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Purpose: Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. Materials and Methods:Materials and Methods: A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 coun-tries were analyzed descriptively, with a Delphi process used for expert recommendations. Results:Results: Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoosper-mia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differ-entiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. Conclusions:Conclusions: This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

Purpose: The purpose of this meta-analysis is to study the impact of varicocele repair in the largest cohort of infertile males with clinical varicocele by including all available studies, with no language restrictions, comparing intra-person conventional semen parameters before and after the repair of varicoceles. Materials and Methods: The meta-analysis was performed according to PRISMA-P and MOOSE guidelines. A systematic search was performed in Scopus, PubMed, Cochrane, and Embase databases. Eligible studies were selected according to the PICOS model (Population: infertile male patients with clinical varicocele; Intervention: varicocele repair; Comparison: intraperson before-after varicocele repair; Outcome: conventional semen parameters; Study type: randomized controlled trials [RCTs], observational and case-control studies). Results: Out of 1,632 screened abstracts, 351 articles (23 RCTs, 292 observational, and 36 case-control studies) were included in the quantitative analysis. The before-and-after analysis showed significant improvements in all semen parameters after varicocele repair (except sperm vitality); semen volume: standardized mean difference (SMD) 0.203, 95% CI: 0.129–0.278; p

On 20th September 2022, Uganda declared the 7th outbreak of Ebola virus disease (EVD) caused by the Sudan Ebola strain following the confirmation of a case admitted at Mubende Regional Referral Hospital. Upon confirmation, the Government of Uganda immediately activated the national incident management system to initiate response activities. Additionally, a multi‑country emergency stakeholder meeting was held in Kampala; convening Ministers of Health from neighbouring Member States to undertake cross‑border preparedness and response actions. The outbreak spanned 69 days and recorded 164 cases (142 confirmed, 22 probable), 87 recoveries and 77 deaths (case fatality ratio of 47%). Nine out of 136 districts were affected with transmission taking place in 5 districts but spilling over in 4 districts without secondary transmission. As part of the response, the Government galvanised robust community mobilisation and initiated assessment of medical counter measures including therapeutics, new diagnostics and vaccines. This paper highlights the response actions that contributed to the containment of this outbreak in addition to the challenges faced with a special focus on key recommendations for better control of future outbreaks.

Purpose:Purpose: The success of vasectomy is determined by the outcome of a post-vasectomy semen analysis (PVSA). This article describes a step-by-step procedure to perform PVSA accurately, report data from patients who underwent post vasectomy se-men analysis between 2015 and 2021 experience, along with results from an international online survey on clinical practice. Materials and Methods:Materials and Methods: We present a detailed step-by-step protocol for performing and interpretating PVSA testing, along with recommendations for proficiency testing, competency assessment for performing PVSA, and clinical and laboratory sce-narios. Moreover, we conducted an analysis of 1,114 PVSA performed at the Cleveland Clinic’s Andrology Laboratory and an online survey to understand clinician responses to the PVSA results in various countries. Results:Results: Results from our clinical experience showed that 92.1% of patients passed PVSA, with 7.9% being further tested. A total of 78 experts from 19 countries participated in the survey, and the majority reported to use time from vasectomy rather than the number of ejaculations as criterion to request PVSA. A high percentage of responders reported permitting unpro-tected intercourse only if PVSA samples show azoospermia while, in the presence of few non-motile sperm, the majority of responders suggested using alternative contraception, followed by another PVSA. In the presence of motile sperm, the major-ity of participants asked for further PVSA testing. Repeat vasectomy was mainly recommended if motile sperm were observed after multiple PVSA’s. A large percentage reported to recommend a second PVSA due to the possibility of legal actions. Conclusions:Conclusions: Our results highlighted varying clinical practices around the globe, with controversy over the significance of non-motile sperm in the PVSA sample. Our data suggest that less stringent AUA guidelines would help improve test compli-ance. A large longitudinal multi-center study would clarify various doubts related to timing and interpretation of PVSA and would also help us to understand, and perhaps predict, recanalization and the potential for future failure of a vasectomy.

No abstract available.

Global research collaboration, through partnerships and networks, is an effective way to deliver highly impactful and sustainable research that is collectively owned and promoted for the global good. Many models exist for effective North-South collaborations that are built on trust and balanced benefits. The European & Developing Countries Clinical Trials Partnership (EDCTP) model emphasises capacity development in clinical trials and product-focused implementation research. To ensure effectiveness and sustainability, capacity development requires a long-term perspective, an integrated system-wide approach, and local ownership and leadership from countries experiencing high disease burdens. Guided by these principles, the EDCTP2 programme, established in 2014, has developed and strengthened human capital and institutional capacities in 39 countries in sub-Saharan Africa to undertake high-quality clinical research guided by good clinical and regulatory practices. Projects in these countries have involved 238 African and 163 European institutions. To date, EDCTP has supported 171 Fellows and 232 postgraduate trainees. EDCTP-short-term training activities have equipped 9628 researchers and medical personnel. The EDCTP capacity-building described here includes its Regional Networks of Excellence and its Consortia for public health emergencies which provide the foundation for sustained efforts against emerging and re-emerging global health threats.

Background: Influenza is a highly contagious disease that affects the upper and lower respiratory tract caused by several subtypes of influenza viruses. While vaccination remains the mainstay strategy to protect populations against influenza, there is a global shortage and inequitable access to influenza vaccines. Although influenza vaccine production capacity increased from 500 million doses in 2006 to 1.5 billion doses in 2013, little is known about the global distribution of these vaccines albeit its introduction. We assessed the global status of influenza vaccine introduction. Research design and methods: We analyzed data from the World Health Organization (WHO) Joint Reporting Form, a publicly available source of immunization data from 194 countries of all six WHO regions. We used 2017 data, available as of July 2018. Results: By December 2017, 117 of 194 (60%) WHO Member States had introduced the seasonal influenza vaccine. European and American regions accounted for 70% (82/117) of the total number of countries that had introduced influenza vaccine. The other four regions account for only 30%. Ninety-four percent (50/53) of countries in the European region and 91% (32/35) in the American region had introduced influenza vaccine. In the Eastern Mediterranean and Western Pacific regions, 67% (14/21) and 52% (14/27), respectively, had introduced the vaccine. Yet only 27% (3/11) and 9% (4/47) in the Southeast Asian and African regions, respectively, had introduced the vaccine. Among countries (n = 117) that had introduced the vaccine, children (56%), older adults (87%), and risk groups (99%) were prioritized and given the vaccines. Conclusions: Introduction of influenza vaccine in the African and Southeast Asian regions remains suboptimal. This critically underscores the need for financing mechanisms and having countries in the regions that are lagging behind to prioritize seasonal influenza vaccine.

Introduction: Enuresis (or Nocturnal Enuresis) is defined as discreet episodes of urinary incontinence during sleep in children over 5 years of age in the absence of congenital or acquired neurological disorders. Recommendations: Suggestions and recommendations are made on the various therapeutic options available within a South African context. These therapeutic options include; behavioural modification, pharmaceutical therapy [Desmospressin (DDAVP), Anticholinergic (ACh) Agents, Mirabegron (β3-adrenoreceptor agonists), and Tricyclic Antidepressants (TCA)], alternative treatments, complementary therapies, urotherapy, alarm therapy, psychological therapy and biofeedback. The role of the Bladder Diary, additional investigations and Mobile Phone Applications (Apps) in enuresis is also explored. Standardised definitions are also outlined within this document. Conclusion: An independent, unbiased, national evaluation and treatment guideline based on the pathophysiological subcategory is proposed using an updated, evidence based approach. This Guideline has received endorsement from the South African Urological Association, Enuresis Academy of South Africa and further input from international experts within the field. Keywords: Enuresis, Nocturnal Enuresis, South African, Therapeutic options, Mobile Phone Applications (Apps), Treatment guideline, Expert consensus

Background: Strengthening immunisation programmes in Africa remains a key strategy of improving vaccine coverage. Research plays a vital role in the design and implementation of strategic immunisation plans for improving vaccination coverage, in turn providing context specific evidence to inform policy and practice. We therefore updated an evidence map describing the types and quality of available literature on childhood immunisation in Africa from 2011 to 2017. Methods: PubMed and Africa Wide databases were searched for English studies on childhood immunisation in Africa published from January 2011 to September 2017. Studies had to be conducted in humans and the reported information needed to be on either: vaccines; immunisation programmes; immunisation policies; or epidemiology of vaccine preventable diseases targeted by Expanded Programme on Immunisation vaccines. Results: Out of 5567 studies retrieved, 797 studies from 165 journals met the inclusion criteria. During 2011–2017, 42 African countries contributed to research on childhood immunisation. Most studies were carried out in multiple countries (15.1%). Five countries contributed 41% of the total research output. Nigeria and South Africa contributed the highest proportion of studies at 12% and 11.4% respectively. The quantity of research output increased progressively from 2011 to 2015 after which there was a significant decline. Conclusion: There was a remarkable increase in childhood immunisation research in the period 2011 to 2017 when compared to the initial assessment. However, the reason for decline in research output from 2015 requires further investigation. Most childhood immunisation research was still generated by five countries as previously observed, highlighting the critical need for strategic investment in research capacities and improved collaboration between countries in Africa. Keywords: Children, Immunisation, Research, Vaccine preventable diseases, Africa

Agreed international development standards underpin high quality de novo clinical practice guidelines (CPGs). There is however, no international consensus on how high quality CPGs should 'look'; or on whether high quality CPGs from one country can be viably implemented elsewhere. Writing de novo CPGs is generally resource-intensive and expensive, making this challenging in resource-poor environments. This paper proposes an alternative, efficient method of producing high quality CPGs in such circumstances, using existing CPGs layered by local knowledge, contexts and products.We undertook a mixed methods case study in South African (SA) primary healthcare (PHC), building on findings from four independent studies. These comprised an overview of international CPG activities; a rapid literature review on international CPG development practices; critical appraisal of 16 purposively-sampled SA PHC CPGs; and additional interrogation of these CPGs regarding how, why and for whom, they had been produced, and how they 'looked'.Despite a common aim to improve SA PHC healthcare practices, the included CPGs had different, unclear and inconsistent production processes, terminology and evidence presentation styles. None aligned with international quality standards. However many included innovative succinct guidance for end-users (which we classified as evidence-based summary recommendations, patient management tools or protocols). We developed a three-tiered model, a checklist and a glossary of common terms, for more efficient future production of better quality, contextually-relevant, locally-implementable SA PHC CPGs. Tier 1 involves transparent synthesis of existing high quality CPG recommendations; Tier 2 reflects local expertise to layer Tier 1 evidence with local contexts; and Tier 3 comprises tailored locally-relevant end-user guidance.Our model could be relevant for any resource-poor environment. It should reduce effort and costs in finding and synthesising available research evidence, whilst efficiently focusing scant resources on contextually-relevant evidence-based guidance, and implementation.

Hematopoietic stem cells (HSCs) obtained from patient-derived human induced pluripotent stem cells (iPSCs) are a promising tool for curing various hematological disorders. We previously demonstrated that enforced expression of the LIM-homeobox transcription factor Lhx2, which is essential for mouse embryonic hematopoiesis, leads to generation of engraftable and expandable hematopoietic stem cells (HSCs) from mouse iPSCs. However, it remained unknown whether Lhx2 can induce HSCs from human iPSCs. Here, we investigated the effect of Lhx2 overexpression on hematopoietic differentiation of human iPSCs. Unexpectedly, Lhx2 severely inhibited proliferation of human iPSC-derived hematopoietic cells. Thus, Lhx2 exhibited differential effects on mouse and human hematopoietic cells. Further studies implied that the inhibitory effect of Lhx2 on human iPSC-derived hematopoietic cells was due to insufficient transcriptional activation ability. Therefore, we modified Lhx2 to strengthen its activity as a transcriptional activator. This modified Lhx2 could induce ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells (HSPCs). We believe that these findings will facilitate the development of a method to efficiently produce HSCs from human iPSCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kitajima, Takahashi, Ando, Shingai, Hamasaki, Tanikawa, Kanokoda, Nakajima, Nishito and Hara.)

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future., Importance: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation., Competing Interests: The authors declare no conflict of interest.

Recent advances in cell engineering technologies enable immune cells to be utilized for adoptive cell transfer (ACT) immunotherapy against cancers. Macrophages have the potential to directly and indirectly exterminate cancers and are therefore an attractive option for therapies. To develop new ACT therapies using macrophages, a great number of macrophages are required. Human induced pluripotent stem cells (iPSCs) are expected to be a source of macrophages; therefore, a system to efficiently produce macrophages from human iPSCs is needed. Here, we demonstrated that human iPSCs were robustly differentiated into macrophages by enforced FMS-like tyrosine kinase-3 (FLT3) signaling via the introduction of exogenous FLT3 into iPSCs and the addition of its ligand FLT3L to the macrophage induction culture. These iPSC-derived macrophages were identical to those obtained by standard differentiation induction methods. Thus, our novel system enables the preparation of scalable macrophages from human iPSCs. We believe that this system will be useful to develop a novel ACT therapy using macrophages., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases., (© 2024. The Author(s).)

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process. Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different. Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Noguchi, Kajimoto, Kumamoto, Shingai, Narasaki, Urabe, Imamura, Harada, Hide, Tanaka, Yanase, Nakamura, Tsutsumi and Sakai.)

Although influenza virus infection has been shown to affect lipid metabolism, details remain unknown. Therefore, we elucidated the kinetic lipid profiles of mice infected with different doses of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) by measuring multiple lipid molecular species using untargeted lipidomic analysis. C57BL/6 male mice were intranasally infected with PR8 virus at 50 or 500 plaque-forming units to cause sublethal or lethal influenza, respectively. Plasma and tissue samples were collected at 1, 3, and 6 days post-infection (dpi), and comprehensive lipidomic analysis was performed using high-performance liquid chromatography-linear trap quadrupole-Orbitrap mass spectrometry, as well as gene expression analyses. The most prominent feature of the lipid profile in lethally infected mice was the elevated plasma concentrations of phosphatidylethanolamines (PEs) containing polyunsaturated fatty acid (PUFA) at 3 dpi. Furthermore, the facilitation of PUFA-containing phospholipid production in the lungs, but not in the liver, was suggested by gene expression and lipidomic analysis of tissue samples. Given the increased plasma or serum levels of PUFA-containing PEs in patients with other viral infections, especially in severe cases, the elevation of these phospholipids in circulation could be a biomarker of infection and the severity of infectious diseases., (© 2023. Springer Nature Limited.)

Viral infections are usually accompanied by systemic cytokinemia. Vaccines need not necessarily mimic infection by inducing cytokinemia, but must induce antiviral-acquired immunity. Virus-derived nucleic acids are potential immune-enhancers and particularly good candidates as adjuvants in vaccines in mouse models. The most important nucleic-acid-sensing process involves the dendritic cell (DC) Toll-like receptor (TLR), which participates in the pattern recognition of foreign DNA/RNA structures. Human CD141 + DCs preferentially express TLR3 in endosomes and recognize double-stranded RNA. Antigen cross-presentation occurs preferentially in this subset of DCs (cDCs) via the TLR3-TICAM-1-IRF3 axis. Another subset, plasmacytoid DCs (pDCs), specifically expresses TLR7/9 in endosomes. They then recruit the MyD88 adaptor, and potently induce type I interferon (IFN-I) and proinflammatory cytokines to eliminate the virus. Notably, this inflammation leads to the secondary activation of antigen-presenting cDCs. Hence, the activation of cDCs via nucleic acids involves two modes: (i) with bystander effect of inflammation and (ii) without inflammation. In either case, the acquired immune response finally occurs with Th1 polarity. The level of inflammation and adverse events depend on the TLR repertoire and the mode of response to their agonists in the relevant DC subsets, and could be predicted by assessing the levels of cytokines/chemokines and T cell proliferation in vaccinated subjects. The main differences in the mode of vaccine sought in infectious diseases and cancer are defined by whether it is prophylactic or therapeutic, whether it can deliver sufficient antigens to cDCs, and how it behaves in the microenvironment of the lesion. Adjuvant can be selected on a case-to-case basis.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hayashida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Among inactivated influenza vaccines, the whole virus particle vaccine (WPV) elicits superior priming responses to split virus vaccine (SV) in efficiently inducing humoral and cellular immunity. However, there is concern for undesired adverse events such as fever for WPV due to its potent immunogenicity. Therefore, this study investigated the febrile response induced by subcutaneous injection with quadrivalent inactivated influenza vaccines of good manufacturing grade for pharmaceutical or investigational products in cynomolgus macaques. Body temperature was increased by 1 °C-2 °C for 6-12 h after WPV administration at the first vaccination but not at the second shot, whereas SV did not affect body temperature at both points. Given the potent priming ability of WPV, WPV-induced fever may be attributed to immune responses that uniquely occur during priming. Since WPV-induced fever was blunted by pretreatment with indomethacin (a cyclooxygenase inhibitor), the febrile response by WPV is considered to depend on the increase in prostaglandins synthesized by cyclooxygenase. In addition, WPV, but not SV, induced the elevation of type I interferons and monocyte chemotactic protein 1 in the plasma; these factors may be responsible for pyrogenicity caused by WPV, as they can increase prostaglandins in the brain. Notably, sufficient antibody responses were acquired by half the amount of WPV without causing fever, suggesting that excessive immune responses to trigger the febrile response is not required for acquired immunity induction. Thus, we propose that WPV with a reduced antigen dose should be evaluated for potential clinical usage, especially in naïve populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Since it became possible to differentiate human pluripotent stem cells (hPSCs) into hematopoietic cells in vitro, great efforts have been made to obtain highly potent hematopoietic stem/progenitor cells (HSPCs) from hPSCs. Immunophenotypical HSPCs can be obtained from hPSCs, but their repopulating potential in vivo is low. Here, we developed a novel hematopoietic differentiation method for human-induced pluripotent stem cells (hiPSCs) to determine why the existing hPSC differentiation systems are inadequate. hiPSC-derived CD45+CD34+ cells in our system were mostly CD38- immunophenotypical HSPCs. The vast majority of human CD45+CD34+ cells in umbilical cord blood, fetal liver, and bone marrow are CD38+ hematopoietic progenitor cells (HPCs); therefore, the poor production of CD38+ HPCs was indicative of a systematic problem. hiPSC-derived CD45+CD34+ cells did not express FLT3, a receptor tyrosine kinase. Exogenous FLT3 activity significantly enhanced the production of CD38+ HPCs from hiPSCs. Thus, poor production of CD38+ HPCs was due to a lack of FLT3 expression. Interferon-γ upregulated expression of FLT3 and increased the number of CD38+ HPCs among hiPSC-derived CD45+CD34+ cells. These results suggest that the poor production of CD38+ HPCs with hPSC differentiation systems is due to a lack of FLT3 expression, and that the addition of interferon-γ can solve this problem., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak., Competing Interests: The authors have declared that no competing interests exist.

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2., Competing Interests: Declaration of competing interest This work was partly supported by grants for the Japan Program for Infectious Diseases Research and Infrastructure (JP21wm0225003 and JP21fk0108104j) from Japan Agency for Medical Research and Development (AMED) and fund from NEC Solution Innovators, Ltd., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Hiroshi Kida reports financial support was provided by the Japan Agency for Medical Research and Development (AMED). Kazumasa Ogasawara reports financial support was provided by the Japan Agency for Medical Research and Development (AMED). Hiroshi Kida reports financial support was provided by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Yasushi Itoh reports financial support was provided by Japan Society for the Promotion of Science.]., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Despite the use of vaccines, seasonal influenza remains a risk to public health. We previously proposed the inactivated whole virus particle vaccine (WPV) as an alternative to the widely used split vaccine (SV) for the control of seasonal and pandemic influenza based on the superior priming potency of WPV to that of SV. In this study, we further examined and compared the immunological potency of monovalent WPV and SV of A/California/7/2009 (X-179A) (H1N1) pdm09 (CA/09) to generate immune responses against heterologous viruses, A/Singapore/GP1908/2015 (IVR-180) (H1N1) pdm09 (SG/15), and A/duck/Hokkaido/Vac-3/2007 (H5N1) (DH/07) in mice. Following challenge with a lethal dose of heterologous SG/15, lower virus titer in the lungs and milder weight loss were observed in WPV-vaccinated mice than in SV-vaccinated ones. To investigate the factors responsible for the differences in the protective effect against SG/15, the sera of vaccinated mice were analyzed by hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) assays to evaluate the antibodies induced against viral hemagglutinin (HA) and neuraminidase (NA), respectively. While the two vaccines induced similar levels of HI antibodies against SG/15 after the second vaccination, only WPV-vaccinated mice induced significantly higher titers of NI antibodies against the strain. Furthermore, given the significant elevation of NI antibody titers against DH/07, an H5N1 avian influenza virus, WPV was also demonstrated to induce NA-inhibiting antibodies that recognize NA of divergent strains. This could be explained by the higher conservation of epitopes of NA among strains than for HA. Taking these findings together, NA-specific antibodies induced by WPV may have contributed to better protection from infection with heterologous influenza virus SG/15, compared with SV. The present results indicate that WPV is an effective vaccine for inducing antibodies against both HA and NA of heterologous viruses and may be a useful vaccine to conquer vaccine strain mismatch.

Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires.

Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research.

Influenza remains a world-wide health concern, causing 290,000-600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms., (© 2021. The Author(s).)

Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza., (© 2021. The Author(s).)

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)