Introduction: Amidst the evolving COVID-19 pandemic, understanding the transmission dynamics of the SARS-CoV-2 virus is key to providing peace of mind for the community and informing policy-making decisions. While available data suggest that school-aged children are not significant spreaders of SARS-CoV-2, the possibility of transmission in schools remains an ongoing concern, especially among an aging teaching workforce. Even in low-prevalence settings, communities must balance the potential risk of transmission with the need for students' ongoing education. Through the roll out of high-throughput school-based SARS-CoV-2 testing, enhanced follow-up for individuals exposed to COVID-19 and wellbeing surveys, this study investigates the dynamics of SARS-CoV-2 transmission and the current psychosocial wellbeing impacts of the pandemic in school communities.Methods: The DETECT Schools Study is a prospective observational cohort surveillance study in 79 schools across Western Australia (WA), Australia. To investigate the incidence, transmission and impact of SARS-CoV-2 in schools, the study comprises three “modules”: Module 1) Spot-testing in schools to screen for asymptomatic SARS-CoV-2; Module 2) Enhanced surveillance of close contacts following the identification of any COVID-19 case to determine the secondary attack rate of SARS-CoV-2 in a school setting; and Module 3) Survey monitoring of school staff, students and their parents to assess psycho-social wellbeing following the first wave of the COVID-19 pandemic in WA.Clinical Trial Registration: Trial registration number: ACTRN12620000922976 The first cases of COVID-19, a novel infection caused by the virus now designated SARS-CoV-2, were notified in December 2019 . Three qCOVID-19 prevalence in Australia has been low relative to the global context, with just over 27,500 cases reported to October 30, 2020. The state of Western Australia (WA), with a population of ~2.5 million, has experienced only 775 cases, 9 deaths and no confirmed community transmission since April 2020. After a period of low school attendance and school holidays (March 15–April 29), during which parents were encouraged to provide home learning to their children, all students in WA were required to return to school from April 29, 2020. This call to return students to school sparked community concern about student and school staff safety. While existing data indicates the COVID-19 burden and rate of transmission in children and young people is much lower than adults –10, therIn addition to gaining an understanding of SARS-CoV-2 transmission in children and young people, the influence of social distancing measures such as school closures on their psychological health and wellbeing needs to be explored . EvidencIn late April 2020, the WA government instigated the DETECT Schools Study in partnership with the Telethon Kids Institute to assess the prevalence and impact of COVID-19 in WA schools. The study, detailed here, seeks to quantify and characterize the transmission of SARS-CoV-2 in WA school settings while investigating the wellbeing of students, teachers, and parents during the pandemic.2. Data collection will initially be conducted for 6 months and may be extended beyond this depending on the evolving COVID-19 situation.The DETECT Schools Study is a prospective observational cohort surveillance study currently underway in 79 WA government schools, including education support settings and residential colleges. These schools are distributed across our large state, over an area of 2.6 million kmThe overall aim of the DETECT Schools Study is to better understand the burden, transmission, and psycho-social factors associated with COVID-19 in WA school communities. To this end, the study comprises three modules with distinct objectives:Module 1 (asymptomatic SARS-CoV-2 testing): to investigate the prevalence of asymptomatic SARS-CoV-2 infection in a cohort of randomly selected school students and staff (in 40/79 participating schools).Module 2 (SARS-CoV-2 index case response): to determine the secondary attack rate of any SARS-CoV-2 infections detected in a WA school setting (in 40/79 participating schools).Module 3 (wellbeing factors associated with the COVID-19 pandemic): to elicit self-reported data on the physical, social and emotional wellbeing of students, their parents, and staff .Modules 1 and 3 of the study are in progress, and Module 2 will be initiated as needed.The DETECT Schools Study is a WA Government initiative, coordinated by the Telethon Kids Institute in partnership and consultation with the WA Departments of Health and Education. Study design, operational oversight and data interpretation is led by the Telethon Kids Institute study team. School selection and management is coordinated by the Department of Education. Swab testing is coordinated by the Department of Health: samples are collected by the Child and Adolescent Health Service (CAHS) and the WA Country Health Service (WACHS), and SARS-CoV-2 testing is performed by public laboratory service provider PathWest.Community involvement and advice was actively sought in the design and preparation of this study and will inform the analysis and dissemination of findings. A National Community Advisory Group for COVID-19 Research, convened by the Telethon Kids Institute, reviewed and provided feedback on procedures and resources including consent forms and surveys to aid in the development of content most appropriate for potential study participants, including children with disabilities. This panel comprised community members from across Australia, including Aboriginal members. Telethon Kids Institute Kulunga Aboriginal Research Development Unit provided advice regarding communications with Aboriginal families who may be involved in the study, including culturally-secure and informed consent processes and measures for supporting Aboriginal families during Module 2, which requires swabbing and symptom checking to take place within the home. Further, advice was sought from the Department of Education and the four participating education support schools to develop visual communication tools to increase accessibility of the survey for students in these settings.While considerable time was spent refining the DETECT Schools Study protocol and resources, the study proceeds with an acute awareness that implementation may well bring to light additional considerations or challenges. As such, consultation with these advisement bodies is ongoing and will continue to be critical in informing the trial as it progresses.Testing team members are trained in swab collection and management by a pediatric infectious disease specialist (AB). This training includes online and/or face-to-face training sessions, and access to swab collection training videos and Standard Operating Procedures for swab collection and transport. Testing team members are also trained in the use of the REDCap database for data collection during in-school swabbing. REDCap training and a School Coordinator Guide are also provided to a School Coordinator from each participating school to aid in survey data collection.a priori criteria. The potential for bias due to this non-random selection will be addressed during data analyses, where sample weightings will be employed to adjust for under- or over-representation across the geographical and demographic characteristics of each school, as determined from Australian Bureau of Statistics (ABS) census data.Eighty schools were selected for participation by the Department of Education, ensuring the inclusion of broad representation across state geography, primary and secondary schools, and specialist facilities including residential colleges and education support settings. A variety of practical considerations with respect to maximizing school participation and project feasibility were considered. Consequently, school selection did not follow a strict randomization procedure from all schools state-wide, although care was taken to minimize biased selection based on any The eighty participating schools were stratified into 10 groups by geographical region and school classification .Within each stratum, schools were allocated to either the swab testing or non-swab testing groups by a simple randomization procedure. Forty schools were randomly allocated to the swab testing group and were invited to participate in Modules 1 (asymptomatic SARS-CoV-2 testing), 2 (SARS-CoV-2 index case response) and 3 (wellbeing survey). The remaining 40 schools were randomly assigned to the non-swab testing group and were invited to participate in Module 3 only. Following recruitment and randomization one non-swab testing metropolitan secondary school opted out of the study, leaving 79 participating schools.Distinct consent was sought for each study module. Invited participants consented to the three study modules (swab testing group) or Module 3 only (non-swab testing group) at the beginning of the study through one online consent process supported by the REDCap platform . ParticiModules 1 and 2 require active consent from participants, collected from staff and students' parents/guardians at the 40 swab testing schools. Consenting individuals (or guardians) complete a consent form in the study database and may subsequently withdraw at any time.For Module 3, a passive consent approach is adopted for students: parents are required to actively “opt-out” their child from participating and the survey is offered to all students who have not opted out. Previously, application of an opt-out consent framework nationally by the Australian Early Development Census (AEDC) resulted in a minimum participation rate of 95%, serving to minimize selection bias and maximize the reliability and generalisability of the research data collected. The use of student passive consent for this module was supported by the consumer reference group and ethics committees. Parents and staff provide active consent to participate but may opt out of participation at any time.Module 1 entails combination throat and nose SARS-CoV-2 swabbing of asymptomatic students and staff to quantify how much (if any) asymptomatic infection is present in schools. Students at school on the day of testing are assumed to be asymptomatic, as the Department of Education has adopted a policy supporting families to keep any children with COVID-19 symptoms at home. From the consented population, up to 150 randomly selected students and staff are swabbed in each school per round, with staff comprising 10% of the sample.Participants are excluded from swabbing if they have experienced any of the following:More than one nosebleed per week.Recurrent vomiting, particularly if triggered by a gag reflex.Large/problematic tonsils awaiting tonsillectomy.Significant anxiety about medical procedures.During the consent process, parents were asked to confirm that their child(ren) did not meet the exclusion criteria. Staff were asked to self-assess against the exclusion criteria.Three to six rounds of swabbing will be conducted, approximately monthly. Of students and staff who provided active consent, 150 will be randomly selected from each school in each round. Student grade level groups will be stratified into two strata per school . Randomization will be conducted within the REDCap database using a simple random number algorithm, generating a list of 200 consenting participants: 45% students from each stratum and 10% staff. If a randomized student or staff member is not available or does not assent to testing on the day, they will be replaced by the next available consenting participant from their stratum until 150 swabs are collected.SARS-CoV-2 swabbing will be carried out by a team of nursing staff. The participant's information will be verified to ensure consent has been obtained, and if for any reason identification and therefore consent cannot be confirmed , then swabbing will not continue with that individual. All participants will be asked to verbally assent prior to swabbing being performed. An oropharyngeal (OP) swab  and subsequent nasal (Na) swab   will be The combined oropharyngeal and anterior nasal (OP/Na) swab employed for this study is not the same as the nasopharyngeal swab commonly used for clinical SARS-CoV-2 diagnosis. OP/Na swabbing is significantly less invasive and therefore less likely to cause discomfort. This allows for the swabbing of students without a parent present, minimizes discomfort and optimizes the rate of assent amongst participants. Recent research indicates that OP/Na swabbing has comparable specificity and sensitivity to nasopharyngeal swabbing for SARS-CoV-2 detection . This swPolymerase chain reaction (PCR) testing of all swabs for SARS-CoV-2 will be carried out at PathWest using an in-house PCR platform of high analytical sensitivity and specificity with appropriate positive and negative controls. To reduce the chance of a false positive result, any sample which returns a positive result will undergo confirmatory testing with the GeneXpert SARS-CoV-2 PCR platform . Testing of asymptomatic participants has the greatest risk of returning an unexpected false positive, and use of a second platform with robust internal controls will minimize the risk of reporting a false positive result.Under the Public Health Act 2016 of WA, COVID-19 is a notifiable disease. Any negative SARS-CoV-2 results will be communicated to participants directly by text message from PathWest, in line with standard clinical care notification procedures. In the instance of a positive SARS-CoV-2 result, PathWest will notify the WA Health Communicable Diseases unit, who will initiate standard contact tracing procedures with the confirmed case and liaise with the key contact for the relevant school to ensure appropriate public health action. The Department of Education recommends closure and deep cleaning of any school in which an index case presents, and this will be actioned by the Department of Health. At all times, the identity of the positive case will be kept confidential.For study data analysis, all SARS-CoV-2 results will be communicated to the study team by means of a secure and confidential file transfer.The study sample size  is calculated with intent to achieve as broad and representative a sampling frame as possible. Taking into account practical considerations such as constraints on school participation and feasible total number of samples per month, and given the potential sources of bias and imprecision inherent with these constraints and the likely very low prevalence rate, the task of estimating state-wide infection prevalence among school populations does not lend itself to simple statistical analysis. As such, this study will employ a sophisticated approach validated using simulated data, which explores its performance in a range of different community transmission intensity scenarios.This study seeks to design a robust yet efficient estimator for the prevalence of SARS-CoV-2 infections amongst the general population of school children and staff in WA based on Module 1 testing data. The challenge will be to bound the range of plausible sampling distributions that might arise from observation through the given survey design of the epidemic spread of this virus, especially given the many uncertainties that remain concerning SARS-CoV-2 transmission dynamics.Under these uncertain epidemic conditions, standard approaches to prevalence estimation based on adjustment by estimated design effects for representing intra-cluster correlation may perform unfavorably. A solution for this unique situation is to adopt an estimator designed specifically for robustness against model misspecification. Analysis of Module 1 data will employ a weighted likelihood bootstrap with Bayesian regularization. This nominal model supposes a Poisson sampling distribution with a Gamma distribution prior on the unknown rate equivalent to a single prior observation of one positive case amongst 6000 (40x150) individuals.Module 2 entails enhanced surveillance of the close contacts of any COVID-19 index case identified in a DETECT swab testing school, with a view to identifying any onward transmission and thus elucidating the role that schools may play in the transmissibility of SARS-CoV-2 between students and staff. In tandem to routine public health surveillance, Module 2 activities will include further swabbing and monitoring of students and staff consistent with existing methodologies such as the World Health Organization's First Few Hundred (FFX) Cases Methodology . Module In the event of a positive SARS-CoV-2 result, the WA Health Communicable Diseases unit will be informed. Routine public health follow-up by this unit will begin immediately, including contact tracing to identify close contacts of the case. A close contact is defined as anyone with >15 min of face-to-face contact or >2 h in the same room with a confirmed case.A list of all individuals at the school who have consented to Module 2 participation will be merged with the Public Health unit's close contact list by secure file transfer, identifying all close contacts of the case who have consented to participation in Module 2.Study investigators will telephone each consented close contact to confirm their participation in Module 2, emphasizing the distinction between routine (compulsory) public health instruction and voluntary participation in the study. Sample collectors will attend the participant's residence to collect an OP/Na swab twice during their 14-day isolation period–between days 4–8 and days 11–14–regardless of whether the participant is symptomatic or asymptomatic. Swabs will be processed, and results conveyed to participants, as described for Module 1. Any positive results will be immediately notified to the WA Health Communicable Diseases unit.Routine public health surveillance requires COVID-19 close contacts to self-monitor for symptoms during their 14-day isolation. Close contacts recruited to Module 2 will also be invited by SMS to complete a daily online symptom diary on days 4–14 of isolation, monitoring the participant's health and for symptoms of COVID-19. Participants who indicate symptoms consistent with the COVID-19 case definition [fever ≥38°C or history of fever  or acute respiratory infection ] will be advised to notify the WA Health Communicable Diseases unit immediately.Module 2 participants, regardless of whether they experience COVID-19 symptoms, will be offered a serology test (testing a blood sample for antibodies which indicate a previous SARS-CoV-2 infection) 1 month after their exposure to the index case. If participants give consent for this test, they will attend a PathWest collection center for serology testing between days 28–42 post-exposure. Blood samples will be centrifuged within 24 hours of collection, and detection of antibodies (IgG and IA) carried out by PathWest using the EUROIMMUN serological assay . ParticiModule 3 is a quantitative, quasi-experimental, cross-sectional pulse survey administered in all 79 study schools immediately following the first round of swab testing, with follow-up in Term 4, 2020. It is designed to identify psycho-social wellbeing factors associated with the COVID-19 pandemic among school-aged children, their parents and school staff in WA. Specifically, the survey aims to investigate:How COVID-19 public health interventions are differentially associated with students' and their parents' and teachers' physical, social, and emotional wellbeing across sub-populations  and geography.How COVID-19 social interventions are differentially associated with students' learning and schools' operations across sub-populations  and geography.How students, school staff and parents in swab testing group schools (40/79 participating schools) felt about the COVID-19 screening activities of Module 1.All 79 DETECT study schools will participate in Module 3. Surveys will be administered to all students aged 9–18 years (Grades 4–12) who have not been opted out, and parents of children aged 3–18 years (Grades Kindergarten-12) and school staff who actively consented to participate. As participation requires completion of a self-report survey in English, participating schools have been consulted to understand the cultural diversity of their community and where possible support will be provided to allow those who do not speak English as a first language to participate. Parents who do not have internet access will not be able to complete the survey, and as such are not included in the study, although provision will be made for paper consent. Some sample loss is inevitable due to individuals choosing not to participate, however measures taken to promote participation include:Targeted and informative communications to parents of eligible children and school staff, explaining the value of participation.Development of separate surveys for different age groups with regard for developmental capabilities and educational settings.Ensuring all responses are anonymous and confidential, with no data to be published in a way that could risk identification of an individual or school.In total, five survey instruments were designed specifically for this study: for primary school students (Grades 4-6); high school students (Grades 7-12); school staff; parents of primary school children (Kindergarten-Grade 6); and parents of high school children (Grades 7-12). For pragmatic and budgetary reasons, surveying children below Grade 4 was outside the scope of this study. All survey instruments contain a set of Hogben questions allowing for the linkage of baseline and follow-up responses while maintaining anonymity .Surveys utilize validated scales including the Child Health Utility Index (CHU9D)  and the Wellbeing surveys will be administered to participants online using REDCap survey software . Email aThe first cycle of survey collection (Phase 1) will be conducted following the completion of the first round of Module 1 swabbing. A second cycle of surveys (Phase 2) will be completed before schools close for the year. Given the dynamic nature of schools' responses to state requirements, COVID-19 prevalence, and broad COVID-19 social policy, differential timing of survey administration has the potential to confound results. To address this, schools are “matched” (one “swab testing” school with one “non-swab testing” school) according to school size, type and geography. Matched schools will complete the survey at the same time.Purposeful selection of participating schools by the Department of Education may dispose the study to bias. To mitigate problems that may arise in generalization, participating schools in both conditions will be pooled and compared to the WA Department of Education estimates based on all WA Government Schools. Comparisons will be made with respect to student composition, school size, and National Assessment Program-Literacy and Numeracy (NAPLAN) performances.Without identification of individual schools, aggregated Phase 1 results will be analyzed using descriptive statistics, including frequencies and prevalence estimates, to determine whether any differences in demographics  exist for child and parent wellbeing, resources for children learning at home during COVID-19, experience of schools handling COVID-19, learning choices during COVID-19, or involvement in the DETECT study.Multivariate multi-level  models will be employed to determine which factors may be associated with changes in key outcome measures, including demographic predictors  of child and parent wellbeing, parent self-efficacy, parent or staff positive and negative emotions, parent or staff stress, total amount of resources for home learning, positive experience of schools handling COVID-19, testing concerns, learning choices, reasons for keeping children at home, and reasons for sending children to school. These models will account for the clustered nature of the data and control for school-related variables such as school size and socioeconomic status (SES).Phase 2 results will be analyzed in the same manner. In addition, comparisons will be made to identify any significant differences between Phase 1 and Phase 2 wellbeing survey results and between swab testing and non-swab testing schools.Raw data will be securely stored in REDCap on a password protected server, to which only researchers associated with this project have access. Data will be encrypted prior to distribution between investigators and shared through secure online portals. Each collection's data (each school and each student/parent/staff within a school) will be stored in different areas of the database, with segregation of identifying data where possible. Analyses will only be conducted on de-identified datasets.This study is designed to inform policy and practice in public health, disease screening and education settings. Interim (post-Phase 1 survey collection) and final (post-Phase 2 survey collection) reports will be delivered to Government stakeholders outlining the findings of the study. A series of academic publications will be published, focussed on key findings and subsets of the data collected. While schools will remain anonymous in reporting and publication, findings from the wellbeing surveys will be used to inform school- and community-based intervention as necessary: as such, school-specific results will be shared with each participating school to inform policy and practice.The COVID-19 pandemic has had high rates of infectivity , seriousWith SARS-CoV-2 now a widespread respiratory virus, we must learn how to live with COVID-19 in a way which allows for children's safe and continued education –while trWith parents instructed to send their children to school if they are well, Module 1 of this study is designed to detect asymptomatic SARS-CoV-2 infection in schools. Other studies have investigated asymptomatic SARS-CoV-2 incidence in pediatric settings, and found low prevalence among those attending pre- and primary schools in the UK  , AmericaThe DETECT Schools approach to SARS-CoV-2 swabbing diverges from the nasopharyngeal sampling commonly used for COVID-19 testing in clinics. Nasopharyngeal swabbing is uncomfortable, leading to a high rate of non-compliance, and emerging data suggests that various non-nasopharyngeal samples are appropriate for the detection of SARS-CoV-2 , 44. DatMacartney et al. have reported that students and staff who attended schools in New South Wales (NSW), Australia while infectious with SARS-CoV-2 did not contribute significantly to COVID-19 transmission through their school attendance . Module While much remains to be learnt about the transmission dynamics of this new virus, the impact of the pandemic on the population's psychosocial wellbeing is also not well understood . Much ofWA children have witnessed the wide-spread impact of COVID-19. They have also experienced unprecedented disease prevention measures including optional school attendance and unpredictable changes in schooling, parental employment, and income and housing security. Gaining an understanding of the impact this may have had on their wellbeing will help to guide nimble local and systemic psychosocial policy and intervention, particularly regarding the maintenance of school attendance. This insight may well be applicable to other low prevalence settings and will be complementary to other contrasting research which focusses on the epidemiological and psychosocial implications of COVID-19 in areas of high prevalence. Further to this, the complementary roll-out of Module 1 and Module 3 activities will allow for assessment of the impact that school-based screening has on community anxiety, informing thoughtful approaches for future track and trace endeavors.Across the globe, cross-disciplinary partnerships have proven vital in society's rapid response to the COVID-19 pandemic . In an AThis protocol describes the activities of the DETECT Schools Study. Initiated by the WA state government, this study seeks to understand the extent of asymptomatic transmission of SARS-CoV-2 in schools, to extend the public health contact tracing response to COVID-19 close contacts by determining the SARS-CoV-2 secondary attack rate in school settings, and to investigate the psycho-social wellbeing factors associated with the impacts of the COVID-19 pandemic in school communities.This study was approved by human ethics review committees at the Child and Adolescent Health Service (PRN RGS0000004059) and the WA Aboriginal Health Ethics Committee (PRN 993), and is conducted in partnership with the WA Departments of Health and Education. Study findings will be shared with stakeholders through regular reporting, and with the academic, medical, and broader communities through scientific publications, presentations, and media releases. Testing numbers and results will be shared iteratively with the public through social media. Written informed consent was obtained from the minor's next of kin for the publication of any potentially identifiable images or data included in this article.HT contributed to design of the study protocol and drafted the manuscript. MM coordinated the conception and design of the protocol and contributed to critical revision of drafts. ME, JM, NM, AW, KL, SA, TB, AL, and MH contributed to design of the study protocol and critical revision of drafts. FM, SZ, PG, JC, DC, and AB contributed to conception and design of the study protocol and critical revision of drafts. LL, MC, PS, and EC contributed to design of the data analysis plan. AM facilitated community and consumer engagement and contributed to evaluation of the study protocol. All authors contributed to the article and approved the submitted version.Telethon Kids Institute authors report grants from the Western Australian Department of Health during the conduct of this study. DC and AB report grants from the Western Australian Department of Health outside the submitted work. AB is an employee of the Western Australian Department of Health. The remaining 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.


During the ongoing pandemic of Coronavirus Disease 2019 (COVID-19) allergic patients need to continue their constant and proper treatment, including allergen-specific immunotherapy. These patients are expected to be at a higher risk for exacerbation of lung inflammation during viral infection. We investigated the putative interplay existing between allergen-specific immunotherapy and COVID-19 infection in a Hymenoptera venom–allergic population. We evaluated the frequency and severity of COVID-19 infection in a cohort of 211 subjects referring to our center for the regular administration of venom immunotherapy (VIT). Our result showed that the median age of our cohort is similar to the one that in our region has been associated with a high incidence of COVID-19 infection, increased hospitalization, and mortality rates. We reported only an isolated positivity of COVID-19 in the overall group; whereas none suffered from upper airway symptoms associated with COVID-19 . Even though the demographic characteristics pose a substantial risk for such a population, we suggest that a regular administration of VIT may help in the development of an immunological milieu able to down modulate the Th1/Th17 environment that has been linked to inflammatory manifestations of COVID-19. To the best of our knowledge, this is the first description of the incidence of COVID-19 infection in Hymenoptera venom allergic patients treated with VIT, suggesting indirectly that venom immune tolerance-inducing treatment may be capable of reducing the aberrant inflammatory response induced by the virus in this specific population. To the Editor,The ongoing pandemic of Coronavirus Disease 2019 (COVID-19) has become a global emergency for public health and medical communities.,Allergic patients and subjects suffering from chronic airway diseases were expected to be at a higher risk for exacerbation of lung inflammation during viral infection. Nonetheless, recent studies have shown a lower prevalence of allergic asthma in COVID-19 populations, suggesting that allergic diseases do not represent a potential comorbidity in the case of severe COVID-19-related diseases.In this regard, limited data are available about the effect of AIT on COVID-19 infections, although it is well established that allergen-specific desensitization plays a role in the changes of cellular population and inflammatory signalling taking place in affected organs.We evaluated the frequency and severity of COVID-19 infection in a cohort of Hymenoptera venom–allergic patients treated with VIT at our Allergy and Clinical Immunology Unit from the end of February until May 20, 2020. As shown in Following strict evaluation of eventual positivity of COVID-19 swab (according to our preventive measures for COVID-19 contagion), all subjects accessed the Allergy Unit of the hospital every 4–10 weeks for the VIT administration. Each patient completed a form with information regarding symptoms  and eventual close COVID-19 contacts in the previous 14 days. None demonstrated to be suffering from upper airway symptoms associated with COVID-19. Fifty-three out of 211 patients (25%) confirmed the eventuality of contact with a positive subject. An isolated case of COVID-19 positivity was registered in a patient with preexisting cardiovascular disease and working as a health care professional who developed bilateral COVID-19-related pneumonia. The subject required hospitalization, an antiviral treatment (lopinavir/ritonavir), and hydroxychloroquine together with high-flow nasal cannula oxygen therapy and anti-IL-6 therapy . Upon treatment, the patient had a complete recovery in clinical laboratory parameters and lung imaging.A considerable share of 171 subjects (81%) showed no evidence of a potential professional exposure, while 5 patients resulted to be health workers, thus being virtually at a higher biological risk; as mentioned above, 1 of these subjects developed COVID-19 associated pneumonia. About a half of our patients were retirees (48%) showing a median age of 69 years (between 65 and 75 years) which in our region has been associated with a high incidence of COVID-19 infection and increased hospitalization and mortality rates. Specifically, the average age of subjects who tested positive at COVID-19 swab and required hospitalization in the geographic area of Treviso was 72 ± 14.5 years, comprising 37 health care professionals (3.9%). Concerns exist that angiotensin-converting enzyme inhibitors (ACE-i) and angiotensin receptor blockers increase susceptibility to SARS-CoV-2 and the likelihood of severe COVID-19 illness. As shown in + T cells that have been associated with a venom-specific IgG4/IgE antibody ratio shift capacity and with a modulatory role. In particular, a subpopulation of B cells — the so-called Breg cells — regulates excessive inflammation via the release of IL-10, inhibits proinflammatory response and increases IgG4 production.Among its main effects, VIT contributes to an overall immunological downregulation secondary to the expression of IL-10 and the increase in circulating Treg cells, i.e. Foxp3+ CD4Only one patient in the overall group had a mild course of COVID-19-related inflammatory syndrome, even though the demographic characteristics pose a substantial risk for such a population living in a geographical area where hospitalization and mortality associated to COVID-19 were higher in comparison to the national sample. Our data represent the first description of the incidence of COVID-19 infection in Hymenoptera venom allergic patients treated with AIT and our results indirectly propose that VIT may represent a putative protective factor in this specific population. We suggest that a regular administration of VIT may have an immunoregulatory role, favoring the development of an immunological milieu able to down modulate the Th1/Th17 environment. The greater limitation of a population study conducted on VIT patients lies mainly in the inclusion criteria for allergen-immunotherapy itself. As a matter of fact, VIT is generally proposed to healthy individuals since its contraindications among others list neoplastic diseases and autoimmune disorders non-responding to treatment. However, the study has the merit of sampling a cohort with a median age that constitutes a major risk factor for inflammatory manifestations of COVID-19,On this basis, we are planning to conduct an extensive retrospective data analysis of allergic patients referring to our center and undergoing any kind of allergen-specific immunotherapy in order to investigate if an interplay exists between desensitizing immunotherapy and COVID-19 resistance.No funding to declare.A.D: provided the design of the study, supplied the acquisition of data, drafting of manuscript and analysis and interpretation; C.A. and F.B.: provided the conception and design of the study, acquisition of data, analysis and interpretation of data, revised it critically for important intellectual content and final approval of the version to be submitted; F.C., R.S., M.R., L.T and E.F.: supplied the design of study and gave final approval of the version to be submitted.Approval was obtained from the local ethics committee.The data that support the findings of this study are available on request from the corresponding author, A.D. The data are not publicly available due to their containing information that could compromise the privacy of research participants.Authors provided formal written Consent to Publish.The authors declare that there is no conflict of interest.


Lipomas are common benign mesenchymal tumor that arise from the adipose tissue. Differential diagnosis between the benign and malignant form (liposarcoma) must be made to prevent complication and recurrence.We reported a 63-year-old man who presented with recurrent large-sized mass on the right forearm with the first presentation approximately 28 years before being treated in our center. Main complaints were discomfort, social embracement, and difficulty in dressing. There were no compression symptoms observed. We performed marginal excision and sent the sample for histopathological examination. The work has been reported in line with the Surgical Case Report (SCARE) criteria.During one year of post-operative period, no recurrence was detected and a complete relief of symptoms was as expected.Diagnostic challenge in such a large-sized (giant) lipoma is in differentiating it with the malignant form. Surgical excision is the choice of treatment. However, long-term follow up is needed due to the risk of recurrence. It commonly occurs subcutaneously, however its appearance on any other plane has been reported. Large-sized lipoma or ‘giant lipoma’ is considered if the size is greater than 5 cm in diameter. We presented a case of a 63-year-old male with giant lipoma on the flexor compartment of his right forearm which was treated with complete removal of the lipomatous mass with marginal excision. Post-operative histopathological diagnosis was well-differentiated liposarcoma lipoma-like. No recurrence had been observed on the 1-year follow up. Our work has been reported in line with the Surgical Case Report (SCARE) Guidelines [2A 63-year old male was referred to orthopedic outpatient clinic at our institute with a large lump on the right forearm region. The lump initially appeared 28 years ago and had re-appeared for three times after three times removal. The latest removal was 7 years before admitted to our center and the latest recurrence was in the next 2 years. The patient was only complaining about mild discomfort and difficulty in wearing clothes. The non-tender lump was increasing in size slowly from the size of quail eggs with well-defined border around the wrist until it occupied the whole flexor compartment of the right forearm. The right forearm circumference was 20 cm compared to 7 cm on the contralateral . There wThe laboratory markers showed no abnormality except for high blood glucose since the patient has diabetes mellitus since the past 11 years. He also had a hyperlipidemia with increase of both total and LDL cholesterol level.Patient underwent radiograph assessment with plain x ray which suggest lipomatous mass  and MagnSurgical exploration was therefore performed under general anesthesia and with the application of a hemostatic tourniquet. A lazy-S design was made approximately one cm lateral to the antecubital fossa and extended distally until distal wrist crease. The incision started on the ulnar-volar side on the distal forearm where the initial mass appeared and where it most superficially located under the skin. After skin incision and entering the subcutaneous plane on the wrist region, there was a lipoma-like lesion which continued sub-muscularly underneath the FDS muscle. Blunt dissection through muscle fiber confirmed a deep-seated lipoma lying over the Flexor Pollicis Longus (FPL) muscle. Running through the lipoma was a shiny cord-like structure, which was identified as the median nerve. The mass was carefully dissected off the median n. until releasing the transverse carpal ligament to ensure that the nerve was left intact. The mass was found to compress the surroundings; however, it was relatively easy to remove due to the pseudo-capsule and the intermuscular location .Fig. 4YeThe 15 × 8 × 5.5 cms mass was sent for histopathological analysis. The mass was well-vascularized as observed that there were big vessels that gave blood supply to the mass and these vessels infiltrated the mass especially at the superior pole, where it gave a yellow-red appearance on that region. The mass consistency was generally rubbery with a firmer consistency at the superior pole. .Fig. 5LoHistopathological analysis suggested a lipomatous tumor, low grade, most suitable with well-differentiated liposarcoma (WDL)-lipoma like subtype .Fig. 6HiDuring routine follow-up in the outpatient clinic at one and three months, the wound had healed well and there were no neurological deficit, functional impairment or recurrence of the lump observed. At one-year follow-up the scar healed nicely without recurrence of the mass .Fig. 7Cl3The etiology and pathogenesis of lipomas remain unknown, although genetic endocrine and traumatic factors have been suggested. Our patient had diabetes mellitus in the past 11 years which was treated by oral anti-diabetic drugs. He also had a long-standing hyperlipidemia with the increase of total cholesterol and LDL cholesterol level.Typical lipoma occurs more often in young adults, women (thigh), and in overweight populations. They vary in size ; when thAlthough the definitive diagnosis of giant lipoma can be made only by histopathological examination, the differential diagnosis between giant lipoma and liposarcoma is of great importance. Once suspected, other investigations must be conducted to provide more information about the tumor such as ultrasonography (USG), computed tomography (CT) scan and magnetic resonance imaging (MRI) as well as technetium-99 diethylenetriaminepentaacetic acid scanning ,3,5.In our case, the plain radiograph showed a radiolucent soft tissue mass along the shaft of radius ulna on the volar part of the right forearm. The MRI showed homogeneous hyperintense fat signal on T1-weighted MR images whereas T2-weighted images showed hypointense fat-suppression signal. The mass appeared inter-muscularly between FCR and FDC with well-defined margin and no infiltration to surrounding structures, raising the susceptibility of a lipomatous tumor with benign characteristics.Surgical is the mainstay treatment for giant lipoma due to the size, recurrence risk and potential malignant transformation. Complete surgical resection, with preferably wide-margin, is recommended to prevent dedifferentiation and recurrence. Adjuvant radiation therapy may benefit in large high-grade liposarcoma and in any other liposarcoma when the wide resection cannot be obtained. The value of chemotherapy is still debatable as adjuvant to surgical therapy .In our case due to the complicated structure and relatively narrow space at the forearm, we could not perform wide resection with much negative margins. Intraoperatively, we found that the mass was deep-seated, well-vascularized, well-circumscribed, lobulated and had a rubbery consistency. Those characteristics were suitable for the lipomatous mass but not specifically present liposarcoma. The cut surface was yellow with more hyperemic areas on the superior pole of the mass. A pseudo-capsule which was formed by the continuous pressure on surrounding tissue made the dissection and enucleation of the mass uncomplicated. Since the mass was intermuscular, we were able to remove the tumor without affecting the adjacent muscular tissue while preserving the neurovascular bundle, in this case, the median nerve particularly ,4,7.Post-operative histopathological exam revealed this giant lipomatous mass as the well differentiated liposarcomas (WDL) lipoma-like subtype. Account for 40–45 % of liposarcomas, WDL usually diagnosed at the 5th decades of life with slight male predominance. WDL are locally aggressive mesenchymal tumors composed of mature adipocytes and stromal cells with at least focal cytologic atypia. WDL are considered as low grade lipogenic tumors with morphologic subtypes dependent on the adipocytic component and background cellularization characteristic. Several histologic subtypes have been described: lipoma-like, sclerosing, inflammatory, mixed, lipo-leiomyosarcoma, WDL with low grade osteosarcoma-like and spindle cell subtype. Lipoma-like subtype, being the most common form of WDL, is grossly indistinguishable from lipoma. It frequently contains lipoblast and scattered atypical cells may be diffuse or extremely rare .In this report, surgical excision resulted in complete relief of symptoms and significant esthetic also became evident in the affected forearm. There was no recurrence in the 1-year follow up. However, we still closely observe this patient for any recurrence since the possible risk of recurrence in the WDL as well as inter-muscularly located lipomatous mass.4The slow growing nature and radiological characteristic of lipoma brought confusion with the benign lesion. However, the giant size and other unusual characteristics  raised the awareness of malignant lipomatous mass. Appropriate diagnosis and complete surgical resection of this tumor provided symptoms relief and esthetic improvement on the affected limb. Long-term evaluation must be conducted in order to detect recurrence. We plan for the wide excision followed by radiotherapy for possible recurrence mass in the future.The authors declare no conflicts of interest.The authors report no external source of funding during the writing of this article.Ethical approval was not required in the treatment of the patient in this report.Written consent has been received from the subject.Dina Aprilya contributes to the study concept or design, data collection and writing the paper.Wildan Latief contributes in the study concept or design, data collection, analysis and interpretation, oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team.Wahyu Widodo contributes in the study concept or design, data collection, analysis and interpretation, oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team.Not required.Wahyu Widodo is the sole guarantor of this submitted article.Editorially reviewed, not externally peer-reviewed.


COVID-19 has dramatically impacted healthcare delivery around the world. As hospitals systems prepared for the actual or perceived onslaught of COVID-19+ patients, measures were implemented that effectively discouraged or restricted patient access to outpatient care, and diagnostic and therapeutic cardiac procedures deemed elective. Furthermore, many governments adopted national lockdowns aimed at reducing the spread of the disease. National or regional lockdowns varied in scope and application from country to country but invariably caused restrictions in movements of people, workers and services, causing massive disruptions and economic loss rd most affected by the pandemic, and Pays-de-la-Loire, one of the least affected). Another important difference between these 2 provinces is their approach to educating the public. Hauts-de France conducted a province-wide educational campaign emphasizing the importance of appropriate use of healthcare services during the pandemic whereas Pays-de-la-Loire conducted no such campaign.In this issue of the journal Van Belle et al. rd most affected province (36% versus 18%).From January to mid-May 2020 both provinces witnessed a decline in MI incidence rates (≅ 8%). Both provinces saw their declines in MI rates exacerbate further (≅ 20%) during the French national lockdown (March 18–May 10) but Hauts-de-France province normalized their MI numbers, relative to 2019, faster than Pays-de-la-Loire despite the fact that the former had 4 times the number of COVID-19 deaths. When missing MIs are reported as a proportion of COVID-19 deaths, a surrogate marker of disease burden and healthcare system overload, the province least affected by the pandemic had a higher percentage of missing MIs relative to the 3By comparing comprehensive data from 2 provinces in the same country with very different COVID-19 disease burden, including public educational approaches, the authors add granularity to our understanding of this important public health issue that can inform policymakers going forward, as most of the United States and Europe enter a second and third wave of the pandemic. Public fear of contracting COVID-19 in the hospital system, not disease burden or healthcare system overload, appears to be the main driver of the reduction in AMI presentations during the pandemic. In fact, dramatic decreases in emergency department volumes were also reported For those patients who did present to the hospital system with AMI during the pandemic, the higher in-hospital mortality for STEMI patients (9.1% in 2020 vs. 6.2% in 2019) despite access to the cardiac catheterization laboratory is troublesome, and likely multifactorial. Previous observations in the US have reported longer door-to-balloon times during the pandemic Some limitations of the study are worth mentioning. The investigators only captured AMI patients who received invasive angiography and therefore any practice changes that might have occurred during the pandemic, such as a shift from mechanical to pharmacological, or no reperfusion, would have been missed. In China, such a shift led to delays in reperfusion, increased mortality and risk of heart failure National lockdowns were disruptive in nature but efforts to educate the public and maintain timely access to emergency care were important to minimize morbidity and mortality for patients with cardiovascular disease. Lockdowns instituted during the first wave of the pandemic failed to emphasize to the public the differences between hospitals  and non-essential services such as bars, restaurants and gyms. Also, the recommendation to “self-quarantine” for 2 weeks when symptoms of COVID-19 were present (many indistinguishable from heart disease such as dyspnea and cough) likely led many patients to delay or forgo needed medical care. The SCAI “Seconds Still Count” national public relations campaign likely contributed to the ‘recovery’ of STEMI activations in the United States. For governments considering additional lockdowns, approaches that preserve essential healthcare services and expand telemedicine capabilities, to include rural and institutionalized communities, are needed to avoid repeating the mistakes of the past.Santiago Garcia: writing of first draft of the manuscript.Timothy D. Henry: Editing and critical revision of the manuscript.Dr. Henry has nothing to disclose. Dr. Garcia reports grants and personal fees from Edwards Life-sciences, grants and personal fees from Medtronic, grants and personal fees from Abbott Vascular, grants from Boston Scientific, outside the submitted work.


Agricultural intensification is one of the major drivers of biotic homogenization and has multiple levels ranging from within-field management intensity to landscape-scale simplification. The enhancement of invertebrate assemblages by establishing new, semi-natural habitats, such as set-aside fields can improve biological pest control in adjacent crops, and mitigate the adverse effect of biotic homogenization. In this study we aimed to examine the effects of ecological intensification in winter wheat fields in Hungary. We tested how pests and their natural enemies were affected at different spatial scales by landscape composition , configuration (presence of adjacent set-aside fields), and local field management practices, such as fertilizer (NPK) applications without applying insecticides. We demonstrated that at the local scale, decreased fertilizer usage had no direct effect either on pests or their natural enemies. Higher landscape complexity and adjacent semi-natural habitats seem to be the major drivers of decreasing aphid abundance, suggesting that these enhanced the predatory insect assemblages. Additionally, the high yield in plots with no adjacent set-aside fields suggests that intensive management can compensate for the lower yields on the extensive plots. Our results demonstrated that although complexity at the landscape scale was crucial for maintaining invertebrate assemblages, divergence in their response to pests and pathogens could also be explained by different dispersal abilities. Although the landscape attributes acted as dispersal filters in the organization of pest and pathogen assemblages in croplands, the presence of set-aside fields negatively influenced aphid abundance due to their between-field isolation effect. Landscape simplification has been suspected to influence the local patterns of species richness and abundance, because of the reduced capacity to support large species-pools, and the lack of opportunity for spill-over between different habitats . NeverthInsect pollinators, natural enemies, and soil decomposers are key factors for production in the various economically important crop systems . These gIn this study, we tested whether the enhancement of beneficial invertebrate assemblages, as pest control providers could contribute to the improvement of crop yields through reduced level of management intensity. We also looked for clues whether the number of newly established, semi-natural habitats adjacent to winter wheat fields could act as proxies for EI. We also tested whether fertilizers, as a key part of intensified crop management, interacted with landscape composition and configuration on selected pathogens, aphids and their natural enemies. We hypothesized that (i) a higher proportion of semi-natural habitats in the landscape can mitigate the negative effects of local (intensive) field management practices on the within-field abundance of natural enemies. Thus, enhanced connectivity between the habitat patches might lead to more natural enemies at the landscape level, and (ii) at the between-field scale, winter wheat fields with adjacent set-aside fields would have a higher abundance of natural enemies and enhanced natural pest control. At the plot level, we assumed that (iii) intensive inorganic fertilizer use in poor soils (acidic soil pH with low soil organic content) would enhance wheat yields, as well as the abundance of pathogens and aphids; while fertilizer usage in good soils  would not have such an effect.High Nature Value Area in North-Eastern Hungary. This region is one of the major target areas of agri-environmental schemes in Hungary (Medicago sativa) and two grass species . During the maximum time of the 3-year set-aside management period, the use of agrochemicals was prohibited, and the fields were mown once a year in the second half of June  and pH. A total of 15 samples were taken per fertilizer control plots at the 10+ stage , urban areas and water bodies were measured using a GIS database . We consSitobion avenae, Hemiptera: Aphidoidea), coccinellid adults and larvae (Coleoptera: Coccinellidae), syrphid larvae (Diptera: Syrphoidea), chrysopid larvae (Neuroptera: Chrysophidae), and the number of parasitized aphids (including mummies). We also visually assessed the major pathogens, including leaf spots , mildew and rust (Puccinia spp.) on the leaves. Plant surveys were conducted on three occasions in April , May  and June .The abundance of aphids, their natural enemies and wheat pathogens was assessed by visual plant surveys in each plot along two parallel transects perpendicular to the margin, 5 m from each other at 5, 10 and 20 m from the edge of the wheat fields adjacent to the set-aside field, which were towards the habitat core. We randomly selected 17 wheat tillers per transect per distance  of the surface-active natural enemies, four pitfall traps were installed along the transects  in each 2 in total. Harvested ears were transported in cotton bags and dried at 24 °C for 48 h in a climate chamber under 10% RH. After threshing, we measured the total fresh grain mass per replicate batch, as well as fresh and dry mass of sub-samples in order to calculate the total grain yield (g/m2) (standardized to a moisture content of 14%); the grain and the ear mass were used for further analyses.In June, all experimental plots were hand-harvested. Two sub-samples of 0.58 × 0.58 m area were taken at each distance , resulting in a harvest sample of 1 mWe used linear mixed models to test the responses of pests (aphid abundance), pathogens , natural enemies (abundance of ground beetles and spiders) and winter wheat yields (estimated as mass of wheat- ear and grain) to soil properties, nitrogen use, landscape complexity and configuration. To achieve a normal distribution of the residuals, all response variables were square-root transformed. We created sets of single-argument models on each response variable, to avoid collinearity and maximize the predictive power of explanatory variables, only one explanatory variable was considered in each model . As for The presence of adjacent set-aside fields significantly affected aphid abundance. There were significantly more aphids in winter wheat fields without adjacent set-aside fields than in those with set-aside fields , Fig. 2AOur results did not indicate significant impact on the abundance of the two studied groups of ground-dwelling predators by any studied factors , Fig. 3.Ear and grain mass were significantly higher in fields without adjacent set-aside fields , 2D but We compared the effects of EI on the relationships between pests and their natural enemies at different spatial scales, from within- to between-field differences and up to landscape scale effects. We found that adjacent set-aside fields and landscape complexity seemed to be the drivers of the reduction in aphid abundance and the prevalence of pathogenic fungi. However, fertilizer use had no direct effect either on pests or their natural enemies.Agricultural landscapes are not static and may influence disease dynamics not only through its structure but also through its own dynamics . We founWe observed that less grain aphids were on plots that had adjacent set-aside habitats, while the natural enemies seemed unaffected, thus our hypothesis (ii) was just partially supported. This could be explained by the spillover of natural enemies into crop fields, resulting in better pest control . Any incsensu latoWe observed no effect of the presence of an adjacent set-aside field on the abundance of natural enemies. The available evidence  suggestsAlthough we demonstrated no effect of fertilization on the abundance of pests, pathogens or their natural enemies, we found that the grain yield was higher in plots without adjacent set-aside than with them. In addition, the yield remained unaffected by soil pH or SOC. These results partially supported our hypothesis (iii), pointing to below- and above-ground ESs. At the below ground, the unaffected yield by soil parameters might be caused by the increased nitrogen loss rate in calcareous soils and the less efficient N utilization in the soil with intensive fertilizer application . A good The fact that we did not detect any direct effect of the fertilizer on crop yield might lead to the conclusion that high soil fertility itself was more important than the external inputs. The lack of a relationship between the yield and any soil parameter such as pH and SOC could also be explained by the fact that while the best soils were farmed by intensive methods, the owners used their less fertile land as set-aside fields. We also found high yields in fields without adjacent set-aside fields. This could be explained by between-site variability in soil properties, which were independent of the proportion of arable land in the landscape, and being masked by intensive management practices . Crop yiIn conclusion, landscape features such as the presence of set-aside areas were the most important factors in determining the abundance of aphids in our studied cereal fields. We found that the lack of fertilization is not directly beneficial for spiders or ground beetles, suggesting that such extensive farming might not boost these natural enemies, due to the probability of increased between-field isolation caused by high compositional diversity at a landscape scale . Further10.7717/peerj.8746/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8746/supp-2Supplemental Information 2The ESRI *.shp file can be opened in google earth or any GIS editing software. The attribute table includes the variables used in the analyses. The *.zip file should be extracted before use.Click here for additional data file.10.7717/peerj.8746/supp-3Supplemental Information 3Referred to as ”(data = aphids)” in the R code fileClick here for additional data file.10.7717/peerj.8746/supp-4Supplemental Information 4Referred to as ”(data = spots)” in the R codeClick here for additional data file.10.7717/peerj.8746/supp-5Supplemental Information 5Referred to as “” in the R codeClick here for additional data file.10.7717/peerj.8746/supp-6Supplemental Information 6Referred to as “(data = harvest)” in the R codeClick here for additional data file.10.7717/peerj.8746/supp-7Supplemental Information 7Click here for additional data file.


In clinical chemistry, frequent monitoring of drug levels in patients has gained considerable importance because of the benefits of drug monitoring on human health, such as the avoidance of high risk of over dosage or increased therapeutic efficacy. In this work, we demonstrate that the drug doxycycline can act as an Au nanoparticle (doxy-AuNP) growth and capping agent to enhance the response of a surface plasmon resonance (SPR) biosensor for this drug. SPR analysis revealed the high sensitivity of doxy-AuNPs towards the detection of free doxycycline. More specifically, doxy-AuNPs bound with protease-activated receptor-1 (PAR-1) immobilized on the SPR sensing surface yield the response in SPR, which was enhanced following the addition of free doxy  to the solution of doxy-AuNPs. This biosensor allowed for doxycycline detection at concentrations as low as 7 pM. The study also examined the role of colloidal stability and growth of doxy-AuNPs in relation to the response-enhancement strategy based on doxy-AuNPs. Thus, the doxy-AuNPs-based SPR biosensor is an excellent platform for the detection of doxycycline and demonstrates a new biosensing scheme where the analyte can provide enhancement. It is important in the analytical community to develop sensors for frequent monitoring of drugs. In particular, clinical diagnosis and therapeutic procedures demand sensors to assess drug levels in patients. There is a need to obtain accurate test results in a short time for a large number of samples to decide on the course of medical treatments . As suchDoxycycline is a wide-spectrum drug belonging to the tetracycline family of antibiotics, which has been a drug of choice for treatment of several types of bacterial infections . ProteasAccording to FDA, safe dose of doxycycline is 200 mg on first day of treatment followed by 100 mg per day. In case of prolonged high dose, patient may suffer from gastrointestinal and renal diseases. It is a relatively low-toxicity drug and has been recommended for human use for a long time. However, the long-term use of doxycycline may lead to some side effects. Elzeinová et al. reported the adverse effects of doxycycline on testicular tissue and sperm parameters in CD1 outbred mice . AccordiCurrently, methods used for doxycycline detection involve analytical techniques such as high-performance liquid chromatography (HPLC) , sequentSurface plasmon resonance (SPR) biosensor is an optical technique that measures the binding events quantitatively in real time without labeling the interacting molecules . The phyDespite the many advantages of SPR biosensors, the binding of small molecules to the sensor surface typically results in small shifts, which constitutes a limitation of SPR biosensors. Most portable and small SPR instruments are not sensitive enough to assess such small refractive index changes, which make them unfit to use for ultrasensitive detection of small organic drugs . To overAuNPs are extensively applied in SPR biosensors, as such the effect of size of nanoparticles on SPR interactions is increasingly well understood. According to Kelly et al., size and shape of the nanomaterials could be effectively used to control the plasmonic characteristics as well as the electromagnetic field amplification of AuNPs . ComparaTo further build on the use of AuNPs-enhanced SPR for drug detection, we report on the fabrication of a doxy-AuNPs-based SPR biosensor for fast and sensitive detection of doxycycline. The use of the analyte to trigger AuNPs overgrowth is used as a novel sensing principle, where the signal of the SPR sensor is proportional to the concentration of doxycycline, in opposition to the usual competition assays resulting in a reduced response of the SPR sensor with concentration. Synthesized doxy-AuNPs were characterized by UV–VIS, X-ray diffraction (XRD), FT-IR, and transmission electron microscopy (TEM). SPR analysis was performed to demonstrate the detection of doxycycline. Various conditions were optimized to improve the SPR response. In this study, doxy-AuNP containing sodium chloride (NaCl) was employed as reagent providing further increase in the biosensor response. Thus, the doxy-AuNPs-based SPR biosensor is an excellent platform for the ultrasensitive detection of doxycycline.Gold (III) chloride trihydrate, doxycycline hyclate, N-hydroxysuccinimide (NHS), 16-mercapto-hexadecanoic acid (16-MHA), 11-mercapto-1-undecanol, ethanolamine hydrochloride, and sodium chloride were purchased from Sigma Aldrich . Sodium hydroxide and N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Fluka chemicals. Protease-activated receptor (PAR-1) was purchased from Cedarlane .To prepare doxy-AuNPs, aqueous solutions of 4 mL gold chloride (0.4 mM) and 2 mL doxycycline (0.8 mM) were added in a conical flask. To the mixture, 3 mL sodium hydroxide (0.01 M) was added. The mixture was continuously stirred for 3 min. After 3 min, a ruby red color was observed . A UV–ViThe crystalline nature of doxy-AuNPs was confirmed via XRD studies. The solution containing doxy-AuNPs was centrifuged three times at 10,000 rpm for 30 min and washed with deionized water each time. Then, the pellet obtained after centrifugation was left overnight to dry under fume hood. Powder XRD analysis was performed with Bruker D2 Phaser using Cu Kα radiation (1.54 Å λ) in the 2θ region, from 0 to 80°. Transmission electron microscopy (TEM) (FEI Tecnai t12) with voltage 80 kV and final emission 10 µA was used to examine the size and shape of doxy-AuNPs. Moreover, 2k AMT camera was applied to take micrographs of doxy-AuNPs. Sample for TEM was prepared by placing 10 µL of doxy-AuNPs colloidal solution on copper grid coated with carbon and formvar film. The sample was left for 24 h to dry and then analyzed with TEM. The FT-IR is the most widely used technique to study the interaction of biomolecules with the nanomaterials. The appearance of certain spectral changes enables to identify the interaction of certain functional groups with the nanomaterials . The doxSPR measurements were performed on a portable SPR instrument  . A dove −1 and reacted for 15 min. The receptor PAR-1 was covalently attached to the SAM through activated carboxylic acid group from EDC/NHS. Subsequently, nonspecific binding sites on the sensing surfaces were blocked by injecting 1 M ethanolamine hydrochloride (pH 8.0) for 10 min followed by rinsing with PBS to remove noncovalently attached receptor PAR-1. This procedure was repeated for all SPR experiments.In all SPR experiments, the SAM-modified gold-coated prism was inserted in the SPR instrument. First, Milli-Q water was added into the flow cell and left for 15–20 min for stabilization. Afterwards, the sensing surface was activated with EDC/NHS and left for 5 min until the resonant wavelength was constant. Then, the sensing surface was rinsed with PBS, followed by the injection of the receptor solution of protease-activated receptor-1 (PAR-1) at 5 μg mLTo examine the electrolytic stability of doxy-AuNPs, different concentrations of NaCl (from 50 to 1000 mM) were added in doxy-AuNPs colloidal solutions and their UV spectra were recorded  in the wavelength range of 300–800 nm. To select the suitable electrolytic condition of doxy-AuNPs, which can give larger SPR response, doxy-AuNPs containing varying concentrations of NaCl (from 50 to 1000 mM) were injected in SPR followed by rinsing each time before and after each injection with same concentration of NaCl in water to record the refractive index baseline. A control experiment of doxy-AuNPs without NaCl was also conducted and sensorgrams were recorded in real time.To examine the effect of doxycycline on the growth of synthesized doxy-AuNPs, varying concentrations of doxycycline (from 1 nM to 1 mM) were added in suspensions of doxy-AuNPs and their UV spectra were recorded in the wavelength range of 300–800 nm. Furthermore, to carry out detection of doxycycline with the SPR system, varying concentrations of doxycycline (0.1 nM to 100 μM) were added in the colloidal suspension of doxy-AuNPs and injected sequentially in flow cell at room temperature for 30 min followed by rinsing each time before and after each injection with 100 mM NaCl in water for 5 min to record baseline. Interaction between biological receptor PAR-1 and doxy-AuNPs was measured as binding shift by SPR biosensor in real time. Control experiments were performed by injecting doxy-AuNPs (containing optimized NaCl concentration) without adding free doxycycline in SPR system. Origin software was used to process the data utilizing the minimum wavelength finding algorithm. From the sensorgram, last 50 data points of 100 mM NaCl steps before and after the doxycycline sensing steps were used to calculate the binding shift from sensorgram. The logarithm of doxy concentration was plotted against the binding shift to find the correlation between concentration of doxycycline and binding shift. Triplicate measurements were carried for all conditions. Reproducibility was obtained from the triplicate SPR measurements of 100 nM doxycycline and measured as a coefficient of variation resulting from the ratio of the standard deviation and the mean response, in percentage.Doxycycline is a broad-spectrum drug with its antimicrobial as well as antitumor activities ,8,10,11.4− with H+ transfer and AuNPs formation. Recently, He et al. has reported the similar phenomena [4 into AuNPs at elevated temperature, which means the reducing capability of such compounds are weaker than the one of the polyphenolic compounds, such as doxycycline. Immediately after addition of doxycycline and sodium hydroxide in gold chloride solution, AuNPs were synthesized in 3 min with characteristic ruby red color, similar to another synthesis previously reported for L-methionine [A wet chemical reduction method was used to synthesize the doxy-AuNPs . Doxycychenomena . The b-athionine . Synthesthionine , thus cothionine .To examine the electrolytic stability of synthesized doxy-AuNPs, different concentrations of NaCl (from 50 to 1000 mM) were added in doxy-AuNPs colloidal solutions and their UV spectra were recorded. Doxy-AuNPs showed good electrolytic stability with slight red shift (from 520 to 530 nm) and small decrease in intensity up to 100 mM NaCl, whereas in case of higher concentrations from 250 to 1000 mM NaCl, LSPR band of doxy-AuNPs further red shifted from 530 to 540 nm with significant broadening and decrease in intensity d. It was−1 for O-H and N-H bonds but this band completely disappeared in case of doxy-AuNPs. This observation hints about the involvement of these moieties in the modification process. Likewise, the absorption bands in the range of 1700–1500 cm−1 were attributed to α,β-unsaturated carbonyl of amide and ketone functionalities of doxycycline. These signals moved to higher values in case of doxy-AuNPs, although the intensity of signals is low. The shifting to higher values justified the involvement of hydroxyl group resulting into disappearance of C=C conjugation.The interaction of doxycycline in synthesis of AuNPs was studied through FT-IR . In caseA shorter Debye length and, as a consequence, decreased colloidal stability are required for the molecular interaction of target analyte to occur on a surface-bound receptor. The presence of NaCl causes the electrostatic screening of surface charges by dissolved ions and reduces the Debye length, and resulted in the higher SPR response reported previously for a methotrexate assay . To seleSequential SPR analysis was performed for the detection of doxycycline . As the Au salt and doxycycline were reacted in equimolar conditions, it is hypothesized that the reaction is incomplete and thus, leaves unreacted Au ions in solution in addition to the AuNPs. Different concentrations of doxycycline (0.1 nM to 10 μM) were added in the colloidal solution of doxy-AuNPs (containing 100 mM NaCl) and injected sequentially into the flow cell of SPR system. In comparison, a control sample was run in which doxy-AuNPs (containing 100 mM NaCl) were injected in flow cell of SPR system. For the control sample, a 25 nm binding shift was observed with doxy-AuNPs  present in the solution work as nuclei for the conjugation and growth of gold atoms produced as result of reaction between tetracycline and HAuCl4 [We conclude that the growth was likely due to the already present doxy-AuNPs in colloidal solution working as nuclei for the gold atoms remaining from the AuNP synthesis and doxycycline . This caused the rapid growth of seeds of doxy-AuNPs , which cd HAuCl4 . Furtherd HAuCl4 .Calibration of this SPR sensor shows linearity over several orders of magnitude and detection of doxycycline from nanomolar to micromolar c, which An ultrasensitive SPR biosensor based on doxy-AuNPs has been successfully developed for the detection of doxycycline. Variation in size and growth of doxy-AuNPs affected the biosensor response. To overcome the limitation of low molecular weight of analyte, doxy-AuNPs (containing 100 mM NaCl) have been applied as an amplification element to obtain significantly enhanced SPR response. The reported biosensor allowed for the detection of doxycycline as low as 7 pM. The high sensitivity, low limit of detection, excellent signal response time , good stability, and reproducibility make this SPR biosensor an excellent alternative to the conventional methods for the detection of doxycycline. In future, the present biosensor can be applied in different fields of medical diagnostics and environmental monitoring for the detection of doxycycline.


Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis, including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities. These results here open new possibilities for evaluating 3D cellular dynamics in vivo, and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.To date, numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases, but few have attempted to characterize these cellular events under conditions similar to the native environment. To address this challenge, a three-dimensional (3D) multimodal analysis platform was developed for characterizing  These characteristics are all ideal for long-term imaging deep into highly scattering tissue.2The immune system is the most diffuse cellular system in the body, and all activities within this system are tightly regulated, including long-range cell migration and short-range communication by local chemical signaling or cell-to-cell contacts.x, y, z, t of cell mobility and morphology, this wealth of data permits researchers to characterize a variety of parameters associated with cellular dynamics. However, the amount of data generated often produces far more information than can be digested by human observers. Therefore, reliable computational analysis tools present an opportunity to take full advantage of the available data in an efficient and reproducible manner. Numerous computational analysis platforms are available, including Matlab (Mathworks), ImageJ (NIH), and Cell Profiler,3 which are some of the well-known tools that allow researchers to characterize cell motility, including parameters such as mean velocity, displacement, and travel direction.6 Other features that can be quantified include cell morphology parameters, such as area, volume, and shape index.8 More advanced morphology measurements are possible through decomposing cell shape in terms of Zernike polynomials, or based on Fourier analysis, neural networks, or principle component analysis.11 The cell parameters and analytical techniques presented above have been widely utilized in characterizing not only cellular morphology, but also cellular activities.Because multiphoton imaging can yield four-dimensional information .18 Among other techniques, CT-FIRE is especially powerful because it integrates fiber-based preprocessing techniques with fiber extraction methods to improve fiber extraction accuracy, which leads to high sensitivity to changes in collagen alignment.13Collagen fiber structure is another biological feature that has been heavily analyzed, most frequently after imaging using second harmonic generation (SHG) microscopy.21 However, few of the existing methods specifically focus on integrating multimodal information together in a temporally and spatially co-registered manner, primarily because of the limited number of multimodal optical imaging systems that are capable of generating these complex high-dimensional image datasets. In addition, the majority of these techniques focus exclusively on the cells in a relatively small field-of-view (FOV). While the information obtained from these methods provide useful molecular and cellular microenvironment information, these tools lack the ability to provide information from the macroenvironment where co-ordinated events among different tissue (skin) constituents can be better observed.To date, numerous computational analysis tools have been developed to quantify the multimodal cellular dynamics both at the molecular and cellular levels.in vivo cellular environment, including two-photon excited fluorescence (TPF), SHG, and fluorescence lifetime imaging microscopy (FLIM). TPF is utilized for visualizing endogenous and exogenous fluorescence, SHG for collagen networks and organization, and FLIM for cellular metabolic activity.23 The imaging analysis platform developed in this study focuses on characterizing the three-dimensional (3D) datasets in a spatially and temporally co-registered manner, allowing users to quantify the relationship between different tissue constituents at any given time of an event. The applicability of the developed analysis platform was subsequently tested on a time-lapse 3D in vivo dataset acquired from wounded mouse skin.To address these challenges, a multimodal imaging analysis platform was developed, which was designed to analyze time-lapse multimodal imaging data acquired by a custom-built multimodal multiphoton microscope. This microscope integrates several imaging modalities commonly used to study the 2.2.1.in vivo multimodal multiphoton imaging. The analysis platform was designed with Matlab Graphical User Interfaces (GUI), allowing users to control selected parameters to optimize both image segmentation and tracking outcome. The goal of creating this type of point-and-click control application is to provide an environment that can effectively utilize the unique and large amount of information offered by time-lapse 2.2.24 With a faster scanning and detection rate, large FOV volumetric data of TPF, SHG, and FLIM images can be acquired in a spatially and temporally co-registered manner. Most importantly, the imaging acquisition time is significantly shorter by incorporating an 8 kHz resonant scanning mirror (EOPC SC-30) for the fast axis,24 allowing comparably larger FOV time-lapse images to be captured, compared to a previous custom-built system.26 In addition, the signals from the photomultiplier tubes (PMTs) are sent through a high bandwidth transimpedance amplifier (Hama-matsu C5594 – 1.5 GHz bandwidth) and directly digitized using a 12 bit, 1.8 gigasamples per second digitizer (AlazarTech ATS-9360).24 The laser excitation is provided by a mode-locked Ti:Sapphire laser (Spectra-Physics Mai Tai HP). TPF and SHG imaging was performed using 920 nm excitation wavelength, and FLIM images were acquired using 750 nm excitation wavelength. All imaging was performed using a custom-built video-rate multimodal multiphoton microscope.in vivo animal, in vitro cell27 and human skin imaging.An inverted microscope design was implemented, with a removable glass coverslip positioned at the center of a motorized stage in between the imaging sample and the objective . Utilizi2.3.26 The animals used in this representative study were four one-year-old female MacGreen B6N.Cg-Tg(Csflr-EGFP)Hume/J transgenic mice (Jackson Laboratory), and all animal procedures were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign. These mice express enhanced GFP (EGFP) in peritoneal, bone-marrow-derived, and broncho/alveolar macrophages, as well as in Langerhans cells. Unlike other GFP-expressing transgenic mouse models, the MacGreen model expresses GFP specifically in macrophages, and therefore is an ideal model for studying the in vivo dynamics of these immune cells. To prepare animals for imaging, the hair on the dorsal skin was carefully removed with an electric shaver and surgical tweezers under general anesthesia  to reduce background auto-fluorescence during imaging. To wound the skin while the animal was still under anesthesia, the shaved area was first cleaned with rubbing alcohol, after which a full-thickness skin wound was created using a sterile 1 mm biopsy punch , followed by additional cleaning with rubbing alcohol. The wounds were left uncovered, and no analgesic compounds were prescribed during the study, as these would potentially interfere with the in vivo immune response.This study was designed to focus on characterizing immune cell dynamics because this group of cells is essential in wound healing.μm lateral step size and a 5 μm axial step size. A 5 min time gap between volumetric mosaics was used to limit laser exposure and potential heating or injury from the continuous scanning laser exposure. A shorter time gap was not possible due to the time required to save the multimodal image data to the hard disk. Although a shorter time gap would be more ideal for capturing cell motility, one of the objectives of this study was also to perform large FOV analysis. Therefore, a large imaging volume with a slightly longer time gap was chosen empirically. It is important to mention that the 5 min time gap did not affect the 3D cell tracking significantly. This volumetric mosaic covered a region of approximately 1000 × 1000 × 20 μm3. This volume of image data was able to capture a significant amount of cellular activity compared to our previous custom-built system.26 In addition, 6 × 6 FLIM mosaics with 80-frame average per stage location were acquired following the 60 min TPF-SHG time-lapse imaging, covering the same lateral dimensions as the TPF-SHG imaging to ensure spatially co-registered TPF-SHG-FLIM.Time-lapse imaging was performed on the day of wounding (Day 1) and 7 days post-wounding (Day 7). On each imaging day, a 60 min TPF-SHG time-lapse image sequence was captured over a 6 × 6 × 5-frame volumetric mosaic with a 20 frame average per location and a 5 min time gap. During volumetric imaging, the motorized translational stage moved at a 150 3.3.1.The analysis platform includes three sections, which are described below.3.1.1.in vivo environment is highly dynamic, and 2D tracking is often not sufficient to fully quantify cellular activity. In addition, breathing motion from the animals can minutely change the imaging depth during time-lapse imaging, and 3D tracking functionality allows observers to follow single-cell movement as accurately as possible. The first component of this module involves displaying and optimizing the volumetric grayscale images of the TPF cell data. Once the images are optimized, the user can crop the original image to isolate an area-of-interest for further analysis. The x–y coordinates of the cropped location can then be stored and utilized across different imaging modalities to ensure all subsequent analysis is performed at the same location where cells are tracked across all time points. Based on the newly selected area-of-interest, threshold-based cell segmentation is performed, which is followed by identifying detected cells, assigning all with numerical tags, and recording all corresponding 3D centroid coordinates. These locations serve as the initial time points for the time-lapse tracking.One of the key aspects of this platform is 3D tracking of cell motility. This function is crucial because the To perform time-lapse tracking, the module allows the user to select an individual cell to be tracked. Using the recorded 3D centroid coordinates, the Euclidean distances between the cell at the current time point (t1) and all identified cells at the next time point (t2) are calculated. The cell with the smallest Euclidean distance was identified as the cell-of-interest at t2. This procedure was repeated between all following adjacent time points until the final time point indicated by the user. Initial characterization showed that the segmentation module can successfully separate individual cells if the distance between cells is visually separable. We also found the tracking method described here can track most cells reliably over time. Though it is possible that other cells can be mistakenly assigned as the t2 location of the cell-of-interest in high-density areas, this concern can be resolved by using smaller time-gap and axial sampling size during imaging. When completed, this module recorded the 3D centroid locations of all selected cells at all tracked time points. With the above information, the instantaneous velocity of all tracked cells can be determined. In addition, users can utilize these coordinates to perform a variety of analyses, such as the traveling direction of the selected cells. When combining travel direction with other data sources, such as collagen structure and fluorescence lifetime, the correlation among these data can be determined.3.1.2.The second part of the analysis module involves analysis of the SHG signal for collagen structure characterization. The goal is to design a module for this platform that enables collagen structure characterization at regions where tracked cells are located, as identified previously in the cell tracking module. Although collagen is a 3D structure, all analyses done in this module were performed in 2D due to the lower axial resolution of the multimodal microscope compared to the transverse resolution.z-direction of the centroid coordinates, SHG analysis could be performed at the same depth as the tracked cell, as shown in 18 Through CT-FIRE, selected individual collage fibers can be identified, and the orientation angle of each collagen fiber can be calculated, as illustrated in This module first allows the user to optimize the image contrast in a manner similar to the cell tracking module. This function then automatically crops the original volumetric SHG image stack according to the cropping parameters recorded previously in the cell tracking module. The SHG region-of-interest image stack is further cropped into smaller SHG regions based on the locations of the tracked cell for the subsequent CT-FIRE analysis. Because the multimodal data is 3D, it is crucial that each 2D SHG collagen image be retrieved from the same depth as the tracked cell. To achieve this, a 200 by 200-pixel SHG image tile was created and centered around the associated tracked cell based on the 3D cell centroid coordinates. By utilizing the 28 Two-dimensional spatial Fourier transforms (FT) are performed on these images, and the FT magnitude images are converted to binary images based on a specified threshold. An ellipse-fit is then performed on these binary images utilizing the Matlab Imaging Processing Toolbox function. To quantify the collagen structure and alignment around tracked cells, the eccentricity of each ellipse is used, defined as the ratio of the distance between the foci of the ellipse and its major axis length, and resulting in a value between 0 and 1. A value closer to 0 suggests that the collagen fibers in the selected regions are isotropically distributed, and a value closer to 1 suggests that the collagen fibers are more aligned in a particular direction.28 With the collagen parameters collected in this module, one can correlate them back to cell parameters and potentially quantify the relationship between collagen structure and cell motility.An additional function in this module was the Fourier analysis of the same cell-based regional SHG image tiles.3.1.3.31 To effectively utilize the large FOV FLIM images, this module performed overall average NADH lifetime calculations as well as regional area-based NADH lifetime calculations.The last module is designed to perform area-based fluorescence lifetime analysis of intracellular reduced nicotinamide adenine dinucleotide (NADH) on large FOV FLIM images. NADH is an important coenzyme involved in the reduction-oxidation reaction in cells, and FLIM has been widely used to probe the metabolic activity in biological tissues.In this module, both the NADH intensity and lifetime data are imported. As FLIM data was only acquired at a single depth in the epidermal layer of the skin, only 2D analysis was performed. Two types of lifetime calculations can be performed. The first utilizes the region-of-interest cropping information stored from the cell tracking module and calculates the average NADH lifetime across this region. The other method allows the users to select any area in the entire image and calculate the regional average lifetime. The incorporation of area-based analysis provides the users freedom to analyze areas outside the TPF-SHG analysis region. This information can provide evidence of the relationships among regional metabolic state, cell motility, and collagen structure. By combining these multimodal parameters quantified in both a temporally and spatially co-registered manner, additional information may be obtained regarding the biological conditions of living tissue, and the cellular activity can be studied in the native tissue microenvironment.3.1.4.To demonstrate the utility of the multimodal analysis platform presented in this work, analysis was performed on multimodal multiphoton microscopy data obtained from a longitudinal wound healing study. The data collected included dynamics of macrophages captured through TPF, the associated collagen structure near the edge of the wound obtained through SHG imaging, and the functional metabolic tissue state through NADH lifetime imaging of the same region. To the best of our knowledge, these datasets represent the first spatially co-registered TPF-SHG-FLIM images of living skin with such an extended FOV. A variety of the biological parameters were available for analysis in this dataset, including the relationship among cellular motility and orientation, collagen fiber alignment, and NADH lifetime.Using the image analysis platform presented above, noticeable influx of GFP cells toward the edge of wound is visualized on Day 1 after a wound was created. In addition, the corresponding FLIM image shows that the NADH lifetime around the edge of the wound appeared to be noticeably longer than the rest of the surrounding skin further away from the wound bed. In comparison, cellular motility on Day 7 is markedly decreased. These observations are presented in 1 This observation can potentially be a visual indication of cells traveling along connective tissues, but more studies are needed to verify the current hypothesis.In addition, the change in cell population over time was calculated using the information provided by the 3D cell tracking. At Day 1, a noticeable increase in cell population is observed over the time duration of the experiment, which is consistent with visual observation . The insTo characterize the migration of cells toward the edge of the wound, the 3D cell tracking module was utilized. The initial and final centroid locations of each tracked cell were utilized to calculate the total displacement, and the inverse tangent was utilized to calculate the direction of cell migration. Analysis of cell migration on Day 1 reveals that most cells moved toward the direction of the edge of wound. The relationship between cell migration and location of wound is clearly shown in in vivo dynamic microenvironment of wounded skin due to the combination of this multimodal image analysis platform and video-rate imaging system, allowing cellular-level analysis over large spatial scales in vivo. The results here show information in the micro- and macroenvironment that can potentially allow users to connect in vivo cell motility to functional and structural tissue parameters.Interestingly, increased cell migration was observed toward the wound edge, corresponding to these areas of longer NADH fluorescence lifetime. This observation can be best visualized by directly comparing cell migration data in in vivo cell motility and local connective tissue structures. However, no clear relationship between cell motility and collagen fiber structures were observed here due to the short time span of the study.In addition to cell migration relative to NADH lifetime distribution, another biological parameter investigated was cell motility relative to collagen fiber angle. By taking advantage of the spatially co-registered multimodal data collection and analysis, local collagen structures of all tracked cells can be quantified using CT-FIRE in the SHG collagen analysis module. Combining with the time-lapse 3D locations of the tracked cells, users can determine relationships between 4.in vivo biological parameters and phenomena that are challenging to characterize with traditional imaging and analysis techniques. While only a few cellular parameters and measurements are presented here to demonstrate both the importance of large FOV multimodal imaging and 3D characterization, many more can potentially be studied using this multimodal analysis platform. In addition, the study presented here is only a demonstration of the application of both the video-rate multimodal microscopy and the analysis module developed in this study. Additional experiments are needed to evaluate this analysis platform under different experimental conditions. Also, future in vivo studies should be performed on imaging volumes that can better represent the 3D regions, including higher sampling of the z dimension. Furthermore, the imaging volume in future time-lapse cell tracking should cover a larger axial dimension without losing significant amount of lateral dimension and the time-lapse information.The results presented here demonstrate an integrated multimodal analysis platform to investigate 17 With the techniques presented here, the analysis of in vivo cellular dynamics can be transformed. By observing and quantifying biological phenomena in this manner, researchers may gain additional insights leading to a better understanding of specific biological microenvironments in health and disease.The multimodal analysis platform can be improved by including additional imaging modalities and analysis modules to address even more biological parameters. The design of this analysis platform is versatile, and the addition of other imaging modalities and parameters can potentially expand the applicability of this analysis platform to other biological phenomena, including the tumor microenvironment and neural activities.