Background: At the onset of the coronavirus disease 2019 (COVID-19) pandemic, anesthesiology residency programs were impacted differently due to various factors such as the local severity of COVID-19, exposure to patient suffering, and inability to complete rotations. We sought to investigate the impact of local-level pandemic severity on the well-being of anesthesiology residents.Methods: This multi-site study surveyed postgraduate year two residents from 15 United States (US) anesthesiology programs using the Perceived Stress Scale, Mini-Z, Patient Health Questionnaire-9,WHO-5 Well-Being Index,and the Multidimensional Scale of Perceived Social Support before the pandemic (baseline survey) and during the first COVID-19 surge (post survey).Results: A total of 144 (65%) residents responded to the initial baseline survey; 73 (33%) responded to the post survey, and 49 (22%) completed both surveys. There was not a statistically significant difference in any well-being outcomes of participants between the surveys, nor was there a significant difference based on the severity of COVID-19 impact at the program’s hospital. Male participants had higher perceived stress scores  and lower social support from family  at the post survey compared to female participants after controlling for baseline scores. Additionally, married participants or those with domestic partners reported higher perceived social support in the post survey .Conclusion: The local COVID-19 severity at a residency program did not disproportionately impact well-being scores among anesthesiology residents. Those most vulnerable to diminished well-being appeared to be male and single participants. As a result, targeted well-being interventions, including those aiming to increase social support, to higher-risk resident groups may be indicated. Future work is needed to assess the longstanding COVID-19 pandemic impacts on resident well-being. When the coronavirus disease 2019 (COVID-19) pandemic began affecting the United States (US) in March 2020, graduate medical education was significantly impacted, structurally and functionally. In several areas of the country with high COVID-19 disease burdens, some residents were redeployed to diverse and unfamiliar environments throughout hospital systems to provide clinical workforce support. Some hospital systems were so overwhelmed by the pandemic that operating rooms (ORs) were repurposed into intensive care units and resident anaesthesiologists served as bedside critical care providers . The strThe impact of the COVID-19 pandemic on anesthesiology residents has been previously described . HoweverThis multi-site study was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill, North Carolina, United States .Electronic mental health surveys were distributed to 222 postgraduate year two (PGY-2) clinical anesthesiology (CA1) residents at 15 US-based anesthesiology residency programs in September and October of 2019 (Baseline survey) and subsequently in March and April of 2020 (Post survey). Mental health measures used in the surveys included: the Perceived Stress Scale, Mini-Z, Patient Health Questionnaire-9, WHO-5 Well-Being Index, and the Multidimensional Scale of Perceived Social Support -16. ThesIn the fall of 2020, we retrospectively contacted the primary investigators of the participating sites, inviting them to complete a Qualtrics survey  reporting their program’s highest Accreditation Council for Graduate Medical Education (ACGME) COVID-19 stage designation  as of April 1, 2020, which was used as a proxy of COVID-19 severity at the program level .Kruskal-Wallis rank sum test was used to examine changes in well-being from the baseline survey to the post survey for continuous variables. Fisher’s exact tests were used for categorical variables. The normality of distribution for continuous outcomes was checked and linear regression models were used to examine the association between continuous outcomes and predictors and to control for the baseline confounding variables. The coefficient estimate βs and corresponding p-values and 95% confidence intervals (95%CI) were reported. The proportional odds assumptions were checked, and proportional odds models were used for ordinal outcomes. Observations with missing values were excluded from the related analysis. A p-value less than 0.05 was considered statistically significant. R version 3.6.3 was used for all the analyses .In total, 144 (65%) residents responded to the baseline survey, 73 (33%) responded to the post survey, and 49 (22%) completed both surveys. Among the 49 participants who completed both surveys, seven were from the four Stage one residency programs, 34 were from the eight Stage two residency programs, and eight were from the three Stage three residency programs. Table We did not find a statistically significant difference in any of the well-being outcomes between the fall of 2019 and the spring of 2020 after the initial outbreak of COVID-19 among participants who completed both surveys  at post survey on average . However, after controlling for baseline survey WHO-5 scores, the difference was not significant . No difference was identified between programs of varying COVID-19 burden for reported perceived stress, social support, or burnout, with or without controlling for baseline survey scores. The odds of having a more severe depression score were 3.89  times higher in Stage three than in Stage one programs at the post survey. The difference is notable but not statistically significant . After controlling for baseline survey depression severity, the odds decreased to 1.75 .Male participants reported lower friend support than females in the post survey, without controlling for baseline survey perceived friend support . However, the gender difference was no longer significant after controlling for baseline survey values. As shown in Figure Compared to single or separated participants, married participants or those with domestic partners reported higher social support in the post survey , although the magnitude of the difference decreased after controlling for baseline survey perceived social support  as shown in Figure Statement of principal findingsIn this longitudinal survey-based study, we aimed to assess the impact of the early COVID-19 pandemic on the well-being of anesthesiology residents in the US. Surprisingly, there were no statistically significant differences in any of the well-being outcomes between pre-pandemic and the beginning of the pandemic. Equally surprising, there were no statistically significant differences relative to the impact of the pandemic at a given institution based on disease burden.Interpretation within the context of the wider literatureThere are several potential reasons for these results that may be correlated to proactive supportive initiatives put in place by residency and graduate medical education leadership. When the pandemic began, residency programs around the country employed various strategies to protect their residents and minimize their exposure to COVID-19 . At this same time, elective surgeries were canceled nationwide, leading to lower OR workloads for a large proportion of residents. Despite a decrease in workload, resident pay and benefits were not affected (even though some faculty members were furloughed). Also, the ACGME mandated that time off from clinical duties due to COVID-19 illness or quarantine would not be counted as sick time. In addition, the American Board of Anesthesiology Basic Examination was postponed, which may have specifically decreased the stress level for these CA-1 residents. It is also possible that we were in the “honeymoon phase” of disaster at the time of our post survey, in which there was plentiful support and community building for healthcare workers deemed as heroes at that time . InteresWhen comparing well-being differences by gender, we found that male residents reported higher stress levels and decreased levels of family support compared to female residents, which was sustained when controlled for baseline perceived stress. This finding suggests male residents may be a vulnerable population in this setting and may warrant additional program support, especially since males are known to be more successful in suicide attempts ,23. InteMarried residents, or those with domestic partners, reported higher levels of family support compared to single or separated residents, although this finding was no longer significant once controlled for baseline perceived family support. The pandemic proved to be very socially isolating for many people. Perhaps living with a significant other helped to maintain the human connection during this otherwise isolating time.Strengths and limitationsTo our knowledge, this is the first multi-site study that assessed the impact of the COVID-19 pandemic on the well-being of anesthesiology residents in the US using a repeated survey design. However, this study had several limitations. First, a higher response rate would have strengthened the results. Without all residents completing the survey, we do not know if there are any response patterns that could bias the results. Interestingly, individual response rates per residency program for the post survey were not significantly different based on the severity of the program’s COVID-19 burden, indicating that the severity of the pandemic did not impact the willingness to engage with the study. Therefore, the stability of the well-being measures held true regardless of the severity of the pandemic. Nonetheless, the relative paucity of Stage one and Stage three programs at the time when the study was conducted may have limited the generalizability of the study findings. Second, the effective total sample size is small, which may limit the power of the study to detect significant differences. However, a posthoc power analysis showed that, with a significance criterion of α = .05 and power = .80, the minimum sample size needed is n=1362 in order to detect a perceived stress level difference between the baseline survey and the post survey, which suggests a trivial between-survey stress difference. Third, the scales used to assess well-being may not have been sensitive enough to detect differences. To mitigate this limitation, we used five validated scales to assess various aspects of well-being, which should have decreased the likelihood of false detection. However, these surveys are meant to assess well-being and not the impact of an event. Additionally, the data collected was not initially intended for the assessment of well-being changes during a pandemic, but rather for a separate longitudinal study regarding overall well-being and social support interventions. Although the post survey coincided with the first COVID-19 surge, this may not have captured the full picture of the stressors or events unfolding at a given residency program or to a given resident at that point in time. It is possible that the well-being scores may have shifted if the assessment had been repeated after surgical volumes returned to pre-pandemic levels, to coincide with the future COVID-19 surges, or simply as the stress of the pandemic’s impact continued over time. Finally, data were collected early in a longstanding pandemic. At this time, the pandemic was a shock to many people with various unknowns. Although conclusions regarding the overall impact of the COVID-19 pandemic on anesthesiology residents cannot be drawn, the initial responses and feelings from residents during this study period may parallel those felt during personal or global crises that our trainees may face.Implications for policy, practice, and researchIn contrast to our hypothesis, the COVID-19 disease burden at an anesthesiology residency program did not appear to significantly impact well-being scores compared to pre-pandemic levels based on our survey data. Residents most vulnerable to the impact of the COVID-19 pandemic on well-being appeared to be male and single participants. As a result, targeted well-being interventions, including those aiming to increase social support, for higher-risk resident groups may be indicated while making sure not to do this at the expense or to the detriment of other groups. Future research is warranted to provide insight into how this longstanding pandemic has affected residents’ work life and wellness, with particular attention to the dynamic nature of pandemic severity.The local COVID-19 severity at a residency program did not appear to disproportionately impact well-being scores among US PGY2 anesthesiology residents. Those most vulnerable to diminished well-being appeared to be male and single participants. Findings from this multi-site repeated survey study should be interpreted with caution and in combination with evidence from other relevant studies due to the low response rate. Future work is needed to assess the longstanding COVID-19 pandemic on resident well-being.


Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) play a main role in the regulation of angiogenesis and lymphangiogenesis. Furthermore, they are implicated in the onset of several diseases such as rheumatoid arthritis, degenerative eye conditions, tumor growth, ulcers and ischemia. Therefore, molecules able to target the VEGF and its receptors are of great pharmaceutical interest. Several types of molecules have been reported so far. In this review, we focus on the structure-based design of peptides mimicking VEGF/VEGFR binding epitopes. The binding interface of the complex has been dissected and the different regions challenged for peptide design. All these trials furnished a better understanding of the molecular recognition process and provide us with a wealth of molecules that could be optimized to be exploited for pharmaceutical applications. The development of molecules able to modulate the vascular endothelial growth factor (VEGF)-dependent angiogenic response is crucial to treat diseases depending on excessive vascularization, such as rheumatoid arthritis and psoriasis, degenerative eye conditions and tumor growth and metastasis, or on insufficient angiogenesis, such as ulcers and ischemic heart disease. Furthermore, molecules able to stimulate appropriate vascular support are also essential in tissue regenerative applications.Several types of molecules have been proposed to modulate (mainly to inhibit) the angiogenic response: antibodies; small organic molecules, especially tyrosine kinase inhibitors (TKIs); and peptide-like molecules. This last class of molecules has been largely explored as it presents the optimal features for targeting large protein–protein interaction surfaces as in the case of molecules targeting the complex VEGF/VEGF receptors (VEGFRs).For these reasons, the research of novel peptides for modulating the biological activity of the VEGF/VEGFR molecular system has been a fervent field in the last few years. Several approaches have been used: screening of linear protein sequences, phage-display peptide library, synthetic libraries and structure-based design. In this review, we intend to highlight the work conducted to develop molecular binders by structure-based design of peptides mimicking VEGF/VEGFR binding epitopes. The structure-based design of peptides targeting VEGF or its receptors has been mainly based on the identification of protein binding regions which can involve protein secondary structure motifs or loops, including the hot spot residues. These binding epitopes are the molds for modeling the peptide structures to obtain the binders which could be conformationally constrained to induce molecular stability and optimal tridimensional arrangements of the interacting side chains for target recognition.VEGF and its receptors play a pivotal role in the regulation of angiogenesis and lymphangiogenesis in vertebrates, driving the sprouting of new blood/lymph vessels from existing ones not only in physiological but also in pathological conditions including cancer. Angiogenesis is a process particularly active during embryogenesis, while during adult life, it is quiescent and limited to physiological phenomena such as the ovarian cycle, wound healing and tissue growth and repair . In factAll VEGF family members also interact with other types of membrane receptors: neuropilin-1 and -2 . NRPs lack cytoplasmic enzyme activity and, consequently, direct signaling capabilities and act as co-receptors of VEGFRs. Recently, NRP-1 has also been discovered as a co-receptor for SARS-CoV-2 viral entry, along with ACE2, and has thus become one of the COVID-19 research foci ,12.Considering the wide scenario involving the complex VEGF molecular system and the biological processes by which angiogenesis is activated, in the last few years, a growing interest has arisen in the predominant role of VEGF in processes other than the well-established physiopathological conditions such as cancer, ocular diseases and wound healing. Recently, the role of VEGF/VEGFRs in cellular rejuvenation and aging , as a neTo date, several pieces of structural information on VEGF/VEGFR family members have been reported based on NMR, X-ray crystallography, small-angle X-ray scattering and single-particle electron microscopy, elucidating how VEGF ligands bind to the Ig-like domains present in the extracellular portion of the receptors . These dTherapeutic angiogenesis is sought as the ultimate intervention to solve chronic ischemia and cardiovascular diseases in those conditions that cannot be treated alternatively. Furthermore, advances in therapeutic angiogenesis have been realized through the use of engineered biomaterials or by delivering angiogenic molecules as recombinant proteins . Its conCurrently, there are two major antiangiogenic therapeutic approaches: neutralization of VEGF/VEGFRs by monoclonal antibodies or engineered proteins that mimic VEGFRs and blockage of VEGF receptor kinase activity with small molecule inhibitors (TKIs) . The firIn the last few decades, pharmaceutical companies invested increasing resources in the development of peptide-based therapeutics, glimpsing the extraordinary potential of peptide molecules as a modern platform for drug development . The ava50, only if a titration was performed against the target) and the reference to the original work.In the following paragraphs, the peptide design of molecules targeting VEGF or VEGFRs is reported. The described molecules are summarized in Several peptides targeting VEGF have been reported , but theStarting from the complex of VEGF/VEGFR-2, the receptor residues within 4 Angstrom distance from the ligand were identified and reported in the linear sequence NGIDFNRDKFSGL. This sequence was submitted to the AntiCP webserver and then to the peptide ranker server with the aim of deriving a bioactive anticancer peptide. A molecular docking analysis was performed to find the more stable peptide–VEGF complex. The anti-VEGF peptide NGIDFNRDKFLFL was the best performer. To improve the conformational stability of the linear peptide, the selected sequence was grafted into the loop of disulfide-rich cyclic peptides SFTI-1 and MCoTI-II . However3-residues and cyclic β-residues to improve the stability of the structural motif. A 39-mer peptide was obtained with a high binding affinity (0.11 μM) and was able to reduce VEGF-stimulated EC proliferation -NHCH 621 μM) . The low 621 μM) . Howevertermined .50 of 52 μM (displacement test) and inhibits ERK1/2 activation and EC migration in vitro.A 25-mer bicyclic peptide was designed for the simultaneous reproduction of three VEGF regions: helix 16–26, loop 61–68 and the β-strand 102–107 . The seqPeptides reproducing the discontinuous binding site of VEGF involving the α1 helix and the β-hairpin region 79–93 were also described. These two regions belong to different monomers in the natural ligand, but their N-termini are in close proximity . The two native binding regions were covalently linked by spacers of diverse lengths. The biological characterization showed that these peptides showed a proangiogenic profile, and the most active compound, EP6, was characterized by NMR .A peptide able to block VEGFR-1 and VEGFR-2 was designed in consideration of the VEGF binding interfaces involved in the molecular recognition of both receptors. In particular, loop 3 (residues 83–91) and residues 61 to 64 of loop 2 were considered important . The pepThe VEGF/VEGFR molecular system has attracted great interest in drug discovery over the last few decades, and several molecules able to interact with either partner have been described, mainly antibodies, small organic molecules and, more recently, peptides. Here, we report a comprehensive overview of the peptide molecules targeting VEGF/VEGFRs developed so far by structure-based design approaches and the strategies pursued to develop them. The design and characterization of peptides mimicking the binding interface of the VEGF/VEGFR molecular complex allowed the accomplishment of structural and biological studies that deepened our understanding of the key elements responsible for the VEGF/VEGFR molecular recognition. Noteworthily, we expect that the design of even more sophisticated peptides targeting VEGF/VEGFRs will produce valuable drug candidates expected to enter clinical trials in the near future, providing novel options for biomedical applications both in the therapeutic and the diagnostic field.


Objectives: Pancreatic undifferentiated carcinoma accounts for 2%–7% of pancreatic carcinomas. We aimed to investigate the pathological and genetic characteristics of pancreatic undifferentiated carcinoma with osteoclast-like giant cells and the key points of treatment.Methods: The clinical data and follow-up results of four patients diagnosed with pancreatic undifferentiated carcinoma with osteoclast-like giant cells between May 2015 and May 2020 at the First Affiliated Hospital of Xi’an Jiaotong University were retrospectively analyzed.Results: Chief complaints included “pain and discomfort in the upper abdomen” (2/4), “nausea and vomiting” (1/4) or no symptoms (1/4). Preoperative mildly elevated tumor markers included carcinoembryonic antigen (1/4) and CA19-9 (1/4). The tumors were located in the tail of the pancreas in three patients and the head and neck in one patient. Tumor metastasis was found in pancreatic adipose tissue in two patients and lymph node metastasis in one patient, with microscopic heterogeneous mononuclear cells and scattered osteoclast-like giant cells of various sizes. One patient (1/4) had a mucinous cystic tumor of the pancreas, and two patients (2/4) had adenocarcinoma of the pancreatic duct. Only one patient received postoperative gemcitabine combined with albumin-bound paclitaxel chemotherapy.Conclusion: Currently, treatment guidelines are lacking for PUC-OGC, and prognosis varies markedly. More cases must be reported to clarify its origination. The long-term follow-up of diagnosed patients and genetic mutation testing can also contribute to improving treatment and prognosis of this disease. Pancreatic undifferentiated carcinoma with osteoclast-like giant cells (PUC-OGC), characterized by monocytes and osteoblast-like giant cells in the tumor tissue and first reported by Rosai in 1968  of all pClinical data of four patients diagnosed with PUC-OGC in the First Affiliated Hospital of Xi’an Jiaotong University from May 2015 to May 2020 were collected and retrospective analysed, including the patients’ general conditions , results of adjuvant examinations  of the pancreas, preoperative and postoperative tumor markers), histological characteristics , treatment  and follow-up information.The study had been performed according to the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University.The patient was a 71-year-old man hospitalized due to nausea and vomiting with progressive jaundice within 40 days. He underwent laparoscopic pancreatic body and tail excision with splenic modular resection and partial jejunostomy anastomosis at a local hospital. Macroscopically, an intraoperative occupying lesion at the duodenal papilla was seen. On 24 May 2017, he underwent whipple & splenectomy & radiofrequency ablation of liver metastases as palliative surgery in our hospital. A 3 cm oval-shaped hard mass of pancreatic origin was palpable in the lesser curvature of the stomach. Histomorphologically, the tumor exhibited PUC-OGC . Immunohistochemical staining demonstrated the following : CK , and the results demonstrated . Immunonstrated  at 3.35 nstrated  and negaPUC-OGC often occurs in elderly patients with no sex differences. Previous reports have indicated that risk factors for PUG-OGC are different from those for pancreatic ductal adenocarcinoma, with little relevance to smoking or alcohol consumption. PUC-OGC most commonly originates from the body or tail of the pancreas. Pathological features of PUG-OGC commonly include massive hemorrhage and necrosis, occasionally with visible calcification with clear margins. The clinical manifestations of PUC-OGC are non-specific and include abdominal pain, a palpable mass, weight loss, etc. The initial symptoms of the patients in this study were abdominal distension, abdominal pain, nausea, vomiting, progressive jaundice and other discomfort . One patThe histological origin of PUC-OGC has been controversial. Mesenchymal cells, epithelial cells, acinar cell progenitor cells, ductal cells, undifferentiated progenitor cells, stem cells, and even mucinous cystic neoplasm origins have been considered. However, most cases of PUC-OGC are currently believed to originate from pancreatic ductal epithelium changes in the interepithelial tissue. It has also been reported that PUC-OGC coexists with ductal adenocarcinoma. The latest WHO tumor classification standards regard PUC-OGC as a special subtype of pancreatic ductal adenocarcinoma. PUC-OGC is usually negative for cytokeratin and p53 but positive for vimentin, leukocyte common antigen, and macrophage markers such as KP1. Immunohistochemistry showed the positive expression of Vim, CD68 and CK in the 4 cases investigated in this report. Due to some pathologists’ subjective selection of immunohistochemically labeled antibodies, the source of OGC still needs to be further explored. Establishing the origin of PUC-OGC would be of great significance in guiding the choice of treatment.The increasing applications of serum tumor markers in PUC-OGC patients are helpful for screening patients; however, more specific diagnostic testing is needed. Among the four patients in this study, only one patient showed a slight increase in preoperative carcinoembryonic antigen, and one patient had a slight increase in preoperative CA19-9. The remaining tumor markers were negative. Preoperative abdominal ultrasonography and enhanced CT examination are helpful to make a definite diagnosis of malignancy. CT scan images can clearly show the size and location of the mass. Typical PUC-OGC can be observed by evident enhancement in the arterial phase, continuous enhancement of irregular solid and cystic solid masses in the portal vein phase , weak orWaddell et al.  classifiAccording to TCGA database , the KRAS gene has a mutation rate of 86%–95% in pancreatic cancer. It was reported that KRAS gene mutations in pancreatic cancer are found in more than 85% of all cases . At thisp = 0.032), which was more significant in individuals carrying the wild-type p53 gene (p = 0.021). According to the genetic test results of the patient in case 4 we reported, KRAS and TP53 mutations are the main driving forces leading to downstream signaling pathway activation and highly malignant disease. In addition, it shows that KRAS and TP53 mutations in PUC-OGC and PDAC can lead to the activation of oncogenes, thus targeted drugs for KRAS and TP53 oncogenic mutations are urgently needed.The TP53 gene encodes the tumor suppressor protein p53, which contains the N-terminal transactivation domain, the DNA-binding core domain, the tetramer domain and the C-terminal regulatory domain. The p53 protein inhibits the formation of urea by downregulating the key enzymes CPS1, OTC and ARG1 of the urea cycle, leading to the accumulation of ammonia, which affects the synthesis of polyamines and thus inhibits the proliferation and growth of tumor cells . Accordip = 0.04). The expression of PD-L1 was associated with a poor prognosis. Multivariate analysis confirmed that the all-cause mortality rate of PD-L1-positive PUC-OGC was three times that of PD-L1-negative PUC-OGC   is generally defined as the total number of somatic non-synonymous mutations per million bases in the genome of each tumor patient. These new mutations produce tumor-specific antigens and are an important factor in activating tumor-specific T cell responses . The disnal PDAC . ClinicaGiven the low disease incidence and paucity of available clinical outcome data, evidence-based guidelines for the treatment of PUC-OGC do not exist, even as part of broader PDAC treatment guidelines. In the United States, neither the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for PDAC nor the ASCO guidelines for curable or metastatic PDAC offer guidance on PUC-OGC treatment . HoweverPUC-OGC is relatively rare in clinical practice. It has been reported that PUC-OGC has a significantly different prognosis, with a median survival time of 11 months, while that for non-resectable cases decreases to 6.5 months ,30. We sAccording to our study, the median survival time of the 4 patients was 9 months, and all cases of them were resectable. It has been reported that the prognosis of resectable PUC-OGC is better than that of undifferentiated carcinoma without OGC, which may be due to the slow rate of metastasis and minimal metastasis to the lymph nodes. The degree of malignancy and prognosis of the tumor are not related to the number of giant cells. The factors that affect prognosis are still being investigated. If the tumor does not invade other organs and has no lymph node metastasis, the selection of an appropriate surgical method and individualized and precise adjuvant treatment after operation may prolong the survival time to a certain extent.via molecular diagnosis, surgery, chemoradiotherapy, targeted therapy, and immunotherapy among other aspects.Early diagnosis of PUC-OGC is difficult because of its occult onset, and the lack of specific clinical manifestations and blood tumor markers. Pathological analysis is essential to the diagnosis and treatment of cancer. Knowledge of the different histological types of pancreatic carcinoma and the targeted treatment after a clear diagnosis can contribute to prognosis of pancreatic carcinoma patients. In this article, we not only summarize the pathological and genetic characteristics of PUC-OGC but also provide a deeper understanding of pancreatic cancer for future reference. With the development of molecular biology and the continuous advancement in gene detection technology, it may be possible to uncover the genetic characteristics of PUC-OGC in the future, uncover its mystery, and create a foundation to bring new opportunities to manage for PUC-OGC


While effective lay-health worker models for mental health care have been demonstrated through efficacy trials, there is limited evidence of the effectiveness of these models implemented in rural LMIC settings.To evaluate the impact of a volunteer community-led intervention on reduction in depression and anxiety symptoms and improvement in functioning, and social participation among people living in rural Gujarat, India.Stepped-wedge cluster randomized controlled trial was used to assess the effectiveness of delivery of psychosocial intervention across 645 villages in Mehsana district of Gujarat, India between April 2017 and August 2019. The primary outcome was an improvement in depression and/or anxiety symptoms assessed using GHQ-12 at 3-month follow-up. Secondary outcomes were improvement in (a) depression and anxiety , Generalized Anxiety Disorder (GAD-7) & Self-Reporting Questionnaire-20 (SRQ-20); b) quality of life (EQ- 5D); c) functioning (WHO-DAS-12), and social participation . Generalized linear mixed-effects models were used to assess the independent effect of the intervention.Out of a total of 1191 trial participants (608- intervention & 583-control), 1014 (85%) completed 3-month follow-up. In an adjusted analysis, participants in the intervention condition showed significant recovery from symptoms of depression or anxiety  at the end of 3-months, with effects sustained at 8-month follow-up . Intervention participants had improved scores on the PHQ-9 (Adjusted mean difference (AMD) –1.8; 95%CI -3.0 to -0.6), and SRQ-20 , at 3-months and PHQ-9, GAD-7, SRQ-20, EQ-5D and WHO-DAS at 8 months follow-up.Atmiyata had a significant effect on recovery from symptoms of depression and anxiety with sustained effects at 8-month follow-up.Findings suggest that Trial registration details. The trial was registered prospectively with the “Clinical Trial Registry in India” (registry number: CTRI/2017/03/008139). Although depression and anxiety disorders affect approximately 6% and 4% of the Indian population , there aAtmiyata is a community-led intervention using non-specialized community volunteers for identification, support, treatment, and referral for persons with common and severe mental disorders [Atmiyata intervention aims to (i) reduce the treatment gap for common and severe mental disorders; (ii) improve mental health outcomes for people with common mental disorders; (iii) improve quality of life among people with mental disorders and (iv) improve access to social benefits for people with mental disorders. The intervention was previously piloted in 41 villages in Nashik district of the Indian state of Maharashtra from 2013–2015 [isorders . The Atm013–2015 . BuildinAtmiyata intervention on symptomatic improvement for people with depression and anxiety symptoms through a stepped-wedge cluster randomized controlled trial [This study presents findings on the effectiveness of the ed trial  in GujarThe stepped wedge trial followed a cross-sectional design , 11, witParticipants were adult community members residing in the Mehsana district with depression and anxiety symptoms. Inclusion criteria were persons aged 18–65 years with a score of ≥3 on the General Health Questionnaire (GHQ-12), indicating a ‘case’ of possible CMD. Exclusion criteria included people unable to provide informed consent, people with terminal medical conditions, and people presenting with suicidal ideation or suicidal behavior at the intake interview.As primary health centers (PHC) in India serve distinct villages, we used PHCs to identify discrete geographical areas, consisting of all villages under a PHC, which was used as a unit of clustering for the trial. In Gujarat, how PHCs are distributed geographically limits the movement of people between PHCs, which helps to minimize contamination of the intervention A PHC in Gujarat covers, on average, a catchment area of 25000–30000 people which spans across 12–13 villages. At the time of the study, there were 56 PHCs in the Mehsana district, and we created 4 equal groups of 14 PHCs . These groups of clusters  were allocated to sequential steps in 5-month intervals. This study used a cross-sectional design, where outcome data is derived from different participants at each period. A random sample of 56 participants was selected from each cluster for each period. Details of blinding and contamination are mentioned elsewhere [Champions were trained to identify people with CMD based on observed symptoms described by a participant during an unstructured interview. When a Champion identified a person, they thought had a CMD and intended to provide counseling sessions to the person, then they informed their supervisor and provided details of that person. The supervisor informed these details to the project manager, who created a screening list. Screening lists of each Champion were merged to create a master screening list, which formed the sampling frame for the intervention periods. Participants were randomly selected from the master screening list using a computer-generated random method. The trained data collection staff screened the participants using the GHQ-12. The participants who scored ≥3 were recruited for the trial until the target sample of 56 participants was reached for each group of clusters and each period.nth number being selected by the statistician. Since the prevalence of CMD is 4–8% [Participants in the control condition were selected from electoral rolls using a systematic random sampling method with a pre-decided random start and random interval, with every  is 4–8%  a screen is 4–8% .Enhanced Usual Care (EUC) was offered to all participants in the control condition who scored ≥3 on GHQ-12. EUC provided information on the impact of distress on physical and mental health and relevant information on available public mental health care services, including services by the District Mental Health Program (DMHP), and helplines for mental health support in and around the Mehsana district. The EUC also provided active support to participants in crisis. A crisis was defined as the participant revealing a recent self-harm attempt or expressing thoughts of self-harm during data collection. Such participants were encouraged to seek help immediately and data collectors sought participants’ consent to inform their family member or a friend about the crisis and mobilize social support to manage the crisis. This condition was considered as enhanced, as usual care provided in the community in these districts does not routinely provide information on mental health and distress or active support during a mental health crisis.Atmiyata intervention consists of community volunteers involved in the identification and delivery of mental health and social care support to people with CMDs. The intervention has been described extensively elsewhere [The lsewhere .Atmiyata community volunteers (Champions) are trained to (i) identify persons with CMD and provide 4–6 counselling sessions; (ii) raise community awareness on social issues impacting mental health, by ‘narrow-casting’ four 10-minute films dubbed in Gujarati on commonly experienced social issues in the community ; (iii) refer cases of severe mental illness (SMD) to mental health services in the public health system and (iv) facilitate access to social benefits.Champions receive 40 hours of training over 3 weeks, including practice-based counseling sessions. Champions are mentored and supervised by trained mental health professionals  every fortnight. Each Community Facilitator is responsible for 40–50 Champions .Champions deliver counseling sessions over 6 to 12 weeks, with no set time between sessions. Sessions lasted 20–40 minutes, based on three evidence-based techniques used in other similar programs , 4: actiThe number of sessions delivered is left to the champion’s discretion and participant consent. All sessions were in the local (Gujarati) language and Champions were expected to deliver the sessions as per protocol. Champions delivered the intervention at the participant’s home or a location preferred by the participant . As this was an implementation research study, we wanted to assess the intervention as implemented in ‘real-life’ settings where the number, duration, and frequency of sessions will vary between the different provider (Champion) and participant dyad. Broad guidelines and recommendations were therefore provided on the number, duration, and frequency of sessions to Champions  which both mimics what is likely to happen in real-life clinical settings and allows exploration of dose-response effects.Baseline data collection started in all groups of clusters in April 2017. The first cluster group (Group A) entered the intervention in September 2017 and the last (Group D) in December 2018.The primary outcome was the proportion of participants with symptomatic improvement in CMD in the intervention condition compared to the control condition, measured by the validated Gujarati version of GHQ-12 at a 3-month follow-up. GHQ-12 scores were expressed as both a continuous outcome   and as aSecondary outcomes include the accuracy of identification of CMD cases by champions and improvement in health-related quality of life using the EQ-5D . Euro quData on serious adverse events  was collected and reported to the ethical review committee in India (Indian Law Society Ethics Committee) and to the funding organization based on a protocol for reporting adverse events developed by the project team.The sample size was estimated using the stepped wedge function of STATA with the Hemming method . The plad and their respective 95% confidence intervals), while binary outcomes were analysed using logistic mixed regression models . Missing data was assumed to be missing at random and hence no imputation was performed, since multilevel mixed models inherently handle such missing data appropriately for statistical inference.Intervention effects were estimated from the model using methods described by Hussey and Hughes . Data weet al. [Demographic variables were assessed cross-sectionally between arms only at baseline, while primary and secondary outcomes were assessed between arms cross-sectionally at baseline, at the 3-month follow-up and at the 8 month follow-up separately , as described by Hemming et al. .As pre-specified in the protocol, we also analyzed model extensions: 1) random cluster by period effect, 2) random cluster by treatment effect, and 3) Interaction between intervention and time period. Details are described in the supplementary material. The model extensions were used for secondary analyses purpose. Each model extension makes implicit modifications to the assumptions about the correlation structure.STATA version 14 was used for all statistical analyses . All results are reported as per the CONSORT extension for stepped wedge cluster randomized trial guidelines .A total of 1191 participants were recruited (583 started in the control condition and 608 started in the intervention condition)  into theParticipant demographic characteristics at baseline are shown in Baseline covariates for GHQ were adjusted in the final regression analysis as the mean scores for GHQ were significantly higher in the intervention condition compared to control .Average GHQ scores were on par or above the recommended cut-off of 6 for primary-care attendees in India , and theThese effects were sustained at 8 months . Similarly, continuous GHQ-12 scores were significantly lower among participants in the intervention condition at the end of 3 months (adjusted mean difference (AMD): -0.1; 95% CI: -1.6 to -0.3; effect size 0.2) and 8 months   ICC for We performed several additional model extensions. First, when allowing for different time points across clusters, the estimated treatment effect increased  and the The champions’ case detection rate  as compared to actual GHQ-12 scores (cut-off score of 3 or more) was 58% for all the clusters and periods in the intervention condition.Data on counseling sessions attended was available for 89% (N = 543) of the intervention sample. Participants attended a mean of 5.2 (SD = 1.2) and a median of 6  sessions. At the 3-month follow-up, 2% (n = 11) of participants were referred to public health services (either the PHC or the DMHP) by the champions. In the control group, 52% (n = 244) were similarly referred to public health services.PHQ-9 depression scores were significantly lower among intervention condition participants compared to those in the control condition, with an adjusted mean difference (AMD) of -1.8  at 3 months and AMD of -4.4  at 8 months (p<0.05). PHQ-9 scores among intervention condition participants changed from moderate depression scores of 10–12 at baseline to mild depression scores of 5–9 (range of adjusted means) at 8-month follow-up (p<0.05). On anxiety symptoms as measured by the GAD-7, the intervention group did not show improvement  at 3 months whereas showed significant improvement -3.5  at 8 months follow-up (p<0.05). Among participants in the intervention condition, GAD scores changed from moderate anxiety symptoms at baseline to mild to none at 8-month follow-up. There was no significant improvement at the 3-month follow-up on functioning scores, as assessed by the WHO-DAS whereas showed improvement at 8 month . WHO-DAS scores increased significantly at 8 months follow-up in intervention participants with AMD of -3.2 (-5.9 to -0.4 effect size 0.2), -3.0  and -2.8  for W1, W2, W3 respectively. Compared to baseline, SRQ scores improved at 3 month  and at 8-month follow-up . Health-related quality of life assessed by EQ-5D, showed significant improvement 0.06  at 8 month follow-up .Atmiyata intervention at the end of 8 months.On the Client Satisfaction Questionnaire, the average score was 24.0 ±5.2 demonstrating a high satisfaction level with Considering the loss to follow-up rate was greater than 10% at the end of 3 months follow-up and 8 months follow-up, baseline measures between the participants who continued the intervention and those lost to follow-up in each condition were analyzed separately. For data at 3 months follow-up in the intervention condition, no significant differences were observed between the baseline values on the GHQ-12, PHQ-9, GAD-7, and SRQ-20) among participants who remained in the intervention compared to those lost to follow-up. In the control condition, the baseline values were significantly higher among participants who remained in the intervention (p<0.05).At 8-month follow-up, mean baseline values of measures were similar (p>0.05) in those who remained in the trial versus those lost to follow-up in both control and intervention conditions.Moreover, the power to detect observed differences between the intervention and control condition remained at 80% at the end of 3 months. For an 8-month follow-up, data were considered until period 4 so based on the observed difference with the sample size of 276 in intervention and 217 in control with 3 steps and 48 clusters the power remained 80%.The overall results across primary and secondary outcomes demonstrate that the intervention was effective, with effects sustained and improving over time, including at 8 months follow-up after counseling sessions ended.Atmiyata intervention is effective in reducing symptoms of common mental disorders and effects are sustained over time. Intervention participants with a common mental disorder were twice as likely to improve at a 3-month follow-up and three times more likely to improve at an 8-month follow-up compared to individuals receiving enhanced usual care. The intervention was also effective in reducing symptoms of depression and anxiety and improving quality of life at 3 months, and improving social participation and improving functioning at 8-month follow-up. In addition, Atmiyata Champions were reasonably skilled (58 percent specificity) at identifying persons with a common mental disorder compared to a structured measurement instrument (GHQ-12) administered by research staff.To our knowledge, this is the first study to systematically evaluate the large-scale implementation of a trained community volunteer-led intervention for common mental disorders using a pragmatic implementation research design in a low and middle-income country. Our findings show that the Our data shows that improvements in the primary outcome are enhanced over time, as indicated by the odds of improvement of depression and anxiety symptoms at 8 months follow-up being higher than at 3-month follow-up. Counseling sessions were completed before the 3-month follow-up data collection and no additional sessions were delivered afterward. This may suggest the intervention’s benefits continue even after the counseling sessions end. As expected, improvement in social functioning and reduction in disability is seen at 8 months but absent at 3 months, suggesting a lag between symptomatic improvement and functional improvement.Atmiyata intervention used community volunteers as compared to many other studies or programs of NSHW interventions which use either paid lay counselors or public health community workers to deliver mental health interventions. From a health system perspective, the effectiveness of community volunteers in improving depression and anxiety symptoms, as shown in this trial, is important for several reasons. First, in most LMICs, primary healthcare staff are overburdened with multiple health tasks and may lack the capacity to take on additional mental health tasks. Our intervention taps into existing community social capital and increases the pool of people available to deliver mental health interventions. Second, using community volunteers and a social model of mental distress to explain symptoms improved the acceptability of the intervention and reduced the stigma associated with seeking help for mental health problems. Third, the Atmiyata intervention included participants who had symptoms of depression or anxiety and required support but were not seeking or receiving care at PHC facilities. Studies have also found that most people seeking help in PHC for physical health symptoms displayed somatic symptoms, had low awareness of depression and few had a previous diagnosis of depression and/or received psychological treatments or anti-depressants [Atmiyata intervention locates distress in the context of difficulties in day-to-day living and social interactions rather than using a bio-medical health paradigm for understanding mental health distress. In our experience, we find this paradigm is closely aligned with the participants’ explanatory models and thus improves the acceptability and adherence to the intervention offered by Atmiyata Champions. Finally, using community volunteers reduces the intervention cost and thus, improves the sustainability of the intervention. Atmiyata is currently examining the feasibility and effectiveness of additional specific intervention components where there is significant community demand for support) and for specific sub-groups (e.g. women with post-partum depression). Additional funding has been secured to continue the Atmiyata program in the Mehsana district for three more years. We have also initiated a program of arranging local site visits for civil society organizations and policymakers across the country to interact with beneficiaries and gain first-hand experience of the effectiveness of the program. We plan to launch an online training module for policymakers to learn more about the Atmiyata program and encourage widespread adoption in diverse communities in India and other LMICs.The ressants , 26. TheAtmiyata program developed and used manuals outlining criteria to assess the suitability of potential Champions when selecting volunteers. There is also significant emphasis on activities to retain the motivation of volunteers including weekly and monthly support meetings and public recognition of their contribution to society.There are several challenges to implementing an intervention using community volunteers. First, any training or capacity building needs to emphasize the need to maintain confidentiality, especially when such volunteers are drawn from the same community where they are likely to have more than a passing acquaintance with those seeking help. Other challenges include identifying suitable volunteers and maintaining their motivation over time. The Atmiyata intervention provides evidence of the effectiveness of a non-specialist delivered mental health intervention for improving symptoms related to common mental disorders in an LMIC setting. This study contributes to the scarce implementation research evidence base for NSHW interventions in LMICs. The findings and the process of implementation of this intervention may support policymakers in India and other countries in decision-making related to what type of interventions to employ for addressing depression and anxiety in their context.The Atmiyata intervention may therefore not be a suitable standalone intervention but better placed as a core component of informal community care, with close linkages to primary and secondary mental health care for addressing more specialized care needs. Although a stepped wedge design has advantages over a parallel cluster design specifically related to constraints related to resources and the challenge of getting a complex intervention ready for implementation, it is challenging to ensure all the clusters are ready to implement on schedule, complicated to analyze as it requires certain assumptions and is dependent to time points if outcomes are already improving. However, the large sample size and a large number of clusters and steps in this trial allowed us to estimate and adjust for time trends. Another challenge is maintaining treatment fidelity when using community volunteers. Further, participants in this trial are community members who are not necessarily seeking treatment from a health provider and thus may be different in some respects from those presenting for healthcare at primary care clinics. We did not conduct a diagnostic interview at baseline (the gold standard) but used a locally validated screening tool (GHQ-12) and locally validated cut-off measures to determine ‘caseness’, or the presence of a common mental disorder [Atmiyata Champions as having a common mental disorder as compared to control participants, who were identified using the GHQ-12 as a screening instrument with randomly selected subjects from an electoral register. We interpret these differences at baseline as evidence that Champions are accurately identifying persons with significant mental health problems (moderate depression and anxiety), which may provide indirect evidence of the efficacy of training received. These baseline differences were adjusted during analysis. Despite the limitations, the pragmatic nature of this trial and limited exclusion criteria  enhances the generalizability of the findings.There are several limitations of this study. Not everyone improved; even at the end of 8 months, nearly 30% of those who received the intervention had significant symptoms of depression and anxiety. The tool GHQ- and locadisorder  recommendisorder  criteriadisorder . It is iThis study highlights several future research and practice directions. First, more evidence generated through pragmatic trial designs such as stepped wedge trials is needed in LMICs. Second, more evidence on the sustained implementation of community-volunteer-led models is needed, particularly evidence or models that consider aspects that impact sustainability such as incentives for community volunteers, waning motivation levels over time, and evolution of social dynamics in villages. Related to sustainability, Atmiyata is testing whether a social franchising model  might beCorrelation structures in an SW-CRCT design are more complicated than a parallel CRT where measurements are taken at a single cross-section only. While Hussey and Hughes’s model is commonly used for the analysis of SW-CRCT trials, it makes several assumptions such as the same underlying time trend across all the clusters, exchangeable or uniform correlation structure (observations within any cluster share the same correlation structure irrespective of how far apart in time measurement are taken) and intra-cluster correlation (ICC) is independent of time period of measurement. However, these assumptions may be untenable, and correlations might decay with increasing separation in time between observations, which can be studied in the alternative discrete-time decay model by Hooper (model extension 1). Thus, to handle the complex correlation structure in our study we performed model extensions.Our study may be regarded as a multi-centric study as there are many clusters and each cluster is exposed and unexposed to intervention therefore, treatment-effect heterogeneity can be investigated by including treatment by cluster interaction (model extension 2) This model allows the variability between clusters to differ between intervention and control periods. In addition to treatment heterogeneity across clusters, treatment effect might vary with time as SW-CRCT runs over multiple time periods and can be investigated using a treatment-by-time interaction (model extension 3). This model explores the interaction between intervention and time period, and modifies fixed effect components only with no modifications to the correlation structure. We present data on these model extensions exploring the sensitivities to deviations from the basic assumptions in Hughes and Hussey’s model around time points, treatment effect, and correlation structure.S1 Checklist(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Compared to the subjects in the control group, all subjects with prior COVID-19 had significantly higher levels of total IgG in induced sputum. In the group of patients who had had severe infection, total IgG in saliva was also higher (p < 0.05). A direct statistically significant correlation was also detected between the levels of total IgG in all studied samples and the levels of specific IgG antibodies against SARS-CoV-2 in the serum. A significant correlation was observed between total IgG levels and the parameters of physical and social activities, mental health, and fatigue levels. Our study demonstrated long-term changes in the humoral mucosal immune response, which were most pronounced in health care workers with a history of severe or moderate-to-severe COVID-19, and an association of these changes with certain clinical signs of post-COVID-19 syndrome.Coronavirus disease (COVID-19) has generated interest in the assessment of systemic immune status, but existing knowledge about mucosal immunity is clearly insufficient to understand the full pathogenetic mechanisms of the disease. The aim of this study was to evaluate the long-term effects of novel coronavirus infection on mucosal immunity in the postinfection period among health care workers (HCWs). A total of 180 health care workers with and without a history of COVID-19 who ranged in age from 18 to 65 years were enrolled in this one-stage, cross-sectional study. The study subjects completed the 36-Item Short Form (36) Health Survey (SF-36) and the Fatigue Assessment Scale. Secretory immunoglobulin A (sIgA) and total immunoglobulin G (IgG) levels were quantified in saliva samples, induced sputum samples, and nasopharyngeal and oropharyngeal scrapings by an enzyme-linked immunosorbent assay. Specific anti-SARS-CoV-2 IgG antibodies were quantified in serum samples by chemiluminescence immunoassay. Analysis of the questionnaire data showed that all HCWs with a history of COVID-19 reported health problems that limited their daily activities and negative changes in their emotional health three months after the disease, regardless of its severity. The following shifts were detected in the adaptive arm of the immune response in different mucosal compartments. Among subjects who had severe or moderate-to-severe COVID-19, salivary sIgA levels were significantly higher than those in the control group ( Over the past two decades, new viral diseases have emerged and become a major public health threat worldwide. In December 2019, an outbreak of a new highly contagious infection caused by SARS-CoV-2 emerged in Wuhan, China, which subsequently swept the world and took the form of a pandemic. Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, which is characterized by high contagiousness and a severe course. HCWs are at high risk of contracting COVID-19. Our data are based on the results of a survey conducted among doctors and nurses working in one of the hospitals in Moscow.4. It was shown that COVID-19 dampened the circadian variation in core body temperature, circulating leukocytes and neutrophils, shifted the circadian variation in heart rate and caused uraemia5.Clinical manifestations of COVID-19 have been extensively researched. A rich semiology is reported in the literature. In addition to the cardinal signs of COVID-19 , cough (68–85%), and sputum (23–41%)), several other signs have been reported: anorexia (84%), asthenia (70–80%), headache (52–55%), ageusia (43%), anosmia (37%), abdominal pain (25%), neuromuscular involvement (19%), dyspnoea (18–85%), odynophagia (19–20%), myalgia (15–48%), confusion (15%), chest pain (6–20%), stroke (6%), rhinitis (5%), nausea/vomiting (4–5%), diarrhoea (4–15%), and haemoptysis (0–5%)9. Currently available serology tests assess immunoglobulin M (IgM) and IgG antibodies against the spike (S) and nucleocapsid (N) proteins of SARS-CoV-2. However, protective antibody levels are still under investigation11. The appearance of antibodies is paralleled by a gradual reduction in viral load, but serum antibodies can neutralize the virus in the blood, which is not sufficient for pathogen elimination12. The humoral mucosal immune response plays a crucial role in fighting COVID-1915.The new disease has generated a great deal of interest in the assessment of systemic immune status, but existing knowledge about mucosal immunity is clearly insufficient to understand the full pathogenetic mechanisms of the disease. The role of antibodies in neutralizing SARS-CoV-2 and offering protection from reinfection is well established. Humoral immunity against SARS-CoV-2 has been evaluated mainly at the system level in patients with a history of novel coronavirus infection (COVID-19) both with acute disease and in the convalescent phase17. The adaptive arm of mucosal immunity contributes greatly to viral eradication, but these processes have rarely been studied18.SARS-CoV-2 primarily affects the upper respiratory tract. The immune response is initiated in the nasal and oropharyngeal mucosa, where nonspecific factors of innate immunity fight pathogens, particularly SARS-CoV-2. The immune response at the mucosal level is implemented through a single structured and highly specialized system, mucosa-associated lymphoid tissue (MALT), which consists of epithelial cells, lymphoid structures and submucosal immune molecules. The main components of mucosal immunity include saprophytic microflora, the epithelial cell layer, keratinization and salivary processes, antimicrobial peptides , the complement system, lymphoid cells of innate immunity, natural killer cells, sIgA, IgG production and other factors19. Preliminary studies have demonstrated high secretory levels of specific neutralizing sIgA in adults with acute COVID-1922. Immunoglobulin G is also found on the mucosal surfaces of the upper respiratory tract and is mainly derived from the blood circulation by passive leakage in the area of the gingival crevicular epithelium, but some is produced locally23. This class of immunoglobulin is known to have a high affinity, play a certain role in the recruitment of phagocytes and natural killer cells, activate the complement system, block the active centres of pathogens, and neutralize toxins24.Secretory immunoglobulin A is the main antibody class found on mucosal surfaces and produced by local plasma cells, primarily as an IgA dimer. It confers protection against various pathogens, including viruses, which is mediated by its neutralizing properties as well as its ability to prevent adhesion of pathogens to the mucosal surfaces and to opsonize pathogenic microorganisms, thus hindering their penetration into epithelial cells25. In recent studies among convalescent COVID-19 patients, a wide variety of persistent systemic symptoms have been reported, which has resulted in the introduction of new terms such as post-COVID-19 syndrome, long COVID-19, and chronic COVID-19. In October 2021, the World Health Organization (WHO) proposed a consensus definition for what they referred to as the “post-COVID-19 condition”. The condition was defined as the presence of symptoms lasting for at least 2 months in individuals with a history of probable or confirmed SARS-CoV-2 infection26. According to the WHO, this condition usually manifests 3 months from the onset of acute illness but cannot be explained by an alternative diagnosis. It is now clear that long COVID-19 has become a meaningful public health concern, given that it now affects millions of people worldwide27. Is there a correlation between immunological and clinical parameters in the post-COVID-19 period? Can shifts in humoral immunity be regarded as a sign or a component of post-COVID-19 syndrome? To date, there have been few studies on changes in the mucosal immune response, particularly the protective role of sIgA and IgG in subjects with a history of COVID-19.The absence of SARS-CoV-2 in the post-COVID-19 period does not necessarily mean complete recoveryThe aim of this study was to evaluate the effects of novel coronavirus infection on the humoral factors of mucosal immunity in health care workers. The study objectives included an assessment of clinical condition using quality-of-life and fatigue questionnaires; an evaluation of the levels of total sIgA and IgG in the respiratory tract mucosa against the severity of the prior disease; and the investigation of a correlation between sIgA and IgG levels in different compartments of the upper and middle respiratory tract and between sIgA and IgG levels and clinical signs of post-COVID-19 syndrome.Participation in the study was offered to 220 medical workers of the D.D. Pletnev hospital, Moscow, who ranged in age from 18 to 65 years who had had COVID-19 between April 2020 and May 2021 and those with no history of this infection. Voluntary informed consent to participate was signed by 202 subjects, thus the response rate was 91.8%. The exclusion criteria were recorded in 22 subjects, the main ones were: taking immunomodulatory or antiviral drugs within the last six months prior to the start of the study (n = 12 (54.5%)), immunization with any vaccine within 30 days prior to enrolment (n = 7 (31.8%)), presence of autoimmune disorder (n = 3 (13.6%)) or severe chronic diseases (n = 2 (9.1%)).29. One hundred thirty-six subjects with a history of COVID-19 were divided into three groups depending on the severity of the prior30 disease: (1) severe disease (n = 16); (2) moderate-to-severe disease (n = 71); and (3) mild or asymptomatic disease (n = 49). The control group consisted of 44 medical workers who had no history of COVID-19  were included in this one-stage, cross-sectional study 125.1 ± 71.1 days after disease onset. The Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants of the virus prevailed in Russia at the specified timeThis study included two visits but did not have a follow-up period.At Visit 0 (Day 0), all subjects voluntarily signed the informed consent form, which met the requirements of the World Medical Association Declaration of Helsinki (2013), the Universal Declaration on Bioethics and Human Rights adopted on October 19, 2005, and the Universal Declaration of Human Rights adopted by the United Nations General Assembly on December 10, 1948. The study protocol was approved by the local ethics committee at Pirogov Russian National Research Medical University . All research was performed in accordance with relevant guidelines/regulations.31.At Visit 1 (Day 1), clinical assessments were performed, including a detailed medical history and complaints and physical examination. The study subjects completed the SF-36 quality-of-life questionnaire and the FAS questionnaire. The Charlson comorbidity index was used to assess all the subjects. Salivary samples and nasal and oral mucosal epithelial scrapings were obtained. Samples of induced sputum were collected after inhalation of 5% hypertonic saline (fixed concentration) for up to 30 min in accordance with the standardized procedureThe following inclusion criteria were applied: age 18–65 years; history or no history of COVID-19; and voluntary signed written informed consent form.The exclusion criteria were refusal to participate in this study; severe birth defects or severe chronic diseases; a history of cancer; a positive HIV or hepatitis B or C test in anamnesis; use of immunoglobulin or blood transfusion within the last three months prior to the start of the study; prolonged use (more than 14 days) of immunosuppressive, immunomodulatory or antiviral drugs within the last six months prior to the start of the study; any surgery or oral inflammation within one month prior to enrolment; any known or suspected immunosuppressive, immunodeficiency or autoimmune disorder; chronic alcohol abuse and/or a history of drug use; immunization with any vaccine within 30 days prior to enrolment; prior immunization with an experimental or approved SARS-CoV-2 vaccine; resolved acute infection within less than a month prior to the start of the study; pregnancy or lactation; simultaneous participation in another clinical study; and inability to comply with the study protocol requirements.32, i.e., an assessment of direct items, where higher scores indicated a greater influence of the item on quality of life , and reverse items, where higher scores indicated a lower influence of the item on quality of life . Interpretation of the FAS data included an assessment of physical and mental scores as well as the total score, with values of the total score > 22 indicating substantial fatigue33.Clinical assessments were performed, including a detailed medical history and complaints and physical examination and analysis of chest computed tomography data. The primary data from the SF-36 questionnaire were interpreted using a predefined methodLaboratory studies were carried out as samples were received from June 19, 2020, to October 22, 2021. Levels of immunoglobulins in mucosal secretions were measured by enzyme-linked immunosorbent assay  based on a two-step sandwich enzyme immunoassay using monoclonal antibodies (mAbs) against IgG and the secretory component linked to the alpha chain of IgA. The tests were performed strictly in accordance with the manufacturer's recommended protocol.Standards with known concentrations of sIgA and the samples were added to the wells of a plate coated with an anti-sIgA mAb. The plate was then incubated according to the test kit instructions. The intensity of the developed colour was proportional to the concentration of sIgA in the sample. The concentration of sIgA was calculated using the standard curve and measured optical density values.First, standards with known concentrations of total IgG and samples were added to the wells of a strip plate coated with an anti-human IgG gamma chain mAb and then incubated. Next, the bound IgG in the plate wells was treated with a conjugate of peroxidase and anti-human IgG lambda and kappa light chain mAbs.The resulting immune complexes consisting of immobilized mAbs, IgG, and the conjugate were detected by an enzymatic reaction with tetramethylbenzidine as a substrate. The intensity of the developed colour was proportional to the concentration of total IgG in the sample. The concentration of total IgG was calculated using the standard curve and measured optical density values.Quantitative determination of IgG antibodies to SARS-CoV-2 was based on a two-step sandwich immunoassay (SARS-CoV-2 IgG (CLIA), Shenzhen Mindray Bio-Medical Electronics Co., Ltd., China).First, serum specimens were incubated with paramagnetic particles coated with a SARS-CoV-2 antigen. A monoclonal mouse anti-human IgG alkaline phosphatase conjugate in MES buffer was then added to the reaction vessel. After incubation, the conjugate became bound to the anti-SARS-CoV-2 IgG antibodies captured on the paramagnetic particles. Finally, the chemiluminescent signal was measured and expressed in relative light units (RLUs). The chemiluminescence intensity in RLUs was proportional to the number of anti-SARS-CoV-2 IgG antibodies in the sample. The concentration of anti-SARS-CoV-2 IgG antibodies was determined against the standard curve.Statistical analysis was performed by parametric and nonparametric methods using IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. software.p < 0.05. The correlation between two parameters was evaluated by Spearman correlation analysis.Descriptive statistics were used to analyse numerical variables. The normality of the distribution of variables was determined using the Shapiro–Wilk test. Parametric criteria were used for normally distributed variables. In this case, the significance of differences in quantitative parameters between the groups was determined using the unpaired Student’s t test. In the absence of a normal distribution of variables, the significance of differences was assessed using the nonparametric Mann–Whitney U test. Depending on the type of distribution, the following measures of central tendency and dispersion were computed: means and root mean square (standard) deviations (M ± SDs) or medians and 25th and 75th percentiles . The differences were considered statistically significant at This study was conducted 125.1 ± 75.0 days following the onset of the first symptoms. The health care workers with a history of severe or moderate-to-severe novel coronavirus infection were older and had a higher body mass index (BMI) and Charlson comorbidity index than subjects with a history of mild or asymptomatic disease and those with no history of COVID-19  and between the levels of sIgA in the oropharynx and salivary gland secretions . A direct interrelation was detected between sIgA levels in induced sputum and salivary gland secretions . An inverse correlation was demonstrated between the levels of total sIgA in all mucosal compartments and the number of days between the onset of disease and the study period .A tendency towards correlation was observed between the levels of sIgA in the oropharynx and nasopharynx , on the other hand Table .Table 5C34.Analysis of the relationship between sIgA and IgG demonstrated a direct interrelation between these parameters in the same mucosal compartments and in matched samples of glandular secretions from the upper and middle respiratory tract. A direct correlation was also detected between the levels of total IgG in all mucosal compartments and the serum levels of specific IgG antibodies against SARS-CoV-2 Table . In cont35) than the rate of mortality in the general population reported in the literature, better overall health and care among HCWs, different age demographics, and other factors could explain these differences. However, due to exposure to numerous infected individuals, HCWs, if infected, could be characterized by a higher viral load, which is associated with worse clinical outcomes36.Our study population included HCWs who were at high risk of contracting novel coronavirus infection, becoming asymptomatic carriers and therefore passing the virus on to patients or colleagues. Although the mortality rate among HCWs is lower  of the S protein measured long after recovery (up to 115 days) were higher than those in the control group34. Randad et al. also evaluated the correlation of results obtained from saliva vs. serum and determined the sensitivity and specificity for each diagnostic medium, stratified by antibody isotype, for the detection of SARS-CoV-2 infection based on COVID-19 case designation for all specimens; matched serum and saliva SARS-CoV-2 antigen-specific IgG responses were significantly correlated43.We found that, unlike sIgA, total IgG was elevated not only in saliva but also in samples of induced sputum from patients who had severe or moderate-to-severe COVID-19. This is consistent with the data reported by Isho B. et al., who showed that anti-SARS-CoV-2 IgG levels were strongly correlated across paired serum and saliva samples, indicating that saliva could be used for monitoring the immune response to SARS-CoV-2 infectionLevels of total IgG correlated with those of sIgA in the same mucosal compartments, anti-SARS-CoV-2 IgG, and some parameters of general health. Levels of IgG in secretions and mucosal scrapings from the upper respiratory tract did not correlate with the number of days post-disease onset. However, we did not measure titres of specific mucosal antibodies.44. Upon recovery, asymptomatic patients exhibited sustained salivary IgG antibodies against SARS-CoV-245. The nasal mucosa is the entry point for SARS-CoV-2. Our study demonstrated that subjects who had mild or asymptomatic disease had significantly (twofold) higher levels of immunoglobulins, which is evidence for strong protection from reinfection. Subjects who recovered from severe or moderate-to-severe COVID-19 had three- or fivefold lower levels of the studied parameter, which may be a sign of immunodeficiency associated with damage to epithelial cells by the virus, as well as, probably, age-related atrophy of the nasal mucosa epithelium, given the predominance of the older category of persons in these groups. The obtained results are not described in the literature and require further study.Interestingly, subjects with a history of severe COVID-19 had lower titres of IgG in the nasopharynx. The study by Alkharaan H. et al. showed that salivary IgG antibody responses in patients with a history of mild COVID-19 were detectable up to 9 months after recovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in the blood and saliva of most patients. Salivary IgA was rarely detected at this time point. Scientists have also demonstrated the tolerance of salivary IgG to temperature and detergent pretreatmentsOur study has a number of limitations. First, it was a one-stage study, which means that we could not follow up on the changes in clinical signs or levels of mucosal immunoglobulins. Nevertheless, the study results suggest the presence of long-term changes in post-COVID-19 syndrome. Second, the absence of strain detection among our participants is also a limitation of our study. Finally, we measured only total sIgA, but it is highly likely that its elevation is a sign of a specific anti-SARS-CoV-2 immune response in the respiratory mucosa. Therefore, this opens up new possibilities for using these laboratory tests to assess the level of mucosal immunity without the need for costly methods. The same is true for evaluating total IgG in different mucosal compartments.46 and may contribute to the understanding of post-COVID syndrome in the context of immune changes.Quantification of salivary IgA and IgG antibodies can elucidate mucosal and systemic immune responses after natural infection in the post-COVID-19 period before or after vaccination. Noninvasive saliva-based SARS-CoV-2 antibody measurement may be a complementary alternative to conventional blood serology. The preliminary findings suggest that the measurement of salivary and serum IgG and IgA merits further investigation as markers of current or recent SARS-CoV-2 infectionWe investigated only a few parameters of mucosal immunity, it was found that individuals who had had a severe and moderate course of infection had elevated levels of total sIgA in saliva samples, as well as levels of total IgG in all areas of the mucous membranes of the respiratory tract, with the exception of the nasal cavity. Elevated levels of immunoglobulins three months after the infection may indicate the persistence of inflammatory reactions, which also correlates with the manifestations of post-COVID syndrome and requires further study. Local immunodeficiency in the nasal cavity may indicate viral damage to epitheliocytes, which might lead to frequent respiratory infections, and probably requires additional immunocorrective approaches aimed at enhancing mucosal immunity, for example, nasal form of medication containing bacterial ligands or recombinant interferons.Although the upper respiratory tract mucosa is where the virus replicates and the immune response is initiated, there are few publications on anti-COVID-19 mucosal immunity, its impact on the course of the disease, and its status in the post-COVID-19 period. Our study demonstrated long-term changes in the humoral mucosal immune response, which were most pronounced in health care workers with a history of severe or moderate-to-severe COVID-19, and an association of these changes with certain clinical characteristics of health care workers with post-COVID-19 syndrome. Studying the humoral immunity of the respiratory mucosa is likely to improve the understanding of the immune response to this emerging disease, help contain the pandemic develop approaches for immunorehabilitation of persons who have had COVID-19 and effective vaccines aimed at protecting the mucous membranes as a major gateway for viral infections.


Social stigma associated with infectious diseases existed throughout the history of pandemics due to fears of contagion and death. This study aims to assess social and self-stigma resulting from COVID-19 infection and other associated factors in Egypt during the pandemic.A cross-sectional study was conducted on 533 adult Egyptians via an online questionnaire. The questionnaire included social stigma toward current and recovered COVID-19 patients and the negative self-image of being a COVID-19 patient.The mean calculated overall COVID-19-related stigma score for the studied sample was 4.7±3.1. The highest reported stigma category was mild stigma: Social stigma towards current COVID-19 patients (88.2%), Social stigma toward recovered COVID-19 patients (64.2%), Negative self-image for being a COVID-19 patient; perceived self-stigma (71.6%) and total stigma score (88.2%) respectively. The overall stigma score was negatively associated with a higher level of education and getting information from healthcare workers and positively associated with getting information from social networks.Social and self-stigma related to COVID-19 infection was mild from the Egyptian perspective but found in a large proportion of the population and mainly affected by getting information from healthcare workers or through social media and being more among those with lower education levels. The study recommends more legislative control on social media for disseminating health-related information and conducting awareness campaigns to counteract these adverse effects. The social stigma associated with diseases has been defined as a negative social judgment that results in refusal and/or rejection. This negative judgment may be directed towards those suffering from diseases or self (self-stigma) . StigmatOver the last decades, social stigma has been associated with various mental disorders and sexually transmitted diseases . HoweverThere have been outbreaks of Ebola and Zika viruses in the past ten years, in addition to an influenza A (H1N1) in the 2009 pandemic, where an associated stigma was reported . This inRecently, the COVID-19 pandemic has affected the whole world; it is caused by the SARS-CoV-2 virus and has had multiple genetic variants and a myriad of related symptoms in addition to a rising toll of fatality . The feaThe double burden of previous quarantine enforcement by law and both dis/misinformation on social media are also direct causes of stigma ; other dMost research reported experiencing stigmatization in individuals not only with current COVID-19 infection and in those recovered patients and their families, Asian minorities, and healthcare workers (HCWs) who have experienced COVID-19-associated stigma , 13–15. A cross-sectional study was conducted in July 2021 among 553 adult Egyptians. A sample size of 334 participants was calculated at a 95% confidence level and an alpha error of 5% using Epi Info 7 software based on the prevalence of stigma among Egyptian healthcare workers (HCWs) who are involved in treating COVID-19 patients as they were the closest population in culture and perceived attitudes to our target population .An online questionnaire was designed following the standard approach suggested in the behavioural insights research for COVID-19 by guidance to the Member States in the WHO European Region , the modThe online questionnaire was distributed through various social networking packages using the snowball sampling method, starting from known individuals, and then they were asked to share the survey with others. The estimated time to fulfil the questionnaire was about 10–15 minutes.The questionnaire started with an introduction that explained the study’s rationale and objective, explaining that participation is voluntary, ensuring the confidentiality of responses and that completing the questionnaire implied the participants’ consent to participate in the study .The questionnaire was designed in the native language of Egypt; Arabic, and was formed of 3 sections:Sociodemographic questions: the section included questions about age, gender, education, and residence.Sources of Knowledge regarding COVID-19: using multiple options formats, which included social networks, the Egyptian Ministry of Health and Population website, Health care workers, Television and radio, posters and brochures, and Newspapers and magazinesQuestions about COVID-19 infection stigma, formed of 3 sub-scales: social stigma toward current COVID-19 patients (12 questions), Social stigma toward recovered COVID-19 patients (6 questions) and negative self-image if being a COVID-19 patient (perceived self-stigma) (7 questions). The respondents were then given a three-point Likert scale . For each response denoting stigma, a score of one was given, while the responses denoting no stigma scored 0.The total score was calculated by adding up the responses denoting stigma in each sub-scale  and then converting it into a percent-score (by division by the maximum score and then multiplying by 100). Then, using the same method, the three sub-scales were added to calculate the total COVID-19-related stigma score.The current study followed the methodology of previous studies methods in categorizing stigma due to the lack of universal cut-off points for stigma scores , 19–21. The content validity of the questionnaire was evaluated by ten experts of different disciplines: family medicine, public health and tropical medicine, Suez Canal University. After analysing the views of the experts, the authors revised language and questions that were unclear, confusing, or perhaps unacceptable to the participants. The questionnaire was then examined for face validity in a pilot research with 15 participants to ensure the questions’ clarity, appropriateness, and simplicity, as well as their multiple-choice format. Minor modifications of a few questions followed feedback from the pilot study.Confirmatory factor analysis (CFA) was conducted to assess construct validity. The CFA findings revealed a statistically significant association between the survey items. The Cronbach’s alpha values were used to assess the questionnaire’s internal consistency. Cronbach’s alpha = 0.753 for the full scale was found in the current study’s pilot trial, showing acceptable reliability . HoweverData were coded, entered, and analysed using Microsoft Excel version 2016. Data analyses were performed using IBM-SPSS software version 22.0. Sociodemographic characteristics, sources of Knowledge regarding COVID-19 and answers to stigma questions were presented using descriptive statistics, including frequencies and percentages (%). The scores for the stigma total and subscales were presented using mean ±SD.A Chi-square test was used to test the statistical significance of categorical data. Fisher’s exact test was used whenever Chi-square test assumptions were violated . Multivariable regression analysis was used to assess factors affecting the overall COVID-19-related stigma score. P-value < 0.05 was considered statistically significant.The study was approved by the Research Ethics committee (REC) of the Faculty of Medicine, Suez Canal University, with a code of 4203. The study participants provided electronically written informed consent after being informed about the purpose of the study and the importance of the online form before data collection.A total of 553 participants were studied. Most of the participants were within the age range of 18–65 years old. Most of them  were females, and with university level of education . The most reported Source of Knowledge regarding COVID-19 in order was through social networks , the website of the Egyptian Ministry of Health and Population  and healthcare workers , while the least reported source was Newspapers and magazines  .As regards social stigma toward current COVID-19 patients, the most reported situations were: not dealing with people coming back from abroad even while observing precautionary measures , being afraid of dealing with people who just come back from abroad , not supporting a close friend who has been infected with COVID-19 , being afraid of a neighbour, a close relative or friend working in the medical field  and victims of COVID-19 should be buried away from usual burring places . However, there was no stigmatization directed towards poor and uneducated people. Also, the least reported stigmatizing situations were: COVID-19-infected persons should feel ashamed  or be blamed for their illness .As regards social stigma toward those currently infected with COVID-19, the most reported s situations where: after recovery, they were not able to usually deal with COVID-19 patients released from quarantine  or allow their family members to do so either . However, all other situations were reported less than 10%.Regarding negative self-image regarding being a COVID-19 patient (self-stigma), the most reported situations where it would be a catastrophic situation if people suspected their infection with COVID-19  they would keep it secret if one of their family members were infected , they would also keep it secret if they have infected themselves , in addition to feeling guilty if got the infection and blame it on their careless behaviours and not following social distancing . They would also keep it secret if one of a family member died due to their infection . However, all other situations were reported with less than 10%.The mean overall COVID-19-related stigma score was 4.7±3.1. The mean scores for the subscales were: 1.35±1.6 for social stigma toward current COVID-19 patients, 1.1±1.5 for social stigma toward recovered COVID-19 patients, and 2.1±1.4 for negative self-image if being a COVID-19 patient. The highest reported stigma category, for sub-scales and total, was mild stigma as the following: Social stigma toward current COVID-19 patients , Social stigma toward recovered COVID-19 patients , negative self-image for being a COVID-19 patient; perceived self-stigma  and total stigma score  respectively .The relation between general characteristics and total stigma score was not statistically significant  . The relAs regards the multivariable regression models for predicting the total stigma score, participants’ overall COVID-19-related stigma score was negatively associated with higher level of education , positively associated with using social networks for information  and negatively associated with getting information from health care workers . These relations were all statistically significant  .The results of the current study reported stigmatization of mild degree among more than three-quarters of the studied participants toward current COVID-19 patients (88.2%), about two-thirds toward recovered COVID-19 patients (64.2%), and about three-quarters for negative self-image for being a COVID-19 patient; perceived self-stigma (71.6%) respectively.This was consistent with what was found by Sangma et al.  in theirThis relatively lower degree of stigma but in large proportion scale of the studied population could be explained by the fact that the pandemic, by the time of data collection, was at its second year with widely distributed active cases all over the country . This maIn Egypt, a recent survey conducted by the Central Agency for Public Mobilization and Statistics (CAPMAS) reflected the effect of the slowdown in economic and business activity, as more than a quarter of respondents stated losing their jobs due to the COVID-19 pandemic sequences. This uprising of financial hardship due to increased unemployment has increased grievances and social stigma .Stigma towards COVID-19 is different from the case of HIV in one significant aspect. COVID-19-related stigma is mainly due to the fear of its ease of transmissibility, high spread, and fatality among the population, while that related to HIV is linked to the unacceptable break of religious boundaries in Egypt, where the main religions are Islam, followed by Christianity , 25. ThiCOVID-19-related stigma was negatively associated with a higher level of education in the current study. This association is consistent with the finding of Abdelhafiz et al.  study onThe higher level of education provides enhanced access to correct and precise information and, as a result, builds a well-established stone of Knowledge about infectious diseases, infectiousness, and preventive measures. This, in turn, facilitates their ability to discriminate between accurate information and misinformation. On the contrary, those with poor Knowledge, as a disadvantage given by the lower level of education, got misled by the factitious news and wrong information .Conversely, COVID-19-related stigma was positively associated with using social networks for information and negatively associated with getting information from healthcare workers in the current study. That finding agreed again with Yuan et al.  systematSocial media provides both an opportunity and a challenge for the public during the COVID-19 pandemic. The opportunity is that it allows information about the disease and prevention but puts them on a great challenge of being affected by misinformation, especially when they are not competent enough to distinguish it, becoming a direct source of fear, anxiety, and stigma .The current study findings should be viewed in light of some limitations. The study’s observational nature was conducted to explore the situation in this new area of inquiry. It was not used to infer causal relationships. And due to the COVID-19 critical situation to achieve social distance, the researchers used the online data collection method. Consequently, the researchers recommend conducting further studies using face-to-face interviews using a probability sampling technique.Social and self-stigma related to COVID-19 infection were mild from the Egyptian perspective but found in a large proportion of the population. However, the perceived stigma was mainly affected negatively by the source from where the population got their health-related information. On the other hand, getting this information from healthcare workers better impacts perceiving stigma. Finally, being with lower education levels makes the population more vulnerable to stigmatizing COVID-19 cases.Based on the current study’s findings, we recommend more legislative actions for the control of social media regarding disseminating health-related information and to increase awareness campaigns to counteract the adverse effects of getting such information from social media instead of the suitable channels for this.S1 Appendix(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.