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.

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.

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

Severe Pneumonia Caused by Toxigenic Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans infection was recently recognized as a zoonosis. We present 2 cases of severe pneumonia complicated by diffuse pseudomembrane formation on the bronchus caused by C. ulcerans-producing diphtheria toxin. Our purpose is to alert medical professionals to the virulence of Corynebacterium species other than C. diphtheriae.

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.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction.

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.

Genetic characterization and comparison of Clostridium botulinum isolates from botulism cases in Japan between 2006 and 2011.

Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.

Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile.

The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 × 10(2) colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.

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.

Complete Genome Sequence of Corynebacterium ulcerans Strain TSU-28, Harboring Two Diphtheria Toxin Genes, Isolated from a Patient with Diphtheria-Like Symptoms.

Diphtheria toxin-producing Corynebacterium ulcerans is an emerging zoonotic pathogen that causes severe disease in humans. Here, we report the complete genome sequence of C. ulcerans strain TSU-28, harboring two diphtheria toxin genes, which was isolated from the throat of a patient with diphtheria-like symptoms in 2019 in Japan.

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

BACKGROUND: Corynebacterium 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. RESULTS: We 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. CONCLUSION: Taken 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.

Rapid detection and discrimination of potentially toxigenic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis by multiplex real-time PCR and amplicon melting curve analysis.

We developed a multiplex real-time PCR assay with amplicon melting curve analysis to rapidly discriminate Corynebacterium ulcerans from Corynebacterium pseudotuberculosis and detect the bacterial diphtheria toxin gene. This assay should be a valuable tool for identification of potentially toxigenic C. ulcerans.

Potential risk of SARS-CoV-2 infection among people handling linens used by COVID-19 patients before and after washing.

The risk of SARS-CoV-2 infection when people handle linens is uncertain. We examined the presence of SARS-CoV-2 on linens, in the air, and on personal protective equipment (PPE) to assess potential infection risk among individuals who handle linens used by SARS-CoV-2-infected people. Patients in a hospital and an accommodation facility who tested positive for SARS-CoV-2 participated in this study in 2020. Linen samples before washing or disinfection, rinse water after washing or disinfection, air in the workplace at the hospital and an accommodation facility, and the PPE worn by linen-handling people were tested for SARS-CoV-2 RNA and viable viruses. Among 700 samples from 13 SARS-CoV-2-infected participants and their surrounding environment, SARS-CoV-2 RNA was detected from 14% (52/362) of the linens used by COVID-19 patients (cycle threshold [Ct] value: 33-40). SARS-CoV-2 RNA was detected from 8% (2/26) of rinse water after washing or disinfection, from 15% (16/104) of air samples in the workspace, and from 10% (5/52) of gowns worn by linen-handling people, all with high Ct values (> 36). No SARS-CoV-2 was isolated from any samples. The potential risk of SARS-CoV-2 infection from handling linens used by SARS-CoV-2-infected people exists but appears to below.

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.

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.

Transmission of toxigenic Corynebacterium ulcerans infection with airway obstruction from cats to a human.

BACKGROUND: Infections caused by Corynebacterium ulcerans, a zoonotic pathogen, have been reported worldwide. This microorganism is known to produce the diphtheria toxin and cause diphtheria-like illness. CASE PRESENTATION: A 63-year-old woman with a history of diabetes and hypertension developed cold and flu-like symptoms, which gradually progressed into respiratory distress. Therefore, the patient was intubated for dyspnea with pseudomembrane formation. A toxin-producing strain of C. ulcerans was identified, also detected in the patient's domestic cats. Multilocus sequence typing confirmed all strains, including the patient's isolate, as ST337. CONCLUSION: Multilocus sequence typing revealed zoonotic transmission of C. ulcerans from domestic cats to a human.

Genome organization and pathogenicity of Corynebacterium diphtheriae C7(-) and PW8 strains.

Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(-) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(-). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(-) genome. Despite this, the C7(-) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

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.