Hospital workers are at high risk of contact with COVID-19 patients. Currently, there is no evidence-based, comprehensive risk assessment tool for healthcare-related exposure; so, we aimed to identify independent factors related to COVID-19 infection in hospital workers following workplace exposure(s) and construct a risk prediction model. We analyzed the COVID-19 contact tracing dataset from 15 July to 31 December 2021 using multiple logistic regression analysis, considering exposure details, demographics, and vaccination history. Of 7146 included exposures to confirmed COVID-19 patients, 229 (4.2%) had subsequently tested positive via RT-PCR. Independent risk factors for a positive test were having symptoms , participating in an unprotected aerosol-generating procedure , duration of exposure >15 min , personnel who did not wear a mask , exposure to aerodigestive secretion , index patient not wearing a mask , and exposure distance <1 m without eye protection . High-potency vaccines and high levels of education protected against infection. A risk model and scoring system with good discrimination power were built. Having symptoms, unprotected exposure, lower education level, and receiving low potency vaccines increased the risk of laboratory-confirmed COVID-19 following healthcare-related exposure events. Healthcare workers are at high risk for exposure to COVID-19, both in the community and in the workplace when caring for patients . The earThe exposure risk assessment and management system is currently mainly based on expert opinion, because only a few studies have addressed this problem, and there is the significant heterogeneity of operational definitions for variables that influence exposure risk, such as the measurement of contact duration, distance, the use of a face mask versus a respirator with eye protection, and differing vaccine regimens and efficacies ,6,7,8,9.In the third quarter of 2021, Siriraj Hospital, a 2300-bed referral center in Bangkok with more than 16,000 employees, conducted more than 200 SARS-CoV-2 genetic tests per day for its personnel. Adapted from USCDC, WHO, European and Thailand public health interim guidelines, the hospital risk assessment and management system classified the risk of exposure and recommended appropriate testing times, work restrictions, and quarantine for those who were exposed to confirmed patients with COVID-19 ,14,15,16The objectives of this study are to identify independent factors associated with SARS-CoV-2 infection detected via RT-PCR in hospital workers following exposure(s) to confirmed positive patients and to build an evidence-based quantitative risk model and risk score for healthcare-related exposure.This study is a retrospective cohort analysis. From July 2021 to January 2022, during the increase in the number of cases of COVID-19 caused by the Delta variant, the hospital implemented a contact tracing and risk evaluation system based on exposure characteristics and immunization status to guide risk-specific SARS-CoV-2 tests, work restriction, and quarantine recommendations . HospitaData collection was completed by exposed hospital workers or their representatives directly into a computer spreadsheet . Completeness and accuracy were validated using mandatory field entry, data validation, and logic checks with feedback confirmation by responsible infection control officers. If personnel had multiple exposures to the same index person, the risk would be assigned to the highest risk event, and recommendations would be arranged according to the latest significant exposure. The classification of exposure risk  according to the consensus of the experts of the hospital detailed in The variables of interest that were not included in the initial dataset  and those subject to recall errors (immunization record) were provided by the hospital informatics and data innovation center. Missing and conflicting data were manually imputed based on available electronic hospital records.COVID-19 vaccination at least 14 days before exposure was considered to exert a full protective effect and was defined as the completion of the last dose. Due to the wide variety of vaccine combinations among Thai health workers , we clas®, Seoul, Korea). The cycle threshold of <40 for the E and N gene and <42 for the RdRp gene was considered positive. To resolve the discrepancies between different genes tested, infectious disease specialists would define the status of the case based on their history and subsequent test(s).COVID-19 was diagnosed via SARS-CoV-2 genetic detection from respiratory samples using a real-time RT-PCR test, Allplex™ 2019-nCoV Assay . All analyses were performed using SPSS™ software version 26.0  and Microsoft Excel™ software version 2203 .Continuous variables were reported as means with standard deviation and medians with interquartile range, while categorical data were reported using frequencies and percentages. The variables between groups were compared using the independent sample T test or Pearson’s chi-square test , with statistical significance defined as a al model . Model fThe study flow diagram is illustrated in Of the 7146 exposures of 5449 hospital workers, 299 (4.2%) cases of COVID-19 infection were confirmed. The incidence of included events and COVID-19 detection gradually decreased during the study period . The basp < 0.001). Most exposures (98.1%) came from personnel who had received at least one dose of the vaccine. The median interval from the last vaccination to the day of exposure was 72 days (range 14 to 236). More than half of the hospital workers (54.2%) received two doses of CoronaVac , 17.5% received an additional ChAdOx-1 , 15.2% received an additional BNT162b2  vaccination as a booster, and 11.2% had other vaccine combinations. The remaining 136 exposures came from hospital workers who were not vaccinated at the time of exposure (1.9%).All events were classified into four exposure risk categories: low (31.9%), moderate (24.1%), high (38.9%), and insignificant risk (but being tested due to COVID-19-related symptoms) (5.1%). This risk classification was highly correlated with the SARS-CoV-2 detection rate , with 90% of all detections occurring within 11 days from the last exposure . No mortAfter prescreening with univariate logistic regression, twelve factors entered the preliminary main effect model , and ninHaving a fever or other COVID-19-related symptoms was the strongest risk factor for SARS-CoV-2 genetic detection . Other strong risk factors included performing an aerosol-generating procedure without full protection , prolonged duration of contact , and personnel not wearing a mask . Direct contact with aerodigestive secretion, the infected person not wearing a mask, and close contact without proper eye protection carried smaller risks. Vaccination was protective against infection: aOR 0.05 (high-potency combinations), aOR 0.17 (moderate-potency combinations), and 0.3 (low-potency combination). Hospital workers with higher levels of education level were less likely to be infected.p 0.346). The discrimination power of the final logistic model and the risk scoring system accessed via ROC curves are depicted in The model fit was confirmed using the Hosmer and Lemeshow test , provided the least protection against infection, while the second most common regimen, heterologous boosted inactivated vaccines (moderate potency), provided slightly better protection but much less when compared with the viral vector-mRNA combination (high potency). This is consistent with the previous report from Sritipsukho et al.  which unAlthough symptoms related to COVID-19 should be considered as a consequence of infection rather than a risk factor for infection, our data support that all symptomatic health workers with an exposure history during the epidemic should be tested, regardless of contact risk and immunologic status, provided that this policy does not overwhelm laboratory testing capacity. A significant portion of infected hospital workers tested positive before the initial recommended test date(s), which implied the benefit of the early test (and early detection) triggered by symptoms. This contrasts with other studies on symptomatic patients presenting at health services which demonstrated poor diagnostic accuracy of signs and symptoms ,27. An eConsistent with a 2020 study by Chadeau-Hyam et al., the level of education of the hospital workers was inversely correlated with the risk of testing positive . This coMost of the COVID-19 risk calculators available provide a very crude risk estimate based primarily on location, the nature of the activity, and the safety measures being taken . Our risThis study has several limitations. We did not include the severity of cases that got infected . Due to the retrospective nature of the observational study, some demographic information may have been missed. Furthermore, most of the data were entered by various staff with different levels of health knowledge. Therefore, misclassification may be an issue. The external validation of the risk model was also difficult to perform due to the rapid shift in the variants of concern and vaccine-induced immunity over time.Having symptoms of COVID-19, inadequate personal protection, low education level, and not receiving a vaccine or receiving a low-potency vaccine regimen were found to be the main risks for COVID-19 infection among all healthcare-related exposures. Our quantitative exposure risk model and risk score have good predictive value and could help combat further spread among hospital workers according to their actual probability of infection.


Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds  in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential. HoweverCyanobacteria and microalgae have attracted more attention in the last few years as an alternative supply for carbohydrates to support industrial fermentative processes . In compA number of cyanobacterial species have been effectively engineered to produce and secrete large amounts of sucrose by taking advantage of cyanobacterial sucrose biosynthesis pathways and heterologous co-expression of sucrose permease CscB,  to exporThis review focuses on the current knowledge of the sucrose synthesis and degradation pathways in cyanobacteria as well as the list of genetic modifications in sucrose metabolic pathways that have been found to increase the production and secretion of this sugar. While other sugars can be produced phototrophically from cyanobacteria . GAP can be interchangeably converted to dihydroxyacetone phosphate (DHAP), and the condensation of GAP and DHAP through the activity of the enzyme fructose 1,6-bisphosphate aldolase (FBA), leads to the formation of fructose 1,6-bisphosphate (FBP). FBP is then further transformed into other hexose phosphates, such as fructose 6-phophate (F6P) and glucose 6-phosphate (G6P). G6P can be used to form nucleotide sugars such as uridine diphosphate glucose (UDP-Gluc).Sucrose is a disaccharide [α-d-glucopyranosyl (1 → 2) β-d-fructofuranoside], whose synthesis pathway appears to be nearly universal among cyanobacteria, as predicted by the presence of sucrose synthesis genes in most of the known genome sequences available so far . Sucrosein vivo  and sucrose phosphate phosphatase SPP; . NDP-Gluin vivo . SuS is in vivo .2.1.1.Thermosynechococcus elongatus was resolved, showing that this enzyme has 16 α-helices and 14 β-sheets, with UDP and S6P bound at the interface of the aforementioned A-and B-domains  is located in the N-terminus of SPS and has been hypothesized to include residues necessary for defining the F6P binding site (7-E) is highly conserved within the C-terminus of SPS and SuS enzymes; its flanking Glu residues play a catalytic role in the reaction by binding to UDP-Gluc  of the bidomainal SPS encoded in Synechococcus elongatus PCC 7942  that disrupted sucrose synthesis, specifically preventing S6P formation  of SPS contains two motifs that are highly conserved across glucosyl-transferase family enzymes . Motif Iing site . Motif IUDP-Gluc . The fircatalyst . Both Glormation .2.1.2.Synechocystis sp. PCC 6803  related to the active site of the phosphohydrolase domain PHD, . All thr motif I . Motif I]X3[D/N] ,B, inclu]X3[D/N] . In a red D473A; . This suNostoc sp. PCC 7119 , and several unicellular cyanobacteria , but which have not been classified as SPP due to key distinctions in conserved domains. These SPP-like proteins are frequently annotated as (putative) HAD-superfamily hydrolases subfamily IIB, and while they contain the three motifs that define SPP (see above) they possess mutations in conserved residues in these motifs that distinguish them from classically defined SPPs ; (ii) the second includes the SPP-like protein of S. elongatus PCC 7942 and the second of the two SPP-like protein paralogs encoded in the genome of members of the order Nostocales, and; (iii) SPP-like proteins that dominate in marine cyanobacteria, such as Prochlorococcus spp.  the first includes the SPP-like protein of cus spp. . In contcus spp. . For insactivity . It has activity , as it hactivity .Bacteroides thetaiotaomicron VPI-5482 , which irreversibly hydrolyzes sucrose into the monosaccharides, glucose and fructose . PhylogeTrichormus variabilis  are grouped in the same transcriptional unit with fructokinase and AMS encoding genes (AMS (EC 2.4.1.4) is a glucosyltransferase that catalyzes the hydrolysis of the glycosidic bond in sucrose, leading to the release of glucose and fructose. Then, the released glucose is used to form α-1,4-linked linear insoluble glucans  is a glucosyltransferase that can catalyze both the synthesis and cleavage of sucrose, but appears to be active principally in the cleavage reaction in vivo . The revCC 29413  and NostPCC 7119 . SuS hasPCC 7119 , 2008. OPCC 7119 . SuS hasPCC 7119 , 2007, aPCC 7119 .Gloebacter violaceus PCC 7421 and Microcystis aeruginosa NIES-4325, might be due to a more recent lateral gene transfer event  that pass through the cell membrane under conditions of high external ionic pressure  concentrations in the cytoplasm without interfering with the cellular metabolism . In cyane solute . In addil as GGA .3.2.S. elongatus PCC 7942, SPS7942 activity is regulated by inorganic ions, Na+ and Cl−, which activate the SPS domain of this bifunctional protein, but have relatively little impact on the enzymatic activity of the SPP domain  to cope with environmental stressors . As a bi4.1.S. elongatus PCC 7942 accumulates nearly 300 mM intracellular sucrose , representing a significant portion of the cell biomass  from Escherichia coli ATCC 700927  accumulated nearly twice as much intracellular sucrose than its wild-type counterpart , in contrast to SPS6803 which is also bidomainal, but has a non-functional PHD domain and is regulated distinctly from SPS7942 , so it is possible that this observation is related either to the manner in which salt-ions can regulate the function of some SPS domains  either had no effect on, or decreased sucrose productivity in S. elongatus PCC 7942 or Synechococcus elongatus UTEX 2973 , were sucrose levels increased in comparison with SPS6803-only strain, albeit marginally , and is more tolerant of high-light and high-temperature conditions , the expression of a heterologous pathway may act as an additional “sink” and bypass downstream limitations of cell growth and division. While this remains a speculative possibility, the relaxation of acceptor-side limitations on photosystem I suggests that sucrose secretion pathways (or other heterologous metabolic sinks) may utilize “excess” light energy that might otherwise be lost to photosynthetic inefficiencies under certain conditions  of Synechococcus sp. PCC 7002 led to an accumulation of three times more sucrose than wild-type under hypersaline conditions (glgC), was downregulated in sucrose-secreting S. elongatus PCC 7942, there was only a minor or insignificant increase in sucrose  and can direct electrons from an over-reduced photosynthetic electron transport chain to oxygen . The flackground . Furtherckground .4.4.S. elongatus PCC 7942, Qiao and colleagues identified genes indirectly linked to sucrose productivity, glycogen accumulation, and photosynthetic activity (2+ uptake (rpaB) reproduced a growth-arrest phenotype in S. elongatus PCC 7942  as an alternative to traditional environmentally unsustainable processes . Multipl2 inputs, efficient carbon fixation) and paired with other microbes that have demonstrated efficiency in transforming simple carbohydrates into a desired end product. Additionally, because the co-culture output can be changed by swapping the “heterotrophic module” , some steps to optimize synthesis for one product  may be transferable to achieve enhanced synthesis across many distinct cyanobacteria-heterotroph pairings. In practice, sucrose-secreting cyanobacteria have already been used as the basis for engineered microbial communities with numerous heterotrophic species and for a variety of end products , a class of biological polymers with comparative qualities to petroleum-based plastics. PHAs have the advantage of being both compatible in blends with commonly used petroleum-based polymers while also exhibiting superior biodegradation properties . Additio. coli W ; Table 2 periods .Pseudomonas putida is a model organism that naturally accumulates medium chain length PHAs (mcl-PHAs) granules in response to starvation, primarily under low-nitrogen and high-carbon conditions  and a sucrose operon repressor (cscR) further improved sucrose utilization , a common precursor molecule, exhibited higher efficiency when the two species were engineered to display complementary surface proteins , and 2,3-butanediol , which a-culture . Interesproteins . The autproteins , an intrP. putida, β-carotene with E. coli or the yeast Yarrowia lipolytica, and violacein by E. coli  degradation  the co-evolution of bidomainal SPS with and without SPP activity among cyanobacterial species; (ii) the role (s) and substrate (s) of SPP-like proteins in cyanobacteria; and (iii) the alternative roles of sucrose in cyanobacteria apart from its osmoprotective functions. Increasing the knowledge in all these areas will not only be useful to understand the regulation and evolution of different sucrose enzymes in cyanobacteria, but also to further engineer these enzymes to obtain high sucrose yields.Cyanobacteria hold considerable potential as cell factories to produce sucrose, yet the development of commercially viable applications of this strategy will require a significant amount of additional research and optimization. Importantly, while yields of sucrose from cyanobacteria could theoretically exceed production from traditional plant crops at scale, significant barriers to translate results from the lab to the field are evident. For instance, deployment of outdoor cultivation would require strains that exhibit resilience to the dynamic fluctuations of temperature, light, diurnal cycles, and abiotic stresses . FurtherA much tighter integration of the signaling and metabolic exchanges between cyanobacterial and heterotroph co-culture partners might suppress contaminating species through competition and exclusion. Adaptative laboratory evolution could be a useful strategy to domesticate increasingly stable co-cultures by better integrating and adapting the partners to one another . Rationa7.7.1.Nostoc sp. PPC 7120 . Each protein in this query list was used to search for homolog sequences in the genome of 121 cyanobacterial genomes, and hits with an E-value less than or equal to 10−15, an identity less than or equal to 35% and a coverage less than or equal to 80% were considered true homologs.The protein sequences of the orthologues of the different enzymes involved in the sucrose biosynthesis and degradation were obtained from NCBI and Uniprot databases. These sequences were retrieved using BLAST tools in both databases; enzymes with a well-established role in these pathways in cyanobacteria were used a queries: SPS unidomainal from CTC 2957 , and INV7.2.Multiple sequence alignment analyses were performed using MEGA X  and visu7.3.p-distance substitution method including both transitions and transversions, uniform rates among sites, and pairwise deletion treatment. Support for each node was tested with 1,000 bootstrap replicates.Unrooted neighbor-joining phylogenetic trees were generated using MEGA X after the multiple sequence alignments of the sequence of SPP proteins and SPP-like proteins using ClustalX with a BLOSSUM matrix and a bootstrap trial of 1,000. The graphical representations of the trees were created using FigTree. The neighbor-joining tree of SPP-like and SPP sequences was generated using the 7.4.Thermosynechococcus vestivus , and the National Science Foundation and the Division of Molecular and Cellular Bioscience (Grant: 1845463). LY was supported by a fellowship from the Plant Biotechnology for Health and Sustainability Training Program at Michigan State University (Grant: NIH T32-GM110523).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has claimed millions of lives worldwide. India also launched a COVID-19 vaccination drive, and clinical trials for a pediatric COVID-19 vaccine are in development.ObjectivesThe study aims to assess the acceptance and attitude of parents regarding the COVID-19 vaccine for children in India. The study also aims to find the association between selected demographic variables and acceptance and attitudes in parents regarding the COVID-19 vaccine for children.Materials and methodsWe conducted a cross-sectional descriptive study with 204 participants, and data were collected online using Google Forms. The study participants were parents of children aged two to 15 years. A self-structured tool was used to assess parents' acceptance regarding vaccinating their children, and a modified vaccination attitudes examination scale was used to assess parents' attitudes toward pediatric vaccination against COVID-19. We used IBM SPSS Statistics for Windows, Version 20.0  to analyze the data. Demographic data were represented as frequency and percentage. Binary logistic regression analysis was used to determine the associations between sociodemographic data and parents' levels of acceptance and attitude. For all the data, p<0.05 was considered significant.ResultsThe majority of the participants (85%) reported acceptance of the COVID-19 vaccine for children. More than 80% of parents agree that vaccines are essential to halt the COVID-19 pandemic and are mandatory for children. Most parents (62%) also believed that complementary medicine is better than vaccines for children. While most parents (95%) reported trusting the vaccine, but more than half (59%) reported concerns regarding the unknown future effects of the vaccine.Mothers (odds ratio (OR), 2.963; p=0.015) and parents of children who received routine vaccination  were willing to vaccinate their children when a COVID-19 vaccine became available. Mothers  and respondents whose family member or close relative suffered from COVID-19  accepted the COVID-19 vaccine irrespective of the child's age. Study participants who had previously tested positive for COVID-19  believed vaccines for children were necessary to halt the COVID-19 pandemic.ConclusionWe sought to assess parents' acceptance and attitudes regarding the COVID-19 vaccine for children in India. According to our results, while parents have a high acceptance of a pediatric COVID-19 vaccine, they also have a few apprehensions. Therefore, for a successful mass vaccination drive among the pediatric age group, there should be rigorous communication regarding the vaccine and staunch health campaigns to create more awareness and acceptance toward the COVID-19 vaccine for children. The appearance of coronavirus disease 2019 (COVID-19) quickly became a pandemic. COVID-19 disrupted daily life in India and presented a serious challenge to physicians and scientists across the globe. Thankfully, children and adolescents seem to develop less severe diseases than adults. The World Health Organization (WHO) reported that global incidence among children under age five, children ages five to 14, and young people aged 15 to 24 is only 2%, 7%, and 15%, respectively [India launched a vaccine against the virus responsible for COVID-19, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on January 16, 2021 . As of tThis study aimed to explore the acceptance and attitude of parents regarding the COVID-19 vaccine for children in India. The findings may help policymakers develop strategies that can reduce vaccine hesitancy prior to the launch of the pediatric COVID-19 vaccine in India.Study design, setting, and sampleWe conducted a cross-sectional descriptive study via online surveys of 204 parents of children aged two to 15 willing to participate and understand Hindi or English. The data were collected from July to September 2021. The surveys were converted to online forms using Google Forms, and a link was sent to subjects through email and WhatsApp. The completed forms that were received within the study window were coded and analyzed.Study toolsSociodemographic ProformaWe collected sociodemographic data from parents, including their age, gender (as father or mother), education, and occupation. We also collected data on past positive COVID-19 test results, the diagnosis of a family member with COVID-19, and any chronic illnesses or lifestyle-related diseases. We collected the age, gender, routine immunization status, and chronic illness or congenital disease history of the children.Questionnaire for Vaccine AcceptanceA self-structured questionnaire consisting of eight items was used to assess the parental acceptance of the COVID-19 vaccine for their children. It used a five-point Likert scale, with responses ranging from strongly disagree (scored as a 1) to strongly agree (scored as a 5). The eight-item acceptance scale had high internal consistency, with a Cronbach's alpha of 0.806. Parents' responses were coded as "nonacceptance" of the COVID-19 vaccine for their children if marked as strongly disagree, disagree, and neutral, and the responses marked as agree and strongly agree were coded as "acceptance." The coding was reversed for the scoring of the question, "For children, naturopathy/Ayurveda is a better option than a vaccine."Vaccination Attitudes Examination ScaleWe used the standardized vaccination attitudes examination scale (VAX) to assess parental attitudes regarding the COVID-19 vaccine for their children . This scData collection procedureThe validated data collection tools were converted to Google Forms. The questions were prepared in Hindi and English. The link was created and circulated via email and WhatsApp. All respondents provided informed consent. The study design received ethical clearance from the Institutional Ethical Committee (AIIMS/IEC/2021/3540).Data analysisWe used IBM SPSS Statistics for Windows, Version 20.0  to analyze the data. Demographic data were represented as frequency and percentage. Binary logistic regression analysis was used to find the association between sociodemographic variables and parental acceptance and attitude toward the vaccine. For all associations, p<0.05 was considered significant.Demographic characteristics of participantsThe study included responses from 204 participants, most of whom were younger than 40-years-old (83%). The mother-to-father ratio was approximately 1:1 , and 50% of respondents had two children. Under half of the participants graduated from university (42.6%); nearly 43% were employed by the government, and just under half (48.5%) were nursing personnel. The majority of parents (87.7%) were vaccinated against COVID-19. Twenty-eight percent of respondents had tested positive for COVID-19. Most respondents (92.2%) had no chronic or lifestyle-related illnesses. Over half of respondents (68%) reported their children had received routine vaccinations, and 94.6% reported their children had no congenital or chronic diseases . These concerns have been found in other studies and are worth addressing by policymakers ,15. PaulRegarding their opinion of policy, most parents (81%) in our study believed vaccinations should be mandatory for children, but another study did not share this majority. Babickie et al. reported that only 40.4% of parents felt vaccination should be mandatory . RegardlThe government should facilitate easy and safe access to vaccines and launch a campaign to encourage vaccination. Praveen et al. suggested that focusing on public fears before launching a mass vaccination program against COVID-19 will aid the campaign's success . CarefulOur study had a few significant limitations. The sample size was small and ethnically homogeneous, which potentially limits the generalizability of our findings. Future studies should be conducted with larger samples and in various ethnic groups to understand the mass acceptance and attitude of parents toward the COVID-19 vaccine for children.This study explored the acceptance and attitude of parents regarding the COVID-19 vaccine for children in India. While most parents were willing to vaccinate their children against COVID-19, we identified several characteristics of parents who were not willing to vaccinate. An unwillingness to vaccinate children against COVID-19 was common among parents who either themselves or loved ones had not been diagnosed with COVID-19, did not accept routine vaccinations, and had a strong belief in naturopathy/Ayurveda. Also, respondents older than 40 and those who were not highly educated and had chronic or lifestyle-related diseases were less willing to vaccinate their children against COVID-19. A common concern among those unwilling to vaccinate their children against COVID-19 was a fear of future unknown adverse effects from the vaccine. Policymakers should consider these findings while developing strategies to reduce vaccine hesitancy prior to the launch of the pediatric COVID-19 vaccine in India.


In critically ill patients with acute kidney injury, renal replacement therapy (RRT) modality and treatment protocols may affect kidney recovery. This study explored whether RRT modality and treatment protocol affected RRT dependence in the ‘Randomized Evaluation of Normal versus Augmented Level of RRT’ and the ‘Acute Renal Failure Trial Network’ (ATN) trials.Primary outcome was 28-day RRT dependence. Secondary outcomes included RRT dependence among survivors and in different SOFA-based treatment protocol groups. We used the Fine-Gray competing-risk model sub-distribution hazard ratio (SHR) to assess the primary outcome. Analyses were adjusted for confounders.p = 0.570) or hospital mortality  However, among survivors, CRRT-first was associated with decreased 28-day RRT dependence  and more RRT-free days (common OR: 1.38 [95% CI 1.11–1.71]). Moreover, among CRRT-first patient, the ATN treatment protocol was associated with fewer RRT-free days, greater mortality, and a fourfold increase in RRT dependence at day 28.Of 2542 patients, 2175 (85.5%) received continuous RRT (CRRT) and 367 (14.4%) received intermittent hemodialysis (IHD) as first RRT modality. CRRT-first patients had greater illness severity. After adjustment, there was no between-group difference in 28-day RRT dependence (SHR, 0.96 [95% CI 0.84–1.10]; There was no difference in RRT dependence at day 28 between IHD and CRRT. However, among survivors and after adjustment, both IHD-first and the ATN treatment protocol were strongly associated with greater risk of RRT dependence at 28 days after randomization.Trial registration NCT00221013 registered September 22, 2005, and NCT00076219 registered January 19, 2004.The online version contains supplementary material available at 10.1186/s13054-022-04151-5. Acute kidney injury (AKI) treated with renal replacement therapy (RRT) carries a high mortality . Among sA current set of guidelines recommends either continuous RRT (CRRT) or intermittent hemodialysis (IHD) as complementary therapies for severe AKI . HoweverAccordingly, we aimed to compare the rate of persistent RRT dependence (defined as RRT dependence at 28 days after randomization) in patients first treated with CRRT vs patients first treated with IHD among subjects enrolled in the ‘Randomized Evaluation of Normal vs. Augmented Level of RRT’ (RENAL) and the ‘Acute Renal Failure Trial Network’ (ATN) trials . MoreoveWe conducted a pooled analysis of individual patient data from the RENAL  and ATN All patients included in the original trials were considered for inclusion in the present study. Briefly, patients were eligible to participate in the RENAL trial if they were critically ill adults with AKI, were deemed to require CRRT by the attending physician, and fulfilled at least one of predefined inclusion criteria . In the ATN trial, critically ill adults with AKI, deemed to require RRT, and with at least one non-renal organ failure or sepsis were included. The only exclusion criterion in the present analysis was the absence of sufficient information on the first modality of RRT after randomization.We established the exposure, CRRT or IHD, according to the first post-randomization RRT modality as a quasi-equivalent to intention to treat analysis. Illness severity was assessed by Acute Physiology and Chronic Health Evaluation (APACHE) III either directly recorded or derived from APACHE II using predefined formulas .The primary outcome was RRT dependence at day 28 after randomization, defined as the last date of RRT. Secondary outcomes included RRT dependence at day 60, RRT dependence at day 28 and day 60 among survivors, RRT-free days at day 28 , ICU, and hospital length of stay, and ICU, hospital, 28- and 60-day mortality.Categorical variables are presented as numbers and percentages and compared using Fisher exact tests. Continuous variables are presented as median (quartile 25%–quartile 75%) and compared using Wilcoxon rank-sum test. Missingness is presented in Additional file All analyses were adjusted using baseline data  with their 95% confidence interval (CI) and presented as cumulative incidence plots. The same strategy was used for RRT dependence at day 60, ICU, and hospital length of stay. In the competing-risk models, subjects who died remain in the risk set and the RRT-free days at day 28 were compared using cumulative logistic models and presented as a common odds ratio (OR) with 95% CI. In this model, a common OR > 1.00 indicates a greater chance of having more RRT-free days at day 28 (better outcome). ICU and hospital mortality were compared with generalized linear models with binomial distribution and presented as odds ratios (ORs) and 95% CI. 28- and 60-day mortality were compared with Cox proportional hazard model and presented as hazard ratios (HRs) and 95% CI. The proportional hazard assumption was assessed through Schoenfeld residuals.In a sensitivity analysis, to assess the consistency of findings, a covariate-balancing propensity score (CBPS) was used to control for observed confounding factors that may influence outcomes . The CBPTo confirm or refute the findings in different cohorts, the primary and secondary outcomes were re-assessed in the following groups: (1) patients receiving CRRT or IHD exclusively in the first three days; (2) patients receiving CRRT or IHD exclusively during the whole follow-up; and (3) patients with a cardiovascular SOFA score of ≤ 2 at randomization.In addition to study the possible association with trial protocol with renal and patient outcomes in patients who received CRRT first by protocol both in ATN and RENAL, we assessed those patients with cardiovascular SOFA score of ≥ 3 at randomization. In such patients, after CRRT-first start, the RENAL trial protocol prescribed continuing CRRT until day 28 or ICU discharge, while the ATN trial prescribed transition to IHD when vasopressor support was no longer needed.In a final sensitivity analysis, for key secondary outcomes, RRT dependence at day 28 and 60 were also assessed considering only patients alive at latest follow-up. In this group of patients, the comparison of RRT dependence at day 28 and 60 among survivors was done considering a generalized linear model with binomial distribution. We also analyzed the relationship between IHD/CRRT time ratio and RRT dependence at day 28. An IHD/CRRT time ratio defined as the percentage of overall RRT time that the patients spent on IHD and CRRT until day 28 after randomization.For all the models, variables with less than 3% of missing were imputed by the median value of the overall cohort. Missing data in premorbid estimated glomerular filtration rate were imputed based on creatinine levels according to age and sex . All anaThe study trials enrolled a total of 2589 patients. After the exclusion of 16 patients with missing data for first RRT modality, and 31 patients with missing primary outcome, we included 2542 patients with 2175 (85.5%) in the CRRT-first group and 367 (16.5%) in the IHD-first group  for IHD-first patients  (Table p = 0.570).On unadjusted analysis, at day 28, 320 14.7%) CRRT-first patients and 113 (30.8%) IHD-first group were alive and RRT dependent   Table .Fig. 3ReResults of the sensitivity analyses for the primary outcome are shown in Fig. Baseline characteristics of patients receiving CRRT or IHD exclusively in the first three days of follow-up or exclusively during the whole follow-up available for each patient are shown in Additional file p < 0.001)   of remaining RRT dependent at day 28 .After adjustment, treatment of such cardiovascular SOFA score of ≥ 3 CRRT-first patients according to the ATN protocol was associated with greater length of stay in ICU, fewer RRT free days, longer hospital stays, significantly greater mortality, and a greater than fourfold increase in RRT dependence at day 28 and day 60 among survivors This individual patient meta-analysis of the ATN and RENAL trials found that there was no statistically significant RRT dependence at day 28 among IHD- and CRRT-first modality. However, this study found multiple indications that renal recovery was affected by RRT modality: (a) overall, after adjustment, the number of RRT-free days at day 28 was significantly greater in CRRT-first patients; (b) in patients receiving CRRT exclusively in the first three days, there was a lower risk of RRT dependence at day 28 among survivors; (c) among those exclusively treated with CRRT or IHD throughout their ICU stay, there was a significantly lower risk of 28-day RRT dependence, and a higher number of RRT-free days to day 28 in CRRT patients; (d) among patients with a cardiovascular SOFA score of ≤ 2, RRT-free days at day 28, and among survivors, RRT independence at day 28 were greater in CRRT-first patients, (e) among survivors who received CRRT as their first modality, there was a significant decrease in RRT dependence at day 28 and (f) among survivors, a higher IHD/CRRT time ratio was associated with RRT dependence at day 28. Finally, irrespective of first choice of RRT modality, as seen in those patients with a cardiovascular score ≥ 3 who all received CRRT-first therapy in both trials, after adjustment, the different trial protocols were associated with markedly different renal and patient outcomes.Many aspects of RRT in ICU patients remain controversial including the effect of modality on renal recovery –16. Yet,When comparing CRRT with IHD in patients who may transition from one to the other over time, a quasi-intention-to-treat approach may be rational. Our study addressed this issue in this way and provides a comprehensive adjusted analysis. In addition, in those patients who, in both trials, were treated with CRRT first because of a high cardiovascular SOFA score, subsequent care differed according to trial protocol only. This makes it possible to assess the adjusted association of a particular trial protocol with renal and patient outcomes, an analysis that has not been possible until now. A similar approach was taken in a recent study of patients involved in two trials of timing or RRT, where only patients allocated to early intervention were assessed . Such stOur findings imply that in the ATN and RENAL trials, after adjustment, receiving CRRT as the first modality of RRT was associated with faster recovery. They also imply that in those patients who went on to survive to at least 28 days, having received IHD as the first RRT modality was independently significantly associated with a greater probability of persistent RRT dependence, a finding which was robust to multiple sensitivity analyses. Finally, they imply that the treatment protocol had a strong association with renal and patient outcomes.This is a large observational study and the first to use individual patient data from two high-quality large multicenter RCTs from the USA and ANZ. Thus, these findings have a degree of internal and external validity. Moreover, the data were collected independently for the purpose of the trials and without knowledge that this analysis would be performed. Thus, they carry no conceivable ascertainment bias. In addition, the methods used for the analysis considered the effect of death before the event of interest (end of RRT dependence) as a competing risk, adjusted analyses for multiple key baseline characteristics, and included several sensitivity analyses and re-assessments in different cohort, which repeatedly showed the same pattern of delayed and/or decreased recovery. Finally, this study is the first to analyze the association between treatment protocol and renal and patient outcomes in CRRT-first patients.We acknowledge several limitations. First, our study is observational in nature. Thus, unmeasured confounding could have affected the results. We can only report associations, not causation, and the findings can only be considered hypothesis generating. Second, in the RENAL trial, all patients received only CRRT, while, in the ATN trial, they received both CRRT and IHD depending on their hemodynamic state. Thus, all IHD patients are from the ATN trial, creating additional bias. Such possible “trial protocol effect” was supported by the analysis of patients with high cardiovascular SOFA scores. Third, independent of trial design, time from ICU admission to randomization was longer in the ATN trial. However, illness severity scores and laboratory tests at RRT initiation were nearly identical and time-to-intervention difference was adjusted for. Fourth, although we mainly focused on RRT dependence at day 28 as the primary outcome in order to maximize power, we acknowledge that there is no consensus on the definition of persistent RRT dependence after AKI. Finally, the association between treatment protocol and renal and patient outcomes in CRRT-first patient may not reflect just the impact of the treatment protocol itself but also the impact of other unmeasured practice variation differences between the USA and ANZ.In critically ill patients with AKI treated with RRT, compared with the use of IHD as first treatment modality, after adjustment for confounders, use of CRRT as first treatment modality was not associated with decreased RRT dependence at day 28. However, among survivors to at least day 28, use of CRRT as first modality was associated with decrease risk of RRT dependence at day 28 and more RRT-free days at day 28. These findings were supported by multiple sensitivity analyses and appeared to reflect not only the choice of modality but, in CRRT-first patients, the possible impact of trial protocol. These findings provide additional detailed epidemiological information to inform clinical decision-making regarding choice of treatment modality and care protocols in critically ill patients with severe AKI.Additional file 1. Supplementary Methods, Supplementary Tables 1-13 and Supplementary Figures 1-8.


Background: Common diagnostic tools for prostate cancer—prostate-specific antigen and transrectal biopsy—show only low predictive value and poor sensitivity. This study examines circulating miRNA in saliva to explore the possibility of a non-invasive and easy-to-execute diagnostic tool for prostate cancer screenings. Methods: 16 miRNAs were extracted from salivary exosomes and analyzed via the delta-CT method. The presented method enables an application of the test in any health institution and even outpatient sector. Recruited participants were suspected to suffer from prostate cancer due to elevated PSA serum levels. Of these participants, 43 were diagnosed with prostate cancer, while 31 suffered from benign diseases and served as control group. Results: hsa-mir-331-3p and hsa-mir-200b were significantly reduced in prostate cancer patients compared to the control group. ROC curve analysis revealed a reliable differentiation strength (AUC > 0.6) for both miRNAs with positive predictive values of 71% indicating prostate cancer. Differentiation of both groups based on PSA serum measurements was insufficient. The other 14 examined miRNAs showed no significant group differences. Conclusions: The presented method and miRNA are promising non-invasive tools to augment the current prostate cancer screening, thereby improving screening sensitivity and reducing numbers of false positive cancer suspects admitted to further invasive diagnostic and therapeutic steps. Today, prostate cancer is one of the most common cancer types among men . WorldwiCirculating miRNAs offer hope to overcome many drawbacks by virtue of their cancer-specific expression and easy accessibility in a variety of human body fluids such as blood/serum, urine, and saliva ,10,11,12In addition, studies showed different levels of circulating miRNAs between tumor patients and healthy control groups. They identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases . SeveralA signature including miRNA-375 was validated by more than one group, making it a reliable marker of a systemic disease. Additionally, miRNA-375 reached a level of significance when correlated with tumor stage and Gleason score in patients’ sera .Six upregulated miRNAs were identified in urine, making it possible to discriminate between PCa and BPH patients. miRNA-484 is among these identified molecules .Despite the effort, to be able to discriminate men with potential PCA risk with a quick, easy and noninvasive test, this field needs further development . Saliva It was found that therapy-induced neuroendocrine prostate cancer is increasing in incidence with the widespread use of highly potent androgen receptor-pathway inhibitors, most probably through neuroendocrine differentiation. This differentiation seems to be accompanied by key miRNA alterations including downregulation of miR-106a~363 cluster and upregulation of miR-301a and miR-375, as Bhagirath et al. demonstrated most recently . New tecThis study aimed to explore the diagnostic and clinical potential of miRNA in saliva samples as a new and easy to access screening tool to identify men suffering from prostate cancer. Due to the diversity of the molecules under consideration, a thorough literature review was performed to identify the most promising miRNA candidates.We identified 16 circulating miRNAs with promising diagnostic relevance as possible biomarkers from whole saliva samples. Literature research was done according to the Preferred Reporting Items for Systematic Review and Meta-analysis statement . We focuThe concept of extraction of miRNA from salivary exosomes was based on the model of Gallo et al. , see FigPatients were continually recruited from the urology department of the Helios University Hospital, Wuppertal. A signed informed consent for the study was mandatory. The conduction of the study took place from summer 2014 until winter 2018. Included were men of any age with elevated PSA-blood serum levels suspected to suffer from prostate cancer and admitted for result clarification via prostate biopsy. Exclusion criteria were secondary diagnosis, e.g., infectious diseases , Sicca Syndrome, prior interventions such as operations of the salivary gland or prostate, urinary diversion with intestine tissue, radiotherapy of the pelvic region or antiandrogen therapy. The sample size of the cohort was planned to be 100 participants with equal distribution between diagnosed prostate-cancer patients and non-cancer patients. Men with elevated PSA levels without histologic proof of cancer in transrectal prostate biopsy samples (31 participants) were assigned to the no-cancer control group. Besides prostate cancer, other reasons for PSA level elevation were medical issues such as prostatitis, urinary occlusion, urinary tract infection or secondary genesis, but most commonly benign prostatic syndrome .In all cases routine blood and urine samples were taken, a routine urine bacterial culture was acquired and before prostate biopsy preventive antibiotic therapy (Ciprofloxacin) was given. Each patient gave an additional 5 mL whole saliva sample. Patients were instructed to refrain for two hours from smoking, drinking or eating to keep all samples pure, genuine and undiluted. In total, the study involved 86 patients of which 74 patients were able to provide whole saliva samples valid for miRNA concentration level analysis. A total of 43 patients were later diagnosed with histologically verified prostate cancer. Furthermore, 31 patients missed the histological criteria for prostate cancer, presenting other causes for elevated PSA levels, e.g., benign prostatic syndrome. The diagnoses behind these causes were not itemized any further. This cohort represents the study sample for analysis of concentration differences of 16 miRNAs between cancer and non-cancer patients. Only the two authors, both medical doctors responsible for specimen collection, had initial access to personal information of the patients. The collected data were anonymized and retrospective conclusions to single patients were and are not possible.g for 2 min to remove food and cell debris. Two milliliters of the supernatant were centrifuged at 1500 rpm for 10 min (Mini-Spin); additionally, for saliva with high viscosity, it was followed by a centrifugation at 4700 rpm for 10 min (Mini-Spin). The supernatant was then transferred into a new tube and centrifuged at 13,500 rpm for 15 min (Mini-Spin) at first and then at 160,000× g for 60 min at 4 °C in a SS55S Rotor in an MGX-120 Ultracentrifuge . The supernatant was then discarded. One mL of Trizol reagent was added to the pellet and mixed. Then, 200 µL of chloroform were added to the Trizol and vortexed for 30 s, before the mixture was allowed to rest for 3 min and subsequently centrifuged at 13,500 rpm for 15 min (Mini-Spin). The upper, clear phase was transferred into a new Eppendorf tube and 500 µL of Isopropanol was added. The mixture was then incubated at −20 °C for 20 min. After that, the mixture was centrifuged at 13,500 rpm for 15 min (Mini-Spin) and the supernatant was again discarded. The pellet was washed with 1 mL of 75% ethanol, followed by centrifugation at 11,000 rpm for 5 min (Mini-Spin) and discarding of the supernatant. The pellet containing the miRNA was dried for 20 min with the cover lid opened at room temperature, and 25 µL of H2O (RNAse-free) were added to the pellet followed by vortexing. At last, the mixture was incubated at 60 °C for 15 min, followed by a brief centrifugation due to condensed water at the cover lid.Immediately after collection, the saliva samples were frozen at −20 °C in a conventional freezer and later stored at −80 °C in our laboratory facilities. For analysis, the samples were thawed at room temperature and centrifuged at 1000× ® was used. Quantitative real-time PCR (qRT-PCR) was used to detect and quantify sequences of interest. In total, 8 µL Master Mix were combined with 12 µL template RNA, adding up to 20 µL RT-mix for the reverse transcriptase reaction protocol.First, miRNA was transcribed into cDNA via reverse transcriptase (RT) reaction using the miScript II RT Kit 50 from QiagenmiScript SYBR Green PCR Kit from Qiagen® and specific primer assays for the sequences were used. The standardizing assay  was U6 snRNA, which by the time this study was conducted was seen to be one of the state-of-the-art reference genes for this type of investigation and its reliability was proven by former studies  = −2.158, p = 0.031, Mann-Whitney-U) [All sixteen prostate cancer-associated miRNAs were detectable in exosomes from saliva samples processed via the modified sampling method by Wiegand et al. and were therefore part of further comparative analysis. hsa-mir-200b and hsa-mir-331-3p showed statistically significant concentration differences between cancer patients and the non-cancer control group  were significantly reduced in saliva samples from prostate cancer patients compared to the non-cancer control group. The observed group differences from saliva samples correspond with former studies reporting a significant reduction of hsa-mir-200b and hsa-mir-331-3p in prostate tumor cells compared to healthy prostate tissue samples ,44,45,46Hsa-mir-200b is a member of the mir-200-family whose involvement in carcinogenesis and aberrant cell development is documented for many different cancer types. In prostate cancer, reduction or loss of hsa-mir-200b limits the suppression of Bmi-1, which is accountable for abnormal cell proliferation, migration and chemosensitivity . Other rFor hsa-mir-331-3p, former studies revealed an important tumor suppressor role in prostate cancer development. The findings of Epis et al. (2009) suggest a role for hsa-mir-331-3p in the development and progression of prostate cancer while focusing on ERBB-2 as a target of this miRNA . A loss As described under the methods section, saliva processing and analysis were based on the protocol presented by Gallo et al. with modifications established by Wiegand et al. In the original paper of Gallo et al. saliva samples were immediately centrifuged and purified to prevent secondary lysis of cells and/or other cellular residues . However, these first steps especially are the most difficult to execute in smaller healthcare institutions, practices or for non-professional individuals at home. The modified sampling and storage protocol established by Wiegand et al.  freezes Wiegand’s freeze storing modification could enhance the possibility of a widespread use of salivary miRNA analysis in everyday clinical life and even the outpatient sector, since this modified sampling process is much easier to implement. Saliva is naturally contaminated with the remains of cell debris, food and lysed bacteria, which probably account for an unspecific fraction of microvesicles in the body fluid. Freeze thawing of the samples will undeniably add different kinds of intracellular microvesicles to the primarily desired fraction of extracellular salivary exosomes. We hypothesized that miRNA analysis focusing on prostate cancer associated miRNAs would withstand any major interferences such as sample contamination or secondary cell lysis by virtue of the sequence’s disease specific expression. Gallo et al. themselves stated that even though the analysis of only extracellular exosome-derived miRNA enhances the sensitivity of miRNA, it is not mandatory .This study supports their statement and showed that concentration measurements of prostate cancer-associated miRNAs  extracted from salivary exosomes (regardless intracellular or extracellular exosome) show significant group differences between prostate cancer participants and the non-cancer control group with reliable diagnostic value. Thus, the effects of freeze thawing seem to be minor and negligible in case of disease-specific miRNA analysis. The reliability of the results is underpinned by the prostate cancer associated expression of those two miRNA sequences numerously described in other studies ,43,44,45Further investigation of the diagnostic strength of hsa-mir-200b and hsa-mir-331-3p was determined via ROC curve analysis, which additionally allows a determination of a cut off value with an ideal ratio of sensitivity to specificity. Both miRNAs presented moderate but reliable strength in differentiating between cancer and the non-cancer control group (AUC of 0.663 for hsa-mir-200b and 0.648 for hsa-mir-331-3p)  see Fig. This isFor comparison, meta-analysis of PSA serum tests  determined a positive predictive value for prostate cancer of only 30%  [Fourteen other miRNA sequences were examined and analyzed in this study. All sequences have successfully been detected in salivary exosomes but in contrast to has-mir-200b and has-mir-331-3p without significant concentration differences between cancer and non-cancer participants. The reason for this inconsistency remains unclear as they all have been described as prostate cancer-associated sequences in the preliminary literature research ,29,30,31Another reason may be found in the composition of the no-cancer control group of this study. All participants recruited for this study were men suspected to suffer from prostate cancer due to elevated PSA serum levels and admitted to hospital for prostate biopsy to histologically secure or disprove a cancer diagnosis. Men diagnosed with benign diseases of the urogenital tract  later formed the non-cancer control group. Dysregulated miRNA leading to abnormal cell-cycle, cell division or cell metabolism may eventually cause tissue carcinogenesis but may similarly be involved in benign prostate pathologies such as prostatitis or benign prostate hyperplasia, e.g., miRNA-106a in BPH . Thus, tThe present study produced promising results for a saliva-based miRNA test method in prostate cancer diagnostics. The study design included men with elevated PSA serum levels. Half of the patients were diagnosed with prostate cancer; the others were diagnosed with benign diseases of the urogenital tract accompanied by PSA level elevation. The results demonstrated a reliable identification of cancer patients among prostate cancer suspects. This hints at a high potential to identify prostate cancer patients in general prostate cancer screenings. However, healthy individuals were not included in the study and are not part of the comparative analysis which is why a detailed diagnostic value has yet to be examined. Nevertheless, the present study design highlights a problematic aspect of the current prostate cancer screening program and presents salivary miRNA measurement as a possible solution. The current prostate cancer screening produces high numbers of false positive cancer suspects, mainly due to the low predictive value of PSA serum measurements . Other cThis study showed a reliable differentiation of prostate cancer patients and those falsely suspected to suffer from prostate cancer due to elevated PSA levels (no-cancer control group) via non-invasive measurement of hsa-mir-331-3p and/or hsa-mir-200b in exosomes from whole saliva samples. Due to the differentiation ability of those two miRNA sequences, a conspicuous PSA test result could be augmented by this method to verify or refute an uncertain cancer suspicion. Thanks to the modified sampling and processing protocol, this augmentative diagnostic method could be applied in nearly every healthcare institution, in the outpatient sector and even at home. In consequence, hsa-mir-331-3p and hsa-mir-200b concentration measurement in saliva samples could reduce numbers of false positive cancer suspects in the preventive screening program and thereby reduce numbers of falsely indicated prostate biopsies.All recruited patients of the present study were suspected to suffer from prostate cancer due to elevated PSA serum levels in preventive screening procedures. In the end, only half of these suspects were diagnosed with malignant prostate cancer; the others showed elevated PSA levels due to benign reasons of other kinds. A differentiation of both groups based on the standard screening parameters and their assessment by medical experts proved insufficient. The present study demonstrated for the first time an easy-to-execute and non-invasive screening method based on salivary miRNA with the ability to substantiate or contradict prostate cancer suspicion with a prognostic value of 70%.Saliva samples could be easily collected and frozen in any healthcare institution and further processed in centralized laboratories. An easily accessible tool could advise clinicians and/or patients in the decision-making process for further diagnostic steps, potentially reduce the number of false positive prostate biopsies, and in the end, may increase the overall sensitivity and specificity of prostate cancer screenings.These findings suggest that exosome-derived salivary miRNA could serve as a reliable and, most importantly, non-invasive evaluation tool of prostate cancer screening results  under the concept of liquid biopsy .We believe that further investigation of salivary exosomes and miRNA expression assays might reveal more potential candidates of prostate cancer-associated circulating miRNAs enabling a non-invasive, saliva-based test method with high prognostic value to indicate prostate cancer and maybe replace PSA measurements in the long run. In this context, hsa-mir-200b and hsa-mir-331-3p are the first two promising candidates for an additional non-invasive part in prostate cancer diagnostics.


To analyse the effect of hospital pre-admission screening and enhanced precaution strategies on the transmission of severe acute respiratory syndrome coronavirus-2.This retrospective cohort study was conducted over 17 months from 11 May 2020 to 30 September 2021 at a large hospital in Tokyo. Universal DNA amplification tests were conducted during pre-admission screening, and enhanced precaution strategies were implemented for all patients with negative admission tests. The primary outcome was the occurrence of symptomatic coronavirus disease 2019 (COVID-19) in patients after admission. The secondary outcomes were time-series analyses of monthly positive admission test numbers, positive rates, clinical features in positive cases, and clinically confirmed nosocomial transmission.n=32) tested positive and were admitted to a designated COVID-19 ward or were not admitted. Among the five inpatients who developed symptomatic COVID-19 during hospitalization, only two cases were related to a single nosocomial transmission.In total, 32,081 patients were screened pre-admission . Of the asymptomatic patients, 0.11% (Pre-admission test screening was effective in identifying asymptomatic cases of COVID-19. This allowed administrators to quarantine patients or delay hospital admission. The combination of testing and enhanced precaution strategies for asymptomatic cases of COVID-19 may minimize nosocomial transmission. As a strategy to prevent nosocomial transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), symptom-based admission screening, such as symptom interviewing, has been implemented in hospital settings Furthermore, a previous study reported the insufficient effectiveness of admission testing alone This retrospective observational cohort study was conducted to evaluate the effect of pre-admission universal DNA amplification tests for all inpatients and the implementation of enhanced infection precaution strategies during hospitalization. The study was conducted at Tokyo Medical University Hospital (TMUH), a 905-bed tertiary hospital, over 17 months from 11 May 2020 to 30 September 2021. Five waves of COVID-19 occurred in Tokyo during the study period: (a) the Wuhan clone from March 2020 to May 2020; (b) the Wuhan clone from July 2020 to September 2020; (c) the Wuhan clone from November 2020 to February 2021; (d) the Alpha variant from April 2021 to June 2021; and (e) the Delta variant from July 2021 to September 2021.TMUH is located in Shinjuku City, the largest urban centre in Tokyo. The admission rate of TMUH averages more than 26,000 patients per year. Patients with a positive SARS-CoV-2 status are admitted to a designated hospital ward in TMUH, whereas those with a laboratory-confirmed negative test are admitted to the general hospital wards. Regardless of the hospital ward, all patients are admitted to equal pressure rooms, except for haematology patients who require positive pressure rooms. Only patients with COVID-19 requiring intensive care or aerosol-generating procedures are admitted to negative pressure rooms. The standard room designs are single-patient rooms or four-patient rooms without side rooms in both the designated COVID-19 ward and general wards. During the study, the standard room air exchange rate was two times per hour. All patients admitted during the study period were classified retrospectively as symptomatic or asymptomatic.Vaccination against SARS-CoV-2 started in Japan on 17 February 2021, with healthcare workers prioritized over the general population. Vaccination rates for first and second doses were 88.69 million (70%) and 75.04 million (59.3%), respectively, at the end of the study period. For individuals aged ≥65 years, who account for the largest number of hospital admissions, the vaccination rates were 32.35 million (90.5%) for the first dose and 31.9 million (89.2%) for the second dose.First, universal DNA amplification test screening was performed for all patients on admission, regardless of symptoms, to identify all patients with laboratory-confirmed SARS-CoV-2 infection. DNA amplification testing occurred within a maximum of 14 days before admission (mean 3 days), as 14 days was the maximum incubation period for all three variants. Additionally, after admission, if the patient became symptomatic or was identified as being at risk through contact tracing, retests were performed. When a patient was diagnosed with COVID-19 after admission, interviews and contact tracing were performed to judge the risk of transmission between patients and from patients to medical staff. Similarly, when medical staff developed COVID-19, they were surveyed about their work status, personal protective equipment compliance, patient contact, and social activity outside of the hospital to determine the risk of transmission from patients to medical staff or from medical staff to patients.Planned admission patients underwent reverse transcription polymerase chain reaction (RT-PCR) assay , which has 85% test sensitivity and 95% test specificity. The loop-mediated isothermal amplification (LAMP) test , which has 80% test sensitivity and 95% test specificity, was used for emergency admission patients. The thresholds for positive or negative results were based on the manufacturers’ manuals. If the LAMP test showed equivocal results, an RT-PCR assay was performed for confirmation.Second, in all general wards, enhanced infection precaution strategies were implemented in addition to universal NAAT admission screening. The details of the enhanced infection precaution strategies are shown in The primary outcome of the study was defined as the occurrence of laboratory-confirmed and symptomatic COVID-19 in patients after admission with negative admission screening tests. The secondary outcomes were defined as the total number of positive admission tests, time-series analyses of monthly positive admission tests, positive universal admission tests, positive admission tests, clinical features of positive admission cases, and the details of clinically confirmed nosocomial transmission of SARS-CoV-2 between patients and healthcare workers.https://stopcovid19.metro.tokyo.lg.jp/).Additionally, a time-series comparison was performed between positive cases at admission screening at TMUH and COVID-19 notification trends in the whole Tokyo metropolitan area. The COVID-19 notification case numbers in the Tokyo metropolitan area were extracted from official data on the Tokyo Metropolitan Government website (n=32). Among the 2525 inpatients who were symptomatic on admission, 334 received positive DNA amplification test results, resulting in a positive test rate of 13.2% (n=334).In total, 32,081 DNA amplification pre-admission tests were performed at TMUH during the study period. Among the tested patients, 29,556 were asymptomatic and 2525 were symptomatic on admission. Among the 29,556 patients who were asymptomatic on admission, 32 received a positive DNA amplification test result and were admitted to a designated COVID-19 ward or were not admitted. The positive rate among the asymptomatic cases was 0.11% (Whereas 10 of the 32 asymptomatic patients with positive test results (31.3%) remained totally asymptomatic, five (15.6%) developed symptomatic COVID-19 after admission. Twelve patients (37.5%) reported being asymptomatic but showed incidental pneumonia on chest X-ray or computed tomography. This meant they had COVID-19 but did not recognize the symptoms (self-unrecognized patients). Five patients (15.6%) had a previous history of laboratory-confirmed COVID-19 more than 14 days before admission (postinfection patients).Despite negative admission tests on admission, five inpatients developed symptomatic COVID-19 after admission. The details of these five cases  who developed symptoms after admission are as follows: Patient A was an index case who transmitted the virus to another case in the same room, Patient B. This was nosocomial transmission between Patients A and B. Related to Patients A and B, three healthcare workers tested positive on DNA amplification tests. Although a mandatory surgical mask policy was in place and complied with by all medical staff and almost all inpatients, Patients A and B could not wear surgical masks due to respiratory dysfunction. Despite Patients A and B not being able to wear surgical masks, the medical staff caring for these individuals did not wear eye protection. Patients C, D and E were confirmed as other COVID-19 cases that developed after admission, which means they were sporadic cases as no additional clinically confirmed cases of nosocomial transmission were found between patients and/or healthcare workers.Another two patients, who were admitted for a few days for gastrointestinal endoscopy, developed symptoms and tested positive at home within 48 h of discharge. Although patients do not generally receive a follow-up call and are not generally subjected to systematic testing after discharge, the patients reported that they developed symptoms and tested positive. These additional cases raised the possibility of transmission to other patients, and contact tracings were performed. However, laboratory-confirmed symptomatic COVID-19 was not reported in any other patients other than Patients A–E.The number and time-series data of asymptomatic positive DNA amplification cases per month are shown in The occurrence of positive results was almost concomitant with the general population epidemic trends . Based oPrevious studies of universal admission screening have reported positive rates ranging from 0.03% to 14.5%, depending on the study period and the epidemic status of the study site . EpidemiThe patient population that should be prioritized for infection prevention for nosocomial transmission appears to be presymptomatic patients. Previous studies have illustrated that presymptomatic patients, who can have a huge impact on transmission ,11, compA simulation study indicated that routine testing, such as more than twice per week, would be effective and would prevent transmission in high-risk settings, such as healthcare facilities Another negative aspect of highly test-dependent strategies is the detection of postinfectious status ,17. EpidThe criteria for the adaptation and withdrawal of test admission screening at each hospital remain an unsolved problem. The present study indicated fluctuating positive cases per month from 0% to 0.52%, almost coinciding with the prevalence in the community. The minimum number of COVID-19 daily notifications was 92 cases, and the weekly average was 8.93 per 100,000 population in the Tokyo metropolitan area among dates with positive NAAT admission screening. A Swiss study reported that universal admission testing did not identify a substantial number of asymptomatic cases in a low-prevalence setting  This study has some limitations. First, although admission test screening was performed comprehensively, follow-up DNA amplification screening (retesting), such as 4–7 days after admission, was not performed. This may result in underdetected transmission of COVID-19. However, during the 17 months of this study, no clinically confirmed nosocomial clusters of COVID-19 from underdetected asymptomatic or presymptomatic patients, other than the case recognized, were observed. Second, the effect of COVID-19 vaccination coverage was not included in the analysis of this study. The vaccination status of the study population could affect the effectiveness of pre-admission screening and the enhanced infection control strategies. Third, the effect of the virus variant was not analysed. In the future, changes in transmissibility or clinical symptoms of the virus due to its variants may influence evaluation of the effect of admission test screening. Fourth, serological testing and whole-genome screening were not performed. Test-based judgement and clinical case identification may result in a low estimation of SARS-CoV-2 infectious status because some asymptomatic or mildly symptomatic cases without further DNA amplification testing may develop after admission. Combining serological tests and whole-genome screening can reinforce the data. If whole-genome screening is undertaken, this may be able to detect and validate nosocomial transmission events. Fifth, the study was observational without a comparison group to measure the impact of this intervention. Determination of the effectiveness of admission test screening or combined infection prevention strategies requires further comparative study. Finally, the study results are from the prevalence and surge levels in Japan, and may not apply to other countries and phases. The data and effects of universal admission screening have varied depending on the prevalence level.More than 30,000 DNA amplification pre-admission screening tests detected only 32 cases of asymptomatic or presymptomatic COVID-19. Although five of these cases developed symptomatic COVID-19, enhanced infection control precaution strategies in addition to universal admission screening may have reduced the occurrence of clusters.None.IN: study design, data analysis, drafting the manuscript.YW: data collection, critical revision of the manuscript.HF: data collection, critical revision of the manuscript.TK: critical revision of the manuscript.HW: study design.TI: study design, data analysis, drafting the manuscript.This study was approved by the Ethical Committee of Tokyo Medical University . The need for consent was formally waived by the Ethical Committee of Tokyo Medical University. This study was performed in accordance with the 1964 Declaration of Helsinki and its later amendments.The dataset supporting the conclusions of this article is included in the article.None declared.