Understanding antibody persistence concerning multimorbidity is crucial for vaccination policies. Our goal is to assess the link between multimorbidity and serological response to SARS-CoV-2 nine months post-first vaccine. We analyzed Healthcare Workers (HCWs) from three cohorts from Italy, and one each from Germany, Romania, Slovakia, and Spain. Seven groups of chronic diseases were analyzed. We included 2941 HCWs . Multimorbidity was present in 6.9% of HCWs. The prevalence of each chronic condition ranged between 1.9% (cancer) to 10.3% . Two regression models were fitted, one considering the chronic conditions groups and the other considering whether HCWs had diseases from ≥2 groups. Multimorbidity was present in 6.9% of HCWs, and higher 9-months post-vaccine anti-S levels were significantly associated with having received three doses of the vaccine  and with having a prior COVID-19 infection . Conversely, lower levels were associated with higher age , more time since the last vaccine dose , and multimorbidity . Hypertension is significantly associated with lower anti-S levels . The serological response to vaccines is more inadequate in individuals with multimorbidity. Vaccination against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been one of the milestones in the history of infectious disease prevention. Both the rapid development of vaccines and the speed with which vaccines have been administered have made this possible. Currently, the ω variant is dominant in the world. The disease caused by this variant is less severe and results in fewer hospitalizations and deaths, but it is more transmissible ,2,3,4,5.Healthcare workers (HCWs) are a professional group particularly exposed to SARS-CoV-2 infection . TherefoVaccines against SARS-CoV-2 are effective in preventing severe cases of COVID-19 and reducing mortality ,11. The The long-term immunity induced by vaccines is essential for protection against rapidly emerging mutant strains of SARS-CoV-2. In a previous study conducted in the ORCHESTRA cohort, we demonstrated that, at 9 months, antibody levels showed a decreasing trend over time without analyzing whether this was dependent on previous diseases or multimorbidity . SeveralMultimorbidity, which refers to the presence of two or more chronic conditions in an individual , has beeA previous study has demonstrated that multimorbidity is associated with weaker initial antibody response and cellular immunity, which could impede virus clearance and lead to a more severe disease progression . Other aLittle is known about the changes on humoral response after vaccination, depending on the type of previous disease, and even less is known about how the presence of two or more chronic diseases affects the humoral response. Therefore, understanding whether the humoral response is affected by previous diseases or not is important for future vaccination policies against SARS-CoV-2, as it helps to identify a group of individuals who are more prone to worse outcomes when infected with SARS-CoV-2.Thus, we aim to identify the association of multimorbidity with serological response to SARS-CoV-2 nine months after first vaccine dose in a large European cohort.ORCHESTRA comprises a prospective multicenter cohort including more than 80,000 HCWs employed in hospitals, and Primary Care Health Centers in different European countries . This anHealth professionals  in direct contact with patients during the first, second, or third wave of COVID-19 were recruited between 27 December 2020 and 31 May 2022. Inclusion criteria included available and positive serology results during a 9-month timeframe from first dose administration, defined as an interval of 210–330 days, and available information about chronic diseases and conditions.Only individuals who had antibodies to SARS-CoV-2 were included in the analyses presented in this paper. SARS-CoV-2 antibody levels assays were different between centers . Only in® data collection system, which includes the patient’s unique numerical identifier and the results of serological and immunological tests. The following variables were collected in this database: (a) vaccines ; (b) SARS-CoV-2 infection, considering PCR results and anti-N serologies; (c) chronic conditions, in the following groups: cardiovascular diseases, respiratory diseases, hypertension, diabetes, diseases involving the immune system, cancer, and allergies  collected at the baseline through self-reported questionnaires or electronic health records according to each country’s definition, (d) sex, (e) age, in 4 different categories  and 10-year increases.A database was created for the ORCHESTRA cohort. (1) Data on sociodemographic characteristics, results of PCR testing, and vaccination status, including date of vaccination doses and type, were either abstracted from electronic health records or collected using self-administered questionnaires or ongoing loco-regional databases. (2) Results on the level of anti-S antibodies were either collected from medical records or generated through ad hoc testing. (3) We also implemented a study-specific RedcapAll cohorts included in the ORCHESTRA project have undergone extensive data harmonization .Absolute and relative frequencies were used to describe the cohorts. Anti-S antibody levels underwent a standardization process: first, they were log-transformed to account for the skewness of the distribution; secondly, to consider the heterogeneity in measurement methods, log-transformed results were normalized by dividing them by the center-specific standard errors. This standardization was also used in previous analyses within the ORCHESTRA project  to obtaip < 0.05 was considered significant. All analyses were performed using Stata® software V. 17 .We fitted two multivariate linear regression models to estimate the relative risks (RR) and corresponding 95% confidence intervals (CI) of an increase of one standard deviation (SD) of normalized log-transformed antibody level 9-month post-vaccination. The first model considered the number of (grouped) chronic conditions registered for each HCWs, grouped as 0 or 1, or ≥2. The second considered each chronic condition group as binary variables. The following confounders were considered in each model: sex, age (10-year increases), time since the last vaccine dose, previous COVID-19 infection (detected by PCR or anti-N serology test), and number of vaccine doses received. All tests were two-sided, and a statistical probability of The analysis included 2941 vaccinated HCWs from seven European cohorts included in the ORCHESTRA project. Among these, 78.5% were female, 73.4% were aged ≥40 years old, and 77.5% had no prior COVID-19 infection. Infection rates varied among cohorts, from 5.7% (Germany—Munich) to 50.7% (Spain—Northern Barcelona). Regarding vaccination, 98.3% had received two or more doses. Multimorbidity (≥2 chronic conditions groups) was present in 6.9% of HCWs, although its prevalence varied greatly between cohorts: from 1.7%  to 27.4% (Spain—Northern Barcelona). The prevalence of each chronic condition ranged between 1.9% (cancer) and 10.3% . The description of each cohort can be found in Mean standardized anti-S levels at 9 months after first dose was 4.67, ranging from 2.39 to 10.78 between cohorts see . On averp-value < 0.0001), and having a prior COVID-19 infection . Conversely, anti-S levels significantly decreased with each 10-year increase in age , more time since the last vaccine dose had passed , and when the HCWs had diseases on ≥2 chronic conditions groups . Among the groups of chronic conditions considered, only hypertension showed a significant association with lower anti-S levels . Sex did not have a significant impact on the anti-S levels 9 months post-vaccination.As This pooled analysis included the results of COVID-19 anti-S antibodies at 9 months after the first vaccine dose in 2941 HCWs from seven different European cohorts included in the ORCHESTRA project.We observed that a higher serological response was associated with previous COVID-19 infection and the administration of the third vaccine dose. Additionally, older age and the time elapsed since the first dose were inversely associated with the antibody level. These results are consistent with previous studies conducted in the ORCHESTRA project, albeit these previous analyses included a large number of HCWs than the current study population . Seven gThe progressive decline of vaccine immunity is common and widely acknowledged. Among the factors that influence the immune response to vaccination, which varies significantly among individuals, are intrinsic host factors , perinatal factors , and extrinsic factors  ,32 ThereSeveral studies show that hypertension is associated with a lower antibody response following vaccination with SARS-CoV-2 ,33 IndivIn the case of diabetes, we did not observe a decrease in the serological response in this study, probably due to the small number of participants with diabetes and the age of the study population. However, other studies conducted on individuals over 65 years old have found that diabetes affects the antibody response to SARS-CoV-2, particularly in non-insulinized individuals ,35.Regarding cancer, our study has few patients, and we found no differences in serological response. However, some studies report cancer patients may have a lower serological response to SARS-CoV-2 vaccination, especially those with hematologic malignancies ,37.Regarding individuals with allergies, no increased antibody response was observed after the administration of the SARS-CoV-2 vaccine. However, a study conducted in Spain, also among HCWs, revealed that allergic individuals exhibited a higher serological response following infection . This coFor the other disease groups analyzed—cardiovascular diseases, respiratory diseases, and diseases involving the immune system—we have not found comparable studies, namely, articles that considered these grouped diseases and their relationship with the serological responses to vaccines. Thus, it is important to note that all studies that analyze serological response and diseases have different patient populations, methodologies, and low statistical power for these conditions, so the findings may not be directly comparable.The relationship between multimorbidity and COVID-19 was identified in early studies. However, establishing causality, determining underlying mechanisms, and understanding the clinical implications are more complex due to the multitude of confounding factors  and the individual characteristics of patients that contribute to significant variability. Several physio-pathological mechanisms have been described that affect different phases of the disease and its sequelae, known as post-COVID syndrome. On 5 May 2023, the WHO declared that the health emergency had ended; however, the threat to public health continues. Therefore, it is now necessary to continue conducting studies that take into account host genetics as a pathway for causal inference by eliminating many sources of confounding. The ongoing investigation of the disease–host interactions and disease states will be crucial in informing the stratification of therapeutic approaches and improving outcomes for patients .The investigation of the serological response to SARS-CoV-2 vaccination in individuals with multimorbidity is a subject of scientific exploration. Current research aims to elucidate how multimorbidity may impact the immune response triggered by the SARS-CoV-2 vaccine. Several studies indicate that individuals with underlying health conditions or multiple chronic illnesses may exhibit a diminished immune response to vaccination compared to those without such conditions. Consequently, this could potentially affect the generation and longevity of vaccine-induced antibodies .Analyzing these associations would help to understand the determinants influencing the serological response in individuals with specific diseases and multimorbidity, apart from identifying the most advantageous conditions for these individuals before, during, and after vaccination to achieve an optimal antibody response. Many unresolved issues regarding potential factors affecting vaccine immunogenicity, including vaccine type, number of doses administered, revaccination intervals, and hyperglycemia in patients with diabetes, should be addressed in future studies research. Moreover, we would like to highlight that the study population consists of healthcare workers, which might be healthier than the general population Further investigations are necessary to achieve a more comprehensive understanding of how multimorbidity influences the serological response to SARS-CoV-2 vaccination.The present study has several notable strengths. Firstly, it employed a prospective design, allowing for the collection of data over time, which enhances the validity of the findings. Additionally, the study benefited from a large sample size, achieved by combining data from seven cohorts of healthcare workers from four European countries. This large sample size increases the statistical power and generalizability of the study’s results.To address the variability in blood analysis methods among the cohorts, a crucial step was taken to standardize the measurement of antibody levels. This standardization ensured that the results obtained were comparable across the cohorts, enabling the identification of genuine differences in antibody levels based on various characteristics and factors predictive of a robust immune response at 9 months post-vaccination. Standardization has been successfully utilized in previous studies and serves as an effective approach for addressing heterogeneity in testing methodologies when comparing studies conducted in diverse populations.The overall consistency of our results with previous literature supports the robustness of the analysis. Furthermore, internal validity was checked through various sensitivity analyses, made possible by the large number of subjects included in the analysis. Finally, this study is part of a series of periodic updates on serological data from vaccinated healthcare workers in the joint ORCHESTRA study. ORCHESTRA, which has already produced results at 6 and 9 months post-vaccination ,32, willA limitation of the study is the lack of data on multimorbidity for all the cohorts included in the ORCHESTRA project, which would have facilitated a larger study size and, consequently, greater statistical power . AnotherThe results of this study, although limited, support the hypothesis that individuals with chronic diseases such as hypertension may also have a lower serological response to vaccines administered for SARS-CoV-2, often accompanied by other cardiovascular diseases or other prevalent diseases. Consequently, this criterion should be considered in guidelines for revaccination against SARS-CoV-2. However, before making these decisions, it would be necessary to conduct longer-term serological studies as the serological response is influenced by various factors such as age, sex, smoking, and medication. This would need segmentation of the population to propose personalized clinical recommendations.This information is important for public health policies as it can help identify populations at higher risk of reduced vaccine effectiveness and guide vaccination strategies, such as the need for additional doses or tailored approaches for individuals with multimorbidity.


BackgroundCase reports have raised the possibility of an association between coronavirus disease 2019 (COVID-19) and spontaneous cervical artery dissection (sCeAD), yet no large studies have examined this association. We hypothesized that adults with confirmed COVID-19 would have an increased risk of sCeAD over the subsequent six months compared to test-negative controls after adjusting for confounding variables.MethodsWe obtained data from a United States medical records network  of >106 million patients, providing adequate power needed for this rare outcome. We identified two cohorts of adults meeting the criteria of (1) test-confirmed COVID-19 or (2) non-COVID-19 test-negative controls, from April 1, 2020, to December 31, 2022. Patients with previous COVID-19 or conditions predisposing to sCeAD were excluded. Propensity matching was used to control for variables associated with sCeAD and markers of healthcare utilization.Results = 0.7576). Both cohorts had a median of five healthcare visits during follow-up.The number of patients reduced from before matching  to after matching, resulting in 491,115 patients per cohort. After matching, there were 22 cases of sCeAD in the COVID-19 cohort (0.0045%) and 20 cases in the non-COVID-19 cohort (0.0041%), yielding a risk ratio of 1.10 (95% CI: 0.60-2.02; PConclusionsOur results suggest that COVID-19 is not a risk factor for sCeAD. This null finding alleviates the concern raised by initial case reports and may better direct future research efforts on this topic. A cervical artery dissection is a tear and hematoma of the arterial wall affecting either one of the paired carotid arteries in the anterior neck or paired vertebral arteries in the posterior neck . In the Several cases of sCeAD during or following severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2), also called coronavirus disease 2019 (COVID-19), have been described in the literature -7. COVIDAside from COVID-19, multiple studies have found an association between respiratory tract infections and sCeAD , a findiAlthough case reports have proposed a potential association between COVID-19 and sCeAD, this has remained unexplored by larger study designs. We aim to examine this possible association using large United States (US) cohorts, with the hypothesis that adults with confirmed COVID-19 will have an increased risk of sCeAD over the subsequent six months compared to test-negative controls.Study designWe used a retrospective cohort study design with active comparator and new user features to reduce bias, which we registered a priori . We inclSetting and data sourceThis study used data from the US TriNetX research network , which includes linked, aggregated, de-identified medical records, medical claims, and pharmaceutical claims data. TriNetX includes routinely collected data from over 106 million patients attending 76 healthcare institutions relating to patient care, diagnoses, and test results, and includes both insured and uninsured patients. The dataset may be searched using standardized coding systems such as the International Classification of Diseases, 10th Edition (ICD-10). With regards to COVID-19, the data are harmonized and must meet several requirements, including standardization of coding of COVID-19 testing data and at least 80% data completeness for demographics .ParticipantsWe included individuals of at least 18 years of age who underwent COVID-19 testing. To optimize the accuracy of baseline medical records with respect to sCeAD risk and ensure patients were actively engaged in the healthcare system, we required patients to have any healthcare visit and blood pressure measurement between one month and two years preceding the index date of their COVID-19 test. To limit loss to follow-up, ensure greater validity of our test-negative case definition, and limit crossover of test-negative controls, we required that all patients had at least one healthcare visit within six months following the index COVID-19 testing date.The COVID-19 cohort included adults at their first instance of a confirmed clinical diagnosis of COVID-19 illness (ICD-10: U07.1) or positive ribonucleic acid (RNA) COVID-19 test result occurring in the dataset, using a custom curated TriNetX code, which includes several RNA-based COVID-19 testing methods . The active control non-COVID-19 cohort consisted of adults included at the first instance of a negative RNA COVID-19 test (TriNetX: 9088). It is important to clarify that we did not use COVID-19 antibody tests to distinguish between cohorts, since antibodies may arise as the result of vaccination rather than illness and variable access to antibody testing.We excluded patients with a previous diagnosis of cerebrovascular disease, such as hemorrhage, infarction, and occlusion or stenosis of the cerebral or cervical arteries, and those with previous dissection or aneurysm of the carotid, vertebral, or basilar arteries, or aorta (Appendix 1: Exclusion codes) . We exclTo account for test selection bias and the potential simultaneity of COVID-19 and sCeAD due to universal COVID-19 testing in acute care settings, we excluded patients registering a positive or negative COVID-19 test on the same date of an inpatient, critical care, hospital observation, or emergency visit. As a related exclusion, we began the follow-up window the day after COVID-19 testing. This helped ensure patients’ medical records were accurate at the time of the index testing date.VariablesWe used propensity matching to balance confounding variables between cohorts and reduce bias. Variables present within 365 days preceding and including the index date of COVID-19 testing were eligible for matching. Variables were selected for matching based chiefly on a positive or negative association with sCeAD, including (Appendix 2: Propensity-matched variables) adverse socioeconomic factors  , demograWe examined for the occurrence of any sCeAD from the day after the index date of COVID-19 testing through the following six months, based on previous research showing that vascular endothelial dysfunction may persist amongst those with COVID-19 for six months . ICD-10 Statistical methodsWe compared between-cohort baseline characteristics using a Pearson chi-squared test or independent samples t-test. Standardized mean differences (SMD) > 0.1 represented residual imbalance. We used logistic regression to calculate propensity scores, matching with a 1:1 ratio, greedy-nearest neighbor algorithm, and caliper width of 0.1 pooled standard deviations (SDs). Risk ratios (RRs) were calculated by dividing the incidence proportion of sCeAD per cohort . Significance was evaluated at p < 0.05. We did not make imputations for missing data. We plotted cumulative incidence and propensity score density using R version 4.2.2  and the ggplot2 package.As a sensitivity analysis, we examined time-to-event data to better understand the timing of sCeAD throughout the follow-up period. We graphed the cumulative incidence of sCeAD per cohort over the six-month window, following propensity matching, to identify any inflection points that may have corresponded to periods of higher or lower incidence of sCeAD. For another sensitivity analysis, we examined the instances of any healthcare visit during follow-up after propensity matching as a metric of healthcare utilization. We compared the mean and median number of visits per patient per cohort and corresponding standard deviations.Considering there was a limited amount of data regarding sCeAD and COVID-19, we used epidemiological data regarding sCeAD to estimate a sample size . We calcParticipantsWe identified patients from approximately 50 healthcare organizations per cohort . Before propensity matching, there were 491,592 patients in the COVID-19 cohort and 1,472,895 patients in the non-COVID-19 cohort. After matching, there were 491,115 patients per cohort . Before matching, the COVID-19 cohort had a lower frequency of patients with cardiovascular medications and anti-hypertensives . After matching, the frequency of “unknown” demographic variables was similar between cohorts for unknown ethnicity , unknown race , unknown sex (0% per cohort), and unknown age (0% per cohort). A graph of the propensity score densities revealed that the propensity scores were sufficiently matched  and eight per 100,000 person-years (non-COVID-19 cohort), both of which are comparable to the expected incidence of sCeAD as described previously in the literature  suggests that our analysis included patients who were actively engaged in the healthcare system rather than those individuals who remained at home and did not present for health care. This is a marker of the success of our strategy to require patients to have preceding/follow-up visit(s) and propensity match for measures of healthcare utilization. Overall, we suggest that our findings are unexplained by between-cohort differences in healthcare utilization.There are several explanations as to why COVID-19 would not increase the risk of sCeAD. In general, sCeAD is complex and multifactorial , and sevWe suggest that excluding predisposing conditions such as Ehlers-Danlos syndrome would not have affected our findings, considering the comparable incidence of sCeAD in our study to the known literature  . In addiOne study from Austria reported a reduction in cases of sCeAD during the COVID-19 pandemic compared to preceding the pandemic . This leIt remains unclear why other respiratory infections such as influenza-like illness have been found to increase the risk of sCeAD ,12, whilWhile there is some overlap between the risk factors for stroke and sCeAD , it appears that COVID-19 specifically increases the risk of ischemic stroke  but not LimitationsAs an observational study, there is a possibility of unmeasured confounding due to the variables and constraints of the study dataset. We were unable to control for the known variants of COVID-19 , COVID-19 disease severity, and had a limited ability to control for social determinants of health and genetic variants related to sCeAD and non-COVID-19 respiratory infections. It was impractical to exclude individuals with respiratory infections relating to an unspecified organism, or those with acute respiratory infections such as the common cold. In addition, viral respiratory infections are often not recorded as patients may not seek medical attention . We wereOur study involving over 982,230 matched US adults provides robust evidence that COVID-19 is not a meaningful risk factor for sCeAD. These findings were not likely explained by differential healthcare utilization or other confounding variables. Despite previous case reports suggesting a temporal association between COVID-19 and sCeAD, our findings indicate that these cases may represent a coincidental rather than a causal relationship. Further research is needed to better clarify the association between other respiratory infections, sCeAD, and intervening medications.


Chemical exchange saturation transfer (CEST) is a molecular magnetic resonance imaging (MRI) method that can generate image contrast based on the proton exchange between labeled protons in solutes and free, bulk water protons. Amide proton transfer (APT) imaging is the most frequently reported amide-proton-based CEST technique. It generates image contrast by reflecting the associations of mobile proteins and peptides resonating at 3.5 ppm downfield from water. Although the origin of the APT signal intensity in tumors is unclear, previous studies have suggested that the APT signal intensity is increased in brain tumors due to the increased mobile protein concentrations in malignant cells in association with an increased cellularity. High-grade tumors, which demonstrate a higher proliferation than low-grade tumors, have higher densities and numbers of cells (and higher concentrations of intracellular proteins and peptides) than low-grade tumors. APT-CEST imaging studies suggest that the APT-CEST signal intensity can be used to help differentiate between benign and malignant tumors and high-grade gliomas and low-grade gliomas as well as estimate the nature of lesions. In this review, we summarize the current applications and findings of the APT-CEST imaging of various brain tumors and tumor-like lesions. We report that APT-CEST imaging can provide additional information on intracranial brain tumors and tumor-like lesions compared to the information provided by conventional MRI methods, and that it can help indicate the nature of lesions, differentiate between benign and malignant lesions, and determine therapeutic effects. Future research could initiate or improve the lesion-specific clinical applicability of APT-CEST imaging for meningioma embolization, lipoma, leukoencephalopathy, tuberous sclerosis complex, progressive multifocal leukoencephalopathy, and hippocampal sclerosis. Chemical exchange saturation transfer (CEST) is an important molecular magnetic resonance imaging (MRI) technique that can generate image contrast based on the proton exchange between labeled protons in solutes and free, bulk water protons ,2. AmideThe APT effect is measured as a reduction in bulk water intensity due to the chemical exchange of water protons with labeled backbone amide protons of endogenous mobile proteins and peptides in tissue ,4. Thus,All patients were examined with a 3-T MRI system  equipped with a 48 channel receiver array coil. APT-CEST images were acquired before contrast administration using single-shot, fast spin echo sequences with the following parameters: field of view = 220 × 220 mm; matrix = 128 × 128; spatial resolution = 1.7 × 1.7 mm; slice thickness = 8.0 mm; repetition time/echo time (TR/TE) = 3000/26.6 ms; and number of slices = 1.Twenty-nine saturation frequency offsets from −7.0 to 7.0 ppm in increments of 0.5 ppm were used to attain a sufficient signal-to-noise ratio within the clinical time frame.1 radiofrequency field equivalent to a continuous RF power level of 2.0 µT (microtesla).The APT imaging consisted of RF saturation (one pulse with a duration of 2000 ms) and an average BThe water frequency shift due to field inhomogeneity was measured in a separate image acquired using the water saturation shift referencing method with 11 offset frequencies ranging from −1.875 to 1.875 ppm at intervals of 0.375 ppm. One reference image was acquired without a saturation RF pulse, resulting in a full Z-spectrum within the offset range. The water saturation shift reference image was acquired with a TR/TE of 3000/26.6 ms, an RF saturation amplitude of 0.5 µT, and a total duration of 2000 ms with a continuous wave. The total acquisition time for both the APT and water-saturation shift reference images was 2 min 9 s.asymMTR) was also calculated. According to the shift-corrected data, the asymMTR values at ±3.5 ppm with respect to water frequency were calculated as follows [satS is the SI with selective imaging and S0 is the SI in the absence of RF for imaging SI normalization. B0 inhomogeneity was corrected on a pixel-by-pixel basis with a water saturation reference map.The APT data were analyzed in MATLAB . The magnetization transfer ratio asymmetry  grade II and III oligodendrogliomas ,16 Figu. The 2016 WHO classification of diffuse astrocytic tumors indicates three grades with different aggressiveness . AlthougGlioblastoma (GBM) is the most common malignant brain tumor in adults. With a designation of WHO Grade IV, it is also the most lethal primary brain malignancy, with a median survival time of only 15 months ,21 Figu.APT-CEST imaging shows an iso-intensity or mild punctate hyperintensity in low-grade gliomas which is significantly lower than what is seen in high-grade gliomas . There aIntracranial lymphoma may present as essentially primary central nervous system (CNS) B-cell non-Hodgkin lymphoma (B-cell PCNSL), metastatic CNS lymphoma, intravascular lymphomatosis, T-cell PCNSL, and intracranial Hodgkin’s lymphoma ,23. PrimPrimary central nervous system lymphomas show more homogeneous APT SI than high-grade gliomas. The maximum APT SI in primary central nervous system lymphomas was lower than in high-grade gliomas . In our Brain metastases (BMs) are associated with a poor prognosis irrespective of the primary tumor they originate from. Lung cancer, breast cancer, and melanoma are the most common causes of BM, accounting for 67–80% of cases . A previMeningioma is the most frequently diagnosed primary brain tumor in adults ,30. MeniPreoperative transcatheter arterial embolization (TAE) of meningiomas with polyvinyl alcohol microparticles has often been performed and is considered a safe, efficient, and cost-effective method with few complications ,35. HoweParanasal sinus mucocele is defined as the accumulation and retention of mucoid secretion within the sinus, leading to the thinning, distension, and erosion of its bony walls. Sphenoid sinus mucocele is pathologically benign and comprises 1–2% of all paranasal sinus mucoceles . To the The solitary fibrous tumor (SFT) is a new combined entity for grade I–III soft-tissue tumors. It was introduced in the 2016 World Health Organization classification of tumors of the central nervous system . In our Schwannoma is a benign tumor and develops from the Schwann sheath.The cranial nerve that is most often affected (in 90% of cases) is the vestibulocochlear nerve , followed by the trigeminal nerve . Acoustic schwannomas grow into the cerebellopontine angle, displacing the brainstem and cerebellum. In most cases, these originate from within the internal auditory canal, the dilation of which is an early radiological sign of tumor growth .This lesion is a benign tumor, and we expected that the APT SI would be lower than that of malignant tumor because the proliferation ability of schwannoma is expected to be low. However, schwannoma showed a high APT SI in our case . A recenIntracranial lipomas are rare growths that represent less than 0.1% of all brain tumors. Most intracranial lipomas are asymptomatic and are found incidentally on imaging while assessing other conditions ,40,41,42The occurrence and extent of radiation necrosis depends on the age of the patient receiving radiotherapy and the lesion volume . A previToxic leukoencephalopathy is characterized by progressive damage to the white matter. Its causes include a wide spectrum of diseases that may injure and cause structural alteration to the white matter. The insults may be due to toxic metabolites secondary to chemotherapy or immunosuppressive therapy, environmental, or infectious in origin . There aTuberous sclerosis complex (TSC) is an autosomal dominant disorder with high clinical variability. It shows various features on brain imaging, including subependymal nodules, radial bands, cortical hamartomas, and subependymal giant cell astrocytomas .In our case, this lesion was suspected to be cortical hamartoma. There are no reports on APT-CEST imaging of TSC and hamartoma. The lesions are benign, and we expected that the APT SI would be lower than that of malignant tumor because the proliferation ability of hamartoma is expected to be low. In our case, the suspected hamartoma showed a low APT SI .Progressive multifocal leukoencephalopathy (PML) is a rare but often fatal brain disease caused by the reactivation of the polyomavirus JC (JCV) . PML almIn systemic lupus erythematosus, risk factors for CNS infection include disease activity, current and cumulative corticosteroid dose, and the use of other immunosuppressants ,49 There are no reports on APT-CEST imaging of PML. However, this lesion is not a tumor, and we expected the APT SI would be low because the proliferation ability of PML is expected to be low. In our case, PML showed a low APT SI .Hippocampal sclerosis (HS) is the most common histopathologic abnormality found in adults with drug-resistant temporal lobe epilepsy . There aRecent studies on gliomas have demonstrated a positive correlation of hte APT SI with the cell proliferation index ,8,11. ThThe concentration of mobile proteins and peptides per cell may increase with the grade of glioma. In the study of Togao et al., tumors with necrosis showed a higher APT SI than those without necrosis. Although it was difficult to confirm whether this was a direct relationship, highly concentrated mobile proteins and peptides in the extracellular space, such as microscopic necrosis or fluid collection in the microcystic space, might also increase the APT SI in tumors. The alternation in tissue pH might affectthe APT SI. ,4,6Our case also showed that high-grade tumors tended to have a higher APT SI than low-grade tumors, and that demyelinating disease and tumor-like lesions tend to have a low APT SI.TAE is a standard pre-operative procedure for meningiomas aimed at reducing intra-operative bleeding and facilitating dissection. In our case of atypical meningioma after TAE, contrast-enhanced, T1-weighted sequences showed no apparent change to the meningioma, but APT-CEST imaging showed a decrease in the SI . After tAPT-CEST imaging may be used as an indicator of therapeutic effects on tumors. Moreover, APT-CEST imaging does not involve the injection of a contrast agent and can be used in patients with renal failure and those who show adverse reactions to contrast media.There have been few reports on APT-CEST imaging of benign lesions. In our case, they tended to show low APT SIs. However, schwannoma  showed aSimilarly, the non-contrast region of GBM  also shoAPT-CEST imaging can provide additional information on intracranial brain tumors and tumor-like lesions over the information provided by conventional MRI methods. This which may help indicate the nature of lesions, differentiate between benign and malignant lesions, and determine therapeutic effects. Future research could initiate or improve the lesion-specific clinical applicability of APT-CEST imaging for meningioma embolization, lipoma, leukoencephalopathy, TSC, PML, and HS. For these purposes, we may have to answer specific research questions and suggest guidelines to allow the development of APT-CEST imaging to reach its full potential.


Risk factors for psychological impairment were being a nurse , a health assistant , or having had an infected family member . Compared to Time 1, gender and experience in COVID-19 units lost significance with psychological symptoms. Conclusions: data over more than 24 months from the pandemic onset showed improvement of HCWs’ mental health; our findings suggested the need to tailor and prioritize preventive actions towards healthcare workforce. Background: Research has shown the substantial impact of the COVID-19 pandemic on healthcare workers’ (HCWs) mental health, however, it mostly relies on data collected during the early stages of COVID-19. The aim of this study is to assess the long-term trajectory of HCWs’ mental health and the associated risk factors. Methods: a longitudinal cohort study was carried out in an Italian hospital. At Time 1 (July 2020–July 2021), 990 HCWs took part in the study and completed the General Health Questionnaire (GHQ-12), the Impact of Event Scale (IES-R), and the General Anxiety Disorder (GAD-7)questionnaire. McNemar’s test measured changes in symptoms’ trajectories, and random effects models evaluated risk factors associated with scores above the cut-off. Results: 310 HCWs participated to the follow-up evaluation . At Time 2, scores above cut-offs were significantly lower ( A large amount of literature is now available to attest to the burden of the COVID-19 pandemic on Health Care Workers’ (HCWs) mental health. Early evidence following the pandemic’s onset showed a severe psychological impact resulting in a high level of sleep disorders, anxiety, depressive and post-traumatic symptoms among HCWs ,3,4. TheIn addition to the pandemic-related risk factors, HCWs may have been exposed to traditional psychosocial risk factors such as high demands, lack of resources, inadequate staffing, and irregular and prolonged shift works . Since pMost research on the impact of COVID-19 on HCWs’ mental health relies on cross-sectional data collected during the early stages of COVID-19, and longitudinal studies are mainly focused on the first year of the pandemic ,19,20,21Italy  was the first European country to be affected by COVID-19. On 12 March 2020, shortly after the first confirmed SARS-CoV-2 infections, a national strategy for the COVID-19 response was developed and restrictive measures  were implemented; each region and related public health services developed specific plans according to the allocated resources, facing severe difficulties in fighting an unprecedented healthcare emergency ,24. HCWsSince the onset of the pandemic in February 2020, Italy experienced four major waves of infection  with an overall 25,791,569 cases of confirmed SARS-CoV-2 infection and 186,643 COVID-related deaths . The COVID-19 vaccine was available from late December 2020. HCWs were among the first to receive the vaccine and Italy was the first European country making vaccination mandatory for healthcare staff. About 90% of the Italian healthcare workforce is now fully vaccinated . In the Italian context, available studies carried on until mid-2021 showed different trends in mental health outcomes, with higher rates of anxiety, depression and burnout in 2021 compared to 2020 found by Lasalvia et al.  in repeaGiven the paucity of research on the long-term impact, we are not aware of the quality of HCWs’ mental health during the second year of the pandemic. Although previous pandemics such as SARS may not be comparable to COVID-19 in its clinical, social and psychological widespread impact, results from previous experience showed HCWs might be suffering from long-term psychological disturbances . The aim of this study is to assess the long-term trajectory and associated risk factors of anxiety, general discomfort, and post-traumatic symptoms among HCWs through a longitudinal cohort study carried out between July 2020 and July 2022 at a university hospital in northern Italy. A multi-step process to evaluate and monitor workers’ mental health was developed in June 2020 at a large university hospital in Milan, Italy. The entire process was conducted jointly by the Occupational Medicine and Psychiatry units.The process aimed to evaluate HCWs’ mental health through a two-level screening and to offer psychological support and psychiatric treatment to those who showed psychological distress. For an extensive description of the methodology adopted for this process, see Fattori et al. .At baseline , 990 HCWs took part in the study and completed a multi-step psychological assessment within the occupational health medical surveillance. Results related to the first-year assessment  were published elsewhere .Follow-up data was collected between July 2021 and July 2022 (Time 2). Workers who participated at Time 1 (T1) were invited at Time 2 (T2) through phone calls by Occupational Medicine Unit staff; a subsequent email was sent to reach out to those who did not answer two phone calls. Baseline participants had been informed of the 12-month recall during T1 data collection; as for baseline, follow-up participation was on a voluntary basis.Occupational physicians within the medical surveillance required by Italian Legislation in terms of occupational safety  performed follow-up evaluation. –The General Health Questionnaire 12  in the –Impact of Event Scale—Revised  to eval–The Generalized Anxiety Disorder questionnaire  to screDuring follow-up evaluation, participants completed, for the second time, the Time 1 psychometric scales: Participants were also asked about exposure, concerns and health beliefs related to COVID-19 in the previous six months through multiple choice questions . Specifically: having been positive for COVID-19, having family members positive for COVID-19, having fear for their personal safety or fear of infecting family members, experiencing social discrimination for being a HCW or changes in their families’ habits, and having thoughts about changing their job.Occupational physicians updated and recorded subjects’ occupational data , as well as pathological events and ongoing medical treatments.We analysed differences between Time 1 and Time 2 measurements  using Student’s t test for paired data. In addition, such differences were compared with the “Minimum Clinically Important Difference” (MCID), defined as the smallest change in an outcome perceived as clinically meaningful; MCID was calculated as half the standard deviation of the scores measured at Time 1 ,41.Possible differences between groups  were graphically explored, representing the average scores for each category at Time 1 and Time 2 and testing mean differences. For categorical outcomes , we applied McNemar’s chi-squared test and calculated odds ratios, risk ratio and risk difference  to analyse paired contingency tables obtained from crossover binary response data. For McNemar’s test only discordant pairs  are informative and their ratio gives McNemar’s odd ratio, an estimate of the proportion of change of the outcome variable from one level to the other.We used random effects logistic models to evaluate variables associated with the probability of scoring above the cut-off. We ran three random effects logistic regressions considering the binary variable indicating whether the score is greater than cut-off or not as outcome. For time-dependent explanatory variables , the interaction terms with time were included in the models and if not significant they were removed.Personal concerns and health beliefs about COVID-19 were graphically explored, distinguishing their distribution at the two different time points. To study their effect on scores in terms of risk factors, they were converted into dichotomous variables  and put one by one in the random effect models.Among the 990 subjects enrolled at baseline (Time 1), 310 (31%) completed the follow-up evaluation (Time 2). The follow-up sample was similar to the baseline sample concerning gender, age and occupational role . Particip < 0.001, t-test for paired samples), indicating an overall improvement in mental health at all scales. The difference in the two measurements was substantial for IES-R and it may be considered clinically meaningful according to MCID, while GAD-7 showed the smallest decrease. Stratification by groups  gave similar decreasing trajectories of average scores for each category and means resulted far from the cut-offs at Time 2 . The McNemar’s Chi-square test provided small p-values, indicating statistically significant difference in the distributions at Time 1 and Time 2 for all the scales. Few workers scored above cut-off at Time 2 and did not at Time 1 ; additionally, 34%, 17% and 61% of subjects obtained scores below cut-off of GHQ-12, IES-R and GAD-7, respectively, after scoring above cut-offs at Time 1. Thus, McNemar’s odd ratios were large  and their confidence intervals did not contain 1, indicating that the remittent condition  was much more plausible than the incident condition . Similarly, risks ratio  and risks difference  showed a higher risk for scoring above the cut-offs at Time 1 than at Time 2.Scores below or above cut-offs at Time 1 and Time 2 were matched in Random effects models for repeated measures investigated possible risk factors for scoring above cut-off for all scales. Females showed greater risk of surpassing the GHQ-12 and GAD-7 cut-offs, and the working experience in a COVID-19 area caused a higher risk of general distress (GHQ-12) and post-traumatic symptoms (IES-R), although without statistically significant differences.COVID-19-related fears and overall concerns decreased over time, especially worries of infecting family members . After cIn this longitudinal cohort study, we assessed the long-term trajectory and associated risk factors of anxiety, general discomfort and post-traumatic symptoms in a sample of different healthcare professionals working in a large Italian hospital during the COVID-19 pandemic. To our knowledge, this is the first study to report longitudinal data of HCWs’ mental health beyond 24 months from the pandemic onset, assessing psychological symptoms with repetitive measurements. Overall, mental health gradually improved over time although with different trajectories depending on the psychological symptoms considered. At Time 2, according to the scales’ cut-offs, we found reduction rates ranging from 8% for GAD to 25% for GHQ; IES-R showed the smallest proportion of scores above the cut-off compared to GHQ-12 and GAD-7, suggesting a general remission for post-traumatic symptoms related to the COVID-19 pandemic. These results may be consistent with existing literature on long-term psychological disturbances in disaster-affected populations, indicating a gradual remission in disorders albeit with differences, as post-traumatic symptoms generally decrease in the years following the traumatic event while anxiety and depression can persist further . Follow-up data collection was conducted after the third wave and during the fourth (Omicron) wave of contagions. During these phases of the COVID-19 pandemic, related hospital admissions and deaths declined considerably, both in the northern regions and throughout Italy ,44. MoreInterestingly, in our study the most relevant decrease was observed for IES-R, whose items specifically asked participants for post-traumatic responses to the COVID-19 emergency, whereas both GAD-7 and GHQ-12 assessed symptoms, which may be related to broader distressing episodes or to circumstances beyond the pandemic .Although psychological improvement in our sample was substantial, about 10% of participants had persistent impairment and a smaller proportion (from 3% for IES-R to 9% for GHQ-12) experienced discomfort only in the second year, suggesting possible late onset of mental distress. To this regard, it is essential to maintain ongoing mental health monitoring to support long-lasting suffering as well as to prevent late-onset mental illness. In addition to psychological and psychiatric support, HCWs’ wellbeing could be sustained through organizational intervention, such as reducing shift lengths, providing effective training for unfamiliar tasks, safety procedures and stress management . Most previous follow-ups conducted in Italian hospitals after 12–15 months from the pandemic onset  had already shown an overall decrease in distress, anxiety, depressive and post-traumatic symptoms ,48; althThe lack of literature on HCWs’ mental health during the second year of the COVID-19 pandemic hampers the comparison between our sample and others from different hospitals and countries. To this regard, it is fundamental to consider studies conducted with similar time points  since mental health impairment may increase during periods with stricter measures and higher death/incidence rates . Regarding the risk factors for psychological symptoms, we found different results when considering both Time 1 and Time 2 outcomes compared to Time 1 outcomes only. Being female and younger were not significant risk factors for impaired mental health in contrast with previous results and pandemic-related literature, which showed women at higher risk for mental health disturbances and quality of life disruption beyond occupational role ,8,51,52;Differently from Time 1, working experiences in COVID-19 areas did not increase the risk for psychological impairment. This is consistent with research conducted during the second year of the pandemic, showing that working in COVID-19 areas might no longer be a massive stressor for health impairment, although workers were exposed to adverse conditions such as high emotional demands ,55. ThisOur study has several limitations. Participation rate at Time 2 was lower than at Time 1 and this may entail a self-selection bias. However, follow-up rates did not differ among subgroups as both baseline and follow-up samples were similar by gender, age and occupational role . We shouAnother important limitation is the lack of data on the pre-pandemic mental health of our sample; this hampers the estimate of the pandemic impact as the literature suggests that a history of mental disorder may be a prominent risk factor for impaired mental health during pandemics ,58. FinaNotwithstanding the declared limitations, our findings from data collected in the second year of the pandemic through a longitudinal cohort study may be of great interest. We found that mental health among HCWs gradually improved compared to the previous year; nonetheless, being a nurse or a health assistant, having a family member positive with COVID-19 in the previous six months and previous experience of moral distress still acted as significant risk factors for adverse psychological outcomes.Our results showed the need to tailor and prioritize preventive actions towards the healthcare workforce, as nurses and health assistant may be at a greater risk of developing impaired mental health conditions compared to colleagues. This result is coherent with data from previous studies conducted during COVID-19 pandemic, suggesting these professionals experienced persistent fatigue and burden . Beyond the pandemic, it is essential to carry on preventive interventions in the healthcare sector through a multi-level approach, as the literature has shown that HCWs experienced chronic exposure to occupational stress and substantial psychological impairment also in the pre-pandemic era ,17,59; tFurther results will provide better insights into long-term symptoms’ trajectories and interventions’ effectiveness . Longitu


Third-line treatment with trifluridine/tipiracil (FTD/TPI) is recommended for patients with metastatic colorectal cancer (mCRC) or gastric/gastroesophageal cancer (GC) who have progressed beyond first- and second-line therapy. We describe a patient with long-term survival following treatment with FTD/TPI. The patient, a 70-year-old woman diagnosed with right-sided mCRC (T3/N1) with metastases to the aortocaval and precaval lymph nodes, received first-line panitumumab and capecitabine for 6 months, followed by second-line bevacizumab and oxaliplatin. She had disease progression following 9 months of second-line therapy and began third-line treatment with FTD/TPI (50 mg bid). Three months after treatment initiation, lymph node involvement was reduced, and following 12 months of FTD/TPI treatment, her disease had stabilized, and she reported no treatment-related adverse events. She remained on the same dose of FTD/TPI for more than 27 months after initiating treatment, with maintenance of stable disease. This patient with mCRC demonstrated a survival benefit with FTD/TPI beyond those reported in published clinical trial data and real-world studies. Colorectal cancer (CRC) is among the most frequently diagnosed malignancies in the world and presents a substantial health burden . For patA 70-year-old woman  with hypertension was diagnosed with right-sided mCRC (T3/N1) on February 2019, with metastases to the aortocaval and precaval lymph nodes . She had KRAS-wild-type mCRC (and no other detected mutations associated with CRC) with carcinoembryonic antigen (CEA) levels of 35 ng/ml. The patient underwent a hemicolectomy and commenced treatment with panitumumab (400 mg by intravenous infusion every two weeks) and capecitabine (2 x 500 mg tablets every 12 hours) but dose was reduced by 25% due to her fragility. Assessed with positron emission tomography (PET) scans and CEA evaluations, the patient had a regressive course after 3 months of treatment according to a stabilized lymph node size, reduced SUV (5.5) and reduction of CEA levels to 9.7 ng/ml. However, 6 months after starting therapy, the lymph node size had increased  and CEA levels had increased to 176 ng/ml indicating disease progression.The patient commenced second-line therapy with bevacizumab (400 mg by intravenous infusion every two weeks) and oxaliplatin (100 ml by intravenous infusion every two weeks) in September 2019. Disease regressed 3 months later: aortocaval lymph node was no longer visible, the precaval lymph node had decreased in size , and CEA levels had decreased to 39 ng/ml. Disease remained stable after a further 3 months of treatment with bevacizumab and oxaliplatin , but 3 months after this  the existing lymph node involvement increased in size , new lymph node involvement was detected and CEA levels increased slightly to 24 ng/ml. As a result, treatment with bevacizumab and irinotecan was initiated (June 2020), but was discontinued after one cycle due to alopecia, neutropenia and general weakness.In July 2020, she began third-line treatment with FTD/TPI (50 mg bid), to control disease progression. After 3 months of treatment, lymph nodes had decreased in size  and CEA levels had almost normalized at 2.8 ng/ml. Following a further 6 months of treatment with FTD/TPI (with no dose delays or reductions), all lymph nodes had disappeared, with the exception of the precaval lymph node , but CEA levels had increased. By July 2021 (12 months receiving FTD/TPI as third-line treatment) her disease had stabilized and CEA levels were 58 ng/ml. The patient experienced no adverse reactions related to FTD/TPI treatment. The patient remains on the same dose of FTD/TPI >27 months after initiating this treatment and continues to have stable disease. As of March 2022, CEA level was 11.7 ng/ml, and the patient remains on FTD/TPI treatment as of October 2022. All treatments and outcomes are summarized in In this case study, an Egyptian patient with mCRC had an impressive survival benefit with FTD/TPI, in excess of those reported in published clinical trial data and real-world studies. She received first- and second-line treatment for 17 months after diagnosis, but then achieved a partial pathological response with FTD/TPI and was still alive, responding and receiving FTD/TPI more than 27 months later. FTD/TPI has demonstrated long-term efficacy in other case reports of mCRC , and othThe outcome with FTD/TPI treatment may potentially be influenced by genetic alterations in, for example, the TYMP gene, which encodes thymidine phosphorylase, the enzyme that is primarily responsible for FTD metabolism and that is inhibited by TPI . MutatioThis patient did not experience any adverse events and did not require any dose delays or reductions with FTD/TPI. The reasons for the absence of any of the adverse events that are frequently observed with FTD/TPI treatment, such as neutropenia and leukopenia , are notChoosing the right treatment from the start  was oneThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ME assumes final responsibility for the work, had access to all data, contributed extensively to the writing of this case report, and gave full approval to submit this work for publication.


The prevalence of burnout and anxiety is constantly increasing among health profession students worldwide. This study evaluates the prevalence of burnout and its relationship to anxiety and empathy during the COVID-19 pandemic among health profession students in the main governmental institution in Doha, Qatar using validated instruments.A cross-sectional survey of health profession students using validated instruments was employed. The Maslach Burnout Inventory-General Students Survey (MBI-GS(S)) to measure burnout; The Generalized Anxiety Disorder (GAD-7) to measure anxiety; and Interpersonal Reactivity Index (IRI) to measure empathy were utilized. Descriptive statistics and multivariable linear regression were used.Of the 1268 eligible students, 272 (21.5%) completed the online survey. Burnout was found to be prevalent amongst the students. The mean scores for the MBI-GS(S) subscales of emotional exhaustion, cynicism, and professional efficacy were 4.07, 2.63, and 3.97, respectively. Anxiety was found to be a strong predictor for burnout and burnout was positively associated with empathy.Findings from this study demonstrated relationships between health profession students’ burnout, anxiety, and empathy. These findings might have an impact on the development of curriculum interventions to enhance student well-being. More burnout awareness and management programs that cater to the specific needs of health profession students are needed. Furthermore, findings of this study may have implications for future educational interventions during times of crisis or how this can be used to improve student experiences in normal times. Burnout has been characterised as a psychological syndrome and a state of continuous stress . It invoGenerally, burnout is a growing concern among health profession students and can have negative impact on their mental health, professionalism, and empathy due to the high demand of the different medical and health education which can be long lasting , 8–11. IFurthermore, the emotional exhaustion and cynicism subscales of burnout have been shown to positively relate to anxiety symptoms, meaning that anxiety can be a predictor of burnout in some students . AnxietyWhen the global COVID-19 pandemic commenced, everyone was affected, including university students who were expected to quickly adapt to online learning and to study in isolation of their peers. University students were particularly vulnerable to the impact COVID-19 on their life, academic performance andsense of belonging and socioeconomic status –27. FurtAt the beginning of the global pandemic, Qatar University, the main governmental institution in Qatar, suspended all classes until further notice as part of preventative measures to reduce the spread of the coronavirus and announced the educational process will continue virtually. The impact of this emergency shift to remote teaching increased faculty and students’ academic demands through emergency curriculum changes, financial burdens due to lockdown, and intensified fear of the pandemic itself adversely affecting students’ academic performance, mental health and well-being . StudentAn online cross-sectional study was conducted among Qatar University's four health colleges students. These students comprised those from the College of Medicine (CMED), College of Pharmacy (CPH), College of Dental Medicine (CDM) and the College of Health Sciences (CHS). The CHS includes the departments of Public Health, Biomedical Sciences, Human Nutrition, and Physical Therapy and Rehabilitation Science. These four health colleges are under the umbrella of QU Health which was launched in 2017 guided by Qatar’s National Vision 2030 to form an integration between the health colleges  who work together to prepare competent health profession graduates capable of improving the health sector in Qatar .Email invitations to participate in the study were sent to all undergraduate and graduate students in QU Health in October 2020 to complete the online survey using SurveyMonkey®. The survey was open from October 2020 until December 2020 and was conducted in English. The survey consisted of five sections: (1) participant’s sociodemographic data; (2) COVID-19 related questions; (3) Generalized Anxiety Disorder 7-item scale (GAD-7); (4) Maslach Burnout Inventory-General Students Survey (MBI-GS(S)); and (5) Interpersonal Reactivity Index (IRI).Sociodemographic data included gender, age, college and department, year of study (year 1 to year 6), marital status , employment status , degree level (undergraduate or graduate), nationality (Qatari or Non-Qatari), rating of physical and mental health , number of courses dropped in Spring semester 2020, and the grade point average (GPA) for Spring 2020 and Fall 2019 semesters.Using a 5-point Likert scale ranging from “not concerned at all” to “extremely concerned”; students were asked to rate COVID-19 related questions pertaining to the level of concern about personal safety and security; fear of contracting COVID-19; concerns about loved ones contracting COVID-19; number of new COVID-19 cases; and COVID-19 pandemic duration. The extent of following hygiene and social distancing recommendations was measured on a 4-point Likert scale ranging from “not at all” to “very closely”.Students’ burnout was measured using the MBI-GS(S). MBI scale is a validated and reliable instrument that was introduced in 1981 and is the gold standard for measuring burnout , 33, andGeneralized Anxiety Disorder 7-item scale (GAD-7) was used to measure generalized anxiety symptoms severity. GAD-7 is a reliable and validated instrument that is used widely as a screening tool for anxiety disorders , 36. A cEmpathy was measured using the Interpersonal Reactivity Index (IRI) . IRI hasDescriptive statistics were used to summarize characteristics of participants and COVID-19 related variables using mean with standard deviation (SD) for continuous variables and numbers with percentages for categorical variables. For MBI-GS (S), the scores were reported as means for each MBI subscale including MBI-EX, MBI-CY, and MBI-PE. The MBI does not have any cut-off points as it did not show any diagnostic validity. GAD-7 score was categorized into mild (0–5), moderate (6–10) and severe (11–15)  MultivarOf the 1268 eligible students, 272 (21.45%) completed the online survey. As shown in Table The means (SDs) of BMI-EX, MBI-CY, and MBI-PE were 4.07 (1.56), 2.63 (1.53), and 3.97 (1.22), respectively.The main factors independently associated with MBI-EX were GAD symptoms severity, year of study, marital status, physical health rating, course withdrawal status, and the extent of following physical distancing measures. Students with severe, moderate, or mild GAD symptoms had higher MBI-EX scores than students with minimal GAD symptoms severity by 0.81 95% CI 0.32, 1.30), 1.12 , and 1.66 , respectively. As compared to students in year 1, students in years 2, 3, 4, and 6 had higher MBI-EX scores ranging between 0.60  for year 4 students and 0.91  for year 6 students. However, this was not statistically significant for year 5 students  and 0.56 , respectively Table . As compThe main factors independently associated with MBI-PE were GAD symptoms severity, study program, extent of following physical distancing measures, and sense of personal safety and security. Students with moderate GAD symptoms had lower MBI-PE scores than students with minimal GAD symptoms severity by 0.72 . However, this was not statistically significant for students with severe or mild GAD symptoms severity Table .Table 5FMBI-EX was significantly and independently associated with empathetic concern and personal distress subscales of empathy. Increase in MBI-EX by 1 score was associated with increase in empathetic concern and personal distress scores by 0.76  and 0.77 , respectively. However, no statistically significant associations were found between MBI-EX and perspective talking and fantasy subscales of empathy (Table p < 0.01 (two-tailed) for both EE and CY [The COVID-19 pandemic has disrupted many aspects of life, especially education. This study aimed to assess how COVID-19 pandemic affected the students’ life in regard to the level of burnout and the relationship between anxiety and burnout, and burnout and empathy amongst QU Health students. In contrast to many of literature findings, gender was not associated with higher levels of burnout , 43. QU E and CY .This study found that burnout was positively associated with empathy which is contradictory to the literature findings that suggest that burnout reduces empathy of students , 46, 47.Burnout has a negative influence on the students’ quality of life, and if persisted, it can lead to detrimental consequences on the students’ mental and physical health as well as their careers . As a reThis study was the first to assess and correlate burnout, anxiety, and empathy amongst students and to assess it amongst health profession students. Another strength of this study is the use of multiple logistics regression which has high statistical power in determining the presence of associations/relationships. The main limitation of this study was the low response rate (21.45%) which could be due to the length of the survey. Another potential limitation is the possibility of social desirability bias which can occur in any cross-sectional study. In addition, the study was conductedat a single institution which limits its generalizability to other academic health institutions in the country such as Weill Cornell Medicine- Qatar, University of Calgary- Qatar and University of Doha for Science and Technology Another limitation is that most participants were female students (93%). Although this reflects the current proportion of female students in colleges within QU Health, the findings of this study may not be generalized to male students. However, consistent with the findings of other studies, female gender has been shown to be a predictor of burnout , 53 and Findings from this study demonstrated relationships between health profession students’ burnout, anxiety, and empathy. Anxiety was found to be a strong predictor for burnout and burnout was positively associated with empathy. These findings might have an impact on the development of curriculum interventions to enhance student well-being. More burnout awareness and management programs that cater to the specific needs of health profession students are needed to help reduce the prevalence and consequences of burnout, as well as, to increase the availability of mental health services for students. Furthermore, it may have implications for future educational interventions during times of crisis or how this can be used to improve student experiences in normal times.


DanioCTC is an approach that tackles a significant hurdle in cancer research: the scarcity of circulating tumor cells (CTCs) in animal models. These CTCs are crucial in understanding how cancer spreads, especially in metastatic breast cancer. To overcome the challenge of their xenotransplantation, the authors combined several advanced technologies to enrich and isolate CTCs from patients. These isolated CTCs were then injected into zebrafish embryos, allowing the researchers to study their dissemination pattern in vivo in a pilot study. Experiments showed that DanioCTC successfully enabled the xenografting of CTCs from patients. CTCs were primarily localized in the head and trunk regions of the zebrafish embryos. Overall, DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, paving the way for a better understanding of the biology of metastatic breast cancer and the development of targeted interventions.Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked  into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases. Breast cancer metastasis is a complex process involving tumor-cell migration, intravasation, survival in the bloodstream, adhesion, extravasation, and colonization at secondary sites ,2. MetasCirculating tumor cells (CTCs) are shed into the blood by tumor tissue and are considered precursor cells for metastasis formation . DespiteOne technology to increase the number of informative cancer patient samples containing high numbers of analyzable CTCs is diagnostic leukapheresis (DLA), a density-based blood separation that facilitates the safe collection of large CTC numbers from liters of patient blood . DLA enaThe limitations of the current mouse xenotransplantation models for the study of early tumor dissemination have prompted the need for alternative animal models. Zebrafish have emerged as a valuable tool for investigating human diseases, including cancer metastasis, owing to their genetic similarity to humans, ease of maintenance, and high fecundity ,16,17. Z2; EGFP-labeled MDA-MB-231 cells were cultured in low glucose DMEM  with 10% fetal bovine serum (Gibco) in a humidified incubator at 37 °C with 5% CO2. Cultured cells were harvested at a confluence of approximately 80% for staining and spike-in experiments .MDA-MB-231 cells were obtained from the American Type Culture Collection ; EGFP-labeled MDA-MB-231 cells were kindly provided by Manja Wobus/Martin Bornhäuser . MDA-MB-231 cells were cultured in RPMI 1640 medium, containing 10% fetal bovine serum, 25 mM HEPES  and 100 units/mL penicillin-streptomycin in a humidified incubator at 37 °C with 5% CODanio rerio) strains were maintained according to national law and under standardized conditions as previously described [kdrl:EGFP) [osx:mCherry) [Tg(OlSp7:mCherry)zf131] for initial characterization of MDA-MB-231 cancer cell distribution in zebrafish embryos  at 100–150 cells/nl. Glass needles for injection were pulled from glass capillaries with filament  with a P-97 Flaming/Brown micropipette puller . Zebrafish embryos  were manually dechorionated, anesthetized using 0.02% tricaine and transferred into a petri dish which was covered with a thin layer of 1.5% low melting agarose in E3 , in order to avoid adhesion of the embryos to the dish. The labeled MDA-MB-231 cell suspension was loaded into a pulled glass capillary and 1–2 nL were micro-injected into the blood circulation of Tg(at 33 °C . Based oDiagnostic leukapheresis (DLA) was performed at the Department of Transplantation Diagnostics and Cell Therapeutics, Duesseldorf, Germany, as previously described ,13. AliqCryopreserved DLA samples were rapidly thawed in a 37 °C water bath and filtered through a fine sieve (mesh size 100 µm). The filtered DLA sample was diluted 1:20 in PBS. For spike-in experiments, MDA-MB-231 cell line cells were added to diluted negative control DLA samples. Therefore, cultured MDA-MB-231 cells were trypsinized using trypsin–EDTA (0.05%), neutralized using complete medium, centrifuged at 1200 rpm for 5 min and resuspended in PBS. Subsequently, 5000 MDA-MB-231 cell line cells were transferred into diluted negative control DLA samples. Viable CTCs of a MBC DLA sample or spiked cell line cells of negative control DLA samples were enriched with the FDA-approved Parsortix system . The Parsortix system was used according to the manufacturer’s instructions. Following the protocol, filtration cassettes with 6.5 µm gaps were used and 100 mbar above ambient pressure were applied ,27.TM Technologies, Vancouver, BC, Canada, AF488), MUC1 , Her2  and CD45  at 37 °C for 60 min for identification by flow cytometry. EpCAM-mediated enrichment of CTCs for xenotransplantation has previously been used , 50 pre-labeled MDA-MB-231 cells were injected with the CellCelector, resulting in a total of 11 embryos. The zebrafish embryos were incubated and the injected cells were monitored at 1 and 3 dpi. The MDA-MB-231 cells disseminated to different areas in the head, trunk, and tail regions in varying numbers  in the next step and processed it according to the workflow depicted in  numbers A–D. On a numbers D. At 3 d numbers C. This fA cryopreserved 2 mL DLA aliquot obtained from a patient with MBC was used for CTC enrichment using the Parsortix system, following our workflow . A totalAfter injection, CTCs disseminated to different areas in the head, trunk, and tail , with anHere, we introduce DanioCTC, a seamless workflow that allows for the injection of low numbers of patient-derived CTCs into zebrafish embryos. This technique enables real-time studies of the differential metastatic traits of individual CTCs in vivo, despite their scarcity in cancer patients’ blood. Unlike previous methods which rely on high cell numbers and retrograde loading of cells into glass capillaries, our approach overcomes this technical limitation by integrating the injection process, viable CTC isolation, labelling, and detection into a reproducible workflow. In general, DanioCTC provides the methodology to investigate cells with a low prevalence in zebrafish embryos, as shown here with patient-derived CTCs. The workflow allows for the selection of cells of interest down to the single-cell level and their subsequent injection. Consequently, the method paves the way for studies investigating the dissemination of cells in vivo, along with studies focusing on the molecular characterisation of cells after injection. With DanioCTC, we aim to preserve the competency of CTCs to metastasize by using directly isolated CTCs. Our workflow combines the detection of CTCs from DLA products with ex vivo labelling techniques and direct injection using the automated micromanipulation system CellCelector, which we have modified and extended. DanioCTC facilitates the investigation of CTCs during the process of metastasis by enabling the injection of low numbers of patient-derived CTCs into zebrafish embryos. This approach provides a good basis for modelling the low frequency of CTCs typically found in cancer patients’ blood. The zebrafish embryo is an ideal in vivo model for studying CTC metastasis due to its transparency, fast development, high progeny number, and lack of adaptive immunity in initial life stages. In this transparent model, the capacity of CTCs to arrest and adhere to the endothelium, which may involve cell rolling and other processes that can be observed in tumor cells and immune cells found in the circulation , can be We present the results of injecting MDA-MB-231 cells at both high and low cell numbers into zebrafish embryos, where they were frequently found in the head (including the CNS) and tail (CHT), which is consistent with the high incidence of brain and bone marrow metastases in triple negative breast cancer . The relOur study, for the first time, employs a semi-automated single-cell micromanipulation system to demonstrate that CTCs from breast cancer patients can be directly injected into zebrafish embryos and monitored for several days by live imaging. While traditional mouse models have been utilized to mimic different stages of the metastatic cascade, zebrafish models offer a promising alternative. Previous research has showcased the potential of zebrafish models in investigating the biology of non-patient CTCs and CTC-clusters, indicating their utility in understanding the metastatic nature of cancer cells ,26,31. FDespite those advancements, cell lines as ‘surrogate CTCs’ have been used in all previous studies to investigate the differential behavior of single CTCs and CTC clusters in zebrafish embryos, due to limitations in the injection process. It is important to note that subtle differences have been observed between cell line-derived surrogate CTCs and patient-derived CTCs ,38,39 anHere, we provide a proof-of-concept study on the DanioCTC workflow that allowed us to inject isolated CTCs from a single MBC patient into zebrafish embryos, to the best of our knowledge, for the first time. This case study, along with the established setup, will allow for further experiments on isolated CTCs, accompanied by ongoing improvements in the equipment and software. Future enhancements in the procedure and setup will reduce the processing time and increase the applicability of the method in personalized medicine approaches. Our case study using DanioCTC paves the way for future investigations of MBC-patient cohort CTCs by studying and characterizing the distribution of these cells in vivo.In summary, our study establishes an innovative workflow for the injection of MBC-derived isolated CTCs into zebrafish embryos, enabling live imaging of patient-derived CTC dissemination in xenografts over time. This approach may become a valuable tool in further studies exploring targeted medicine approaches and the in-depth exploration of CTC metastatic biology, given its capability to work with only a few dozen cells per embryo.


Unexplained infertility accounts for 25% of infertility causes in the UK. Active intervention methods, such as intrauterine insemination (IUI) or in vitro fertilisation (IVF), are often sought. Despite the National Institute for Health and Care Excellence (NICE) recommending IVF for unexplained infertility, this recommendation has generated an ongoing debate, with few fertility clinics discontinuing the use of IUI as the first-line management of choice. In contrast to NICE, recent guidance released from the European Society for Human Reproduction and Embryology (ESHRE) in August 2023 supports the use of IUI as first-line. High-quality evidence behind such interventions is lacking, with current literature providing conflicting results.This review aims to provide a literature overview exploring whether IUI or IVF should be used as first-line treatment for couples with unexplained infertility, in the context of current guidelines.The primary outcome used to assess efficacy of both treatment methods is live birth (LB) rates. Secondary outcomes used are clinical pregnancy (CP) and ongoing pregnancy (OP) rates. A comprehensive literature search of 4 databases: Ovid MEDLINE, EMBASE, Maternity & Infant Care and the Cochrane Library were searched in January 2022. Upon removal of duplications, abstract screening, and full-text screening, a total of 34 papers were selected.This review highlights a large discrepancy in the literature when examining pregnancy outcomes of IUI and IVF treatments. Evidence shows IUI increases LB and CP rates 3-fold compared to expectant management. Literature comparing IUI to IVF is less certain. The review finds the literature implies IVF should be used for first-line management but the paucity of high-quality randomised controlled trials (RCTs), coupled with heterogeneity of the identified studies and a lack of research amongst women > 40 years warrants the need for further large RCTs. The decision to offer IUI with ovarian stimulation (IUI-OS) or IVF should be based upon patient prognostic factors. We suggest that IUI-OS could be offered as first-line treatment for unexplained infertility for women < 38 years, with good prognosis, and IVF could be offered first to those > 38 years. Patients should be appropriately counselled to enable informed decision making.The online version contains supplementary material available at 10.1186/s12905-023-02717-1]. Infertility affects an estimated 1 in 7 couples across the UK, of which 25% is due to unexplained causes . ExpectaGlobally, there is a discrepancy between the use of IUI or IVF as first-line treatment for unexplained infertility . While ITherefore, in the context of currently conflicting guidelines, this review aims to explore current evidence on the practise of IUI versus IVF as first-line treatment for unexplained infertility via comparison of pregnancy outcomes. The primary outcome is live birth (LB) rates as the preferred outcome for evaluation of infertility treatment recommended by the ESHRE , 13. SecThe PICO framework was used to develop this review question and the search strategy Table . A liter6 papers evaluating the effects of IUI upon LB rates were identified, with 1 randomised controlled trial (RCT) and 5 retrospective observational studies –21. The McLernon et al. reported couples with IUI-OS to be 3 times more likely to conceive than those with EM . It was Although studies reporting cumulative LB rates have comparable results (26.5% versus 28.5%) , 19, incMultiple studies have explored the optimum number of IUI-OS cycles leading to a LB. The consensus is that an average 2 cycles of IUI-OS are required –21. MultThe NICE guidelines state that OS agents should not be offered to women with unexplained infertility , althougUnlike the NICE 2013 guidelines , the recThe literature search identified 12 papers addressing IUI with CP and/or OP rates, including: 3 RCTs, 7 cohort studies, 1 observational study and 1 prospective analysis –28. The The literature suggests that IUI-OS is an effective treatment for women with unexplained infertility as compared to EM , 18, 22.A large retrospective cohort study (n = 851) concluded IUI-OS to be well-tolerated, with 37.4% CP rate over 3 cycles and patient preference for IUI-OS over the invasiveness of IVF . TherefoSeveral studies did not examine differences between forms of OS; further trials would benefit from exploring forms of OS on the effects of increasing pregnancy rates with IUI , 23. It 15 studies compared IUI and IVF with LB rates, with 8 RCTs, 2 cohort studies, 2 longitudinal studies, 1 cross-sectional study and 2 retrospective analyses –45.5 RCTs and 1 cohort study concluded that IUI-OS should be offered as first-line treatment over IVF –45. Rath41The Fast Track and Standard Treatment (FASTT) trial, a 2010 RCT, compared participants initially undergoing 3 cycles of IUI-OS with CC before embarking on treatment pathways of either: 3 cycles IUI-OS with FSH before 6 cycles of IVF (n = 247) or solely 6 cycles of IVF (n = 256) . AlthougRetrospective studies and RCTs with smaller sample sizes have attempted to corroborate these initial FASTT recommendations for the early, direct implementation of IVF rather than IUI-OS , whether in a combined or stand-alone approach –36. UnfoThe ESHRE guidelines , 11 also13 studies reported CP and/or OP rates with IUI-OS and IVF, with 7 RCTs, 4 retrospective cohorts and 2 prospective cohorts 38, 48, 49.All RCTs favouring IUI-OS found no difference between IVF and IUI-OS but argued IUI-OS is less invasive and less expensive with higher patient tolerance , 41, 44.Follow-up length and number of cycles differed between studies, which may account in part for the variation in results. Tjon-Kon-Fat et al. published a high-quality, multi-centre RCT with no difference in OP nor LB rates between treatments but had limited their trial to a 12-month analysis post first treatment . This maThe cohort studies were limited in quality by their design and retrospective nature , 48, 49.Similarly to LB rates, the ESHRE guideline also finds that 1 cycle of IVF is associated with higher CP rates compared to 3–6 months of EM  based on 2 RCTs, but due to its very low quality, the evidence was deemed inconclusive , 11, 50.Concerns over IUI-OS as first-line treatment is mainly attributed to its risk of MP, which is associated with considerable maternal and neonatal morbidity, in addition to substantial financial burden .Evidence of varying quality demonstrated comparably low MP rates following IUI-OS and IVF, in which most were twin pregnancies , 45, 51.The literature identified suggests acceptably low MP rates can be achieved with IUI-OS, with the implementation of strict cancellation criteria. However, future research and clinical practice should look to determine more standardised criteria across countries worldwide. No significant differences in multiple pregnancy rates were reported between IVF and IUI-OS treatment in systematic reviews by Pandian et al.  , and NanThis review has highlighted several limitations within the literature identified by our search. Firstly, there was no standardisation in the definitions of unexplained infertility and pregnancy outcomes between samples. This is particularly evident for pregnancy rates where definitions vary widely. Further heterogeneity was also observed amongst the diagnostic and inclusion criteria of the selected studies with international comparisons being difficult to make. The literature identified is skewed towards cohort studies, mainly of a retrospective design, emphasising the lack of high-powered RCTs. Studies associated with fertility treatments often have high dropout rates, of which emotional burnout and time commitments were frequently cited as reasons for discontinuation , 39. GivThis review itself has limitations with regards to subsets of assisted reproductive techniques, and EM with OS was not addressed. This work was also unable to evaluate IUI without OS as literature regarding its efficacy upon pregnancy outcomes was minimal. Additionally, it may have been useful to standardise the following methodologies: agents and doses used in OS, timing of insemination within the menstrual cycle, acceptable fertility prognosis as well as algorithmic methods and tools used to calculate this. A variety of maternal and neonatal outcomes were also not addressed, such as incidence of ovarian hyperstimulation syndrome, mode of delivery and preterm birth rates. Evaluation of cost-effectiveness was outside the scope of this review and discrepancies between national healthcare models may yield varying analysis, although widely agreed that upfront costs of IVF are higher , 41, somUpon a review of the existing literature, IUI-OS largely demonstrates improved LB, CP and OP outcomes when compared to EM. Studies comparing IUI-OS and IVF were altogether inconclusive; with some studies reporting increased LB, CP and OP outcomes with IVF and others finding no difference between the interventions. Yet, seminal research to support IVF is over 15 years old and more recent research concludes that IUI-OS should be favoured, albeit due to higher patient tolerance and lower cost rather than differences in efficacy. As supported by systematic reviews and meta-analyses, further RCTs are warranted . Future Future work may support emerging evidence from ESHRE regarding the use of IUI-OS as first-line treatment. This review identified a paucity of information regarding the use of active treatments amongst women above the age of 40 which warrants further study in the contexts of unexplained infertility. Additionally, this review identified the risk of multiple pregnancy and the importance of implementing risk counselling as important considerations for the clinical applicability of these interventions. However, in the interim of awaiting more conclusive evidence, clinics should continue to offer both interventions, especially as we await further review of NICE guidance in light of the emerging literature presented by ESHRE and presented in this review. The decision to offer IUI-OS or IVF should be based upon factors such as the woman’s age, duration of infertility, existing pathologies, previous fertility treatments and previous pregnancies. We suggest that IUI-OS could be offered to women with unexplained infertility who are less than 38 years with good prognosis, and IVF could be offered first to those greater than 38 years of age. Patients should be appropriately counselled to enable them to make an informed decision.Below is the link to the electronic supplementary material.Supplementary Material 1