Motohide Takahashi

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of Bordetella pertussis Infection

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) method to detect Bordetella pertussis infection. This LAMP assay detected B. pertussis with high sensitivity, but not other Bordetella species. Among nasopharyngeal swab samples from subjects with suspected pertussis, LAMP results showed a high level of agreement with results of conventional PCR. This method is a rapid, sensitive, and specific method for diagnosis of B. pertussis infection even in clinical laboratories with no specific equipment.

Corynebacterium ulcerans Diphtheria in Japan.

To the Editor: Corynebacterium ulcerans causes a zoonotic infection similar to diphtheria, which is caused by C. diphtheriae. Studies indicate that signs and symptoms of a diphtheria-like illness caused by C. ulcerans are milder than those caused by C. diphtheriae. However, some strains of C. ulcerans produce potent diphtheria toxin and may cause severe symptoms similar to those caused by C. diphtheriae (1). We report a case of a diphtheria-like illness caused by C. ulcerans infection. A previously healthy 52-year-old woman first noticed hoarseness approximately 3 days before admission to the hospital. On February 16, 2001, severe dyspnea and fever developed, and the patient was referred to the emergency room of the Asahi General Hospital by her private practitioner. Physical examination indicated a large stridor, which could be heard without using a stethoscope. Cyanosis was not observed. The endoscopic examination showed a thick white coat covering the nasopharynx and laryngeal vestibulum, and subglottic constriction was also observed. A chest x-ray showed diffuse infiltrates in both lungs. Pertinent laboratory findings on admission included leukocyte count of 16.8 x 103/μL and C-reactive protein of 20.0 mg/dL. The serum level of liver transaminase was normal, and both Wassermann reaction and anti-HIV antibody tests were negative. Pharyngolaryngitis and pneumonia was diagnosed in the patient. Because of severe dyspnea, intubation was performed, which caused sudden and unexpected exacerbation of the condition. Severe cyanosis subsequently developed. Extubation was immediately performed, and a thick white material was found to be filling the lumen of the endotracheal tube. Reintubation was performed, and dyspnea subsided. The patient was hospitalized in the intensive-care unit. Sulbactam sodium/ampicillin sodium (6 g per day) was intravenously administered for 4 days; however, the symptoms were not much improved. The symptoms were most consistent with those of diphtheria. Therefore, the patient was subsequently placed on erythromycin (1.0 g/day) and quickly responded to this treatment without administration of diphtheria antiserum. Erythromycin was intravenously administered at 1 g per day for 9 days, then orally administered at 1,200 mg per day for the next 14 days. Throughout the hospitalization, no complication occurred, and no abnormalities were noted in the electrocardiograms or in the patient’s neurologic status. The patient was discharged uneventfully, and no serious sequelae were noted for 20 months. History of immunization for diphtheria was not known. After the hospitalization for this acute illness, a laboratory report showed that C. ulcerans was cultured from the thick white coat of the throat. No other bacteria were found. The National Institute of Infectious Diseases in Tokyo later confirmed identification of the bacteria. By using Elek’s test, Vero cell toxicity, and polymerase chain reaction for toxigene, this strain of C. ulcerans was proven to produce diphtheria toxin identical to C. diphtheriae (2–4). Although administering appropriate antibiotics as well as antitoxin is a standard of care for patients with diphtheria, antitoxin was not given to this patient because of her rapid response to the erythromycin therapy. C. ulcerans infections in humans occur after drinking unpasteurized milk or coming in contact with dairy animals or their waste (5,6). However, person-to-person transmission of C. ulcerans has not been reported, and in some cases, the route of transmission is not clear (7). Recently, C. ulcerans-producing diphtheria toxin was isolated in the United Kingdom from cats with nasal discharge (8). Our patient did not have direct contact with dairy livestock or unpasteurized dairy products; however, more than 10 dairy farms are scattered around her home. Moreover, she kept nearly 20 cats in her house and had been scratched by a stray cat a week before illness developed. This stray cat, which had rhinorrhea and sneezing, had wandered into her house. The stray cat died before the patient became ill, and no further investigation could be made. Stray cats might well be one of the possible carriers of C. ulcerans and might have transmitted the bacteria to this patient. To our knowledge, a case of human infection caused by C. ulcerans has never been reported in Japan. On the basis of current experience, this bacterium does exist in Japan and can potentially cause a serious diphtheria-like illness in humans.

Novel Corynebacterium diphtheriae in domestic cats.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Human Fulminant Gas Gangrene Caused by Clostridium chauvoei

ABSTRACT The first human case of fulminant gas gangrene caused by Clostridium chauvoei, a pathogen causing ruminant blackleg, was confirmed for a 58-year-old man suffering from diabetes mellitus. The patient developed conspicuous emphysematous gangrene in the right chest wall as well as intravascular gas entrapments and died 2 h after hospital arrival.

Production and immunogenic efficacy of botulinum tetravalent (A, B, E, F) toxoid

A tetravalent (type A, B, E and F) toxoid was produced and its efficacy and safety were assessed. The toxoid preparation was inoculated from two to five times to 15 healthy adult volunteers participating in botulinum toxin research. The serum samples taken from the toxoid recipients were titrated for the antitoxin potencies by enzyme-linked immunosorbent assay (ELISA) and the neutralization test. The neutralizing and ELISA titers were too low to correlate each other. The mean neutralization titer of four recipients in 9 months after three doses of toxoid was about 0.1IU/ml for each of the four types, whereas, the one receiving five doses possessed a higher titer. Since the amount of the toxin handled in laboratory work is usually not so large, three or more doses of the present toxoid will bestow sufficient immunity on the workers participating in botulinum research. Nevertheless booster injections might be desirable to those at higher risk, handling the toxin of a high concentration.

Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage

BackgroundCorynebacterium ulcerans can cause a diphtheria-like illness, especially when the bacterium is lysogenized with a tox gene-carrying bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon phage lysogenization is a common feature of C. ulcerans and C. diphtheriae. However, because of a lack of C. ulcerans genome information, a detailed comparison of prophages has not been possible between these two clinically important and closely related bacterial species.ResultsWe determined the whole genome sequence of the toxigenic C. ulcerans 0102 isolated in Japan. The genomic sequence showed a striking similarity with that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of C. diphtheriae. The 0102 genome contained three distinct prophages. One of these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same structural order as for tox-positive C. diphtheriae prophages. However, the primary structures of the individual genes involved in the phage machinery showed little homology between the two counterparts.ConclusionTaken together, these results suggest that the tox-positive prophage in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae NCTC 13129.

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD(50) test (ip LD(50)). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay. This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD(50), an effect that cannot be detected using the mouse ip LD(50) assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations.

Two Japanese Corynebacterium ulcerans isolates from the same hospital: ribotype, toxigenicity and serum antitoxin titre

Two toxigenic Corynebacterium ulcerans isolates recovered from pharyngeal swabs of two patients from the same hospital in Japan during 2001-2002 were characterized by PFGE and ribotyping. Toxin production in different culture media was examined and serological analysis of patient sera was performed. The two isolates could not be distinguished by PFGE; however, their ribotypes were distinguishable. One of the isolates could represent a novel ribotype. Analysis of toxin production in different culture media demonstrated that the two isolates produced varying amounts of the diphtheria toxin. Serological analysis showed a greater than sevenfold increase in the serum antitoxin titre during the course of infection in one patient.

Interferon and natural killer cell activity in patients with exanthem subitum.

Early immune response was studied by assessing interferon (IFN) and natural killer cell activity in 13 patients with exanthem subitum asociated with human herpesvirus 6 infection during the acute and convalescent phases. Only IFN-alpha showed a significant increase in the plasma of patients during the acute febrile phase compared with the convalescent period. The inhibitory effect of IFN-alpha and IFN-beta on human herpesvirus 6 replication was demonstrated in vitro with cord blood mononuclear cells. Natural killer cell activity was also significantly augmented in the acute phase, especially in the exanthem period, rather than in the convalescent phase (P < 0.01). These results suggest that the enhanced IFN-alpha response and natural killer cell activity in the acute early phase of the disease may play pivotal roles in the recovery from exanthem subitum.

Assay in mice for low levels of Clostridium botulinum toxin

When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LD50 was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed. If this symptom is taken as a marker, 1.0 mouse intraperitoneal LD50 can be detected within 6 h and 0.1 LD50 within 24 h. The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity. Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LD50, a linear relationship was obtained between the log dose and the score. By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LD50 can be titrated accurately and easily.