Standardization of the Japanese National Standard, Equine Botulinum Antitoxin Type A, and Factors Affecting Standardization.

Equine botulinum antitoxin is one of the most popular countermeasures for human botulism. The unitage of the antitoxin product is defined according to national minimum requirement or pharmacopoeia in each country by referring to national standard antitoxins for four types (A, B, E, and F). With the expected depletion of the national standard antitoxins, replacement national standard antitoxins are produced and standardized through collaboration of the National Control Laboratory and other participants, including manufacturer(s). Therefore, Japanese National Standard Botulinum Antitoxin Type A, Equine, was replaced according to the results of a collaborative study involving the National Institute of Infectious Diseases and KM Biologics Co., Ltd. The unitage of the replacement material was determined through mouse neutralization tests, which involved toxin-antitoxin mixture injection at pH 7.0. Potency value of 440 units/vial was obtained. However, the Japanese Minimum Requirement for Biological Products was revised, and the neutralization reactions were repeated at pH 6.0, for which considerably different potency value (656 units/vial) and survival profile of mice were obtained. In September 2021, the replacement material, Japanese National Standard Botulinum Antitoxin Type A, Equine, lot 2, was established with potency value of 656 Units/vial. The impact of pH-dependent change in potency on antitoxin quality control is discussed.

Clostridioides difficile infection in thoroughbred horses in Japan from 2010 to 2021.

We encountered 34 Clostridioides difficile (C. difficile) infection (CDI) cases among Thoroughbred horses in Japan from 2010 to 2021. Among them, 79.4% (27/34) either died or were euthanised. The risk factors associated with CDI and mortality among Japanese Thoroughbred horses remain unclear. We used genetic methods to examine C. difficile strains and their relationships with prognosis. Twenty-two (64.7%) cases were hospitalised at the onset of colitis. Outcomes were balanced for hospitalisation rates at the onset of colitis. The mortality rates of cases treated with metronidazole (65.0%) were significantly lower than untreated cases (100%). The predominant genotype of C. difficile isolate was polymerase chain reaction ribotype (RT) 078, isolated from 12 cases (35.3%), followed by RT014 (six cases, 17.6%). Binary toxin (C. difficile transferase [CDT])-positive strains, including all RT078 strains, were isolated from 16 horses. Mortality rates in RT078 strain (75.0%) or CDT-positive strain (83.3%) cases were comparable to that in cases of other types. Sufficient infection control is needed to prevent CDI in Thoroughbred horses. A timely and prompt CDI diagnosis leading to metronidazole treatment would improve CDI outcomes.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

An ELISA system for tetanus toxoid potency tests: An alternative to lethal challenge.

For a long time, a widely used method for tetanus toxoid (Ttd) potency has been the challenge test, in which animals are immunized and then challenged with tetanus toxin in lethal or non-lethal way. In the context of animal welfare, an alternative is desired because the method causes unsustainable distress to animals. We aimed to replace the system for describing test results, in which scores are assigned to symptoms exhibited by challenged animals, with scores assigned to antibody ELISA titers in immunized mouse sera. The potency values and confidence intervals calculated by the absorbance score system were equivalent to those calculated by the symptom score system. We also attempted to utilize the raw ELISA absorbance instead of the assigned absorbance score and obtained similar results. ELISA may serve as an alternative to the lethal challenge for Ttd potency tests, not only in Japan but also in other countries in which mouse challenge tests are employed.

Gene expression analysis during the conversion from a viable but nonculturable to culturable state in Vibrio cholerae.

We previously reported that Vibrio cholerae in a viable but non-culturable (VBNC) state can be converted to a culturable state by treatment with catalase. This finding enabled us to develop an assay system to observe the time course of the conversion from VBNC to culturable in V. cholerae. VBNC cells began to convert to culturable cells as early as 2 h after catalase supplementation. Gene expression in VBNC cells during catalase treatment was analyzed using RNA microarray. Many ribosomal DNA genes were stimulated 6 h post catalase exposure, suggesting that the conversion-driving signal started prior to 6 h. Focusing on the period prior to cell proliferation, we found that 16 genes might be involved in the conversion mechanism in V. cholerae, and they showed enhanced expression at 2 h and 4 h after catalase addition. These upregulated genes included phage shock proteins (pspA, B, and C), alternative sigma factor (rpoE) and its negative regulator (rseA), cobW C terminal domain-containing protein, damage-inducible helicase (dinG), cholerae toxin secretion protein epsM, HTH-type transcription regulator (iscR), mechanosensitive ion channel family protein, anthranilate synthase component I, fructose-specific IIBC component, molybdenum import ATP-binding protein (modC), LysE family translocator, putative organic hydroperoxide resistance protein, and a hypothetical protein. This study identified genes involved in the catalase-induced conversion of V. cholerae VBNC cells to a culturable state and provided valuable insights into the mechanisms involved in the conversion process.

Seroepidemiology and Carriage of Diphtheria in Epidemic-Prone Area and Implications for Vaccination Policy, Vietnam.

In 2019, a community-based, cross-sectional carriage survey and a seroprevalence survey of 1,216 persons 1-55 years of age were conducted in rural Vietnam to investigate the mechanism of diphtheria outbreaks. Seroprevalence was further compared with that of an urban area that had no cases reported for the past decade. Carriage prevalence was 1.4%. The highest prevalence, 4.5%, was observed for children 1-5 years of age. Twenty-seven asymptomatic Coerynebacterium diphtheriae carriers were identified; 9 carriers had tox gene-bearing strains, and 3 had nontoxigenic tox gene-bearing strains. Child malnutrition was associated with low levels of diphtheria toxoid IgG, which might have subsequently increased child carriage prevalence. Different immunity patterns in the 2 populations suggested that the low immunity among children caused by low vaccination coverage increased transmission, resulting in symptomatic infections at school-going age, when vaccine-induced immunity waned most. A school-entry booster dose and improved infant vaccination coverage are recommended to control transmissions.

First confirmed case of infant botulism caused by Clostridium botulinum type A(B) in a 10-month-old infant in Hanoi, Vietnam.

Infant botulism is a rare but sometimes life-threatening toxemia caused by ingestion of Clostridium botulinum spores. Although cases of infant botulism have probably occurred in Vietnam in the past, they have never been diagnosed and reported. Herein, we report the isolation of C. botulinum type A(B) from the stool of a 10-month-old infant during hospitalization.

First report of foodborne botulism due to Clostridium botulinum type A(B) from vegetarian home-canned pate in Hanoi, Vietnam.

Even one case of foodborne botulism constitutes a public health emergency. We report a series of cases with delayed treatment due to delayed diagnosis. Clostridium botulinum type A(B) was isolated from vegetarian home-canned pate, but not from stool samples. These are the first recorded cases of foodborne botulism in Hanoi.

Molecular epidemiology of endemic Clostridioides difficile infection in Japan.

We reviewed the molecular epidemiology of Clostridioides difficile infection (CDI) in Japan by reviewing articles in which typing analysis was performed on recovered C. difficile isolates. Most of the multicenter studies showed that the major prevalent PCR-ribotypes (RT018-related type, RT014, RT002, RT369, and RT017) accounted for more than 75% of clinical isolates in Japan, which has not changed significantly since the late 1990s. Within the RT018-related isolates, a shift from RT018 to RT018'' (QX239) and the persistence of high levels of antimicrobial resistance were observed. Among toxin A-negative, toxin B-positive C. difficile, RT017 was replaced by RT369, which was more resistant to fluoroquinolone. The isolation rate of binary toxin-positive isolates was low (2-6%), except in one study (10%). Isolation of RT027 and RT078 was rare in endemic settings, while there was a first report of a nosocomial outbreak due to RT027 C. difficile in 2019. Notably, the vast majority of RT027 isolates, including the epidemic strain responsible for the outbreak, were susceptible to moxifloxacin, suggesting that Japanese RT027 represents the pre-epidemic RT027 genetic background. To understand the CDI burden in Japan, a nationwide strain-based surveillance system is imperative.

Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae.

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

Surface Layer Protein A Expressed in Clostridioides difficile DJNS06-36 Possesses an Encephalitogenic Mimotope of Myelin Basic Protein.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Recent studies suggest that migration of Th1 and Th17 cells specific for enteric bacteria from the gut to the CNS may lead to the initiation and/or exacerbation of autoimmune diseases including MS. Human leukocyte antigen (HLA)-DR15 is an MHC class II (MHCII) haplotype highly associated with the development of MS that contains the two HLA-DRB* genes, DRB1*1501 (DR2b) and DRB5*0101 (DR2a). To identify enteric bacteria which harbor antigenic epitopes that activate myelin-specific T cells and drive CNS inflammation, we screened for enteric bacteria which express cross-reactive epitopes ('mimotopes') of an immunodominant myelin basic protein 89-98 (MBP89-98) epitope. Based on known MHCII HLA-DR2a amino acid binding motifs and cultivation with splenic T cells isolated from MBP-T cell receptor (TCR)/DR2a transgenic (Tg) mice, we discovered that a certain variant of surface layer protein A (SLPA), which is expressed by a subtype of Clostridioides difficile, contains an amino acid sequence that activates MBP89-98-reactive T cells. Furthermore, activation of MBP-specific T cells by SLPA upon active immunization induced experimental autoimmune encephalomyelitis (EAE) in MBP-TCR/DR2a Tg mice. This study suggests that a unique strain of C. difficile possesses an encephalitogenic mimotope of MBP that activates autoreactive, myelin-specific T cells.

Fatal fulminant Clostridioides difficile colitis caused by Helicobacter pylori eradication therapy; a case report.

A 74-year-old male was referred to our critical care department for refractory severe watery diarrhea with advanced leukocytosis (over 70,000/µl) after multiple administrations of eradication therapy against Helicobacter pylori (HP). He was diagnosed as having fulminant colitis due to Clostridioides difficile after antimicrobial eradication therapy. He was given intravenous metronidazole and oral vancomycin. He also received supportive therapy including continuous hemodiafiltration for severe metabolic acidosis. However, despite emergency open sigmoidectomy, he died. The C. difficile isolate recovered was PCR-ribotype 002, which was positive for toxins A and B but negative for binary toxin. HP eradication therapy for prevention of chronic gastritis and stomach cancer is now in widespread use. Although such secondary severe complications are rare, we consider it to be necessary to pay sufficient attention when administering HP eradication therapy.

Performance of laboratory tests for detection for Clostridioides difficile: A multicenter prospective study in Japan.

BACKGROUND: The optimal and practical laboratory diagnostic approach for detection of Clostridioides difficile to aid in the diagnosis of C. difficile infection (CDI) is controversial. A two-step algorithm with initial detection of glutamate dehydrogenase (GDH) or nucleic acid amplification test (NAAT) alone are recommended as a predominant method for C. difficile detection in developed countries. The aim of this study was to compare the performance of enzyme immunoassays (EIA) detecting toxins A and B, NAAT detecting the toxin B gene, and GDH compared to toxigenic culture (TC) for C. difficile as the gold standard, in patients prospectively and actively assessed with clinically significant diarrhea in 12 medical facilities in Japan. METHODS: A total of 650 stool specimens were collected from 566 patients with at least three diarrheal bowel movements (Bristol stool grade 6-7) in the preceding 24 h. EIA and GDH were performed at each hospital, and NAAT and toxigenic C. difficile culture with enriched media were performed at the National Institute of Infectious Diseases. All C. difficile isolates recovered were analyzed by PCR-ribotyping. RESULTS: Compared to TC, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of EIA were 41%, 96%, 75% and 84%, respectively, and for NAAT were 74%, 98%, 91%, and 92%, respectively. In 439 specimens tested with GDH, the sensitivity, specificity, PPV, and NPV were 73%, 87%, 65%, and 91%, and for an algorithm (GDH plus toxin EIA, arbitrated by NAAT) were 71%, 96%, 85%, and 91%, respectively. Among 157 isolates recovered, 75% of isolates corresponded to one of PCR-ribotypes (RTs) 002, 014, 018/018", and 369; RT027 was not isolated. No clear differences in the sensitivities of any of EIA, NAAT and GDH for four predominant RTs were found. CONCLUSION: The analytical sensitivities of NAAT and GDH-algorithm to detect toxigenic C. difficile in this study were lower than most previous reports. This study also found low PPV of EIAs. The optimal method to detect C. difficile or its toxins to assist in the diagnosis of CDI needs further investigation.

Clostridioides (Clostridium) difficile infection burden in Japan: A multicenter prospective study.

Clostridioides (Clostridium) difficile is the leading cause of healthcare-associated infectious diarrhea in the developed world. Retrospective studies have shown a lower incidence of C. difficile infection (CDI) in Japan than in Europe or North America. Prospective studies are needed to determine if this is due lack of testing for C. difficile or a true difference in CDI epidemiology. A prospective cohort study of CDI was conducted from May 2014 to May 2015 at 12 medical facilities (20 wards) in Japan. Patients with at least three diarrheal bowel movements (Bristol stool grade 6-7) in the preceding 24 h were enrolled. CDI was defined by positive result on enzyme immunoassay for toxins A/B, nucleic acid amplification test for the toxin B gene or toxigenic culture. C. difficile isolates were subjected to PCR-ribotyping (RT), slpA-sequence typing (slpA-ST), and antimicrobial susceptibility testing. The overall incidence of CDI was 7.4/10,000 patient-days (PD). The incidence was highest in the five ICU wards (22.2 CDI/10,000 PD; range: 13.9-75.5/10,000 PD). The testing frequency and CDI incidence rate were highly correlated (R2 = 0.91). Of the 146 isolates, RT018/018″ was dominant (29%), followed by types 014 (23%), 002 (12%), and 369 (11%). Among the 15 non-ICU wards, two had high CDI incidence rates (13.0 and 15.9 CDI/10,000 PD), with clusters of RT018/slpA-ST smz-02 and 018"/smz-01, respectively. Three non-RT027 or 078 binary toxin-positive isolates were found. All RT018/018" isolates were resistant to moxifloxacin, gatifloxacin, clindamycin, and erythromycin. This study identified a higher CDI incidence in Japanese hospitals than previously reported by actively identifying and testing patients with clinically significant diarrhea. This suggests numerous patients with CDI are being overlooked due to inadequate diagnostic testing in Japan.

Community-acquired fulminant colitis caused by binary toxin-producing Clostridium difficile in Japan.

We report a case of community-acquired fulminant colitis caused by Clostridium difficile in Japan. A 46-year-old woman was diagnosed with severe infectious enterocolitis and was admitted at another hospital. The stool culture was positive for toxigenic C. difficile. Since the patient presented with fulminant C. difficile infection (CDI) with toxic megacolon, respiratory insufficiency, and circulatory failure, she was transferred to Kyorin University Hospital for intensive care. Intubation and antibiotic therapy were performed. The general condition improved with conservative treatment, and she was discharged without sequelae. While the recovered isolate was toxin A and B-positive and binary toxin-positive, it was identified as polymerase chain reaction (PCR) ribotype ts0592 and slpA sequence type ts0592. The isolate was different from PCR ribotype 027 epidemic in Europe and North America. In Japan, binary toxin-producing strains are rare and have not caused an epidemic to date. Furthermore, there are few data on community-acquired CDI in Japan. In this case, a non-elderly woman with no major risk factors such as antibiotic use, administration of proton pump inhibitor and history of gastrointestinal surgery developed community-acquired fulminant CDI caused by the binary toxin-positive strain, and ICU treatment was required. Further studies focusing on the role of binary toxin-positive C. difficile in the severity of community-acquired CDI are necessary.

Development of vaccine for Clostridium difficile infection using membrane fraction of nontoxigenic Clostridium difficile.

Although standard antibiotic therapy is performed for diarrhea and pseudomembranous colitis caused by Clostridium difficile, a high recurrence rate of C. difficile infection (CDI) remains a major problem. We previously showed that a membrane fraction of nontoxigenic C. difficile (ntCDMF) was effective as a vaccine antigen by in vitro experiments. In this study, we examined whether ntCDMF had an in vivo effect in animal challenge experiments. By intrarectal immunization with ntCDMF, the number of C. difficile cells in feces of mice was decreased approximately 99% compared to the control mice. In addition, survival rate of C. difficile-challenged hamsters was increased almost 30% by immunization with ntCDMF. These results showed that ntCDMF could be a practical vaccine candidate.

Tetanus in a partially immunized child.

A 13-year-old boy developed tetanus, although he had protective antitoxin antibody raised by three doses of tetanus toxoid vaccine. Four days after injury, he presented with muscle rigidity of his posterior neck, excessive diaphoresis, and risus sardonicus and was subsequently diagnosed with tetanus. Tetanus is rare in developed countries, particularly during childhood, but must be promptly diagnosed based on clinical symptoms.

Fecal microbiota transplantation for recurrent Clostridium difficile infection in a patient with ulcerative colitis.

Fecal microbiota transplantation (FMT) has been reported as a safe and effective therapy in patients with refractory and recurrent Clostridium difficile infection (CDI). FMT has also been reported as a promising therapy in patients with ulcerative colitis (UC). Both, CDI and UC, are believed to be caused by dysbiosis, such as altered compositions or decreased diversity of the intestinal microbiota. This report describes a patient with UC in remission with a second recurrent episode of CDI, who was treated with FMT. A single FMT performed via colonoscopy completely resolved the patient's diarrhea and eradicated C. difficile bacteriologically without any severe complications. Molecular biological analysis of the patient's fecal microbiota showed that FMT could dramatically change the altered composition of intestinal microbiota and restore its diversity. Despite the restoration of the intestinal microbiota, FMT could not prevent a relapse of UC in this patient. However, it improved the intestinal symptoms of CDI and could prevent further recurrences of CDI.

Predominance of PCR-ribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains?

Global spread and evolutionary links of an epidemic Clostridium difficile strain (PCR-ribotype 027) have been noted in recent decades. However, in Japan, no outbreaks caused by type 027 have been reported to date. A total of 120 C. difficile isolates from patients at 15 hospitals during non-outbreak seasons between 2011 and 2013 as well as 18 and 21 isolates collected from two hospitals in 2010 and 2009, respectively, in outbreak periods in Japan, were examined. Among these 120 isolates, Japan-ribotypes smz and ysmz (subtype variant of smz) were the most predominant (39.2 %) followed by Japan-ribotype trf (15.8 %). Types smz/ysmz and trf were also concurrently predominant at two hospitals in the outbreak settings. Out of the five binary toxin-positive isolates observed, only one was PCR-ribotype 027 and another PCR-ribotype 078. Type smz was later found to correspond to PCR-ribotype 018. High rates of resistance against gatifloxacin, moxifloxacin, erythromycin and clindamycin were observed in the PCR-ribotype 018 isolates. Interestingly, all trf isolates were toxin A-negative, toxin B-positive, but they did not correspond to PCR-ribotype 017, thus being assigned a new ribotype (PCR-ribotype 369). In conclusion, PCR-ribotypes 018 (smz) and 369 (trf) were identified as major circulating strains in both outbreak and non-outbreak settings in Japan. Given their epidemiological relevance, molecular investigations are warranted to clarify potential evolutionary links with related strains found elsewhere, such as PCR-ribotypes 018 and 017 from Europe and North America.

[Infectious diseases in Asia/International collaboration in bacterial infectious diseases].

Proactive approaches to collect precise information are necessary to control infectious diseases in the whole world. A collaborative research for infectious diseases with institute or university of countries that infectious diseases occur is one of the good approaches. In this paper, we introduce collaborative researches on infectious diseases with Asian countries, such as Vietnam, Philippines, Thailand, and India.