笹原 鉄平

In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices.

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

An 88-year-old man was treated with high-dose systemic steroid therapy for COVID-19 and idiopathic interstitial pneumonia months before admission to the hospital because of swelling and redness in his left arm. Cryptococcus neoformans was detected in his blood sample on day eight of admission, and despite antifungal therapy, he died on day 43. Clinicians should be vigilant about the risk of prolonged immunosuppression as a side effect of high-dose systemic steroid usage for COVID-19.

We describe a case of probable prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Alpha(B.1.1.7) variant shedding for 221 days from the diagnosis, in a healthy 20-year-old Japanese pregnant woman with a normal delivery. To our knowledge, this is the longest duration of SARS-CoV-2 shedding reported in an immunocompetent individual to date.

目的:近年途上国へ派遣される労働者が増加している。途上国は衛生環境が悪いことが多く、健康管理(感染症対策)が重要となる。赴任前に健康指導を受ける労働者、指導を行う産業医の認識を調査し、海外派遣労働者のニーズに合った、効果的な健康管理を提案することを目的とした。方法:途上国へ派遣された経験を持つ労働者を対象にインターネット調査を行い、属性、業種、滞在期間、勤務企業概要、赴任前推奨ワクチン種、ワクチン費用負担、感染症事前教育の有無・役立ち度・理解度、持参薬の有無、自社の感染症対策への満足度を収集した。また、指導する側として、産業医資格取得講習参加者を対象に、アンケートにて属性、日常業務における途上国赴任予定労働者からの相談の有無、日常業務における途上国赴任予定者に対する感染症に関する指導の有無、途上国赴任に際し推奨するアドバイス項目を取得した。結果:労働者が事前に得ることで、自社の感染症対策満足度に寄与する要因として、会社規模に加え、事前教育、相談窓口の情報提供が挙げられた。これらを多くの産業医資格取得講習参加者は、提供すべきと回答したが、情報提供に自信がないと考えていることが明らかとなった。結論:海外派遣労働者には、海外派遣前に、企業と連携し、事前教育、相談窓口の情報を、積極的に提供すべきである。また、産業医など指導する側は、渡航医学についての知識や情報源についての理解を深める必要がある。(著者抄録)

Background Detection of coronary artery lesions (CALs) at initial echocardiography can aid in diagnosing Kawasaki disease (KD) and inform primary adjunctive treatments. We aimed to characterize patients with KD with CALs detected at initial echocardiography. Methods and Results We analyzed data from the nationwide Japanese KD survey that contained information on 103 222 population-based patients diagnosed with KD across Japan during 2011 to 2018. Patients with CALs detected at initial echocardiography were assessed by age, day of illness, and number of principal KD signs (≥3). Multivariable logistic regression analysis was performed to evaluate factors independently associated with CAL detection. Overall, 3707 (3.6%) patients had CALs detected at initial echocardiography. Patients aged <12 and ≥60 months were associated with CAL detection (adjusted odds ratio [95% CI], 1.28 [1.18‒1.39] and 1.32 [1.20‒1.45], respectively; reference, 12‒59 months). Patients with delayed hospital visits were increasingly at higher risk for CAL detection (days 7‒8, 1.84 [1.63‒2.08]; days 9-10, 4.30 [3.58-5.15]; and days ≥11, 9.12 [7.63‒10.90]; reference, days 1-4). Patients with 3 or 4 principal KD signs were independently associated with CAL detection (1.75 [1.63‒1.88]). These patients were significantly more likely to be aged <12 months but were not associated with delayed hospital visit. Younger patients visited at earlier days of illness. Conclusions Timely diagnosis could be beneficial for patients with KD. However, even when the hospital visit occurred early in the course of illness, patients with 3 or 4 principal KD signs, especially younger patients, were at higher risk of CAL detection at initial echocardiography.

INTRODUCTION: Hand hygiene is crucial for infection control in long-term care facilities for elderly (LTCFEs), because it can be easily implemented in the low-resource settings of LTCFEs. This study investigated the actual status of hand hygiene adherence in LTCFEs, identified the factors inhibiting its appropriate implementation, and evaluated the effectiveness of a hand hygiene promotion program. METHODS: In this before-and-after study, participants were staff members (n = 142) at two LTCFEs in Gunma Prefecture, Japan. We modified the World Health Organization's "five moments for hand hygiene" and assessed participants' hand hygiene adherence rates in four situations: (1) Before touching around a resident's mucous membrane area; (2) Before medical practice or clean/aseptic procedures; (3) After body fluid exposure/risk or after touching around a resident's mucous membrane area; and (4) After touching a resident's contaminated environments. The study was divided into four phases. In Phase 1, participants self-assessed their hand hygiene adherence using a questionnaire. In Phase 2, we objectively assessed participants' pre-intervention adherence rates. In Phase 3, an intervention comprising various hand hygiene promotion measures, such as education and hands-on training on hand hygiene practices and timings, was implemented. In Phase 4, participants' post-intervention adherence rates were objectively measured. RESULTS: Although most participants reported high hand hygiene adherence rate in the self-assessment (93.1%), the pre-intervention evaluation revealed otherwise (16.8%). Participants' post-intervention adherence rates increased for all four situations (77.3%). CONCLUSION: The intervention program helped increase participants' hand hygiene adherence rates, indicating its effectiveness. Similar interventions in other LTCFEs may also improve adherence rates.

BACKGROUND Randomized controlled trials previously provided different conclusions about the superiority of adding corticosteroids to initial intravenous immunoglobulin treatment for the prevention of coronary artery abnormalities in patients with Kawasaki disease (KD). To further assess this issue, we analyzed large-scale data from nationwide KD surveys in Japan, where combination treatment (corticosteroids added to initial standard intravenous immunoglobulin treatment) has become commonly used for patients at high risk for KD. METHODS AND RESULTS Standard intravenous immunoglobulin treatment and combination treatment were compared using data from time periods with and without combination treatment. Outcome measures were coronary artery abnormalities and initial intravenous immunoglobulin treatment failure. Hospitals where ≥20% of patients received combination treatment were identified, and treatment and control groups were selected via matching by age, sex, illness day at initial treatment, and KD recurrence. Matched group selection and subsequent analyses were conducted 1000 times to minimize sampling bias and potential confounders (bootstrapping). From 115 hospitals, 1593 patients with KD in the treatment group and 1593 controls were selected for each of the 1000 sample iterations. The median proportion of patients who developed coronary artery abnormalities among the treatment group and controls were 4.6% (95% CI, 3.8%-5.8%) and 8.8% (95% CI, 7.5%-10.0%), respectively: an estimated risk ratio of 0.53 (0.41-0.67). A median of 14.1% (95% CI, 12.4%-15.9%) of the patients in the treatment group and 21.7% (95% CI, 19.8%-23.4%) in the controls had treatment failure: an estimated risk ratio of 0.65 (0.56-0.75). CONCLUSIONS Combination treatment reduced coronary artery abnormality risk by an estimated 47% and treatment failure by 35%. Multiple-dose corticosteroids may provide benefit in selected patients at high risk for KD.

BACKGROUND: Previous studies demonstrated that coronary artery lesions (CALs) resulting from Kawasaki disease (KD) can improve over time. However, limited information is available on sub-acute outcomes of CALs detected at admission during KD illness. METHODS: The nationwide Japanese KD survey contained substantial information on KD patients with CALs detected at admission and who received standard IVIG treatment within 10 days of disease onset. Coronary outcomes were evaluated by changes in CALs from admission to the first assessment at 30 days from disease onset in three categories: improved, unchanged, and progressed. Ordinal logistic regression analysis was performed to evaluate factors associated with the outcomes. RESULTS: Of 2024 patients with CALs detected at admission, improved, unchanged, and progressed outcomes were found in 1548 (76.5%), 390 (19.3%), and 86 (4.2%), respectively. Over 80% of patients with coronary artery (CA) dilatations had improved outcome. Independent factors associated with worse outcomes were larger-size CALs (adjusted ORs [95% CIs]: CA aneurysm = 5.13 [3.65-7.22] and giant CA aneurysms = 7.49 [3.56-15.72] compared with CA dilatation, respectively), age ≥ 60 months (1.45 [1.08-1.94] compared with 12-59 months), recurrent KD (1.57 [1.07-2.29]), parental history of KD (2.23 [1.02-4.85]), and delayed admission (8-10 days from disease onset: 1.76 [1.21-2.57] compared with 1-4 days). CONCLUSIONS: KD patients with larger CALs, ≥60 months old, and with recurrent status or parental history may require more rigorous treatment. In addition, delayed admission may result in worse coronary outcome, indicating that prompt diagnosis and treatment are required.

BACKGROUND: Platelet count is considered as a biomarker for the development of coronary artery abnormalities (CAAs) among Kawasaki disease (KD) patients. However, previous studies have reported inconsistent results. We addressed the controversial association of platelet count with CAAs using a large-scale dataset. METHODS: A retrospective cohort study was conducted using KD survey data from Japan (2015-2016; n = 25,448). Classifying patients by intravenous immunoglobulin (IVIG) responsiveness, we described the trends in platelet count using the lowest and highest values along with the specific illness days. Multivariate logistic regression analysis was performed to evaluate the association between platelet count and CAAs, adjusting for relevant factors. RESULTS: Platelet counts rapidly decreased from admission, reached the lowest count at 6-7 days, and peaked after 10 days. Platelet counts in IVIG non-responders decreased with a lower minimum value than IVIG responders, but subsequently rebounded toward a higher maximum. Compared with patients with normal platelet counts (150-450 × 10/L), patients with abnormally high platelet counts (>450 × 10/L) were more likely to have CAAs at admission (adjusted odds ratio: IVIG responders, 1.50 [95% confidence interval 1.20-1.87] and non-responders, 1.46 [1.01-2.12]). By contrast, IVIG non-responding patients whose counts were below normal (<150 × 10/L) after hospitalization were at higher risk for developing CAAs (2.27 [1.44-3.58]). CONCLUSIONS: Platelet count varied widely by illness day and was confounded by IVIG responsiveness, which might have contributed to previous inconsistent findings. KD patients with abnormally high platelet counts at admission or abnormally low counts after hospitalization were at higher risk for CAAs.

Following publication of the original article [1], the authors spotted errors in their paper concerning the positive rate in the right side in Table 2. (Table Presented).

Bacillus cereus is a spore-forming bacterium. B. cereus occasionally causes nosocomial infections, in which hand contamination with the spores plays an important role. Therefore, hand hygiene is the most important practice for controlling nosocomial B. cereus infections. This study aimed to determine the appropriate hand hygiene procedure for removing B. cereus spores. Thirty volunteers' hands were experimentally contaminated with B. cereus spores, after which they performed 6 different hand hygiene procedures. We compared the efficacy of the procedures in removing the spores from hands. The alcohol-based hand-rubbing procedures scarcely removed them. The soap washing procedures reduced the number of spores by more than 2 log(10). Extending the washing time increased the spore-removing efficacy of the washing procedures. There was no significant difference in efficacy between the use of plain soap and antiseptic soap. Handwashing with soap is appropriate for removing B. cereus spores from hands. Alcohol-based hand-rubbing is not effective.

自治医科大学附属病院では，鋭利物廃棄容器を携帯し忘れて発生する針刺し切創を防止するために，鋭利器材や廃棄容器などを搭載した独自の「血管穿刺支援カート」を企画し2010年に導入した．本研究では，2007–2013年における各年のカート配置総数・針刺し切創報告総件数・廃棄容器を携帯しないために発生した事例数を調査し，カート導入による針刺し切創の減少効果を評価した．針刺し切創報告総件数はカート導入前後で変化しなかったが，廃棄容器を携帯しないために発生した事例はカート導入によって有意に減少した(p<0.01)．また，カート配置総数と廃棄容器を携帯しないために発生した事例数の間には，負の相関が見られた（相関係数r=−0.83，p<0.05）．<br>

Background: Biological monitoring is used to assess toluene exposure in medical examinations. The American Conference of Industrial Hygienists, Japanese Society for Occupational Health and Deutsche Forschungsgemeinschaft have proposed various biological exposure determinants, such as toluene in blood and urine, and o-cresol in urine. Toluene in blood is a common biomarker among them. Toluene is a volatile organic solvent; therefore, sample preservation under appropriate conditions before measurement is necessary. However, little study has been done on the stability of toluene in workers' blood samples under conditions simulating those of a medical examination. Finding: We carried out a pilot study on the stability of toluene in blood from humans, according to different methods of sample preservation. Toluene in blood was analyzed by head space-gas chromatography/mass spectrometry. The sealing performance of the vial was examined by using toluene-added blood and the stability of toluene in blood according to the preservation period was examined by using blood from toluene-handling workers, which was collected with vacuum blood tubes. The sealing performance of the headspace vial used in this study was good for three days and toluene in blood in tubes from workers was stable at least within 8 hours up to blood packing at 4 degrees C. Conclusion: We could propose that the collected blood need only be transferred into headspace vials on the collection day and analyzed within a few days, if the samples are preserved at 4 degrees C. Our data size is limited; however, it may be considered basic information for biological monitoring in medical examinations.

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-alpha and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.

We describe an outbreak of Bacillus cereus bacteremia that occurred at Jichi Medical University Hospital in 2006. This study aimed to identify the source of this outbreak and to implement appropriate control measures. We reviewed the charts of patients with blood cultures positive for B. cereus, and investigated B. cereus contamination within the hospital environment. Genetic relationships among B. cereus isolates were analyzed. Eleven patients developed B. cereus bacteremia between January and August 2006. The hospital linens and the washing machine were highly contaminated with B. cereus, which was also isolated from the intravenous fluid. All of the contaminated linens were autoclaved, the washing machine was cleaned with a detergent, and hand hygiene was promoted among the hospital staff. The number of patients per month that developed new B. cereus bacteremia rapidly decreased after implementing these measures. The source of this outbreak was B. cereus contamination of hospital linens, and B. cereus was transmitted from the linens to patients via catheter infection. Our findings demonstrated that bacterial contamination of hospital linens can cause nosocomial bacteremia. Thus, blood cultures that are positive for B. cereus should not be regarded as false positives in the clinical setting.

病院で使用するタオルには，加熱処理が義務づけられている．しかし，熱に耐性であるBacillus cereusの芽胞が病院タオルを汚染することがある．さらに，病院タオルのB. cereus汚染は医療関連感染の原因となりうる．したがって，病院タオルのB. cereus汚染を定期的に評価する必要がある．しかし，その評価のための標準方法は確立されていない．今回の研究において，我々は4つの測定方法(スタンプ法・スワブ法・単純抽出法・ビーズ抽出法)を用いて，病院タオルのB. cereus汚染の評価を行なった．スタンプ法ではタオル汚染を定量評価することが不可能であった．また，スワブ法では病院タオルから少数のB. cereusしか検出できなかった．一方，抽出法では病院タオルから多数のB. cereusを検出することができた．特に，ビーズ抽出法では単純抽出法の2倍の菌数を検出することができた．以上の結果より，ビーズ抽出法は病院タオルのB. cereus汚染の評価に最も適した測定方法と考えられる．さらに本研究の結果から，タオル中のB. cereusの分布密度が不均一であることが判明した．したがって，タオルのB. cereus汚染を評価する場合，1枚のタオルから複数のサンプル断片を採取して調べる必要がある．