS Corynebacterium diphtheriae specimen

I reconstituted the lyophilised material/specimen from NEQAS with 1ml of nutrient broth, gently mixed and left for 5 minutes. This is then plated on culture media which includes Hoyles Tellurite agar and blood agar. Hoyles Tellurite agar is incubated in air for 48 hours at 35-37^oC while blood agar are incubated anaerobically for 24-48 hours at 35-37^oC. The culture plates where observed at 24 and 48 hours for growth with sufficient growth observed at 24 hours. They were grey/black colonies on the Hoyle’s tellurite agar while on blood agar, the colonies appear grey and granular with irregular edges and small zone of haemolysis. Gram stain was performed on the isolates and they show Gram positive rods with some of them in pairs, lying on each, slightly curved, clubbed end and resembling Chinese letters.

Biochemical test (catalase test) was performed following culture growth and it was positive. The isolate was then identified using Biomerieux VMS (MALDI-TOF) as C. diphtheria. For patient samples, as soon as the isolate is identified as C. diphtheria the consultant microbiologist will be informed and chocolate agar slopes inoculated which will be sent to our reference laboratory for confirmation, biotyping and toxigenicity. Health and safety procedures should be strictly followed when processing a suspected diphtheria specimen including the use of Class I safety cabinet and personal protective equipment.  

It is also important to establish whether the isolated C. diphtheria is toxigenic or not. This is done with the use of ELEK double diffusion precipitin method at our reference laboratory at Health Protection Agency, Respiratory and systemic infections laboratory (RSIL), Streptococcus and Diphtheria Unit, Colindale, London. The test is performed with the use of a filter paper strip impregnated with diphtheria antitoxin which is buried just beneath the surface of a special agar plate before the agar solidifies. Positive and negative known toxigenic strains plus the strains to be tested are streaked on the agar surface in a line across the plate and at a right angle to the antitoxin paper strip. The plate is then incubated for 24 hours at 35-37^oC. Transmitted light is used to examine the plate after incubation for the presence of fine precipitin lines at 45 degrees to the streak. Toxin producing strains reacts with the antitoxin with the formation of precipitin lines.

Diphtheria is an acute contagious disease caused by Corynebacterium diphtheriae usually affecting the upper respiratory tract mucosa. It is characterised by the formation of a fibrinous pseudomembrane. It also causes damage to the myocardial and neural tissue caused by the action of a potent exotoxin. The organism grows in the upper respiratory tract where it is absorbed into the mucous membrane producing exotoxin known as diphtheria toxin which causes destruction of the epithelium and a superficial inflammatory response. Once the exotoxin is absorbed into the mucous membrane, they can be carried in the circulation into distant organs such as heart. There are four recognised biotypes of C. diphtheria which include mitis, gravis, intermedius and belfanti with gravis known to produce the most exotoxin and thus more pathogenic that the rest.