The mouse toxicity bioassay as a laboratory confirmation test for tetanus

Sir, The article entitled ‘Tetanus: a diagnostic challenge in the Western World’ published in the Acta Clinica Belgica describes a case of tetanus in a 65-year-old woman who presented herself to the emergency with pain and motor weakness. The state of the patient worsened in the days that followed the hospitalization and she was finally diagnosed with tetanus on the basis of typical clinical features. The tetanus infection originated from soiled injuries on her leg that were caused several days earlier by bites of a chicken. The patient had not received tetanus immunization for years and was therefore not completely immunized against tetanus. Nowadays, tetanus has become a very rare disease in the Western countries and publishing this case report helps clinicians to adequately diagnose and treat the disease. The authors state (p. 418): ‘The diagnosis of tetanus must be made on clinical findings outlined above, as there are no laboratory tests that can diagnose or rule-out tetanus’. The clinical diagnostic is essential and remains the primary diagnosis, but a laboratory diagnostic test does help in definitive diagnosis. The toxin tetanospasmin is responsible for the clinical features of the disease and is disseminated via the bloodstream and the lymphatic system in generalized tetanus cases. It can therefore be detected in the human serum and/or in Clostridium tetani cultures from infection sites using the mouse toxicity testing (bioassay). The laboratory diagnosis of tetanus involves thus the detection of toxigenicity in the serum by mouse toxicity testing or both the isolation of C. tetani from wounds and the detection of toxigenicity in the isolate by the mouse toxicity test. Serum, wound swabs or surgical specimens can be submitted for the laboratory diagnosis of tetanus. For the two latter materials, an isolation of C. tetani must first occur. The bioassay method consists of injecting mice with filtrated cultures or with the serum. Tetanus antitoxin can be used to perform a seroneutralization test and is injected to control mice. The tetanus antitoxin will specifically neutralize the neurotoxic effects of the tetanospasmin. The induced effects (paralysis and death) in mice are therefore prevented by the injection of mice with tetanus antitoxin whereas large amounts of toxins would tend to kill the mice within 24 hours. Typical paralysis and/or death of the mice with prevention of these effects by the administration of tetanus antitoxin establishes a positive test for tetanus toxin. The turnaround time – tat – of this bioassay is 1 to 4 days. The toxemia is transient since the toxin can be fixed quickly. Therefore, sampling of the serum must be done rapidly after infection (less than 3 days) and before antitoxin is given in order to detect the toxin. Absence of toxin or lack of an isolate does not exclude diagnosis of tetanus. The presence of an isolate does not confirm the diagnosis either, since non-toxigenic strains of C. tetani have been reported. Unfortunately, up to now, more precise quantitative data on the performance of the bioassay (sensitivity, specificity) are not available, to our knowledge. Nevertheless, when positive, the mouse test contributes in a pertinent manner to demonstrate the toxicity of the strain or to demonstrate the presence of the toxin in the serum. We agree that, when negative, no clear conclusion can be given, and the clinical impression remains a priority in the clinical decision process. The toxicity bioassay is the definitive test for the laboratory diagnosis of tetanus. Such a bioassay is available in Belgium and is performed by the NRL C. botulinum and C. perfringens at the Scientific Institute of Public Health (WIV-ISP) which possesses a long experience in this field. In the past the Laboratorium received several human serums for toxin analysis with one positive case in 1990. Currently, serum from dogs, horses and cattle are still being sent for analysis (with several positive results). The Laboratorium also received faeces or isolated strains for which the toxin production was Correspondence to: Dr L. Delbrassinne, Scientific Institute of Public Health, National Reference Centre for C. botulinum and C. perfringens, Laboratorium of zoonotic Clostridia, 642, rue Engeland, 1180 Brussels, Belgium. Email: ldelbrassinne@wiv-isp.be