A strong correlation between increasing age and higher GDF-15 levels was identified . Mean age was different between the groups, and this represents a confounding factor of the measurements. FGF-21 levels did not differ between migraine patients and controls  but were significantly influenced by increasing BMI . Neither biomarker showed correlation with headache frequency. Higher FGF-21 levels were associated with a higher mean intensity of headache attacks, reduced health-related life quality and anxiety. When confounding factors were considered, increased serum levels of FGF-21 and GDF-15 were not detected in migraine patients. However, the results show an age-dependence of GDF-15 in migraine patients, and this should be considered in future studies. Similar findings apply to the relationship between FGF-21 and BMI. Previous studies that did not adjust for these factors should be interpreted with caution.Mitochondrial processes may play a role in the pathophysiology of migraine. Serum levels of two biomarkers, Fibroblast-growth-factor 21 (FGF-21) and Growth-differentiation-factor 15 (GDF-15), are typically elevated in patients with mitochondrial disorders. The study investigated whether the presence of migraine may influence FGF-21 and GDF-15 serum levels considering vascular and metabolic disorders as possible confounders. A cross-sectional study in two headache centers was conducted analyzing GDF-15 and FGF-21 serum concentration in 230 patients with episodic and chronic migraine compared to a control group. Key clinical features of headache were evaluated, as well as health-related life quality, anxiety and depression using SF-12 and HADS-questionnaires. Elevated GDF-15 values were detected in the migraine group compared to the control group (506.65 ± 275.87 pg/mL vs. 403.34 ± 173.29 pg/mL,  Clinical, biochemical and genetic studies indicate the role of mitochondrial dysfunction in the pathogenesis of migraine. It is a complex disorder of sensory processing, and its pathophysiology is far from being fully understood . As migrIn this context, biochemical analyses in patients with migraine showed significantly elevated lactate levels in serum and liquor pointing towards a disturbance in the oxidative metabolism ,3. This From the epidemiologic point of view, several studies show a higher prevalence of migraine than expected in patients with a genetically proved mitochondrial disorder such as MELAS, MERRF (myoclonic epilepsy with ragged red fibers) and LHON (Leber’s hereditary optic neuropathy) ,12,13,14As non-invasive and economical steps in the diagnosis of a mitochondrial disease, the peptide molecule fibroblast-growth-factor-21 (FGF-21) and growth-differentiation-factor-15 (GDF-15) are established biomarkers. Both were found to be elevated in patients with mitochondrial diseases ,20,21. TThis study investigated whether serum concentrations of both FGF-21 and GDF-15 are altered in patients suffering migraine considering disease severity, attack morphology and concomitant disorders.A multi-center controlled cross-sectional study was conducted. All participants were recruited at two tertiary headache centers for all participants suffering migraine and at an outpatient medical office for control subjects. Subjects were eligible if aged between 18 and 70 years. For the migraine group, patients with episodic migraine (EM) with at least one migraine day a month within the last three months and chronic migraine (CM) were eligible. A well-kept headache diary at least for the last three months was necessary for inclusion. The presence of other primary or secondary headache disorders other than tension-type headache (maximum three days per month) was an exclusion criterion in this group. In the control group, an infrequent tension-type headache with no more than one headache day a month was allowed. Headache diagnoses were made by a headache expert at each center. A physical and clinical neurological examination was performed as part of the clinical routine. 2), myopathy, diabetes mellitus, known coronary heart disease and liver disease. If there were anamnestic  or clinical–neurological  indications of a possible mitochondrial disease, the patient was not included in the study. The local ethics committee of the Martin-Luther-University Halle-Wittenberg approved the study. Written informed consent was obtained from all individual participants included in the study. Data collection took place in the years 2018 and 2019.General exclusion criteria were (a) no informed consent could be taken and (b) at least one of the following criteria is met: proved or supposed mitochondrial disorder, obesity  . Headachg for 15 min and plasma was then transferred to polypropylene tubes and stored at −80 °C. Concentrations of FGF-21 and GDF-15 were measured using commercially available kits . The scientist who performed the assays (L.S.) was blinded to the study group the sample belonged to.Plasma concentrations were calculated using an ELISA-based approach. After the headache interview, blood samples were drawn from a peripheral vein  into a 9 mL tube prefilled with EDTA with a final concentration of 1.6 mg/mL . Blood samples were centrifuged at 2500× Explorative statistical analyses were performed using IBM SPSS Statistics 25.0 for Windows . Correlations between the concentrations of the mitochondrial biomarkers and parameters of headache were analyzed using simple and multiple linear regression. For non-dichotomous characteristics such as age, grouped analyses were performed as shown in the respective sections. Non-parametric tests  were used to compare FGF-21 and GFD-15 between groups. The individual statistical tests are indicated within the results section. A total of 328 patients were included in the study, 230 in the migraine group and 98 in the control group. Demographic data and headache-related parameters are presented in The MIDAS score showed a severe disability in 207 patients (91.6%). The results of the SF-12 questionnaire were compared with a norm sample representative for Germany . The phyn = 230) with the control group . In contrast, GDF-15 plasma levels were elevated in the migraine group  compared to controls . No differences were seen comparing EM with CM with regard to both FGF-21 (p = 0.195) and GDF-15 . The results are summarized in All data are given as median and 25–75% quartile range unless otherwise specified. The plasma level of FGF-21 was not different comparing the migraine group . This could not be found for GDF-15 . The results with the appropriate statistical tests are summarized in Furthermore, no correlation could be observed between the concentration of any biomarker and the number of headache days a month, the localization of headache, the presence of aura and family history of migraine . Regardip < 0.001, p < 0.001). In the multiple regression analysis considering possible influencing factors /year). This was reproducible for both recruitment sites. Comparing GDF-15 levels between the control and the migraine group within the defined age groups, no significant differences could be detected .For GDF-15, a strong association of higher values with increasing age was found. Participants were divided into age cohorts (10 years). A difference of GDF-15 values between all groups could be shown . This was true for both recruitment sites. In the multiple regression analysis /BMI). This was reproducible for both recruitment sites. Within the defined BMI groups as stated in p-range 0.188–0.719, Mann-Whitney U tests).FGF-21 levels raise with increasing BMI. For analysis, the participants were divided in BMI groups b. The stanalysis , a positp-values with the corresponding confidence intervals and not the medians are shown. The analysis confirmed the association between BMI and FGF-21 and between age and GDF-15 when the other factors are also included. In addition, the results showed that higher headache intensity was associated with higher levels of both biomarkers. However, taking all these factors into account, no association between the presence of migraine and elevated serum concentrations of the two mitochondrial biomarkers can be demonstrated, as shown in the first row in Multiple regression analysis was performed including potentially influencing factors on the FGF-21 and GDF-15 values collected in this study. All parameters included in the regression analysis are shown in This study investigated the association between serum concentrations of the biomarkers FGF-21 and GDF-15, which have been investigated for mitochondrial diseases, and the presence of migraine. In a non-parametric analysis, elevated serum levels of GDF-15, but not FGF-21, were found in a large cohort of patients with migraine compared with an appropriately sized control group. The migraine group consisted of nearly 50% of patients with chronic migraine and therefore represents a group of severely affected patients at two tertiary headache centers. This is underlined by the results of SF-12 and HADS-questionnaire in the migraine group showing reduced life quality and depressive symptoms in a relevant proportion of the patients. However, in the multiple regression correcting for identified confounders, no association between both biomarkers and migraine was found. They are peptide molecules with pleiotropic effects. Although both parameters (possibly GDF-15 superior to FGF-21) are considered capable of distinguishing patients with mitochondrial diseases from those without , serum cPrevious studies have identified different cut-off values of the two biomarkers for mitochondrial disease in children, young adults, and adults ,15,16. ERegarding clinical characteristics of migraine, no association between the two biomarkers and monthly headache days and no difference between patients with episodic and chronic migraine were detected. In this context, it has already been shown that patients with mitochondrial diseases and migraine do not have an increased attack frequency compared to patients with migraine . With inNo associations with depression, anxiety or decreased quality of life were found for GDF-15 in this study. Anxiety correlates with lower FGF-21 values in migraine patients. This is partially in line with existing data showing reduced FGF-21 levels in cerebrospinal fluid in patients with anxiety symptoms , althoug(a)The determination of FGF-21 und GDF-15 does not allow a valid statement about mitochondrial involvement in migraine. This may also be true in other diseases where mitochondrial involvement is suspected. This is in line with a recently published study, in which plasma levels of GDF-15 assessed in patients with open angle glaucoma were found to be strongly confounded by age and vascular diseases . Due to (b)Studies that investigated FGF-21 and GDF-15 and did not correct for vascular, inflammatory and metabolic disease should be interpreted with caution.(c)Mitochondrial involvement in migraine cannot be excluded. There are still no known biomarkers in migraine that can predict, e.g., a therapeutic response to prophylaxis and/or acute therapy. Further studies, preferably in a younger group, could investigate the effect of vitamin B2 (riboflavin) and coenzyme Q 10 in migraine patients with elevated GDF-15 and/or FGF-21 levels.Our data underline thatLimitations of this work are the exclusive cross-sectional approach and the use of a non-age-matched control group. The biomarkers were collected at different times of the day, and the subjects did not have to be fasting. Life circumstances, e.g., food habits or physical activity, were not considered. Furthermore, metabolic or cardiac diseases were not recorded in detail , except for the exclusion criteria mentioned above. Chronic inflammatory diseases may affect GDF-15 levels, as well as possibly FGF-21, and this was not analyzed in detail. Additionally, despite history and clinical examination, mono- or oligosymptomatic mitochondrial diseases in particular cannot be excluded in the study population. The influence of long-term medication, e.g., migraine prophylaxis, on biomarker values was not studied explicitly. This could be further investigated in a longitudinal study. From the group of anticonvulsants, valproate in particular should be avoided in mitochondrial diseases ,46. SincThe strengths of this work are the well-characterized migraine group with detailed recording of clinical parameters at two tertiary headache centers, the analysis of anxiety and depression symptomatology and the recording of health-related quality of life.A further diagnostic of mitochondrial diseases will be offered to the participants with highly elevated levels of one or both biomarkers.


We focus on the role and current applications of 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography in the management of lymphoma patients through improved staging or treatment response assessment or through early PET-driven therapeutic strategies, leading the way to a novel area of personalized medicine, optimizing disease control and toxicity. We discuss the potential future directions of innovative metabolic metrics that are being developed, notably to assess response to new immunotherapy regimens and to provide an improved prognostic factor for predicting patients’ survival. Finally, we present new radiopharmaceuticals developed following the identification of pathways or specific receptors in lymphomas, providing great opportunities for molecular imaging in treatment evaluation and management.18F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG-PET/CT) is an essential part of the management of patients with lymphoma at staging and response evaluation. Efforts to standardize PET acquisition and reporting, including the 5-point Deauville scale, have enabled PET to become a surrogate for treatment success or failure in common lymphoma subtypes. This review summarizes the key clinical-trial evidence that supports PET-directed personalized approaches in lymphoma but also points out the potential place of innovative PET/CT metrics or new radiopharmaceuticals in the future. Over the past several decades, positron emission tomography coupled with computed tomography using 18F-fluorodeoxyglucose (FDG-PET/CT), a tracer of glucose metabolism, has been considered as state-of-the-art imaging and provided the most significant improvement compared to conventional imaging in staging lymphoma . At baseIn this article, we focus on the current applications and future directions of FDG-PET/CT in managing patients with lymphoma through the development of new metabolic metrics as well as new radiopharmaceuticals.FDG-PET/CT have shown better diagnostic performance than CT, especially for extra nodal involvement, as this functional imaging is able to identify tumor lesions with high glucose metabolism when no significant abnormalities are detected by CT. This led to treatment modification in several cases in which FDG-PET/CT evaluation caused upstaging of lymphoma patients.Interestingly, FDG-PET/CT provides a better evaluation of bone marrow involvement compared to bone marrow biopsy that evaluates a very limited sample of bone marrow, whereas an FDG scan can identify focal involvement in the whole body. Invasive bone marrow biopsy is no longer indicated for the routine staging of Hodgkin lymphomas (HL) and most diffuse large B-cell lymphomas (DLBCL), and FDG-PET/CT has become the gold standard for the assessment of bone marrow invasion ,4,5.The baseline acquisition is also needed as reference to evaluate treatment response and is also used to calculate innovative PET driven metrics that will be described later in this review.Several interpretation criteria have been proposed to assess tumor response in lymphoma. The first FDG-PET/CT trials focused on the characterization of residual masses after end of treatment, as it was known that residual masses in CT were not strongly related to a treatment failure and thus patient’s evaluations were often classified as “Unconfirmed Complete Response” ,7.In 2009, in an international workshop held in Deauville (France), a standardization of these residual masses’ characterization was proposed in order to compare results across trials. This visual 5-point scale (5PS), named the Deauville score, is currently a widely used interpretation criteria in lymphoma and has even been derived to evaluate other solid cancers. The Deauville score has been reshaped in the recent Lugano classification, which introduces tumor response additionally to residual masses’ characterization but still mainly relies on Deauville score definitions. As a result, FDG-PET/CT was formally incorporated into standard staging and response assessment of FDG-avid lymphoma.In the Lugano classification, Deauville scores 1 to 3  are considered as complete metabolic response (CMR), whereas 5PS scores 4 and 5  are considered either no-metabolic-response , partial metabolic response , or progressive metabolic disease . This Lugano classification has demonstrated good prognostic value and excellent inter-observer reproducibility and should now be considered as the method of choice for standardization of FDG-PET/CT reports.Systematic surveillance exams in patients with a negative FDG-PET/CT at the end of treatment, in particular in patients with HL or DLBCL, are no longer recommended since it was found to be associated with false positives and increased radiation exposure with no evidence of benefit in patient outcome ,9.The recent introduction of immunomodulatory agents may complicate response assessment due to the development of a flare reaction or pseudo-progression in response to these agents. Provisional criteria have been proposed  with the introduction of the term “Indeterminate Response” to address such lesions. Renewed imaging after 12 weeks to distinguish pseudo-progression from real progressive disease is recommended . A recenAll these recommendations have improved risk stratification in patients with lymphoma and result in a clearer identification of non-responders with a significant prognostic benefit. This strong clinical impact has been reported in large cohorts of patients with the most frequent lymphoma subtypes  and is also supported by cost-effectiveness analyses ,13.FDG-PET/CT exams allow prediction of outcome after completion of treatment, and they also provide insights for a risk-adapted strategy to distinguish poorly responding patients requiring additional therapy, from good responders who may be exempt from part of the standard therapy, thereby limiting toxicity. The concept of interim PET (iPET) emerged in the last ten years and allows for analyzing a continuous metabolic process during treatment ,15. WhenThe prognosis of HL has been improved with the new therapy regimens established over the last decades, including ABVD  and BEACOPP  ,17. In pTwo response assessment timings are now approved, including iPET during the first line of chemotherapy, and end-of-treatment FDG-PET/CT after the first line of chemotherapy (±radiotherapy).Early treatment evaluation performed after two cycles of chemotherapy by iPET has shown a high prognostic value in several studies ,18,19 wiThe intergroup randomized study H10 , conductIn the H10 trial , iPET2+ The RAPID trial ,28 incluIn advanced stage disease (stages IIB-IV), BEAesc in the HD18 trial  has demoTwo interesting approaches were investigated to limit BEAesc exposure. The first strategy proposed treatment escalation for poor responders after two cycles of ABVD by introducing the BEAesc. In the RATHL Study , after tThe second approach was a reverse strategy evaluating treatment de-escalation for good metabolic PET2− responders after two cycles of BEAesc because the negative predictive value of PET2 is significantly superior to its positive predictive value.p = 0.65) with 87% of iPET2− patients in the experimental arm (vs. 88% in the standard arm) (median follow-up of 50.4 months). This demonstrated the possibility of underexposing treated patients and limiting the risks of immediate and late toxicity, confirmed by the rate of serious adverse events significantly lower in the experimental arm . Furthermore, in both arms, a new time point for FDG-PET/CT decisional evaluation was investigated after four cycles (iPET4), its positivity being considered as a treatment failure and iPET4+ patients being eligible for salvage therapy. Thus, three prognostic groups were identified according to the full PET-driven strategy (iPET2/4) with a 5y-PFS of 90.9% , 75.4% , and 46.5% (iPET4+). This strategy offers an interesting stratification of the patients that is more relevant than the classical biological parameters as prognostic analysis of factors affecting PFS revealed that in multivariate analysis only the results of the complete strategy including iPET2/iPET4 remained significant. The AHL2011 trial ,35 incluThe HD18 trial  includedDuring the past several years, significant prolonged survival in DLBCL has been observed in all age groups, particularly with the addition of a chimeric anti-CD20 monoclonal antibody, rituximab, to intensive cytotoxic chemotherapeutic regimens: cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) . InterimThe randomized phase II DLBCL LNH 2007-3B  includinThe phase III prospective study GAINED  randomizRecently an individual patient data meta-analysis was built to determine the optimal timing and optimal PET positivity criteria for interim PET to predict response in 1977 DLBCL patients from the PETRA database . All patDespite the fact that FDG-PET/CT is accepted as the current gold standard for response assessment, 15–20% of DLBCL patients with metabolic CR will experience disease recurrence. The exploration of additional response assessment approaches is justified. Growing evidence is emerging on the value of next-generation sequencing techniques to identify minimal residual disease and circulating tumor cell DNA to assess response . FDG PETFL represents the most common indolent lymphoma and can have a variety of clinical presentations . About 9Regarding response assessment, the Deauville Score analysis of post-induction FDG-PET/CT seemed to represent, in this histologic subtype also, a helpful prognostic tool. It better identifies patients with no active metabolic disease (complete metabolic responses) compared to the CT-based or IHP response evaluation criteria  with impMantle Cell Lymphoma (MCL) is an aggressive subtype of NHL that represents about 5% of all NHLs . The valp = 0.0003) and OS (p = 0.0003). SUVmax appears to have an even greater prognostic value when combined with clinical and biological biomarkers  fluoro-9-β-D-arabinofuranosyl-adenine) also appears as a good candidate to enhance diagnostic accuracy and therapeutic evaluation especially in low or heterogeneous FDG-avid lymphoma, showing high selectivity for lymphoid cells, regardless of the cell cycle ,100,101.In addition to their widespread use in solid cancers, the performance of lipid tracers also looks promising in lymphoma, although the literature is currently more limited. Tsuchiya et al. have compared the value of 11C-acetate and FDG-PET/CT in a small series of patients  with appThe spread of phenotypic PET imaging using monoclonal antibodies (mAbs) or peptides provides a set of non-invasive options to investigate in vivo targets’ expression and distribution and to acquire robust diagnostic, prognostic, and theranostic information. So far, in lymphoma, the vast majority of studies focusing on phenotypic imaging have evaluated in the context of radioimmunotherapy for mAbs distribution, pharmokinetics, and absorbed radiation doses ,107 targA novel and promising target is CXC chemokine receptor 4 (CXCR4). This transmembrane receptor is involved in the cell migration process and in the homing of hematopoietic stem cells to the bone marrow compartment. Several studies reported the use of 68Ga-Pentixafor that showed a high contrast in CXCR4-expressing lymphomas ,116,117.Fibroblast activation protein inhibitor (FAPI) also emerges as a new and highly promising probe for diagnostic and possibly theranostic application in various neoplasms including lymphoma. FAP is overexpressed by cancer-associated fibroblasts (CAFs) present in tumor microenvironment, which provides a high tumor uptake and a very low accumulation in normal tissues, resulting in excellent signal-to-noise ratios ,120.Finally, another area for future development of PET imaging in lymphoma is the rise of immunomodulatory therapy options, such as immune checkpoint inhibitors and CAR T-cells therapy. Indeed, despite the lack of literature in haematological neoplasia, these applications generate many expectations in view of the importance of these two therapeutic options in NHL. In this way, a recent study reported the feasibility of PET imaging with radiolabeled programmed cell death-ligand 1 (PD-L1) mAbs for the assessment of therapy in solid malignancies .Huge chaFDG-PET/CT stands as the main imaging modality in lymphoma. Compared to computed tomography, FDG-PET/CT improves staging and end of treatment evaluation, including improved residual mass evaluation by discriminating between fibrosis and remaining disease.Early assessment of response to therapy demonstrated strong prognostic value, and a series of recent prospective randomized phase III studies confirmed the early PET-driven therapeutic strategy, leading the way to a novel area of personalized medicine, optimizing disease control and toxicity.New FDG-PET/CT parameters are being developed to assess response to new immunotherapy regimens and provide an improved prognostic factor for predicting patients’ survival. Radiomics in particular is a very challenging area of research. In contrast to histological biopsy-based invasive biomarkers that examine a limited area of tumor, radiomics non-invasively investigates the entire lesion or disease. Because visualizing tumor heterogeneity is crucial for assessing the aggressiveness and prognosis of lymphoma, radiomics might offer tremendous possibilities in the management of these patients.Finally, the identification of pathways or specific receptors in lymphomas has enabled the development of new radiopharmaceuticals providing great opportunities for molecular imaging in treatment evaluation and management. It is unclear whether these radionuclide probes besides FDG will have a part to play in clinical routine diagnostics. However, the tendency for very advanced treatments, which depend on the genetic and phenotypic composition of lymphoma cells, provides exciting opportunities for nuclear medicine in the context of immunotherapy and personalized medicine. Ultimately, molecular imaging could lead to greater cost-effectiveness by allowing candidate selection for expensive targeted therapies, among which CXCR4 is a serious candidate. Other new tracers, such as FAPI, could be used in addition to ¹FDG-PET/CT imaging, specifically in indolent lymphoma patients.


Methods: Data about types of SU activities and admissions were obtained from 10 SUs throughout Italy, certified by the Italian Multidisciplinary Working Group on Syncope (GIMSI), from 10 March 2020 to 31 December 2020 and compared with the same time frame in 2019. Results: A remarkable reduction in overall non-invasive diagnostic tests  and cardiac invasive procedure. Elective cardiac pacing procedures disclosed a significant decrease ; conversely, the decrease of urgent procedures was not significant . There was a significantly increased rate of patients who underwent both telemedicine follow-up visits  and cardiac implantable electronic devices (CIEDs) remote monitoring follow-up visits . Conclusion: The COVID-19 outbreak was associated with a remarkable decrease in all clinical activities of Syncope Units in Italy, including both non-invasive tests and cardiac invasive procedures; conversely, a significant increase in telehealth activities was shown.The aim of our study was to evaluate the impact of the COVID-19 outbreak on Syncope Units (SUs) Activities in Italy.  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic human coronavirus recognized as the cause of coronavirus disease 2019 (COVID-19) . Followip-value less than 0.05 was considered statistically significant. All analyses were performed by SPSS Software, Version 24  and STATA 14.0 software .This is a retrospective multicenter observational study. Data about syncope unit activities including clinical and instrumental non-invasive evaluations, interventional cardiac procedures, type of SU admission and diagnosis were obtained from 10 Syncope Units, certified by the Italian Multidisciplinary Working Group on Syncope (GIMSI) throughout Italy, from 10 March 2020 to 31 December 2020 and compared within the same time frame in 2019. Assessed instrumental non-invasive examinations were all tests currently used for syncope evaluation according to the current guidelines . Assessep = 0.04], whilst both subgroups were equally distributed for sex. The clinical visits for the initial TLOC evaluation were 858 in 2019 vs. 344 in 2020, with a reduction rate of −59% (p = 0.001). Overall non-invasive diagnostic tests were 2.300 in 2019 vs. 752 in 2020, with a reduction rate of −67% (p < 0.001). The type and number of diagnostic tests are graphically represented in One thousand two hundred and seventy-five patients who were referred to SUs for the management of TLOC and related symptoms over the two observation periods were enrolled in the study, of which 377 were during the national lockdown for COVID-19 outbreak and 898 during the same time period in 2019. Patients treated during the COVID-19 lockdown were significantly younger than those treated in 2019 [median age 63 years (IQR: 46–80) vs. 70 years (IQR: 46–80) vs. p < 0.008), followed by a remarkable reduction in both outpatient  and emergency department activities   .p < 0.001). There was a remarkable reduction in both ILR  and PM implantation . Elective cardiac pacing procedures disclosed a significant decrease ; conversely, the decrease of urgent procedures was not significant   .Despite a significant reduction rate in the diagnosis of all types of syncope, no differences of prevalence according to the type were shown across the two observation periods .p < 0.001) and CIED remote monitoring follow-up visits   . PopulatOur findings suggest that the COVID-19 lockdown was associated with a significant reduction rate in all the clinical activities of Syncope Units in Italy, including both non-invasive tests and cardiac invasive procedures. The remarkable changes in the SUs activities were more likely because of the reduction of patients referred from other hospital wards, which were converted into COVID-19 care centers; moreover, we noticed a reduction of outpatient accesses to SUs, mainly due to the Italian government’s measures to contain SARS-CoV-2 diffusion. The reduction of the emergency department admission rate for patients in need of an SU specialist evaluation might be explained by the fear of acquiring COVID-19 infection once hospitalized.Our evidences support and enhance the data which described the reduction in the hospitalization rate for percutaneous coronary intervention ,6 and caThe remarkable increasing use of remote monitoring for the follow-up of CIEDs recipients and the increasing telemedicine follow-up visits for patients who experienced TLOC and related symptoms during the COVID-19 lockdown suggest that telehealth helped us to assure continuous care, reducing disease exposure for staff and physicians ,13. In tThe COVID-19 outbreak caused a remarkable reduction of patients who were referred to SUs for the management of TLOC and related symptoms. This evidence suggests the hypothesis that TLOC episodes may have contributed to the increase in non-COVID-19 out-of-hospital mortality in Italy.


Coronavirus Disease 2019 (COVID-19) represents a complex therapeutic challenge. As the pandemic progresses, patients are presenting with ectopic pregnancies (EPs) and symptomatic COVID-19.We present the management of a patient with multiple medical comorbidities and tubal EP in the setting of severe symptomatic COVID-19 infection where all management options were precluded.Case report with literature review of management of tubal EP in the setting of severe symptomatic COVID-19 infection.After careful consideration of options, the patient underwent successful medical management with methotrexate while receiving supportive care for COVID-19.Methotrexate proved to be the safest therapeutic option in this patient. Management of patients with severe COVID-19 and gynaecologic emergencies should be individualised and carefully reviewed with evolving knowledge of COVID-19. The global pandemic of COVID-19 is caused by infection with enveloped positive-stranded RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may have multiple clinical presentations. Over 80% of patients infected with COVID-19 have mild disease, 14% severe illness, and 5% critical illness with an overall case-fatality rate of 2.3% among those with confirmed illness . Older aEctopic pregnancy (EP) is an unfortunate condition occurring in 2% of pregnancies . When unAs the pandemic progresses, patients are presenting with EPs and symptomatic COVID-19 infection. Consideration of pulmonary comorbidity in the setting of active COVID-19 infection and development of pregnancy may preclude established medical regimens to treat an EP. We present the management of EP with methotrexate (MTX) in the setting of severe symptomatic COVID-19 infection where established treatment regimens were guarded.2. She presented with complaints of worsening dyspnea and chest pain and was found to have COVID-19. She was started on low-flow nasal cannula oxygen therapy to maintain her oxygen saturation above 92%. She also had severe orthopnoea (not tolerating -15° of inclination). Chest X-ray showed a constellation of findings concerning for atypical/viral pneumonia. She was admitted to the medicine service for supportive therapy including oxygen and dexamethasone, albuterol as needed, and clinical monitoring. Her Beta-hCG (B-hCG) serum level was 3,838 mIU/ mL and a transvaginal ultrasound demonstrated an endometrial thickness of 4 mm, with no evidence of an intrauterine pregnancy (IUP), and a right adnexal mass with peripheral vascularity, highly suggestive of a right tubal EP. There was no free fluid noted on the ultrasound. At this time, the patient was experiencing significant COVID- 19 symptoms, which were exacerbated by her underlying asthma and morbid obesity.A 36-year-old Gravida 6 Para 3 at 6 weeks 4 days of gestational age presented to the emergency room of our institution in July of 2020. The patient had history of asthma, chronic hypertension, and morbid obesity class III with a body mass index of 55 kg/mOf note, three days before presenting to our ED, she presented to a different hospital due to vaginal bleeding and abdominal pain. Her B-hCG serum level then was 2,359 mIU/mL and ultrasound failed to identify an IUP. She was discharged with precautions and follow-up for pregnancy of unknown location.Considering the patient’s symptoms, active pulmonary disease, and comorbidities, a medical management of EP was initiated. A two-dose MTX protocol was carried out, with 50 mg/m 2 via intramuscular injection of MTX administered on hospital day (HD) 2 and 6. An appropriate drop in hCG >15% was noted . Mild trEP in the setting of symptomatic COVID-19 infection represents a challenge for the health care team. To the best of our knowledge, this is the first case report detailing EP management in a patient with severe COVID-19 respiratory symptoms. Hansel and Stovall recently published a case report of a patient with asymptomatic COVID-19 who was diagnosed with a non-ruptured EP . The autIn our case, extensive efforts between a multidisciplinary team were made to examine how her comorbidities would affect the management of the EP. Different therapeutic options were considered. Expectant management of an EP is an option for patients who meet specific criteria: 1) low and either decreasing or plateauing serum B-hCG levels, 2) asymptomatic, and 3) understand risk of potential need for surgical intervention if the EP ruptures . SuccessExpectant management in this patient’s case presented was not possible given serially increasing B-hCG serum levels, symptoms, and unacceptable higher likelihood of failure.The possibility of rupture resulting in hemodynamic instability requiring emergency surgery would be detrimental in this patient. She was deemed a high-risk surgical candidate due to her compromised respiratory status as a result of COVID-19 infection and underlying comorbidities of asthma and morbid obesity, which increased the chance of potential surgical/anaesthesia complication. She was strongly considered to be a difficult extubation possibly requiring long-term ventilator support postoperatively.Additionally, increased morbidity and mortality are associated with performing surgery on patients with pulmonary compromise due to COVID-19. The COVIDSurg Collaborative demonstrated 51% of patients with COVID-19 who underwent a variety of surgical procedures experienced pulmonary complications with a 30-day mortality of 38% . More coAlthough uncertain with regards to transmission, consideration was also raised regarding exposure of healthcare personnel to viral particles in the operating theatre within escaping surgical smoke or during intubation or extubation .Lastly, medical management with chemotherapeutic agent MTX was considered. Others have previously suggested medical management as a theoretical option for COVID- 19 patients . CurrentMost common side effects with MTX therapy are gastrointestinal, however, other rare side effects described are pulmonary toxicity, immunomodulation, and myelosuppression. Although not well understood, MTX-induced lung- injury has occurred via pulmonary hypersensitivity reaction , direct Patients with COVID-19 infection are noted to have leukocytosis and lymphopenia . Our patAdverse side effects are more likely with increased dose and duration of MTX. Theoretically, patients with severe symptomatic COVID-19 infection have a higher likelihood of developing side effects if renal function is impaired. Given the intermediate-dosage, limited exposure, and normal renal function in our patient, the risk of MTX toxicity and adverse effects was low.Evidence shows comparable and not statistically different rates of resolution for EP between single- and multiple-fixed dose regimens  as well as single- versus two-dose therapeutic regimen  . The mulAlthough, no cut-off value for serum B-hCG level has been determined to contraindicate medical management, increasing MTX therapy failure rates are reported with rising levels of pre- treatment serum B-hCG levels . One stuTherefore, in our case, a two-dose approach was used to maximize the opportunity of treatment success as this patient’s pre-treatment serum B-hCG value was 4,166.7 mIU/mL. It was concluded despite her comorbidities; this was the safest approach in managing her non-ruptured EP compared with risks posed by surgical and expectant management.This case addressed unanswered questions regarding suitable therapeutic approaches for patients with symptomatic COVID-19 and EP. Expectant management was precluded due to risk for rupture and need for emergent intervention. Surgical intervention would have increased morbidity and mortality. In this case, MTX for EP management was successfully administered.Further investigation is needed on most appropriate means to address EP within the spectrum of COVID-19 presentation. Treating this patient led us to consider the following regarding management. Expectant management with close follow-up should only be considered if there exists evidence of serially decreasing B-hCG levels suggestive of a failing pregnancy. When contemplating medical management, patients should receive evaluation by a multidisciplinary team to identify risk factors for treatment failure and toxicity as well as contraindications to MTX therapy. When considering contraindications, active pulmonary disease should be considered within the entire spectrum of COVID- 19 presentation as asymptomatic patients may also present with underlying pathology and atypical imaging . The COVIf surgical intervention is needed, the patients should be evaluated by all relevant care teams and medically optimised for the procedure. If general anaesthesia is contraindicated, alternative methods of sedation and analgesia may be considered. Efforts to limit exposure of healthcare personnel while effectively managing EP should be made, such as using minimally invasive technique with only necessary staff in the operating theatre. Personnel should wear personal protective equipment during any intervention.Any patient with high suspicion for EP rupture, should have rapid COVID-19 testing and be taken to the operating theatre for emergency surgery. Delaying surgical intervention, when deemed appropriate is strongly discouraged, since the benefits outweigh the risk of increased morbidity/mortality.Overall, patients presenting with EP and symptomatic COVID-19 need individualised and collaborative treatment planning. Patients should be extensively counselled regarding the risks of each management option. When available, multidisciplinary treatment planning should occur to reduce risk of morbidity/ mortality.


Enterobacteriaceae (ESBL-E) among international travelers visiting antimicrobial resistance (AMR) hotspots. Although antibiotic use and travelers’ diarrhea have shown to influence the ESBL-E acquisition risk, it remains largely unknown whether successful colonization of ESBL-E during travel is associated with the composition, functional capacity and resilience of the traveler’s microbiome. The microbiome of pre- and post-travel fecal samples from 190 international travelers visiting Africa or Asia was profiled using whole metagenome shotgun sequencing. A metagenomics species concept approach was used to determine the microbial composition, population diversity and functional capacity before travel and how it is altered longitudinally. Eleven travelers were positive for ESBL-E before travel and removed from the analysis. Neither the microbial richness (Chao1), diversity (effective Shannon) and community structure (Bray–Curtis dissimilarity) in pretravel samples nor the longitudinal change of these metrics during travel were predictive for ESBL-E acquisition. A zero-inflated two-step beta-regression model was used to determine how the longitudinal change in both prevalence and abundance of each taxon was related to ESBL acquisition. There were detected increases in both the prevalence and abundance of Citrobacter freundii and two members of the genus Bacteroides, in association with remaining uncolonized by ESBL-E. These results highlight the potential of these individual microbes as a microbial consortium to prevent the acquisition of ESBL-E. The ability to alter a person’s colonization resistance to a bacterium could be key to intervention strategies that aim to minimize the spread of MDR bacteria.Previous studies have shown high acquisition risks of extended-spectrum beta-lactamase-producing  For example, up to 84% of travelers to India and 42% of travelers to Morocco have been shown to have acquired MDR Enterobacteriaceae (MDR-E) in their intestines upon travel return.7 Considering that the intestinal tract is an open system, which is confronted with a myriad of bacteria from the environment , the human intestinal microbiota is considered the most important reservoir through which travelers acquire and import AMR.8International travel has increased in parallel with globalization, with pre-SARS-CoV-2 pandemic statistics of over 1 billion tourist arrivals occurring each year.9 or when undergoing specific medical procedures,10 intestinal colonization by MDR bacteria substantially increases the risk of MDR infections and consequently mortality. Enterobacteriaceae are the cause of some of the most common nosocomial MDR infections,11 and with the high rates of acquisition of Extended Spectrum Beta Lactamase-producing Enterobacteriaceae (ESBL-E) during international travel, the admission of travelers to hospitals is posing a serious health risk. Moreover, the introduction of MDR bacteria by international travelers also poses a potential risk for public health, as this permits the transfer of MDR bacteria to household members.12 Identifying successful intervention strategies to prevent MDR-E acquisition by travelers would therefore not only directly protect vulnerable groups of travelers that have an increased risk of infections by (translocation of) intestinal (MDR) bacteria  but also reduce the spread of AMR across the globe.Although most travelers will lose the acquired MDR strains within months after their return from travel, the acquisition of intestinal MDR bacteria can pose a direct health threat to the traveler. Specifically, when hospitalized, most significantly within intensive care units that frequently use large amounts of antibiotics,13 and travelers’ diarrhea13–15 have been repeatedly shown to increase the risk of MDR-E among travelers. In addition, travelers with pre-existing chronic bowel disease have a higher risk of acquiring MDR-E.7 Each of these factors are linked to major perturbations to the gut microbiome, suggesting that the microbiome might play a pivotal role in the susceptibility to MDR-E colonization upon travel. Indeed, the role of the intestinal microbiome in providing colonization resistance against incoming opportunistic pathogens has long been established.16 Endogenous microbes can directly or indirectly interfere with pathogen colonization through a variety of mechanisms, including the competition for nutrients and the production of small antimicrobial peptides (bacteriocins) .17–19 Moreover, the stimulation of the host immune system by the commensal microbiome has been shown to interfere with pathogen colonization.20Besides travel destination, the use of antibiotics during travel21 No significant associations between the intestinal microbiota composition before or after travel and the risk of MDR-E acquisition was found. However, the microbiota profiles did significantly differ between individuals who cleared MDR-E within one month and those who were persistently colonized. In another study among 10 international travelers, profiling of longitudinally collected fecal samples by metagenomic next-generation sequencing did not also reveal alterations in microbiome diversity and composition in those acquiring MDR-E.22To uncover the effect of travel on the gut microbiome, a previous study employed metatranscriptomics on fecal samples from 43 travelers visiting tropical regions.These inconclusive results highlight the need for a larger-scale longitudinal study using whole metagenome sequencing (WMGS) to examine if specific microbial taxa and their functional capacities may protect against MDR-E acquisition during travel.Here, we performed WMGS on fecal swabs of 190 travelers before travel and immediately upon return from South Asia, Southeast Asia, North Africa and East Africa. Metagenome sequences were processed via coabundance gene clustering to create taxonomic and metabolic profiles. In order to identify taxa and traits associated with a reduced rate of MDR-E acquisition, the (dynamics in) microbiome community structure, composition and functional profile were compared between travelers who acquired MDR-E during travel and those that did not.E. coli , 9 strains (6.1%) were characterized as Klebsiella pneumoniae and the remaining 3 strains were characterized as Proteus mirabilis, Citrobacter freundii and Klebsiella ornithinolytica.Fecal culturing revealed that 103 (57.5%) out of the 179 travelers who were negative for ESBL-E prior to travel acquired ESBL-E during their trip. From the post-travel fecal samples (T1) of these 103 travelers, a total of 148 morphologically distinct ESBL-E strains were isolated. The vast majority of strains were identified as Fecal swabs were whole metagenome sequenced and processed into coabundance genomes . Upon quality filtering and preprocessing of WMGS data of the pre- and post-travel samples, 37,381 CAGs were generated of which 144 CAGs contained more than 700 genes and were classified as MGS.P = .235) nor when stratified to travel destination .The ability of the baseline microbiome to predict a traveler’s risk of acquiring ESBL-E was determined by the richness, diversity, community structure and taxonomic composition of the MGS observed in pretravel samples of travelers who did or did not subsequently acquire ESBL-E during travel. The observed number of metagenomic species  and estimated number of species Chao1;  were sigWe subsequently examined whether the overall microbial community structure prior to travel, as examined by means of the Bray–Curtis dissimilarity, was associated with ESBL-E acquisition risk. Ordination of pretravel samples in Principal Coordinates Analyses (PCoA) did, however, not show any clustering according to ESBL-E acquisition. Fitting the ESBL-E acquisition status to the ordination, using envfit, confirmed the lack of association between the baseline microbial community structure and the risk of subsequently acquiring ESBL-E during travel ; P = 3.The significance of age, BMI, sex and ESBL-E acquisition in the microbial community structure was determined using the envfit function in vegan. The significance value was determined based on 999 permutations. All P values derived from envfit were adjusted for multiple comparisons using FDR adjustment (Benjamini–Hochberg procedure) (Supplementary Table 2).Microbiome structure and diversity are altered during travel, but mainly associated with the development of travelers’ diarrhea and not the acquisition of ESBL-EP = .95 and P = .85, respectively). Also when stratifying the analyses for travel destination, no associations between these measurements and ESBL-E acquisition were observed during travel (Supplementary Table 1).To examine the association between microbiome stability and the susceptibility to ESBL-E acquisition, longitudinal analyses were performed. When examining the shifts in alpha diversity metrics within travelers, the estimated microbial richness  and micrP < .001). When stratified for travel destination, the negative association with diarrhea was still observed for travelers visiting East Africa and South Asia (Supplementary Table 1).However, a statistically significant decrease in the microbial richness  and diversity  was associated with travelers’ diarrhea (both P = .504). Upon stratification according to travel destination, this lack of significant differences in the stability of the microbial community structure in association with ESBL-E acquisition remained.As for the stability in microbial richness and diversity, the stability in the microbial community structure was also examined among travelers. The within-subject Bray–Curtis distance between pre- and post-travel samples was not significantly different between travelers who did or did not acquire ESBL-E (P = .002). There was also a significantly larger within-subject Bray–Curtis distance between travelers who ate at local street food stalls daily . Upon stratification according to travel destination, this signal remained for travelers eating daily from street food stalls in Southeast Asia (Supplementary Table 2).However, the microbial community structure is less stable among travelers who developed travelers’ diarrhea as the within-subject Bray–Curtis distance was significantly larger among travelers that experienced diarrhea compared to those that did not , and three belonged to the genus Bacteroides . The genes within the remaining two MGSs hits various phyla, and interpretation for theslatter two MGSs should therefore be done with caution as these might be potential artifacts.The shifts in prevalence and relative abundance of individual microbial taxa in association with ESBL-E acquisition were next examined with ZIBR.g travel . Three bBacteroides MGS , had both a 0.42-fold decrease and a 0.33-fold decrease in travelers who did and did not acquire ESBL-E, respectively. Bacteroides MGS-098 had a 1.47-fold increase in abundance in travelers who did not acquire ESBL-E, but a 0.21-fold decrease in travelers who acquired ESBL-E. Additionally, MGS-115, the most prevalent Bacteroides, had a 0.69-fold increase in travelers remaining ESBL-E-negative and a 1.01-fold decrease in travelers who acquire ESBL-E.MGS-091, the most abundant Citrobacter freundii. Both C. freundii MGS showed similar longitudinal patterns; increasing 5.96 and 5.95 fold, respectively, in travelers who did not acquire ESBL-E while decreasing 2.23 and 1.45 fold in travelers who did. The abundance of MGS-016, identified as Klebsiella pneumoniae, has both a 1.28- and 2.02-fold increase in those subjects who did and did not acquire ESBL-E, respectively. However, the increase in its prevalence from 8.9% to 31.7% of subjects is larger in travelers who acquire ESBL-E during travel, compared to 7% to 20.8% of subjects in those who remain negative for ESBL-E.The most pronounced shifts were, however, observed for two MGSs identified as MGS-090 had a 0.04-fold decrease in travelers who remained negative for ESBL-E, but a 0.6-fold decrease in those who acquired ESBL-E. MGS-134 had a 0.94- and 1.16-fold decrease in travelers who remained negative for ESBL-E and acquired ESBL-E, respectively.The metabolic profile of the samples was determined by functionally annotating each gene of each MGS and clustering by the KEGG module. Samples were heirarchically clustered by distance to each other, based on the fold changes of each module, but did not cluster by ESBL-E acquisition . To examine if short-chain fatty acid production might be protective for ESBL-E acquisition, we next examined the abundances of KEGG Orthology genes involved in butyrate, succinate and propionate production. Neither the baseline abundances of these genes nor the abundances in post-travel samples or shifts in abundance between pre- and post-travel were associated with ESBL-E acquisition (Supplementary Tables 4–6).Enterobacteriaceae and genus Bacteroides were significantly associated with ESBL-E colonization risk.The gut microbiome’s role in providing colonization resistance against invading bacteria is of particular relevance in people traveling internationally to countries in which MDR bacteria are endemic. This study aimed to discover which aspects of the microbiome structure – or consortia of bacteria – contribute to colonization resistance against MDR bacteria. Neither the microbial diversity and community structure in pretravel samples nor the longitudinal change of these metrics during travel were predictive of the susceptibility to acquiring ESBL-E. Alternatively, prevalence and abundance changes in individual microbial taxa belonging to the family 21 where reads were mapped to 16S and 23S rRNA databases for identification, and small-scale WMGS on a data set of up to 10 travelers.22 There is therefore a limited scope and depth of current research into the role of the microbiome in this field, so in this study, the WMGS of 179 travelers provides a significant contribution to the closure of a major gap in the literature.Studies focusing on the association between colonization with MDR bacteria and the gut microbiota primarily utilize 16S rRNA gene amplicon methods, which have limitations in their lack of resolution to a species level analysis, and/or cross-sectional studies, which do not capture the longitudinal microbial changes. ESBL-E acquisition in travelers has been researched with meta-transcriptomics,22 Travelers’ diarrhea is known to be associated with an increase in ESBL-E acquisition,7 but ESBL-E acquisition without travelers’ diarrhea seemingly has little effect on the microbiome richness and diversity. This is unlike previous studies that associated increased pathogenic bacteria with a reduced microbial richness and altered microbiome in hospital patients.24 However, samples collected from hospitalized patients are often confounded by antibiotic use, which reduces the diversity and richness measurements, as well as significantly perturbing even stable microbiomes. The results here are therefore suggesting that ESBL-E can colonize a traveler’s microbiome without perturbation and without causing symptoms, but in those who develop travelers’ diarrhea, it is difficult to determine if ESBL-E colonization precedes the onset of diarrhea.In our study, the diversity and community composition did not differ between travelers before travel, or within individuals during travel, in relation to ESBL-E acquisition. Instead, after the development of travelers’ diarrhea, the diversity is significantly reduced and community composition is drastically altered, consistent with the literature.Citrobacter freundii had a statistically significant increase in abundance in travelers who did not acquire ESBL-E. Commensal Enterobacteriaceae, of which Citrobacter is a member, are continuously competing with other commensal or invading members of the family. This is achieved through various mechanisms, such as the secretion of antimicrobial proteins,25 the metabolism of nutrients26 or competition for oxygen.27 These mechanisms may change the host’s susceptibility to colonizing species,28 as it must have the necessary pathways to outcompete. Further studies on the interactions between C. freundii and ESBL-E in the human guts are required to uncover the mechanisms by which ESBL-E colonization might be inhibited by C. freundii.In this study, MGS both identified as Bacteroides was also associated with travelers who remained negative for ESBL-E, whereas a decrease of these MGS as well as an additional Bacteroides MGS was observed in travelers who acquired ESBL-E. The healthy gut microbiome is usually dominated by the strict anaerobe phyla of Bacteroidetes (of which Bacteroides is a member) and Firmicutes, whereas the facultative anaerobes of Enterobacteriaceae populate at a much lower proportion.29 Interestingly, in a recent cross-sectional study on the association between the fecal microbiota and ESBL-E colonization among 200 healthy volunteers living in rural Thailand, the most profound difference was detected in the phylum Bacteroidetes. In particular, the abundance of B. uniformis was significantly lower in ESBL-E carriers as compared to noncarriers.30A statistically significant increase in abundance of two MGS of Bacteroides species exhibit immunomodulatory effects that are beneficial to the host and can increase the colonization resistance to invading bacteria. A major mechanism of this is the inhibition of host inflammation via the secretion of sphingolipids,31 a signaling molecule or outer-member vesicles containing polysaccharide A,32 a molecule that induces the production of T cells to suppress inflammation.33 Low-grade intestinal inflammation perturbs the gut microbiome and can enhance the colonization of E. coli,34 so reducing this is vital to maintain colonization resistance to invading bacteria. Bacteroides species may have further immunomodulatory effects via the improvement of host macrophage’s phagocytic function35 or via an increased xylan degradation,36 which has links to an enhanced immune system and improved health.38 Moreover, Bacteroides species can produce a variety of short-chain fatty acids and organic acids, including acetic acid, propionic acid, isovaleric acid and succinic acid. Short-chain fatty acids have been shown to provide colonization resistance against antibiotic-resistant Enterobacteriaceae, among others, by triggering intracellular acidification.39 To reveal a potential protective role of metabolites, including sphingolipids, xylan breakdown products and short-chain fatty acids, produced by Bacteroides species or other members of the microbiome, future studies on the fecal metabolome are warranted.Various 40 and can lead to increase in abundance of taxa, notable Enterobacteriaceae and Ruminococcaceae, that can grow at room temperature in the presence of oxygen.41 However, as the samples all experienced the same conditions, the effects are not likely to differ between samples; instead, the quantities should not be extrapolated to other sampling methods.A major strength of this study is the use of whole metagenome shotgun sequencing as a representation of the total microbiome. The longitudinal nature of the data set additionally strengthens the study as it allows for temporal associations on the effects of travel on the microbiome, with a large sample size to correct for confounding factors. However, metagenomics limits the resolution at which to confidently identify individual strains. Instead, the taxa-specific analysis should be interpreted as where to focus potential future research. This study has a limitation with the sampling method employed, as the transport at room temperature and use of Cary–Blair medium on fecal swabs can increase the microbial richness and diversityEnterobacteriaeceae. The identification of species associated with ESBL-E acquisition necessitates for higher resolution research. The current analyses cannot easily detect how the commensal Enterobacteriaceae population is interacting with the invading ESBL-producing E. coli, so it would be of significant interest to perform experimental studies to explore the competition between various species and strains within this genus and family. The extent at which each member alters the microbiome’s colonization resistance could be discovered. Alternatively, the knowledge on factors facilitating the elimination of previously acquired ESBL-E is still limited, so research on samples taken on additional time points after travel is worthwhile being examined.The observations of this study highlight the likely importance of discrete species in the dynamics of ESBL-E acquisition, especially within the family Enterobacteriaceae, instead of highlighting the importance of Bacteroides and other members of Enterobacteriaceae. The discovery of taxa that may contribute to colonization resistance will aid in the development of intervention strategies to minimize the acquisition and spread of MDR bacteria.This study emphasizes how the overall microbiota diversity, community structure and functional metabolism play little role in the acquisition of ESBL-producing 42 Briefly, all travelers provided questionnaire data and fecal swabs before travel, immediately upon return and one month after return. Fecal swabs  were incubated in tryptic soy broth supplemented with vancomycin (50 mg/L) and after overnight culture, subcultured onto chromID ESBL agar plates . All morphologically distinct colonies were identified to the species level using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . For all Enterobacteriaceae, antibiotic minimum inhibitory concentrations were measured with the automated susceptibility testing system Vitek 2 (bioMerieux). Phenotypical confirmation of ESBL production was performed by combination disc diffusion tests, according to current national Dutch guidelines.43 Remaining feces was biobanked at −80°C for future analyses.The present study was embedded within the COMBAT study , a longitudinal multicenter study among 2,001 Dutch international travelers for which the design has been described previously.For the present study, we randomly selected paired pre- and post-travel biobanked fecal samples from 190 travelers who visited South Asia, Southeast Asia, North Africa or East Africa and who had not used antibiotics in the three months prior to travel departure. Pretravel samples of 11 travelers were positive for ESBL-E, and both pre- and post-travel samples of these travelers were removed from future analyses in order to study shifts in the microbiome in subjects “at risk” for ESBL-E acquisition. The metadata for the resulting 179 subjects who were negative for ESBL-E and had not taken antibiotics within three months prior to travel are displayed in 44 which includes bead-beating , USA) with 0.1 mm zirconium-silica beads , USA)) followed by purification using QIAamp DNA Stool Mini kit columns . A Qubit® fluorometer dsDNA HS Assay (Invitrogen) was used to quantify extracted DNA, and this DNA was stored at −20°C.Fecal metagenomic DNA was extracted using protocol Q of the International Microbiome Standards Consortium,45 purified using the Agencourt AMPure XP system (Beckman Coulter) and quantified using the Quant-iT PicoGreen dsDNA assay (Invitrogen). 10 nM of DNA from approximately 96 samples was equimolarly pooled (three independent times), for each sequencing lane. Pools were submitted for paired-end 2 × 150 base pair sequencing (llumina NextSeq High-Output platform) with a targeted sequencing depth of 5 million reads per sample. Raw shotgun metagenomic reads have been deposited and released to NCBI SRA under BioProject ID PRJNA688274Diluted DNA (0.5 ng/uL) was prepared for sequencing with a Nextera Library Prep kit as described previously,46 Reads were trimmed and filtered using fastx,47 with a minimum quality cutoff score of 15 and a minimum length cutoff of 60 bp. High-quality reads were assembled using Soap 2.0448 into scaftigs of a minimum length of 500 bp.Samples were processed using the MOCAT software.de novo gene prediction via MetaGeneMark.49 Predicted genes were clustered using BLAT by single linkage.50 Genes were clustered if there was at least a 95% sequence similarity and at least 90% of the shorter gene was covered. This created a nonredundant gene catalog.Scaftigs underwent 50), and a gene abundance table was produced.High-quality reads were screened against the nonredundant gene catalog with SOAPaligner from Soap 2.04 and the parameters of a length cutoff of 30 bp, a percent cutoff of 95% identity, a seed length of 30, a maximum number of mismatches of 5, a random assignment of multiple matches and the – M 4 flag indicating for it to find the best hits. Coverage of each gene per sample was calculated using the soap.coverage script (source code available at Ref. 50) that groups genes by similar abundance patterns across the samples. A canopy was created by grouping genes to a randomly selected gene within a distance of > 0.9 Pearson correlation coefficient and > 0.6 Spearman’s rank correlation coefficient. Multiple canopies were clustered together if their median abundances had a distance of > 0.97 Pearson correlation coefficient. Canopies were considered to be of insufficient quality when containing only 2 genes; at least 90% of their abundance was made up from only 3 samples or were present in less than 4 samples. Canopies of a high quality were identified as coabundance gene groups (CAGs) and CAGs with at least 700 genes contained enough information to potentially be classified as metagenomic species (MGS).The abundance table was analyzed with the canopy clustering algorithm (source code available at51) at a percentage identity of >45% on at least 100 bp and a maximum of 50 hits for each gene were kept. Bacterial taxa that received fewer than 25 hits were filtered out as noise. An MGS was assigned to a species if at least 70% of the total hits matched at a sequence similarity of >95% on at least 100 bp. Parameters were reduced to 60% of hits at >85% similarity to be assigned to a genus.The genes of each MGS were aligned to the blast nucleotide database  using BLASTN  by eggNOG-mapper.54 KEGG annotations were selected from the results, and pathways were grouped into KEGG modules for downstream analysis. KEGG abundance per sample was calculated as the sum of the MGS abundances in which the KEGG module was detected.All genes in each MGS were converted into amino acid sequences with Transeq.Additionally, the initial nonredundant gene catalog was annotated to KEGG Orthology (KO) and KOs belonging to butyrate, succinate and proprionate production were selected. Total microbiome abundances were calculated from the gene catalogs’ equalized gene abundance table, and by summing every instance, a KO was annotated to a gene in each traveler.55 The dissimilarity in the microbial community structure (beta diversity) between samples was calculated by means of the Bray–Curtis dissimilarity index. Associations between the microbial community structure and metadata were determined using Envfit from the package Vegan.Statistical analyses were carried out in R on the entire study population as well as stratified according to travel destination. Alpha diversity measurements of species richness, Chao1 index and effective Shannon index were calculated using Vegan.To examine changes in microbial richness and diversity over time, the delta in alpha-diversity  was calculated. To examine the (in)stability of the microbial community structure, the within-subject Bray–Curtis distance between pre- and post-travel samples was calculated.Univariate analysis consisted of the t-test or Mann Whitney U-test, depending on normality as determined by the Shapiro-Wilk normality test, which was applied to examine associations between ESBL-E acquisition and microbial richness, diversity or SCFA abundance at baseline, as well as longitudinal changes in richness , diversity (effective Shannon), SCFA abundance and community structure (Bray Curtis dissimilarity) measurements. Multivariate analysis consisted of fitting the data to a linear regression model, with the diversity measurement as the outcome and ESBL-E acquisition as the main covariate of interest, while adjusting for the following potential confounding factors: age (years), sex , BMI, travelers’ diarrhea (yes/no) and food consumption at food stalls (never/sometimes/daily).To reduce the number of comparisons, taxa or KEGG modules present in <10% of samples or in which the relative abundance in the top 10% of samples was below 0.1% were removed from further taxonomic and functional analysis.23 was carried out on all remaining taxa; except for taxa absent in less than 10 samples as this is a prerequisite of the zero-inflated aspect of the model. The model was fit using the post-travel CAG as the dependent variable and its pretravel abundance, ESBL-E acquisition, age, sex, BMI and travelers’ diarrhea as covariates for both the logistic and beta components of the model. The time point variable was set to 1 due to the study containing only two time points, and the Gaussian quadrature points was set to 30. Analysis was carried out on all samples collectively and repeated for each travel destination separately.Zero-inflated two-step beta regression (ZIBR) analysis56 was used for the remaining taxa detected in the majority of samples (i.e. absent in less than 10 samples). Post-travel CAG abundance was predicted against ESBL-E acquisition while using pretravel CAG abundance, age, sex, BMI and travelers’ diarrhea as additional covariates. Negative binomial generalized linear models were also used to study changes in KEGG module abundance in association with ESBL-E acquisition.A negative binomial generalized linear modelClick here for additional data file.


On the other hand, LEAP-2 administration markedly inhibited ghrelin-induced food intake in lean, obese (ob/ob and DIO) mice, aged rats and GH-deficient dwarf rats. In contrast, the inhibitory effect on glucose levels can only be observed in some specific experimental models indicating that the mechanisms involved are likely to be quite different. Taken together from these data, LEAP-2 emerged as a potential candidate to be therapeutically useful in obesity.Data gleaned recently shows that ghrelin, a stomach derived peptide, and liver-expressed-antimicrobial peptide 2 (LEAP-2) play opposite roles on food intake. However, the data available with LEAP-2 in relation to in vivo studies are still very scanty and some key questions regarding the interplay among ghrelin and LEAP-2 remain to be answered. In this work, using rats and mice, we study fasting-induced food intake as well as testing the effect of diet exposure, e.g., standard diet and high fat diet, in terms of ghrelin-induced food intake. The anorexigenic effect of LEAP-2 on fasting induced food intake appears to be dependent on energy stores, being more evident in  Ghrelin, a hormone produced in the stomach, is the most potent orexigenic hormone. Chronic ghrelin administration promotes weight gain and adiposity in rodents , as wellAlthough plasma LEAP-2 was positively correlated with BMI and food intake in humans and mice  and LEAPob/ob). We also carried out a comparison of the effects of LEAP-2 in food intake in relation to NPY, the most potent orexigenic neuropeptide known to be involved in the ghrelin orexigenic signaling pathway. Since ghrelin and LEAP-2 also appear to play a dual role on GH secretion, we studied their effect on food intake in genetically GHS-R1a-deficient animals or in the context of physiological GH deficiency as aging. Finally, we also studied the effects of LEAP-2 on glucose levels. Our data showed that LEAP-2 exerted a potent anorexigenic effect in mice and rats in a variety of experimental models. In contrast, the inhibitory effect on glucose levels can only be observed in some specific experimental models indicating that the mechanisms involved are likely to be quite different. Taken together from these data LEAP-2 emerged as a potential candidate to be therapeutically useful in obesity.In this work, we decided to carry out a careful characterization of the in vivo effects of LEAP-2 and ghrelin on food intake and glucose homeostasis. We decided to use a variety of experimental models including rats and mice assessing fasting-induced food intake as well as testing the effect of diet exposure, standard diet and high fat diet (HFD), in terms of food intake. In mice, we studied in detail the inhibitory effect of LEAP-2, in which the orexigenic effect of ghrelin is unopposed by leptin  were used for the experiments. For the experiments, rats and mice were maintained until 24 months of age. The mice and rats were fed with standard chow (STD) or the high fat diet (HFD)  for 12 weeks. The animals were housed with an artificial 12-hour light (8:00 to 20:00)/12 h dark cycle, under controlled temperature and humidity conditions and allowed free access to standard laboratory chow and tap water; animals were housed in air-conditioned rooms (22–24 °C) under a 12:12 h light/dark cycle and fed standard rat chow, or high fat diet and water ad libitum.Adult male Lewis, dwarf rats (GH-deficient rat) as described before  and SpraAfter the experiment, the mice were killed, and all tissues were rapidly explanted (WAT was weighted) and snap-frozen on dry ice. The tissues were collected and frozen at −80 °C until they were used.The animal procedures were conducted according to the principles approved by the Animal Care Research Bioethics Committee from the University of Santiago de Compostela (license 15010/17/002); performed in compliance with the Directive 2010/63/EU and Spanish Royal Decree 53/2013, on the protection of animals used for experimental and other scientific purposes.David Kopf Instruments; Tujunga, CA, USA) under ketamine/xylazine anesthesia. The lateral ventricle was targeted using a 25-gauge needle  a cannula was inserted as previously described [For the central treatments, intracerebroventricular (ICV) cannulae were stereotaxically implanted under ketamine/xylazine anesthesia. Rats or mice were placed in a stereotaxic frame  were given access to 0.15% sodium saccharin rather than water for 30 min. Immediately afterward, one experimental group was injected intraperitoneally with 0.15 mol/L lithium chloride (LiCl) in saline [Five days before the test, mice were allowed 2 h daytime access to water. On the day of the experiment, rats  by means of the tail vein at the different time points.The quantitative measurement of LEAP-2 in plasma samples was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Human LEAP-2 (38–77) ELISA kit, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) according to the manufacturer’s instructions. This kit has been previously validated ,26. Intrn > 7). For normal distributions, a parametric test was conducted; for two-group comparisons, the unpaired t-test was carried out. In the statistical analysis data with a p-value, less than 0.05 were considered statistically significant. For a multiple comparison test, a one-way ANOVA followed by Bonferroni’s post hoc multiple comparison test was performed. Data analysis was performed in GraphPad Prism Software Version 9.0 .Values were plotted as the mean ± SEM for each genotype. To test whether the data follows a Gaussian distribution, a normality test was performed . Both animal models of obesity showed higher levels of LEAP-2 in circulation in comparison to their lean controls  the glucose levels after LEAP-2 administration were lower than in control mice after refeeding  are highly expressed. The interplay between ghrelin, growth hormone and energy homeostasis is also supported by data showing that ghrelin orexigenic effect is at least partially mediated by central GHR, being blunted in animals with genetic deletion of the GHR . Taking ob/ob mice, LEAP-2 administration produced lower glucose blood levels than mice treated with vehicle after 2 hours refeed.As we described before, ghrelin is essential in the counterregulatory action of glucose, increasing blood glucose levels. Therefore, we would expect that LEAP-2 maintains low blood levels and indeed this result was obtained in mice but only under HFD. Additionally, in monogenetic obese model ob/ob animals where genetic deletion of ghrelin resulted in marked improvement in the hyperglycemia produced for leptin-deficiency while deletion of GHSR had the opposite effect, it worsened the hyperglycemia of the ob/ob mice [The relevant role of ghrelin in glucose homeostasis is beyond any doubt. These effects are exerted, both, by acting in peripheral tissues and in the brain by a variety of mechanisms. At the periphery, they include pancreatic hormone secretion, gastrointestinal mobility, intestinal glucose adsorption, glucose metabolism at peripheral tissues or GH and cortisol secretion . In the /ob mice . In cont/ob mice ,42. On tob/ob and DIO), aged rats and GH-deficient rats shows that LEAP-2 is a potent anorexigenic agent and maintains its capacity to blunt ghrelin-induced hyperphagia. Further work assessing its effects following chronic administration is merited in order to establish its potential in terms of drug development.In sum, we extend previous findings regarding the relevance of LEAP-2 as an anorexigenic signal. Specifically, our data using different experimental models: lean, obese (


The COVID-19 pandemic dramatically changed medical care. Healthcare professionals are faced with new issues. Patients who survived COVID-19 have plenty of different continuing symptoms, of which the most common are fatigue and breathlessness. It is not well known how to care for patients with persistent or worsening respiratory symptoms and changes on chest X-ray following COVID-19 pneumonia. In this article, we talk about a subgroup of patients with organizing pneumonia following COVID-19 pneumonia that could be effectively treated with systemic glucocorticoids. It is important that patients with COVID-19 pneumonia be followed-up at least three weeks after diagnosis, in order to recognize early lung damage. We are providing a management algorithm for early diagnosis of lung diseases after COVID-19 pneumonia. The World Health Organization (WHO) had declared the Coronavirus disease 2019 (COVID-19) a pandemic by March 2020. For more than a year, human beings have faced the largest medical challenge. Globally, by the 4 March 2021, there have been 114,853,685 confirmed cases of COVID-19 and 2,554,694 deaths reported by the WHO [The identified pathogen was a novel enveloped RNA beta coronavirus that has now been named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . The criViral pneumonia is the most common indication for hospital admission. More than 80% of patients are hospitalized to general medical wards and only a smaller proportion of patients are admitted to intensive care units (ICU) . COVID-1A total of 200,884 cases of COVID-19 and 3294 deaths due to COVID-19 were reported by the Lithuanian Department of Statistics from March 2020. During the first wave of the pandemic the incidence of COVID-19 and the mortality rate in Lithuania were very low. Only 863 patients with COVID-19 were hospitalized and 102 of them died. However, during the second wave, Lithuania was among the leaders in Europe, in terms of the number of new COVID-19 cases. The rate of hospitalized patients reached 11,673 cases. .Current COVID-19 guidelines mostly cover management of the acute disease, prevention, and vaccination. Less is known how to deal with the persistent symptoms after COVID-19. During the acute COVID-19 period, the extent of lung damage is proportional to the disease severity. However, it is still not completely known what predicts the severe course of COVID-19. Moreover, the severity of acute COVID-19 is not thought to be linked to the likelihood of development of persistent symptoms after the acute COVID-19 period . People There was no clear definition of “long COVID” until now. Recommendations basically are made on experts’ opinion. United guidelines of three organizations “COVID-19 rapid guideline: managing the long-term effects of COVID-19“were recently published in order to help clinicians . Authorsn = 185) of these patients. Most of them—131 (70%) were treated in the ICU. The mortality, associated with SARS CoV2 pneumonia, for patients requiring ICU care was 56% [We are working at the tertiary university hospital . A total of 443 patients with COVID-19 were treated in our university hospital from March until December 2020. Viral pneumonia was diagnosed in 41.7%  significant radiological changes on chest radiograph or computed tomography (CT); or (2) the demand of continuous invasive or non-invasive lung ventilation, high flow oxygen therapy or long-term oxygen therapy. A significant part of the latter group of patients develop chronic respiratory failure. We believe that previous lung disease and condition, concomitant diseases and their treatment are important . COVID-1Patients with COVID-19 have increased risk of thromboembolic complications. The risk of venous thromboembolism (VTE) is higher in those with severe COVID-19, even when prophylactic anticoagulation is used . The SARFinally, we present our management algorithm for patients with early diagnosis of lung damage after COVID-19 pneumonia . This alPatients still requiring inpatient care, who are suspected to have COVID-19 lung damage , should be referred to a pulmonologist for assessment.Patients with COVID-19 pneumonia, who have been discharged from the hospital or were treated as outpatients, should consult their family doctor in the coming in 2–4-week period. Family doctors should undertake clinical assessment and repeat two-views chest X-ray, blood count, C reactive protein, oxygen saturation measurements, electrocardiography. If the chest X-ray changes have not resolved and/or the patient’s respiratory symptoms are ongoing, COVID-19 lung damage is suspected, and hence referral to a pulmonologist should be considered.Patients who are referred to the pulmonologist should proceed to a high-resolution CT scan and CT pulmonary angiogram to assess for the presence of possible interstitial lung disease (ILD) and pulmonary embolism (PE). Patients diagnosed with PE should be treated according to standard guidelines. If there is evidence of ILD, patients should undergo pulmonary function testing , the 6 minutes’ walk test and lung biopsy according to the clinical situation. Finally, the patients are discussed in the ILD multidisciplinary team meeting together with the pulmonologist, radiologist and pathologist if their diagnosis and treatment of ILD is confirmed.If there is no evidence of ILD or PE, another diagnosis should be considered and managed appropriately.Mild COVID-19 patients (without COVID-19 pneumonia) are not included in this algorithm. Follow-up is not recommended if these patients do not have any respiratory symptoms. If patients’ respiratory symptoms continue or worsen they should consult their family doctor. Patients with ongoing radiographic changes but improving clinical symptoms have to follow this algorithm, because radiological changes which were not resolved could be related with lung cancer. The multidisciplinary rehabilitation team should work with these patients.In conclusion, COVID-19 is a novel infection, being the largest medical challenge for more than a year. Lung damage, presenting with different interstitial changes, seen on a CT scan, could follow COVID-19 pneumonia. One of them—organizing pneumonia—is regarded as glucocorticoids sensitive. Patients with post COVID-19 pneumonia have to be evaluated to recognize early lung damage. We are hoping that our proposed management algorithm of post COVID-19 pneumonia could be another step to improve the care of these patients and result in less consequential outcomes.