The ability to predict an ND50 value of ≥118.25, indicating a protective effect, was comparable among different assays. Applying optimal cutoffs based on Youden’s index, the kappa agreements were greater than 0.60 for all assays in the total group. Overall, relatively poor performance was demonstrated in the ChAdOx1 group, owing to low antibody titers. Although there were intra-assay differences related to the vaccination protocols, as well as interassay differences, all assays demonstrated fair performance in predicting the protective effect using the new cutoffs. This study demonstrates the need for a different cutoff for each assay to appropriately determine a higher neutralizing titer and suggests the clinical feasibility of using various assays for estimation of the protective effect.Estimating neutralizing activity in vaccinees is crucial for predicting the protective effect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As the plaque reduction neutralization test (PRNT) requires a biosafety level 3 facility, it would be advantageous if surrogate virus neutralization test (sVNT) assays and binding assays could predict neutralizing activity. Here, five different assays were evaluated with respect to the PRNT in vaccinees: three sVNT assays from GenScript, Boditech Med, and SD Biosensor and two semiquantitative binding assays from Roche and Abbott. The vaccinees were subjected to three vaccination protocols: homologous ChAdOx1, homologous BNT162b2, and heterologous administration. The ability to predict a 50% neutralizing dose (NDIMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic continues to last, despite high COVID-19 vaccination rates. As many people experience breakthrough infection after prior infection and/or vaccination, estimating the neutralization activity and predicting the protective effect are major issues of concern. However, since standard neutralization tests are not available in most clinical laboratories, it would be beneficial if commercial assays could predict these aspects. In this study, we evaluated the performance of three sVNT assays and two semiquantitative binding assays targeting the receptor-binding domain with respect to the PRNT. Our results suggest that these assays could be used for predicting the protective effect by adjusting the cutoffs. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a lengthy pandemic globally, with the emergence of variants with different mutations. While the vaccination rate has reached 85.4% and the booster vaccination rate 49.2% in Korea as of January 2022 , breakth3–6–per se, those targeting the RBD of the spike protein have shown a correlation with the neutralization titer in patients with COVID-19 , use live pathogens, a biosafety level 3 (BSL3) facility is required, which is usually not feasible in clinical laboratories. In addition, the low throughput, long turnaround time, and suboptimal standardization of the PRNT are factors that make it inappropriate for clinical laboratories. To overcome these limitations, a method called the surrogate virus neutralization test (sVNT) has been developed that can be performed in BSL2 clinical laboratories . The sVNCOVID-19 .In this study, we aimed to evaluate the capability of various assays to estimate the neutralization activity compared to the PRNT in vaccinees subjected to different vaccine protocols and to address the titer correlation with respect to the PRNT.50) of ≥20, the sVNT assays showed relatively poor agreement. Among the three sVNT assays, qualitative agreement with the PRNT ND50 value of ≥20 was the highest for GS regardless of the vaccine type . When the different vaccine types were compared, the BNT162b2 group showed a significantly higher agreement  in all assays than the other two groups. While all assays demonstrated high specificities (87.59 to 100.00%), the sensitivities varied by a significant magnitude across the different assays and vaccine types (46.52 to 99.67%). The sensitivities, specificities, and kappa agreements are listed in While the semiquantitative binding assays showed almost perfect agreement with a PRNT 50% neutralizing dose  and SD Biosensor (SD) assays showed better performance in predicting the protective effect in the ChAdOx1 and heterologous groups. When the receiver operating characteristic (ROC) curve for a PRNT ND50 value of ≥118.25 was implemented for each assay and vaccine protocol /mL; and Abbott, 57.48 BAU/mL. The GS, Roche, and Abbott assays showed superior performance in predicting the protective effect using the newly determined cutoffs than that achieved using the manufacturer cutoffs. As the new cutoffs were higher than the manufacturer cutoffs, higher agreement was achieved with higher specificity (77.54 to 94.86%), sacrificing sensitivity (58.00 to 95.42%).To estimate the protective effect using commercial assays, the performance of each assay with respect to a PRNT NDvaluated . When thprotocol , all ass50 value of ≥20, implying emerging neutralizing antibodies. Nonetheless, the sVNT assays, especially the BM and SD assays, were insufficient to suggest positive concordance with the PRNT, which was prominent in the ChAdOx1 group. The samples collected 2 weeks after the second dose in the BNT162b2 and heterologous groups showed nearly 100% inhibition using all three sVNT assays. During the waning period, the results showed a trend similar to those obtained after the first dose, where relatively low antibody titers resulted in false negativity using the BM and SD assays, particularly in the ChAdOx1 group. The BNT162b2 group showed a consistently higher neutralization titer than the ChAdOx1 group across all time points, and the heterologous group had the highest neutralizing activity during the waning period compared to the two homologous groups. See Table S1 in the supplemental material for detailed quantitative results of the assays at each time point and for each type of vaccination protocol.The quantitative results of each assay are depicted in R2) for the GS, BM, SD, Roche, and Abbott assays were 0.51, 0.58, 0.51, 0.49, and 0.56, respectively. The quantitative results of all assays showed strong correlation with the results of the log-converted PRNT. The results are illustrated in The quantitative results of each assay showed moderate to strong correlation with the titers of the PRNT in all vaccine groups, while the correlation coefficient (Spearman’s correlation) was lowest in the ChAdOx1 group. Overall, the coefficients of determination  derived from the convalescent-phase sera of 116 reverse transcription-PCR (RT-PCR)-confirmed patients.Conventionally, an NDThe cutoffs for a protective effect obtained using Youden’s index were higher than the manufacturer cutoffs for all assays except the SD assay. These higher cutoff values provided higher agreement at the expense of sensitivity. Epidemiologically, false positivity rather than false negativity is a greater obstacle, since this could pose a risk to individuals without a sufficient protective effect. Thus, the use of these new cutoffs with higher specificity would be desirable. Regarding the SD assay resulting in an opposite trend with a lower cutoff for the protective effect, we believe that it was difficult to determine an appropriate cutoff because of the poor discriminative power at low antibody titers. However, with adjustment of the cutoff and a higher target titer indicating a protective effect, the performance of the assay was improved.50 value of ≥20 that was greater than that of the sVNT assays. As binding antibodies against the RBD are not mutually exclusive with neutralizing antibodies but rather coexist with affinity and avidity maturation, the binding assays are also feasible for use in predicting the neutralizing effect, aside from the underlying assay mechanism. In addition, affinity maturation is achieved with the decay of antibodies with low affinity, which contributes to increased neutralization potency  of ≥20. Other commercial sVNT and binding assays were conducted following the manufacturer’s instructions at Samsung Medical Center, Seoul, South Korea. The cutoffs of the GS, BM, SD, Roche, and Abbott assays suggested by the manufacturers were ≥30%, ≥30%, ≥20%, 0.82 BAU/mL, and 7.1 BAU/mL, respectively.Six different assays were utilized in this study: one PRNT, three sVNT assays, and two binding assays. The three sVNT assays used in this study were the cPass SARS-CoV-2 neutralization antibody detection kit , the AFIAS COVID-19 nAb kit  , and the standard F SARS-CoV-2 nAb fluorescent immunoassay (FIA)  . The two semiquantitative binding assays utilized in this study were the Elecsys anti-SARS-CoV-2 S kit   and the AdviseDx SARS-CoV-2 IgG II Quant kit  . The PRNT using wild-type SARS-CoV-2 was carried out by the Korea Disease Control and Prevention Agency, and the detailed procedure for the PRNT is described in our previous publication , 11. The50 value of ≥20, the performance of each assay in predicting an ND50 value of ≥118.25 was assessed, since it was the cutoff representing the 50% protective effect. The PRNT ND50 value indicating 50% protective effect was determined using the methods of Khoury et al.  of each hospital, and written informed consent was obtained from each participant. The hospitals are as follows: Samsung Medical Center, Gangman Severance Hospital, Yongin Severance Hospital, Severance Hospital, Seoul National University Bundang Hospital, Asan Medical Center, Chungbuk National University Hospital, Kyungpook National University Hospital, Kyungpook National University Chilgok Hospital, Korea University Ansan Hospital.


Depression is a common and debilitating disease, and even residual symptoms of depression can cause significant functional impairment. To achieve normal functioning, residual symptoms should also be identified and targeted by a competent treatment strategy. Thus, this study aimed to examine residual symptoms of depression and their associated factors among patients with depression.A cross-sectional study surveyed Thai patients with depression at two psychiatric outpatient clinics, Songklanagarind Hospital, and Songkhla hospital; from June to October 2021. The questionnaires inquired about: (1) demographic information, (2) the PHQ-9 Thai version, (3) a questionnaire focusing on depressive symptoms that impacted daily life, and were originally expected to be improved due to antidepressants. All data were analyzed using descriptive statistics, and associated factors concerning depressive symptoms were analyzed by a Chi-square and a logistic regression.Of all 566 respondents, the majority of them were female (75.4%). The overall mean age was 43.8 ± 18.1 years. The depressive symptoms that had high frequency, high impact on daily life, and that the participants expected that they are resolved or get better via antidepressants were: sleeping problems (81.6%), feeling depressed (79.9%), and lack of pleasure (75.4%). Most of the participants (65.7%) received one type of antidepressant, and the most prescribed antidepressants were selective serotonin reuptake inhibitors (51.1%). In regard to objectives, 45.4% of participants reported having residual depressive symptoms which included sleeping problems (71.2%), feeling down (62.6%), lack of pleasure (62.3%), and poor appetite (61.9%). The associated factors relating to residual depressive symptoms were younger age, high education level, and having physical illness.Almost half of patients with depression had residual symptoms, and they showed symptoms with high individual variability. Further to receiving effective treatment, a focused and individualized approach aiming for symptomatic remission, functional recovery, and quality of life improvements is key to recovery. Therefore, shared decision-making, and taking into account drug efficacy based on symptom profiles are both highly recommended. Depression is a common and debilitating disease. It is the largest contributor to global disability. In 2012, the World Health Organization Global Burden of Disease Survey ranked depression as the fourth leading cause of disability worldwide  and are MDD is described as a combination of multiple symptoms with high individual variability; and if left untreated MDD may result in progressive alterations in brain circuit function and morphometry. Recent findings suggest that pharmacotherapy may halt and possibly reverse those effects. A delay in treatment is related with poorer clinical outcomes. Therefore, the need to rapidly treat depression to full recovery should be highlighted . NowadayConcerning residual symptoms, they may meet the criteria for subsyndromal and/or minor depression , or haviThis study aimed to identify the core depressive symptoms that affected daily life and were expected to get better due to antidepressants, among Thai patients with depression. Additionally, residual symptoms, factors associated with residual symptoms, and antidepressant prescriptions were also evaluated. The identification of residual symptoms, which ones are the most frequent and in which occasions they occur, may provide support towards a more individualist intervention program based on symptom profiles.After being approved by the Ethics Committees of the Faculty of Medicine, Prince of Songkla University (REC: 63-522-3-1), this cross-sectional study was conducted at the two listed psychiatric outpatient clinics; Songklanagarind Hospital, which was an 800-bed university hospital serving as a tertiary referral center in Southern Thailand, and Songkhla hospital, which is a 508-bed general provincial hospital in Southern Thailand. All outpatients with depression, who had an appointment and were followed up at two psychiatric outpatient clinics; from June to October 2021, were invited to participate in the study.Patients with the first major episode of depression, as diagnosed by their psychiatrists, were selected in the medical register based on the following criteria: ICD-10 code; F33.0-F33.9; except F33.3, aged more than 18 years, taking antidepressants for at least 12 weeks , acknowlThe research assistant approached all of the eligible outpatients with depression for recruitment and handed them an information sheet, which delineated the rationale for the study and the allotted time to complete the survey. All eligible participants had at least 15–20 min to consider whether to collaborate in the study or not. To ensure that the participants’ identities would be protected, there was no requirement for their signatures. Furthermore, they were informed that their data would remain anonymous and that they could withdraw at any stage of the questionnaire without giving any reasons; and with no impact on their treatment. Participants willing to collaborate were invited to a private location to complete the questionnaires, and were informed that they could stop at any time if they felt uneasy or distressed without them needing to provide any reason. Moreover, if the participants exhibited a high level of distress or worry, advice and/or further clinical management was provided to them.Personal and demographic information consisting of questions related to age, gender, religion, marital status, occupation, income, physical illness, substance usage, types, and length of time of their antidepressants prescription.The Patient Health Questionnaire-9 (PHQ-9) Thai version, a self-rating questionnaire to evaluate depression consisting of nine questions. The score of each question employed a 4-point rating scale, never = 0; rarely = 1; sometime = 2; always = 3. The total score ranged from 0 to 27, with a recommended cut-off score of nine or greater which meant major depression. The questionnaire demonstrated internal consistency; Cronbach's alpha coefficient of 0.79; sensitivity of 0.53; specificity of 0.98 A questionnaire containing a checklist in regard to depressive symptom profiles  and daily life impact, and the symptoms expected to get better due to antidepressants.The questionnaire was reviewed by 3 psychiatrists; the content validity index (CVI) score was 0.8. A pilot study was conducted with 20 volunteers; thus, Cronbach’s alpha was 0.8.Residual depressive symptoms among patients with depression were identified in the case of patients with a first episode of depression, who received antidepressants more than 12 weeks ago  and stilDescriptive statistics, such as proportions, means, standard deviations (SD), median and interquartile ranges (IQR) were calculated. Chi-square or Fisher’s exact tests and logistic regression analyses were used to identify associations between demographic characteristics, and depressive symptom profiles. The analyses were conducted using R version 3.4.1 . Statistical significance was defined as a p-value of less than 0.05.From June to October 2021, 573 patients with depression attended both psychiatric outpatient clinics, and 566 of them agreed to participate and complete the questionnaires. The response rate was 98.8%. The mean age was 43.8 ± 18.1 years. The majority of participants were female (75.4%), Buddhist (73.7%), unmarried (56.5%), and had no history of physical illness and substance uses  . The most common depressive symptoms among young adults were thoughts of being better off dead or of hurting themselves (38.7%), moving or speaking too slowly (37.9%), feeling bad about themselves or failure (34%), trouble concentrating on things, and a diminished ability to think (32.1%), while the main depressive symptoms among the elderly were sleeping problems (24.6%), restlessness (24.2%), and a loss of interest or pleasure (23.2%)  reported having a PHQ-9 score of nine or greater, indicating the presence of residual depressive symptoms Table . MoreoveThe most common residual depressive symptoms were sleeping problems (71.2%), low mood (62.6%), loss of interest or pleasure (62.3%), and poor appetite (61.9%). In regard to age groups, the most common residual depressive symptoms among young adults were thoughts of hurting themselves or suicidal ideation 65%), trouble in concentration 54.7%), feeling bad or low self-esteem (53.8%), and loss of interest (53.5%), while the main residual depressive symptoms among the elderly were low mood (10%), and sleeping problems (9.9%)  participants received antidepressants for less than one year, and half of them received antidepressants for more than 2 years Table . The medOut of all participants, 372 (65.7%) and 162 (28.6%) received one and two types of antidepressants, respectively. The most prescribed antidepressants were selective serotonin reuptake inhibitors (SSRIs) (51.1%); sertraline (27.9%); fluoxetine (15%), and SSRIs plus other antidepressants (13.9%) Table .Table 4Tp-values from the univariate analysis were lower than 0.2 were included in the initial model for multivariate analysis. Multivariate analysis indicated that age, religion, education, and physical illness were statistically significant factors associated with having residual depressive symptoms. The patients with depression aged between 18 to 24, had a higher rate of residual depressive symptoms than the older group, the adjusted odds ratio (AOR) was 12.08, 95% confidence interval (CI) at 6.28 to 23.23. The same was true when comparing them with those whose religion was Islam or Christianity, had higher education, and physical illness; AOR (95% CI) was 1.70 , 1.73 , and 1.55 , respectively , serotonin and norepinephrine reuptake inhibitors (SNRIs), mirtazapine, bupropion, and agomelatine being first-line recommendations for pharmacotherapy for MDD , 22. FurIn regard to depressive symptoms profiles, in this study, we found that sleeping problems, feeling depressed or hopeless, and loss of interest or pleasure were the symptoms presenting with the highest frequency and level of impact on the patients’ daily lives. The depressive symptom profiles from our study feature a number of differences versus a study from Canada  which idConsidering residual depressive symptoms, almost half of the participants (45.4%) reported residual depressive symptoms which included sleeping problems, feeling down, loss of pleasure, and poor appetite. Additionally, this study identified that the mean duration of receiving antidepressants was at 26.9 months, and that 65.7% of participants received one type of antidepressant, and that SSRIs were the most common antidepressants prescribed. In regard to the neurotransmitter model of function in depression, a well-known concept, depression is described as a combination of two components: a lack of positive affect and an increase in negative affect. Negative affect means viewing the world as an unpleasant, disturbing, hostile, and threatening place. Lack of positive affect equals an inability to take pleasure rewards from normal activities. During treatment with antidepressant medication, some patients might experience particularly unresponsive depression-related symptoms with a higher lack of positive affect, while other patients might experience depression with an increase of negative affect, such as symptoms of anxiety . As per In this study, patients with depression, aged 18 to 24, had a higher rate of residual depressive symptoms than the older group, the AOR was 12.08. Based on prior studies, depression was a prevalent and serious mental disorder among youth adults or adolescents and adults, and it was related with suicide, an increase in family problems, substance abuse, absenteeism , and disAdditionally, this study identified that patients with higher education and experiencing a physical illness had a higher rate of residual depressive symptoms than the rest of the group. Higher levels of education could be associated with them having a higher level of work responsibilities and as a result more tension. While having physical illness may be associated with elevated life stress. Some physical symptoms could also produce anxiety, distress, suffering, and sleeping problems. Therefore, these factors may make depression more complicated.Finally, several other factors that were not addressed by our study could have possibly influenced the response to treatment and the presence of residual depressive symptoms, such as alcohol usage, and biological factors; an imbalance of pteridine metabolism in depression . The priThis study had both limitations and strengths worth mentioning. To our knowledge, this is the only study on this topic conducted in southern Thailand during the last decade. However, this study had some limitations as it was a cross-sectional survey and utilized self-administered questionnaires; therefore, some misunderstandings about the intended meaning of the questions may have taken place. Another drawback is that our data were quantitative, the sample size and that the participants were restricted to patients with depression in lower, southern Thailand. Most participants were female in gender and had a high educational level. Hence, these results may not demonstrate the situation or condition of patients with depression in all gender groups, all educational levels, or the whole country in a proportionate manner. It is recommended that future studies feature a larger number of patients with depression from other hospitals in Thailand; and that a more comprehensive, multi-centered research study, with more qualitative or in-depth methods should be employed. Moreover, there are some factors to be aware of, such as alcohol usage or abuse, medication adherence, drug tolerability, psychotherapy, family, and social support, as these may influence the prognosis or the outcome of treatment among patients with depression.Almost half of the patients with depression had residual symptoms, and they showed symptoms with high individual variability. Further to receiving effective treatment, a focused and individualized approach aimed to symptomatic remission, and functional recovery is necessary. Therefore, shared decision-making, and taking into account drug efficacy based on individual symptom profiles are both highly recommended.


Needle-tract seeding (NTS) is a rare but serious complication of Endoscopic Ultrasound-guided biopsy of pancreatic adenocarcinoma. Due to its very low incidence, there is lack of evidence about its management. The aim of our systematic review is to deepen the knowledge about this entity and highlight therapeutic approaches. After a systematic search we retrieved 45 cases, plus one from our center. The majority of patients (87.1%) underwent surgical resection of the primary pancreatic tumor, of which only 55.9% received neoadjuvant and/or adjuvant chemotherapy. NTS occurred at a median of 19 months after primary diagnosis, with a secondary surgical approach in 82.4% of patients and a median overall survival of 26.5 months compared to 15.5 months when treated with chemo/radiotherapy. NTS is rare, generally occurs late and might be treated aggressively with good results. As only a low rate of patients developing NTS underwent (neo)adjuvant chemotherapy, one may speculate that it might be protective.Needle-tract seeding (NTS) has been sporadically reported as complication of Endoscopic Ultrasound (EUS)-guided aspiration (FNA) in pancreatic adenocarcinoma (PDAC). However, the evidence of its treatment and outcome is sparse. Adhering to PRISMA guidelines, we conducted a systematic review of EUS-FNA NTS cases of PDAC and analyzed their management and outcome. Up to September 2022, the search query retrieved forty-five cases plus an unpublished case from our center, for a total of forty-six; 43.6% were male, with a mean age of 68.6 years. Thirty-four patients (87.1%) underwent an initial surgical resection, with only 44.1% and 5.9% undergoing adjuvant and neoadjuvant chemotherapy, respectively, and 5.9% undergoing both. The NTS nodule was mostly located in the posterior gastric wall, developing at a median of 19 months after primary resection; 82.4% underwent surgical resection of the seeding, while for 17.6%, palliative chemotherapy treatment. Follow-up after NTS diagnosis and treatment was reported for only twenty-three patients: when NTS was treated with surgery, the median overall survival was 26.5 months compared to 15.5 if treated with radio/chemotherapy. NTS after EUS-FNA of PDAC occurs late and might be treated aggressively with good results. Interestingly, only a low number of patients developing NTS underwent chemotherapy for the primary cancer, suggesting its possible protective role. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality , and it It is, therefore, not surprising that the seeding of PDAC cells is possible. This phenomenon was first reported after percutaneous radiologically-guided fine-needle aspiration (FNA) , in the Endoscopic ultrasound (EUS)-guided FNA is a well-established method for the tissue acquisition of pancreatic neoplasms, with a sensitivity of 85% and a specificity of 98% . Peri-prNonetheless, the NTS of PDAC in general is a poorly investigated entity, and the management of the seeding localization has been scarcely discussed. While the first reports would consider NTS similarly to disease recurrence or metastatic progression of the disease, offering only palliative support to the patient, the most recent ones started considering a surgical eradication of the new disease site . This exAlso, given the increase in both the incidence of PDAC and the use of EUS-FNA, NTS recognition and the development of its standardized management seem necessary. Another relevant aspect is the association of NTS with perioperative treatments. As the rationale for both adjuvant and neoadjuvant chemotherapy of PDAC is to reduce the risk of micro-metastases presenting as distant recurrence sites after surgery, it is possible to hypothesize that they also reduce the risk of NTS. This is, however, an unexplored issue.The aim of our study was, therefore, to conduct a systematic review of cases of NTS of PDAC associated with the use of EUS-FNA and analyze their clinical features, management, and outcome, including those of an unpublished report from our center.Adhering to 2020 PRISMA guidelines [Two authors (LA and RPDLP) screened all titles and abstracts for eligible articles based on predefined eligibility criteria. Full-text manuscripts were evaluated, and references of the identified manuscripts were examined for further potentially relevant articles not identified by the initial search.This review has been registered in PROSPERO–National Institute for Health Research, ID –CRD42022356314.Inclusion criteria were: (a) case reports or case series concerning patients with a pancreatic lesion undergoing EUS with FNA/FNB; (b) development of a lesion that is highly suspect or cyto/histologically confirmed as deriving from the primary pancreatic tumor; (c) the lesion is described as being located in the trajectory of the previous FNA puncture site, and therefore, highly suggestive for NTS. Exclusion criteria were: (a) pancreatic lesions different from PDAC; and (b) peritoneal recurrences.In the event of duplicate publications, the most recent or more complete one was selected.The following data were retrieved and registered in a dedicated spreadsheet: country of origin, publication year, patient gender and age, PDAC-EUS and surgical specimen size, site, caliber and type of needle used to perform FNA on the primary PDAC, number of FNA passes, puncture route, TNM stage and management of the primary PDAC, histological diagnosis, grading and R status of the resection, localization of seeding, time lapse between EUS-FNA of the primary tumor and diagnosis of seeding nodule, seeding nodule management, and patient outcome. Whenever any of this information was not provided in the manuscript, we attempted to retrieve them by contacting the corresponding author by email.A case of NTS diagnosed at our center was also included.We report the case of a 57-year-old male, referred to our center for the follow-up of alcoholic chronic pancreatitis, who underwent pancreatico-jejunostomy in 2015 for recurrent acute flares. In March 2016, at a follow-up EUS, a 20 mm mass of the pancreatic tail was diagnosed. An EUS-FNA of the lesion with a 25-gauge Menghini-tip needle  was carried out through the posterior gastric wall (2 passes) with a slow-pull technique, and the cytological examination revealed PDAC. The patient had a Karnofsky performance status score of up to 80%, and the pre-operative computed tomography (CT) scan confirmed the 20 × 20 mm lesion of the tail, invading the splenic vessels; levels of Ca19.9 were slightly elevated (175 U/ml). In April 2016, the patient underwent upfront distal pancreatectomy with splenectomy for T2N0M0 R0 G3 (TNM 8th edition) PDAC, with 14 negative lymph nodes removed. Follow-up management included adjuvant chemotherapy with 6 cycles of gemcitabine and radiological surveillance with CT scan every 3 months.®  contrast agent with a hypoenhanced core  were selected from Pubmed with no additional reports from Scopus. Another 3 reports were identified from hand-searching of the references of the selected manuscripts and included, for a total of 33 reports ,45,46,47As there were some missing data in all the retrieved studies, we tried to retrieve them by contacting all corresponding authors of these 33 papers, but we only managed to receive a reply from three of them ,44,45, aThe unpublished newly developed case from our center was added to the 45 literature cases from the 33 reports for a total of 46 cases, described between 2005 and 2022 ,45,46,47Seventeen of the 46 patients (43.6%) were male, with a mean age of 68.6 years. Forty-three cases (93.5%) arose from the pancreatic body/tail, 3 (6.5%) from the pancreatic head; all 46 cases (100%) were histologically diagnosed as Pancreatic Ductal Adenocarcinoma (PDAC).The caliber of the needles used to acquire tissue for the primary cancer diagnosis was 22 G in 27 cases (64.3% of cases), but other needles of 19, 20, 21, or 25 G were also employed; in terms of number of needle passes, these were >1 in 33 (94.3%) of the reported cases and varied between 1 and 5. Puncture route was trans-gastric in 44 cases (95.6%).Thirty-four patients (87.1%) underwent surgical resection of the primary pancreatic lesion, of which only 2 (5.9%) received neoadjuvant chemotherapy, 15 (44.1%) received adjuvant chemotherapy, and 2 (5.9 %) received both. Five patients (12.8%) were not resected and were treated with primary chemotherapy and/or radiation therapy. Thus, the majority of the reported NTS cases were treated with surgery alone on the primary tumor.Time between EUS-FNA and the diagnosis of NTS ranged between a few days to a maximum of 42 months, with a median onset after 19 months. In 42 (91.3%) patients, NTS was reported in the gastric wall, whereas 2 (4.4%) were reported in the remaining pancreas itself, 1 (2.15%) in the duodenum, and 1 (2.15%) in the cardia.Concerning the management, 28 (82.4%) patients diagnosed with NTS underwent a new surgical resection as treatment for the seeding nodule, mostly localized in the posterior gastric wall, while 6 (17.6%) underwent palliative chemotherapeutic treatment, being, therefore, managed as a metastatic spread of the disease instead. For 12 patients (26.1%), no information about NTS treatment was available, and for 24 patients (52.1%), no information about overall survival was available. The corresponding authors of 20 of these patients were contacted, but replies for only 10 patients were received, with most of them replying that no information could be retrieved except for one case .Of the 28 patients undergoing surgical resection of the gastric NTS, disease re-recurrence after NTS removal was described in 6 patients . The median overall survival of NTS treated with surgery was 26.5 months compared to the 15.5 months of survival of patients treated with oncological treatment alone. Although a statistical comparison was not considered methodologically sound, an aggressive treatment seemed to be associated with better outcome.Other information about chemotherapy, surgical treatment, EUS-FNA, seeding nodule, tumor stage, and patient management are reported in NTS is a rare but serious long-term complication of EUS-FNA, with a very low prevalence that has been reported to range between 0.003% and 0.009% . A recenWith the widespread use of EUS with tissue acquisition for pancreatic lesions, NTS localizations may be expected to become more frequent, and their standardized management is therefore advisable. The management of NTS localization is, instead, not standardized and several options, from surgical resection to palliative chemotherapy could be considered. The present systematic review reported data on 46 cases that fulfilled our stringent inclusion criteria for cases in which PDAC seeding on the needle track was deemed extremely likely. In some of these cases, the authors performed molecular analyses of the NTS tissue and compared it to the primary pancreatic tumor to confirm the etiology of the nodule based on the striking similarity of genomic landscape . We exclWe gathered information to homogenize available data from previous sparse reports and hereby present the largest combined analysis on NTS features in 46 patients. Although our aim was to retrieve all of the cases’ missing data by contacting the authors, the long time gap between the first and the last reported cases did not allow us to obtain complete information about the clinical course.Most of the 46 included NTS cases were located in the gastric wall 91.3%), a minority being in the duodenum, in the cardia, and in the pancreas itself. This is not surprising, as most of the seeding cases occurred after trans-gastric biopsies of lesions of the body-tail that would have their stomach preserved when surgically resected ,42,45,46.3%, a miOne may therefore hypothesize that the real incidence of NTS in general may be underestimated and underdiagnosed.An interesting issue arose regarding the timing of NTS diagnosis: there was a wide range, from few days to 42 months (median 19 months), even in patients with similar disease course and treatments. This heterogeneity is likely explained by the variability in terms of growth rate that is also observed in primary PDAC treated with chemotherapy or experiencing recurrence after radical surgery . In thisIn terms of NTS management, it is interesting to highlight how in a minority of the reported cases 17.6%), NTS was considered and, therefore, managed as a metastatic site of disease, usually bearing an expected survival of <12 months and, therefore, receiving either chemotherapy or palliative treatments alone. Most patients (82.4%) were instead treated with secondary surgical excision of the NTS nodule, as their NTS was considered as a de novo T2N0M0 lesion [.6%, NTS A very recent nationwide survey conducted in Japan  reportedThe reported cases of NTS support the concern of EUS being potentially risky for the diagnosis and tissue acquisition of resectable pancreatic neoplasms. Some researchers have indeed highlighted a possible role of EUS-FNA in promoting distant metastasis, analyzing cell-free DNA plasma concentration before and after EUS-FNA of PDAC and showing how EUS-FNA is associated with increased plasma concentration of cell-free DNA and increased detection of mutant KRAS . As a maSince the first publication of reports about NTS after EUS-FNA, several novel methods and devices have been introduced to reduce the number of required punctures . In the While the event of seeding during EUS for PDAC should be kept in mind, we might speculate that this risk may be reduced by neoadjuvant and adjuvant treatments. In fact, notably, only 55.9% of the patients with reported NTS who underwent surgical resection of the primary pancreatic tumor had undergone neoadjuvant and/or adjuvant chemotherapy for the primary tumor, despite current guidelines recommending a perioperative chemotherapy for all fit patients . One mayIn conclusion, the present systematic review gathered available data on NTS after EUS-FNA pooling the largest dataset of reported cases to date, with data confirming that this rare complication usually occurs late (median onset after 19 months from EUS-FNA) and should be distinguished from “typical” distant disease recurrence since, when treated more aggressively with repeated surgery, the overall survival seems longer (26.5 months) compared to that of patients treated with palliative treatments (15.5 months).


Primary pulmonary myxoid sarcoma (PPMS) is an extremely rare malignant mesenchymal tumor of the lung, with only less than 40 cases reported. We described a case of a 64-years-old man with a mass on the medial and lower lobe of the right lung confirmed as a primary pulmonary myxoid sarcoma on biopsy. Diagnosis of this tumor remains challenging because of its nonspecific clinical and imaging characteristics. This study emphasizes CT finding to improve the understanding of PPMS. Primary pulmonary myxoid sarcoma (PPMS) is an unusual entity of malignant mesenchymal lung tumor The clinical symptoms and imaging findings of this tumor were non-specific, making a preoperative diagnosis of this tumor quite challenging ,3. A fewA 64 years old male presented to our center with 2 weeks of coughing prior to hospitalization. He also complained of the right chest heaviness for 2 months and had a history of bloody cough one year before, with exertional dyspnea, anorexia, weight loss, and no fever. The patient was a heavy smoker and was able to consume roughly one pack a day. No history or family account of cancer, diabetes, hypertension, or other pulmonary infection was recorded.On physical examination, his vital sign was normal with a blood pressure of 120/80 mmHg, heart rate of 90 x/m, respiratory rate of 22 x/m, body temperature of 36,4°C, and oxygen saturation of 98% in the room air. A slower movement of the right chest was noted at the chest examination, with dull percussion and a decrease in pulmonary sound in the right lower hemithorax. Another physical examination was within the normal limit. The laboratory result showed no significant abnormality and no increase in any tumor marker.The plain radiograph of the chest showed an opacity on the right hemithorax obscuring the right hemidiaphragm and right anterior and posterior costophrenic angle, resembling a pleural effusion .Fig. 1A This finding was confirmed by CT scan, depicting a solid mass (41 HU), measuring approximately 13.2 × 15.0 × 12.2 cm with fat and cystic component, distinct margin, loculated, septated with 4 mm septal thickness, at the medial and lower lobe of the right lung a, c, d. Afterwards, a bone survey, abdominal ultrasonography, and brain MRI were conducted to investigate the metastatic processes; nevertheless, no metastatic process was discovered. A subsequent open biopsy was performed, resulting in primary pulmonary myxoid sarcoma, confirmed by immunohistochemical staining . The patThe prevalence of PPMS was comparable between female and male patients, with 19 females out of 38 cases reported . The medPatients with this tumor exhibited non-specific signs and symptoms, with several asymptomatic cases discovered upon medical screening ,7. OtherPPMS is a tumor originating from mesenchymal elements of the bronchial wall, vessels, or pulmonary stroma ,8. This The imaging feature of this tumor is uncertain. It frequently occurs in the right lung and is closely related to the bronchus Microscopically, this tumor comprises lobules of delicate, lace-like strands and cords of lightly atypical round and spindle cells within a prominent myxoid stroma No immunohistochemical markers support the diagnosis of this tumor ,11, althMost patients with PPMS require surgical treatments with satisfactory clinical outcomes Our findings on the PPMS case were in line with the previous findings that this is a rare condition with non-specific clinical presentations and CT characteristics. Although the definite diagnosis of primary pulmonary myxoid sarcoma could only be made after pathology examination, CT features remain helpful in specifying the nature, location, and relationship between the tumor and other structures. Most PPMS patients were treated surgically and had an excellent prognosis.


NLRP3) gene. At present, there is no report about the variation of R262W in China.Cryopyrin-associated periodic syndrome (CAPS), a rare genetic autoimmune disease, is composed of familial cold autoinflammatory syndrome (FCAs), Muckle-Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID). MWS is caused by dominantly inherited or de novo gain-of-function mutations in the NOD-like receptor 3 , and C-reactive protein (CRP) and serum amyloid protein (SAA) levels. Whole exome sequencing identified a missense variation c.784-786delinsTGG (p.R262W) in the coding region of the NLRP3 gene in a patient with MWS was first reported in China.A classical variant of the  Cryopyrin-associated periodic syndrome (CAPS) is a rare genetic autoimmune disease with clinical heterogeneity. CAPS includes mild, moderate, and severe phenotypes. The mild phenotype is known as familial cold autoinflammatory syndrome (FCAs), the moderate phenotype is known as Muckle-Wells syndrome (MWS), and the severe phenotype is known as neonatal onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurocutaneous joint syndrome ,2.NLRP3 gene, which encodes cryopyrin, a key protein component of inflammasomes. These variations can lead to excessive production of interleukin-1β (IL-1β) which can lead to the occurrence of inflammatory reaction.CAPS is caused by variations in the NLRP3 gene. This gene variation site was the first to be reported in China.Here we reported a case of MWS, who had recurrent fever, urticarial-like rash, and bilateral conjunctival congestion. Whole exome sequencing identified a missense variation c.784-786delinsTGG (p.R262W) in the coding region of the A 3-year-old boy was first referred to our department in 2020 with complaints of recurrent fever with no apparent cause for more than 10 months. Each episode of fever generally lasted for about 1–5 days, and the interval between each episode was approximately 15 days. During all episodes of fever, the temperature was about 37.5ºC with occasional blips up to 39ºC, accompanied by bilateral conjunctival congestion and urticarial-like rash. There were no fatigue, headache, nasal congestion, runny nose, sore throat, cough and asthma, abdominal pain, vomiting and diarrhea, and joint swelling and pain. Routine blood examination showed elevation in leukocyte count, neutrophil count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) and serum amyloid protein (SAA) levels.NLRP3 gene (c.784-786delinsTGG) in the coding region that resulted in amino acid substitution of arginine for tryptophan at codon 262 (p.R262W), which is a missense variation. The frequency of variation in this database is normal. Through family verification analysis, the parents of the subject had no variation at nucleotide locus 784 in the coding region, suggesting a spontaneous mutation. According to the ACMG guidelines, this mutation was preliminarily determined to be pathogenic. Furthermore, we verified the gene function through two software, and the results are as follows: 1. This variation is predicted to be probably damaging with a score of 1.000 by PolyPhen-2. 2. This variation is predicted to be disease causing with a score of 0.999 by MutationTaster. The mutation was validated using Sanger sequencing  is caused by gene variations that change the protein encoded by them, leading to innate immune dysfunction and subsequent systemic excessive inflammatory response . Most paCAPS is an extremely rare continuum of autoinflammatory disease with severe and persistent inflammation status. This syndrome encompasses a spectrum of three subsets according to different clinical manifestation: FCAS, MWS and CINCA/ NOMID. CAPS was originally considered as three independent diseases. In fact, it is now considered as a disease process with different severity under the same genetic background. The clinical characteristics of CAPS was shown in Table NLRP3)  gene located on chromosome 1q44 [NLRP3 gene encodes the NLRP3 protein (cryopyrin), which can oligomerize and recruit other intracellular proteins such as ASC (apoptosis related spot like protein containing caspase recruitment domain) and several chaperones to form NLRP3 inflammasomes. The formation of this complex activates the proinflammatory protease called caspase-1, which in turn mediates release of proinflammatory cytokines such as IL-1β [NLRP3 gene variation can lead to continuous activation of inflammasomes and excessive production of IL-1, leading to systemic inflammation [https://infevers.umai-montpellier.fr/web/) listed more than 240 sequence variants of NLRP3 gene as of November 2020, and more than 100 are known to be pathogenic/possible pathogenic, and most of which are located in exon 3 [NLRP3 gene was first reported in 2002 and its involvement in MWS and FCU was confirmed, but some clinical symptoms, such as deafness, AA-type amyloidosis, and cold sensitivity, do not always exist, indicating that NLRP3 gene variation is not an independent pathogenic factor, and there may be epigenetic and other factors involved in the pathogenesis. Unknown gene modification may affect its phenotype [NLRP3 gene, which led to the R > W missense variation at amino acid 262. Considering his pathogenesis, clinical and laboratory manifestations, he was diagnosed with MWS. Previous studies have found that MWS is common in the Nordic population. Our findings have expanded the ethnic scope and clinical manifestation spectrum of the disease. The variation site of R260W was reported for the first time in China.CAPS is caused by dominantly inherited or de novo gain-of-function mutations in the NOD-like receptor 3 , positive family history, cold trigger attack, and most of them have experienced chronic disease course . This caThe objective of the treatment of CAPS is to inhibit systemic inflammation, prevent organ injury, and improve the quality of life of patients. Due to the central role of IL-1 in the pathogenesis of CAPS, biologically targeted therapy against IL-1 is recommended for CAPS . At presThe immunoregulatory effect of thalidomide is mainly manifested in the expression of some cytokines and adhesion molecules, as well as the regulation of immune cell activity: 1. Regulatory effect on cytokines. (1) Regulation of TNF-α: thalidomide can specifically reduce the level of TNF-a and regulate the secretion of other cytokines induced by TNF-α . The regulation of thalidomide on TNF-α may be through the regulation of cytochrome C pathway, leading to apoptosis of monocytes, accelerating the degradation of TNF-α mRNA, thus leading to the decrease of TNF-α production, and thus affecting the inflammatory response ,35. (2) As we all know, fever is the most common clinical manifestation in children, and the causes are also diverse. Therefore, the diagnosis and differential diagnosis of fever in children is very challenging. Thus, clinically, children with recurrent fever, rash, musculoskeletal symptoms, and elevated inflammatory indicators, especially those with a positive family history, should be alert to the possibility of CAPS. If CAPS is suspected, the NLRP3 gene should be detected using molecular genetics for the presence of variations. If diagnosis is confirmed, the patients should be treated individually and closely followed up to prevent further organ damage and maximize the quality of life.In this case, MWS was suspected and confirmed by the presence of a classical variation of the NLRP3 gene, which has also been reported in other MWS patients. This variation was reported in China for the first time. Our findings have expanded the ethnic scope and clinical manifestation spectrum of the disease, and equally important, provide a new idea for the diagnosis of recurrent fever. However, more in-depth follow-up and research are needed to optimize the treatment of MWS.


Aceruloplasminemia is a rare autosomal recessive disorder of iron accumulation in the brain. It is one of the subtypes of Neurodegeneration with brain iron accumulation and is characterized by the uniform involvement of all basal ganglia, thalami, dentate nuclei, and cortex. Aceruloplasminemia is the only known iron overload disorder in which brain and systemic iron overload are combined. Here, we present a 53-year-old female who had progressive cognitive disorders with motor deficits. MRI showed extensive and abundant iron deposited in the brain and in the liver. MRI showed iron deposited in the brain and in the liver.Neurodegeneration with brain iron accumulation (NBIA) is a group of diseases characterized by an abnormal accumulation of iron in the brain.A 53-year-old female who presented with extremity tremor, bradykinesia, and plastic rigidity of the limbs. In her medical history, the patient was followed for mild chronic microcytic anemia since childhood, unlabeled, and refractory to iron supplementation. She was also hospitalized for anxiety, mood disorders, and unexplained bizarre behavior at age 45, according to the family . Subsequently, the patient presented a progressive worsening of extremity tremor, bradykinesia, and plastic rigidity of the limbs. She had no other reported pathologies  or toxic habits.T2WI), hypointensity on T1 and diffusion-weighted image in the area that were hypodense on CT. These areas are surrounded by hypointesnity on T2WI and cortex bilaterally . Given the patient’s medical history, with lesions centered on the basal ganglia on CT, an ischemic origin seemed unlikely. Therefore, a brain MRI was performed and showed hyperintensity on T2 , serum copper was relatively normal and 24 h urine copper was 22 μg/24 h, ruling out Wilson’s disease.The biological analysis  showed mAbdominal MRI revealed significant iron deposition in the liver without iron deposition in the spleen . On the 3,4 Ceruloplasmin is a copper transporter that transports more than 95% of copper, it also plays a role in the oxidation of ferrous iron (Fe 2+) into ferric iron (Fe 3+), thus avoiding the deposition of iron in organs .5 ACP was first reported in 1987 in Japan by Miyajima, with prevalence estimated at 1/2,000,000 in Japan.3 It is the only one of the NBIAs in which brain and systemic iron overload is combined.2,4 In the brain, iron deposition is characterized by uniform involvement of all basal ganglia, thalami, dentate nuclei, and cortex, which is unique to this disease.1,2,4ACP is a rare autosomal recessive disorder of iron accumulation in the brain caused by CP mutations that encode CP, which in turn leads to the absence or a strong reduction in CP activity.4,6 In ACP, mild microcytic anemia is the biological sign that appears in childhood, but is rarely diagnosed at this stage.7,8 Vila Cuenca et al proposed a simplified algorithm for the diagnosis of ACP.6 Microcytic anemia with elevated ferritin associated with low transferrin saturation is an indicator of early diagnosis of PCA especially when associated with neurological disorders. This would help prevent irreversible neurological complications. , behavioral disturbances, and extrapyramidal signs observed most frequently in the Caucasian population.9 These non-specific neurological signs can often be underestimated.In the Japanese series, the triad of retinal degeneration, dementia, and diabetes is frequently cited, cerebellar disorders  and involuntary movements  in rare cases.1,2,10 To date, more than 10 disease entities related to NBIA disorders have been identified.10,11 PKAN is the most common form in which iron deposits are often restricted in the globus palludus characterized by the 'eye of the tiger' sign.1,2,12 In our case, the characteristic sign «eye of the tiger» was not observed. On MRI, eye of the tiger sign is the typical manifestation. T2 weighted sequences show hypointensity of the globus pallidus with a central hyperintensity, reflecting an excessive accumulation of iron with gliosis.1,12On brain imaging, all cases of NBIA show iron deposition, but differ in their clinical and neuroimaging features .1,2,10 T1,13 In NFT, iron deposition can be multiple, but not simultaneously and uniform. Furthermore, in NFT there are cystic cavitations and areas of confluent hyperintensity T2 on MRI involving the global pallidus and putamen that are not seen in ACP.1,14 Biologically, in ACP, ferritinemia is high, in contrast to NFT where ferritinemia is normal or low.Two neurodegenerative diseases with iron accumulation in the brain show great similarity: ACP and neuroferritionopathy (NFT) . In ACP,Wilson’s disease was suspected on the basis of clinical features and low level of ceruloplasmin. But the appearance on the brain MRI was not compatible and the 24 h urine copper level was <100 μg/24 h, which ruled out this diagnosis.2,4 Outside the brain, iron deposits are seen in the liver, but rarely result in clinically evident manifestations.7,15 In our case, the patient did not show signs of cirrhosis or hepatocellular insufficiency. Iron deposits have been reported sporadically in the pancreas, other endocrine glands, the heart, and others.4,15ACP is the only known iron overload disorder in which cerebral and systemic iron overload are combined.4,10 However, there are many other conditions with low levels of ceruloplasmin, including Wilson’s disease, Menkes disease, and others that remain differential diagnoses. Genetic study showing a mutation of the ceruloplasmin encoded gene located at the chromosomal locus 3q24-q25 confirms the diagnosis.4,16The diagnosis of ACP is made by the absence or low level of ceruloplasmin, as its name suggests, as well as by clinical and biological signs (anemia+elevated ferritin with low transferrin saturation) and imaging signs (iron deposition).Aceruloplasminemia is a rare disease due to a deficiency or total absence of ceruloplasmin.Clinical manifestations are increasingly variable. Microcytic anemia with neurological manifestations  associated with high serum ferritin and low transferrin saturation should be investigated by brain MRI.Microcytic anemia with elevated serum ferritin and low transferrin saturation associated with neurological manifestations  can indicate aceruloplasminemia.Systemic iron deposits in the brain  on brain MRI should suggest aceruloplasminemia and lead to the determination of ceruloplasmin.


The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and insidiously. Coronavirus disease 2019 (COVID-19) screening is an important means of blocking community transmission in China, but the costs associated with testing are high. Quarantine capacity and medical resources are also threatened. Therefore, we aimed to evaluate different screening strategies to balance outbreak control and consumption of resources.A community network of 2000 people, considering the heterogeneities of household size and age structure, was generated to reflect real contact networks, and a stochastic individual-based dynamic model was used to simulate SARS-CoV-2 transmission and assess different whole-area nucleic acid screening strategies. We designed a total of 87 screening strategies with different sampling methods, frequencies of screening, and timings of screening. The performance of these strategies was comprehensively evaluated by comparing the cumulative infection rates, the number of tests, and the quarantine capacity and consumption of medical resource, which were expressed as medians .Full Screening), if the screening frequency was four times/week, the cumulative infection rate could be reduced to 13% , the miss rate decreased to 2% , and the quarantine and medical resource consumption was lower than higher-frequency Full Screening or sampling screening. When the frequency of Full Screening increased from five to seven times/week (which resulted in a 2581 increase in the number of tests per positive case), the cumulative infection rate was only reduced by 2%. Screening all people weekly by splitting them equally into seven batches could reduce infection rates by 73% compared to once per week, which was similar to Full Screening four times/week. Full Screening had the highest number of tests per positive case, while the miss rate, number of tests per positive case, and hotel quarantine resource consumption in Household-based Sampling Screening scenarios were lower than Random Sampling Screening. The cumulative infection rate of Household-based Sampling Screening or Random Sampling Screening seven times/week was similar to that of Full Screening four times/week.To implement COVID-19 nucleic acid testing for all people (Full Screening would be necessary, but intermediate testing frequency may be more cost-effective in non-extreme situations. Screening in batches is recommended if the testing capacity is low. Household-based Sampling Screening is potentially a promising strategy to implement.If hotel quarantine, hospital and shelter hospital capacity are seriously insufficient, to stop the spread of the virus as early as possible, high-frequency The online version contains supplementary material available at 10.1186/s40249-022-01049-w. The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first reported in South Africa in November, 2021, rapidly became the dominant variant in the current global coronavirus disease 2019 (COVID-19) pandemic –6.The present vaccine may not effectively prevent infection caused by the Omicron variant due to its immune evasion . WithoutPrevious studies have focused on optimizing screening strategies to reduce the time from reporting the results of testing to quarantining contacted individuals and patients –20, explIn this study, we developed a stochastic network-based dynamic model that incorporates real household size, age structure, and contact patterns. It considered NPIs, such as testing, tracing, and isolation, and changes in testing sensitivity due to changes in the viral load of infected individuals over time. By simulating the spread of Omicron variant, 87 different screening strategies with different sampling methods, frequencies, and timings of screening were evaluated in terms of the cumulative infection rate, miss rate, tests per positive case identified, and the consumption of quarantine and medical resources.The transition of individuals between different disease processes was simulated using the Gillespie algorithm, in which the probabilities of possible events (transition between compartments) were computed, and one event was executed accordingly at each time step for a randomly selected node . At eachWe constructed a community containing 2000 people based on the age structure of the urban population and household size in 2020 , among wFull Screening, screening all people in the community; (2) Household-based Sampling Screening, selecting the individual with the most external connections in each family; once selected, they were screened on each screening day; and (3) Random Sampling Screening, randomly sampling a proportion of people in the community and then screening them on each screening day. Here, we set the sample size to be equal to the number of families. Second, we designed the frequency of screening to range between daily and biweekly. Furthermore, we also established different scenarios that screened three or four times/week. Finally, we assumed that weekly screening can be conducted in batches, which allows the individuals to be allocated into different batches and each batch is screened on different days. We also considered the Shanghai Strategy , Shenzhen Strategy , and Beijing Strategy . These strategies were implemented every 2 weeks in practice.We designed 87 whole-area nucleic acid screening strategies, considering different sampling methods, frequencies, and timings of screening  varied within 30 days after infection , NPIs would be implemented . Screening with different strategies was carried out simultaneously, and the screening results were reported 1 day after the testing day. Each strategy was simulated 1000 times over of period of 100 days.We conducted a sensitivity analysis on the basic reproduction number  to 14% , and the miss rate decreased from 32%  to 27% . Similar trends also occurred in Household-based Sampling Screening and Random Sampling Screening.The higher the frequency of screening, the lower the cumulative infection rate    Fig. a, b. Whe6%) Fig. d, e. In Full Screening as an example, as the frequency increased from once biweekly to daily, the number of tests per positive case increased from 6  to 4252 . It was notable that if screening was increased from four times/week to seven times/week, the increase in the number of tests per positive case identified was as high as 3895. When screening in batches, there was a slight increase in number of tests per positive case identified with increasing number of batches  were lower than those of Random Sampling Screening ; Full Screening seven batches/week  were substantially lower than those of Full Screening once per week . The complete results of sensitivity analysis were shown in Additional file The results with the different parameters were similar except for entclass1pt{minimaLarge-scale screening is a key measure in China’s control of COVID-19 . A recenFull Screening with high frequency—is necessary. (2) If the testing capacity is quite low, screening the targeted population in many batches is recommended. For example, the tests consumed by Full Screening the population once a week in seven batches was considerably less than the tests consumed by Full Screening four times/week, although the Full Screening does not need to be very high because the marginal effect of increasing frequency on epidemic control decreases with increasing frequency. In the case of Full Screening four times/week is a reasonable compromise. Besides, high-frequency sampling strategies also work under the restriction of testing, quarantine and medical resources. Household-based Sampling Strategy is better than Random Sampling Screening because the infection rates, miss rates, tests per positive case identified, and hotel resource consumption of the former were all lower. The outbreak mitigation effect of Household-based Sampling Screening seven times/week was comparable to Full Screening four times/week in our simulation.Our results showed that (1) if the quarantine and medical resources are seriously insufficient, the strict screening strategy—Household-based Sampling Screening is potentially promising in practice. Due to the extremely contagious nature of the Omicron variant, family cluster epidemics were common, and the secondary attack rate among households was over 50% [Household-based Sampling Screening was also convenient to organize in the actual implementation process, and since it screened fewer individuals, it not only reduced the potential risk of disease spread caused by gathering during nucleic acid screening, but also greatly reduced the demand for testing, medical, and other resources during the whole-area screening, ensuring the sampling quality and testing accuracy. In addition, Household-based Sampling Screening was conducive to the epidemic control measure implemented—quarantining close contacts—because quarantining infected individuals and their household members together improved the efficiency of transfer and quarantine which in turn helps breaks transmission and improves control the epidemic. Household-based Sampling Screening is a form of stratified sampling.We believe that over 50% , 32. TheIn the future, the prevention and control of COVID-19 will gradually turn to “normalization”, which allows for more flexible “stratification” according to key areas and key populations. A unit consists of a group of people with a similar risk of infection due to close contact with each other. For example, a school, a dormitory room, class, or grade can be considered a unit. A certain number of people from each unit are selected for testing, and if a positive test is found, the entire unit is quarantined.With a certain frequency of screening, the screening efficacy can be improved by optimizing the timing of screening. We recommend scheduling screenings evenly during a screening cycle. The Shanghai Strategy is an example of testing with a higher frequency at the beginning of a screening cycle, while the Beijing Strategy is closer to evenly distributed screening, the number of infected people in the former being more than that of the latter. Dividing once only screening into multiple batches is an extended way of arranging the tests evenly during a certain time period. The more batches, the more even the distribution. Furthermore, screening above the testing capacity is likely to extend the turnaround time of transferring people to the quarantine or medical places, reduce the quality of sampling and testing, and influence the efficiency of close contact tracing and quarantine . So, scrThis study has several limitations. First, reinfection with COVID-19 was not considered. Previous studies have indicated that the reinfection rate of COVID-19 is approximately 0.22–0.32% , which iHousehold-based Sampling Screening are recommended with the goal of mitigating the outbreak and restricting the consumption of resources. The specific strategy design should be determined according to the virus transmission feature and actual resource constraints considering cost-effectiveness, feasibility and sustainability.Intermediate screening (such as four times/week for a fast-spreading outbreak), screening in batches, and Additional file 1: Supplements of the methods.Text S1. Stochastic network-based dynamic model (P2). Text S2. Community network structure (P7). Table S1. Parameters related to disease transmission and control (P4). Table S2. Screening strategy (P6). Table S3. False negative rate of nucleic acid testing (1-test sensitivity) (P8). Table S4. Network parameters (P10). Table S5. Sensitivity analysis parameters (P11). Fig. S1. Compartmental structure used to describe the progression of disease states (P11). Fig. S2. A. Random network degree distribution; B. Network degree distribution after increasing social distance (P12).Additional file 2: Supplements of the results.Fig. S3. The number of infections in 100 days for each strategy (P2). Fig. S4. The cumulative infection rates of 1000 simulations (P3). Fig. S5. The number of people quarantined in the hotel in 100 days for each strategy (P4). Fig. S6. The number of people quarantined in the shelter in 100 days for each strategy (P5). Fig. S7. The number of hospitalized cases in 100 days for each strategy (P6). Fig. S8. Cumulative infection rate, miss rate, and tests per positive of screening at different times (P7). Fig. S9. Cumulative and the maximum number of quarantine and hospitalized people of screening at different times (P7). Fig. S10. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S11. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S12. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S13. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S14. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S15. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S16. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S17. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S18. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when Fig. S19. Cumulative infection rate, miss rate, tests per positive, the number of quarantining and hospitalization when


Monitoring measurable residual disease (MRD) is crucial to assess treatment response in Multiple Myeloma (MM). Detection of MRD in peripheral blood (PB) by exploring Extracellular Vesicles (EVs), and their cargo, would allow frequent and minimally invasive monitoring of MM. This work aims to detect biomarkers of MRD in EVs isolated from MM patient samples at diagnosis and remission and compare the MRD-associated content between BM and PB EVs. EVs were isolated by size-exclusion chromatography, concentrated by ultrafiltration, and characterized according to their size and concentration, morphology, protein concentration, and the presence of EV-associated protein markers. EVs from healthy blood donors were used as controls. It was possible to isolate EVs from PB and BM carrying MM markers. Diagnostic samples had different levels of MM markers between PB and BM paired samples, but no differences between PB and BM were found at remission. EVs concentration was lower in the PB of healthy controls than of patients, and MM markers were mostly not detected in EVs from controls. This study pinpoints the potential of PB EVs from MM remission patients as a source of MM biomarkers and as a non-invasive approach for monitoring MRD. Treatment of Multiple Myeloma (MM) has improved substantially, demonstrated by the current sequential and integrative approach using drugs of different classes in combination, which has clear benefits for patients . In receA deep and sustained complete response (CR) to treatment is the ultimate objective in any phase of MM treatment. Nonetheless, achieving CR is not enough to ensure longer survival in MM. Indeed, the persistence of very low levels of plasma cells (PCs) in patients’ bone marrow (BM), as measurable residual disease (MRD) after treatment, will constitute the soil for a subsequent relapse and are a major cause of drug resistance. Quantifying MRD allows for the assessment of chemotherapy efficacy and clinical outcome, the identification of patients at high-risk of recurrence, and provides important information for therapy decisions ,4. MRD eIndeed, MRD constitutes the most significant predictor of clinical outcome ,10. In l−6 using next-generation flow (NGF) cytometry [−6. Both NGF and NGS lack standardization over different laboratories but yield similar results [MRD evaluation can be divided into two approaches: measuring intramedullary disease by multiparameter flow cytometry (MFC) immunophenotyping or by high-throughput/next-generation sequencing (NGS) molecular assessment of immunoglobulin gene rearrangements; or by quantifying extramedullary disease (EMD) using functional imaging methods ,22,23. Uytometry ,25,26. C results ,28. The  results ,30. MRD  results . MM is c results . When us results . These lExtracellular Vesicles (EVs) are a group of lipid bi-layer circular particles with different biogenesis that are heterogenous in size and content. EVs include exosomes , microvesicles (with plasma membrane origin and sizes ranging from 50 to 2000 nm), or even apoptotic bodies (with both cytosolic components and nuclear fragments and sizes ranging from 50 to 5000 nm) ,33,34. IIn MM, EVs play a crucial role in mediating the mutual crosstalk between the MM PCs and cells from the tumor microenvironment by transferring active molecules such as lipids, proteins, and regulatory RNAs, thus contributing to MM pathobiology ,51. AnotConsidering the flow of communication between MM-EVs and other cells in the BM microenvironment, as well as the dynamic quantity of EVs in relation to disease severity, stage, or treatment phase ,58,59,60EVs from the PB and BM of MM patients and EVs from the PB of healthy controls were isolated by Size Exclusion Chromatography (SEC) followed by ultrafiltration (UF) and were characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Western Blot (WB) in terms of size distribution, morphology, and the presence of well-known protein EV markers to confirm the identity and purity of the isolated EVs.Our results show the possibility of isolating EVs from the BM and PB of patients with MM. Choosing the optimal strategy to isolate EVs is a critical step in achieving enough of a yield of EVs with minimal contamination by non-vesicular proteins/particles in order to assure their accurate study ,61. SEC Finally, the presence of several EV markers  was found by WB in fractions 4 to 10 , which presented mean EV sizes between approximately 40 and 100 nm B, withouDifferent immunophenotypic markers of MM were analyzed by WB in the ultrafiltrated pools from SEC fractions 3 and 6 for both PB and BM patient samples at diagnosis and remission status. The MM EV markers analyzed consisted of the well-known MM markers related to specific PC lineages, such as CD38 and CD138, and others used by the Euroflow Consortium for the diagnosis and response evaluation to treatment by flow cytometry: CD45, CD56, CD19, CD81, CD117, CD27, and cytoplasmic immunoglobulin kappa and lambda light chains . ResultsLevels of CD38 and CD138 were analyzed in ten patients. A higher number of CD38 positive samples were observed compared to CD138 and co-presentation of CD38/CD138. This co-presentation was particularly evident in patients 4 and 8. However, other patients, such as patient 1 and 3, did not present CD38+ in EVs at diagnosis. Recently, it was described that low levels of CD38 were found in PCs of patients with EMD, conferring poor response to anti-CD38 monoclonal antibodies. Interestingly, our patients without CD38 in EVs at diagnosis, and CD38-negative by flow cytometry , did preBoth CD38 and CD138, as specific lineage markers, were described to be the best markers to use when identifying the source EVs derived from monoclonal PCs ,57. NoneExpression of CD45 was found in EVs from six patients , and in Restriction for kappa (κ) or lambda (λ) light chains is usually used as a surrogate for clonality in MM, with abnormal ratios suggesting the presence of such a restriction . ImportaOther markers were reported to be increased in MM EVs, although they are not specific to MM and could be used as aberrant markers. For example, increased levels of the transmembrane glycoprotein CD147 were found in MM-derived EVs and appeared to be related to tumor cell growth and MM progression ,80, whilWhen comparing the presence of MM markers between paired PB and BM samples, it was possible to verify that, with some minor exceptions, most of the markers present in the EVs isolated from BM were also present in EVs isolated from PB . HoweverEven though this WB analysis is not a quantitative analysis, and the analysis of results is limited by the lack of a proper EV protein loading control, this suggests that the cargo of immunophenotypic markers on EVs from BM and PB paired samples is not the same. Therefore, the search for these biomarkers in liquid biopsies  from diagnostic samples may not provide similar results to the ones obtained from BM aspirates. Nonetheless, similar levels of immunophenotypic markers are present in EVs from BM and PB at the remission stage, suggesting that the follow up of MM patients by analysis of EVs in liquid biopsies (PB samples) may be representative of the disease markers in the BM. This finding is of major relevance regarding the potential use of EVs as biomarkers in liquid biopsies for MRD monitoring.Detection of MRD in PB mirroring BM and/or EMD would bring obvious advantages to patients and clinicians, allowing frequent and minimally invasive real-time monitoring of the disease ,84,85,86In all the ten patients included in the analysis, there was a lack of consistency regarding the levels of most MM markers found in the cargo of EVs isolated from paired diagnosis or remission samples . NonetheRegarding the comparison of MRD status determined by flow cytometry and the EV MM markers found at remission, it is difficult to establish a definite correlation, even though some patterns can be found . Indeed,Interestingly, patient 8 also presented a disappearance of CD138 in EVs isolated from remission . Results are presented in As expected, IgG kappa and lambda were detected in EVs isolated from healthy donors . HoweverThe absence of MM markers in EVs from healthy controls further suggests that markers such as CD38, CD56, CD117, and CD27 are suitable for use as biomarkers of MM or in response monitoring of MRD.Patient peripheral blood (PB) and bone marrow (BM) samples were collected from Multiple Myeloma (MM) patients at diagnosis and at remission on the 100th day after autologous stem cell transplant (ASCT) at the Clinical Hematology Department from Centro Hospitalar Universitário São João . All patients were newly diagnosed with MM and treated upfront with the triplet combination of bortezomib, thalidomide, and dexamethasone. Treatment response on the 100th day after ASCT was assessed according to the International Multiple Myeloma Group recommendations ,24,106.−5) and the limit of detection (LOD) in each assay was determined according to the formula (20/nucleated cells) × 100. Any patient was considered MRD negative if there was an absence of clonal PCs or if they were present below the LOD achieved in the corresponding sample [All Minimal Residual Disease (MRD) assays were performed on the BM aspirates within 24 h of collection according to the Eight Color EuroFlow panel for MM, combining surface antigens for the identification of phenotypically abnormal clonal plasma cells (PCs)—CD38, CD138, CD45, CD19, CD56, CD27, CD81, and CD117—and cytoplasmic lambda and/or kappa light chains to confirm clonality. MRD was considered negative with a cut-off level of less than 20 clonal PCs in 2 million nucleated cells . The PB and BM samples were transferred to centrifuge tubes using an aseptic technique, and an equal amount of phosphate-buffered saline 1× (PBS 1×) was added to each. The mix was gently transferred to tubes containing Histopaque-1077  and centrifuged for 30 min at room temperature (RT) at 400× g. The Platelet-Rich Plasma (PRP) was centrifuged , resulting in Platelet-Poor Plasma (PPP). The PPP was divided into aliquots and immediately stored at −80 °C until further use. Samples were collected in tubes buffered with 3.8% of sodium citrate (Vacutestw/v) trisodium citrate dihydrate  in PBS , Sepharose cross-linked 2B  was placed in a 10 mL syringe  with its tip filled with a piece of nylon stocking (20 denier). When the compacted Sepharose reached the 11 mL mark, a 3 MM paper filter  was added to the top. The column was kept in 20% ethanol and stored at 4 °C until further use.The isolation of Extracellular Vesicles (EVs) was performed using a previously described method with some alterations . After bFor EVs isolation, the 20% ethanol solution was removed by loading PBS-citrate into the SEC column. The PPP (1 mL) was loaded with the continuous addition of PBS-citrate. Ten sequential 1 mL fractions were collected. Fractions were either used immediately for EVs characterization or stored at −80 °C until further use. ® Ultra-4 Centrifugal Filters Ultracel®—100 K, Milipore, Merck Life Science, Darmstadt, Germany) and centrifuged at 4 °C at 3200× g. The ultrafiltrated (UF) sample was collected, quantified (as described below), and stored at −80 °C until downstream analysis.To concentrate the EVs, around 4 mL from pools of EV-rich and protein-low fractions  was filtered with a 100 kDa cut-off membrane (AmiconThe protein amount of each SEC fraction (either from the EV cargo or protein contaminants) was determined using the Lowry protein assay , according to the manufacturer’s instructions. The absorbance was analyzed in a microplate reader  with a 488 nm excitation wavelength and read at the 655 nm emission.7 to 109 particles/mL). The samples were loaded at a constant rate in a NanoSight NS300  with a 1 mL syringe  using a Nanosight syringe pump  at RT. Three separate 30 s videos were recorded with the following specifications: camera type, sCMOS; laser type, Blue488; camera level, 15/16; slider shutter, 1206/1300; slider gain, 366/512; FPS, 25.0; temperature, 21.1–25.5 °C; viscosity, 0.878–0.974 cP; syringe pump speed, 40. Particles were detected by video analysis using NanoSight NTA Software  with the following settings: detection threshold, 5; blur size, auto; max jump distance, auto (8.3–16.2 pixels). The mean, mode, and median vesicle size (nm) and estimation of the particle concentration (particles/mL) were determined. The data obtained by NTA were then analyzed using GraphPad (Prism 8).Particle concentration and size distribution were obtained by Nanoparticle Tracking Analysis (NTA). The SEC fractions and the UF sample were pre-diluted  in 0.22 µm of filtered PBS-citrate  sucrose , mounted in Formvar-carbon-coated electron microscopy grids  for 2 min in the dark at RT, and dried with a filter paper. TEM grids were then counterstained with 5% uranyl acetate and visualized under the transmission electron microscope  with an acceleration voltage of 80 kV. The data were obtained by the Histology and Electron Microscopy Service, i3S, Porto, Portugal. Representative TEM photographs were acquired and the EV size for each SEC fraction was measured by image analysis using ImageJ software.EVs size, morphology, and integrity were visualized by Transmission Electron Microscopy (TEM) using negative staining. Each sample was resuspended in PBS-citrate or in a 1:2 solution of PBS-citrate and HEPES 20 mM  +4%  and transferred to a nitrocellulose membrane  using a Mini Trans-Blot® cell system . After the transfer, the proteins were stained with Ponceau S Solution  and images were acquired with Chemidoc XRS+ System equipment . The membranes were blocked for at least 2 h at RT in a blocking solution consisting of 5% (w/v) non-fat dry milk  in TBS-T . After blocking, membranes were incubated with primary antibodies  diluted in the blocking solution in accordance with the manufacturer’s instructions . Signal quantification of the bands was normalized to the total protein of the lane, which was obtained by Ponceau staining.The SEC fractions and the UF sample were denatured in loading buffer  and boiled at 95 °C for 5 min. A total of 15 μg of protein from each fraction was separated using SDS-PAGE  for MM-specific markers (such as CD38 and CD138) were used, as well as other antibodies relative to the antigens used in the EuroFlow panel, such as CD19, CD27, CD45, CD81, CD56, CD117, and cytoplasmic lambda and/or kappa light chains  Supplem.t-test which was obtained in GraphPad Prism 8.0 software. Statistical significance was considered whenever p < 0.05.The statistical analysis was performed using the two-tailed unpaired In the present study, we demonstrated the possibility of isolating EVs from the BM and PB of MM patients at distinct disease stages (diagnosis and remission) and compared the MRD-associated markers between paired BM and PB EVs at diagnosis and remission stages. Firstly, our results demonstrated that EVs from BM and PB samples, both from diagnosis and remission, were successfully isolated using the SEC method, concentrated by UF, and characterized by NTA, TEM, and WB in terms of size distribution, morphology, and the presence of well-known EV protein markers. Secondly, EVs isolated from PB or BM at diagnosis present specific MM markers, such as CD38 and CD138, confirming the potential of EVs as MM biomarkers. Thirdly, by analyzing alterations in the levels of MM markers in EVs isolated from paired diagnosis and remission samples, it was possible to verify differences between those markers, enhancing the possibility of using EVs to monitor MRD. Fourthly, even though in the diagnostic samples the levels of MM markers were different in several PB and BM samples, in the remission samples those levels were very similar, thus suggesting that EVs have the potential to monitor MRD in liquid biopsies from remission samples. Finally, the much lower levels of MM markers in the EVs isolated from the PB of healthy controls supports the possibility of using those markers as specific MM biomarkers to non-invasively monitor MRD. Nonetheless, some caveats are exposed in our study. It was not powered to provide definite clinical advice; thus, further studies should be attempted to extend this study to a larger sample of MM patients and integrate this approach with NGS or NGF at different disease timepoints. Moreover, even if the SEC method proved to be efficient in isolating EVs and the protocols to characterize those EVs were well described, overall, the methodology is time consuming and needs to be further optimized to become reproducible and easier to use in clinical practice. Furthermore, there is no standardization in the interpretation of results, which relies on sample-by-sample analysis by experienced operators.Overall, this study suggests the possibility of isolating EVs from PB samples of MM remission patients for use in the monitoring of MRD. Our results warrant a prospective and larger study with more samples from MM patients to confirm and reproduce the herein presented data, to optimize the methodology in order for it to become easier to use in clinical practice, and to determine its sensitivity regarding MRD detection so that it can possibly assume its role as a method for analyzing non-invasive liquid biopsies of MM.