Clostridium tetani – Toxin detection (experimental inoculation and neutralization test); IgG antibodies; Molecular diagnosis (PCR and sequencing).

Information 15-07-2018.

Tetanus is a potentially fatal disease of the nervous system caused by tetanus toxin, produced by Clostridium tetani, a ubiquitous anaerobic bacterium found in the soil. Once the nervous system captures this toxin, the inhibitory synapses are blocked, causing characteristic muscle stiffness, spasms and autonomic instability. Fortunately, the inclusion of a tetanus toxoid vaccine in childhood vaccination programs in developed countries has made it a rare disease. However, in countries with low resources it continues to be a major disease.

The genus Clostridium is heterogeneous and consists of more than 200 species of saprophytic, gram-positive and spore-forming anaerobes. The bacteria of the genus Clostridium are found mainly in the soil, and are frequent in the human or animal intestinal flora. About 20 species of Clostridium are toxigenic and are associated with pathology, such as C. botulinum, C. perfringens, C. difficile and C. tetani. These species produce one or more characteristic exotoxins, which cause disease to the host and, eventually, can cause death. Within the C. tetani species, only the toxin producing strains produce disease in humans or animals.

Clostridium tetani is a gram-positive bacillus, thin, obligate anaerobic and spore-forming. The bacterium Clostridium tetani is found in soil, dust, and the intestinal tract of humans and animals. This bacterium and its spores are ubiquitous in the environment, although they are found more frequently in hot and humid climates, and in agricultural areas, where the soil is rich in organic matter. However, spores of C. tetani have also been detected in street dust, carpets, etc. When subjected to adverse conditions, C. tetani forms terminal rounded spores with a larger diameter than the rest of the bacillus, giving it the general appearance of a drumstick. Although the organism is sensitive to heat and oxygen, its spores are highly resistant to desiccation, heat and exposure to oxygen, so C. tetani can survive in environments such as the soil for prolonged periods. In addition, the spores are able to resist boiling and are very resistant to many chemical agents, including household disinfectants.

For germination and multiplication of the bacteria, C. tetani requires low oxygen tensions. In well oxygenated healthy tissue, if spores are introduced, germination does not occur and spores are eliminated by phagocytes. However, if the spores are inoculated into a damaged tissue, together with adjuvants such as soil, feces, chemicals or other bacteria, the local oxygen tensions decrease and favorable conditions are created for germination and vegetative growth. Therefore, any violation of the skin's defenses by wounds, burns, animal bites, human bites, and even insect bites may result in the inoculation of the spores. Also, infection cases have been documented through surgical procedures, intramuscular injections, composite fractures, and dental infections. In general, C. tetani enters an organism through an open wound, where the conditions for its germination and multiplication are given. During the growth phase, C.tetani produces two toxins, tetanospasmin or tetanus toxin, and tetanolysin. Although the role of tetanolisin is not fully understood, it is believed that it contributes to the establishment of infection at the site of inoculation by causing tissue necrosis, which can serve to decrease tissue oxygenation and promote bacterial growth. Tetanospasmin is a potent neurotoxin, responsible for the manifestations of tetanus. It is encoded in a plasmid of 75 kB, which explains the existence of toxigenic and non-toxigenic strains, according to the presence or absence of this plasmid. This toxin is produced as a single chain of 150 kDa that undergoes post-translational cleavage to form a heavy chain (100 kDa) and a light chain (50 kDa) connected by a disulfide bridge. When C. tetani synthesizes tetanospasmin in wounds, the neurotoxin is released into the surrounding tissues. Tetanus toxin can enter the nervous system from the adjacent muscle, or spread through lymphatic vessels and blood. When the neurotoxin penetrates the motor neurons, it is transported retrograde to the central nervous system. Once there, it enters the inhibitory interneurons and blocks the release of neurotransmitters (glycine and GABA) in the presynaptic terminals, after the proteolytic cleavage of the SNARE VAMP2 protein. The reduction of inhibition results in the disinhibition of the motor neuron discharge, which gives rise to the characteristic symptoms of muscle stiffness and spasms. The muscle groups with the shortest neuronal pathways are affected first, therefore, trismus (the constant and involuntary contraction of the muscles of the jaw) and dysphagia (difficulty swallowing) are common early symptoms.

Tetanus is a usually self-limiting disease, but of great mortality, especially in the extreme ages of life, in neonates and the elderly. The overall mortality rate from tetanus has decreased considerably due to intensive vaccination campaigns. However, despite widespread immunization programs, a large number of cases continue to be reported in developing countries. In developed countries, cases of tetanus occur in unvaccinated people or in elderly people who have decreased their immunity over time. Intravenous drug users are also a sector of risk, due to the possible use of contaminated needles or drugs. The incubation period of the disease can last from 1 to 60 days, but is, on average, around 7 to 10 days. The severity of symptoms depends on the distance from the site of infection to the central nervous system, with more pronounced symptoms associated with shorter incubation periods. The symptoms can last from weeks to months with a mortality rate of 10% of those infected, being higher in those without previous vaccination. In addition, frequent long-term, neuropsychiatric and motor complications have been documented in survivors, many of whom recover completely.

There are four forms of tetanus based on clinical findings: generalized, neonatal, local and cephalic tetanus. Generalized tetanus is the most common form, occurring in approximately 80% of cases. Patients present a descending pattern of muscle spasms, presenting first trismus or jaw closure. Simultaneously, dysphagia and muscular rigidity appear that progressively affects the muscles of the neck, shoulders, thorax, back, abdomen and proximal muscles of the limbs. Another characteristic sign, but less frequent, is a fixed or rigid smile called “risus sardonicus”. These spasms can occur for up to 4 weeks, taking months to complete recovery. The most severe cases present with autonomic instability, whose manifestations are diverse: fever, arrhythmia, blood pressure and unstable heart rate, respiratory difficulties, and even premature death. The second type, neonatal tetanus, is a generalized form of tetanus in newborns of unvaccinated or insufficiently vaccinated mothers due to the unsanitary manipulation of the umbilical cord. Without treatment, it has a high mortality. Local tetanus and cephalic tetanus are the rarest forms of tetanus, and both can progress to the generalized form. Local tetanus is the persistent contraction of the muscles near the site of the injury, which may persist for weeks and has a good prognosis. Tetanus cephalic is limited to the muscles and nerves of the head, and occurs after a head injury, laceration of the head, eye injury, dental procedures, or otitis media. It presents rigidity in the neck, dysphagia, trismus, retracted eyelids, deviant gaze and risus sardonicus. It is generalized rapidly and its mortality is high.

The treatment of tetanus is based on the severity of the disease. However, early debridement of the wound, antibiotic therapy, intramuscular or early intravenous administration of human antitetanus immunoglobulin (HTIG) and neuromuscular blockade should be performed in all patients. The first-line therapy includes HTIG that eliminates the free tetanospasmin toxin, but nevertheless, does not affect the toxin that is already attached to the central nervous system. Debridement of the wound will control the source of toxin production. Although toxins are the main cause of the disease, it has been shown that metronidazole, an anaerobic antibiotic, slows the progression of the disease.

In addition to humans, animals can also suffer from tetanus. All livestock species are susceptible to C. tetani infection. This bacterium is often found as part of the intestinal microflora of animals. Horses are the most susceptible domestic species, so vaccination of these animals is recommended. Sheep and goats are more susceptible than cattle. Individual cases may occur or outbreaks may occur in flocks after castration, ear tagging or dehorning. In addition, the disease occurs also, occasionally in dogs, and rarely in cats.

The diagnosis of tetanus is usually clinical, without the need for a laboratory test. The microbiological diagnosis is based on the culture of the wounds, although the isolation of the microorganism by culture is only obtained in 30% of the cases. In addition, routine bacteriological studies do not indicate whether a strain of C. tetani is a carrier of the toxin plasmid. The molecular diagnostic techniques developed in recent years allow the detection of the presence of toxigenic C. tetani strains, by using primers targeted to the gene that produces the tetanus toxin. In addition, by inoculating mice with patient's serum or plasma, or with a filtrate obtained from the culture of the bacteria, the presence of toxins in the serum or in the culture can be detected. However, if the mice are affected and die, to confirm that they have died from the tetanus toxin, the neutralization test should be performed using specific antiserum of the tetanus toxin (neutralization test), or the presence of the producing genes should be detected of the tetanosesmin of C. tetani in the culture medium (molecular detection by PCR).

Tests carried out in IVAMI:

  • IgG antibodies. 
  • Molecular diagnosis of C. tetani (PCR) and of toxigenic strains of C. tetani (PCR). 
  • Detection of tetanus toxin by inoculation in mice (MIT test). 
  • Detection of tetanus toxin by inoculation in mice and confirmation by neutralization test. 

Recommended samples:

  • Molecular diagnosis (PCR): biopsy or aspiration of the wound. 
  • Detection of free toxin, in humans or animals, by inoculation to mice: blood, plasma or serum. 
  • Detection of the microorganism or its spores in environmental samples: soil, feces of animals, samples of surfaces or environments. 

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days. 
  • Frozen: more than 2 days. 

Delivery of results:

  • Molecular diagnosis (PCR) of C. tetani strains and toxigenic strains: 24 to 48 hours. 
  • Detection of free tetanus toxin by inoculation in mice (MIT): 7 days. 
  • Detection of tetanus toxin by inoculation in mice and confirmation by neutralization test: 14 days. 

Cost of the test:

  • Molecular diagnosis (PCR) of toxigenic C. tetani strains: consult 
  • Detection of tetanus toxin by inoculation in mice (MIT): consult 
  • Detection of tetanus toxin by inoculation in mice and confirmation by neutralization test: consult