Background and objectiveThe coronavirus disease 2019 (COVID-19) pandemic has highlighted the shortcomings worldwide in terms of preparedness protocols related to epidemics. A key area of research that is evidently overlooked across the globe is the mental health of family caregivers taking care of patients with COVID-19. In light of this, this study aimed to engage in a comparative analysis between the two worst affected countries, India and the United States of America (USA), which differ considerably in their demography, socio-epidemiological factors, and health system efficiency.MethodsA cross-sectional study was conducted among 1,250 family caregivers of patients with COVID-19 in India and the USA to assess their stress, anxiety, and sleep disturbance levels using the 10-item Perceived Stress Scale (PSS-10), the 7-item Generalized Anxiety Disorder (GAD-7) scale, and the Pittsburgh Sleep Quality Index (PSQI), respectively. Psychological assessment questionnaires were administered through online mode, which gathered demographic information and responses on several self-reporting scales. The main outcome measures were self-reported ratings on PSS, GAD-7 scale, and PSQI.ResultsWe found that 75.4% of the family members of COVID-19 patients suffered from mental health issues. The scores of all three scales were higher in caregivers from the USA than in India, more evident and pronounced in caregivers of hospitalized patients. The test scores were statistically significant (p<0.05) indicating a negative impact of having a dependent member in the family, being married, being of younger age, and having a longer duration of COVID-19 infection. Vaccines were found to have a life-enhancing effect.ConclusionOur findings highlight that the mental health of family caregivers is an ignored aspect and must be addressed. We recommend the implementation of well-researched and appropriate legislation, treatment programs, and health policies that involve not only the patients but also their families. The coronavirus disease 2019 (COVID-19) emerged as a pandemic in 2019 and was declared a public health emergency of international concern (PHEIC) by the WHO on January 30, 2020 .In addition to its economic impact, COVID-19 has also affected people's short- and long-term health, including their mental health . The psyUnderstandably, COVID-19 infection in any family member results in psychological trauma due to social isolation, increased economic stress, and stigmatization by society. Therefore, it is important to acknowledge and understand the role that caregivers have played in this pandemic and its subsequent effects on their mental health. Studies have shown that informal caregivers of patients who were discharged from hospitals can be classified into two categories: (1) caregivers who are motivated by altruism and have a positive impact on the patient and their health and (2) those who provide care based on familial norms. Both types of caregivers, especially those directly involved in the care of patients, are pushed toward physical and mental burnout and hence experience depression, increased anxiety, and deteriorating physical health. Unsatisfactory supportive care and lack of health information are the major contributors to anxiety, stress, and sleep disturbances among caregivers .The economic and social impact of COVID-19 has been the greatest on India and the USA. The USA, being a developed country, has a stronger healthcare infrastructure with a doctor-to-population ratio of 2.6/1,000, whereas India, an emerging and developing country (EDC) with a doctor-to-population ratio of 0.9/1000, has a healthcare system that is encumbered . Also, tAlthough recent studies have looked into the impact of the COVID-19 pandemic on mental health, they have largely focused on the suffering of the patients and very few have focused on the family members and caregivers of the patients. To date, there has been no study highlighting the prevalence of symptoms of stress, anxiety, and disturbances in sleep quality among the family members of patients with COVID-19 while also comparing the variations based on demographic, environmental, physical, social, and economic factors. Also, there have been no attempts to compare the aforementioned deleterious effects on a developed (USA) versus an emerging and developing nation (India). Our study aims to address this gap in the literature by exploring the effects of the pandemic on the mental health of family caregivers of COVID-19 patients in India and the USA.Study design and settingA community-based cross-sectional study was conducted to estimate the prevalence of stress, anxiety, and sleep disturbances among family members of patients with COVID-19. The study was conducted among family members of patients who suffered from COVID-19 in India and the USA, the two countries worst affected by the pandemic. We employed purposive sampling to distribute the questionnaire over WhatsApp and email to all the participants, covering rural as well as urban areas.Participant selectionParticipation in the study was voluntary and anonymous. Participants were selected from both countries as per well-defined inclusion and exclusion criteria.Inclusion and exclusion criteriaWe included family members or informal caregivers who were either directly or indirectly involved in the care of the patient. We excluded the respondents who had a history of pre-existing mental illness, as these illnesses can act as confounders that can alter self-reports and misrepresent the effect of COVID on the mental health of caregivers. Data collection and study instrumentsPsychological assessment questionnaires were administered to all the subjects. The study objectives and the risks and benefits of participation were clearly explained, and detailed informed consent was taken online before providing the questionnaire. The main outcome measures were self-reported ratings on stress, anxiety, and sleep quality. Data were collected from 1,272 participants from India and the USA, with data collection stopped upon reaching a sample of 625 from each country. Among the total participants who filled out the form, 98.3%  gave consent and filled the form further while 1.7% (n=22) denied it and landed on the "thank you" page directly. The questionnaire, administered via Google Form, had 12 sections including consent, basic demographic details, possible confounders, means of handling stress, and psychosocial assessment scales, i.e., scales for stress, anxiety, and sleep. The language of the survey was confined to English, to ensure uniformity and the validity of the scales used.The psychological responses from family caregivers were assessed using three established and globally validated self-reporting psychological instruments/scales:The 10-item Perceived Stress Scale (PSS-10)It is a 10-item self-reported scale used to measure global perceived stress during the preceding month. Scores range from 0 to 40 with higher scores indicating a higher risk for future distress .The 7-item Generalized Anxiety Disorder (GAD-7) scale It is a 7-item self-administered scale used as a screening measure of anxiety. The scores of 5, 10, and 15 are taken as indicators of mild, moderate, and severe anxiety, respectively .The Pittsburgh Sleep Quality Index (PSQI)It determines sleep quality in the previous month, using seven assessment indicators. Scores per item range from 0 to 3 points, and total scores range from 0 to 21 points. A total PSQI score <5 indicates good sleep quality, while a score ≥5 indicates poor sleep quality .Study size and sampling methodologyA purposive sampling model was used for data collection with the questionnaire distributed online to people in various parts of the two countries, allowing for the inclusion of a comprehensive representative sample. The sample size of approximately 1,250 (625 from each country) was calculated using the G*Power statistical analysis software using the following parameters: effect size: 0.2, alpha error probability: 0.03, power (1-beta): 0.95, and allocation ratio N2/N1: 1.Statistical analysisThe collected data were analyzed primarily based on scores of scales, and individual responses to questions of self-reporting scales were also analyzed qualitatively to gain a deeper insight. Quantitative variables were analyzed using SPSS Statistics  version 27.0 for Windows. All comparative analyses were done using standard statistical tools by applying parametric and nonparametric tests comparing all variables and confounders. The data were first qualitatively assessed using demographic analysis and normality assumptions (Shapiro-Wilk test); qualitative variables were analyzed using diagrammatic charts and quantitative data were then analyzed using the Student's t-test and the analysis of variance (ANOVA) test contrasting the scores of the implicated scales.Ethical approval and consentThe study was approved by the Ethics Committee of the Faculty of Medicine - Alexandria University with IRB no. 00012098 and FWA no. 00018699, and all participants provided online written informed consent.The study involved 1,250 participants, 625 from each country. Demographic information was collected to account for its impact on stress, anxiety, and disturbances in sleep and to eliminate any confounding variables such as gender, age, income, and place of residence that can impact a person’s health. Of the 1,250 participants, 55% were male (689) and 44.9% were female (561). Most participants were young adults, aged 18-29 years (53.4%) and were either graduates (49.2%) or undergraduates (24.2%). Economically, most people reported belonging to the middle class (77.7%), with fewer in the lower (10.1%) and upper classes (12.2%). Most participants lived in urban areas (79%), with fewer in rural (11.7%) and suburban (9.3%) areas. This study found that 75.4% of caregivers of COVID-19 patients suffered from some form of mental health distress.StressModerate to high stress was found in 95.68% of the study population from the USA and 81.60% of the Indian subjects Figure .AnxietyA higher prevalence of moderate (34.08%) and severe anxiety 19.68%) levels were observed among respondents in the USA, whereas mild anxiety levels (36.96%) were more prevalent in India  with 86.72% reporting poor sleep quality while only 48.8% reported poor sleep quality in India  and sleep disturbances (p=0.001) with vaccinated individuals scoring lower on these self-reporting scales. Similarly, the levels of anxiety (p=0.007) and sleep disturbances (p<0.001) were found to be lower in groups with vaccinated patients Table .The main concerns expressed by family members were fear of infection in other family members (58.5%), progression of disease in the patient (56%), fear of the death of the patient (53.5%), and the availability of health services (48.7%). The most common concern among family members of the Indian population was fear of infection to other family members (62.24%) while that from the USA was fear of death of patients (60.32%) Figure .In these challenging times, people have adopted various methods to cope with anxiety, stress, and sleep disturbances. About 20% of the respondents have chosen conversation with family members as their main coping mechanism Figure . Other iTo summarize the results, we found that men were more prone to anxiety and sleep disturbances; however, women were more affected by stress. The US subjects exhibited much greater levels of anxiety, stress, and sleep disturbances. Respondents who were directly involved in the care of patients had considerably higher levels of anxiety, sleep disturbances, and stress. Similarly, fatalities in the neighborhood or in the family were associated with higher levels of anxiety. Anxiety, sleep disturbances, and stress levels were higher in sole earners, small families, families with dependent members, and patients with a history of chronic disease Table .Various factors had an effect on GAD-7, PSS, and PSQI scores among families of COVID-19 patients. These included being married, younger age, and prolonged duration of infection, which were among the leading factors for anxiety, stress, and sleep disturbances. Profound stress and sleep disturbances were found among respondents from lower-income groups. News sources and social media were shown to have a negative impact on mental health leading to anxiety, sleep disturbances, and stress. The vaccination status of respondents also had a statistically significant impact on anxiety and sleep disturbance levels, with vaccinated people scoring lower on these self-reporting scales. Anxiety and sleep disruptions were also shown to be lower in groups caring for vaccinated patients Table .COVID-19 has highlighted several gaps in the healthcare systems globally, including poor emergency response protocols. Several studies have shown that pandemics result in adverse psychological outcomes among the public ,21. ThisOur study found that among family members of COVID-19 patients, 44% had moderate to severe anxiety, 88.6% had moderate to severe stress, and around 67% of respondents had poor sleep quality; these findings are in line with the study by Khubchandani et al., whose findings revealed a general prevalence of anxiety (42%) and psychological distress (39%) during the pandemic. The study also indicated that the rate of serious mental health issues such as anxiety and stress has more than doubled during the pandemic .India and the USAPsychological health varies across regions and countries, especially during a massive disease outbreak owing to differences in outbreak severity, the strength of the national economy, government preparedness, and availability and accessibility of medical facilities and health information. Furthermore, the evolving nature of the outbreak and the legislative response in each region also affect the psychological state of the people, especially those involved in patient care . A betteBeing a family caregiver during COVID-19COVID-19, in general, has been seen to have adverse effects on the mental health of the population ,7, and tFactors affecting mental healthWe also explored the possible factors affecting mental health and sleep. Higher stress, anxiety, and sleep disturbances were directly influenced by the patient's health condition as suggested by the presence of a comorbid chronic illness, longer duration of COVID-19 illness, and disease severity . This is because the patient being hospitalized means worsening disease severity, which causes greater trauma among the caregivers. This finding aligns with the study conducted by Paul et al., which concluded that the relatives (of patients in ICU) experienced negative physical and psychological impacts due to unidentified needs and this impact might transgress far beyond the acute phase of the illness . MoreoveHigher stress, anxiety, and sleep disturbances were also related to some external factors; anxiety and sleep quality were worse in married patients while stress and poor sleep quality were more prevalent in economically backward groups. Other factors included deaths in the neighborhood, news and media, family education, and socioeconomic status Table . These fLimitations The study's limitations essentially pertain to the self-reported data collected via electronic mode; an offline mode of data collection through clinician-administered scales can often show differences in results. Another limitation relates to its cross-sectional study design, which lacks the ability to demonstrate a temporal association between exposure and outcome, which could be better portrayed by having a matched control group as in a case-control study design. Furthermore, the questionnaire was administered only in the English language, which might have limited the number and type of participants. Lastly, purposive sampling was used in the study due to the lack of access to national data on COVID-19 patients and their family members.Future prospectsWe hope to pave the way for future researchers to further explore the effect of pandemics on the mental health of caregivers. More studies exploring the effects of cultural, ethnic, regional, economic, and environmental differences need to be conducted. Governments can initiate need-based interventions for vulnerable groups, and provide telepsychiatry consultations and emergency assistance for psychological and behavioral issues. Hopefully, after the identification of these needs, various solutions will be crafted, especially in the domain of public health legislation and policies to address mental health.The importance of the mental health of family members of COVID-19 patients cannot be overstated. This research highlights the magnitude of the problem and provides a comparative analysis of the impact of the pandemic between a developed country and an emerging and developing nation. Although measures are being put in place to assess the psychological status and prevent sleep and mental disorders, more work needs to be done to ensure adequate response to increasing cases of anxiety, stress, and poor sleep quality. The indicative survey revealed the association of the issue with factors such as the severity of the disease, isolation, gender, healthcare systems or lack thereof, and the role of mass media, all of which critically influence the psychological and physiological well-being of these family members, and could be a compass for the development of intervention programs to resolve such issues. These issues can also be effectively mitigated by proper health promotion and public health education. Emphasis should be placed on improving healthcare systems and equipping hospitals with sufficient resources to manage future pandemics. Well-researched and appropriate legislation, treatment programs, and health policies should be implemented to integrate healthcare services and introduce psychological care at the earliest and at all levels.


Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses , a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of “flavescence dorée” phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of “flavescence dorée”, makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years. Vitis vinifera L.) is one of the most widespread woody crops with significant economic importance, much work has been carried out on various pathogens, including viral agents. Eighty-six viruses have been confirmed to infect grapevine by 2020, with varying adverse effects ranging from unknown to very important and associated with substantial economic losses [Collections of plant viruses and virus-like agents, in vivo in their host plants, are a crucial prerequisite for the study of multiple aspects of the pathogen, especially for the development of robust detection methods and for epidemiological and ecological studies. Plant collections occupy a special position when dealing with pathogens that cannot be cultured on artificial medium, such as viruses and viroids. Since grapevine , 368 grapevine accessions were selected based on virus significance, infection status (single or mixed), site of origin, and cultivar; later, their rooted cuttings were included in the grapevine virus collection .Most of the grapevines (35%) were from the Kaštela region, as this region and its autochthonous cultivars were found to be highly infected with various combinations of viruses. Overall, ArMV, GFLV, GFkV, GLRaV-1, 2, 3, GVA, and GVB were present in 12 (3.3%), 61 (16.6%), 87 (23.6%), 131 (35.6%), 29 (7.9%), 312 (84.8%), 203 (55.2%), and 22 (5.9%) grapevine accessions, respectively, which roughly reflects the sanitary situation found in the original sites/vineyards from which the cuttings were collected. According to the number of viruses in each accession, eight (2.2%) plants were free of viruses tested, 85 (23.1%) had a single virus infection, 114 (30.9%) were infected with two viruses, 109 (29.6%) with three, 42 (11.4%) with four, eight (2.2%) with five, while one (0.3%) grapevine accession was infected with six viruses. A detailed overview of all grapevine accessions included in the collection and their sanitary status as determined by ELISA can be found in As part of the information gap regarding GRSPaV infection, one of the most widespread grapevine viruses, screening of 26 accessions from the collection was performed by Western blot, RT-PCR, cloning, and Sanger sequencing. For this purpose, plants from different locations in the coastal region were selected, and a comparison of detection with two primary antibodies (As7-276 and As2003) in the Western blot and two primer sets (RSP13/RSP14 targeting the helicase gene and 48V/49C targeting the coat protein gene) for detection by RT-PCR was performed. Western blot with antibodies As7-276 developed a clear signal in 20 samples, with two other samples showing a weak signal , while As2003 clearly detected GRSPaV in 21 samples, with another sample showing a weak signal (Vlaska 137). In the same three samples , the virus was not detected by either antibody, while Babic 113 gave a weak signal with As7-276 and no signal with As2003. Including the weak positives, the detection rate for As7-276 and As2003 was 23/26 (88.5%) and 22/26 (84.6%), respectively.The primer pair RSP13/RSP14 used in RT-PCR confirmed the presence of the virus in all accessions tested, while the primer pair 48V/49C failed to detect the virus in Plavac mali 234, giving a diagnostic sensitivity of 96.2%. A detailed overview of the detection efficiencies using different antibodies in Western blot and different primer sets in RT-PCR can be found in Cloning and Sanger sequencing of the 299 nts (99 aa) long segment of the helicase gene and the 289 nts (96 aa) long segment of the coat protein gene was performed. Excluding the primer sequences, sequence analysis of the helicase segment revealed pairwise identity in the range of 87.62–98.99% and 94.94–100% at the nt and aa levels, respectively. The same analysis of the coat protein segment revealed pairwise identity in the range of 94.11–100% and 95.83–100% at the nt and aa levels, respectively .Comparison of Croatian GRSPaV isolates with sequences available in GenBank showed high similarity with other isolates from other parts of the world, especially with those from South Africa, in both the replicase and the coat protein regions. A detailed overview of GRSPaV isolates from other countries most similar to those found in Croatia can be found in Phylogenetic analysis showed the Tamura-3 parameter model with invariant sites (I) and Kimura-2 parameter with gamma distribution as the best models for nucleotide substitution for the partial helicase and the partial CP region, respectively. The phylogenetic tree of helicase poisoned isolate Babic 015 within the GRSPaV-BS group, isolate Nincusa 108 within the GRSPaV-SG1 group, and Plavac mali 085 and 234 within the GRSPaV-1 group, whereas the other isolates formed a separate group. According to the CP phylogenetic tree, isolates Marastina 058, Nincusa 108, and Plavac mali 128 clustered within the GRSPaV-SG1 group, Posip 076 and Plavac mali 085 clustered within the GRSPaV-1 group, while three other isolates  clustered separately .To gain better insight into the composition of viruses and viroids in grapevines from the collection, 22 accessions were selected for screening by HTS on the Illumina NextSeq 500 platform. For selected grapevines, results from HTS confirmed ELISA results obtained ca 10 years ago and detected the presence of 10 additional viruses not included in the original screening: GVE, GVF, GVG, GVT, GLRaV-4 , GRSPaV, GDefV, GRLDaV, GRVFV, and GSyV-1. On the other hand, there were differences in the presence of some viruses that were not determined by ELISA, but were detected by HTS, in particular GLRaV-1 , GVA , GFLV , and GFkV . HTS analysis confirmed the high incidence of GRSPaV infection in the collection (81.8% of samples screened by HTS), which was already detected in other grapevines by Western blot and RT-PCR. In addition, the following viroids were identified in HTS-screened samples: hop stunt viroid (HSVd) and grapevine yellow speckle viroids 1 and 2 (GYSVd-1 and GYSV-2) .After the first appearance of “flavescence dorée” (FD)-like symptoms in the collection during the 2020 growing season, followed by rapid spread, symptomatic plants were tested for the presence of FD in September 2020 and 2021 by LAMP. The presence of FD was confirmed in six (40%) of the fifteen samples tested. Two grapevine accessions were tested in two consecutive growing seasons, one resulting as negative (Vlaska 143) and the other positive  in both seasons. Grapevine accessions Grk 003 and Mladenka 148 showed FD-like symptoms in 2020, but did not show symptoms in 2021, while Plavac mali 272, Vlaska 131, and 150 were lost during the winter 2020/21 .Following the practice of other countries, where viticulture is an important economic sector, a collection of grapevine viruses was established in 2009 at the experimental field of University of Zagreb Faculty of Agriculture. Initially, the collection served as a source of positive and negative controls in diagnostic tests for economically important viruses, but over the time it has also been used for other experiments and investigations, such as studies on genetic diversity, vector transmission, and alternative hosts. Consequently, this grapevine collection has allowed cooperation between different scientific institutions  in the form of material exchange that can greatly improve current knowledge and diversity of grapevine pathogens.After the establishment of the Croatian grapevine virus collection and ELISA screening for eight viruses, further studies showed a high infection rate with GRSPaV, a virus with known global distribution . The priThe Sanger sequencing results revealed the substantial genetic diversity of the Croatian GRSPaV. Interestingly, the isolates from cv. Plavac mali  were found to be identical in the helicase region, and 085 and 128 were found to be identical in the partial coat protein, although each came from a different region . Compared with the other sequences in GenBank, the Croatian GRSPaV isolates showed the greatest similarity in the helicase region with isolates from South Africa, Italy, Canada, Slovakia, and France. Interestingly, in the case of Plavac mali 128, the most similar isolate was from Italy, derived from the cultivar Pagadebit, which is considered synonymous with Plavac mali. In the coat protein region, Croatian GRSPaV isolates had slightly lower diversity, comparable to isolates from South Africa, Tunisia, Brazil, China, Italy, and France. The high genetic diversity of some isolates was also confirmed by phylogenetic analyses of the part of the helicase gene, which placed five Croatian isolates  in a separate group, while three isolates  formed a separate group according to the partial coat protein region. This is consistent with the results of other studies confirming the high diversity also found in isolates of this virus from different parts of the world ,11,12.Xiphinema index cannot be excluded, especially in the plants Babica 122, Marastina 016, Posip 123, and Vugava 036, since their neighboring plants were infected with GFLV  and, as the grapevine grows on its own roots, phylloxera. Evidence of the very demanding care is that in the 13 years after establishment, 207 out of 368 (56%) grapevine accessions were lost for various reasons . In ordeIn 2007 and 2008, several extensive studies focused on clonal and sanitary selection of autochthonous grape varieties were conducted in the Croatian coastal viticultural region. In these field surveys grapevines were tested for eight different viruses  using ELISA kits from Agritest . The sample source was 0.1 g of phloem scrapings taken from well-wooded cuttings collected during dormancy, which were pulverized in a sterile mortar with a pestle and the addition of liquid nitrogen. The pulverized tissue thus obtained was diluted with extraction buffer and all other ELISA steps were performed according to the manufacturer’s instructions. The OD values for each sample were determined using the EL 800 spectrophotometer  at a wavelength of 405 nm two hours after addition of the substrate p-nitrophenyl phosphate . Samples/grapevines with an OD value that were at least two times the average value of the negative controls were considered positive. Based on the ELISA results, 368 grapevines were selected according to their sanitary status , place of origin, and cultivar, and their cuttings were placed in plastic containers filled with planting medium/peat to be rooted under the greenhouse conditions. A year later, the potted accessions were moved to an open area, and in 2009, the grapevines were planted at the current site, part of the experimental field located in the vicinity of the campus of University of Zagreb Faculty of Agriculture.To cover a broader region and potential wider genetic variability, this part of the study was conducted on a total of 26 grapevines/plants from 26 different sites/vineyards comprising 12 autochthonous cultivars  from the collection. Mentioned grapevines were subjected to grapevine rupestris stem pitting-associated virus (GRSPaV) analysis by Western blot and RT-PCR followed by cloning and sequencing. In both tests (Western blot and RT-PCR), lyophilized tissue of cv. Refošk infected with GRSPaV  was used as positive control, while tissue from cv. Babic free from mentioned virus was used as a negative control.Western blot was performed according to the standard protocol: proteins from 100 mg of young leaf petioles collected during the beginning of vegetation (March) were isolated according to the previously described method . BrieflyIn the case of RT-PCR, total RNA was extracted from 0.1 g of cortical scrapings taken from well-wooded canes using the RNeasy Plant Mini Kit . The RNA was used for one-step RT-PCR detection with two sets of primers: RSP13/RSP14, which amplify 339 bp fragment of the helicase domain , and 48VTotal nucleic acid (TNA) extracts were prepared from 22 grapevine accessions as previously described . TNA wasScreening for the presence of FD phytoplasma was completed in 2020 and 2021. Samples were selected according to the symptoms typical for grapevine yellows: yellowing/reddening discoloration and downward rolling of laminae. Extraction of DNA was performed from leaf petioles and main veins of symptomatic leaves using the Plant Material Lysis Kit  from seven grapevine accessions in 2020 and from 10 accessions in 2021. Detection of FD was performed as previously described  using co


Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, and Verrucomicrobiota. The richness and diversity of gut microbiota increased from 6 to 24 months old . Beta diversity revealed 61 gut microbiota genera that were significantly different in relative abundance among the three age groups. Among the 61 gut microbiota genera, 30 distinct trajectory curves (DTCs) were generated by group-based trajectory models after log2 transformation of their relative abundance. Subsequently, Spearman correlation analysis revealed that only five gut microbiota DTC were correlated with the BMI_Z DTC. Therefore, we further examined the association between the five gut microbiota genera DTC and BMI_Z DTC using generalized estimation equation models. The results revealed a significant association between the DTC groups of Parabacteroides and that of BMI_Z . The results of this study validated the hypothesis that the richness and diversity of gut microbiota developed with age in twins. Moreover, participants with a higher DTC of log2-transformed Parabacteroides had a higher BMI_Z DTC during the first 2 years of life. Further studies are needed to confirm the association between Parabacteroides and BMI_Z in other populations.The gut microbiota undergoes rapid and vital changes to microbial community structure and the microbial-immune crosstalk during the first 3 years of life, which is thought to be involved in the pathobiology of later-life disease. Compared to single-born children, little is known about the gut microbiota of twins in early childhood. Based on the Wuhan Twin Birth Cohort study, 344 stool samples from 204 twin families were analyzed to investigate the difference in gut microbiota composition at 6, 12, and 24 months of age. Furthermore, this study evaluated the association between gut microbiota development curves and body mass index z-score (BMI_Z) curves at 6, 12, and 24 months of age. The predominant microbiota phyla identified in twins were  The human gut hosts a dynamic microbial ecosystem, namely, the gut microbiota, which has an extensive metabolic repertoire and is increasingly recognized to provide essential benefits to its host. These include dietary energy extraction, development and maintenance of the immune system, and protection against pathogens . During Staphylococcus, followed by Enterobacteriaceae, Enterococcus, Lactobacillus, and Bifidobacterium. These are gradually replaced by obligate anaerobic bacteria, such as the Firmicutes, Clostridiaceae, and Lachnospiraceae  , 344 stoTwins were generated from the WTBC study born between October 2016 and December 2020, conducted in Wuhan Children’s Hospital , Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The including criteria of twins in the study were: (1) born at the study hospital without congenital disease; (2) had at least two follow-ups with stool samples or anthropometric data collected at 6, 12, and 24 months of age; (3) no antibiotic used during the first year of life. This study was approved by the Ethics Committee of Wuhan Children’s Hospital  (IRB number: WHFE2016050).Face-to-face interviews were conducted within 30 days of the twins reaching 6 months of age and within 90 days of the twins reaching 12 and 24 months of age. At each follow-up, fresh stool samples were collected in a sterile plastic container 1 or 2 days before the interview by caretakers and were immediately frozen at –20°C at home and transferred into the –80°C freezer within 1 h after arriving at the study hospital. During the interview, the twins’ weight and height were measured by trained nurses, with weight measured to the nearest 50 g and height measured to the nearest 0.1 cm. Both weight and height were measured in duplicate, and the mean of the values was used in the analyses. Information about the maternal educational level, delivery age, pre-pregnant BMI, delivery mode, gestational age at delivery, twins’ sex, birth weight and height, feeding pattern at 1 and 6 months old, and antibiotics intake during the first year of life was recorded.2), and BMI z-scores were calculated by using the age- and sex-specific BMI reference from WHO Child Growth Standards (2006)  divided by the square of the height (ms (2006) . The mat1) (2) (3 was used based on the MUSCLE classifier (Version 3.8.314) algorithm to annotate taxonomic information  was used to extract DNA from stool samples, and pooled DNA ≥ 200 μg was used for Illumina sequencing. The primer pair 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACHVGGGTWTCTAAT) were used to amplify the V3–V4 region of the 16S rRNA gene. TruSeq DNA PCR-Free Sample Preparation Kit  was adopted to generate the sequencing libraries, and the library quantity was determined by Qubit 2.0 Fluorometer . Then, the libraries were sent for sequencing by Illumina NovaSeq6000  at Novogene Co. Ltd (Beijing China). The raw data generated from the Illumina NovaSeq6000 platform were paired-end reads. To merge reads of the same DNA fragment, FLASH (Version 1.2.7) was used to identify the splicing sequences . Based o1) . To cont1) . Sequenc1) (2) . Sequencormation .For epidemiological data, the descriptive characteristics of the participants were expressed as mean (SD) for normally distributed variables, and as frequency (percentage) for categorical variables. Variance analysis (ANOVA) was used to determine the difference of normally distributed variables among the three age groups, and the chi-square test was used to evaluate the significance of categorical variables.p < 0.05; ANOVA) was visualized using a heatmap.To study the phylogenetic relationship of different OTUs and the predominant species in different age groups, a Venn plot was used to identify the difference in OTUs, and a bar plot was used to illustrate the top 10 or 20 microbiota in phylum or genus taxa, respectively. Furthermore, the changes in predominant microbiota among each age group were illustrated in a Sankey diagram, which was conducted using the MUSCLE software . Hierarchical clustering of the 35 most abundant genera that were significantly different among the three age groups in relative abundance  and Shannon diversity index  with QIIME (version 1.7.0). As non-normal distribution data, the Chao1 index was log2-transformed to obtain a normal distribution. Linear mixed-effect (LME) models were used to make inferences for the age-specific longitudinal alpha-diversity data in SAS 9.4. In LME models, the twins’ family was set as the random effect, while the twins’ zygosity, sex, birth weight, gestational age, feeding pattern at 1 month old, maternal delivery age, and educational level were the controlling variables.p < 0.05 following false discovery rate (FDR) correction.Beta-diversity analysis was measured based on Bray–Curtis distance matrices for individuals sampled at each age group using the Kruskal–Wallis test. Principal coordinates analysis (PCoA) was performed to discriminate principal coordinates and visualize signatures among the three age groups. Similarity percentage analysis  was adopted to further identify the gut microbiota at the genus level that showed significant differences in relative abundance among the three age groups with a contribution rate for difference > 0.001 with simple function in vegan package, R software. Linear discriminant analysis Effect Size (LEfSe) was used with R software to identify the predominant microbiota genera at different ages, considering biological consistency and effect relevance. Significance was accepted as Based on the results from SIMPER and LEfSe analyses, gut microbiota genera that were significantly different among the three age groups were included in the association analysis with the twins’ body mass index z-score. Subsequently, the relative abundance of each candidate microbiota genera at each time point were log2-transformed to obtain a normal distribution. Then, group-based trajectory models (GBTMs)  were usep < 0.05 in our analyses.The statistical significance was defaulted as A total of 344 stool samples were collected from 204 twins in 129 families (75 families with both twins and 54 families with only one twin included), among which 149, 130, and 65 stool samples were collected at 6, 12, and 24 months of age, respectively. The basic characteristics are summarized in Firmicutes, Proteobacteria, Actinobacteriota, and Bacteroidota were the most abundant phyla in relative abundance, while Bifidobacterium, Escherichia-Shigella, Bacteroides, and Veillonella were among the most abundant genera in relative abundance  and the Shannon diversity index (diversity measure) increased from 6 to 24 months old . Considep = 0.006). Results from the Kruskal–Wallis test showed significant differences in beta-diversity dissimilarity indices among the three age groups  with age in the studied twins. Significant differences in beta diversity were also detected among the three age groups, as expected. In association analyses, an association between the high log2-transformed Bifidobacterium  in National Genomics Data Center (Nucleic Acids Research 2022), the China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA007379) that are publicly accessible at This study was approved by the Ethics Committee of Wuhan Children’s Hospital  (IRB number: WHFE2016050). Written informed consent to participate in this study was provided by the participants’ parents.HM, RL, FX, HZ, AP, and YT coordinated the Wuhan Twin Birth Cohort study and collected the data. HM, SY, RL, AZ, and HX designed the study and obtained funding. HM and YZ analyzed the data. HM, YZ, JZ, AZ, and HX interpreted the results and wrote and edited the manuscript. All authors critically reviewed the manuscript and approved the final version for submission.


To explore the correlation between postpartum hepatitis and changes of plasmacytoid dendritic cells’ (pDC) function and frequency in hepatitis B e antigen (HBeAg)-positive pregnant women with chronic hepatitis B virus (HBV) infection.Pregnant women with chronic HBV infection receiving antiviral treatment (treated group) or not receiving antiviral treatment (untreated group) were enrolled and demographic information was collected before delivery. Clinical biochemical, virological serology, pDC frequency and functional molecular expression were tested before delivery and at 6, 12, 24 weeks after delivery.+ pDCs and CD86+ pDCs antepartum had no significant difference from any time postpartum. The frequencies of CD83+ pDCs, CD86+ pDCs in the treated group antepartum were significantly higher than those in the untreated group , and the same at 12 weeks postpartum . The frequency of CD86+ pDCs in the treated group was significantly higher than that in the untreated group at 24 weeks postpartum .90 eligible pregnant women were enrolled, 36 in the untreated group and 54 in the treated group. 36 patients developed postpartum hepatitis, including 17  in the untreated group and 19  in the treated group (χ2 = 1.304 p=0.253), and 22 cases of hepatitis occurred at 6 weeks postpartum, 12 at 12 weeks postpartum, and 2 at 24 weeks postpartum. The alanine transaminase (ALT) levels at any time postpartum were significantly higher than that of the antepartum, especially at 6 weeks and 12 weeks postpartum. However, the frequencies of pDCs, CD83+ pDCs and CD86+ pDCs in pregnant women with chronic HBV infection.Postpartum hepatitis in HBV infected women mainly occurs at 6-12 weeks postpartum. Antiviral treatment during pregnancy can significantly increase the frequencies of CD83 The rate of HBV MTCT maintained 7%-11% in newborns of HBeAg-positive mothers despite combined active-passive immunization with a hepatitis B vaccine and hepatitis B immunoglobulin (HBIG) shortly after birth. HBeAg positive and high HBV DNA level  with pleiotropic effect during viral infection, and they have a strong ability to activate the initial immune response and activate natural killer (NK) cells and T lymphocytes to further activate and regulate the antiviral immune response . They plThe elevation of postpartum ALT is a common phenomenon. It is believed that due to the decrease of steroid hormone levels after delivery, the immunosuppressive state during pregnancy is rapidly relieved, triggering the immune response to HBV and mediating liver tissue injury and hepatocyte necrosis in patients with chronic hepatitis B (CHB). Hepatocyte necrosis leads to elevation of ALT and indicates that these women have entered the immune clearance period or the reactivation period , which iHowever, there are few studies on the changes of postpartum immune cell function of HBeAg-positive pregnant women with chronic HBV infection and its association with postpartum hepatitis, and few reports about the effects of antiviral treatment during pregnancy on postpartum immune cell function and postpartum hepatitis. Therefore, we designed a prospective case-cohort study and recruited HBeAg-positive chronic HBV infected pregnant women after informed consent. We measured the frequency of pDCs and the expression of functional molecules in peripheral blood antepartum and at 6, 12, 24 weeks postpartum to explore the frequency and functional changes of pDCs after delivery.5 IU/ml; 3. Liver function remained normal; 4. With or without antiviral treatment at 24-28 weeks of gestation as they wish. Exclusion criteria: 1. History of amniocentesis during pregnancy; 2. History of hereditary diseases in one or both families of the couple; 3. Fetal abnormalities detected before enrollment by imaging or other prenatal tests, including genetic testing; 4. Coinfection with hepatitis C virus, hepatitis D virus, human immunodeficiency virus, syphilis, toxoplasmosis, rubella, or cytomegalovirus; 5. History of two or more spontaneous abortions; 6. Previous delivery of a child with deformity; 7. Twin or multiple pregnancies; 8. The mother had a history of cancer or autoimmune diseases.Pregnant women with chronic HBV infection were enrolled in the study from January 2016 to January 2018. Inclusion criteria: 1. Between the ages of 20 and 40; 2. HBsAg and HBeAg positive over 6 months, and serum HBV DNA > 2.0×10Pregnant women were classified into treated group  and untreated group . Antiviral therapy was stopped immediately after delivery and all women were followed up at 6, 12 and 24 weeks postpartum.All subjects signed an informed consent. The study was approved by the ethics committee of Beijing Ditan Hospital, Capital Medical University (JDL-2017-004-01), and registered at clinicaltrials.gov .2 ml of peripheral venous blood of all pregnant women was collected by EDTA anticoagulant violet tube at the time of antepartum (baseline) and 6, 12, 24 weeks postpartum, cells were collected by BD FACS Calibur within 4 hours of blood collection.Virological indexes  and ALT, glutamic oxaloacetic transaminase (AST), total bilirubin (TBIL), albumin (ALB) were tested by the laboratory of Beijing Ditan Hospital, Capital Medical University.pDCs were detected with Lineage1-FITC, HLA-DR-PerCP, Anti-CD123-APC, Anti-CD83-PE, Anti-CD86-PE antibodies, and CD83MFI and CD86MFI data were analyzed by FlowJo7.6 software.Data were analyzed by SPSS 19.0 software. All data were presented as mean ± standard deviation (mean ± SD) or median, interquartile interval [median ], box-and-whisker plots or percentage. Student t-test was used for normal distribution data and Mann-Whitney test was used for skewness distribution data between groups. P < 0.05 was considered statistically significant.90 pregnant women were enrolled, including 54 cases with antiviral treatment (treated group) and 36 cases without antiviral treatment (untreated group). There was no difference between the two groups in age, gestational weeks, pregnancy complications, neonatal weight, length, Apgar score at 5 minutes and neonatal malformations (P>0.05). The number of gravidity and parity in the untreated group were significantly higher than those in the treated group . The level of TBIL was 7.05±2.18umol/l in the treated group and 8.18±2.73umol/l in the untreated group (P=0.033), but TBIL in both groups was within the normal range. The levels of HBsAg, HBeAg and HBV DNA antepartum in the treated group were significantly lower than those in the untreated group (P<0.05), which was related to antiviral therapy. The basic information is shown in + pDCs and CD86+ pDCs in all pregnant women enrolled were not significantly different from those at 6, 12 and 24 weeks postpartum (P > 0.05)  as the diagnostic criterion for the occurrence of hepatitis according to the Asian-Pacific clinical practice guidelines on the management of hepatitis B . The ALT > 0.05) Table 2.+ pDCs antepartum was significantly higher than that of the 24 weeks postpartum . In the untreated group, the frequency of CD83+ pDCs antepartum was significantly lower than that of the 6 weeks postpartum  . In the treated group, the frequency of CD83p=0.040] Table 3.+ pDCs and CD86+ pDCs in the treated group were significantly higher than those in the untreated group before delivery . The frequencies of CD83+ pDCs and CD86+ pDCs in the treated group at 12 weeks postpartum were significantly higher than those in the untreated group . The frequency of CD86+ pDCs in the treated group at 24 weeks postpartum was significantly higher than that in the untreated group . It is suggested that antiviral treatment in the third trimester of pregnancy can significantly increase the frequencies of CD83+ pDCs and CD86+ pDCs in peripheral blood antepartum and postpartum (The frequencies of CD83stpartum Table 4.Dendritic cells (DCs) can be divided into pDCs and myeloid dendritic cells (mDCs) according to the source of precursor cells. According to specific functions, DCs can be divided into immature DCs and mature DCs, most of which are immature DCs under normal circumstances and have a strong ability to capture antigens , 12. HowRecently, the function of pDCs has attracted extensive attention in the field of chronic HBV infection. As the only innate immune cells with antigen presenting ability, which can activate non-sensitized initial T cells, pDC is the main effector cell of human antiviral immunity and plays a double-acting role in antiviral treatment, which can mediate both immune response and immune tolerance , 14, 16.+ pDCs and CD86+ pDCs in pregnant women with chronic HBV infection before delivery and 12 weeks postpartum. This might indicate that antiviral therapy in late pregnancy increases the proportion of mature pDCs, the immune activity and antiviral function of pDCs, which may improve the success rate of postpartum hepatitis treatment. However, this study had a small sample, and only involved the changes of pDCs in peripheral blood, without discussing the changes of the immune function in the liver, therefore, if we want to elucidate the above causal relationship, we need to further enlarge the sample and investigate the changes of the immune function in liver, as well as in peripheral blood, which would shed new light on the prevention and treatment of postpartum hepatitis in HBeAg positive chronic HBV infected pregnant women.Studies have suggested that HBsAg and HBeAg in patients with chronic HBV infection can inhibit the function of DCs, which leads to a decrease in the frequency and function of pDCs in patients with chronic HBV infection , 21. OurThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The ethics committee of Beijing Ditan Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study.FW, GW, LZ, ML, and YX contributed to study concept and design. MS, YH, YLi, XB, YLu, TJ, WD, SyW, FS, ZZ, GS, RL, MC, SlW, YG, HH, MX, XC, and LH performed the data collection. FW, GW, LZ, ML, and YX conducted data analysis. FW, MS, YH, LY, and XB wrote the first draft. FW edited the English version. GW, LZ, ML, and YX approved the submitted version after modification. All authors contributed to the article and approved the submitted version.Project supported by Beijing Science and Technology Commission (Z211100002921059). National Science and Technology Major Project of China . The Capital Health Research and Development of Special (2022–1–2172). The Digestive Medical Coordinated Development Center of Beijing Hospitals Authority (XXZ0302 and XXT28). Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (XMLX 202127). High-level Public Health Technical Personnel Training Program of Beijing Municipal Health Commission (2022–3–050).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer XW declared a shared affiliation with the authors to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to control chickens, those fed with the synbiotic throughout the productive cycle generally exhibited less histopathological lesions and had higher villi at intestinal level, and ultimately performed better in terms of body weight gain, feed conversion efficiency and liveability. The composition of the caecal microbial ecosystem was also studied, proving that synbiotic supplementation influenced the abundance of several bacterial populations. To fully understand the exact implications of these changes, further studies are required, which will be informed and facilitated by the present data.Since the use of antibiotics as growth promoters has been banned, the poultry sector is searching for alternatives to support production efficiency. Synbiotics, which consist of a mixture of prebiotics and probiotics, appear to be a promising way to do so by sustaining chickens’ gut health. In this study, the synbiotic PoultryStar® sol to broilers in drinking water. To this purpose, three Ross 308 broiler flocks, representing separate progenies of a breeder flock which was treated with the same synbiotic, were housed in separate farms, divided into treatment and control groups, and followed throughout the productive cycle. Synbiotic administration was shown to improve gut health even in absence of a challenge, with limited changes in terms of macroscopic intestinal lesions and more overt differences related to histopathological scores and villi length. Synbiotic-fed chickens performed consistently better in terms of body weight gain, feed conversion ratio and survivability. Lastly, the evaluation of the caecal microbiome through next-generation sequencing highlighted the effects of synbiotic supplementation on the composition of the bacterial population, the implications of which will, however, require further studies to be better comprehended.In recent years, the applicability of prebiotics, probiotics and their mixtures, defined as synbiotics, in poultry production has received considerable attention. Following the increasing regulation of antibiotic use, these nutraceuticals are seen as an alternative way to sustain production efficiency and resistance to pathogens and stressors by modulating birds’ gut health. The aim of this study was to evaluate the benefits provided under field conditions by administering the multi-species synbiotic PoultryStar Salmonella spp. and Campylobacter spp. are the most common causes of human foodborne bacterial diseases linked to poultry [For decades, antibiotics have been utilized in the poultry industry to prevent and treat diseases and promote growth. Despite the significant improvements in technology and hygienic practices made at all stages of poultry production, bacterial diseases remain a persistent threat not only to animal health, but also to humans, with recent reports showing that  poultry ,2. Due t poultry .Since antibiotic growth promoters (AGPs) were crucial to controlling dysbacteriosis and enteropathogens , novel aClostridium perfringens [Campylobacter spp. [Salmonella spp. [The studies conducted on synbiotics, which rely on a synergism between probiotics and prebiotics, have shown particularly promising results. The supplementation of different synbiotics to broilers was demonstrated to improve body weight (BW) gain and feed efficiency , reduce fringens , Campyloter spp.  and Salmlla spp. .The benefits of synbiotics in terms of performance and intestinal health, along with their modulatory action on the microbial enteric composition , appear ® sol (PS) , as detailed in Prentza et al. [2. PS was administered in one of the two, while the other acted as control. In detail, depending on the house size, 8160 chicks were housed in the treatment house of farm 1, while 14,280 were placed in the control house; for farm 2, 11,730 and 10,200 birds were set up in the treatment and control houses, respectively; in farm 3, 15,198 chicks were placed in each of the houses.The present study was conducted on the broiler progenies of a broiler breeder flock that was treated with the synbiotic product PoultryStara et al. . Day-oldad libitum. The lighting program started at 7 days of age, providing 4 hours of darkness and 20 hours of light. Birds were vaccinated against infectious bursal disease (IBD), infectious bronchitis (IB) and Newcastle disease (ND) at the hatchery following the local vaccination program.To ensure flock health and welfare and achieve good flock performance, management conditions followed the official guidelines for broiler birds . Wheat sPS, which is a synbiotic containing patented probiotic strains plus prebiotic fructooligosaccharides, was administered in one of the two houses in each of the three broiler farms, based on the manufacturer’s guidance. Specifically, a daily dosage of 20 g/1000 birds was administered in clean drinking water consecutively for the first three days of age, as recommended following the chicks’ placement, and then once a week till slaughter age was reached.To evaluate the chickens’ intestinal health and the presence of dysbiosis, the integrity of the intestinal wall was visually evaluated at three different time points  on ten birds randomly picked from different points of each house of each farm by applying a macroscopic lesion scoring system consisting of ten different parameters, which were scored 0 when absent and 1 when present. The individual scores were summed and divided by 2.5, yielding a total score ranging between 0 and 4 ,22.Specimens from different intestinal tract segments were collected at 38 doa from ten birds randomly picked from different points of each house of each farm for histopathological and morphometrical evaluations. In detail, 3 cm long segments were collected from the duodenum, jejunum, ileum and caecum, keeping the collection sites consistent for each tract, and placed in 10% neutral buffered formalin as described by Hoerr . One mm The morphometry of the intestinal villi and crypts were examined in each section, performing optical capture and measurement with Image Pro-Plus v.6.0 software . The selection of the villi followed the criteria proposed by Gava et al. , namely To evaluate potential growth differences, 100 randomly selected chickens from each house of each farm were weighed longitudinally from 0 to 30 doa. The final BW was recorded when birds were loaded onto the trucks at 42 doa, and the feed conversion ratio (FCR) was calculated by dividing feed intake by the total BW gain. In addition, the carcass weight of 100 randomly selected birds from each group was measured at the slaughterhouse. Mortality was recorded daily throughout the whole cycle.To evaluate the caecal microbial composition, Next Generation Sequencing (NGS) was performed on 60 caecal content samples taken at 38 doa from 10 randomly selected birds of each of the two houses of the three farms. The analyses, conducted using an Illumina MiSeq System  at LGC Genomics GmBH , targeted the V3 region of the 16s rRNA gene, generating 2 × 300 paired-end sequences. Following a preliminary quality evaluation with FastQC 0.11.9, the forward and reverse sequences were trimmed at 195 bp and 220 bp, guaranteeing a minimal Phred score of 28 and allowing for a maximum expected error of 2 bases for each read. DADA2  was usedbetadisper function of the vegan R package [Alpha diversity was evaluated using the Simpson, Shannon, Chao1, and Observed species indexes. Permutational ANOVAs on the euclidean distances among samples were performed for significance testing between groups, after verifying that group dispersions were adequately homogeneous using the  package . The abssurvival package. All statistical analyses were performed in R (version 3.3.2) [p < 0.05, with the sole exception of the differential abundance analysis of microbial populations, for which the significance level was set to p < 0.01.The existence of significant differences in terms of BE score, histopathological lesion score, villi and crypts height which may have been ascribed to treatment, farm effect or sampling age  were investigated using the non-parametric Kruskal–Wallis test followed by post-hoc Mann–Whitney test with Bonferroni correction. The treatment and farm effects on the carcass weight was investigated with a two-way ANOVA followed by post-hoc Tukey’s test. Log-rank test was used to compare the Kaplan–Meier survival curves with the n 3.3.2)  setting p < 0.0001). Significant differences between PS-fed and control birds were found at 10 (p = 0.0228) and 38 doa (p = 0.0495) and when considering all ages together (p = 0.0162). Since the farm effect was also shown to be significant (p = 0.009), each farm was also assessed individually. The differences between synbiotic-treated and control groups were found to be limited to farm 3, where they were again significant at 10 (p = 0.0077) and 38 doa (p = 0.0108) and when considering all ages together (p = 0.0009). No statistically significant differences were observed in farm 1 and 2.The results obtained in terms of BE score  showed tp = 0.0023), with farm 2 scoring worse than both farm 1 (p = 0.0005) and farm 3 (p = 0.04265), and at caecum level (p = 0.01499), which seemed mostly ascribable to differences between farm 2 and farm 3 (p = 0.03127).More overt differences were found in terms of histopathological lesions scores . The avep < 0.0001 for all intestinal tracts) and crypts  length.The average villi and crypts lengths measured at 38 doa in PS-treated and control flocks, along with the villi/crypts (V/C) ratio, are shown in p < 0.0001) and in each individual farm, while the farm effect and the interaction between treatment and farm effect were not.Throughout the cycle, the average live BW measured in synbiotic-treated flocks was consistently higher than in control ones . The comp < 0.0001). Since the farm effect was found to be significant (p < 0.0001), each farm was also considered separately. In detail, mortality was 4.8% in the control group and 2.5% among treated birds in farm 1 (p < 0.0001); 3.7% and 2.8% in farm 2 (p = 0.0002); 6.9% and 4.7% in farm 3 (p < 0.0001)  .As shown in the dendrogram based on euclidean distances provided in The differences between treatments within the same farm was also made visible by some of the alpha diversity measures, namely the Observed species and the Chao1 indexes, although were less evident according to the Shannon and Simpson indexes . The effA more comprehensive overview of diversity is provided by p = 0.228) a permutational ANOVA test was subsequently performed, revealing siginificant differences between synbiotic-treated and control chickens at overall level (p = 0.002) and in each of the three farms .Since the beta dispersion within treatment group proved adequately homogeneous (p < 0.0001), but not with any other considered parameter. Considering the design of the study, these results allowed us to isolate the effect of the synbiotic treatment on bacterial composition. Out of 9530 ASVs, 65 had a significant differential abundance on an alpha level of 0.01 (after Benjamini–Hochberg multiple testing correction). By setting out the obtained adjusted p-values for each ASV against the respective fold changes (p-values are shown in Intercorrelation analysis revealed a significant Spearman correlation between the treatment and the length of caecal villi (ρ = −0.6195;  changes , 40 ASVsThe evaluation of macroscopic intestinal features and histological measurements allowed us to assess the chickens’ gut health, and how it was impacted by the synbiotic treatment. Although the BE and histopathological lesion scores suggested good intestinal health in all treatment and control groups, likely due to the absence of a challenge, some differences in favor of the treated chickens could still be noted. In particular, significantly lower BE scores were observed in synbiotic-fed birds only in farm 3, while more overt differences were found in terms of histopathological lesions in most intestinal tracts of chickens raised in all farms. These results were in line with those observed after administering PS to the group of broiler breeders that birthed the investigated progenies, which also pointed at a general intestinal eubiosis in both treatment and control groups, with the former scoring better overall .The BE scores measured in this study allowed us to demonstrate a significant age effect, showing a biologically plausible increase in subsequent time points. In addition, between-farm differences were present according to both scores. Such an effect, which was observed also for other evaluated parameters, could easily be ascribed to environmental factors related to the individual farms, such as differences in management, stockmanship, housing, farm location and others. These variations should be considered inherent to the field conditions in which the study was conducted to reproduce the real-life application of the tested synbiotic.The evaluation of villi and crypts length at 38 doa provided additional insights on the effect of PS. Longer villi are considered an indicator of a greater surface area and thus a greater adsorption capability ,32, whilBy promoting good intestinal health and preventing any undesired condition of dysbacteriosis or inflammation, synbiotic administration should ultimately promote production efficiency. In this study, synbiotic-treated flocks performed significantly better according to several parameters, including feed conversion ratio, carcass BW and daily cumulative mortality rate. These results agree with several other studies in which other synbiotics were tested ,40,41,42The impact of PS administration on caecal bacteriome was also investigated through high-throughput sequencing. Caeca represent the enteric tract with the highest microbial density, mainly composed of obligate anaerobes belonging to the phyla Firmicutes and Bacteroidetes . The stuLachnospiraceae, a family of cellulolytic bacteria capable of metabolizing non-starch polysaccharides that are among the earliest colonizers of the caecum of broiler chickens [Faecalibacterium, which are common inhabitants of the caeca involved in butyrate production and in the anti-inflammatory response [Monoglobus, a less abundant component of the intestinal microbiome capable of degrading pectin [Clostridia UCG-014. Overall, based on alpha diversity measures, PS chickens exhibited a lower species richness than control ones.A clear treatment effect was visible in all farms, significantly impacting the abundance of 65 ASVs. Among the most influenced were representatives of chickens ,47, whicresponse ,49, and g pectin , were unConsidering the complexity and variability of the gut microbiota, it is hard to ascertain the implications of the observed changes. An increasing number of studies on synbiotics are using molecular assays to investigate their effects on gut bacterial populations, reporting different results. For instance, Baffoni et al.  and Pine® sol in broilers, which improved the productive performance in terms of FCR, carcass weight and mortality rate. Treated chickens also exhibited a better intestinal health, having lower histopathological lesion scores and longer villi across most intestinal tracts. In addition, synbiotic supplementation was shown to influence the caecal microbial ecosystem, causing some taxa to be more or less abundant in synbiotic-fed flocks. The exact implications of these changes will, however, require further studies to be better understood.The presented results support the benefits achieved by the administration of PoultryStar


Background: Legg-Calvé-Perthes disease (LCPD) is an idiopathic pediatric hip disorder associated with avascular necrosis of the femoral head. Although there is no standardized and optimal treatment protocol for patients with LCPD, there are three primary treatment strategies: symptomatic treatment, nonsurgical containment using orthotic devices, and surgical containment.Objective: This study aimed to describe the demographic characteristics, management and outcome of pediatric patients with LCPD at our center between 2005 and 2015.Material and Methods: In this retrospective study, 23 patients with LCPD who represented all patients with LCPD treated at King Abdul-Aziz Medical City, Jeddah, Saudi Arabia between 2005 and 2015 were enrolled. Their demographic and clinical characteristics, treatment methods, and outcomes were evaluated.Results: Descriptive statistics showed that most of the patients were males (87.0%), with a mean age of 7.1±2.4 years. None of the patients had a family history of LCPD. Approximately two-thirds of the patients reported hip pain as the chief concern. LCPD was observed in the left hip in 60.9% of patients, the right hip in 21.7% of patients, and both hips in 17.4% of patients. Approximately half of the patients (55.3%) were treated with physiotherapy, and 10.6% were treated with orthotics. Despite that, 10 patients (43.4%) required surgical management after the conservative approach, and six of them underwent pelvic Salter innominate osteotomy.Conclusion: These results highlight the experience of a single center in managing patients with LCPD. Treatment was different based on patient age; non-surgical treatment, mainly physiotherapy, was predominant in younger children. However, regardless of the type of treatment, the earlier interventions have proven to provide better outcomes in patients with this health condition. Legg-Calvé-Perthes disease (LCPD) is a pediatric hip disease of unknown cause that may lead to avascular necrosis (AVN) of the femoral head ,2 and waResearchers have indicated that the classification of LCPD corresponds to the disease stage, outcome, and predictors of outcome . UnderstIn the context of treatment, there is strong evidence that early intervention has the most favorable outcomes . OverallStudy design, population and study settingThis study was a retrospective cross-sectional study. All patients included in this study were aged below 15 years, had LCPD, and visited the pediatric orthopedic department at King Abdul-Aziz Medical City, Jeddah, Saudi Arabia, between 2005 and 2015. Patients with other causes of AVN were excluded.Data collection methodsThe data collection sheet for this study was filled by the authors of the present study using information from the medical records database, which comprised two sections. The first section included the following information: age at diagnosis, date of diagnosis, and gender. The second section included the following clinical data: medical history which included history of breech presentation, oligohydramnios, cesarean section, neonatal intensive care unit (NICU) admission, family history of the same disease, consanguinity, chief complaint and associated symptoms, trauma, hip fracture, past medical and surgical history, and treatment modalities whether conservative, surgical, or both and their outcomes.Study planThis study planned to recruit pediatric patients who fulfilled the inclusion criteria between 2005 and 2015. Data collection began after obtaining permission from the ethical and scientific committee of King Abdul-Aziz Medical City with approval reference number RJ16/068/J and date 20-02-2021. We scrupulously guarded the confidentiality of the participants' data throughout the study. Prior to data collection, the participants provided informed consent for study participation and publication of data.Data management and statistical analysisThe SPSS statistical software package for Windows  was used for data entry and statistical analysis. Quality control was performed at the coding and data entry stages. Data are presented using descriptive statistics in the form of frequencies and percentages for qualitative variables and means and standard deviations for quantitative variables.General characteristics of patients with Legg-Calvé-Perthes diseaseDuring the study period, 23 patients (27 hips) had LCPD. These 23 patients comprised 20 (87.0%) males and three 13.0%) females. The age range was four to 14 years, and the mean age was 7.1±2.4 years. Of these patients, 16 (69.6%) were younger than eight years, and seven (30.4%) were older than eight years. Eight (30.8%) patients had a history of trauma, and nine (39.1%) had a previous medical illness; however, none had a previous history of surgery. Only one patient (14.3%) out of seven was born by cesarean section; however, none were born by breech delivery. None of these children had oligohydramnios exposure, required NICU admission, or had a family history of LCPD; however, two (8.7%) reported consanguinity between first cousins  were treated with physiotherapy, including range of motion exercises and non-weight-bearing activities (Figure Most patients showed radiologic improvements, as well as improvements in the range of movement, with patients who underwent surgical treatment showing greater improvement than those treated without surgery Table . MoreoveChildren diagnosed with LCPD have a high risk of early osteoarthritis as adults. Methods of preventing this risk include surgical and non-surgical modalities that mainly aim to restore a contained spherical femoral head . TherefoClinically, patients with LCPD usually present with pain, limited hip movement, and gait disturbance . More thFor prediction purposes, orthopedic surgeons use the recommended classification system to indicate functional outcomes and recommend treatment. However, an effective classification system should be reproducible, depend on indicators related to the disease process, and allow the grouping of similar hips, thereby enabling comparisons of treatment outcomes . AlthougThe type of treatment depends on two main factors: patient age and the extent of the femoral head involvement . ChildreSymptomatic therapy has been recommended for patients of all ages with group A LCPD and patients aged under seven years with group B LCPD . HoweverThe results of the current study showed that physiotherapy was the most common non-surgical treatment. Physiotherapy included range of motion and non-weight-bearing exercises followed by orthotics. A study reported that treatment with Scottish Rite orthosis for 12 months did not reduce the power applied to the hip joint but reduced the angle of hip joint abduction while walking . AbductiThe primary surgical method was pelvic Salter innominate osteotomy in the current study, which showed radiological and range of movement improvements in 50% of patients. In agreement with this, previous studies showed that femoral osteotomy surgery improved outcomes in 58% of patients , and innLastly, the present study has several limitations, including its retrospective design, which may have resulted in a loss of information. Moreover, the small number of patients, combined with various pathologies and treatments, limited the ability to generalize the results of this study among this population. Additionally, many patients were lost to follow-up, thus limiting our ability to determine the long-term effects of treatment. Finally, the range of motion and patient scoring systems have not been adequately documented. The present study shows the experience of a single center in managing patients with LCPD. A detailed clinical evaluation is essential and should always be correlated with radiological assessment. The prognosis of LCPD regressed to different prognostic variables, included age at presentation and the femoral head sphericity, and congruence. Designing a public health program may help to increase the awareness and will encourage parents to visit the clinic in the early stages of the disease.


Monkeypox (MPX) has been regarded as a neglected tropic disease of Western and Central Africa since the early 70s. However, during May 2022, an unprecedent outbreak of MPX has involved most of European Countries, as well as North and South America. While the actual extent of this outbreak is being assessed by health authorities, we performed a pilot study on specific knowledge, attitudes, and practices (KAP) in a sample of Italian medical professionals , focusing on Occupational Physicians (OP), Public Health Professionals (PH), and General Practitioners (GP), i.e., medical professionals more likely involved in the early management of incident cases. More specifically, we inquired into their attitude on the use of variola vaccine in order to prevent MPX infection. From a total of 566 questionnaire (response rate of 5.5%), 163 participants were included in the final analyses. Knowledge status was quite unsatisfying, with substantial knowledge gaps on all aspect of MPX. In turn, analysis of risk perception suggested a substantial overlooking of MPX as a pathogen, particularly when compared to SARS-CoV-2, TB, HIV, and HBV. Overall, 58.6% of respondents were somehow favorable to implement variola vaccination in order to prevent MPX, and the main effectors of this attitude were identified in having been previously vaccinated against seasonal influenza , and being favorable to receive variola vaccine . In summary, the significant extent of knowledge gaps and the erratic risk perception, associated collectively stress the importance of appropriate information campaigns among first-line medical professionals. Orthopoxvirus [Cynomolgus monkeys recently imported to Singapore [Monkeypox virus (MPX) is a complex DNA virus that belongs to the Poxviridae family, Chordopoxvirinae subfamily, genus poxvirus ,2,3. Firingapore ,5, unlikingapore , and wasingapore . Since tingapore ,9. The fingapore . Since tingapore ,11,12, singapore ,14, but ingapore ,18,19,20Since 7 May 2022, an unprecedented, but not unexpected, outbreak of MPX across Europe, Americas, and Australia occurring in subjects with no established travel link to endemic areas ,21,22,23MPX is usually characterized by a lower case-fatality-ratio (CFR) than smallpox, the latter historically averaging 30% overall in unvaccinated individuals . DependiMore precisely, we report on three medical categories that could be reasonably be involved in the early management of incident cases: (a) General Practitioners (GP), as they may represent the first medical professionals to be referred by patients asking for consultation ,33,35,36A cross-sectional online survey was performed between 24 May 2022 and 31 May 2022 according to the STROBE  initially contacted the group administrators, preventively requesting their authorization to share the study invitation. The latter included a summary of the aims of the survey and a direct link to the first page of the questionnaire , that, in turn, included the full informed consent. Participants were then asked about their consent for study participation through a specific dichotomous question . Even though all individuals receiving the questionnaire and agreeing with the participation were able to complete the survey, only respondents who accurately compiled the questionnaire and fulfilled the main inclusion criteria  were included in the following analyses.The survey was anonymous, and no personal data  were requested, saved, or tracked. Even though no monetary compensation was offered to the participants, they were preventively guaranteed that at the end of the questionnaire a full explanation of all items would be provided, representing an educative opportunity on MPX.Questionnaire items were specifically designed for this study through an extensive literature review ,46,47,48Main demographic data: age, gender, seniority as medical professional, medical background ; the Italian region where the professional mainly worked and lived.Knowledge Test. Participants received a total of 24 statements on MPX . A summary score  was then calculated by adding +1 to a sum score for every correct answer, whereas a wrong indication or a missing/“don’t know” answer added 0 to the sum score .MPX) and the perceived frequency (FMPX) of MPX in Italian population by means of a fully labeled 5-point Likert scale (range: from “not significant” to “very significant”). According to Yates [MPX and FMPX .Risk perception. Participants were requested to rate the perceived severity  to 5 . Medical professionals were then requested to similarly rate how important they perceived the capability of the vaccine to avoid natural infection and complications, and about their willingness to pay, both as a personal expense , and from a Public Health point of view . Respondents were then requested to rate through a full Likert scale 1  to 5  whether they perceived or not MPX as a likely occurrence during daily activities in the following months, whether they perceived or not MPX as potentially affecting daily working activities, and whether they were confident of not to be able to recognize a MPX case. Respondents were then asked to rate how difficult they perceived the management of different infectious diseases in the Italian settings, and more precisely: MPX, seasonal influenza virus (SIV), SARS-CoV-2, Hepatitis B virus (HBV), tuberculosis (TB), Human Immunodeficiency Virus (HIV). All of the aforementioned disorders were rated 1 (not difficult) to 10 (very difficult). Eventually, participants were requested to report whether they had received or not VARV vaccine , SIV vaccine during previous influenza season , and SARS-CoV-2 vaccine (at least 2 doses).The final questionnaire is available as The questionnaire and retrieved data were handled anonymously and confidentially through an anonymous, observational design. Participants were preventively briefed about the aims and design of the study before giving their consent to the survey and guaranteed that individual participants could not be identified based on the presented material. As this study plausibly caused no harm or stigma to the participants, and did not include clinical data about participants, a preliminary evaluation by an Ethical Committee was not forcibly required according to the Italian law .In order to preventively remove duplicate questionnaires from the analyses, when two or more entries were characterized by the very same year of birth, year of graduation, reported gender, region where the professional lived and worked, or reported occupational profile, only the first entry was retained and included in the analyses, whilst the other ones were removed.Continuous variables were initially reported as average ± SD, while categorical values were reported as percent values. Cumulative scores  were initially normalized as percent values, and then dichotomized as high vs. low estimates by median value. Likert scales were similarly dichotomized, with scores for “agree” and “totally agree” aggregated as “somewhat agreeing” vs. scores from “totally disagree” to “neutral” aggregated as “somewhat disagreeing”.p-value < 0.10, and variables were therefore compared through Mann–Whitney or Kruskal–Wallis tests for multiple independent samples. On the other hand, when variables passed the normality check , they were compared by means of Student’s t-test or ANOVA, where appropriate. Association between variables was similarly assessed through Pearson’s correlation coefficient or Spearman’s rank correlation coefficient, for variables either passing or not the normality test.Continuous variables were initially tested for normal distribution . Normality distribution was assumed as rejected for Distribution of categorical variable was reported by outcome variable of being favorable or not to promote VARV vaccine in the general population in order to avoid MPX infection, and initially analyzed through chi-squared test. Internal consistency of the knowledge sections was measured through calculation of the Cronbach’s alpha. Cronbach’s alpha is a measure of how closely related a set of items are as a group, being usually considered a scale of reliability. In general, a score ≥ 0.7 is considered acceptable.p < 0.05) with outcome variables were included in a multivariable model of binary logistic regression analysis in order to calculate adjusted odds ratios (adjOR) and their respective 95%CI.All categorical variables that at univariate analysis were significantly associated .As shown in Among the respondents included in the analyses , the majp = 0.109)  was calculated. Interestingly, the distribution of the cumulative score passed the normality check  a, with n= 0.109) . NeverthThe detailed results of knowledge test are reported in Interestingly, the majority of participants acknowledged the potential transmission of MPX through the respiratory system, by means of respiratory droplets, but also through direct contacts and body fluids (78.5%), and that standard preventive measures may be sufficient to avoid infection (74.8%). However, only 44.4% understood MPX as a pathogen circulating among various hosts, not only primates, and 42.3% had knowledge of the long survival of MPX on contaminated surfacesThe most significant uncertainty among the respondents was represented by the inappropriate understanding of actual global incidence of MPX during the last decade . Even though nearly half of participants acknowledged that MPX does not evolve in uncomplicated influenza-like illness (48.5%), the high rate of systemic complications was largely overlooked (20.9%), particularly in children compared to adults (34.4%). Moreover, only 28.2% of participants were aware that the skin rash associated with MPX is typically synchronous rather than asynchronous, and even though 60.1% of respondents reportedly knew that an effective  vaccine against MPX is available, with 51.2% acknowledging the availability of effective drugs, only 32.5% had knowledge that individuals previous vaccinated against VARV still require further vaccination shots. Moreover, only 17.8% of respondents were aware that smallpox has a case-fatality ratio ranging between 30% and 40%, while the majority of them (72.4%) correctly reported the lower lethality of MPX .Eventually, two hypothetical clinical cases were presented to the participants. While a large share of respondents (77.9%) properly acknowledged a case with (a) atypical skin rash; (b) lymph nodal involvement ; (c) history of travel to countries endemic for MPX, as a probable MPX case, greater uncertainties were reported when facing a case characterized by generalized or localized skin rash, either maculopapular or vesiculopustular, umbilicated skin lesions, and lymphadenopathy (42.3%).p = 0.389; Overall, 30.1% of participants perceived that MPX would become a likely occurrence during daily activities, and 32.5% that it could potentially affect them: when asked to rate the perceived health threat represented by MPX, an overall score of 5.42 ± 2.18  was reported, and the correspondent distribution passed the normality check (D’Agostino–Pearson p < 0.001), with no substantial differences among the sampled groups of medical professionals . The distribution of the score was visually and statistically skewed  and avoid systemic complications (90.8%).When dealing with the reported willingness to pay for a candidate MPX vaccine, 31.9% of respondents were not interested, 23.9% would rate as “acceptable” a total cost < 10€ per shot, 20.2% would consider a cost of 10 to 49€ per shot, 12.9% a cost ranging from 50 to 99€ per shot, and 11.0% would agree to pay 100€ or more per shot. On the other hand, when focusing on the optimal price, the majority of respondents reported that it should be offered at no cost for the recipients (65.6%), with 16.0% recommending a cost < 10€ per shot, 14.7% acknowledging a payment between 10 and 49€ per shot, and only 3.7% of respondents agreeing for a total payment exceeding 50€ per shot .p = 0.031), while the former scored a not significantly lower KS estimates that the latter ones . In correlation analyses, not only synthetic scores were each other not correlated , but KS was not correlated with either age  or seniority  of respondents, while RPS was negatively correlated with both demographic data . In other words, professionals with greater age and seniority were less concerned towards MPX than the younger and less experienced ones.Professionals reportedly favorable to the delivery of VARV vaccine in order to prevent MPX infections exhibited a substantially higher RPS that those against , TB , HBV , HIV , but it was similar to estimates for SIV .Focusing on the perceived potential health threat represented by MPX , it was p = 0.018). Similarly, having a seniority as Physician > 20 years , having been vaccinated against VARV , and being not interested in paying for a putative MPX vaccine  were more frequently reported in professionals agreeing with VARV vaccine than in those somewhat disagreeing.In univariate analysis for dichotomous variables , while gp = 0.019), a more favorable attitude towards being vaccinated , and having been vaccinated against SIV  were more frequently reported among participants favorable to the implementation of VARV vaccine against MPX than in those against.On the contrary, higher risk perception , and having been vaccinated against SIV  were identified as positive effectors.In order to properly and quickly respond to the requirements of a MPX outbreak, front-line Italian HCWs should be able to recognize and correctly manage incident cases of this previously uncommon disorder ,33,35,36The reasonable explanations of these uncertainties are quite straightforward. Firstly, with a mean age of around 43 years, the majority of the respondents were born in a “Variola-free” world and did not receive a specific University-level formation on this pathogen ,35,41,52In fact, when dealing with MPX, clinical expertise and high suspicion index are instrumental in guaranteeing ad appropriate and timely diagnosis, as laboratory exams other than PCR may be somehow misleading. Orthopoxviruses share several common antigenic features, with a substantial cross-reactivity of elicited antibodies ,42,55,56Not coincidentally, and most notably, the very same risk perception on MPX and the potential consequences of its spreading have been substantially overlooked by study respondents, even compared to similar studies performed well before the current outbreak ,35,36. DEven though the early reports on the 2022 MPX outbreak seemingly rule out that this pathogen could follow the paths of SARS-CoV-2 infection in 2020 ,21,62, iThe reasons of this features have been identified in some specificities of MPX. On the one hand, the conservative surface antigens guarantee some efficacy against MPX of VARV vaccines ,36,42,52Therefore, our study not only inquired the actual knowledge status of study participants, but specifically inquired them about their acceptance of VARV vaccine as instrumental in avoid MPX infection, and the corresponding results were somewhat unexpected. First of all, even though the majority of respondents was somehow favorable towards the implementation of this policy (58.9%), neither knowledge nor risk perception were in fact associated with the attitude on vaccination. In fact, multivariable analysis identified two positive effectors, represented by having been vaccinated against SIV in the previous season , and being either favorable or highly favorable to receive VARV vaccination against MPX . Again, such attitude is somewhat consistent with previous report on the KAP of HCWs towards vaccination, where a generally positive attitude towards a certain vaccine or a group of vaccines usually results in a better acceptance of new interventions  ,50,68.Nonetheless, a positive attitude of HCWs towards vaccines is not granted ,71,72,73Second, there is clear evidence that HCWs are quite effective in promoting vaccine acceptance in the general population. Unfortunately, when HCWs are themselves affected by vaccine hesitancy, they may also spread this attitude among the people they care for ,76,79,80Limits. Despite its novelty and its potential significance, our study is affected by several limits. For one, in order to quickly reach a proper sample size, we opted for a web-based survey, and this design is affected by a series of implicit shortcomings, and particularly the extensive “self-selection” of participants [icipants ,81,82. Iicipants ,84, the Second, our sample was based on a small, convenience study group of 163 medical professionals , which could be hardly considered fully representative of the national level. In fact, as previously suggested by Harapan et al. in a similar study on Indonesian GP, a conservative assumption that 50% of sampled professionals would have a good knowledge of MPX, a target sample of 382 professionals should be preferentially achieved assuming a 5% margin of error, and a confidence interval of 95% ,35,85. HThird, even though discussion groups involved in the recruitment of the study participants usually performed a preventive selection , that allowed to estimate cross-inscriptions and actual number of individual profiles potentially reached by the original invitation, we cannot rule out that some of the respondents did not fully adhere to our selection criteria , with a further impairment in the representativity of the sample.TM that reports the users’ web interest in a specific keyword through a normalized value ranging from 0 to 100, in turn proportional to the ratio between the keyword-related queries and the total of web queries) and both RPS  and GKS  for Monkeypox from Google Trends™  ,63,87. FOur results should, therefore, be regarded as a pilot study, whose most significant contribution to the Public Health effort in containing the MPX outbreak is represented by the prompt availability of a rapid assessment of the actual knowledge and behavior of Italian Physicians on the eve of the first out-of-Africa MPX outbreak. On the other hand, our results should be validated and more properly defined in the following months.In conclusion, despite some significant limits, our study suggests that Italian physicians potentially involved in the early management of MPX would require a more specifically tailored formation in order to guarantee their capability to cope with the requirements of their patients during the ongoing MPX outbreak. Despite the limit of the present study, and particularly the reduced sample size, our methodology could be implemented in future studies monitoring the knowledge status of HCWs towards MPX and similarly emerging pathogens.