Color Transferability from Solution to Solid Using Silica Coated Silver Nanoparticles.

The interpretation of color change in sensors and tests can be linked to incorrect conclusions if the intrinsic color changes are not accounted for. In this work, we study the intrinsic color change associated with the process of embedding nanoparticles in a polymer to create nanocomposite films. We present a safer, faster method to coat silver nanoparticles with silica and employ a seven-factor Plackett-Burman design to identify critical factors in the synthesis. Silver nanodisks with increasing thicknesses of the silica shell showed a decreasing sensitivity of their localized surface plasmon resonance (LSPR) toward changes in the refractive index surrounding the nanoparticle. A color shift of up to 72 nm was observed when bare nanoparticles were embedded in poly(vinyl alcohol), but no color change was perceived when nanoparticles were coated with a 25-nm-thick silica shell. Understanding the origin of color changes intrinsic to the preparation of polymeric nanocomposites aids in the design and correct use of plasmonic sensors.

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.

Fecal microbiota transplantation as therapy for recurrent Clostridioides difficile infection is associated with amelioration of delirium and accompanied by changes in fecal microbiota and the metabolome.

Recurrent Clostridioides difficile infection (rCDI) is a frustrating condition that may affect a person's quality of life for months. Microbiome-based therapy such as fecal microbiota transplantation (FMT) has been effective for the treatment of rCDI by correcting the imbalance of the gut microbiota. Appropriate antibiotic treatment is recommended for at least two recurrences before offering FMT. Here, we report the case of a 92-year-old woman who experienced five recurrences of Clostridioides difficile infection (CDI) (six episodes in total) complicated by dementia and delirium, both of which were dramatically improved by FMT, which was associated with alterations in fecal microbiota and the metabolome. Analyses of whole microbial communities and metabolomic analyses were performed on stool specimens collected from the patient on the first episode, the third episode, the day of FMT (before FMT), and 2, 8, and 23 weeks after the FMT and from the donor. The patient had various fecal dysbioses on the first and third episodes and on the day of FMT. Two weeks after FMT, diversity of the gut bacteriome as well as the virome increased dramatically and was reflected in a positive clinical outcome for this patient. Metabolomic analysis revealed that short-chain fatty acids, which have been reported to be associated with improved memory function, were increased after FMT.

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.

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.

A childhood-onset intestinal toxemia botulism during chemotherapy for relapsed acute leukemia.

BACKGROUND: Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children. CASE PRESENTATION: We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy. CONCLUSION: This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.

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.

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.

Fulminant pseudomembranous colitis caused by Clostridium difficile PCR ribotype 027 in a healthy young woman in Japan.

In the past two decades, Clostridium difficile polymerase chain reaction ribotype 027 strain has rapidly emerged as the leading cause of antibiotic-associated colitis in North America and Europe; however, it has been reported only occasionally in Japan. We report a case of fulminant pseudomembranous colitis caused by this strain in a healthy young woman in Japan without any previous medical history. The strain isolated from our patient was susceptible to both gatifloxacin and moxifloxacin, thus representing a historic strain. The acquisition of fluoroquinolone resistance was reported as the important key genetic event linked to the virulence of this strain. It is noteworthy that the fluoroquinolone-susceptible 027 strain caused fulminant colitis in a healthy young woman in a non-endemic area. Our experience suggests that C. difficile PCR ribotype 027 has the potential virulence factors that are not associated with a fluoroquinolone resistance-conferring mutation.

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.

Redefining Clostridioides difficile infection antibiotic response and clinical outcomes.

With the approval and development of narrow-spectrum antibiotics for the treatment of Clostridioides difficile infection (CDI), the primary endpoint for treatment success of CDI antibiotic treatment trials has shifted from treatment response at end of therapy to sustained response 30 days after completed therapy. The current definition of a successful response to treatment (three or fewer unformed bowel movements [UBMs] per day for 1-2 days) has not been validated, does not reflect CDI management, and could impair assessments for successful treatment at 30 days. We propose new definitions to optimise trial design to assess sustained response. Primarily, we suggest that the initial response at the end of treatment be defined as (1) three or fewer UBMs per day, (2) a reduction in UBMs of more than 50% per day, (3) a decrease in stool volume of more than 75% for those with ostomy, or (4) attainment of bowel movements of Bristol Stool Form Scale types 1-4, on average, by day 2 after completion of primary CDI therapy (ie, assessed on day 11 and day 12 of a 10-day treatment course) and following an investigator determination that CDI treatment can be ceased.

Identification of a novel virulence factor in Clostridium difficile that modulates toxin sensitivity of cultured epithelial cells.

Two glucosylating toxins named toxins A and B play a role in the pathogenesis of Clostridium Difficile infection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of other C. difficile factors remains unknown. We utilized culture filtrate of C. difficile with a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed "actin aggregate," at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate of C. difficile ATCC 9689 and was renamed Srl. Purified glutathione S-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity of C. difficile toxins.

Evaluation of a simultaneous detection kit for the glutamate dehydrogenase antigen and toxin A/B in feces for diagnosis of Clostridium difficile infection.

Rapid detection kits for toxin A/B in feces are widely used as a diagnostic tool for Clostridium difficile infection (CDI). Their low sensitivity, however, has been considered a problem. In this study, we evaluated a new rapid diagnostic kit for simultaneous detection of the glutamate dehydrogenase (GDH) antigen and toxin A/B, C. DIFF QUIK CHEK COMPLETE. A total of 60 stool specimens from 60 patients with antibiotic-associated diarrhea were examined. Using C. difficile culture as the reference method, the GDH portion of this kit indicated a sensitivity, specificity, and negative predictive value of 100, 93.3, and 100%, respectively. The toxin A/B portion showed a sensitivity and specificity of 78.6 and 96.9%, respectively, compared to the culture results of toxin B-positive C. difficile (toxigenic culture). Of the 23 specimens that showed "dual positives" for GDH and toxin A/B, 22 were toxigenic culture positive, whereas C. difficile culture was negative in all the 28 specimens that showed "dual negatives" for GDH and toxin A/B. Of the nine "GDH-positive and toxin A/B-negative" specimens, six exhibited positive results by toxigenic culture. Results showing "dual positives" and "dual negatives" for GDH and toxin A/B can be reported as "true positive" and "true negative," respectively, whereas additional testing for confirmation, such as toxigenic culture, is required for specimens with discrepant results. Diagnostic algorithms, utilizing the simultaneous detection kit for GDH and toxin A/B as an initial screening test, may be useful for accurate and efficient diagnosis of CDI as well as the control of healthcare-associated infections.

A retrospective study of the epidemiology of Clostridium difficile infection at a University Hospital in Japan: genotypic features of the isolates and clinical characteristics of the patients.

Clostridium difficile is a major cause of antibiotic-associated diarrhea and frequently results in healthcare-associated infections. The epidemiology of C. difficile infection (CDI), including the prevalent polymerase chain reaction (PCR) ribotypes and the clinical characteristics of the patients, is not well known in Japan, compared to the situation in the United States and Europe. We performed PCR ribotyping of C. difficile isolates from 71 consecutive patients with CDI at a University Hospital over a 3-year period and investigated the clinical features of those patients. CDI was diagnosed when a patient with diarrhea or colitis was found to have toxin B-positive C. difficile with no other enteropathogenic microorganisms. Toxin A-positive, toxin B-positive, binary toxin-positive (A(+)B(+)CDT(+)) strains; toxin A-positive, toxin B-positive, binary toxin-negative (A(+)B(+)CDT(-)) strains; and toxin A-negative, toxin B-positive, binary toxin-negative (A(-)B(+)CDT(-)) strains were isolated from 4, 58, and 9 patients, respectively, indicating that infections with binary toxin-positive strains were uncommon (5.6%). PCR ribotyping of the isolates demonstrated that among the 71 strains, 20 different PCR ribotypes were identified and that types smz, yok, and hr were predominant (19, 14, and 13 isolates, respectively), all of which were A(+)B(+)CDT(-). No specific time periods or wards were found to be associated with the three types; PCR ribotyping analysis clearly showed that the three types spread almost evenly in all wards for the 3 years studied. Comparative analysis of the clinical characteristics of patients harboring the three C. difficile types indicated that the duration of CDI was longer in the yok group than in the hr group. PCR ribotyping, which is easy to perform, appears to give us useful information to trace CDI cases in clinical settings. Further, the analysis of a large number of CDI cases may allow evaluation of the possible relationship between specific C. difficile types and the clinical features of patients.

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.

[Antibiotic-associated diarrhea due to Clostridium perfringens].

A 40-year-old man undergoing allo-hematopoietic stem cell transplantation for chronic myelogenous leukemia and developing diarrhea was administered prophylactic antibiotics including levofloxacin, fluconazole, cotrimoxazole, and vancomycin. Stool specimens were positive for toxin A in enzyme immunoassay but negative for toxin B in cell culture assay with a neutralization test, indicating that toxin A detection was false-positive. Stool culture yielded enterotoxin producing Clostridium perfringens, not Clostridium difficile. Polymerase chain reaction (PCR) detected the gene encoding C. perfringens enterotoxin in DNA extracted from stool specimens, but not the toxin B gene. Laboratory tests for enterotoxic C. perfingens may therefore be necessary for diagnosing antibiotic-associated diarrhea when culture for C. difficile is negative.

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.