Rapid Detection of Clostridium tetani by Recombinase Polymerase Amplification Using an Exo Probe.

Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of C. tetani. Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic C. tetani strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.

The Role of the Gastrointestinal Tract in Toxigenic Clostridium tetani Infection: A Case-Control Study.

Tetanus arises from wound contamination with Clostridium tetani, but approximately one fifth of patients have no discernable entry wound. Clostridium tetani is culturable from animal feces, suggesting the gastrointestinal tract could be an endogenous reservoir or direct-entry portal, but human data are lacking. In this study of 101 Vietnamese adults with tetanus and 29 hospitalized control subjects, admission stool samples were cultured for C. tetani. Anti-tetanus toxin antibodies were measured by ELISA. Clostridium tetani toxigenicity was evaluated using polymerase chain reaction and sequencing. Toxigenic C. tetani was cultured from stool samples in 50 of 100 (50%) tetanus cases and 12 of 28 (42.9%) control subjects (P = 0.50), and stool samples of 44 of 85 (52.4%) tetanus cases with clinically identified wounds compared with 6 of 15 (47.6%) patients without clinically identified wounds (P = 0.28). Nine of 12 (75%) control subjects with toxigenic C. tetani in their stool samples lacked protective antibody concentrations. These findings fail to show evidence of an association between gastrointestinal C. tetani and tetanus infection, but emphasize the importance of increasing vaccination coverage.

Novel neutralizing human monoclonal antibodies against tetanus neurotoxin.

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)-reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix-reactive antibodies provided enhanced protection compared to the use of each antibody alone.

Tetanus outbreak in a sheep flock due to ear tagging.

Tetanus is an acute, often fatal, infectious neuromuscular disease in all farmed mammals caused by Clostridium tetani. The disease is sporadic but outbreaks of tetanus have been described, as a result of wound contaminated with spores of C. tetani, which sporulates to the vegetative form and produce toxins. The present study reports an outbreak of tetanus in a sheep flock, shortly after ear tagging. Three sheep from a large flock (with a population of 1000 sheep) were presented with signs of: convulsion, limb stiffness, incoordination and trismus ("lock jaw"). There were wounds and scabs in most livestock where ear tags had been attached 1 week prior. Clinical examination revealed tachycardia, dyspnoea with dilated nostrils, mild fever, erected ear pinnae, teeth grinding, mild bloat, muscles rigidity, prolapse of third eyelid and anxiety. According to the history stated by the owner, the case fatality rate of the disease from the beginning was 50% during the outbreak. Necropsy did not reveal any significant finding. Gram-positive bacilli with terminal spores representing C. tetani were isolated in anaerobic cultures which were taken from ear wounds. Procaine penicillin G was administrated at 20 000 IU/kg BW for 5 days, but antiglobulin was not available to treat affected animals. Mortality significantly declined one day after onset of treatment. In this report, the organism was probably introduced by contaminated instruments which were used for ear tagging of sheep. Wound exudation and adhesion following rubbing, created a favourable anaerobic condition for the spores to germinate with production of neurotoxin. Vaccination can protect animals against tetanus, but it does not preclude the need to apply standard hygienic principles when performing management procedures causing wounds. In pasture holding system, many pathogens are present in environment, so tetanus should be considered important in farm animals, because of its high fatality rate and the long course of convalescence.

Achieving neonatal tetanus elimination in Nigeria: undisclosed challenges and prospects.

The global initiative for neonatal tetanus elimination was launched in 1989 following the unacceptably high neonatal morbidity and mortality from tetanus. Since then tremendous progress has been made evidenced with a 94% reduction in mortality. Despite this impressive progress, two global target dates have been missed, the latest being in 2005; which led to a further target of 2015 as the global neonatal tetanus elimination date. This target date has probably been missed again as there are still 21 countries yet to be validated. Nigeria is one such country and contributes two-thirds of the burden of neonatal tetanus globally. What are the prospects and challenges of neonatal tetanus elimination in Nigeria? This paper discusses these and other relevant issues regarding neonatal tetanus elimination and sustaining clearance.

Generalized Tetanus in a Gyrfalcon ( Falco rusticolus ) with Pododermatitis.

A 2-yr-old male gyrfalcon ( Falco rusticolus ) was presented for severe and generalized muscle spasticity and pododermatitis. The falcon had been treated for pododermatitis over the previous 4 mo. Muscle rigidity and spasms involved the entire bird but were more severe on the right leg. The bird was also tachypneic and hyperthermic at 45 C. While the plantar pododermatitis lesions had healed, there was still a small abscess on the lateral aspect of the right foot. Clinical signs were consistent with tetanus. Several bacteria were isolated from the abscess including Clostridium tetani . The isolate was confirmed to be toxigenic by PCR. Attempts to detect tetanus toxin in the bird's plasma were unsuccessful. The abscess was debrided. The gyrfalcon received equine tetanus antitoxin, intravenous metronidazole, methocarbamol, midazolam, a constant-rate infusion of Fentanyl, active cooling, and supportive care. Inhalant anesthesia with isoflurane was the only treatment that would lower the body temperature and reduce the clinical signs. The gyrfalcon died a few hours after admission. The characteristic clinical signs and isolation of toxigenic C. tetani from a wound were strong supportive evidence for a diagnosis of tetanus. This case constitutes the first reported natural occurrence of tetanus in an avian species. Further information is needed to determine whether gyrfalcons are more susceptible to tetanus than are other avian species and whether pododermatitis lesions may be risk factors.

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin.

Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin.

[Tetanus in Ho Chi Minh City, Vietnam: epidemiological, clinical and outcome features of 389 cases at the Hospital for Tropical Diseases]. / Le tétanos à Hô-Chi-Minh-Ville, Vietnam : épidémiologie, clinique et pronostic, à propos de 389 cas à l'Hôpital des maladies tropicales.

The objective of this study is to describe the different aspects of tetanus during the past recent years in southern Vietnam: epidemiology, clinical picture, management, and death risk factors. It is a retrospective study concerning 389 cases admitted in 2007 and 2008 at the reference Hospital for Tropical Diseases in Ho Chi Minh City. 93% of all cases were generalized tetanus, and 50% were severe cases. A majority of patients were adult males (medium age 43, M/ F sex-ratio 2.9). Half of them underwent tracheotomy and 39% assisted ventilation. Case fatality rate was 6.4%, the lowest reported rate worldwide in the last ten years. Fatalities resulted mainly from neuro-vegetative disorders, essentially cardiogenic shock (28% of all deaths) [OR = 16.95; p < 0.001], sepsis (24%) [OR = 3.25; (p < 0,114], and acute renal failure (16%) [OR = 7,22; p < 0.004]. Age over 60-year [OR = 4.53; p < 0.0001] and a leukocyte count>12.000/mm(3) [OR = 2.32; p < 0.020] were significantly associated with fatal outcome, contrarily to incubation and extension phase durations, or delayed serum administration. Systematic extension of vaccination to all adult males, and improved access to post exposure sero-immunization at all levels of health centres throughout the country may further reduce tetanus burden in Vietnam.

Isolation and Antibiogram of Clostridium tetani from Clinically Diagnosed Tetanus Patients.

Clostridium tetani, the etiologic agent of tetanus, produces a toxin that causes spastic paralysis in humans and other vertebrates. This study was aimed for isolation, identification, and determination of antimicrobial susceptibility of C. tetani from clinically diagnosed tetanus patients. Isolation was done from deep-punctured tissues of the foot and arm injuries of 80 clinically diagnosed tetanus patients from the Pakistan Institute of Medical Sciences hospital. We successfully screened out five C. tetani isolates out of 80 samples based on the strain-specific characteristics confirmed through biochemical testing and toxin production. A disc diffusion method was used for antimicrobial susceptibilities and C. tetani isolates showed susceptibility to cefoperazone, chloramphenicol, metronidazole, penicillin G, and tetracycline, but were found to be resistant to erythromycin and ofloxacin. During animal testing, all the infected mice developed symptoms of tetanus. The results showed that identification of C. tetani is possible using biochemical and molecular tools and that the strains of C. tetani isolated had not developed resistance against the antibiotics most often used for the treatment of tetanus.

Three cases of neonatal tetanus in Papua New Guinea lead to development of national action plan for maternal and neonatal tetanus elimination.

Maternal or neonatal tetanus causes deaths primarily in Asia and Africa and is usually the result of poor hygiene during delivery. In 2011, three neonatal tetanus cases were investigated in Papua New Guinea, and all three cases were delivered at home by untrained assistants. The babies were normal at birth but subsequently developed spasms. A neonatal tetanus case must be viewed as a sentinel event indicating a failure of public health services including immunization, antenatal care and delivery care. The confirmation of these cases led to the drafting of the Papua New Guinea National Action Plan for Maternal and Neonatal Tetanus Elimination. This included three rounds of a tetanus toxoid supplementary immunization campaign targeting women of childbearing age (WBCA) and strengthening of other clean delivery practices. The first immunization round was conducted in April and May 2012, targeting 1.6 million WBCA and achieved coverage of 77%. The government of Papua New Guinea should ensure detailed investigation of all neonatal tetanus cases reported in the health information system and perform subprovincial analysis of tetanus toxoid coverage following completion of all three immunization rounds. Efforts also should be made to strengthen clean delivery practices to help eliminate maternal and neonatal tetanus in Papua New Guinea.

[Clostridium tetani isolated from patients with systemic tetanus].

Clostridium tetani is widely distributed in ground or mud, especially in field and pond-shore surface layers. C. tetani is rarely isolated from specimens of patients with tetanus, and is generally diagnosed based on clinical symptoms such as trismus or general tonic spasms. This means that positive C. tetani infection is rarely diagnosed bacterially. Using gram straing, we identified C. tetani in specimens from patients suspected of C. tetani infection brought to the Kitasato University Hospital emergency center. Rapid gram staining information in the bacteriology laboratory is expected to improve recovery from C. tetani infection. It is therefore necessary to ensure clinical specimen quality control, and to keep standard strains of rare bacteria for isolation and identification.

Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

The utilization of a commercial soil nucleic acid extraction kit and PCR for the detection of Clostridium tetanus and Clostridium chauvoei on farms after flooding in Taiwan.

Clostridial diseases are zoonoses and are classified as soil-borne diseases. Clostridium chauvoei and Clostridium tetani cause blackleg disease and tetanus, respectively. Since bacteria and spores are re-distributed by floods and then, subsequently, contaminate soils, pastures and water; the case numbers associated with clostridial diseases usually increase after floods. Because Taiwan is often affected by flood damage during the typhoon season, possible threats from these diseases are present. Thus, this study's aim is to apply a combination of a commercial nucleic acid extraction kit and PCR to assess the prevalence of Clostridia spp. in soil and to compare the positivity rates for farms before and after floods. The minimum amounts of Clostridium tetanus and Clostridium chauvoei that could be extracted from soils and detected by PCR were 10 and 50 colony forming units (cfu), respectively. In total, 76 samples were collected from the central and southern regions of Taiwan, which are the areas that are most frequently damaged by typhoons. Noteworthy, the positive rates for Clostridium tetanus and Clostridium chauvoei in Pingtung county after the severe floods caused by a typhoon increased significantly from 13.73 and 7.84% to 53.85 and 50.00%, respectively. This study for the first time provides the evidence from surveillance data that there are changes in the environmental distribution of Clostridium spp. after floods. This study indicates that screening for soil-related zoonotic pathogens is a potential strategy that may help to control these diseases.

[Characteristics of Clostridium tetani and laboratory diagnosis of tetanus]. / Charakterystyka Clostridium tetani i laboratoryjna diagnostyka tezca.

The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans.

Neonatal tetanus elimination in Pakistan: progress and challenges.

Pakistan is one of the 34 countries that have not achieved the neonatal tetanus (NT) global elimination target set by the World Health Organization (WHO). NT, caused by Clostridium tetani, is a highly fatal infection of the neonatal period. It is one of the most underreported diseases and remains a major but preventable cause of neonatal and infant mortality in many developing countries. In 1989, the World Health Assembly called for the elimination of NT by 1995, and since then considerable progress has been made using the following strategies: clean delivery practices, routine tetanus toxoid (TT) immunization of pregnant women, and immunization of all women of childbearing age with three doses of TT vaccine in high-risk areas during supplementary immunization campaigns. This review presents the activities, progress, and challenges in achieving NT elimination in Pakistan. A review of the literature found TT vaccination coverage in Pakistan ranged from 60% to 74% over the last decade. Low vaccination coverage, the main driver for NT in Pakistan, is due to many factors, including demand failure for TT vaccine resulting from inadequate knowledge of TT vaccine among reproductive age females and inadequate information about the benefits of TT provided by health care workers and the media. Other factors linked to low vaccination coverage include residing in rural areas, lack of formal education, poor knowledge about place and time to get vaccinated, and lack of awareness about the importance of vaccination. A disparity exists in TT vaccination coverage and antenatal care between urban and rural areas due to access and utilization of health care services. NT reporting is incomplete, as cases from the private sector and rural areas are underreported. To successfully eliminate NT, women of reproductive age must be made aware of the benefits of TT vaccine, not only to themselves, but also to their families. Effective communication strategies for TT vaccine delivery and health education focusing on increasing awareness of NT are strongly suggested. It is imperative that the private and government sectors work cooperatively to report NT cases and improve routine TT vaccination coverage.

Tetanus as cause of mass die-off of captive Japanese macaques, Japan, 2008.

In 2008 in Japan, 15/60 captive Japanese macaques died. Clostridium tetani was isolated from 1 monkey, and 11 had tetanus-specific symptoms. We conclude the outbreak resulted from severe environmental C. tetani contamination. Similar outbreaks could be prevented by vaccinating all monkeys, disinfecting housing areas/play equipment, replacing highly C. tetani-contaminated soil, and conducting epidemiologic surveys.

A case of otogenic tetanus.

The authors are presenting our experience of managing an interesting case of a 12-year-old girl who presented to our clinic with otorrhea for 3 months and trismus for 1 week. Examination showed bilateral ear discharge with central perforations in tympanic membranes, palatal paralysis and trismus. Systemic examination revealed only mild stiffness of hand muscles. CT-scan head and neck was done to look for intracranial complications of otitis media. However; it revealed only decreased pneumatisation of mastoid cells. She was admitted in the hospital and started on intravenous and local antibiotics after sending ear swab and blood cultures. But she showed no improvement in 48 h. So on the clinical suspicion (trismus and stiffness of hands) remote possibility of otogenic tetanus was considered and she was given tetanus toxoid and immunoglobulins. She gradually showed improvement in her symptoms. Thereafter, culture from ear discharge was also reported positive for Clostridium tetani.

The effect of magnesium sulfate on uncontrollable contractions in a tetanus case.

Tetanus is a rare disease caused by the exotoxins of Clostridium tetani. Higher mortality rates have been reported among the elderly and in the newborn. In this report of a tetanus case, the treatment and prognosis of contractions resistant to diazepam, midazolam, and atracurium infusion has been evaluated.

Postoperative tetanus after laparoscopic obturator hernia repair for strangulated ileus: report of a case.

This report presents the case of an 84-year-old woman who developed tetanus 3 days after the resection of a gangrenous small intestine caused by obturator hernia incarceration. The diagnosis of tetanus was clinically made after the appearance of generalized spastic contractions with opisthotonus. Clostridium tetani organisms residing in the gastrointestinal tract were presumed to have been endogenously inoculated into the strangulated intestine, where it produced tetanospasmin, causing tetanus. The patient successfully recovered after aggressive intensive care. There have been 16 case reports of tetanus occurring after gastrointestinal surgical procedures. Primary care physicians should thus be aware of the fact that, although extremely rare, C. tetani residing in the gastrointestinal tract can provide a possible endogenous source of tetanus infection.