Several studies have suggested that depression is associated with an increased risk for fracture; however, the results are conflicting. This study aimed to conduct a meta-analysis of cohort studies assessing the association between depression and the risk for fracture.Relevant studies were identified by a search of Web of Science, PubMed, Embase, China National Knowledge Infrastructure and WanFang database to Feb 2018. Cohort studies on the relationship between depression and the risk for fracture in the general population were included in the meta-analysis. Data collection was in accordance with the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines, and the quality of the included studies was assessed using the Newcastle–Ottawa scale. Two independent investigators screened the abstracts and full texts of the studies, extracted data, and assessed the quality of the study. Either a fixed-effect or random-effects model was used to compute the pooled risk estimates when appropriate.P < 0.001 for heterogeneity; random-effects model). In the subgroup analyses conducted in terms of study region, the pooled HR for the studies conducted in Europe was higher  than that in America and Asia, with a significant difference between the groups (P = 0.036).In total, 16 cohort studies with 25 independent reports that included 414,686 participants during a follow-up duration of 3–14 years were included in the analysis. The pooled hazard ratio (HR) for total fracture was 1.24  contains supplementary material, which is available to authorized users. Osteoporotic fracture is a critical health problem worldwide because it causes severe pain, disability, decreased quality of life, and increased mortality and health costs , 2. MoreDuring the past decades, several studies have assessed the association between depression and the risk for fracture. However, the results were conflicting –9. Some This review was conducted in accordance with the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines , with reThe following search terms were used to identify relevant citations:  and  along with . In addition, the reference lists of the original and relevant review articles were also assessed to further identify relevant studies. Papers published in English or Chinese were considered.Studies were included in the meta-analysis based on the following inclusion criteria: (1) the cohort comprised non-institutionalized adults; (2) the exposure of interest was depression; (3) the outcome was fracture; (4) the risk estimates with the corresponding 95% confidence intervals (CI) of depression related to fracture were reported. Studies were excluded if (1) the study had a case control design or retrospective design; (2) Reviews, letters, commentaries were excluded; (3) Lack of any information that allowed to calculate effect estimates and corresponding 95% CI. For cohorts with several reports, we tried using data from non-overlapping follow-up periods of each report, or publications with the longest follow-up periods were selected.We extracted the following information from each eligible study: last name of the first author, year of publication, country where the study was performed, number of participants, characteristics of the participants , follow-up time, depression measures, fracture type, and covariates adjusted in the multivariable analysis.Quality assessment was performed according to the Newcastle–Ottawa scale (NOS) , which iP value < 0.10 was considered an indication of statistically significant heterogeneity) and the I2 statistic  [Hazard ratio (HRs) and their 95% CI were used to quantify the association between depression and fracture, and the reported relative risk (RRs) were considered equivalent to HRs. Any study results stratified by sex and fracture type were considered as independent reports. The heterogeneity of HRs across the studies was evaluated using the Cochran Q test (ctively) , 15. A fctively) .Subgroup and sensitivity analyses were conducted to explore potential heterogeneity across studies, and the differences among subgroups were tested via meta-regression analysis. Publication bias was assessed via visual inspection of the funnel plot and using the Begg  and EggeNo Patients were involved in determining the research question or the outcome measures or in developing plans for the design or implementation of the study. In addition, no patients were required to provide advice on the interpretation or writing of the results. There were no plans of disseminating the research results to the study participants or the relevant patient population.Initially, we retrieved 209, 677 and 568 citations from Web of Science, PubMed, and Embase, respectively. After removing duplicates and reviewing the titles and abstracts, we identified 87 potentially relevant articles. After assessing the full text of articles that may be relevant, 16 eligible studies met the inclusion criteria and were finally included in our meta-analysis. The results of literature search and selection are presented in Fig. Additional file P = 0.000, I2 = 56.5%).Results from the random-effects meta-analysis of depression and the risk for fracture are presented in Fig. n < 5000). When applying study regional categories, the pooled HR for the studies conducted in European countries was higher  than that in America and Asia, with a statistically significant difference (P = 0.036). No significant difference was found between the groups in terms of other variables.Table P = 0.000; Begg, P = 0.018). We used the trim-and-fill method to assess the impact of any potential publication bias, and results showed that eight studies may be needed to obtain funnel plot symmetry for fracture . Therefore, the pooled HR did not substantially change after the correction for potential publication bias.To examine the impact of a single study on the results, we omitted a study at each turn and pooled the results of the remaining studies. The pooled HR did not substantially change, ranging from 1.24 (95% CI: 1.14–1.35) to 1.29 (95% CI: 1.19–1.42). Visual inspection of the funnel plot revealed some asymmetry , which was slightly higher than that of a previous meta-analysis of 10 studies published before 2009  [Our subgroups analyses identified an important finding. That is, the associations between depression and fractures varied between populations when subgroups analyses conducted by continents. The association was stronger in individuals from European countries than those from America and Asia. This may be attributed to the variation in health care systems in different geographic regions, availability of health services and other factors that are currently unknown. Since the studies included in the current meta-analysis were conducted in high-income countries(areas), such as those in Europe, North America, and Taiwan (research from Asian countries included only two studies conducted in Taiwan), these results should be cautiously generalized to developing countries. To apply this finding to a wider population, more studies must be conducted in other populations from Asia, Africa, and South America.Depression may contribute to fracture via several possible mechanisms. First, depressive disorders are associated with the deregulation of the hypothalamic–pituitary–adrenocortical axis , chronicThis meta-analysis has several limitations. First, a moderate level of heterogeneity across studies was observed, which might result from the differences in the characteristics of the participants, sample sizes, depression measures, and statistical adjustments for potential confounders. Although moderate heterogeneities were still observed in some subgroups, the pooled HRs consistently showed positive associations in all subgroups and the prediction interval remained significant. Second, the funnel plot indicated a possible publication bias; however, the trim-and-fill method was used to correct the bias, which did not materially change the positive association. Third, we conducted only limited subgroups analyses because most original studies included in this meta-analysis did not adjust for other confounders such as social-economic status, medical comorbidities or ethnicity; information about these factors was not provided in most original reports. Finally, because the measurement of depression was mainly based on self-reported symptom scales, misclassification of exposure was inevitable, and this might bias the actual association between depression and fracture.In conclusion, this meta-analysis provides strong evidence that depression is significantly associated with an increased risk of fracture, particularly in individuals in Europe. Given the high prevalence of depression and osteoporotic fractures in the general population, the observed association between depression and fractures have substantial implications, both clinical and preventive. Mental health is closely related to bone health. It is greatly important for primary care practitioners and mental health care workers to take depression account into the prevention and clinical treatment of osteoporotic fractures.Additional file 1:Table S1. Characteristics of studies included in the meta-analysis. (DOCX 18 kb)


AbstractAraneae) community assembly in Macaronesian islands. To obtain the data, we applied the sampling protocol COBRA (Conservation Oriented Biodiversity Rapid Assessment), in twelve 50 m x 50 m native forest plots and five dry habitat plots on the island of Madeiraand in 5 dry habitat plots on the island of Porto Santo. Through this publication, we contribute to the knowledge of the arachnofauna of the Madeiran archipelago.Here we present the data obtained from the samples collected as part of a large research project (MACDIV) which aims at understanding the drivers of spider species. Out of the 105 recorded (morpho)species, 34 were endemic, 26 native non-endemic, 22 introduced and 23 species of unknown origin. We report seven new records of possibly recently introduced species in the Madeiran archipelago. We also present 21 new records for Madeira island and 32 for Porto Santo (33 for the whole archipelago).From the samples that we collected, we obtained a total of 14,902 specimens, of which 49% were adults . We identified these specimens to 87 species and 18 morphospecies (undescribed), belonging to 26 families. Species of the family  The north Atlantic archipelago of Madeira is composed of the volcanic islands of Madeira, Porto Santo and the Desertas, as well several islets. At approximately 700 km from the African coast and at more than 400 km from the Canary Islands, it is the second most isolated Macaronesian archipelago, after the Azores. This intermediate distance, combined with an geological age of 5-27 My, has allowed frequent colonisation rates and enough diversification time to generate diverse species communities . Since iThe laurisilva is indeed a unique and iconic ecosystem, that is often seen as representative of the Macaronesian archipelagos as a whole and contains a great deal of endemic species . HoweverThe need for an update in many taxa is exemplified by spiders. Although, according to the last available checklist , the spiThis publication is the second of a series on Macaronesian spider fauna see  and provClethraarborea, Laurusnovocanariensis, Ocoteafoetens and Perseaindica, were dominant Clethraasalicina  Fig. 3)ClethraaWe applied two versions of the optimised and standardised COBRA protocol (Conservation Oriented Biodiversity Rapid Assessment) : the proMadeira and Porto Santo islands, Madeira, Macaronesia, Portugal32.66138 and 33.0921 Latitude; -17.15871 and -16.30453 Longitude.Data range: 2016-8-01 – 2017-4-30.Sampling in the native forest occurred in August 2016. Sampling in dry habitats occurred in April 2017.Dalberto Teixeira Pombo insect collection at the University of AzoresDTPAll specimens were preserved in 96% ethanolDalberto Teixeira Pombo insect collection at the University of Azores (Curator: Paulo A. V. Borges)Open Data Commons Attribution LicenseMACDIV_COBRA_Madeira_Forest_and_Dryhttp://ipt.gbif.pt/ipt/resource?r=spiders_madeirahttp://islandlab.uac.pt/software/ver.php?id=381MACDIV_COBRA_Madeira_Forest_and_DryDarwin Core Archive62http://ipt.gbif.pt/ipt/resource?r=spiders_madeiraversion 1The following data table includes all the records for which a taxonomic identification of the species was possible. The dataset submitted to GBIF is structured as a sample event dataset, with two tables: event (as core) and occurrences. The data in this sampling event resource have been published as a Darwin Core Archive (DwCA), which is a standardised format for sharing biodiversity data as a set of one or more data tables. The core data table contains 562 records (eventID). One extension data table also exists with 3281 occurrences. An extension record supplies extra information about a core record. The number of records in each extension data table is illustrated in the IPT link. This IPT archives the data and thus serves as the data repository. The data and resource metadata are available for downloading in the downloads section.We collected a total of 14,902 specimens – of which 49% were adults 7,263) - belonging to 105 (morpho) species and 26 families  (introduced), Porrhoclubionadecora  (native non-endemic), Neosconacrucifera  (introduced), Microlinyphiajohnsoni  (native non-endemic), Cryptachaeablattea  (introduced), Cheiracanthiumalbidulum  (endemic), Ceratinopsisacripes  (native non-endemic), Meta stridulans Wunderlich, 1987 (endemic), Echinotheridiongibberosum  (native non-endemic), Episinusmaderianus Kulczyński, 1905, (native non-endemic) and Paidiscuraorotavensis  (native non-endemic). The five most abundant species were Zodarionstyliferum  , Episinusmaderianus Kulczyński, 1905 , Diplocephalusgraecus  , Tenuiphantestenuis   and Macaridionbarreti  .The most widespread (morpho)species were endemic, native non-endemic, introduced and unknown.In this section, we point out, explain and discuss the identification of some of the species and morphospecies found – the ones for which information is limited and ordered as in Tables 2, 3 and 4 – and their classification as SynaphrissaphrynisThis species is known from Spain and Selvagens Islands. It is also present on Bugio island (Desertas), as one of the authors (L. Crespo) identified a specimen collected by M. Boieiro et al. in 2011/12 (unpublished record). The limited number of available records does not allow us to infer its total distribution with certainty.HaplodrassusomissusHaplodrassusomissus had not been recorded previously in Madeira, it does not come as a surprise to be present there, given its known distribution across the Mediterranean basin and the Canary Islands.Although Haplodrassus sp. 164Haplodrassus MS 164 belongs to the dalmatensis group.This morphospecies is present in Porto Santo and, possibly, in other locations of the Madeira archipelago. Haplodrassus sp. 158Haplodrassus MS158 belongs to a different group from that of Haplodrassus MS164, as its genitalia is more similar to species such as H.deserticola, which is present in the Canary Islands or H.minor, which has been recorded from Europe to Turkey, according to the World Spider Catalogue. It is possible that more species of this group are present in the Madeira archipelago.The morphospecies MacarophaeuscultiorMacarophaeuscultior may be a single-island endemic from Madeira. Although the World Spider Catalog and the Banco de Datos de Biodiversidad de Canarias . Therefore, we considered this species as introduced.Theridionhannoniaeunknown biogeographic category, given the impossibility of knowing whether it arrived at the Madeiran archipelago naturally or with human intervention.This species has a Mediterranean distribution and, in the Madeiran archipelago, it is usually found in dry, relatively disturbed areas. We classified this species as of Theridion sp. 89This morphospecies is most likely a new species, so we considered it to be single-island endemic, probably from laurel forest.Misumenacf.nigromaculataMisumenanigromaculata, whose female has been described only once and whose male is unknown.Here we considered this morphospecies to be a new endemic species given the little information on XysticusnubilusXysticusnubilus has a Mediterranean distribution and is usually found in dry habitats in the Madeiran archipelago. Therefore, as with Theridionhannoniae, we classified it as of unknown biogeographic category in Madeira.HyptiotesflavidusAlthough the distribution of this species is largely Mediterranean, given that it is usually found in laurel forests, we classified it as native to Madeira.


Bottled water is one of the sources of drinking water in many arid and semi-arid countries, including Iran. The greatest concern is the health effects of exposure to excessive nitrate concentrations in drinking water due to the development of methemoglobinemia in children. Therefore, the present study was aimed at determining the concentration of nitrate and its risk assessment in drinking water bottled in Iran. 71 different bottled water brands were identified in this study. The nitrate concentration in water samples was then measured using an Ion Chromatography No. 4110 in accordance Standard Methods for the Examination of Water and Wastewater. The hazard quotient (HQ) of nitrate was calculated using the formula based on input variables including nitrate concentration, water per capita, body weight and reference dose. The results showed that the concentration of nitrate in bottled water was in the range of 0.146–50.1 mg/L (average 10.55 mg/L) in one of which, the concentration of nitrate was higher than the WHO guideline. The mean EDI values for nitrate in different groups of infants, children, teenagers and adults were 0.0795, 0.5633, 0.3976 and 0.3186 mg/kg, respectively. The mean HQ values for nitrate in different groups of infants, children, teenagers and adults were 0.0528, 0.3737, 0.2638 and 0.2114, respectively. In general, the hazard quotient (HQ>1) for the population consuming bottled water, appropriate strategies should be considered in order to reduce the concentration of nitrate in bottled water. Specifications TableValue of the data•Bottled drinking water is one of the source of nitrate absorption into the body •The results of the study indicated that the nitrate concentration in many brands of bottled water is lower than the standard limit, therefore, the consumption of bottled water does not create a threat to the health of consumers.•Based on risk assessment and data analysis, the highest percentage of HQ>1 was associated with the age group of children, thus the sensitivity should be applied to the selection of drinking water brands for this age group.1The parameters used to calculate the nitrate risk assessment in bottled water are shown in 2In this study, 71 different brands of high-consumption bottled water in Iran were randomly selected, the nitrate concentration of all bottled water samples was measured using an Ion Chromatography No. 4110 in accordance with the method described in the book “Standard methods: For the examination water and wastewater, 22nd edn” EDI: Estimation of daily nitrate consumptionCf: Nitrate concentration in drinking waterCd: Average daily drinking water intakeBw: body weightFirst, the daily nitrate consumption in bottled water was estimated according to Eq. EDI: Estimated Daily intake (mg/kg d)RFD: Reference doseThe reference dose for nitrate is 1.6 mg/kg d [14]Then, HQ risk contribution was calculated to predict non-carcinogenic risk of exposure to nitrate using Eq.


The E-FABP concentration in tears tended to be significantly lower in patients with SS  than healthy control subjects . The E-FABP concentration in tears significantly correlated with the results of dry eye parameters. Conclusion: The E-FABP concentration in tears appears to be related to ocular surface epithelial damage and tear stability and may be a promising novel biomarker in the diagnosis of SS.Purpose: Sjögren syndrome (SS) is a chronic inflammatory autoimmune disease of the lacrimal and salivary glands. This study compared the concentrations of epidermal fatty-acid binding protein (E-FABP) in the saliva, serum, and tears of SS patients with dry eye and dry mouth, with those of healthy adults to investigate the usefulness of E-FABP as a diagnostic marker for SS. Design: Prospective, observational case series. Participants: The subjects were 11 new patients with untreated Sjogren syndrome and 12 healthy control individuals. Methods: The diagnosis of SS was in accordance with the Ministry of Health, Labour and Welfare (Japan) Diagnostic Criteria (1999). Saliva, serum, and tear specimens were collected during internal medicine, dental, and ophthalmological examinations. The ophthalmological tests included the Dry Eye-related Quality of life Score (DEQS), tear break-up time (BUT), vital staining with fluorescein (FS) and lissamine green (LG), and the Schirmer test-1. The E-FABP concentration in the tears, saliva, and serum was measured by enzyme-linked immunosorbent assay (ELISA). Main outcome measure: The E-FABP concentrations were compared between patients and controls. Results: There were significant differences between the patient and healthy control groups in all ophthalmological test results. There were no significant differences between the groups in the E-FABP concentrations in the saliva ( Sjögren syndrome (SS) is a chronic inflammatory autoimmune disease of the lacrimal and salivary glands . AccordiBecause ophthalmological testing forms the basis for the diagnosis of SS, which presents with a variety of systemic symptoms, ophthalmologists play a major role in the diagnosis of the disease. Previous research into the mechanism of onset of SS identified the appearance of multiple autoantibodies, as well as the presence of autoreactive lymphocytes infiltrating organs, and as a result it is thought that an autoimmune response is the cause of the disease. However, as the cause remains poorly understood, continuous research is being conducted for a method of early diagnosis and markers that are specific to disease activity.We have recently shown oxidative stress induced lacrimal gland and keratoconjunctival damage in patients with SS . Recentlp = 0.0812 by Mann–Whitney U test) or sex ratio (p = 0.5901 by Fisher’s exact test).This was an observational case series study of 11 newly diagnosed patients with untreated dry eye or dry mouth  who were examined at Tokyo Dental College Ichikawa General Hospital and 12 healthy control subjects . The study was approved by the institutional ethics review board of Tokyo Dental College Ichikawa General Hospital  and was registered at University Hospital Medical Information Network (UMIN) Center. Written informed consent was obtained. The subjects in the patient group underwent the same diagnostic testing protocol at the departments of ophthalmology, internal medicine, and dental and oral surgery; their diagnoses of SS were conducted in accordance with the Japanese Ministry of Health, Labour and Welfare (MLHW) Revised Diagnostic Criteria (1999) . No statp = 0.0136) (p = 0.1513) (p = 0.4799)  . The E-F 0.1513) . The E-F 0.4799) , indicatp = 0.0136)  .p = 0.001)  was significantly higher in the patient group (40.09 pts) than in the healthy control group (7.67 pts) (= 0.001) .p < 0.0001) (The results of tear break-up time (BUT), ocular surface vital staining, and the Schirmer test-1 are shown in  0.0001) .r = 0.342, p = 0.0179), FS , LG  (The results of the Spearman’s rank correlation tests between the E-FABP concentration in tears and the results of the DEQS and tear function examinations are shown in = 0.001) .Although the existing diagnostic criteria for SS are continually being revised, improvements can still be made in the areas of early diagnosis and assessment of disease activity. We believe that biomarkers that can be collected using minimally invasive techniques can be used for these purposes. In recent years, many studies have reported the use of the concentrations of proteins in serum, saliva, and tears as a comprehensive screening tool, and these have been predicted to be useful ,20,21,22RM) cells utilize exogenous free fatty acids in the beta-oxidation that occurs in mitochondria, which in turn suggests that FABP4 and FABP5 are involved in increased uptake of free fatty acids [RM cells have been reported in non-barrier tissues, such as the cranial nervous system, glandular system, lymph tissue, liver, kidneys, pancreas, and the joints, and there is a strong suspicion that they are involved in the pathological expression of chronic inflammatory diseases and autoimmune diseases [RM cells have also been reported to exist in cases of human dry eye [E-FABP, which was the focus of our study, has been found not only in epithelial cells, where they are mainly present, but also in a variety of biological tissues other than epithelial cells, such as the brain, liver, and kidneys as well as in adipocytes and in the central nervous system. E-FABP has also been reported to be related to the immune system, oxidative stress, the inflammatory response of tissues, cell differentiation and apoptosis, and malignant diseases ,31,32,33ty acids . In addidiseases ,43,44,45 dry eye . However, in the pathology of SS, the expression of multiple genes that regulate lipid deposition in the lacrimal and salivary glands as well as lipid metabolism has been observed. This is thought to be one major potential area ,49,50,51Research on the relationship between SS and FABP includes a study by Baldini et al. which found, through the use of proteome analysis of human saliva, that there was a highly significant difference in FABP5 expression in the SS patient group compared with the healthy volunteer group, and they mentioned the possibility that FABP5 can be used as a specific marker that reflects the disease activity of SS and its severity . It shouOur previous work showed an elevation of lipid peroxidative stress markers in the tears and conjunctiva of patients with SS, concluding that oxidative stress has a role in the pathogenesis of dry eyes associated with SS .In the present study, we compared the E-FABP concentrations in tears, saliva, and serum specimens. To the best of our knowledge, our study is the first to report E-FABP levels in tears. Although we did not find significant differences between the E-FABP concentrations in saliva and serum, we did find that the E-FABP concentration in the tears of patients with dry eye was lower, and in particular, that there was a significant difference between the SS and the healthy control groups. However, it should be remembered that our preliminary results represent 11 untreated patients and further clinical trials on a larger number of subjects will be needed.The possible advantages of this new biomarker include its detectability in tears by enzyme-linked immunosorbent assay (ELISA), which is a non-invasive method compared to lachrymal gland biopsies. Another advantage is the possibility of the biomarker being an early diagnostic marker, although this needs to be detected in further trials on a large numbers of subjects. The patients in this study underwent initial examination for untreated disease, where our findings may reflect the initial stages of untreated SS in contrast to the patient population in the study by Baldini et al. The current observation, revealing a significant difference of FABP in tears only, suggests that FABP and lipid metabolism in the lacrimal glands and appendages of the eye may be specialized, and also that, r because all the patients in this study were undergoing initial examination for untreated disease, the findings may reflect the initial stages of SS. The finding of correlations between the E-FABP concentration in tears and dry eye parameters suggests that the E-FABP concentration in tears can be used to assess the epithelial damage in the ocular surface, the decline in tear stability and its quality. However, there remains a need to investigate the physiological functions of FABP, and in particular, the mechanism of action of FABP on lipid metabolism and the immune system using histological findings obtained from the non-obese diabetic (NOD) and the FABP-KO mice and from further human studies. Our initial work in the NOD SS model mice shows decreased E-FABP tear levels which appear to be correlated with the level of inflammation in tears and the lacrimal gland, and the extent of lipid oxidative stress (data not shown). E-FABP is known to bind to free fatty proteins, downregulating Nos2, Cxcl10 and IL-6 genes causing an activation of NF-κB signaling pathways . TherefoSaliva, serum, and tear specimens were collected from all patients and control subjects, and the E-FABP concentrations in the specimens were measured. Ophthalmological testing consisted of the Dry Eye-related Quality of life Score (DEQS) obtained by a dry eye questionnaire sheet, tear break-up time (BUT) and the Schirmer test-1 for tear function testing, and vital staining with fluorescein (FS) and lissamine green (LG) for ocular surface testing. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Tokyo Dental College .Tears, saliva, and serum were collected from the patients in conjunction with the normal tests performed during an initial examination. Twenty microliters of tears (per subject) were collected from the lateral canthus using a glass capillary tube. Saliva was collected when the amount of saliva secretion was measured. Serum was collected using the standard blood collection procedure. The collected specimens were immediately separated by centrifugation at 3000 rpm and 4 °C for 5 min, and the E-FABP concentration in the resulting supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The specimens were stored at −80 °C until analysis.All specimens were thawed, subjected to vortex, and centrifugally separated, and finally, all precipitates were removed prior to assaying. The assays were performed using the FABP5 ELISA Kit (Human)  based on the standard sandwich ELISA technique and following the manufacturer’s instructions. The DEQS, (dry eye questionnaire sheet developed in Japan) was carried out in all subjects. The mean DEQS for patients with dry eye and control subjects has been reported to be 33.7 and 6, respectively .Standard BUT measurement was performed. First, 2 µL of a solution with 1% FS (without preservatives) were introduced into the eye with a micropipette. The subjects were asked to blink a few times. Three measurements were taken during the time at which the first corneal black spot appeared after the last complete blink. The mean of these three measurements was used as the tear film break up time.The ocular surface staining scores were assessed using FS and LG dyes. In each case, 2 µL of a solution of 1% FS and 1% LG (without preservatives) was introduced into the eye with a micropipette to stain the subject’s ocular surface. The staining scores were converted to numerals for the purpose of assessment using a 0- to 9-point scale . The Schirmer test-1, which does not involve the use of a local anesthetic, was utilized. Standardized strips of filter paper  were placed on the lateral canthus of the eye, not touching the cornea, and the subjects were instructed to keep their eyelids closed for 5 min. After 5 min, the length (mm) of the filter paper that was wetted by tears was measured. p value of less than 5% was considered as statistically significant.Fisher’s exact test was used to compare the male-to-female ratios in the subject groups , and the Mann–Whitney U test was used to compare age distributions and test results. Spearman’s rank correlation coefficient was used to calculate the correlations between test pairs. A


These results suggest that controlled breathing protocols to induce vagal activation should consider the SBR, being limited to values moderately lower than the baseline.Controlled breathing maneuver is being widely applied for cardiovascular autonomic control evaluation and cardiac vagal activation through reduction of breathing rate (BR). However, this maneuver presented contradictory results depending on the protocol and the chosen BR. These variations may be related to the individual intrinsic profile baseline sympathetic tonus, as described before by others. In this study, we evaluated the effect of controlled breathing maneuver on cardiovascular autonomic control in 26 healthy subjects allocated into two protocols: (1) controlled breathing in three different rates  and (2) controlled breathing in rates normalized by the individual spontaneous breathing rate (SBR) at 100, 80, 70, and 50%. Our results showed autonomic responses favorable to vagal modulation with the lower BR maneuvers. Nevertheless, while this activation was variable using the standard protocol, all participants of the normalized protocol demonstrated an increase of vagal modulation at 80% BR (HFnu 80 = 67.5% vs. 48.2%,  Controlled breathing maneuver is a methodology widely used to evaluate cardiovascular autonomic control  and an improvement of vagal component in faster breathing pattern (0.2 Hz)  and baseline sympathetic modulation was already demonstrated , fixed or normalized to spontaneous breathe. A standard protocol, based on the normalization of individual SBR, can enhance vagal activation and this standardization could improve the efficiency and clinical applicability of vagal modulation protocols.Twenty six healthy subjects, age between 24 and 38 years old, were enrolled into the study. Recruitment and selection were done at two clinical research centers participating in this study. The participants had not been diagnosed for hypertension, diabetes, chronic respiratory disease, autonomic dysfunctions, as well as to use tobacco, alcohol or other drugs.N = 10) or Normalized Protocol .All participants were requested to not ingest caffeine, alcohol or practice exhaustive physical activity 12 h before the protocol. The protocol was approved, according to the ethical guidelines of the 1975 Declaration of Helsinki, by the local Committee for Ethics in Research and all subjects signed an informed consent form. After fulfill inclusion criteria, the subjects were allocated in two groups: Standard Protocol  and illumination ambient. The electrocardiogram (ECG) was recorded at 300 Hz and the pulse pressure signal was continuously and noninvasively acquired at 1,000 Hz, in the supine position . The participants of the SP were asked to breath at specific breathing rates usually found in the literature. First, the subjects should stay at rest, breathing spontaneously for 10 min while ECG would be registered. Afterwards, they should breathe following the sound generated by a digital metronome, to control breathing at different rates: 0.17 Hz , 0.25 Hz  e 0.33 Hz . The blocks R10, R15 and R20 were set randomly. A 10-min register was performed for each protocol, with a 5 min interval between each other.The subjects were evaluated in the Laboratory of Clinical Investigation, Cardiology Institute, in a silent, controlled temperature (±23°C) and illumination ambient. The pulse pressure signal was continuously and noninvasively acquired at 1,000 Hz, in the supine position .In the Normalized Protocol, controlled breathing maneuvers were determined by normalization of individual SBR. First, the subjects were asked to be at rest for 10 min, while BP signals were acquired, and the SBR was observed. Afterwards, they should follow the sound generated by a digital metronome, based on individual observation, set randomly: 100, 80, 70, 60, and 50% of SBR. A 10-min register was acquired for each protocol, with a 5 min interval between each other.In the SP, the time series of heart rate (tachogram) were obtained from the interval between two consecutive peaks of RR interval (ECG records). In the NP, the time series of heart rate were generated by detection of the systolic peaks records of blood pressure. Stationary sequences of 200–300 beats, and coincident in both protocols, were selected   and normalized units (nu). In the HRV spectrum LF and HF bands represent sympathetic and parasympathetic modulation, respectively. The ratio between LF/HF components is related to the cardiac sympathovagal balance  represents the standard deviation of normal-to-normal (NN) heart beats. The pNN50 index corresponds to percentage of adjacent NN intervals that differ from each other by more than 50 ms. The RMSSD index is calculated by the root mean square of successive differences between normal heart beats (RMSSD).post-hoc test. For nonparametric data Friedman ANOVA test with repeated measures was applied. Pearson correlation was used to assess the association among variables. P < 0.05 was considered significant. Significant effects of the controlled breathing protocol on HRV parameters were accessed by a Linear Mixed Model (LMM). Estimation of fixed effects and covariance parameters was performed using the Restricted Maximum Likelihood (ReML) method. Null hypotheses were tested using Type III F-statistics and rejected if p < 0.05. Main effects of multiple-levels factors were compared using Bonferroni's adjustment of confidence intervals. Dependent variables were modeled including a fixed factor associated to the group , a random factor associated with the intercept for each subject, in order to handle the unbalanced repeated measures, and a random subject-specific effect of the respiratory maneuver. This additional random factor allows the variance of dependent variables to differ across groups. Finally, residual covariances were assumed diagonal and homogeneous.Data are shown as mean ± standard deviation. Statistical analyses were performed using one-way ANOVA followed by Tukey p = 0.022, p = 0.015, and p = 0.011, respectively), and in normalized (p = 0.041) values  blocks. There was no change in the parameters of VPA and its spectral components, as well as in the α index, related to spontaneous baroreflex sensitivity.Ten healthy volunteers, age between 24 and 32 years old, were recruited and allocated into the SP. The subjects had a mean SBR = 14.5 ± 1.7 breathes/min. Statistical analysis between the groups of men (four) and women (six) showed no significant differences in autonomic parameters. The maneuvers did not induce changes in the values of HR and SBP  and women (10) showed no significant differences in autonomic parameters. Blocks with imposing percentage of respiratory frequencies lower than the SBR generated displacement of HF spectral component toward the LF range. Statistical analysis between SP and NP groups at rest, showed no significant differences in autonomic parameters. Figure p < 0.0001), reduction of LFnu component (p < 0.0001) and of LF/HF index (p = 0.01) of all VC sessions in relation to baseline. There were no changes in the spectral BPV components for any sessions to which subjects underwent. However, when evaluated individually it was observed that the responses did not occur homogeneously in every situation.There were no changes in HR and SBP, as well as in the values of HRV and BPV components. The assessment of autonomic control demonstrated changes in the LF and HF components, related to sympathetic and cardiac vagal modulation, respectively Table , 5. FurtThe effect of the NP on the HFnu component of all ventilation blocks for each subject individually is observed in Figure The controlled ventilation maneuver lead to vagal activation and it is indicated in the treatment of hypertension, in order to reduce blood pressure and improve the clinical profile (Radaelli et al., Our results highlight for the very first time the importance of considering individual spontaneous respiratory rate and its effect on basal sympathetic tone and vagal activation. Although we observed a significant increase in vagal activation at 10 breaths/min Figure  shows thIt is known that slow breathing induces a generalized decrease of excitatory pathways in the regulation of cardiovascular and respiratory systems (Joseph et al., Recent study evaluated the effects of gradual increase and decrease of breathing rate on healthy subjects and appointed that, in the few subjects who had significant trends, sympathetic burst areas could change directly or inversely with breathing frequency (Stankovski et al., Protocols that impose very slow breathing rates can induce physiological changes in lung function in search of adaptation to prolonged ventilation. Such changes may occur acutely and are deleted when the protocol involves training with evaluation of chronic effects. Both hypoxia and hypercapnia increase ventilation and can increase sympathetic tone, HR and SBP (Somers et al., A possible increase in cardiac sympathetic component has importance both as investigative clinical character once controlled ventilation has been applied as drug intervention in several diseases such as in hypertension (Radaelli et al., In conclusion, we have shown the importance of SBR in the evaluation for the application of maneuvers controlled ventilation. We demonstrate the behavior of the autonomic control with the variation of RF, by proposing a standard baseline for each subject protocol. The use of the maneuver of controlled ventilation at 80% of SBR evoked increase in cardiac vagal component in all subjects assessed, enhancing its application in protocols designed to improve vagal activation in healthy individuals, in view of the importance of applying a method with known effect and the most reliable possible, and motivating the study of such a protocol in pathological situations.As perspectives, the standard protocol could be applied to populations of different pathologies for both assessments for chronic and acute effect study by adaptively training, seeking the construction of individual models for more efficient responses. Furthermore, the study of the acute effect of slow BR with methodological rigor, evaluating directly the sympathetic tone and respiratory function, could provide more precise information about the effect of FR at 50% of SBR on cardiovascular autonomic control.We admit as an important limitation of this study, the lack of respiratory function evaluation, such as spirometry. We understand that this evaluation could lead to more detailed analysis on the possible physiological adaptations during maneuvers. In addition, we could be more accurate in the HRV analysis if we also had ECG records.KC, MI, and NM conceived and designed this study. LD, JF, AS, DD, ET, SG, and AP collected the data and LD, and AC analyzed it. LS, AC, CS, and KC drafted the article. DD, AC, CS, NM, MI, and KC made the critical revision of the article. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ticks are important ectoparasites of horses that can affect animal welfare and vector several infectious, including zoonotic, diseases. In order to investigate the species distribution, epidemiology and seasonal dynamics of ticks infesting horses in Israel, 3267 ticks were collected from 396 horses in 24 farms across the country from July 2014 to June 2015.Hyalomma being the most prevalent genus (70% of ticks). Pasture was the most prominent risk factor for tick infestation , and is represented here by two areas with a Mediterranean climate that differ in their environmental characteristics: the Golan Heights ; and the Carmel mountain ridge . Although these two sites are less than 100 km apart, the composition of the tick populations infesting horses differed significantly between them. In GH the most abundant tick species was Hyalomma excavatum (P < 0.001), while in CMR it was Hyalomma marginatum (P < 0.001). The GH also hosted a more diverse tick fauna than the CMR, including Haemaphysalis parva  and Rhipicephalus turanicus , which were not found at the other sites. A few Rhipicephalus bursa, Hyalomma rufipes and Hyalomma turanicum were also found on horses.Ticks were found on 50% of the farms and on 25% of the horses, with The current findings can be used in epidemiological studies assessing the risk of tick-borne equine diseases in the area. Further analysis is needed to determine the specific distribution and habitat preferences of each tick species.The online version of this article (10.1186/s13071-018-3093-0) contains supplementary material, which is available to authorized users. Ticks are important ectoparasites of horses that can affect animal welfare and vector several infectious, including zoonotic, diseases. In recent years there has been a growing interest in assessing the climate niches of different tick species in order to better understand the relationship between pathogens, vectors and hosts . The surwww.gideononline.com/ebooks/country/infectious-diseases-of-israel/). Equine piroplasmosis (EP) is highly prevalent in Israel [Hyalomma, including several possible vectors of EP and CCHF. However, none of the previous studies focused on horses as a host, and all were performed more than four decades ago [Israel is situated in the Mediterranean region, with a climate ranging from Mediterranean to extreme arid Fig.  and provn Israel –9, and zn Israel –13. Seveades ago –16.Fig. The present study was designed as a large-scale survey of the horse population in Israel in order to describe equine tick infestation, identify risk factors, and explore the environmental and climatic factors associated with different tick species.Ticks were collected from horses at 24 farms selected to represent different geographical and climatic areas in Israel. Collections were performed every three months to represent all seasons: summer, July-August 2014; autumn, November-December 2014; winter, February-March 2015; and spring, May-June 2015. On each occasion 14–21 farms were visited, all available horses were inspected thoroughly, and all ticks were removed manually and stored in absolute ethanol until processing. During sampling, data were collected regarding the characteristics of each horse, the management of the farm , and the site of the body of the horse from which ticks were removed.The sex, stage and species of each tick were determined according to its morphological characteristics –19.https://ims.data.gov.il/) for each sampling period comprised average rainfall, number of rainy days, and maximum daily rainfall, as well as minimum, maximum and mean ambient temperature, average moist temperature, average dew-point temperature, average humidity and average wind speed. Data of the soil type were obtained from the Israel government agency for mapping website (http://www.govmap.gov.il/).Geographical and environmental characteristics of each farm were analyzed using the geographical information system (GIS). Data obtained using remote sensing MODIS (moderate resolution imaging spectroradiometer satellite) technology comprised altitude, average salinity of soil, two vegetation indices  and enhanced vegetation index (EVI), and minimum, maximum and mean land surface temperature during the day (LSTD) and night (LSTN) . Data obχ2 or Fisher’s exact test, as appropriate. Relative abundance of each tick species was calculated as the seasonal percentage of the species collected throughout the year. (ii) Data pertaining to each farm, the average number of total ticks and specific tick species per horse , in each season were used to determine associations between tick infestation and farm location, geographical area and housing of the horses. Associations with climatic parameters were analyzed only for farms with pasture. Associations between categorical parameters and tick load were determined by Student’s t-test or analysis of variance (ANOVA), as appropriate. Correlations with climatic quantitative parameters were calculated using Spearman’s rho (ρ). (iii) Data presenting the tick load of each of the examined horses housed on pasture were used to analyze associations between tick burden and horse age, sex, breed and color. Associations between categorical parameters and tick load were determined by Student’s t-test or ANOVA, as appropriate, and odds ratios (OR) were calculated with their 95% confidence intervals (CI). All parameters that significantly correlated with total tick load were included in a multivariable forward stepwise logistic regression model, as well as a Generalized Estimating Equation (GEE), with the farm as a random variable. Statistical significance was set at P < 0.05. The analyses were performed using SPSS 22.0® and WinPepi 11.43® statistical software.Data were analyzed in three ways: (i) Data for each individual tick were used to determine tick species and sex distribution, and its association with factors such as season and location on the horse. Associations between categorical parameters and each specific tick species were analyzed using For the study, 396 horses were sampled on 24 farms: 218 horses on 14 farms during the summer; 293 horses on 20 farms during the autumn; 317 horses on 21 farms during the winter; and 268 horses on 19 farms during the spring. Of these, 156 horses were sampled for all four seasons, 93 horses were sampled for three seasons, 47 horses were sampled for two seasons, and 100 horses were sampled only once, making a total of 1096 samples  were mares, 193 (48%) were geldings, and 12 (3%) were stallions. Most of the horses  were mixed-breed, while the rest were pure-bred, comprising 68 (17%) Quarter horses; 10 (2.5%) Paint horses; 13 (3%) Ponies; 10 (2.5%) Arabians; and several other breeds (fewer than 10 horses of each). Horses were of various colors: 54% were dark , 29% were light , and 17% were spotted .F = 8.408, P < 0.001). Seven of the 24 examined farms kept horses on pasture and were located in three geographical regions: two in the Galilee, two on the Carmel mountain ridge (CMR) and three on the Golan Heights (GH) = 8.598, P < 0.001). The number of infested horses at each farm is provided in Table A total of 3267 ticks were collected during the study period. Ticks were found on 50% 12/24) of farms and on 25% (274/1096) of horses , with Hy. excavatum being the most prevalent species in the GH  and Hy. marginatum the most prevalent in the CMR . Haemaphysalis parva and Rhipicephalus turanicus ticks were found only in the GH, with Ha. parva peaking in the autumn  and Rh. turanicus peaking in the spring . Rhipicephalus (Boophilus) annulatus was found mainly on two farms (one in the CMR and the other in the GH), in which the horses were used for herding cattle . Hyalomma turanicum was rarely found, and was almost exclusive (6/7 ticks) on one farm located in the Great Rift Valley, near the Dead Sea.Eight species of ticks were found to infest horses and the total number of ticks and species distribution varied among farms and seasons Fig. . HyalommHyalomma spp. and Ha. parva were more abundant than males in this tick’s peak season .Almost all ticks found were adults , 59% male (1940 ticks) and 41% female 1322)  annulatus, infested mares more than male horses . Spotted horses  had lower tick burdens than solid colored horses, but this burden was not different for Hy. marginatum and Rh. (B.) annulatus infestation. Horse age was not associated with tick infestation. Both breed  and sex  of the horse were found to be significant predictors of tick infestation in the multivariable GEE model.The host parameters significantly associated with tick infestation in the univariable analysis are summarized in Table Hyalomma ticks were found mainly on the rear parts of the horse: under the tail, in the inguinal area and hidden in the prepuce or between the udders ; Haemaphysalis parva ticks were found mostly on the proximal parts of the horse, including the neck, chest and trunk ; and Rh. turanicus ticks were found in both the ears and inguinal areas , but mainly on the rear  part of the horse .The distribution of ticks on different body parts of the horse varied among tick species Fig. . HyalommFarms were selected to represent the distribution of the equine population in Israel, with more farms in the north and center of the country and fewer in the south. Most of the farms were located in areas of Mediterranean climate 15 farms), and the rest in areas ranging from semi-arid to extreme arid  were located further south, at an altitude of approximately 420 m and with an average rainfall of 625 mm.The Carmel is a limestone mountain ridge located near the Mediterranean Sea. It peaks at 546 m, and features a Mediterranean climate and vegetation. The soil there is mainly terra-rossa and rendzina. One of the farms had 47 horses and was located 320 m above sea level, while the other had 18 horses and was located at an altitude of 119 m. The environmental characteristics of both areas are provided in Table P < 0.001 for all associations), while maximum, minimum, and average ambient temperature, as well as moist temperature and dew point, positively correlated together  and negatively correlated with the rain parameters (ρ < -0.69 and P < 0.002).Rainfall per season, number of rainy days, and maximum daily rain had a strong positive correlation (ρ > 0.86 and P < 0.001).The relative abundance of each tick species in the different seasons is provided in Table Ha. parva infestation moderately and negatively correlated with all ambient temperature parameters. Hyalomma excavatum infestation moderately and negatively correlated with mean and minimum NDVI and EVI, mean and minimum LSTN (land surface temperature in the night), minimum LSTD (land surface temperature in the day) and soil salinity and moderately and positively correlated with mean and maximum LSTD, altitude, and wind speed. Hyalomma marginatum infestation negatively correlated with all rain parameters, and positively correlated with all temperature parameters, except the minimum ambient temperature. Rhipicephalus (B.) annulatus demonstrated a moderate negative correlation with multi-annual average rainfall and maximum LSTD and altitude, and a moderate positive correlation with the minimum LSTN.Specifically, Rhipicephalus turanicus infestation had a moderate negative correlation with mean and minimum NDVI and EVI and soil salinity, and a moderate positive correlation with maximum EVI and mean LSTD.Rh. annulatus, which is a one-host tick, and thus all stages are expected to be found on the same host. The seasonal abundance of each species concurs with previous data on its peak season [The tick species infesting horses found in this survey represent species that are prevalent on livestock throughout the Mediterranean area –25, and k season . Tick seThe vast majority of ticks were recovered from horses on pasture, which is the most prominent risk factor for tick infestation. Naturally, the chance of tick-host encounter increases on pasture environment, and most of the other studies of equine ectoparasites have focused on horses on pasture , 28. AltHy. marginatum and the predominant species in the GH was Hy. excavatum. Although a similar number of horses was examined in both areas, the number of ticks found in the GH was 3-fold higher than that in the CMR, and the tick species found in the GH were more diverse. While all species found in the CMR were also found in the GH, Rh. turanicus and Ha. parva were found only in the GH  in the CMR area [Theileria and Babesia species. Moreover, climate change have led to the spread of Hy. marginatum to new niches in Europe [Hy. excavatum and Hy. marginatum are established vectors of EP [d Africa , and wasd Turkey , 29, 30.d horses , as wellCMR area . This tiCMR area , which mn Europe , 6, 24. rs of EP , which irs of EP , 8.Hyalomma turanicum and Hy. rufipes are closely related to Hy. marginatum [Hy. turanicum ticks were found in this study, six of which were on one farm located near the Dead Sea, in the Great Rift Valley area, which is characterized by a desert arid to extreme arid climate and low altitude (361 m below sea level). This species prefers steppe and desert climates [Hyalomma rufipes vectors CCHF and several Rickettsia and Babesia species, and has been found on migratory birds in Israel and elsewhere [Hy. rufipes ticks were found in this survey in both the CMR and the GH. This number seems to be somewhat low considering that Israel is a major path for migratory birds between Africa, Asia, and Europe, and features a suitable climate for this species, although its adults are rarely found outside the Afrotropical region.rginatum , 24. Onllsewhere . Only fiRhipicephalus. Rhipicephalus turanicus was the most prevalent species of this genus, and was found only in the GH and in the spring. It is found on a large variety of hosts and of climates [Rhipicephalus (B.) annulatus was found only on horses working and grazing with cattle. It is a one-host tick with a clear preference for cattle, but can occasionally be found on horses [About 15% of the recovered ticks were of the genus climates , 31, 34 n horses , 24, 29.n horses , but sinHa. parva, and were found only in the GH in the autumn and winter. This species was documented in Israel to infest mostly sheep and goats, but was also reported in horses and is found mostly in the cold season [Almost 15% of the ticks were d season , 29, 36.Hyalomma spp. were collected mostly from the hindquarters; Ha. parva was found mostly on the neck and chest; while Rhipicephalus spp. were found on both the hindquarters and ears. These preferences have been previously documented [Different tick species were found in specific places on the horse: cumented , 25, andHost parameters, such as sex, breed, and color that were found to be associated with tick infestation, mainly reflect the horse populations found on farms with access to pasture. Most pure-bred horses, as well as stallions, are kept in stalls and one of the farms with pasture mainly raises mixed-breed appaloosas.Hyalomma species in the two niches studied. The climate niches in the CMR and the GH differ in many of the inspected parameters, including altitude, soil, the range of both ambient and land surface temperature and rainfall. While many of these parameters correlated with the abundance of different tick species, since data were collected only at four time points, many climatic factors correlated together; and since most ticks were collected in one season, the information we can deduct from these correlations is limited. A more elaborate study or a comparison with high-resolution data from other regions is needed in order to better identify the major climatic and geographical components that influence the preferences of the different species.Several environmental factors, including rain, temperature and vegetation, have been described to affect tick activity and life-cycle, with species-specific and also sub-population preferences , 5. ThisHyalomma genus was the most prevalent tick observed to affect horses, and the two dominant species are known vectors of EP. Haemaphysalis parva and Rh. (B.) annulatus are not known as vectors of equine diseases, and their potential to transmit infectious agents between horses and other hosts should be further explored.This study is the first large-scale survey of ticks infesting horses in Israel, and provides an annual picture of horse-tick interactions. Pasture was found to be the main factor influencing exposure to ticks. Consequently, horse owners as well as veterinarians should consider a practice of parasite control, especially during the peak seasons of spring and summer. The CMR and the GH areas were shown to be distinct habitats in regard both to equine tick abundance and diversity. Additional file 1:Table S1. Ticks collected at each farm and season. For each farm the geographical coordinates are specified, along with the number of horses examined for each season, the number of ticks collected of each species, and the average number of ticks per horse. (XLSX 16 kb)


Background: Convincing evidence on Virtual Reality (VR) exposure for phobic anxiety disorders has been reported, however, the benchmark and golden standard for phobia treatment is in vivo exposure. For direct treatment comparisons, the control of confounding variables is essential. Therefore, the comparison of VR and in vivo exposure in studies applying an equivalent amount of exposure in both treatments is necessary.Methods: We conducted a systematic search of reports published until June 2019. Inclusion criteria covered the diagnosis of Specific Phobia, Social Phobia, or Agoraphobia, and a randomized-controlled design with an equivalent amount of exposure in VR and in vivo. We qualitatively reviewed participants' characteristics, materials, and the treatment procedures of all included studies. For quantitative synthesis, we calculated Hedges' g effect sizes for the treatment effects of VR exposure, in vivo exposure, and the comparison of VR to in vivo exposure in all studies and separately for studies on each diagnosis.Results: Nine studies (n = 371) were included, four on Specific Phobia, three on Social Phobia, and two on Agoraphobia. VR and in vivo exposure both showed large, significant effect sizes. The comparison of VR to in vivo exposure revealed a small, but non-significant effect size favoring in vivo (g = −0.20). Specifically, effect sizes for Specific Phobia (g = −0.15) and Agoraphobia (g = −0.01) were non-significant, only for Social Phobia we found a significant effect size favoring in vivo (g = −0.50). Except for Agoraphobia, effect sizes varied across studies from favoring VR to favoring in vivo exposure.Conclusions: We found no evidence that VR exposure is significantly less efficacious than in vivo exposure in Specific Phobia and Agoraphobia. The wide range of study specific effect sizes, especially in Social Phobia, indicates a high potential of VR, but also points to the need for a deeper investigation and empirical examination of relevant working mechanisms. In Social Phobia, a combination of VR exposure with cognitive interventions and the realization of virtual social interactions targeting central fears might be advantageous. Considering the advantages of VR exposure, its dissemination should be emphasized. Improvements in technology and procedures might even yield superior effects in the future. Phobic anxiety disorders (ICD-10 F40) are listed as a subcategory of anxiety disorders in the ICD-10  or in real live (exposure in vivo). Exposure therapy in other anxiety disorders differs slightly from the procedure in phobic anxiety disorders. In panic disorder, interoceptive exposure to internal stimuli in the form of bodily sensations like heartbeat or dizziness are mainly applied   technology represents an option with the potential to overcome such barriers. VR exposure therapy (VRET), also called exposure therapy in vivo treatments for fear of flying), and requires less organization . Furthermore, there are fewer difficulties concerning safety and insurance arrangements.By bringing virtual phobic stimuli into the therapist's office, VR exposure has many structural advantages. It is less time consuming in its application , cost-effective  , relaxation techniques with imaginal exposure, and computer aided exposure as active controls, and particularly to exposure-based interventions including imaginal and in vivo exposure as active control groups. Chesham et al.  and head tracking, and with dependent samples. Since Carl et al.  checklist and protocol provided by the PRISMA Group  no combination of the VR or in vivo exposure therapy with a specific psychopharmacological treatment. The outcomes inclusion criterion was that (7) studies examined the reduction of phobic anxiety as the primary outcome. To ensure an high internal and external validity, the inclusion criteria for study design were (8) a minimum number of 10 participants per group, (9) a randomized assignment of the participants to one of both exposure conditions, (10) an equivalent amount of exposure in both conditions and equivalency concerning additionally applied interventions alongside exposure treatment, (11) a pre and post measurement of phobic anxiety (12) with a symptom specific, standardized questionnaire or interview, and (13) sufficient statistical values (means and standard deviations in outcome parameters for each group).Only original studies published until June 2019 were included. The language inclusion criterion was (1) a report written in English or German. The population inclusion criterion was (2) an ICD or DSM diagnosis for Agoraphobia, Specific Phobia, or Social Phobia. The intervention inclusion criteria were (3) a treatment for Agoraphobia, Specific Phobia, or Social Phobia, (4) exposure therapy in virtual reality using immersive systems  and head tracking  in the experimental group, (5) exposure therapy A literature search in PubMed, PsychInfo and Web of Science was conducted in October and November 2017 and was updated in November 2018 and in June 2019. We also asked experts in the field of VR therapy to provide possible eligible studies.We searched for the keywords “virtual” and “phobia” in the PubMed, PsychInfo and Web of Science databases. Moreover, we conducted a search on the term “social anxiety.” We did not set a time limit for the period in which the studies were conducted. Depending on the different databases' search template structure, we used slightly different search strategies. In PubMed the connector ‘AND’ was used to search for “Virtual AND Phobia” as well as “Virtual AND anxiety AND social” in titles and abstracts. In PsycInfo we searched for “virtual” in title and “phobia” in abstracts, as well as for “virtual” in title and “anxiety” and “social” in abstracts. In Web of Science we searched for “virtual” in title and “phobia” in the topic, as well as for “virtual” in title and “anxiety” and “social” in the topic.n = 1,126) and other source (n = 3), obvious duplicates (n = 143) were removed first. The titles and abstracts of the remaining reports (n = 986) were then screened against the eligibility criteria. If a title or abstract provided the information that at least one eligibility criterion was not fulfilled, the record was excluded (n = 944). All remaining records with no evidence for a violation in eligibility criteria within the abstract were passed on for full-text screening (n = 42). During this process, all 13 eligibility criteria were assessed, and reports not fitting every eligibility criterion were excluded (n = 33). We contacted two authors to check on dependent samples in different records to avoid including data twice. One author provided the information that there was no overlap in the samples of two flight phobia studies  age of the participants as means, standard deviations and range, (3) distribution between the sexes of the participants, (4) medication of the participants, (5) treated disorder , (6) number of total treatment sessions in the VR and in vivo condition , (7) amount of exposure in the VR and in vivo condition, assessed in form of (7a) the number of exposure sessions and (7b) the duration of exposure sessions in minutes, (8) exposure strategy, (9) type of HMD with (9a) resolution and (9b) field of view, (10) information on movement mode and further stimulation of senses alongside the sense of sight in VR, (11) description of the VR exposure environment, (12) description of the in vivo exposure environment, (13) therapeutic interventions used for pre- and post-processing and to accompany exposure, (14) sample on which the calculation of means and standard deviations was carried out on (intent-to-treat vs. completer sample), (15) type of standardized measurement for symptom specific anxiety, and (16) means and standard deviations of the pre- and post-symptom measurement for the VR and in vivo group. If more than one symptom measurement was applied, the measurement that assessed the anxiety symptoms of the treated phobia the most specifically was collected.The following data were extracted: (1) number of participants in total and in the VR and Risk of bias in the individual studies was assessed using a tool for bias detection in randomized trials from the Cochrane Collaboration .As qualitative synthesis of all included studies, we conducted a qualitative review. A qualitative review provides a structured presentation and assessment of central characteristics of the included studies. For this purpose, we examined and summarized the participants' characteristics, diagnostic measures, study methodology, and treatment materials and procedures to provide an overview and a basis for discussions on effect sizes and future research perspectives. In the examination of the treatment materials and procedures, we particularly considered information on the exposure strategy, visual VR devices , movement mode in VR, devices for further stimulation of senses alongside the sense of sight in VR, VR and in vivo exposure therapy from the included studies on phobic anxiety disorders, we performed a quantitative meta-analysis. In this regard, we calculated pre- to post-effect sizes for VR exposure, in vivo exposure, and the comparison of VR to in vivo exposure for the individual studies and then synthesized them for all included studies. In addition, we separately calculated synthesized effect sizes of all studies on Specific Phobia, Social Phobia and Agoraphobia. We used Microsoft Word Excel 2016 as the software tool for the statistical analysis.To provide a statistical summary of the results on the efficacy of VR and VR exposure therapy and in vivo exposure therapy. As a first step, we calculated the pre- to post-effect sizes for (1) the VR exposure treatment and (2) the in vivo exposure treatment of the individual studies included in the meta-analysis. We therefore computed the standardized mean difference between pre- and post-measurement separately for the VR group and in vivo group of each study using Cohen's d for studies that use pre-post-scores according to Borenstein et al. , medium (0.5), and large (0.8)  Ellis, .Comparison of VR and in vivo exposure therapy. As a second step, we calculated the pre- to post-effect sizes for (3) the comparison of VR to in vivo exposure therapy for the individual studies included in the meta-analysis. For each study, we calculated the standardized mean difference by subtracting the pre to post change in the in vivo group from the pre to post change in the VR group, and then divided the result by the pooled pre-test standard deviation  the comparison of VR and in vivo exposure from all included studies. During the calculation of each model according to Borenstein et al.  VR exposure, (2) n et al. , an estiin vivo exposure, and for the comparison of VR to in vivo exposure therapy separately for studies on Specific Phobia, Social Phobia, and Agoraphobia. Because the estimate of the between-studies variance, which is necessary to calculate the random-effect model, has a poor precision if the number of studies is very small  the comparison of VR to in vivo exposure therapy separately, for all included studies on (1) Specific Phobia, (2) Social Phobia, and (3) Agoraphobia. During the calculation of each fixed-effect model, the effect size of each individual study was weighted by the inverse of its own variance. The weighted mean was then calculated as the sum of the weighted effect sizes, divided by the sum of the weights. In addition, we computed the variance, standard error, 95% confidence interval, Z-value and two-tailed p-value for the summary effect using equations according to Borenstein et al.  studies that do not show statistically significant effects remain unpublished . All studies included participants with the ICD or DSM diagnosis of a phobic anxiety disorder. In the two studies on flight phobia as a Specific Phobia, also patients with an Agoraphobia with flying as the main feared stimulus were included . The number of total treatment sessions includes exposure sessions as well as additional sessions, for example with interventions like psychoeducation or relapse prevention . The duration of one exposure session ranged from 20 to 90 min with a mean duration of 54.29 min (SD = 22.81) – though two studies did not give information on this treatment procedure characteristic  refers to the view or the surroundings that a human eye can see without eye movements  in particular for the VR condition but not for the in vivo condition. Anderson et al.       and thereby below a small effect (g < 0.20)  and in vivo exposure therapy (g = 1.07) showed a large, significant overall effect size, when synthesizing the nine included studies (n = 371) on phobic anxiety disorders using a random-effect model (see g = 0.68), and a large, significant effect size in Social Phobia (g = 1.17) and Agoraphobia (g = 0.99). in vivo exposure therapy also yielded a medium, significant effect size in Specific Phobia (g = 0.72), and a large, significant effect size in Social Phobia (g = 1.19) and Agoraphobia (g = 0.90)  see .in vivo exposure therapy in all nine included studies on phobic anxiety disorders (n = 371), using a Hedges' g random-effects model, we obtained a mean overall effect size estimate of Hedges' g = −0.20, SE = 0.18, p = 0.271, 95% CI   and therefore very small, and not significantly different from zero. Accordingly, we found no evidence for a significant difference in the efficacy of VR and in vivo exposure therapy over all studies on phobic anxiety disorders.For the comparison of the treatment effect of VR exposure therapy and .16] see . The negin vivo exposure therapy for the three subtypes of phobic anxiety disorders, we applied fixed-effect models (see results in n = 153) showed a non-significant result in favor of in vivo exposure that was below the level of a small effect (g < 0.20), g = −0.15, SE = 0.16, p = 0.333, 95% CI . This means that we found no significant difference in the efficacy of VR exposure and in vivo exposure in Specific Phobia. The pooled effect size of three studies on Social Phobia (n = 148) showed a medium and significant effect size favoring in vivo exposure, g = −0.50, SE = 0.17, p = 0.003, 95% CI . Accordingly, VR exposure therapy was found to be significantly less efficacious than in vivo exposure therapy in Social Phobia. The pooled effect size for two studies on Agoraphobia (n = 70) yielded a non-significant result close to zero, g = −0.01, SE = 0.23, p = 0.959, 95% CI , not favoring one of the treatment conditions. This indicates similar treatment effects of VR and in vivo exposure therapy in Agoraphobia.To separately compare the treatment effects of VR exposure and Visual inspection of a funnel plot see  showed ain vivo exposure, this meta-analysis synthesized nine randomized-controlled trials with altogether 371 participants, comparing the pre to post treatment effects of VR and in vivo exposure therapy in phobic anxiety disorders applied with an equivalent amount of exposure and with equivalent additional interventions alongside exposure in both conditions. VR and in vivo exposure both yielded large effect sizes concerning the reduction of phobic fear. For the comparison of VR and in vivo exposure therapy, we found a small, but non-significant effect size  favoring in vivo exposure over VR exposure . However, this effect size was even below the level of a small effect. An earlier meta-analysis by Powers and Emmelkamp (in vivo exposure therapy (g = 0.34). Those differences of our results to former meta-analyses comparing VR to in vivo exposure might be due to factors like the number and type of studies included, the selected outcome measures, or the data analysis strategy. Powers and Emmelkamp  . On the other hand, the variance could result from differences in the specific materials and procedures of exposure therapy in the individual studies . Furthermore, it could be due to differences in the efficacy of VR exposure for different kinds of phobic anxiety disorders. We discuss these factors in the paragraphs that follow.Considering the results for the individual studies included in our meta-analysis, the effect sizes for the pre to post treatment efficacy of VR in comparison to .53) see , with son = 4), we found a negative effect size in favor of in vivo exposure (g = −0.15)  see , which wl et al. , who parl et al. , the metin vivo exposure were found for two studies, one on fear of spiders  distinctly points to a similar efficacy of VR and in vivo exposure, as the synthesized effect size is close to zero and non-significant (g = −0.01) , which was, however, non-significant. Carl et al. (in vivo exposure) in the control group, and VR exposure to different environments as the main intervention in the experimental group  see . Here, tl et al.  synthesil et al.  additionl et al. . The twog = −0.50) in favor of in vivo exposure in the synthesis of studies comparing VR and in vivo exposure in Social Phobia (n = 3)  for Social Anxiety. Also, Carl et al.  see . This inm et al.  found anl et al.  found a l et al.  and Chesl et al. , we exclin vivo exposure over VR exposure only in Social Phobia but not in Agoraphobia and Specific Phobia, indicating that it might be more difficult to create virtual social environments for Social Phobia exposure than virtual spiders, heights and airplanes for Specific Phobia exposure, or virtual shopping malls, subways, or tunnels for Agoraphobia exposure. In addition, Social Phobia is considered to be a more complex disorder with high comorbidity, chronicity, and impairment  in vivo than in VR. In general, the creation of avatars, agents, and social interactions for VR settings is an issue which is challenging not only for psychological but also for computer science research. As an example, the degree of naturalism required for virtual agents is being intensively discussed, which seems to not be linearly related to the users' acceptance of the agent   in the study which included the youngest participants   showed a result in favor of VR exposure therapy (g = 0.27) , in one case shows a similar efficacy of VR to in vivo exposure (g = 0.02)    anxiety disorders than the target disorder of the particular study. Those were, in particular, patients with Agoraphobia in the two studies on fear of flying  and head tracking were included, leading to a relatively homogeneous application of VR stimuli. Nevertheless, the studies did not all use the same type of hardware, such as different types of HMDs see  and diffin vivo condition was required according to the eligibility criteria. Nevertheless, the exposure environment in VR and in vivo was not always equal (see in vivo condition (Rothbaum et al., Concerning the treatment procedure, a similar amount of exposure in the VR and qual see . For exaA limitation of the included data is that the original studies differed in providing either data on an intent-to-treat sample, a completer sample, or both. If available, intent-to-treat data was used for synthesis, but if not provided, data on completer samples was included see . This shin vivo exposure therapy in different phobias and allow a discussion on potential mechanisms behind differences in effect sizes for the comparison of VR and in vivo exposure therapy between different phobic anxiety disorder.A limitation concerning the synthesis of data is the use of fixed-effect models for pooling a smaller number of studies on one specific kind of phobic anxiety disorder. A fixed-effect model requires functionally identical studies (Borenstein et al., in vivo exposure.Furthermore, the applied statistical tests are only valid for testing differences between groups, but not for proving equivalency. Therefore, the non-significant results have to be interpreted cautiously and the relevance of effect sizes has to be considered. Future meta-analyses based on a larger number of trials should also draw on non-inferiority or equivalence tests (Piaggio et al., Finally, we could not conduct a statistical analysis of potential effective factors of VR exposure therapy due to the small number of available studies. As soon as more original studies have been published, this will also be an important research question for future meta-analyses.In general, this meta-analysis and the original studies were conducted by researchers in the field of VR. This is a potential source of bias, especially as no pre-registration of this systematic review and meta-analysis protocol was conducted. However, we comprehensively disclose our methods and results. Furthermore, as there are no original studies available from field-independent researchers, we provide a comprehensive and objective description of the materials and procedures of all studies included in this meta-analysis. In this regard, we want to enable the reader to capture information for an independent assessment and interpretation of the results and thereby attenuate the bias caused by non-independent researchers.in vivo exposure as the golden standard of treatment for phobias, synthesized from methodologically comparable studies with an equivalent amount of exposure in VR and in vivo, and with equivalent interventions applied alongside VR and in vivo exposure in phobic anxiety disorders, especially in Agoraphobia and Specific Phobia. For Social Phobia, the synthesized effect size points to a superiority of in vivo exposure, but the wide range of effect sizes for the individual studies also shows the high potential of VR exposure in this phobic anxiety disorder.This meta-analysis provides results on a head to head comparison of VR exposure and in vivo exposure, the individual effect sizes for the studies on Specific Phobia and Social Phobia ranged from inferiority to equivalency and even superiority of VR exposure. Studies that yielded an equivalent or even superior effect of VR exposure combined an exposure of agoraphobic situations in VR and in vivo with cognitive interventions and interoceptive exposure (Botella et al., in vivo and allowed patients to move in VR by walking around freely in a demarcated space (Emmelkamp et al., in vivo exposure in Social Phobia and applied social interactions realizing rejection experiences in the VR condition (Bouchard et al., in vivo exposure.While the individual effect sizes for the studies on Agoraphobia both indicate equivalency between VR and in vivo exposure in clinical practice (Neudeck and Einsle, Considering the results of this meta-analysis, and because there are barriers in conducting in vivo exposure therapy could be realized in future. This is because VR has the possibility to create ideal environments for exposure, for example virtual rejection experiences targeting central fears of Social Phobia patients as for example achieved in the study conducted by Bouchard et al. (As there were only a few studies with sufficiently homogenous materials and procedures published on the examined research question, this meta-analysis is still based on a small number of studies. A proportionally high level of internal validity can be expected for the results, but the generalizability should be verified in an updated meta-analysis as soon as more studies are published in the following years. In addition, future research should focus on the effective factors of VR exposure therapy and further examine mechanisms enhancing the treatment effects that may be applicable using VR exposure. Examples are the impact of cognitive strategies (see the study conducted by Bouchard et al., d et al. , and alsd et al. . The creAM and TW developed the idea of the research question. FK performed the systematic literature search to identify published studies, and conducted the title, abstract and full-text screening. TW also conducted abstract and full-text screening, selecting eligible studies. AM was consulted to discuss disagreements during the screening process. TW and FK performed the data extraction. TW conducted the statistical analyses with contributions from FK and AM. TW and AM interpreted the findings and wrote the manuscript draft.AM is stakeholder of a commercial company that develops virtual environment research systems. 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.