Clinical phase II and III studies of an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform.

BACKGROUND: We have developed an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform (KD-295). OBJECTIVES: In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double-blind, randomized, parallel-group comparison study and the phase III study was conducted in an open-label, non-randomized, uncontrolled study. METHODS: Healthy adult volunteers aged 20 - 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD-295 intramuscularly twice with a 21-day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 µg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 µg HA + 1/2AS03), HA (7.5 µg HA + AS03), and HB (7.5 µg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated. RESULTS: In the phase II study, all four vaccine formulations were well-tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well-tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines. CONCLUSIONS: These data indicate that the MA formulation of KD-295 was well-tolerated and highly immunogenic and it can be considered a useful pandemic and pre-pandemic influenza vaccine.

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.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

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.

Impact of regional functional ischemia on global coronary flow reserve in patients with stable coronary artery disease.

BACKGROUND: Global coronary flow reserve (g-CFR) provides powerful prognostic information. The relationship between g-CFR and the regional physiological indices of fractional flow reserve (FFR), coronary flow reserve (r-CFR), and the index of microcirculatory resistance remains undetermined. This study aimed to assess the relationship between regional and global physiological indices and determinants of cardiovascular magnetic resonance imaging (CMR)-derived g-CFR. METHODS: A total of 151 patients with single de novo intermediate to stenotic epicardial lesions referred for diagnostic invasive coronary angiography who underwent phase-contrast cine CMR of the coronary sinus (CS) were included. g-CFR was calculated as the ratio of hyperemic and resting CS flow (CSF). Regional and global physiological parameters were compared, and determinants of g-CFR were assessed. RESULTS: There was a weak linear relationship between FFR and g-CFR (R2=0.04, p=0.013), while r-CFR and g-CFR, or combinations of the other regional-global indices were not significantly correlated. When patients were divided into two groups by FFR of 0.80, there were also no significant differences in global physiological indices between the groups (FFR≤0.80 vs. FFR>0.80; g-CFR: 2.73 vs. 2.61, p=0.48; hyperemic CSF: 3.32 vs. 3.52ml/min/g, p=0.84). Higher high-sensitivity cardiac troponin-I (hs-cTnI) and higher resting CS flow were independently associated with impaired g-CFR, and the combination could efficiently identify patients with g-CFR<2.0. CONCLUSIONS: Given weak relationship among global and regional physiological indices, these indices may provide complementary efficacy for prognostication in patients with single-vessel stable coronary artery disease. Combination of hs-cTnI and resting CS flow could estimate g-CFR without pharmacological hyperemic induction.

Optical Coherence Tomography-Defined Plaque Vulnerability in Relation to Functional Stenosis Severity and Microvascular Dysfunction.

OBJECTIVES: This study sought to investigate the relationship of unstable plaque features with physiological lesion severity and microvascular dysfunction. BACKGROUND: The functional severity of epicardial lesions and microvascular dysfunction are both related to adverse clinical outcomes. METHODS: We investigated 382 de novo intermediate and severe coronary lesions in 340 patients who underwent optical coherence tomography, fractional flow reserve (FFR), and index of microcirculatory resistance (IMR) examinations. Lesions were divided into tertiles based on either FFR or IMR values. The optical coherence tomography findings were compared among the tertiles of FFR and IMR. Each tertile was defined as follows: FFR-T1 (FFR <0.74), FFR-T2 (0.74 ≤ FFR ≤0.81), and FFR-T3 (FFR >0.81); and IMR-T1 (IMR ≥25), IMR-T2 (15 < IMR <25), and IMR-T3 (IMR ≤15). RESULTS: No significant relationship was observed between FFR and IMR. The prevalence of optical coherence tomography-defined thin-cap fibroatheroma (TCFA) was significantly greater in IMR-T1 than in IMR-T2 and IMR-T3. An overall significant difference in the prevalence of TCFAs was detected among FFR tertiles, although no pairwise comparison revealed statistical significance. The prevalence of ruptured plaque was significantly greater in IMR-T1 than in IMR-T2 and IMR-T3, although no significant difference was observed between FFR tertiles. Multivariate analysis showed that FFR and IMR were independent predictors of the prevalence of TCFAs (odds ratio: 0.036; 95% confidence interval: 0.004 to 0342; p = 0.004; and odds ratio: 1.034; 95% confidence interval: 1.014 to 1.054; p = 0.001, respectively). CONCLUSIONS: Lower FFR and higher IMR values were independent predictors of the presence of a TCFA in angiographically intermediate-to-severe stable lesions or nonculprit lesions in acute coronary syndrome.

Impact of Elective Percutaneous Coronary Intervention on Global Absolute Coronary Flow and Flow Reserve Evaluated by Phase-Contrast Cine-Magnetic Resonance Imaging in Relation to Regional Invasive Physiological Indices.

BACKGROUND: Few studies have documented changes in global absolute coronary blood flow and global coronary flow reserve after percutaneous coronary intervention (PCI) in relation to regional physiological measures. Phase-contrast cine-magnetic resonance of the coronary sinus is a promising approach to quantify global absolute coronary blood flow. We aimed to assess the impact of elective PCI on global absolute coronary blood flow and global coronary flow reserve by quantifying coronary sinus flow (CSF) using phase-contrast cine-magnetic resonance in relation to regional physiological indices. METHODS AND RESULTS: We prospectively studied 54 patients with stable angina undergoing elective PCI for a single proximal lesion. Phase-contrast cine-magnetic resonance was used to assess CSF and CSF reserve at rest and during maximum hyperemia, before and after PCI. Regional physiological indices were obtained during PCI. A complete data set was obtained in 50 patients. Hyperemic CSF increased significantly after PCI (pre-PCI, 230.2 [167.4-282.8] mL/min; post-PCI, 267.4 [224.1-346.2] mL/min; P<0.01), although 12 patients (24.0%) showed a decrease, despite successful PCI and improved fractional flow reserve. CSF reserve numerically, albeit not statistically significant (P=0.19), increased from 2.65 (1.95-3.96) to 2.98 (2.13-4.32). Patients with decreased CSF after PCI were associated with significantly greater pre-PCI hyperemic CSF, lower global coronary vascular resistance, lower regional microcirculatory resistance, and higher fractional flow reserve (all P<0.01). CONCLUSIONS: Fractional flow reserve-guided PCI in patients with single de novo lesions was associated with increased absolute hyperemic CSF, although 24% of patients showed decreased hyperemic CSF, despite successful and uncomplicated PCI. The present approach combining regional and global physiological assessments may provide a novel insight into the dynamic behavior of the coronary hemodynamics and microvascular function after PCI.

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.

Absolute Myocardial Blood Flow After Elective Percutaneous Coronary Intervention Evaluated on Phase-Contrast Cine Cardiovascular Magnetic Resonance Imaging.

BACKGROUND: Few studies have documented changes in myocardial blood flow (MBF) after percutaneous coronary intervention (PCI). Phase-contrast cine cardiovascular MRI (PC-CCMR) of the coronary sinus (CS) is a promising approach to quantify MBF. The aim of this study was to quantify CS flow (CSF) on PC-CCMR as a measure of volumetric MBF before and after elective PCI.Methods and Results:We prospectively studied 34 patients with stable angina undergoing elective PCI for a single de novo lesion. Breath-hold PC-CCMR of CS was acquired to assess CSF and coronary flow reserve (CFR) at rest and during maximum hyperemia both before and after PCI (median, 3 days before PCI and 10 days after PCI, respectively). In total, hyperemic CSF increased significantly after PCI (before PCI, median, 2.3 mL/min/g [IQR, 1.5-3.2 mL/min/g] after PCI, 3.0 [1.8-3.7] mL/min/g), although 13 patients (38.2%) had a decrease despite successful PCI and fractional flow reserve (FFR) improvement. Global CFR also significantly increased from a median of 2.5 (IQR, 1.5-3.5) to 3.4 (IQR, 2.1-4.2), whereas 12 patients had decreased CFR after PCI. Pre-PCI hyperemic CSF was the only independent factor of change in CSF following PCI. CONCLUSIONS: Serial PC-CCMR of CS as a measure of change in absolute MBF is feasible. Uncomplicated PCI does not necessarily increase hyperemic global MBF, despite regional FFR improvement.

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.

Relationship between optical coherence tomography-derived morphological criteria and functional relevance as determined by fractional flow reserve.

BACKGROUND: Although several previous studies have indicated that optical coherence tomography (OCT)-derived minimal lumen area (MLA) correlates with fractional flow reserve (FFR) severity, other morphologic criteria for functionally significant coronary stenosis assessed by FFR have not been fully elucidated. This study aimed to identify OCT-based morphological predictors of physiologically significant ischemia assessed by FFR in angiographically intermediate coronary lesions. METHODS: We investigated 194 de novo intermediate coronary lesions in 178 patients with stable angina pectoris who underwent OCT imaging and FFR measurement. The lesions were divided into two groups according to an FFR threshold: ischemia group, FFR <0.75 (n=69) and non-ischemia group, FFR ≥0.75 (n=125). Clinical characteristics, angiographic, and OCT findings were compared between these two groups. Predictors of significant ischemia defined as FFR <0.75 were identified by logistic regression analyses. RESULTS: Patient age, statin use, lesion in left anterior descending artery (LAD), OCT-derived MLA, and lipid volume index (LVI) (averaged lipid arc multiplied by lipid length) were independent predictors of FFR <0.75 in multivariate logistic regression analysis. Receiver operating characteristic analysis suggested that age <64 years old, OCT-derived MLA ≤1.39mm2, and LVI ≥733 are the best cut-off values for predicting FFR <0.75. Multiple logistic regression models, including the MLA combined with the LAD location, LVI, age, and statin use, provided superior predictive efficacy for physiologically significant ischemia compared with the model that only employed MLA. CONCLUSIONS: Information on OCT-derived LVI and lesion locations facilitates better identification of coronary lesions that cause ischemia than OCT-derived MLA analysis alone.

Prognostic values of blood pH and lactate levels in patients resuscitated from out-of-hospital cardiac arrest.

AIM: Early prediction of prognosis after out-of-hospital cardiac arrest (OHCA) remains difficult. High blood lactate or low pH levels may be associated with poor prognosis in OHCA patients, but these associations remain controversial. We compared blood lactate and pH levels in OHCA patients transferred to our hospital to measure their prognostic performance. METHODS: We investigated the associations between blood lactate and pH levels on admission and neurological outcomes in 372 OHCA patients who had a return of spontaneous circulation. RESULTS: Of the 372 OHCA patients, 31 had a favorable neurological outcome. Blood lactate levels were lower in patients with a favorable outcome than in those with an unfavorable outcome, but this difference did not reach statistical significance (82 ± 49 vs. 96 ± 41 mg/dL). However, pH levels were significantly higher in patients with a favorable outcome than in those with an unfavorable outcome (7.26 ± 0.16 vs. 6.93 ± 0.19, P < 0.001). The relative cumulative frequency distribution curve analysis showed the optimal cut-off points of lactate and pH to be approximately 80 mg/dL and 7.05, respectively. Sensitivity and specificity to predict a favorable outcome were 61% and 64% for lactate <80 mg/dL and 84% and 80% for pH >7.05, respectively. Areas under receiver-operating characteristic curves were significantly larger for pH than for lactate levels (P < 0.001). In multivariate analysis, pH >7.05 was an independent predictor for a favorable outcome. CONCLUSION: After OHCA, patients with a favorable outcome had lower lactate and higher pH levels than those with an unfavorable outcome, but pH level was a much better predictor for neurological outcome than lactate levels.

A clinical phase I study of an EB66 cell-derived H5N1 pandemic vaccine adjuvanted with AS03.

BACKGROUND: We conducted a phase I clinical trial of a cell culture-derived AS03-adjuvanted influenza vaccine containing HA antigen (A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2) derived from EB66 cells (KD-295). METHODS: Healthy male adult volunteers (20-40 years old, N=60) enrolled in the study were divided into 3 groups, the MA group (3.8 µg of HA+AS03), HA group (7.5 µg of HA+AS03), and 1/2 MA group (half the volume of the MA group), and received KD-295 intramuscularly twice with a 21-day interval. After administration of KD-295, adverse events, clinical laboratory parameters, and immune response to the vaccine strain and heterologous virus strains were evaluated. RESULTS: No severe adverse events leading to discontinuation of vaccine administration occurred. The vaccine was well-tolerated. There was no dose dependency in the rate, timing, or duration of the adverse events. Immunogenicity of the vaccines was evaluated by HI (hemagglutination inhibition) assay, which confirmed that the antibody response to the vaccine strain and heterologous strain in all groups met the three criteria for immunogenicity described in the Japanese guidelines for development of a pandemic prototype vaccine. We also measured the neutralizing antibody titers against several virus strains, and confirmed a significant rise in antibody levels to both the vaccine strain and heterologous strains. CONCLUSION: The EB66-derived H5N1 influenza vaccine adjuvanted with AS03 elicited a broad cross-reactive antibody response among H5N1 strains with acceptable reactogenicity. Therefore, KD-295 can be considered a useful pandemic and pre-pandemic influenza vaccine candidate.

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.

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.

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.

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.

Effects of simultaneous immunization of Haemophilus influenzae type b conjugate vaccine and diphtheria-tetanus-acellular pertussis vaccine on anti-tetanus potencies in mice, guinea pigs, and rats.

Haemophilus influenzae type b vaccine conjugated with tetanus toxoid (HibT) was licensed for use in childhood immunization in Japan in 2007. As adsorbed diphtheria-tetanus-acellular pertussis (DTaP) combined with HibT vaccine has not been introduced in Japan, DTaP and HibT vaccines are injected at separate sites with a similar immunization schedule. There are various interfering or stimulatory effects between components of combined vaccines contained in DTaP and HibT vaccines. In this study, we investigated the effect of HibT containing combination vaccines on anti-tetanus potencies by using animal models (mouse, guinea pig, and rat). HibT vaccine and HibT components of imported DTaP-HibT vaccine alone showed comparable or higher anti-tetanus potency than DTaP vaccine and DTaP-containing components of combination vaccines. Mixing these components before injection resulted in potencies greater than the sum of individual potencies. Injecting individual components at separate sites in animals resulted in potency roughly equivalent to the sum of the individual potencies. These results provide useful information regarding the use of HibT-containing multivalent vaccines in childhood immunization.

Experimental manufacture of equine antivenom against yamakagashi (Rhabdophis tigrinus).

Yamakagashi, Rhabdophis tigrinus, is a natricine snake widely distributed in eastern Asia. Severe bite cases, some with fatal outcomes, occur regularly in Japan. Because previous production of R. tigrinus antivenom in rabbits and goats was quite effective, we considered the experimental manufacture of a new antivenom against R. tigrinus in horses. This new antivenom could be used in emergency treatment of snakebite victims. Two horses were immunized with venom extracted from about 500 snakes. After an adequate increase of the antivenom titer, serum was collected and subjected to standard purification procedures for the manufacture of equine antivenoms. The purified immunoglobulin fraction was freeze-dried in 1,369 vials under optimum conditions for therapeutic use. This antivenom proved to be very potent in neutralizing the coagulant and hemorrhagic activities of the snake venom. In cases of severe bites, this antivenom was used and recognized as effective even after the occurrence of severe symptoms.