New hospitalizations after first episode psychosis (FEP) may be viewed as an indicator of instability in a psychotic disorder. In the current study we wanted to analyse long term risk for psychosis hospitalizations after FEP. We also wanted to analyse predictors for late hospitalizations, with focus on early antipsychotic medication.First episode psychosis cases were recruited to the Swedish Parachute project in 1996–1997. The program offered highly available and continuous psychosocial support and a cautious use of antipsychotic medication for 5 years from inclusion. Longitudinal data from population registers on psychiatric hospitalizations up to 14 years after inclusion were analysed. One hundred and sixty-one cases were included of the original 175 in the project. Associations with possible early predictive factors from the original project data were analysed with COX regression.p value < 0.001).A majority of the cases (67%) had hospitalizations in the first year after inclusion in the study. The cohort then diverged into a group (46%) with new hospitalizations for psychosis after the first year, most of them multiple times, and another group (54%) without new hospitalizations for psychosis, many without any late antipsychotic medication. Forty-two percentage of the cases had antipsychotic medication by month 12, and it was significantly associated with later psychosis hospitalizations (HR = 2.5, The study demonstrates that a large part of FEP cases have a good outcome as measured by absence of new hospitalizations for psychosis, and that many cases may terminate antipsychotic medication within a year of FEP onset without later relapses needing hospitalizations. First episode psychosis (FEP) has a heterogeneous outcome –5. Many Many factors affect thresholds for psychiatric hospitalizations such as availability of hospitalization services, quality of outpatient care, and individual preferences, and the indications for hospitalizations may vary between countries or regions. Even so, it is reasonable to consider psychiatric hospitalizations in the years after FEP as an indicator of remaining vulnerability and instability in psychiatric states . There aThe Parachute project was based on a 5-year program for early intervention in first episode psychosis. The program offered high availability for social and psychological support for patients and relatives as well as psychoeducational components, delivered in small multidisciplinary FEP teams , in a siThe overall aim of the current study was to investigate long-term stability of psychotic disorder in the Parachute cohort, as measured by hospitalizations recorded in population registers, after the first year of treatment. In contrast to previous population-based register studies of rehospitalizations after a first hospitalization for FEP, we investigated new hospitalizations for psychosis after the first year of care. The rationale for starting the second year was that many cases have repeated hospitalizations in the often chaotic period around the onset of the FEP, followed by later stabilization in psychiatric states to various extents, and a lower risk for hospitalizations from the second year onwards , 24. TheThe following research questions were investigated: first, what is the rate and volume of hospitalization during a long-term follow-up spanning years 2–14 after initial diagnosis; second, what are early predictors of hospitalizations in the long-term follow-up and third, to what extent does the early use of antipsychotic medication predict time to hospitalization?The current paper presents an exploratory, naturalistic long-term follow-up study of a cohort of FEP cases from the Parachute project. The Parachute project has previously been described in detail . The genIn 1994 the psychiatric clinics in Sweden were invited to participate in the project. After evaluation of interested clinics, 17 clinics throughout Sweden were included to participate, representing a catchment area of about 1.5 million inhabitants, or about one-sixth of Sweden’s population. All clinics were publically financed and had responsibility for the psychiatric care in defined geographic areas. As no private care for early psychosis patients was available in Sweden, we consider the cohort to be rather complete regarding treated incidence of FEP. All the participating clinics offered regular psychiatric inpatient care. In addition, ten of the clinics also offered access to crisis homes as a complement to standard inpatient care. The crisis homes were small scale, homelike, low stimulus overnight facilities with low thresholds for admittance. Patients were usually admitted for short stays when they considered they needed the service.The collection of cases started 1 January 1996 and ended 31 December 1997 (24 months).The inclusion procedure and criteria were as follows: patients living in the catchment area, for the first time seeking psychiatric help for psychotic symptoms, age 18–45 years, and without a dominating substance abuse or a diagnosed brain disorder were identified as candidates. During the first week every candidate patient was diagnosed with a SCID interview (axis 1) according to DSM-IV—usually performed by a responsible consultant psychiatrist. Cases with a diagnosis of schizophrenia, schizophreniform psychosis, schizoaffective psychosis, delusional disorder, brief psychosis, psychotic disorder not otherwise specified, or affective disorder with non-congruent psychosis were offered the opportunity to participate in the study.Two hundred and fifty-three patients were considered to fulfil the criteria for FEP. Based on an estimation of expected incidence of schizophrenia of 0.97 per 10,000 person-years in a general population , the expTen of the 175 patients were recorded as dead prior to 2011. Complete records until 2011 in the population registries were retrieved for 161 cases. For four cases complete records were not retrievable, likely due to emigration. In the current study, the 161 cases were described and analysed. A flow chart of the number of cases in different stages of the process has previously been published .The program had a policy of using the lowest effective doses of antipsychotic medication “as needed” based on symptom severity and clinical judgement. Maintenance medication for the purpose of preventing relapse after remission of positive symptoms was not encouraged. Antipsychotic polypharmacy was generally avoided. Attempts were made to avoid antipsychotic medication during the first 1–2 weeks if possible. Benzodiazepines were used for anxiety or insomnia during this period. Types and doses of antipsychotic medication were chosen based on clinical judgement. All patients were offered psychodynamically oriented psychotherapeutic support during the entire project stay. The method aimed at creating a trustful alliance, supporting cases to verbalize problems, supporting sound self-confidence, confronting stigma, and help patients understand stressful events or feelings. The method was trained within the teams and at regular conferences for the participating clinics. After the 5-year project time, with access to the FEP team, patients needing continuous care were referred to general psychiatric outpatient services, often with less accessibility to support.All patients underwent thorough psychiatric and medical examinations, including a checklist of background variables at the inclusion. The checklist included data on the highest educational achievement and an assessment of the highest GAF score in the year before onset of FEP, which were used in the current analyses. Data collections were carried out at 5 time points in the first year and after year 3 and year 5 after inclusion, with checklists of current situation and rating scales. In the current study, the following data from the month 12 assessments were used: antipsychotic medication in the 2 weeks preceding the assessment, current symptom ratings with BPRS, used to assess symptomatic remission, and a retrospective SCID-I based diagnosis for the first psychotic episode. The data collection has been described in detail previously . Month 1Remission from psychotic symptoms was estimated based on BPRS assessments. The BPRS based criteria proposed by Andreasen et al. , but witThe Inpatient Care Diagnoses Database from the Swedish National Board of Health and Welfare includes all individuals admitted to psychiatric or general hospitals , 28 withSeven of ten small-scale crisis-homes managed by clinics in the study, were classified as inpatient care and delivered data on admissions, discharges, and diagnoses to the national registries. A few cases thus may have had care in crisis-homes, not recorded in the registers.The Causes of Death Database comprises information on all deaths of Swedish residents .The Swedish Prescribed Drug Database comprises information on all dispensed prescriptions in Sweden from 1 of July 2005 and onwards . HoweverFor analyses of antipsychotics, ACT codes N05A were used, with the exceptions of N05AN (lithium), N05AA02 (levomepromazine), N05AD03 (melperone), and N05AF03 (chlorprothixene) since these medications are usually used for other indications than psychotic symptoms.Amounts of dispensed medication are reported in the register as number of “defined daily doses” (DDD) for every dispensation. Definitions of one DDD according to the WHO ATC/DDD index , for somThe longitudinal integration database for health insurance and labor market studies (LISA) from Statistics Sweden includes data on income from employment, payments from sickness benefits and of student aides. Data on income from employment were available for the period from the year of inclusion in the study until year 14 after.For the outcome analyses, the cohort was dichotomized into cases with and without any hospitalizations for psychosis in the years 2–14 after FEP.Descriptive analyses, Wilcoxon signed-rank tests, odds ratios (OR), Cox regression analysis and Kaplan–Meier survival graphs were made with the R software . SignifiThe counting of cases with hospitalizations in a certain period included all cases with days in hospital in that period. An episode of hospitalization could then be counted several times if it spanned several counted periods.For the Cox regression analysis of psychosis hospitalization outcome, eight dichotomized factors were used as indicated in Table p value < 0.1 in the univariable analysis were analysed. Stepwise modelling based on the Akaike information criterion (AIC) resulted in the second 4 factor model which was deemed optimal.Stepwise forward multivariable Cox regression was performed as follows: first a univariable analysis of all the eight factors was performed. In a first multivariable step, seven factors with Cases with missing data on a variable were excluded from analyses involving the variable.Demographic data for the cohort have previously been described , 34. 55%Retrospective SCID-1 based diagnoses for the first psychotic episode were recorded 12 months after inclusion for 153 cases, and missing for eight cases.Forty cases (26%) with schizophrenia (DSM-IV 295.1/.2/.3/.6/.9).Thirteen cases (8%) with schizophreniform disorder (DSM-IV 295.4).Eleven cases (7%) with schizoaffective disorder (DSM-IV 295.7).Seventeen cases (11%) with delusional disorder (DSM-IV 297.1).Twenty-one cases (14%) with brief psychosis (DSM-IV 298.8).Thirty cases (20%) with psychosis NOS (DSM-IV 298.9).Twelve cases (8%) with manic of bipolar disorder with non-congruent psychotic symptoms (DSM-IV 296.0/4/5/6/7/8).Nine cases (6%) with major depression with non-congruent psychotic symptoms (DMS-IV 296.2/3).The distribution of retrospective diagnoses for the 153 patients were as follows:Twenty-nine additional cases were given a schizophrenia, schizophreniform, or schizoaffective diagnosis (ICD10 F20 or F25) for the first time at in-patient or out-patient care in the years 2–14 (based on population registers). In total 93 cases  were given a schizophrenia, schizophreniform, or schizoaffective diagnosis at in-patient or out-patient care within 14 years of follow up. Cases with a first time schizophrenia related diagnosis later than by month 12 were not included in the schizophrenia category in the analyses of outcome prediction.Of the 161 cases included in the study, 106 (67%) had any in-patient care in the first year. Fifty cases (31%) had more than one hospitalization in the first year.The proportion of the study population with hospitalizations declined over time already during the first year. Of the 106 cases, 89 (55%) had any in-patient care in the first month after inclusion, 57 (35%) in the second or third months after, and 42(26%) in the 4th–12th months after .The median number of days of in-patient care in the first year for cases with any in patient care was 29 days with 25% and 75% quartiles at 13 and 76 days.p value = 0.32 with Wilcoxon signed-rank test).The median age at inclusion for cases with in-patient care the first year was 29 years compared to 27 years for cases without in-patient care. The difference was not statistically significant .Fifty-one percentage of the cases with any psychiatric hospitalization in the first year were women compared to 39% of the cases without. The difference was not statistically significant .Twenty-six percentage of the cases with in-patient care the first year did not have a high school exam compared to 15% of the cases without in-patient care. The difference was not statistically significant  had hospitalizations for only other psychiatric disorders than primary psychotic disorders. The most common diagnoses in those cases were manic episodes or bipolar syndrome (ICD10 F30/ F31) in ten cases, and unipolar depression in three cases (F32/ F33). Seven of the cases with affective diagnoses also had specifiers for psychotic symptoms.p value < 0.001).We investigated if there was any association between hospitalizations and work record in the late period, and found a strong association between no hospitalizations for psychosis in the years 2–14 and work record in the same period. Fifty-three of the 88 cases (86%) without any hospitalization for psychosis in the period had incomes from regular work higher than 100,000 Swedish kronor  per year for more than 3 years in the period, compared to 20 of 73 cases (27%) with any hospitalization for psychosis in the period. This difference was highly significant : not having a high school degree, a prescription of antipsychotic medication by month 12 :p value < 0.001). The median amounts dispensed in the years 11–14 was 273 DDD per year in the group with antipsychotics month 12, and 246 DDD per year in the group without antipsychotics month 12 (only counting cases with late dispensing).There was a strong correlation between antipsychotic medication by month 12 and late antipsychotic medication in the years 11–14. Eighty-three percentage of the cases with a prescription of antipsychotics by month 12 had any dispensing of antipsychotic medication in the years 11–14, compared to 43% of the cases without antipsychotics by month 12. There was a strong correlation between antipsychotic medication by month 12, and a schizophrenia-related diagnosis by month 12. .In the group of cases with a schizophrenia-related diagnosis (DSM-4 295) by month 12, there was a non-significant indication of a difference in risk for later hospitalizations in the years 2–14 between cases with and without antipsychotic medication by month 12. Twenty-six of 40 cases (65%) with antipsychotics by month 12 had later hospitalizations for psychosis, and 11 of 24 cases (46%) without antipsychotics by month 12 .There was a strong association between any hospitalization for psychosis in the years 2–14 and any antipsychotic medication in the years 11–14. Sixty-two of 72 cases (86%) with hospitalizations had any dispensing of antipsychotics in the period, compared to 33 of 88 cases (38%) without any hospitalization for psychosis in the years 2–14. The different was highly significant (OR = 9.0, Based on population register data, we studied outcome after FEP in terms of hospitalizations for psychosis or not after the first year of treatment. We found that around half of the cases had hospitalizations for psychosis after the first year, most of these cases with multiple hospitalizations, and that the other half did not. Cases without any hospitalizations for psychosis in the period we think represent a group with more stable psychiatric states in terms of psychosis symptoms, even though there is room for substantial psychiatric symptoms without needing hospitalizations.Considering that there is a substantial variation between countries in the proportion of cases that are hospitalized in the years after FEP , dependiIn the 10 years outcome study of the Danish OPUS project , there wWe found several possible predictors for late hospitalizations for psychosis. As previously described, there was a strong association between a low educational level and late hospitalizations . We beliAs in previous studies, low age at onset of FEP was associated with later hospitalizations . This faWhile gender has been associated with other outcome measures after FEP , 37, 38,We found a strong association between antipsychotic medication by month 12 after FEP and later hospitalizations for psychosis. This finding is in contrast to some previous studies that have found a high risk for new hospitalizations after discontinuation of antipsychotic treatment after FEP , 39, 40.A schizophrenia related diagnosis by month 12 was actually also a predictor for later hospitalizations in the univariable HR analyses in the current study, antipsychotic medication by month 12 correlated with a schizophrenia related diagnosis, but had a stronger correlation than a schizophrenia related diagnosis to late hospitalizations in the univariable analyses. The medication factor likely absorbed the diagnosis factor in the multivariable analysis.In the Parachute project there was a policy of cautious treatment with antipsychotic medication. The psychiatrists would encourage cases to stop using antipsychotics if they found them recovered from acute symptoms. Another possible explanation for the association between medication month 12 and later hospitalizations is then that in the Parachute project, medication by month 12 reflects severity in the psychotic disorder, and do it with better predictive power than a schizophrenia related diagnosis, at least as measured with later hospitalizations as outcome.Another hypothetical factor is that long term use of antipsychotic medication may increase the risk for later hospitalizations after termination. Hypotheses around super-sensitivity of dopamine receptors or other slow adaptations of the dopamine system to antipsychotic medication have suggested such a connection . Some epThat long term antipsychotic treatment is undoubtedly indispensable for many cases with schizophrenia and other psychotic disorders has been demonstrated not least in large population register based studies , 42, 43.In conclusion, the study demonstrates that a large part of FEP cases have a good outcome as measured by the absence of new hospitalizations for psychosis, and that many may terminate antipsychotic medication within a year of FEP onset without later relapses needing hospitalizations.There were several limitations to this study. The sample size was rather small which limited the power for analyses on sub-groups. There was no randomization to different treatment arms for antipsychotic medication, which for example means that the choice of antipsychotic medication at month 12 was dependent partly on uncontrollable factors. The register data on medications was only available from July 2005, which limited the knowledge of the use of antipsychotic medication in the years 2–10 under study.


Over the last decade, virtual reality (VR) has emerged as a cutting-edge technology in stroke rehabilitation. VR is defined as a type of computer-user interface that implements real-time simulation of an activity or environment allowing user interaction via multiple sensory modalities. In a stroke population, VR interventions have been shown to enhance motor, cognitive, and psychological recovery when utilized as a rehabilitation adjunct. VR has also demonstrated noninferiority to usual care therapies for stroke rehabilitation.The proposed pilot study aims to (1) determine the feasibility and tolerability of using a therapeutic VR platform in an inpatient comprehensive stroke rehabilitation program and (2) estimate the initial clinical efficacy (effect size) associated with the VR platform using apps for pain distraction and upper extremity exercise for poststroke neurologic recovery.d effect sizes with corresponding 95% CIs will be calculated for upper extremity neurologic recovery, hand dexterity, and pain. The proportion of participants who achieve minimal clinically important difference after using the VR platform will be calculated for each clinical outcome.This study will be conducted in the Comprehensive Integrated Inpatient Rehabilitation Program at the James A Haley Veterans’ Hospital. Qualitative interviews will be conducted with 10 clinical staff members to assess the feasibility of the VR platform from the clinician perspective. A prospective within-subject pretest-posttest pilot design will be used to examine the tolerability of the VR platform and the clinical outcomes  in 10 veteran inpatients. A VR platform consisting of commercially available pain distraction and upper extremity apps will be available at the participants’ bedside for daily use during their inpatient stay (approximately 4-6 weeks). Clinician interviews will be analyzed using qualitative descriptive analysis. Cohen This study was selected for funding in August 2020. Institutional review board approval was received in October 2020. The project start date was December 2020. The United States Department has issued a moratorium on in-person research activities secondary to COVID-19. Data collection will commence once this moratorium is lifted.Our next step is to conduct a large multi-site clinical trial that will incorporate the lessons learned from this pilot feasibility study to test the efficacy of a VR intervention in inpatient rehabilitation and transition to home environments. When VR is used in patients’ rooms, it serves to provide additional therapy and may reduce clinician burden. VR also presents an opportunity similar to home-based practice exercises. VR can be implemented in both clinical settings and people’s own homes, where engagement in ongoing self-management approaches is often most challenging. This unique experience offers the potential for seamless transition from inpatient rehabilitation to the home.PRR1-10.2196/26133 Over the last decade, virtual reality (VR) has emerged as a cutting-edge technology in stroke rehabilitation. VR is defined as a type of user-computer interface that implements real-time simulation of an activity or environment, allowing user interaction via multiple sensory modalities . VR inteUpper limb deficits occur in up to 85% of stroke survivors and they significantly affect performance of activities of daily living . The litThree recent randomized controlled trials (RCTs) ,18,19 ofP<.001) [A recent multi-site study (N=546) found a 30% prevalence of pain across the acute, subacute, and chronic poststroke stages . CognitiP<.001) . VR may P<.001) . Sense oP<.001) . A rapidP<.001) . A meta-P<.001) . Thus, iDecades of animal research and recent research in human subjects provide compelling evidence that the adult brain affected by stroke can reorganize itself in response to experience and training, with sufficient repetition playing a critical role -31. In pNonimmersive VR environments are projected on 2D screens . Nonimmersive VR can facilitate stroke symptom improvement ,18,19, bIn a pilot study ,42 in thThe proposed feasibility pilot project will address the RR&D goal of maximizing functional recovery by pilot testing an immersive VR intervention designed to increase exercise dosage for the upper limb and decrease pain for inpatient veterans after stroke without increasing therapist time . The VR Our proposal is innovative in 4 distinct ways. First, we will use immersive 3D rather than the more typically used 2D VR. Immersion and the resulting “presence” within the virtual environment are thought to be the principal mechanisms of positive change ,32. SecoSpecific aim 1: Determine the feasibility and tolerability of using a therapeutic VR platform in an inpatient comprehensive stroke rehabilitation program.RQ 1.1: What is the feasibility of using the VR platform from the clinician perspective?RQ 1.2: What is the tolerability for poststroke inpatients using the VR platform?Specific aim 2: Estimate the initial clinical efficacy or effect size associated with the VR platform using apps for distraction and upper extremity exercise for veterans after the stroke.RQ 2.1: What are the estimated effect sizes and degree of precision for the outcomes of upper extremity neurologic recovery, hand dexterity, and pain?RQ 2.2: How clinically responsive are dexterity and upper extremity neurologic recovery (primary) outcomes to early stroke rehabilitation using a therapeutic VR platform?Our methodological framework is based on the work by the Virtual Reality Clinical Outcomes Research Experts committee . We willThere are 2 populations for the proposed project. The first population consists of veterans (n=10) who have been diagnosed with an acute ischemic or hemorrhagic stroke and are admitted to JAHVH inpatient rehabilitation after a stroke. Inclusion criteria are as follows: (1) age 18-80 years and (2) stroke diagnosis verified by brain imaging. Exclusion criteria are as follows: (1) unable to follow instructions or participate in immersive VR therapy due to significant cognitive impairment and (2) history of seizures. The second population consists of occupational therapists and rehabilitation nurses (clinician champions) working in the Comprehensive Interdisciplinary Inpatient Rehabilitation Program, who will provide data on the feasibility of using VR in an inpatient environment (RQ 1.1).All patients admitted to the Comprehensive Interdisciplinary Inpatient Rehabilitation Program at JAHVH (a designated Primary Stroke Center) with a diagnosis of acute ischemic or hemorrhagic stroke will be considered for inclusion in the study. A minimum of 5 beds will be designated for study participants. The Comprehensive Interdisciplinary Inpatient Rehabilitation Program admits 3.5 stroke patients per month, that is, 42 per year. We feel this is a sufficient subject pool from which to enroll the target sample size of 10 patients (16% of the patients admitted over the 18-month enrollment period). We have found that the technology is motivating for patients, which will help retention [The VR intervention uses off-the-shelf technology: Oculus Quest HMD and commercially available apps specifically developed or adapted for Oculus Quest . App selPrior to beginning the intervention, clinician champions  and the project manager will be instructed with regard to the use of the HMD and VR apps by our technologist. Staff will have the opportunity to practice with the HMD and apps for 2 weeks prior to using the apps with patients. Following institutional review board approval and funding on site, potential subjects will be identified by the admitting physician, principal investigator, or the project manager in the absence of the principal investigator. The project manager will use a Health Insurance Portability and Accountability Act waiver to check the inclusion and exclusion criteria.Once patients are enrolled, the project manager will collect the baseline data and administer the preintervention outcome measures . FurtherPatients will be instructed in the use of the HMD with VR apps by a project occupational therapist. It is anticipated that subjects may need 1-3 sessions of instruction. VR dosage will be 2 half-hour sessions per therapy day, facilitated by an occupational therapist and clinician champions, overseen by the principal investigator. The timing of VR sessions will vary based on the patient’s therapy schedule. During the VR session, the patient will be reclining or seated in bed with both bed rails raised. The clinician champions will bring the VR HMD to the bedside and assist the patient with donning the device. Once the patient is comfortable using the HMD with VR apps, the clinician champion will begin each session by setting the patient up and making sure that they are successfully engaging with the app. The clinician champion will return 30 minutes later to remove the VR HMD from the room. This process will be repeated a second time each therapy day. Patients can initiate the use of a more challenging app (blue category) that gradually includes hand/arm movement. Some patients may progress to the pink category in which hand/arm coordination is required.The average length of stay in the acute inpatient rehabilitation unit at JAHVH is 4-6 weeks. Accordingly, postintervention data will be collected at week 5 or at the end of week 4 if the veteran is being discharged. RQ 1.2 tolerability data will be collected throughout the subjects’ participation in the study.Once all veterans have completed the study, RQ 1.1 feasibility data will be collected from clinician champions.Feasibility is the degree to which the VR treatment can be successfully integrated within the flow of usual care . FeasibiHand dexterity will be measured using the Action Research Arm Test ,46. The The Intake and Discharge Questionnaires from the Pain Outcomes Questionnaire-VA (POQ-VA) will be utilized to assess pain-related treatment outcomes . SpecifiA data set was used during the first month of the study by using Microsoft Excel software as Excel is easily imported into the statistical analysis system for analysis. We have chosen to use Excel on our local research server rather than VINCI because this is a prospective cohort of new admissions and a relatively small sample. Data will be collected and entered into the database by the project manager. Data entry will be verified by the principal investigator. Data will be stored on the secure JAHVH Research Service R-drive. With the proposed pilot study design, the overall analytic goals are to (1) determine the feasibility and tolerability of using a therapeutic VR platform in an inpatient comprehensive stroke rehabilitation program and to (2) estimate, with reasonable precision, the effect sizes of upper extremity neurologic recovery, hand dexterity, and pain reduction outcomes.Qualitative descriptive analyses  will be t test to determine statistical significance.For RQ 2.1, the primary outcomes will consist of preintervention to postintervention changes on 2 physical measures of stroke recovery: the Action Research Arm Test  and the For RQ 2.1, since the effect sizes to be calculated are standardized measures, corresponding results across these outcomes will be directly comparable. However, these metrics do not necessarily translate to meaningful clinical differences (improvements). Therefore, for those outcome measures with published metrics for MCID , resultsThis study was selected for funding by VA RR&D in August 2020. The approval for the study from the University of South Florida Institutional Review Board and the JAHVH R&D Committee (Protocol STUDY001075) was received in October 2020. The project start date was December 2020. All VR equipment for this study has been purchased and inventoried. Clinical staff are currently being trained to use the VR equipment in the clinic. The United States Veterans Health Administration has issued a moratorium on all in-person VA research activities secondary to COVID-19. Data collection will commence once this moratorium is lifted and will follow the projected study timeline presented in If the aims of this research are achieved, VR will be used in combination with established pain management strategies to improve neurologic recovery and hand dexterity and to decrease pain. The short-term goal of this project is to determine the feasibility of conducting an RCT to determine the effectiveness of using VR as an adjunct to usual care therapy to enhance upper extremity neurologic recovery and hand dexterity and to decrease pain. Our long-term goal is to provide veterans with an exercise and pain reduction modality that can serve as an adjunct to scheduled therapy and assist with the clinic to home transition. VR has the advantage of being easily implemented both within VA health care settings as well as veterans’ own residences, where engagement in ongoing self-management approaches is often most challenging ,42.As this pilot study will employ a within-subject design to evaluate the magnitude of stroke rehabilitation over 3 weeks with the use of VR technology, there will be no control condition to judge rehabilitation results to that which might be expected from time alone and natural history of stroke recovery. Therefore, as described for RQ 2.1, we will place a premium on evaluating rehabilitation results by using MCID, which is highly relevant to patients and generally would not be expected to be achieved simply from time alone (4 weeks).Dissemination will be led by the principal investigator. Channels for dissemination include (1) annual progress and final summary reports to VA RR&D service, (2) bulleted briefings to our Program Partner, (3) presenting findings at national and local research meetings/conferences and VA cyberseminars and Military Health System Speaker series, and (4) submitting manuscripts to relevant peer-reviewed journals.Examining the feasibility of this immersive VR intervention will be beneficial for veterans, clinicians, and policy makers. The health care market size of extended reality  technology utilization is projected to grow from nearly US $2.1 billion in 2019 to roughly US $8-11 billion by 2026-27 ,54. Desp


Cryptococcal meningitis in HIV-infected patients in sub-Saharan Africa accounts for three-quarters of the global cases and 135,000 deaths per annum. Current treatment includes the use of fluconazole and amphotericin B. Recent evidence has shown that the synergistic use of flucytosine improves efficacy and reduces toxicity, however affordability and availability has hampered access to flucytosine in many countries. This study investigated the evidence and cost implications of introducing flucytosine as induction therapy for cryptococcal meningitis in HIV-infected adults in South Africa.A decision analytic cost-effectiveness and cost impact model was developed based on survival estimates from the ACTA trial and local costs for flucytosine as induction therapy in HIV-infected adults with cryptococcal meningitis in a public sector setting in South Africa. The model considered five treatment arms: (a) standard of care; 2-week course amphotericin B/fluconazole (2wk AmBd/Flu), (b) 2-week course amphotericin B/flucytosine (2wk AmBd/5FC), (c) short course; 1-week course amphotericin B/flucytosine (1wk AmBd/5FC) (d) oral course; 2-week oral fluconazole/flucytosine  and e) 1-week course amphotericin B/fluconazole (1wk AmBd/Flu). A sensitivity analysis was conducted on key variables.The highest total treatment costs are in the 2-week AmBd/5FC arm followed by the 2-week oral regimen, the 1-week AmBd/5FC, then standard of care with the lowest cost in the 1-week AmBd/Flu arm. Compared to the lowest cost option the 1-week flucytosine course is most cost-effective at USD119/QALY. The cost impact analysis shows that the 1-week flucytosine course has an incremental cost of just over USD293 per patient per year compared to what is currently spent on standard of care. Sensitivity analyses suggest that the model is most sensitive to life expectancy and hospital costs, particularly infusion costs and length of stay.The addition of flucytosine as induction therapy for the treatment of cryptococcal meningitis in patients infected with HIV is cost-effective when it is used as a 1-week AmBd/5FC regimen. Savings could be achieved with early discharge of patients as well as a reduction in the price of flucytosine. Cryptococcal infection is the most common cause of meningitis in adults living with HIV in sub-Saharan Africa. Cryptococcal meningitis (CM) is a serious opportunistic infection, which mainly affects persons with severe immunodeficiency. Most cases are observed in patients with CD4 T lymphocyte (CD4) cell counts < 100 cells/μL. In 2014, an estimated 280,000 people were reported to be positive for serum cryptococcal antigen globally and 220,000 new cases of cryptococcal meningitis occurred, of which sub-Saharan Africa contributed three-quarters of the cases and 135,000 deaths [Generally, cryptococcal meningitis in HIV-infected patients in South Africa is treated initially with intravenous amphotericin B either alone or in combination with oral fluconazole . FluconaLack of availability and access to 5-flucytosine is a common problem, especially in low-middle income countries. Also, the current international price renders it unaffordable in such countries , 6. In SFindings from the Advancing Cryptococcal Meningitis Treatment for Africa (ACTA) trial  and a reA cost-effectiveness analysis from the perspective of the public payer, the South African government, was conducted using a decision tree analytic model measuring Life Years (LY) and Quality Adjusted Life Years (QALY) over a period of 25 years with a discount rate of 5%. Direct costs relating to treatment, hospital stay, pathology tests and managing adverse events were measured in South African Rands (ZAR) for 2018 and converted to USD. The model was developed based on survival estimates from the ACTA trial, a randomised controlled trial of flucytosine as induction therapy in HIV-infected adults with cryptococcal meningitis. The details of this study are available elsewhere . BrieflyA simple decision tree model was built in Microsoft Excel 2016), using survival probabilities from the main ACTA trial at time points of 2 and 10 weeks  and then6, using The population of interest in this analysis were HIV-infected adults presenting with cryptococcal meningitis to a public health facility in South Africa. In general, these patients are treated at either a secondary  or tertiary and quaternary  facility. Patients are usually treated in a general ward rather than an ICU or high-care setting.The study perspective was that of a provider, in this instance the South African Government, and therefore costs and outcomes were considered from the viewpoint of the public healthcare system. Direct costs such as medicines, hospital stay, consultations, adverse events, diagnostics and laboratory tests were considered.The decision analytic model considered five treatment arms: (a) standard of care; 2-week course amphotericin B/fluconazole (2wk AmBd/Flu), (b) 2-week course amphotericin B/flucytosine (2wk AmBd/5FC), (c) short course; 1-week course amphotericin B/flucytosine (1wk AmBd/5FC) (d) oral course; 2-week oral fluconazole/flucytosine  and e) 1-week course of amphotericin B/fluconazole (1wk AmBd/Flu).The time horizon of 25 years for this model was based on the average life expectancy of an HIV-infected patient at 35 years of age who is receiving ART from estimates from a recent South African collaborative study with a weighted average of CD4 counts and ratio of male: female demographics . A sensiA discount rate of 5%, based on the South African Pharmacoeconomic Guidelines  was usedhttp://ghdx.healthdata.org/gbd-results-tool) [The model uses mortality rates from the full ACTA trial at at 2 and 10 weeks  and 6- ats-tool) .Utilities were obtained from the Merry et al. cost-effectiveness study, whereby a utility of 0.5 was allocated to patients ill with CM during induction therapy (week 1–2), 0.8 to patients in the maintenance phase of CM (week 3–52) and 0.95 to patients well with HIV (year 2 onwards) . A sensiAll costs were determined for 2018. If necessary, prices or tariffs from previous years were adjusted by the average annual Consumer Price Index to bring them up to 2018 prices. All costs are presented in US dollars (USD) based on an average South African Rand to US dollar exchange rate for 2018 (Average R13.25)  treatment costs for 2018 were obtained from a recent systematic review of per patient costs for HIV care in South Africa [The cost impact analysis was based on 7,497,774 HIV-infected patients in 2018  with a CFor managing relapses or recurrences, the WHO Guidelines recommend restarting the induction phase as per the initial recommendations. This model did not specifically include relapses or recurrences; however, these were included in the total count of cases of CM per year and assumed to have similar costs to first infections. Although paradoxical cryptococcal immune reconstitution inflammatory syndrome does occur with an estimated frequency of 10–50%  in patieIt was assumed that patients had one diagnostic LP and one therapeutic LP regardless of which regimen they were treated with. In the ACTA trial patients received on average 3 LPs, at baseline and on days 7 and 14, however this was conducted under clinical trial conditions and the WHO guidelines do not recommend routine follow up LPs in resource limited countries [Generally, there is no vial sharing for amphotericin B for patients, so it was assumed that 2 vials per dose were used. This was based on expert opinion of an estimated patient weight of 60 kg at a dose of 1 mg/kg/day . Although the average weight of a patient is usually assumed to be 70 kg, in the ACTA trial the average weight range was 50-53 kg across treatment cohorts. Differences in weight and the opportunity for vial sharing were tested in the sensitivity analysis.For the base-case it was assumed that all patients stayed in hospital for an average length of stay of 17 days  based on the utilisation data from the ACTA trial .There is little published information on the use of antibiotics as treatment of neutropenia in patients with CM so assumptions were based on availability of these medicines on the Essential Medicines List. It was assumed that all patients who experienced neutropenia requiring IV antibiotics were still in hospital for CM treatment and therefore did not incur additional hospital costs, only those of the antibiotic treatment.When the proportion of patients alive at each time point in each arm is taken into consideration, the highest total treatment costs were in the 2-week AmBd/5FC arm, followed by the 1-week AmBd/5FC, then the 2-week oral regimen and standard of care with the lowest total cost in the 1-week AmBd/Flu arm. Monitoring costs are highest in the 2-week Am/5FC arm followed by the standard of care. Supportive medicine costs are lowest in 1-week courses with no additional costs for the oral regimen. The 1-week AmBd/5FC arm has the highest ART costs due to more people being alive throughout the model  also has the poorest outcomes. If the treatment arms are compared to the lowest cost, lowest QALY regimen then the 1-week AmBd/5FC has the lowest ICER at USD119/QALY, followed by the standard of care Table . The oraWhen a sensitivity analysis was conducted to assess the uncertainty in the model it was found that varying most parameters did not have a substantial impact on the outcomes and the ICERs mostly remained below USD300/QALY. The sensitivity analysis focused mainly on the 1wk AmBd/5FC regimen compared to the 1wk AmBd/Flu as this reflected the lowest ICER in the analysis. The model is most sensitive to cost of infusions fees, hospital length of stay, life expectancy and probability of survival at 12 months in the lowest cost arm. The model was less sensitive to the inclusion of annual ARV treatment costs and then the cost of flucytosine  or neutropenia (antibiotics) does not impact substantially on the ICER outcomes, nor the inclusion or exclusion of use of potassium or magnesium supplementation. Increasing the number of LPs had very little impact on the ICERs. Increasing patient weight resulted in higher costs but only slightly increased the ICER. When the annual ART costs are removed, the 1wk AmBd/5FC still remains the most cost-effective although the ICER is reduced to USD55/QALY.The model is sensitive to the price of flucytosine with the costs of the 1wk AmBd/5FC and oral arm nearing those compared to standard of care at a 50% price discount, although the extended dominance over the oral regimen and standard of care remains.When the cost of infusion fees is increased beyond USD 37, both the 1-week flucytosine and oral course are dominant over the standard of care although not at the standard cost of tertiary level facility infusion fees (USD17 per infusion). Although other published cost-effectiveness analyses included an infusion fee as a separate cost item, in South Africa it most likely included in the hospital daily rate. The impact of excluding the infusion fee cost was assessed and found to reduce the ICER but not to less than USD104/QALY. Varying the discount rate from 0 to 10% also had little impact on the ICER.Due to the trial protocol in ACTA the average length of stay (LOS) in the trial was 17 days regardless of which treatment arm patients were in. It is possible that patients who are well enough to be discharged may leave sooner if they are on the 1-week AmBd/5FC or oral regimen. This has an impact on the model outcomes so that if the length of stay in hospital is reduced to 10 or 7 days for either the 1-week or oral flucytosine course, the ICERs are further reduced with a lower cost and higher QALYs compared to the 2-week courses of treatment.The incremental costs for all the related costs  for the first year of treatment were presented as well as the impact if only medicine costs are considered. Assuming the base case inputs, modelling the same LOS (17 days) for all treatment arms, and if up to 60% patients were to switch to that particular regimen in the first year, the greatest total cost impact for 2018 is with the 2-week AmBd/5FC course of treatment , followed by the 1-week course , the oral regimen , the current standard of care  with the 1 week fluconazole course having the lowest cost impact at USD7 825,941. Although the 1-week AmBd/Flu course has the lowest cost, it was decided not to include it in the cost impact analysis due to the higher mortality and the fact that the 2-week AmBd/Flu course is the current standard of care.If 60% of patients were switched to the flucytosine regimens, the average additional cost per patient  over what is currently spent on standard of care would be USD293 per patient per year for the 1-week flucytosine course Table . The incIn addition, the model is sensitive to whether an infusion fee is included or not with the incremental cost increasing slightly for the 1-week course when an infusion fee is not included. As the infusion fee increases so the cost of the 1-week flucytosine course becomes cost neutral. The greatest impact is seen when the LOS is reduced for the 1-week AmBd/5FC regimen assuming that patients may be discharged sooner. If the LOS is reduced to 10 days or 7 days, the 1-week courses become increasingly cost saving.The introduction of flucytosine as the mainstay of induction therapy for the treatment of cryptococcal meningitis has been hampered by issues of affordability and sustainable access, particularly in LMICs in Africa . This stWhilst the 1-week AmBd/Flu (currently lowest cost per patient) would also incur lower costs due to a shorter hospital stay, it has the highest mortality rate of all the treatment arms. Theoretically this presents a possible decision that could be made to choose this regimen over the other arms despite the mortality loss in favour of a significant savings, although it is unlikely the decision-making committee would recommend such an option.The model was sensitive to reductions in length of stay for the short course flucytosine treatments. Data on length of stay in patients on flucytosine from a study in the Eastern Cape, South Africa, indicates that the average LOS post introduction of flucytosine was reduced to a median of 14 days IQR 14–27) from 20 days (IQR 9–21) in the pre-flucytosine cohort, however this was not significant  from 20 . The iniAmphotericin has widely been implicated in the development of nephrotoxicity and anaemia , 29. TheA limitation of our model was the assumption that there would be no additional changes in health or costs in the following years. However, relapses do occur, particularly in patients who default on their fluconazole maintenance therapy or who stop their ARVs , 32. TheCost-effectiveness thresholds vary by country and in many cases are not explicit. The commonly used WHO threshold of one to three times per capita GDP has been increasingly refuted  and moreThe outcomes and resource utilisation in a general clinical practice setting such as that in the public sector in South Africa may differ from other countries and it is recommended that if flucytosine is included on the Essential Medicines List in South Africa, a monitoring and evaluation study is conducted to verify these outcomes and costs.This updated cost-effectiveness analysis in a public health setting in South Africa confirms that the addition of flucytosine as induction therapy in the treatment of cryptococcal meningitis in patients infected with HIV is cost-effective when used as a 1-week regimen with an ICER of USD119/QALY, compared to the lowest cost fluconazole regimen. The model is most sensitive to changes in costs rather than outcomes with the greatest impact seen where cost reductions can be achieved by reducing the hospital length of stay and infusion fee costs.Although the incremental cost of flucytosine compared to current standard of care is in the region of USD600 000 per annum, savings could be achieved with early discharge of patients as well as a reduction in the price of flucytosine.Improved access to flucytosine at a reasonable cost in South Africa, and particularly with the introduction of the 1-week course, has the potential to reduce the length of stay from 14 days to 7 days of treatment with an associated reduction in mortality. With the availability of robust evidence and cost-effectiveness data, access to flucytosine in LMICs and UMICs as an essential medicine is imperative and affordability can be achieved with appropriate price negotiations.


Vocal communication of woodpeckers has been relatively little studied so far, mostly because majority of species use drumming to communicate. Our recent study on the Middle Spotted Woodpecker revealed that a call which is specific for floaters is individually distinctive and functions as a vocal signature of unpaired individuals. The aim of the current study is to investigate whether a contact call of paired territory owners of the same species enables discrimination of individuals and their sex. Acoustic analyses revealed that the call is individually distinctive and experimental approach confirmed that woodpeckers are able to distinguish between a contact call of their partner and a stranger. We also found that the contact call shows significant sex differences. Interestingly, the acoustic parameter enabling sex identification is different than the parameters coding individual variability of the call. The design of a call so that its first part would code the identity of an individual and the second part would code its sex presents an effective and fine-tuned communication system. The results of our study also suggest that the contact call of paired Middle Spotted Woodpeckers may be useful for conservation biologists as a tool supporting other census methods. There is a vast evidence of between-individual variability in vocal traits of individuals belonging to the same species or even population7. This variability exists in many aspects of bird vocal behavior and may have evolved for different aims, such as quality advertisement10 or individual recognition12. In the latter case vocal distinctiveness results from slight differences in a syrinx and airsacks structure or the length and movements of a beak15. A certain combination of physical traits often produces vocal signatures of individuals. Numerous social interactions depend on proper recognition of individuals, for example mates20, neighbours26 or offspring29. It also helps to maintain dominance hierarchies31 and to avoid inbreeding32. Thus, one may infer that vocal signatures and the recognition of individuals using spectral characteristics of their voice should be widely prevalent in avian taxa due to numerous benefits both for signalers and receivers.Vocal communication plays an important role across bird species. It helps to resolve territory conflicts and defend boundaries against rival males as well as to attract females35. Having said that, calls may play an important role in woodpeckers communication37 and some species, like for example the Middle Spotted Woodpecker Dendrocoptes medius, drum rarely and use mainly calls to communicate.As it comes to woodpeckers, their vocal communication has been relatively little studied so far, mostly because majority of woodpecker species use drumming in mate attraction and territory defence12. Call-1 and Call-2 are characteristic for paired individuals, whereas Call-3 is typical for floaters. Call-1 is generally produced by individuals sitting in a tree. Territorial pairs use Call-1 as a contact call and an initial response to any disturbance within a territory. Call-2 is frequently uttered by territory owners during flight toward intruder and it goes along with territorial conflicts and trials to remove a rival from a territory. It frequently precedes a direct fight. Call-3, typical for unpaired individuals, is a persistent vocalization without any obvious stimulus. Spectral characteristics of call-3 proved to be individually distinctive and play-back experiments revealed that woodpeckers were able to distinguish between calling floaters—calls of familiar floaters induced a reduced aggression of territory owners compared to calls of stranger floaters. This led to the conclusion that Call-3 functions as a vocal signature of unpaired individuals.Our recent study, based on observation of spontaneous interactions between individuals and playback experiments, revealed that the Middle Spotted Woodpecker produces three call types of different spectral characteristics and functions12, thus it seems logical that mates should be able to distinguish calls of their partner from the vocalization of neighboring Middle Spotted Woodpecker pairs or intruders. We test the hypotheses that (i) between-individual differences in spectral characteristics of Call-1 are greater than within-individual variation in the production of this vocalization, (ii) individuals respond differently to Call-1 produced by their mate and unfamiliar Middle Spotted Woodpecker and (iii) spectral characteristics of Call-1 show significant sex differences and enable to tell males from females. The latter assumption, if confirmed, would be a valuable tool for researchers studying this species because sexual dimorphism is hardly visible in the Middle Spotted Woodpecker and males can be distinguished from females only by experienced field workers by slight differences in the length of the red crown38. We also inquire which structures of the contact call of woodpeckers convey information about identity and sex of calling individuals. We hypothesize that even a quite simple waveform pattern used by the Middle Spotted Woodpecker as a contact call is sufficient to encode a multidimensional message.Our current study aims to test whether calls of paired Middle Spotted Woodpeckers are also individually distinctive and enable recognition of particular conspecifics. We have analyzed bioacoustics parameters of Call-1 produced by different territorial individuals to find out whether this call may function as a vocal signature of paired individuals. We have also investigated the reaction of pair-members to a play-back of Call-1 uttered by their partner and by a stranger. Call-1 is often used as a contact call between pair members when they forage separately in different parts of a territoryVisual analysis of sonograms of Call-1 of different individuals initially supported our hypothesis that between-individual differences in spectral characteristics of Call-1 are greater than within-individual variation in the production of this vocalization  differed significantly between individuals  showed high accuracy of proper classification of 310 calls to 31 individuals based on the selected eight acoustic parameters of Call-1. Cross-validated procedure resulted in the correct assignment of calls to individual woodpeckers in 80.3% of all cases. This value is well above random assignment 3.2%), which shows that Call-1 is individually distinctive and may function as a vocal signature of paired woodpeckers. The lowest score of correct classification of calls to an individual was 60%, and the highest score was 100% , the change in the frequency of the first part of the dominant frequency (13.8%) and frequency of the mid-part of the dominant frequency (12.5%), which together explained 90% of differences  with cross-validated procedure demonstrated a correct assignment of calls to sex in 79.2%. Proper classification of female calls was 80% and male calls 78.6%. Obtained values show that Call-1 differs between sexes. Post-hoc analysis revealed that the acoustic parameter explaining 100% between-sex variability in Call-1 was the change in the frequency of the second part of the dominant frequency Fig. B. NoticeIn DFA for sex we used only one call per individual to avoid pseudoreplication of the data and violation of the assumption of independence (explained in Methods section). To test the accuracy of classification to sex of all calls in our dataset  we run a Logistic Regression, which showed 75.8% of correct classifications . The outcomes of the regression using multiple calls per individual were similar to those obtained in DFA conducted on a single call per individual (75.8% vs. 79.2%), demonstrating notable sex differences of Call-1 irrespective of the method used in analysis.A play-back experiment in which 27 paired individuals of the Middle Spotted Woodpecker were presented either with Call-1 produced by their mate or Call-1 produced by a stranger revealed that territory owners responded differentially to both types of playback Fig. .Figure 412. In only one case (which translates to 3.7%) a play-back of mate Call-1 was followed by an aggressive response with Call-2. On the other hand the play-back of stranger Call-1 triggered the response with both Call-1 (51.8%) and Call-2 (74%), and the latter one was more frequent. Bearing in mind that Call-2 warns the intruder about a territory owner’s readiness to a direct fight12, the results of the experiment indicate that territory owners discriminated between mate and stranger Call-1. The type of play-back  significantly affected the response with Call-1  and Call-2  of pair members.A play-back of mate Call-1 in 81% of cases resulted in a vocal response with Call-1, which seems natural as Call-1 function as a contact call between pair members12. Together these results show that, although the repertoire and the structure of Middle Spotted Woodpeckers calls is rather simple, it allows for coding identity and sex of a calling individual. Furthermore, the experimental approach revealed that woodpeckers make use of this information and distinguish between familiar individuals and strangers.Our study revealed that Call-1 of the Middle Spotted Woodpecker, which is the most frequent vocalization of pair members, is individually distinctive, exhibits significant sex differences and enables mate recognition. These results extend our previous findings demonstrating that Call-3, a characteristic vocalization of Middle Spotted Woodpecker floaters, is a vocal signature of unpaired individuals and enables their recognition by territorial neighbours39, the area of woodpecker Piciformes vocalization is still almost unexplored. The studies exploring vocal communication of woodpeckers not only are scarce but also they are descriptive40 and often involve observations of only few individuals41. Bioacoustic analyses and the experiment in our current study show that Call-1, which functions as a contact call between mates and a signal of a general arousal12, is individually distinctive and pair members respond differently when they hear a play-back of their mate Call-1 and stranger Call-1. In the first case they answer with Call-1, simply keeping in touch with the calling partner. This changes when it comes to a play-back of stranger Call-1, which mainly involves the response with aggressive and territorial Call-2 followed by Call-1. The presence of the latter one in the response towards a stranger may be explained by the fact that woodpeckers use Call-1 to express anxiety during any disturbance within a territory12 and also they might have called their mate to inspect the intruder. However, the response with Call-1 to the play-back of stranger Call-1 was significantly less frequent than a response with Call-1 to the play-back of mate Call-1.Although much has been written about vocal communication in birds42. It was demonstrated not only in songbirds but also in species using less complex vocalisation46. A few studies described individual recognition by calls also in woodpeckers, including the Downy Woodpecker Picoides pubescens47 and the Accorn Woodpecker Melanerpes formicivorus37. Interestingly, woodpecker drumming was also demonstrated to be individually distinctive, however it was not tested whether it was used by birds for the recognition of conspecific48. Discrimination of individuals is beneficial because it enhances communication of mates over longer distances as well as it limits unnecessary, repetitive aggressive interactions between neighbours during the breeding season49. Thus one can expect that majority of vocalisation systems, including calls in woodpeckers, should provide the possibility of coding vocal signatures. We were not able to classify single calls to an individual with a 100% accuracy but our results are similar to those observed for call classification in other bird species, for example in African wood owls Strix woodfordii50, European nightjars Caprimulgus europaeus46, Corncrakes Crex crex51, or Woodcocks Scolopax rusticola52. It seems that call classification around 80% is a typical outcome in species possessing vocal signatures. Noteworthy, DFA in our study was conducted on single calls but during woodpecker vocalization Call-1 is often uttered in bouts, which likely rises the probability of individual identification of calling woodpeckers by their conspecifics to higher levels than indicated by DFA based on classification of single calls.Individual recognition based on vocal characteristics is widely described in to-date studiesBubo bubo and the Great horned owl Bubo virginianus encode information both on sex and ID of calling individual, however the acoustic parameters responsible for individual and sex differences were mostly the same and referred to frequency and temporal variables describing a call54. The study on Kittiwake Rissa tridactyla revealed that greeting calls produced by individuals are sexually dimorphic and individually distinctive and in this species both cues are included in different features of the call – sex of individual is encoded in the value of fundamental frequency of the call while ID in a set of temporal and frequency parameters of the call55. In many species of petrels, especially those active at night, calling is restricted to a single signal which conveys information about individual identity, sex and even a population to which the individual belongs56. In the Cory’s shearwater Calonectris diomedea female calls are characterized by lower fundamental frequency than male calls while ID of individuals is encoded in chronological ordination of syllables in a call56. In a play-back test birds discriminated both mates from strangers and males from females. Similarly, calls of the Manx shearwater Puffinus puffinus are sex and individually specific57. Sexual differences are encoded in the frequency spectrum, which is broader for females than for males. Individual differences include a set of temporal and frequency parameters of the call. Individual and sexual features were also demonstrated in begging calls of the Barn owl Tyto alba nestlings58. Interestingly, call features coding ID and sex became more pronounced with nestling hunger and increasing competition for food. In some species, like for example the White-faced whistling duck Dendrocygna viduata, differences between sexes were expressed significantly more strongly than the individual differences59. Also, the species were reported, in which individual features were absent in vocalization, however sexual identification of individuals was possible based on call parameters60. Above examples show that a number of solutions have been evolutionary designed to convey the information about an individual during vocalization. Our study adds to this variety by describing the system of coding ID and sex in the Middle Spotted Woodpecker. However, it should be underlined that further play-back experiments involving digital modification of woodpecker contact calls are needed to confirm our preliminary evidence that individual ID and sex are likely coded in separate acoustic parameters of a single call. Our study also lacks repeated recordings of the same individuals to confirm that vocal characteristics of woodpeckers encoded in a contact call are stable over a period of time. Thus further studies are needed to address above issues.Our study also revealed that Call-1 uttered by males and females exhibits significant sex differences. Intersestingly, the acoustic parameter enabling sex identification was different than the parameters coding individual variability of Call-1, which shows that the structure of Call-1 is sufficient to transmit multidimensional information between individuals. The design of the call so that its first part would code the identity of an individual and the second part would code individual’s sex presents an effective and fine-tuned communication system. The results published by other researchers show that evolution of vocal communication in birds has led to a similar solution in a number of avian species. For example, the calls of the European eagle owl Dryobates minor, the Great Spotted Woodpecker Dendrocopos major and the Syrian Woodpecker Dendrocopos syriacus . It needs further investigation to test whether their function is similar, but if confirmed, it would reveal a simple and universal way of coding ID and sex across various woodpecker species.Taking into account that a waveform pattern of Call-1 is rather simple, one may expect that the number of combinations of features coding identity and sex is not endless, thus the efficiency for a proper ID coding may decrease with the increasing number of individuals tested. However, the Middle Spotted Woodpecker is rare, sedentary and does not live in high densities. Additionally, due to its habitat preferences towards old woods dominated by oaks it lives in small and isolated populations. It seems that in such systems Call-1 provides sufficient possibilities for proper individual recognition and identification. Interestingly, calls of a very similar structure to Call-1 of the Middle Spotted Woodpecker are present in vocal repertoires of other woodpecker species, for example the Lesser Spotted Woodpecker 61. Sexing by voice could be conducted in the Middle Spotted Woodpecker only with some degree of probability because there is no acoustic parameter which does not overlap in ranges of values between sexes, however counting individuals based on spectral characteristics of their calls seems reliable.The results of our study also suggest that Call-1 of the Middle Spotted Woodpecker, which is individually distinctive and shows notable sex differences, may be useful for conservation biologists as a tool supporting other census methodsThe contact call of the Middle Spotted Woodpecker is individually distinctive and woodpeckers are able to distinguish between a contact call of their partner and a stranger, suggesting that the call involves a vocal signature of paired individuals. The contact call also exhibits significant sex differences, although sexual features are less expressed than individual ones. The acoustic parameter enabling sex identification is different than the parameters coding individual variability of the call. Our study shows that the structure of the contact call is sufficient to transmit multidimensional information between individuals. The design of the call so that the first part will would code the identity of an individual and the second part would code its sex presents an effective and fine-tuned communication system. The results of our study also suggest that the contact call of paired Middle Spotted Woodpeckers may be useful for conservation biologists as a tool supporting other census methods.63. It is sedentary, socially monogamous64 and territorial in the spring65. Both parents share breeding duties64. Each year they excavate a new cavity and raise a single brood66. A relatively large red crown is characteristic of both males and females but is longer in males62 which enables sexing individuals by an experienced observer.The Middle Spotted Woodpecker is a medium-sized woodpecker (20–22 cm long) of the Western Palaearctic that inhabits deciduous forests, especially areas with old oaks Quercus, hornbeams Carpinus and elms Ulmus, as well as a patchwork of clearings, pasture and dense woodlandThe study was conducted in the riverine forest of Warta River Valley in Central Poland near Czeszewo  in the years 2009–2010. The study plot consists of 222 ha of woodland with trees of the genera Quercus, Fraxinus, Ulmus, Fraxino and Ulmetum in flooded areas and Quercus, Carpinus, Stallario and Carpinetum on higher areas. The whole study area, of which about 40% is covered by mature, nearnatural forest stands, has been protected as the Czeszewski Las Nature Reserve since 2004.67. To make surveys more efficient we used the play-back of Middle Spotted Woodpecker calls. All territories were mapped and confirmed during subsequent surveys. We distinguished between breeding and unpaired individuals based on an array of behavioral cues, such as: reaction to play-back (defence of a territory vs. retreat), presence/absence of a partner and a nest hole, presence/absence of persistent vocalisation typical for unpaired Middle Spotted Woodpeckers63. For more information on territory mapping and woodpecker status please refer to Węgrzyn & Leniowski12.Each year at the beginning of the breeding season (March/April) the entire study area was surveyed three times to search for breeding pairs and unpaired individualsCall-1 of Middle Spotted Woodpeckers was recorded using a shot-gun microphone AKG Blue line (SE 300 B + CK 98) with a windshield and a portable recorder Edirol R-09HR. Recordings were made in 2009–2010. We recorded Calls-1 during spontaneous vocalization of individuals. Call-1 is typically produced by individuals sitting on a branch or a trunk. In terms of social context it is used as a contact call with a mate without any obvious stimuli when pair-members forage separately in different parts of a territory, but it may also signal arousal due to any disturbance within a territory . Recordings were made between 9.30 a.m. and 2.00 p.m. as during the early spring this is the time of the highest vocal activity of Middle Spotted Woodpeckers (they become active with temperature rise). Calls produced by given individual were recorded on the same day but not in the same interaction. In analyses we used 10 calls per individual which were randomly selected from the calls recorded during our single visit to the territory.68. We used the following acoustic measures  = 25, window = Hamming and temporal overlap = 87.5%. This gave a 244 Hz bandwidth with 46 Hz frequency and 2.67 ms time resolution12 .We used ANOVAs (with Bonferroni probability adjustments) to test if there were significant differences in the eight measured Call-1 parameters within and between individuals. We used a discriminant analysis (DFA) to determine whether Call-1 could be assigned to the correct individual based on the eight acoustic parameters described above. In the DFA we applied the cross validated classification and a leave-out-one test. DFA for ID was run on 310 calls from 31 individuals .69. In the case of purely nested data comprising only two factors (like ID and sex) the solution is to use only a single randomly selected call per each subject or to use a Logistic Regression which is less restrictive than DFA with regard to assumptions, permits the analysis of multi-factor data sets, and also allows for a classification of cases70. However, Logistic Regression does not allow for a stepwise method which reveals the parameters that explain the highest level of between-class variability. As we wanted to know which call parameters differ the most between the sexes and at the same time we also wanted to test the accuracy of classification to sex of all calls in our dataset we run both analyses .To test if Call-1 exhibits significant sex differences we compared the eight measured acoustic parameters of Call-1 produced by males and females using t-test. Subsequently we conducted DFA using the same parameters  to determine the probability of the assignment of Call-1 to the correct sex. However, DFA for sex was run on a single call per individual  because using multiple calls from a given individual when trying to classify the calls into some category other than individual ID (such as sex) can bring misleading results. If one tries to use traditional DFA in this scenario, the assumption of independence will be violated and the results may be misleading. Doing so would pseudoreplicate the data and hence violate the assumption of independence12 as close as possible). Bouts of calls used in the play-back experiment were natural recordings of Call-1 adjusted to the length of 15 calls per bout by deleting final calls if an original bout exceeded 15 calls. We did not elongate too short bouts by inserting additional calls as we wanted to minimize manipulation. Bouts were separated by 5 s of silence . We put in effort to do play-backs in such a way as to avoid the subject’s mate presence during the experiment. It was possible because in the studied population of the Middle Spotted Woodpecker each mate forages in a different part of the territory of the pair69. We did play-backs within a subject’s foraging range and the distance between the speaker and the tested woodpecker was short (30–40 m), so the subject bird was always the first who responded to the play-back. In all cases we were able to see the subject before and during the play-back. The play-back was turned off as soon as a subject individual started responding to avoid masking the response by the play-back. To test whether the play-back of mate Call-1 and stranger Call-1 triggers a different response from territory owners we conducted a Linear Mixed Model with REML method and main effects design. We used repeated measures approach with the type of a playback as a fixed factor, the sex of an individual as a random factor and the type of a response  as dependent variables. A repeated measure procedure was applied because each woodpecker was tested twice .The aim of the experiment was to test the response of paired individuals to Call-1 produced by their mates and by strangers. The experiment was conducted in the year 2010 before egg laying stage (March/April) when the vocal activity of the Middle Spotted Woodpeckers is the highest. We conducted play-backs of Call-1 of a pair-member and a stranger in 27 breeding territories occupied by Middle Spotted Woodpecker pairs. Stranger play-backs were prepared using vocalization of individuals that occupied distant territories from the subjects of the experiment. The study plot consisted of 222 ha of woodland and Middle Spotted Woodpeckers are sedentary, so an individual from a distant territory  was considered a stranger for the territory owner to whom the play-back was presented. Responses to play-backs were recorded, transformed to sonograms using Avisoft-SASLab Pro software and analyzed. To prepare play-backs we used recordings of Call-1 collected earlier in the same population. The playbacks of mate Call-1 and stranger Call-1 presented in the same location were separated by an interval of at least 72 h. The play-back of mate Call-1 and stranger Call-1 were presented in alternating order in subsequent locations. To avoid pseudoreplication, each woodpecker received a play-back of Call-1 of a stranger prepared from the recording of a different individual. Playbacks of Call-1 consisted of five bouts, 15 calls in each bout (to mimic a spontaneous production of these calls described in Węgrzyn and Leniowski


The immunobullous condition Pemphigus Vulgaris (PV) is caused by autoantibodies targeting the adhesion proteins of desmosomes, leading to blistering in the skin and mucosal membrane. There is still no cure to the disease apart from the use of corticosteroids and immunosuppressive agents. Despite numerous investigations, the pathological mechanisms of PV are still incompletely understood, though the etiology is thought to be multifactorial. Thus, further understanding of the molecular basis underlying this disease process is vital to develop targeted therapies. Ample studies have highlighted the role of Desmoglein-3 (DSG3) in the initiation of disease as DSG3 serves as a primary target of PV autoantibodies. DSG3 is a pivotal player in mediating outside-in signaling involved in cell junction remodeling, cell proliferation, differentiation, migration or apoptosis, thus validating its biological function in tissue integrity and homeostasis beyond desmosome adhesion. Recent studies have uncovered new activities of DSG3 in regulating p53 and the yes-associated protein (YAP), with the evidence of dysregulation of these pathways demonstrated in PV. The purpose of this review is to summarize the earlier and recent advances highlighting our recent findings related to PV pathogenesis that may pave the way for future research to develop novel specific therapies in curing this disease. The autoimmune disorder, Pemphigus is a blistering disease that results in intraepithelial blisters affecting predominantly the mucosa and skin. It is mediated by circulating pathogenic autoantibodies that attack the keratinocyte cell surface proteins leading to acantholysis . AcanthoClinically, PV presents as multiple flaccid blisters limited to the suprabasal layer in the epidermis or mucosal membrane, whilst the keratinocytes in the top layers maintain their cell cohesion. Interestingly, the basal cells also maintain their adhesion to the basement membrane, thereby histologically appearing like a ’row of tombstone’ . On the The desmosomal cadherin DSG3 was first identified as an isoform of the desmoglein subfamily and the auto-antigen in PV . FollowiDSG3 have been indicated as a key mediator involved in desmosome remodeling, epidermal proliferation and differentiation, cell migration and apoptosis, and thus validating that DSG3 acts as a signaling molecule that has a major impact on tissue integrity and homeostasis. The binding of PV IgG to DSG3 has been demonstrated to stimulate the phospholipase C signaling pathway which causes calcium release and PKC activation leading eventually to the desmosome and cell surface DSG3 depletion and the loss of intercellular cohesion and formation of blisters . Later sAn in vivo transgenic mouse study has shown increased keratinocyte proliferation and altered terminal differentiation associated with misexpression of Dsg3 in the suprabasal layers of the skin suggesting this basal cell isoform of DSG is involved in regulating cell proliferation . FindingOwing to the widespread expression of DSG3 at the plasma membrane beyond the desmosomes it is thought that the non-junctional pool of DSG3 may be responsible for cell signaling and is the primary target of PV IgG . A previThe pathological mechanisms of PV are controversial and remain a hot topic under debate in the field. Ample studies support the concept that PV is caused by the pathogenic IgG autoantibodies directed against DSG3 as well as DSG1. However, there is evidence also suggesting pemphigus blistering is not solely caused by autoantibody targeting to DSG3 but rather results from intracellular signaling mediated by non-DSGs targeting autoantibodies that leads to shrinkage of cell and apoptosis ,21,22. NThis long-standing theory states that the binding of autoantibodies to the extracellular domain of DSGs causes loss of intercellular adhesion and pemphigus acantholysis ,24,25. HThe different distribution patterns of DSG1 and DSG3 in the skin and oral mucosa support this theory. It describes that if both DSG1 and DSG3 are present and only one is inactivated by autoantibodies then the other will compensate and provide adhesion, but if only one DSG is present and is attacked by the autoantibodies, then the acantholysis will occur ,28.Various signaling pathways are at play in the development of pemphigus acantholysis. The phosphorylation of DSG3 is caused by the binding of pathogenic PV IgGs to DSG3 followed by its dissociation from plakoglobin and then endocytosis-mediated degradation . PKCα shThis theory explores new mechanisms with the findings obtained by independent groups. Early studies have proposed that apoptosis may be responsible for pemphigus acantholysis ,37. The basal cell shrinkage hypothesis, to explain the pathogenesis of acantholysis. After the pathogenic PV autoantibodies bind to the keratinocyte receptor(s), a series of signal-transduction pathways trigger the rupture of the cytoskeleton, resulting in the collapse and shrinkage of the keratinocytes. This theory also vividly links to cell apoptosis and basic pathological features, emphasizing that apoptotic enzymes contribute to acantholysis development in terms of both molecular events and chronologic sequence [In 2009, Grando et al. proposed a new terminology ‘apoptolysis’- sequence . Althougsequence ,44, therRehman et al. have identified DSG3 acting as an anti-stress protein by counterbalancing p53 in the maintenance of normal epithelial homeostasis and demonstrated disruption of this regulation in PV  Figure . The stuSome signaling pathways that are activated in PV ,45,46 arWAF1/CIP1 in these double knockdown cells compared to the respective controls [It is worth noting that the Dsg3/p53 pathway in keratinocytes established by Rehman et al. seems to be unique. To address the specificity of this pathway, Rehman et al. have performed the RNAi knockdown experiments on other junctional proteins, such as desmoplakin (a marker of desmosomes) and E-cadherin . They found that neither desmoplakin knockdown nor E-cadherin knockdown gave rise to p53 induction (even when exposed these siRNA transfected cells to UVB) similar to what had been observed in the DSG3 knockdown cells . Furthercontrols . These fcontrols . This dicontrols ,56. Indecontrols . In accocontrols .Reports about the clinical association of PV with cancer are few and vary. Several earlier studies indicated a favorable effect of DSG3 reduction in carcinogenesis with its decrease correlated with a more aggressive cancer phenotype ,60,61. ITaken together, the above-discussed studies link together several unrelated pathways and open new avenues of research to investigate the pathogenesis and stress-induced tissue damage in PV. It also suggests a potential new direction of novel approaches in the diagnosis and treatment of not only PV but also other diseases including cancer.2O, O2, etc.) or preventing their formation. The imbalance of oxidant/antioxidant is identified in various autoimmune diseases including rheumatoid arthritis and lupus erythematosus [Increased accumulation of reactive oxygen species (ROS) or free radicals can lead to oxidative stress. Although ROS is indispensable at moderate concentrations to defend from microorganisms involved in the cellular signaling systems , excess ematosus ,72,73,74ematosus ,78,79,80ematosus ,79. HoweA recent study by our group has demonstrated that elevated ROS production can be evoked by treating keratinocytes with PV patient’s sera as well as a pathogenic mouse monoclonal antibody AK23, with concomitant accumulation of Yes-associated protein (YAP) especially in the cytoplasm of antibody-treated cells . ImportaAnother recent study by our group has determined that DSG3 is capable of regulating YAP independent of antibody binding to DSG3 ,86. RNA 2+/Mn2+-ATPase isoform 1) can prevent blister formation in the neonatal mouse skin of both the passive transfer model and skin explant [Although autoantibodies against DSGs are essentially the cause for the disease initiation, many non-DSG antibodies have also been implicated in blister formation in PV ,88. For  explant . However explant ,100,101.The pathological mechanism in PV is triggered by autoantibodies and the immune system is involved in the disease’s onset and progression. Various studies have suggested an increased risk of pemphigus in those with a family history of other autoimmune disorders, as well as the presence of specific genotypes associated with HLA and non-HLA genes ,103. To The pathological mechanisms underlying the pemphigus acantholysis are complex and remain not completely clear. Over the past three decades, significant advances have been made in our understanding of PV pathophysiology. Autoantibodies targeting the N-terminus of DSG3 are likely the initial and critical step that has an impact on its action in anti-stress response and hence triggers various intracellular events leading to blistering in tissues expressing DSG3 that include skin and oral mucosa. The direct inhibition of DSG3′s trans-interactions is not sufficient to induce the complete loss of cell cohesion and the synergistic action of orchestra signaling networks such as p38 MAPK, EGFR, Src, c-Myc, and Rho GTPases are considered to be vital in the pathophysiology of PV. Likely, the non-DSG antibodies also play a role, however, it remains unclear if they are responsible for the PV clinical phenotype ,90. Addi