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Abstract This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan’s Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged

Candidemia, linked to high mortality rates, requires prompt antifungal therapy for better outcomes. Treatment is structured as an action bundle, which is beneficial when followed closely. However, the Japanese action bundle lacks detailed guidance on severe complications like endocarditis or ocular issues. To address this, we adjusted the action bundle and assessed outcomes with and without AFT intervention. We strengthened protocols for blood cultures and organ assessments, and the AFT contacted the primary physician when yeast-like fungi were detected in the patient’s blood culture bottles. Analyzing 204 candidemia cases from 2008–2021, we observed increased adherence and reduced mortality post-AFT intervention. Ophthalmology consultations rose significantly, but many patients had only one visit, suggesting inadequate follow-up. If endophthalmitis is diagnosed, a change in the treatment approach may be necessary. There is a possibility that abnormal ocular findings will be detected during subsequent visits, which highlights the need for improvement in ophthalmology follow-up rates as a future challenge for our AFT activities.

Abstract It has long been known that field-aligned irregularities within equatorial plasma bubbles (EPBs) can cause long-range propagation of radio waves in the VHF frequencies such as those used for TV broadcasting through the so-called forward scattering process. However, no attempt has been made to use such anomalous propagations of VHF radio waves for wide-area monitoring of EPBs. In this study, we investigated the feasibility of monitoring of EPBs using VHF radio waves used for aeronautical navigation systems such as VHF Omnidirectional radio Range (VOR). There are 370 VOR stations in the Eastern and Southeastern Asian region that can be potentially used as Tx stations for the observations of anomalous propagation. We have examined the forward scattering conditions of VHF waves using the magnetic field model and confirmed that it is possible to observe the EPB-related anomalous propagation if we set up Rx stations in Okinawa (Japan), Taiwan, and Thailand. During test observations conducted in Okinawa since 2021, no signal has been received that was clearly caused by anomalous propagation due to EPBs. This is simply because EPBs have not developed to high latitudes during the observation period due to the low solar activity. In March 2023, however, possible indications of EPB-related scattering were detected in Okinawa which implies the feasibility of observing EPBs with the current observation system. We plan to conduct pilot observations in Taiwan and Thailand in future to further evaluate the feasibility of this monitoring technique. Graphical Abstract

Most elderly patients with tuberculosis (TB) have previously been infected with Mycobacterium tuberculosis, which remains dormant in the body for decades and may reactivate when their immunity declines due to underlying diseases. Elderly cancer patients are at a high risk for TB, and the treatment of TB reactivation in these patients is challenging. Among cancer patients, the incidence of TB reactivation is the highest in lymphoma patients. However, the impact of chemotherapy on TB reactivation in lymphoma patients is unknown. We report the case of an immunocompetent elderly patient with primary central nervous system lymphoma (PCNSL) having no prior history of TB, who developed miliary TB during multiagent chemotherapy consisting of rituximab, high-dose methotrexate, procarbazine, and vincristine (R-MPV therapy). Retrospectively, the chest computed tomography showed calcification of the pleura, suggesting that the patient had a latent tuberculosis infection (LTBI) and developed miliary TB from the reactivation of TB triggered by the R-MPV therapy. Our case emphasizes that when chemotherapy is administered to patients with PCNSL, interferon-gamma release assay (IGRA) should be performed if there are findings on chest examination suggestive of LTBI, such as pleural calcification, and if IGRA is positive, chemotherapy should be given concurrently with LTBI treatment.

ABSTRACT Vancomycin-resistant enterococci (VRE) are a significant cause of healthcare-associated infections. In Japan, the estimated isolation rate of VRE is approximately 0.2%; however, sporadic outbreaks continue to occur in healthcare facilities. Despite sporadic cases of VRE at our hospital since the 2007 outbreak, it is unclear whether the same strains from 2007 were still prevalent. Therefore, we performed whole-genome sequencing of stored VRE strains at our hospital and assessed their potential utility as an alternative to pulsed-field gel electrophoresis (PFGE) for infection control. In contrast to previous studies, our analysis focused on examining strain characteristics over time. The results of taxonomic characterization using average nucleotide identity (ANI) values and multilocus sequence typing (MLST) showed good agreement with the PFGE analysis, indicating that ANI values and MLST were viable potential alternatives to PFGE. Notably, many of the strains isolated in 2010 belonged to ST1421. The first reported VRE outbreak in Japan, caused by pstS-null VRE ST1421, occurred in Aomori in 2018. However, the present study demonstrated, for the first time, that the ST1421 strain existed in Japan as early as 2010. In the time series analysis, the observed PFGE pattern did not correlate with the time of strain isolation, particularly for isolates collected after 2012. We also observed cases where isolates sharing the same Tn1546 variant were found on distinct clonal backgrounds. While ANI and MLST may serve as suitable alternatives to PFGE for short-term outbreak evaluations, a plasmid analysis may prove useful for long-term assessments. IMPORTANCE Our study emphasizes the efficacy of whole-genome sequencing (WGS) in addressing outbreaks of vancomycin-resistant enterococci. WGS enables the identification and tracking of resistant bacterial strains, early detection and management of novel infectious disease outbreaks, and the appropriate selection and use of antibiotics. Furthermore, our approach deepens our understanding of how resistant bacteria transfer genes and adapt to their environments or hosts. For modern medicine, these insights have significant implications for controlling infections and effectively managing antibiotic use in the current era, where antibiotic resistance is progressing.

In C1–C2 posterior fixation, the C1 lateral mass and C2 pedicle/translaminar screw insertion under spine navigation have been used frequently. To avoid the risk of neurovascular damage in atlantoaxial stabilization, we assessed the safety and effectiveness of a preoperative computed tomography (CT) image-based navigation system with intraoperative independent C1 and C2 vertebral registration. It is ideal when a reference frame can be linked directly to the C1 posterior arch for C1-direct-captured navigation, but there is a mechanical challenge. A new spine clamp-tracker system was implemented recently, which allows reliable C1- and C2-direct-captured navigation in nine patients with traumatic C2 fractures. In this way, there was no misalignment of C1–C2 screws. C1 lateral mass screws were used except for one case, and translaminar screws were primarily used as an anchor for C2. The C1 lateral mass screw locations, which are 19 mm laterally from the C1 posterior arch’s center, are taken to be constant. However, there is one unusual circumstance in which using a C1 laminar hook instead of a C1 lateral mass screw appears to be a beneficial substitute. The increase of surgical accuracy for posterior C1–C2 screw fixation without cost constraints is significantly facilitated by intraoperative C1- and C2-direct-captured navigation with preoperative computed CT images.

Clinically mild encephalitis/encephalopathy with reversible splenial lesions (MERS) is a mild form of encephalitis/encephalopathy that appears in association with various conditions, including infection. COVID-19 is also known to cause MERS. MERS more commonly occurs in children, and adult cases are relatively rare. Typical head MRI findings include a round lesion in the mid-layer of the corpus callosum with a high signal intensity on diffusion-weighted images. Most improve within a week. Although the exact mechanism by which the cerebral corpus callosum is affected is still unknown, several hypotheses have been proposed, including the involvement of electrolyte abnormalities (e.g., hyponatremia) and inflammatory cytokines (e.g., IL-6). In this report, we describe the first case of COVID-associated MERS with a typical imaging course and hyponatremia, with a review of the relevant literature. When psychiatric symptoms and the disturbance of consciousness appear in COVID patients, MERS should be considered in addition to delirium due to fever and hypoxia.

Abstract Background Ventricular tachycardia (VT) non‐inducibility at the end of ablation is associated with a less likely VT recurrence. However, it is not clear whether we should use VT non‐inducibility as a routine end point of VT ablation. The aim of this study was to evaluate VT recurrence in patients in whom VT non‐inducibility was not achieved at the end of the radiofrequency (RF) ablation and the factors attributing to the VT recurrence. Methods We analyzed that 62 patients in whom VT non‐inducibility was not achieved at the end of the RF ablation were studied. Results Over 2 years, 22 (35%) of the cases had VT recurrences. A multivariate analysis showed that an LVEF ≥35% (HR: 0.19; 95% CI: 0.06–0.49; p

Abstract Background Corticosteroids are widely used in patients with cardiac sarcoidosis (CS). In addition, upgrading to cardiac resynchronization therapy (CRT) is sometimes needed. This study aimed to investigate the impact of corticosteroid use on the clinical outcomes following CRT upgrades. Methods A total of 48 consecutive patients with non‐ischemic cardiomyopathies who underwent CRT upgrades were retrospectively reviewed and divided into three groups: group 1 included CS patients taking corticosteroids before the CRT upgrade (n = 7), group 2, CS patients not taking corticosteroids before the CRT upgrade (n = 10), and group 3, non‐CS patients (n = 31). The echocardiographic response, heart failure hospitalizations, and cardiovascular deaths were evaluated. Results The baseline characteristics during CRT upgrades exhibited no significant differences in the echocardiographic data between the three groups. After the CRT upgrade, responses regarding the ejection fraction (EF) and end‐systolic volume (ESV) were significantly lower in CS patients than non‐CS patients (ΔEF: group 1, 6.7% vs. group 2, 7.7% vs. group 3, 13.6%; p = .039, ΔESV: 3.0 ml vs. ‐12.7 ml vs. ‐37.2 ml; p = .008). The rate of an echocardiographic response was lowest in group 1 (29%). There were, however, no significant differences in the cumulative freedom from a composite outcome among the three groups (p = .19). No cardiovascular deaths occurred in group 1. Conclusion The echocardiographic response to an upgrade to CRT and the long‐term prognosis in patients with CS should be carefully evaluated because of the complex etiologies and impact of immunosuppressive therapy.

Abstract Introduction Many clinical studies have identified significant predictors or risk factors for the severity or mortality of coronavirus disease 2019 (COVID‐19) cases. However, there are very limited reports on the risk factors for requiring oxygen therapy during hospitalization. In particular, we sought to investigate whether plasma glucose and HbA1c levels could be risk factors for oxygen therapy requirement. Materials and Methods A single‐center, retrospective study was conducted of 131 COVID‐19 patients hospitalized at Saitama Medical University Hospital between March 2020 and November 2020. To identify the risk factors for oxygen therapy requirement during hospitalization, a stepwise multivariate binary logistic regression analysis was performed using several clinical parameters commonly obtained on admission, including plasma glucose and HbA1c levels. Results Of the 131 patients with COVID‐19, 33.6% (44/131) received oxygen therapy during hospitalization. According to the logistic regression analysis, male sex (odds ratio [OR]: 8.76, 95% confidence interval [CI]: 1.65–46.5, P

Abstract We carried out a statistical analysis of the propagation characteristics of Es and Medium-Scale Traveling Ionospheric Disturbances (MSTIDs) by combining data of HF Doppler (HFD) sounder and Total Electron Content (TEC) obtained from the GPS receivers of GEONET (GPS-TEC) for 4 years from 2014 to 2017. We made use of Es reflection data from the HFD receivers in Sugito, Saitama (36.0°N, 139.7°E), Fujisawa, Kanagawa (35.3°N, 139.5°E), and Sugadaira, Nagano (36.4°N, 138.3°E) in Japan. By using this triangle observation, we succeeded in deriving the horizontal speed and direction of the motion of Es. In addition, we estimated the phase velocity of MSTIDs observed in the simultaneously obtained maps of GPS-TEC with the same triangle observation procedure. The speeds of Es and MSTIDs were commonly less than 100 m/s in most cases and their propagation direction was predominantly southwestward. This result is consistent with the statistical characteristics of nighttime MSTIDs observed in the previous studies. More importantly, good correspondence between the propagation characteristics of the two phenomena at two different altitudes confirms that Es and MSTIDs move in tandem with each other, further suggesting that Es in the E region plays an important role in the generation and propagation of MSTIDs in the F region. Graphical Abstract

Abstract Background Some of atrial fibrillation (AF) drivers are found in normal/mild late‐gadolinium enhancement (LGE) areas, as well as moderate ones. The atrial wall thickness (AWT) has been reported to be important as a possible AF substrate. However, the AWT and degree of LGEs as an AF substrate has not been fully validated in humans. Objective The purpose of this study was to evaluate the impact of the AWT in normal/mild LGE areas on AF drivers. Methods A total of 287 segments in 15 persistent AF patients were assessed. AF drivers were defined as non‐passively activated areas (NPAs), where rotational activation was frequently observed, and were detected by the novel real‐time phase mapping (ExTRa Mapping), mild LGE areas were defined as areas with a volume ratio of the enhancement voxel of 0% to

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (blaKPC, blaNDM-1, blaIMP-1 group, and blaVIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

We report a case of Talaromyces marneffei fungemia in a patient with HIV infection with a history of travelling to southern China. At first, Pneumocystis pneumonia was considered in this case because chest CT images showed typical ground-glass opacity and elevated β-D-glucan levels. However, PCR testing of sputum for Pneumocystis jirovecii was negative and a filamentous fungus was isolated from blood cultures. The cultured fungus was subsequently identified as T. marneffei, and the patient was considered to have pneumonia caused by this organism. However, skin disease and lymphadenopathy, which are common in T. marneffei infections, were not observed during the disease course. This patient was successfully treated with voriconazole and consequently the chest CT shadow disappeared. In the present case, T. marneffei infection required differentiation from pneumonia with Pneumocystis jirovecii infection.

Abstract High-frequency Doppler (HFD) sounding is one of the major remote sensing techniques used for monitoring the ionosphere. Conventional systems for HFDs mainly utilize analog circuits. However, existing analog systems have become difficult to maintain as the number of people capable of working with analog circuits has declined. To solve this problem, we developed an alternate HFD receiver system based on digital signal processing. The software-defined radio (SDR) technique enables the receiver to be set up without the knowledge of analog circuit devices. This approach also downsizes the system and reduces costs. A highly stabilized radio system for both the transmitter and receiver is necessary for stable long-term observations of various phenomena in the ionosphere. The global positioning system disciplined oscillator with an accuracy of $${10}^{-11}$$ 10 - 11 compensates for the frequency stability required by the new receiving system. In the new system, four frequencies are received and signal-processed simultaneously. The dynamic range of the new system is wider (> 130 dB) than that of the conventional system used in HFD observations conducted by the University of Electro-Communications in Japan. The signal-to-noise ratio significantly improved by 20 dB. The new digital system enables radio waves to be received with much smaller amplitudes at four different frequencies. The new digital receivers have been installed at some of the stations in the HFD observation network in Japan and have already captured various ionospheric phenomena, including medium-scale traveling ionospheric disturbances and sudden commencement induced electric field fluctuations, which indicates the feasibility of SDR for actual ionospheric observations. The new digital receiver is simple, inexpensive, and small in size, which makes it easy to deploy new receiving stations in Japan and elsewhere. These advantages of the new system will help drive the construction of a wide HFD observation network. Graphical Abstract

Abstract We introduce a network observation of anomalous long-distance propagation of aeronautical navigation (NAV) very high-frequency (VHF) radio waves due to sporadic E layer (Es). The system has been operative since May 2019 at 6 stations in Japan. The receiver consists of a log-periodic antenna, air-band filter, software-defined radio and small computer which is capable of recording the strength of radio signals in the frequency band from 98 to 118 MHz with a temporal resolution of 2 s. The receiver covers not only the NAV band including VHF omnidirectional radio range (VOR), instrument landing system localizer (ILS LOC) and ground-based augmentation system VHF data broadcast (GBAS VDB) from 108 to 118 MHz, but also broadcasting channels from 98 to 108 MHz. Soon after the start of the full operation of the network observation, a strong Es event was detected by an ionosonde in Tokyo during the daytime on July 4, 2019, in which foEs (critical frequency of Es) was sometimes higher than 15 MHz. The VHF radio wave monitoring system detected multiple signatures of Es anomalous propagation (EsAP) at all the stations extending from Okinawa to Hokkaido. At some stations, the EsAP signatures continued for a few hours with some brief intervals of disappearance. The observed correspondence between the enhancement of foEs and the occurrence of anomalous propagation confirmed that an extreme electron density enhancement within Es caused the anomalous long-distance propagation of VHF NAV signals. The data from this network observation can be browsed at http://gwave.cei.uec.ac.jp/cgi-bin/vor/vhf.cgi in near real-time basis. This near real-time monitoring capability allows people in the aeronautical operation community such as air navigation service providers, pilots, and airline engineers to check the propagation environment of VHF NAV signals online, which contributes to a mitigation of ionospheric space weather impacts on the aeronautical navigation systems. Not only that the current method for detecting Es in a wide area can be used to visualize the spatial distribution of Es in two-dimensional fashion through a combination of other observation techniques such as ionosondes and total electron content (TEC) measurements using Global Navigation Satellite System (GNSS) signals.

Abstract Background Capnocytophaga canimorsus is a gram-negative bacterium and an oral commensal in dogs and cats, but occasionally causes serious infections in humans. Septicemia is one of the most fulminant forms, but diagnosis of C. canimorsus infection is often difficult mainly because of its very slow growth. C. canimorsus infective endocarditis (IE) is rare and is poorly understood. Since quite a few strains produce β-lactamase, antimicrobial susceptibility is pivotal information for adequate treatment. We herein report a case with C. canimorsus IE and the results of drug susceptibility test. Case presentation A 46-year-old man had a dog bite in his left hand 3 months previously. The patient was referred to our hospital for fever (body temperature > 38 °C), visual disturbance, and dyspnea. Echocardiography showed aortic valve regurgitation and vegetation on the leaflets. IE was diagnosed, and we initially administered cefazolin and gentamycin assuming frequently encountered microorganisms and the patient underwent aortic valve replacement. C. canimorsus was detected in the aortic valve lesion and blood cultures. It was also identified by 16S ribosome DNA sequencing. Ceftriaxone were started and continued because disk diffusion test revealed the isolate was negative for β-lactamase and this case had cerebral symptoms. The patient successfully completed antibiotic treatment following surgery. Conclusions We diagnosed C. canimorsus sepsis and IE by extended-period blood cultures and 16S ribosome DNA sequencing by polymerase chain reaction, and successfully identified its drug susceptibility.

Abstract Background Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan. Methods Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records. Results Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p

Abstract A 41‐year‐old man with persistent atrial fibrillation (AF) underwent radiofrequency (RF) catheter ablation using an online real‐time phase mapping system: ExTRa Mapping. Box isolation could not terminate AF. Subsequently, RF applications on nonpassively activated areas (NPAs), where rotational activations were frequently observed, at the posterior bottom of left atrium outside of box lesion could convert AF to common atrial flutter. Of interest, the NPA near the posterior bottom were located on the patchy fibrotic tissue area assessed by the late‐gadolinium enhancement magnetic resonance imaging. This indicated the possibility of the critical AF rotor meandering through the fibrotic tissue area.

Abstract Background There are no reports on the prevalence of Chagas disease in Japan. Furthermore, screening programs and access to diagnosis and treatment have not been established. This study aimed to clarify the prevalence of Chagas disease among suspected cases in Japan and provide the reference data required for disease control. Methods Seventeen patients with suspected Chagas disease in Japan between 2012 and 2017 were included in the study. Patients were diagnosed with Chagas disease based on the two different serological tests for antibodies to Trypanosoma cruzi. Real-time polymerase chain reaction assay and blood culture techniques were performed to confirm T. cruzi parasitemia. Results Of the 17 patients, 11 (64.7%) were immigrants from Latin America. Ultimately, 6 patients (35.3%) were diagnosed with Chagas disease. Of these 6 patients, median age was 53.5 years, 5 patients were immigrants from Latin American, and 1 was Japanese who had a congenital infection. T. cruzi parasitemia was confirmed in 4 patients (66.7%), and 5 (83.3%) were in the chronic phase (Chagas cardiomyopathy, 4; megacolon, 1). Two patients (33.3%) commenced benznidazole treatment. Conclusion Our study showed that some patients of Chagas disease living in Japan are already in the chronic phase at diagnosis because of substantial diagnostic delays. Further epidemiological studies on the prevalence of Chagas disease and systematic screening programs for the Latin American population are needed.

Background Medium‐dose (25 gray) x‐ray radiation therapy has recently been performed on patients with refractory ventricular tachyarrhythmias. Unlike x‐ray, carbon ion and proton beam radiation can deliver most of their energy to the target tissues. This study investigated the electrophysiological and pathological changes caused by medium‐dose carbon ion and proton beam radiation in the left ventricle (LV). Methods and Results External beam radiation in the whole LV was performed in 32 rabbits. A total of 9 rabbits were not irradiated (control). At the 3‐month or 6‐month follow‐up, the animals underwent an open‐chest electrophysiological study and were euthanized for histological analyses. No acute death occurred. Significant LV dysfunction was not seen. The surface ECG revealed a significant reduction in the P and QRS wave voltages in the radiation groups. The electrophysiological study showed that the local conduction times in each LV site were significantly longer and that the local LV bipolar voltages were significantly lower in the radiation groups than in the control rabbits. Histologically, apoptosis, fibrotic changes, and a decrease in the expression of the connexin 43 protein were seen in the LV myocardium. These changes were obvious at 3 months, and the effects were sustained 6 months after radiation. No histological changes were seen in the coronary artery and esophagus, but partial radiation pneumonitis was observed. Conclusions Medium‐dose carbon ion and proton beam radiation in the whole LV resulted in a significant electrophysiological disturbance and pathological changes in the myocardium. Radiation of the arrhythmogenic substrate would modify the electrical status and potentially induce the antiarrhythmic effect.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner “What's in My Pot?” (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Abstract Background The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. Results A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. Conclusions An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Abstract Background A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. Methods We generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. Results Our LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. Conclusions Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

We have studied the energy recovery effect in high-temperature furnace using a high-emittance fiber (HEF) unit. Previous studies have shown that energy recovery effect occurs as placing the HEF unit perpendicular to the gas flow. However, there are some problems such as clogging and pressure loss. In this study, we examined the energy recovery effect and mechanisms of the effect when HEF unit placed in parallel to the gas stream. In the numerical simulation based on thermo-fluid dynamics, energy recovery effect was also appeared even though the HEF unit placed parallel to the gas flow. The simulation, confirm that most of thermal radiation through the exhausted pipe from the inside of furnace is absorbed by HEF unit and is emitted back to the furnace. This simulation indicated more than 10% input power reduction is expected. We also reveal that amount of energy conservation is depend on the HEF unit’s length, position, and number of plates in a unit. In the experiment using similar furnace to simulation model, we can also obtain the input power reduction by 3.5% at 1073 K.

Most cases of hypophosphatasia (HPP) exhibit early loss of primary teeth. Results of microcomputed tomography (micro-CT) analysis of teeth with HPP have rarely been reported. The purpose of the present study was to describe the mineral density distribution and mapping of exfoliated teeth from an HPP patient using micro-CT. Four exfoliated teeth were obtained from a patient with HPP. Enamel and dentin mineral densities of exfoliated teeth were measured on micro-CT. The mean values of enamel and dentin mineral densities in mandibular primary central incisors with HPP were 1.61 and 0.98 g/cm3, respectively. The corresponding values in the mandibular primary lateral incisors were 1.60 and 0.98 g/cm3, respectively. Enamel hypoplasia was seen in the remaining teeth, both maxillary and mandibular primary canines and first and second molars. Micro-CT enables nondestructive, noninvasive evaluation and is useful for studying human hard tissues obtained from patients.