Cost-Effectiveness Analysis of Maternal Respiratory Syncytial Virus Vaccine in Protecting Infants from RSV Infection in Japan.

INTRODUCTION: Respiratory syncytial virus (RSV) is one of the major causes of respiratory tract infections among children. Until recently, the monoclonal antibody palivizumab was the only RSV prophylaxis available in Japan. In 2024, the bivalent RSV prefusion F protein-based (RSVpreF) vaccine was approved for the prevention of RSV infection in infants by active immunization of pregnant women. In this study, we assessed the cost-effectiveness of a combined strategy of RSVpreF vaccine and palivizumab in Japanese setting. METHODS: Using a Markov model, we evaluated prevented cases and deaths of medically attended RSV infections from birth to age 11 months for each of the three healthcare settings: inpatient (hospitalization), emergency department visits, and outpatient visits. Incremental cost-effectiveness ratios (ICERs) were calculated from economic outcomes (intervention costs, medication costs, and productivity losses) and quality-adjusted life years (QALYs). Further, we calculated the maximum price of RSVpreF vaccine within which the program would be cost-effective. RESULTS: In comparison with the current prophylaxis (palivizumab alone), a combined prophylaxis of year-round RSVpreF vaccination of pregnant women and palivizumab prescription for premature infants born in < 32 weeks gestational age (wGA) and all infants with high risk prevented 14,382 medically attended cases of RSV (hospitalization, 7490 cases; emergency department, 2239 cases; outpatient, 4653 cases) and 7 deaths, respectively. From a healthcare payer perspective, when the price of RSVpreF vaccine was equal to or less than ¥23,948 (US $182), a combination prophylaxis was cost-effective under the ICER threshold of ¥5 million per QALY. The other combination prophylaxis of year-round RSVpreF vaccination and palivizumab prescription of premature born in < 32 wGA regardless of risk in infants was a dominant strategy (more effective and less costly). CONCLUSION: A combined prophylaxis of year-round RSVpreF vaccine and palivizumab could be a cost-effective strategy to protect neonates throughout the infant stage (< 1 years old) in Japan.

Cost-effectiveness analysis of adult pneumococcal conjugate vaccines for pneumococcal disease in Japan.

BACKGROUND: The 23-valent pneumococcal polysaccharide vaccine (PPSV23) is used in the Japanese National Immunization Program for older adults and adults with increased risk for pneumococcal disease, however, disease incidence and associated burden remain high. We evaluated the cost-effectiveness of pneumococcal conjugate vaccines (PCVs) for adults aged 65 years and high-risk adults aged 60-64 years in Japan. RESEARCH DESIGN AND METHODS: Using a Markov model, we evaluated lifetime costs using societal and healthcare payer perspectives and estimated quality-adjusted life-years (QALYs), and number of prevented cases and deaths caused by invasive pneumococcal disease (IPD) and non-IPD. The base case analysis used a societal perspective. RESULTS: In comparison with PPSV23, the 20-valent PCV (PCV20) prevented 127 IPD cases 10,813 non-IPD cases (inpatients: 2,461, outpatients: 8,352) and 226 deaths, and gained more QALYs (+0.0015 per person) with less cost (-JPY22,513 per person). All sensitivity and scenario analyses including a payer perspective analysis indicated that the incremental cost-effectiveness ratios (ICERs) were below the cost-effectiveness threshold value in Japan (JPY5 million/QALY). CONCLUSIONS: PCV20 is both cost saving and more effective than PPSV23 for adults aged 65 years and high-risk adults aged 60-64 years in Japan.

Cost-effectiveness analysis of 20-valent pneumococcal conjugate vaccine for routine pediatric vaccination programs in Japan.

BACKGROUND: The Japanese National Immunization Program currently includes the pediatric 13 valent pneumococcal conjugate vaccine (PCV13) to prevent pneumococcal infections. We aimed to evaluate the cost-effectiveness of 20-valent PCV (PCV20) as a pediatric vaccine versus PCV13. METHODS: A decision-analytic Markov model was used to estimate expected costs, quality-adjusted life-years (QALYs), and prevented cases and deaths caused by invasive pneumococcal disease, pneumonia, and acute otitis media over a ten-year time horizon from the societal and healthcare payer perspectives. RESULTS: PCV20 was dominant, i.e. less costly and more effective, over PCV13 (gained 294,599 QALYs and reduced Japanese yen [JPY] 352.6 billion [2.6 billion United States dollars, USD] from the societal perspective and JPY 178.9 billion [USD 1.4 billion] from the payer perspective). Sensitivity and scenario analyses validated the robustness of the base scenario results. When comparing PCV20 with PCV13, the threshold analysis revealed an incremental cost-effectiveness ratio that was within the threshold value (JPY 5 million/QALY) at a maximum acquisition cost of JPY 74,033 [USD 563] (societal perspective) and JPY 67,758 [USD 515] (payer perspective). CONCLUSIONS: As a pediatric vaccine, PCV20 was dominant over PCV13 regardless of the study perspective.

Cost-effectiveness analysis of abrocitinib compared with standard of care in adult moderate-to-severe atopic dermatitis in Japan.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a significant clinical, economic, and human burden. The JAK1 Atopic Dermatitis Efficacy and Safety (JADE) program's Phase 3 trials demonstrated that as a treatment for moderate-to-severe AD in adults with previous exposure to immunotherapy, abrocitinib showed superior efficacy and safety compared with standard of care (SoC), consisting of topical corticosteroids. This study assessed the cost-effectiveness of abrocitinib with SoC versus SoC alone for this patient population in Japan from a societal perspective. A hybrid decision tree and Markov model were used to capture the initial treatment and long-term maintenance phases. Clinical inputs at 16 weeks were obtained through a Bayesian network meta-analysis of four pivotal trials from the JADE program. Clinical inputs at 52 weeks were derived from the JADE EXTEND trial. Response-specific utility inputs were obtained from published literature. Resource use, costs, and productivity inputs were gathered from Japanese claims analysis, literature, public documents, and expert opinion. Costs and quality-adjusted life years (QALYs) were discounted at 2.0% per year and incremental cost-effectiveness ratios (ICERs) were calculated. Sensitivity and scenario analyses were performed to validate the base case results and explore a payer perspective. Over a lifetime horizon and with the base-case societal perspective, abrocitinib produced a mean gain of 0.75 QALYs, incremental costs of JPY (¥) 2 270 386 (USD [$] 17 265.6), and a resulting ICER of ¥3 034 514 ($23 076.5) per QALY compared with SoC. From a payer perspective, the incremental costs increased to ¥4 476 777 ($34 044.4), with an ICER of ¥5 983 495 ($45 502.6) per QALY. The results were most sensitive to treatment-specific, response-based utility weights, drug costs, and productivity-related inputs. From a Japanese societal perspective, abrocitinib demonstrated superior QALYs and with a willingness-to-pay threshold of ¥5 000 000 ($38 023.4) per QALY, can be considered cost-effective compared with SoC as a treatment for moderate-to-severe AD in adult patients with previous immunosuppressant exposure.

Cost-effectiveness analysis of COVID-19 booster vaccination with BNT162b2 in Japan.

BACKGROUND: The aim of this study was to evaluate the public health and economic impact of the COVID-19 booster vaccination with BNT162b2 in Japan during an Omicron-dominant period from early 2022. RESEARCH DESIGN AND METHODS: A combined cohort Markov decision tree model estimated the cost-effectiveness of annual or biannual booster vaccination strategies compared to no booster vaccination for those aged 65 years and above, and those aged 60-64 years at high risk as the base case. The societal perspective was primarily considered. We also examined other target populations with different age and risk groups. Sensitivity and scenario analyses with alternative inputs were performed. RESULTS: Annual and biannual vaccination strategies were dominant from the societal perspective in the base case. Incremental Cost Effectiveness Ratios (ICERs) from the payer perspective were JPY 1,752,499/Quality Adjusted Life Year (QALY) for annual vaccination and JPY 2,831,878/QALY for biannual vaccination, both less than the threshold value in Japan (JPY 5 million/QALY). The results were consistent even when examining other target age and risk groups. All sensitivity and scenario analyses indicated that ICERs were below JPY 5 million/QALY. CONCLUSIONS: Booster vaccination with the COVID-19 vaccine BNT162b2 is a dominant strategy and beneficial to public health in Japan.

Degree of Alignment Between Japanese Patients and Physicians on Alopecia Areata Disease Severity and Treatment Satisfaction: A Real-World Survey.

INTRODUCTION: Alopecia areata (AA) is characterized by non-scarring scalp and/or body hair loss and can negatively impact patient mental health. Data are limited on the alignment of patient and physician perceptions of AA severity with each other and with Japanese Dermatological Association (JDA) guideline criteria, and of patient-physician alignment on treatment satisfaction. Therefore, we performed analyses to compare JDA severity groupings with patient-physician alignment on disease severity and to explore treatment satisfaction in AA in Japan. METHODS: Data were drawn from the Adelphi AA Disease Specific Programme (DSP)™, a real-world survey of physicians and patients with AA in Japan conducted January-March 2021. Patients and physicians reported patient AA severity as mild, moderate or severe based on their subjective judgement. Patients were then categorized into five hair loss severity groups according to JDA criteria (S1-5), and patient-physician pairs were matched to assess alignment on severity and treatment satisfaction. RESULTS: Subjective patient- and physician-reported disease severity generally followed JDA severity groupings. The percentage of patient-physician alignment on severity recognition was 76.3% in the overall population. In misaligned pairs, 20.2%, 14.5%, 7.3%, 25.0% and 0.0% of physicians rated disease as more severe than patients in S1, S2, S3, S4 and S5, respectively. Regarding treatment satisfaction, patient-physician alignment was 57.6% in the overall population. In S5, 46.2% of physicians reported being less satisfied than patients. Both physicians and patients cited lack of efficacy as the main reason for dissatisfaction. Of 221 patients, 39.8% and 29.9% were categorized as borderline-abnormal cases for anxiety and depression, respectively. CONCLUSIONS: This study highlights previously unreported patient-physician misalignment on disease severity, level of treatment dissatisfaction and unmet needs due to the lack of effective treatment. Further study on how improvement of the misalignment between physicians and patients could increase both patient and physician satisfaction with treatment and improve the quality of life for patients with AA.

Economic burden of alopecia areata: A study of direct and indirect cost in Japan using real-world data.

Alopecia area (AA) is a common autoimmune disorder, characterized by hair loss. Although its impact on quality of life is fairly well understood, studies on the economic impact of AA are limited. The aim of this study was to quantify the personal and nationwide economic burden of AA in Japan. Data were drawn from the Adelphi AA Disease Specific Programme (DSP)™, a real-world, cross-sectional survey with retrospective data collection, of Japanese physicians and patients with AA. The study was conducted in 2021, before the approval of Janus kinase inhibitors for AA. Physicians and their consulting AA patients completed questionnaires regarding disease severity, treatment, and AA-related costs. The Work Productivity and Activity Impairment questionnaire was used to evaluate the impact of AA on patients' work and activity. Nationwide estimates of cost and productivity loss were extrapolated from collected patient data. A total of 50 physicians provided data on 235 patients; 58.7% were female, mean ± SD age was 41.1 ± 11.8 years, and mean physician-estimated scalp hair loss was 40.4% ± 30.2%. Prescription medication use was high (92.3% of patients), but the use of over-the-counter medication was low, at 8.7%. Mean cost to patients for medication was ¥ 4263 (US$ 32.42) per month. Productivity while at work (presenteeism) was significantly impaired (23.9% ± 25.7%), but absenteeism was low (0.9% ± 2.8%). The total nationwide cost of AA was estimated at 112.7 billion yen (US$ 857 million), of which 88.1 billion yen (78.2%) was due to productivity loss. Over 2 million days per year of activity time were estimated to be lost due to AA. Thus, despite not being a physically limiting disease, AA has a significant impact in terms of cost and time, both on a personal and national level. These data highlight the need for more targeted interventions to reduce the effects of AA on the Japanese economy.

Systematic Literature Review of the Use of Productivity Losses/Gains in Cost-Effectiveness Analyses of Immune-Mediated Disorders.

INTRODUCTION: In light of the lack of an agreed international standard for how to conduct cost-effectiveness analyses (CEAs), including cost-utility analyses (CUAs) from a societal perspective, there is uncertainty regarding to what extent the inclusion of productivity losses/gains in economic evaluations can affect cost-effectiveness results and subsequently decisions on whether to recommend new health technologies. To investigate this, we conducted a systematic review of CEAs and CUAs of drug-based therapies for a set of chronic immune-mediated disorders to understand how cost elements and calculation methods related to productivity losses/gains are used, examine the impact on the incremental cost-effectiveness ratio (ICER) of including productivity costs, and explore factors that affect the inclusion of productivity loss. METHODS: Databases (MEDLINE® In-process, MEDLINE, Embase and Cochrane Library) were searched from January 2010 to October 2020 by two independent reviewers for all CEAs and CUAs in adults with any of the following conditions: ankylosing spondylitis, chronic idiopathic urticaria, Crohn's disease, fibromyalgia, juvenile idiopathic arthritis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and ulcerative colitis. Relevant study data were extracted and evidence was synthesized for both qualitative and quantitative analysis. Productivity cost elements including absenteeism, presenteeism, unemployment/early retirement, premature mortality and informal care were extracted, along with the method used to determine them. A multivariate analysis was performed to identify factors associated with the inclusion of productivity loss. RESULTS: Our searches identified 5016 records, culminating in 198 unique studies from 234 publications following screening. Most of the studies investigated rheumatoid arthritis (37.0%) or psoriasis (32.0%). The majority were CUAs, with some including both a CEA and a CUA (73.0%). Most studies used a payer perspective only (28.5%) or a societal perspective only (21.0%). Of the 49 studies incorporating productivity losses/gains, 42 reported the type of cost element used; all of these used patient absenteeism, either alone or in addition with other elements. Only 16 studies reported the method used to value productivity changes, of which eight used a human capital approach, four used a friction cost approach and four used both approaches. Twenty-eight of the 49 studies (57.1%) reported inclusion of productivity losses/gains as contributing to more favourable cost-effectiveness outcomes and ICERs, while 12 (24.5%) reported no substantial impact. On the basis of a multivariate analysis, rheumatoid arthritis as the target disease had a statistically significant association with the inclusion of productivity loss compared with psoriasis and inflammatory bowel disease. CONCLUSIONS: The results of our review suggest that incorporating productivity cost elements may positively affect cost-effectiveness outcomes in evaluations of therapeutics for immune-mediated disorders. Our work highlights the continued need for clarity when reporting how CEAs and CUAs in this disease area are conducted, in order to better inform healthcare decision-making.

A Systematic Literature Review of Economic Evaluations and Cost Studies of the Treatment of Psoriasis, Atopic Dermatitis, and Chronic Urticaria.

INTRODUCTION: Psoriasis (PSO), atopic dermatitis (AD), and chronic urticaria (CU) are common manifestations of immunological skin and subcutaneous conditions and have been shown to have a substantial impact on the quality of life of patients. The cost of treating those conditions can also be high, as the use of biologic treatments has become more common for moderate to severe patients. In this review, we examine characteristics of economic evaluations and cost studies conducted for the three conditions. METHODS: A literature search was conducted using PubMed, Embase, and the Cochrane Library from January 1, 2016 to October 26, 2020 to identify economic evaluations where the cost of one or more drug treatment was evaluated and cost studies covering any intervention type. Each database was searched using keyword and MeSH terms related to treatment costs (e.g., health care cost, drug cost, etc.) and each condition (e.g., PSO, AD, eczema, CU, etc.). RESULTS: A total of 123 studies were reviewed, including 104 studies (85%) of PSO (including psoriasis, plaque psoriasis, psoriatic arthritis, and psoriasis vulgaris), 14 studies (11%) of AD, and 5 studies (4%) of CU. Seventy-two studies (59%) reviewed reported the inclusion of biologic treatments, 10 studies (8%) did not include biologic treatments, and 41 studies (33%) did not report whether or not a biologic treatment was included. While nearly all studies (98%) included direct costs, only 22 studies (18%) included indirect costs. CONCLUSIONS: Economic evaluations for AD and CU may be needed in order to better understand the value of new treatments. Moreover, a clearer delineation for biologic treatments and indirect costs (i.e., productivity losses and gains) may be required.

Health-related quality of life in patients with alopecia areata: Results of a Japanese survey with norm-based comparisons.

Alopecia areata (AA) is a non-scarring hair loss disorder affecting approximately 2% of the global population. AA is reported to have a significant negative impact on the emotional and psychological well-being of the patients. This study aimed to evaluate the health-related quality of life (HRQoL) of Japanese patients with AA in comparison to the Japanese population norms (national standard values for Japanese) using Short Form Health Survey 36 Item Version 2.0 (SF-36v2). The study also aimed to access the negative effect of AA on patients' daily lives and the proportion of patients having anxiety and/or depression. This cross-sectional, non-interventional web-based survey study included 400 participants aged 17-84 years currently suffering from medically diagnosed AA. The assessment tools integrated in the online questionnaire included SF-36v2, the Dermatology Life Quality Index (DLQI), and the Hospital Anxiety and Depression Scale (HADS). All outcome measures from the tools were evaluated across the study population. SF-36v2 subscale scores for patients with AA revealed lower scores specifically for mental health (45.7 ± 10.1 points), social functioning (45.8 ± 10.9 points), vitality (46.2 ± 9.8 points), and role emotional (46.9 ± 11.6 points) as compared to the Japanese population norms of 50 ± 10 points each. The DLQI questionnaire-based analysis indicated that 32.1% of respondents showed a moderate to extremely large effect on their lives; and HADS-A (anxiety) and HADS-D (depression) scores categorized 46.0% and 41.8% respondents as doubtful-to-definite cases, respectively. Multivariate linear regression revealed that hair loss range, age, comorbidities, and depression significantly worsened DLQI scores. In conclusion, the results of this survey demonstrated that a significant decrease in the HRQoL scores was observed in Japanese patients with AA in comparison with the national norms. Hence, emphasis on mental health is crucial for AA management.

Burden of illness, medication adherence, and unmet medical needs in Japanese patients with atopic dermatitis: A retrospective analysis of a cross-sectional questionnaire survey.

Atopic dermatitis (AD) negatively affects patients' daily lives. Poor medication adherence is a major barrier to treatment success. However, factors causing patients' poor adherence are unclear. This study aimed to identify factors associated with improvement of medication adherence in Japanese patients with AD and to evaluate illness burden and unmet medical needs for AD. We retrospectively analyzed Web-based questionnaire surveys conducted in 2018 in patients with AD aged 15 years and above who had been in- or outpatients within the past year from the survey. Quality of life using the EuroQol 5-Dimension (EQ-5D), and work productivity and activity impairment using Work Productivity and Activity Impairment Questionnaire (WPAI) were compared between patients and matched controls who had not visited a hospital for any disease within the past year. Subpopulation analysis was performed to explore factors affecting medication adherence. Unmet medical needs in AD treatment were identified by the percentage of patients who rated issues on the questionnaire as important but who were unsatisfied with them. In this study, we identified 1739 patients with AD. The scores of EQ-5D and WPAI showed that patients had statistically lower quality of life and higher impairment of work and activities than controls. High medication adherence scores were seen in patients with high health literacy levels and those who were well satisfied with communication with health-care providers, information received from them, or explanations of AD. Current unmet medical needs for AD were medical treatment costs, ease of hospital visits and explanations about disease prognosis. Patients tended to put a higher priority on communication with physicians than on that with nurses and pharmacists. In conclusion, we have identified patients' higher health literacy levels and satisfaction with the communication with their health-care provider as potential factors to improve medication adherence.

A comparative study of PF-06438179/GP1111 (an infliximab biosimilar) and reference infliximab in patients with moderate to severe active rheumatoid arthritis: A subgroup analysis.

AIM: PF-06438179/GP1111 (PF-SZ-IFX) is a biosimilar of reference infliximab (Remicade® ). This analysis compared the efficacy of PF-SZ-IFX and reference infliximab sourced from the European Union (IFX-EU) in patient subgroups from a randomized, comparative study of PF-SZ-IFX versus IFX-EU. METHODS: Patients with rheumatoid arthritis were randomized 1:1 to PF-SZ-IFX (n = 324) or IFX-EU (n = 326); study drug (3 mg/kg) was administered intravenously at weeks 0, 2, and 6, then every 8 weeks thereafter. Subgroup analyses of efficacy endpoints such as American College of Rheumatology criteria for ≥20% clinical improvement (ACR20), change in high-sensitivity C-reactive protein (hs-CRP), and change in Disease Activity Score in 28 joints, four components based on hs-CRP (DAS28-CRP) at weeks 14 and 30 were performed by age, gender, race, region, immunogenicity status, and treatment history. RESULTS: Overall, ACR20 response rates as well as changes in DAS28-CRP and hs-CRP at week 14 were similar between PF-SZ-IFX and IFX-EU within the subgroups of age, gender, race, region, treatment history, and immunogenicity status. Results to week 30 support overall similarity in efficacy between the two treatment arms in all subgroups. CONCLUSION: Overall, PF-SZ-IFX and IFX-EU were similar in efficacy within the analyzed subgroups of age, gender, race, region, treatment history, and immunogenicity status. The efficacy results from these subgroup analyses were aligned with the previously described results for the overall population up to week 30.

A PCR-RFLP assay to detect and type cytolethal distending toxin (cdt) genes in Campylobacter hyointestinalis.

Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

A new variant of cytolethal distending toxin in a clinical isolate of Campylobacter hyointestinalis.

Increasing numbers of Campylobacter hyointestinalis have been isolated from humans and animals with gastroenteritis, although the virulence mechanism of this species remains largely unknown. Here, we show that C. hyointestinalis isolated from a patient with diarrhoea in Thailand produced a novel variant of cytolethal distending toxin (CDT). Sequencing of a 13 965 bp genomic region of C. hyointestinalis carrying the genes coding for Ch-CDT revealed three ORFs of 798, 804 and 537 bp, which code for the Ch-CdtA, Ch-CdtB and Ch-CdtC subunits, respectively. The deduced amino acid sequence of Ch-CdtA showed ∼38.9 % homology with the CdtA of Campylobacter coli, but sequences of Ch-CdtB and Ch-CdtC were homologous to CdtB (65.7 %) and CdtC (33.1 %) of Campylobacter upsaliensis, respectively. Filter-sterilized sonic lysate of C. hyointestinalis demonstrated distension and death of HeLa cells by arresting the cell cycle at the G(2)/M phase and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. Rabbit antiserum raised against recombinant Ch-CdtB was not reactive against the recombinant CdtB protein of Campylobacter jejuni. A reconstituted Ch-CDT holotoxin prepared using each of the recombinant subunit proteins demonstrated distension and death of HeLa cells, suggesting that the C. hyointestinalis isolate indeed produced functionally active Ch-CDT. Furthermore, the immunological distinctiveness of the Ch-CDT produced by C. hyointestinalis and the increasing prevalence of the species in patients and animals with gastroenteritis suggest that this species may be an important emerging zoonotic pathogen.

A PCR-RFLP assay for the detection and differentiation of Campylobacter jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis.

Although Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of human gastrointestinal diseases, other Campylobacter species are also involved in human and animal infections. In this study, we developed a cytolethal distending toxin (cdt) gene-based PCR-RFLP assay for the detection and differentiation of C. jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis. Previously designed common primers, which can amplify the cdtB gene of C. jejuni, C. coli and C. fetus, were used for detecting seven Campylobacter species and differentiating between them by restriction digestion. The PCR-RFLP assay was validated with 277 strains, including 35 C. jejuni, 19 C. coli, 20 C. fetus, 24 C. hyointestinalis, 13 C. lari, 2 C. helveticus, 22 C. upsaliensis, 3 other Campylobacter spp. and 17 other species associated with human diseases. Sensitivity and specificity of the PCR-RFLP assay were 100 % except for C. hyointestinalis (88 % sensitivity). Furthermore, the PCR-RFLP assay successfully detected and differentiated C. jejuni, C. coli and C. fetus in clinical and animal samples. The results indicate that the PCR-RFLP assay is useful for the detection and differentiation of seven Campylobacter species important for human and animal diseases.

Simple PEG conjugation of SPIO via an Au-S bond improves its tumor targeting potency as a novel MR tumor imaging agent.

Gold/iron oxide magnetic nanoparticles are hybrid nanoparticles containing a core of magnetic iron oxide and surface colloidal gold, which allows for various biomaterials to be immobilized on the surface of the iron oxide nanoparticles via colloidal gold. Here, we developed a novel magnetic resonance (MR) imaging agent to broaden the MR tumor-imaging spectrum of superparamagnetic iron oxide nanoparticles (SPIO), e.g., Feridex(), a clinical MR imaging agent for diagnosing liver cancer. Au/Feridex was synthesized by electron beam irradiation, and thiol-modified poly(ethylene glycol) (PEG-SH) was easily conjugated to its surface via an Au-S bond without the need for any chemical reactions. PEG conjugation of Au/Feridex enhanced its accumulation in Meth-A tumor tissue and decreased its accumulation in normal liver tissue. In addition, MRI using PEG-Au/Feridex, in contrast to MRI using unmodified Au/Feridex and Feridex, detected B16BL6 and Meth-A tumor tissues in vivo. This finding indicates that PEG-Au/Feridex is useful for diagnosing various types of tumors. In addition, because the synthesis of PEG-Au/Feridex is simple and high yields are easily produced, PEG-modified SPIO for tumor diagnosis can be prepared on an industrial scale with low cost.

Direct cell entry of gold/iron-oxide magnetic nanoparticles in adenovirus mediated gene delivery.

Gold/iron-oxide MAgnetic Nanoparticles (GoldMAN) imparts useful magnetic properties to various biomolecules. Gold nanoparticles immobilized on the surface of magnetic nanoparticles allow for the conjugation of biomolecules via an Au-S bond. Here, we present a practical application by utilizing GoldMAN and a magnetic field to induce intracellular transduction. This method has great potential for application of the adenovirus gene delivery vector (Ad), widely used for in vitro/in vivo gene transfer, to Ad-resistant cells. We demonstrated that Ad was easily immobilized on GoldMAN and the Ad/GoldMAN complex was introduced into the cell by the magnetic field, which increased gene expression over 1000 times that of Ad alone. The GoldMAN penetrated the plasma membrane directly, independent of the cell-surface virus receptors and endocytosis pathway. This mechanism will contribute to improve the gene expression efficiency of Ad. This technology is a useful tool for extending Ad tropism and enhancing transduction efficiency. GoldMAN also makes possible the effective use of various biomolecules within the cell because of its interesting cell-entry mechanism.