Breastfeeding can be a vital way of acquiring passive immunity via the transfer of antibodies from the mother to the breastfeeding infant. Recent evidence points to the fact that human milk contains immunoglobulins (Ig) against the SARS-CoV-2 virus, either after natural infection or vaccination, but whether these antibodies can resist enzymatic degradation during digestion in the infant gastrointestinal (GI) tract or indeed protect the consumers remains inconclusive. Herein, we evaluated the levels of IgG, IgA, and secretory IgA (SIgA) antibodies against the spike protein of SARS-CoV-2 in 43 lactating mothers who received at least two doses of either an mRNA-based vaccine  or an adenovirus-based vaccine . We also accessed the potential persistence of SARS-CoV-2 IgA, IgG, and secretory IgA (SIgA) antibodies from vaccinated women in the GI tract of the infants by means of a static in vitro digestion protocol. Our data depict that, although slightly reduced, the IgA antibodies produced after vaccination resist both the gastric and intestinal phases of infant digestion, whereas the IgGs are more prone to degradation in both phases of digestion. Additionally, SIgA antibodies were found to greatly resist the gastric phase of digestion albeit showing some reduction during the intestinal phase. The evaluation of the vaccine induced Ig profile of breastmilk, and the extent to which these antibodies can resist digestion in the infant GI tract provide important information about the potential protective role of this form of passive immunity that could help decision making during the COVID-19 pandemic and beyond. The coronavirus disease 2019 (COVID-19) outbreak, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed global public health concern due to its high dispersion rate influencing public health, society, and the economy across the world , one conFor breast-fed infants, human milk is a source of various nutrients  and bio-active components that promote neonatal growth and protect from viral and bacterial infection . In termIt has been previously demonstrated that human milk from mothers who have been infected with SARS-CoV-2 contains antibodies against the virus, and that these antibodies also demonstrate a neutralizing ability ,15,16,17Whichever the antibody profile secreted in milk upon vaccination, it is understood that in order for any such antibodies to play a role in immunoprotection, Igs must resist proteolytic degradation throughout digestion to remain intact and functional in order to bind pathogens in the digestive system and block that route of infection. Additionally, to provide a more systemic protection, these antibodies, must be absorbed into the bloodstream of infants . ImportaIn this study, we first validated the accuracy of a commercially available ELISA assay for the determination of anti-SARS-CoV-2 IgAs and IgGs in breastmilk. We then employed these assays to evaluate the Ab response in breastmilk after vaccination with both doses of either the Pfizer/BioNTech, Moderna or the Astra Zeneca vaccine in 43 lactating participants. In 10 of these participants, we also evaluated the response following a booster dose. Finally, we assessed the persistence of anti-SARS-CoV-2 IgA, IgG, and SIgA antibodies following a static in vitro protocol of infant GI digestion. To our knowledge this is the largest study, to date, performed to explore whether SARS-CoV-2 antibodies withstand digestion.In the present study, 43 lactating women that had no diagnosis of laboratory-confirmed COVID-19 (with an antigen rapid test or RT-qPCR test) and were scheduled to receive a COVID-19 vaccine were recruited . The vaccinated participants had received two or three doses of the mRNA vaccines. Control milk samples were obtained from the same individuals before vaccination and used as controls to establish positive cut-off values for each assay. Samples were also collected 1 day before the second dose, 3 weeks after the second dose, and, in some participants (n = 10), 3 weeks after the booster (3rd dose) dose to determine antibody levels. All samples were collected in sterile tubes, stored immediately at −20 °C and later divided into 1.5 mL aliquots and stored at −80 °C until used. On the collected breast milk samples, anti-SARS-CoV-2 IgA, IgG, and SIgA were evaluated prior and after simulation of infant GI digestion. The study design overview is shown in In addition to breastmilk samples, clinical and demographic characteristics were also collected. Participants received information and gave written informed consent before enrolment. All procedures were approved by the Cyprus National Bioethics Committee (protocol number EEBK/EΠ/2021/24).A − Vo)/VA × 100, where VA is the known value and Vo represents the observed value. The precision of the assays was evaluated using the percentage coefficient of variation (CV), as follows: % CV = Standard deviation (SD)/Average × 100. The recovery of the assays was evaluated via the equation: % Recovery = (MS − MN)/TS × 100 [S is the measured concentration of the spike sample, MN is the measured concentration of the neat samples, and TS is the theoretical concentration of the spiked sample. Unspiked milk was used as a blank in these experiments. The ELISA ab277285 kit was also utilized with modifications to evaluate the levels of SIgA, where the IgA antibody was replaced with the Sheep anti-Human secretory component  antibody in a dilution of 1:1000. This antibody reacts with both bound secretory component (secretory IgA) and with the free SC present in human secretions.We previously optimized the use of commercially available ELISA kits specific for the determination of IgA and IgG immunoglobulins against SARS-CoV-2 in human plasma and serum (Abcam Cat numbers: ab277285 for IgG and ab277286 for IgA)  with somTS × 100 , where Mg for 20 min at 4 °C, fat was removed and skimmed fraction transferred to new tubes and centrifuged again under the same conditions to remove residual fat and cells. Fat-free milk was aliquoted and stored at −80 °C until testing. For the evaluation of the levels of IgA, IgG, and SIgA specific to SARS-CoV-2 spike protein in human milk, commercially available ELISA kits from Abcam were used with modifications as mentioned above. Milk was either undiluted or diluted 1:2 using assay buffer (provided by the manufacturer) (for IgA) or 1:2 (or 1:4) using assay buffer supplemented with 1% w/v BSA (for IgG) and added to the plate. Antibody levels were reported as Units (U) using the following equation (that was provided by the manufacturer) Units = ((Sample Absorbance × 10/Cut-off) × DF)), using, as cut-off, the mean absorbance values of milk samples before vaccination, where DF is the dilution factor. Samples containing > 10 Units of IgA or IgG were considered as positive. Anti-SARS-CoV-2 IgA, IgG, and SIgA were analyzed in the undigested breast milk samples and after the gastric and intestinal stages of simulated in vitro digestion. Milk samples were quickly thawed in a water bath at 37 °C and centrifuged at 1300× A two-step in vitro infant GI digestion method that consists of a 60 min of gastric phase and a 120 min of intestinal phase, as previously described by ,31, was Unless otherwise stated, data are reported as means ± standard deviation (SD). The normal distribution of continuous data was analyzed with the D’Agostino and Pearson omnibus normality test. Antibody levels were log-transformed before all statistical processes. Log values of antibody levels follow a normal distribution and, thus, are presented as mean ± SD.p-values were two-tailed and p-values < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism .The levels of antibodies before and after vaccination were compared via one-way ANOVA with Tukey’s multiple comparison test was used to compare percentage stability of each antibody before and after digestion. All reported Characteristics of study participants and their infants are presented in p < 0.001) the recovery of spiked-IgG in human milk samples from 53.6% to 83.75% (data not shown) and decrease the assay error from ~50% to 18.25% [We have preciously validated two commercially available ELISA kits from Abcam (developed for use with serum and plasma samples), to evaluate the levels of SARS-CoV-2-specific IgA and IgG in human milk samples . Initialo 18.25% . Therefoo 18.25% .We next evaluated the presence of IgG and IgA antibodies against SARS-CoV-2 in breastmilk of 43 participants after vaccination with two doses. Of those participants, 10 (23.3%) also donated a sample after the booster dose, six months after the second vaccine dose, therefore IgG and IgA antibodies were evaluated in those samples as well. Samples were collected one day before the first dose, one day before the second dose and three weeks after the second and booster dose . Fat-frep < 0.05) and IgA antibodies (p < 0.001)  . A furthOf our cohort of 43 lactating participants, 35 of them had donated enough milk in order to further perform digestion analysis. Therefore, 35 samples were subjected to simulated infant GI digestion and concentrations of anti-SARS-CoV-2 IgA, IgG and SIgA were determined using the ELISA assays, as described previously. Vaccine-induced anti-SARS-CoV-2 Ig levels were decreased through the gastric phase and further through the intestinal phase of digestion as compared to basal levels . HoweverIn the present study, we demonstrated that commercially available ELISA kits specific for the detection of anti-SARS-CoV-2 IgG, IgA and SIgA in human serum and plasma can be utilized with some modifications for the detection of these Igs in human breastmilk samples. All three assays were highly accurate and met the typical validation requirements for their intended use. Subsequently, using these assays we detected anti-SARS-CoV-2 antibodies in breastmilk in a Cyprus cohort of 43 lactating participants that were vaccinated with the Pfizer/BioNTech, Moderna or AstraZeneca vaccines. It was shown that anti-SARS-CoV-2 antibodies could be detected in the breastmilk of vaccinated individuals with levels further increasing following the second dose of vaccination. Our findings agree with several other studies that have demonstrated the induction of anti-SARS-CoV-2 antibodies in the breastmilk following vaccination. In this study, we were also able to investigate several samples from 10 individuals who had received a booster vaccination, and showed that antibody levels were further increased, supporting that booster vaccination is useful not only for continuous protection of the individual but potentially also of the breastfeeding infant. To our knowledge, only two other studies have evaluated the presence of SARS-CoV-2 antibodies in breastmilk after a booster dose: a study of four individuals conducted by Leung et al., which also showed that booster dose is able to re-trigger the secretion of neutralizing IgA in breastmilk , and a pSeveral studies have shown that Igs against SARS-CoV-2 in human milk induced after infection or vaccination have a neutralizing ability, suggesting their potentially protective role ,35,36,37Our previous preliminary study was the first to investigate the persistence of anti-SARS-CoV-2 antibodies in breastmilk from vaccinated lactating women by means of an in vitro simulated infant GI digestion . In the The study by Calvo-Lerma et al., is the only other study that has investigated the survival of IgA and IgG specific for SARS-CoV-2 after in vitro simulated digestion . In thisAn important strength of this study was the evaluation of SIgAs in breastmilk samples of vaccinated individuals. The majority of IgAs in human milk are SIgAs, which have a primary role as a protective agent in human milk, protecting mucosal surfaces against various pathogens, including viruses, by inhibiting the pathogen adherence to these surfaces . WhetherSIgA is found as dimers containing the highly glycosylated secretory component and J chain, which make them less susceptible to proteolysis. This may explain our findings that following gastric digestion there is only minimal decrease in sIgA levels. This is indeed the first study that has differentiated the anti-SARS-CoV-2 SIgAs present in breastmilk and has investigated their resilience through digestion. Nevertheless, further studies are required to show whether the digested breastmilk has the potential to neutralize the virus, something that has not been studied to date. In this study, we did not include individuals that had been infected with SARS-CoV-2, therefore conclusions regarding differences in various antibody isotype levels cannot be drawn. Additionally, this study did not measure IgA and IgG concentrations in the serum of the lactating mothers, thus comparisons of antibody titers between serum and milk could not be performed. However, it has been reported that the antibody response to COVID-19 vaccination was rapid and highly synchronized between breastmilk and serum of lactating mothers . FurtherThis study represents the biggest study up to now in Europe reporting specific antibody levels in milk for three different vaccines. Our study provides preliminary data, and it has limitations. Larger prospective studies, bigger samples sizes, and distinct cohorts in different locations and long-term follow-up are needed to better understand the time course of SARS-CoV-2 immunity in milk and the possible differences in the presence of antibodies in breast milk according to vaccine type. In addition, longer follow-up of the antibody levels would be needed in order to determine their persistence. Furthermore, although it has been suggested that antibodies found in breastmilk would exert strong neutralizing effects, no functional assays were performed. In addition, the potential impact on neonatal growth as well as the potential protective effect against infection in the infant remains elusive.In this study, anti-SARS-CoV-2 antibodies were detected in breastmilk samples of a Cyprus cohort of 43 lactating participants that were vaccinated with the Pfizer/BioNTech, Moderna or AstraZeneca vaccines. Antibody levels in milk were shown to be increased following the second dose and booster doses of vaccination. Notably, this is also the biggest study to date to examine the resilience of IgG, IgA and SIgA antibodies via digestion and to show that a number of SARS-CoV-2 antibodies can withstand digestion, with levels remaining above cut-off values, even following the intestinal phase of digestion. Overall, these findings suggest that vaccine-induced anti-SARS-CoV-2 antibodies can be immunologically active throughout infant digestion, and, hence, protective, in the baby’s lumen during breastfeeding. Further studies will confirm these findings.


Introduction: Several studies have demonstrated that the absolute numbers of select surgical interventions for myocardial infarction, stroke, and appendicitis decreased during the COVID-19 pandemic, possibly due to overall decreased hospital presentation. We sought to identify if this pattern was also true for children with hydrocephalus and cerebrospinal fluid (CSF) diversion procedures. We hypothesized that there would be a detectable decrease in CSF diversion procedures performed during the COVID-19 pandemic, as compared to a pre-COVID-19 baseline.Methods: A chart review of all patients that underwent a CSF diversion procedure from March 2019 to February 2021 was performed at Ann and Robert H. Lurie Children’s Hospital of Chicago. The pre-COVID-19 period was defined as March 2019 to February 2020 and the COVID-19 pandemic period was defined as March 2020 to February 2021. CSF diversion procedures included endoscopic third ventriculostomy (ETV), ETV/choroid plexus cauterization (CPC), initial shunt placement, shunt removal/replacement, shunt revision, and temporization procedures. Data included gender, race, ethnicity, insurance type, etiology of hydrocephalus, type of procedure, and whether the procedure was performed due to infection. Results: Overall, there was no significant difference in the absolute number of CSF diversion procedures performed when comparing the pre-COVID-19 and COVID-19 periods . Furthermore, there was no observed difference in the gender, ethnicity, or insurance status of children undergoing a CSF diversion procedure. There was, however, a significant increase in the number of procedures performed due to infection during the COVID-19 pandemic at our institution (p = 0.04).Conclusion: Unlike several other surgical conditions during the COVID-19 pandemic, a statistically significant change in CSF diversion procedures was not observed at our institution. The increased number of procedures for infection at our institution is likely multifactorial and will be investigated further. As the United States entered the COVID-19 pandemic at the beginning of March 2020, a sudden decline in surgical cases, such as orthopedic and breast surgery cases , and ItaPediatric surgical volume had a noted reduction in the United States between 2019 and 2020 ,12. One We sought to identify if this pattern was also true for children with hydrocephalus and cerebrospinal fluid (CSF) diversion procedures at Lurie Children’s Hospital of Chicago. We hypothesized that there would be a detectable decrease in CSF diversion procedures performed from March 2020 to February 2021 (the COVID-19 pandemic), as compared to a pre-COVID-19 baseline. This article was previously presented as an oral presentation at the 17th WFNS World Congress of Neurosurgery Meeting on March 14, 2022.Following institutional review board (IRB) approval (IRB # 2021-4375), a retrospective chart review of all patients that underwent a CSF diversion procedure from March 2019 to February 2021 was performed at Ann and Robert H Lurie Children's Hospital of Chicago. The pre-COVID-19 period was defined as March 2019 to February 2020 and the COVID-19 pandemic period was defined as March 2020 to February 2021, from the start of the Illinois statewide stay at home mandate to the first year of the pandemic. CSF diversion procedures included included endoscopic third ventriculostomy (ETV), ETV/choroid plexus cauterization (CPC), initial shunt placement, shunt removal/replacement, shunt revision, and temporization procedures. Data included gender, race, ethnicity, insurance type, etiology of hydrocephalus, type of procedure, and whether the procedure was performed due to infection. All patients from the COVID-19 pandemic period had their COVID-19 status prior to surgery noted as well. The primary outcome measure was to compare the absolute number of CSF diversion procedures performed before and after the peak of COVID-19. Secondary outcomes focused on the differences in an absolute number of procedures due to infection and acuity of cases, as determined by the neurosurgeon and noted in the operative, whether urgent (within 24 hours) or elective (outside of 24 hours). Differences in categorical variables were analyzed using a chi-square test and significance was determined with a p-value <0.05.CSF diversion procedures represented 29% of 843 surgeries performed in the division of neurosurgery between March 2019 and February 2020 (pre-COVID-19). Following the COVID-19 pandemic, 31% of all 777 surgeries were CSF Diversion procedures . During the COVID-19 pandemic period, the most common infectious cause was Staphylococcus epidermidis (44%) followed by Propionibacterium acnes (12%)  test prior to their scheduled operation. For those who needed to undergo urgent surgeries with a positive COVID-19 test, special precautions were taken to maximize the safety of all staff involved in the operation, representing a commitment to patient care and the providers and hospital staff.During the COVID-19 pandemic, there was a decrease in overall neurosurgical cases from 843 to 777 consistent with other pediatric surgical institutions ,12. PrioA finding of interest is the observed increase in procedures performed due to bacterial infection during the COVID-19 period. The difference between pre-COVID-19 and COVID-19 rate of surgeries done for shunt infection is statistically significant (Figure This study has several limitations, including the inherent limited observational nature of retrospective chart review. The thought process of families in terms of decision-making of timing and need for seeking care is not elucidated well. The timing of onset of symptoms or concern to presentation and surgery is difficult to quantify. Relative delay in care, if present, is not known. Furthermore, we analyzed a subset of pediatric neurosurgical procedures, CSF diversion. We cannot generalize that other categories of pediatric neurosurgical procedure, such as brain tumor resections, would have similar results when comparing the number of procedures pre-COVID-19 and during COVID-19.The COVID-19 pandemic brought drastic changes to healthcare. Institutions saw a decrease in surgical volume as new protocols were established due to the pandemic. Unlike several other surgical conditions during the COVID-19 pandemic, a statistically significant change in CSF diversion procedures was not observed at our institution. The increased number of procedures for infection at our institution is likely multifactorial and will be investigated further.


This study investigated the frequency and patterns of interstitial lung disease (ILD) and their clinical effect on all-cause mortality during the follow-up period in patients with antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitis (AAV) in Korea.  The medical records of 255 AAV patients with ILD were retrospectively reviewed. ILD and its patterns, the usual interstitial pneumonia (UIP) and non-UIP patterns, were confirmed using high-resolution computed tomography both at AAV diagnosis and during follow-up. Forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) were also obtained.  The median age was 65.0 years, and 34.9% were male. ILD occurred in 53 patients, among whom 49.1% developed ILD after AAV diagnosis. Among AAV subtypes, the frequencies of ILD were significantly higher in both patients with microscopic polyangiitis (MPA) and those with AAV having myeloperoxidase (MPO)-ANCA (or P-ANCA) compared to other subtypes. However, there was no statistical significance in AAV subtypes or FVC/DLCO ratio between patients with the UIP and non-UIP patterns. In particular, the cumulative patients' survival rate was lower in patients with AAV and ILD than in those without ILD.  ILD occurred in one-fifth of Korean patients with AAV in this study and was associated with MPA and MPO-ANCA (or P-ANCA). In addition, ILD significantly increased the rate of all-cause mortality in these patients with AAV. Therefore, we suggest the need for more attention and more frequent regular visit for patients with AAV and ILD regardless of the time of ILD occurrence. Antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitis (AAV) is a necrotising vasculitis that affects small-sized vessels, mainly capillaries, venules, and arterioles and occasionally adjacent arteries . Since AAmong nine clinical categories based on version 3 of the Birmingham vasculitis activity score (BVAS) version 3, a pulmonary manifestation of AAV includes wheezing, nodules or cavities, pleural effusion, pleurisy, infiltrate, endobronchial involvement, massive haemoptysis/alveolar haemorrhages, and respiratory failure. Of these, some cases of “pulmonary infiltrate” may be associated with interstitial lung disease (ILD) . ILD is Previous meta-analyses have reported the clinical implications of ILD in patients with AAV: (i) ILD occurs in approximately 23% of patients with GPA and up to 45% of those with MPA; (ii) ANCA positivity might be associated with the concurrent occurrence of ILD in patients with AAV: in particular, MPO-ANCA might be detected in 46-71% of patients with AAV and ILD; (iii) the UIP pattern is predominantly found in patients with AAV ranging from 43% to 83%; and (iv) ILD is likely associated with a relatively high mortality rate in patients with AAV, in particular, those with the UIP pattern . A previHowever, to date, no studies have reported the frequency and patterns of ILD and their clinical effect on all-cause mortality during the follow-up period in Korean patients with AAV. Hence, this study included only patients with AAV and ILD confirmed using HRCT both at AAV diagnosis and during follow-up and investigated the frequency and patterns of ILD and their clinical effect on all-cause mortality during the follow-up period in patients with AAV in Korea.The medical records of 255 patients with AAV enrolled in the Severance Hospital ANCA-associated VasculitidEs (SHAVE) cohort were retrospectively reviewed. The SHAVE cohort is an observational cohort of patients with AAV of a single centre. As described in the previous study , 12, theDemographic data, AAV-related variables, and clinical data at diagnosis were collected and are shown in This study only considered ILD confirmed using HRCT. The UIP and non-UIP patterns were confirmed by a radiologist specialising in lung imaging. Within 2 weeks of HRCT performance, pulmonary function tests (PFTs) were performed in 45 patients with AAV and included forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO).U tests were used to compare significant differences between two continuous variables. Comparison of the cumulative survival rates between the two groups was performed using the Kaplan-Meier survival analysis with log-rank tests. Statistical significance was set as P < 0.05 [All statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0 . Continuous and categorical variables are expressed as medians with interquartile ranges and numbers (percentages), respectively. Significant differences between categorical variables were analysed using chi-square and Fisher's exact tests. The Mann-Whitney P < 0.05 .At AAV diagnosis, the median age was 65.0 years, and 34.9% of patients were male. Among 255 patients with AAV, 138 were classified as having MPA, 52 as having EGPA, and 62 as having GPA. ANCA positivity was reported in 80% of patients with AAV. The medial BVAS and FFS were 12.0 and 1.0. The most common clinical manifestations were renal and pulmonary conditions (60.8% for each). ILD occurred in 53 patients with AAV (20.8%) during a median follow-up period based on ILD of 0 months (an interquartile range of 4.1). Among 53 patients with AAV and ILD, ILD was found at AAV diagnosis in 27 patients (50.9%) and after AAV diagnosis in 26 patients. Furthermore, UIP was identified in 29 patients with AAV and ILD (54.7%). In addition, 32 patients with AAV (12.5%) died owing to any cause during a median follow-up period based on all-cause mortality of 37.2 months (49.1%). The median follow-up period from ILD diagnosis to mortality was 40.5 months.P < 0.001 and P = 0.005); however, there was no significant difference between patients with EGPA and those with GPA. Patients with MPA tended to have higher frequencies of the UIP pattern than those with EGPA and GPA, but the difference was not statistically significant (ILD was identified in 42 patients with MPA (30.4%), 3 patients with EGPA (5.8%), and 8 with GPA (12.3%). Furthermore, the UIP pattern was confirmed in 27 of 42 patients with MPA and ILD (64.3%) and in 2 of 8 GPA patients with ILD (25.0%). However, no patients with EGPA and ILD turned out to have the UIP pattern . Patientnificant .P < 0.001). Conversely, the frequency of ILD was significantly higher in patients with AAV not having PR3-ANCA (or C-ANCA) than in those having PR3-ANCA (or C-ANCA) . However, the frequencies of the UIP patterns did not differ significantly between the two groups based on ANCA type (The frequency of ILD was significantly higher in patients with AAV having MPO-ANCA (or P-ANCA) than in those not having MPO-ANCA (or P-ANCA) . However, 53 AAV patients with ILD showed no difference in the cumulative patients' survival rates between those with and without the UIP pattern , among whom 49.1% developed ILD after AAV diagnosis. Second, among the AAV subtypes, ILD occurred more frequently in patients with MPA than in those with EGPA and GPA. Furthermore, among ILD patterns, the UIP pattern tended to be observed more often in patients with MPA than in those with other subtypes; however, the difference was not statistically significant. Third, patients with AAV having MPO-ANCA (or P-ANCA) showed significantly higher frequencies of ILD compared to those not having MPO-ANCA (or P-ANCA). Conversely, patients with AAV having PR3-ANCA (or C-ANCA) less frequently had ILD compared to those not having PR3-ANCA (or C-ANCA). Finally, during the follow-up period, patients with AAV and ILD exhibited a significantly lower cumulative patients' survival rate than those without ILD but were not applicable to the UIP pattern.In the present study, the frequency of ILD in patients with AAV was 20.8% , which wPrevious studies showed that the UIP pattern is more likely to be associated with all-cause mortality than the non-UIP pattern in patients with AAV and ILD. A meta-analysis reported that patients with AAV and the UIP pattern had a higher risk of poor prognosis, including all-cause mortality (RR 4.36) . HoweverWe propose explanations for this discrepancy. The first is the short follow-up period. Although approximately half of the patients developed ILD after AAV diagnosis, the median follow-up period based on ILD was 0 months, which supports this hypothesis. Future studies are needed to assess the difference in the survival rates between patients with and without the UIP pattern in the longer follow-up periods.P = 0.058 and P = 0.056, respectively). Among 32 deceased patients, 20 of 138 patients with MPA (14.5%), 1 of 52 patients with EGPA, and 11 of 65 patients with GPA (16.9%) died. A comparison of patients with MPA and GPA showed that although the proportion of the non-UIP pattern was higher in patients with GPA, the overall mortality was comparable to that of MPA.The second explanation is the relationship between ILD patterns and AAV subtypes. Patients with MPA tended to present with the UIP pattern more frequently than patients with EGPA or GPA  lung infiltrate , 2. In rWhen only 26 patients with AAV, who had ILD after AAV diagnosis , were considered as AAV patients with ILD, the significant differences in the incidence of ILD between MPA and EGPA patients and that between MPA and GPA patients, which were observed in On the other hand, the cumulative patients' survival rates according to the presence of ILD or UIP patterns in 26 patients who had ILD after the diagnosis of AAV were compared between the two groups. Patients with ILD tended to exhibit a lower cumulative patient's survival rate than those without ILD; however, no statistical significance was observed, whereas there was no significant difference between patients with UIP and those with non-UIP (Supplementary Fig. The strength of this study is that it is the first to investigate the frequency of ILD and the effects of ILD and its patterns on all-cause mortality in patients with AAV in Korea. This study also had some limitations. First, we could not determine the exact time of ILD occurrence in patients with AAV who had ILD at AAV diagnosis. Patients with AAV who might have had ILD before AAV diagnosis were considered to have ILD at AAV diagnosis. As this may alter the follow-up duration from ILD diagnosis to all-cause mortality, it may have influenced mortality outcomes according to the UIP or non-UIP patterns. Only 45 of 53 AAV patients with ILD underwent PFTs, which may have confounded the analysis of the correlation between PFT results and the mortality rate. Additional limitations include the small number of patients with AAV due to the single centre and retrospective study. A future prospective study with a larger number of patients and serious HRCT and PFT results will provide more dynamic and reliable data on the clinical implications of ILD on the prognosis of patients with AAV.ILD occurred in one-fifth of Korean patients with AAV in this study and was associated with MPA and MPO-ANCA vasculitis. In addition, ILD significantly increased the rate of all-cause mortality in these patients with AAV. Therefore, we suggest the need for more attention and more frequent regular visit for patients with AAV and ILD regardless of the time of ILD occurrence.


In vitro, it displayed similar growth kinetics as the parental virus and keeps the luciferase activity and genetic stability after eight rounds of serial passages. The concept-proof test confirmed that RvJX-Nsp2325-HiBiT can be easily used to evaluate the efficacy of antiviral reagents by detecting the reduction of luciferase activity, showing a consistent trend with infectious titers, as well as to set a novel convenient virus neutralization assay based on the intensity of luciferase activity. Last, the viral proliferation, virulence, validity, and HiBiT stability were further investigated in pig inoculation study, showing that the luciferase activity can be directly detected in the tissue samples or indirectly from the MARC-145 cells inoculated with sera from RvJX-Nsp2325-HiBiT-inoculated pigs. Taken together, the results indicate that the HiBiT-tagged virus is a convenient and stable tool for evaluating viral propagation both in vitro and in vivo, which can provide a high-efficient platform for screening and evaluating anti-PRRSV therapies.Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for the global pork industry, characterized for its genetic variation and unsatisfied heterological protection from vaccines. A high-throughput screening platform for developing anti-PRRSV therapies is urgently needed. Here, an 11-amino-acid subunit HiBiT derived from NanoLuc luciferase was inserted into the PRRSV genome at four loci of the Nsp2 coding region or as an additional TRS2 driven open reading frame (ORF) between the ORF7 and 3′-untranscribed region (3′-UTR), respectively, and five recombinant viruses with luciferase activity were successfully rescued. The virological characteristics of the representative virus RvJX-Nsp2IMPORTANCE Luciferase reporter tagged virus is crucial to viral quantification in the study of viral replication, pathogenesis and exploring antiviral reagents. It is urgently needed for PRRSV academia to construct a stable, fast, and high-throughput reporting system, which can be used both in vitro and in vivo. Here, an 11-amino-acid luciferase subunit was successfully inserted into the PRRSV genome; the feasibility, genetic stability, and efficiency for viral quantification both in vitro and in vivo were characterized; and the results demonstrated it has greatly improved the convenience and efficiency for screening the anti-PRRSV reagents. Furthermore, a novel luciferase-based virus neutralization assay was successfully set, which can eliminate the step of sample gradient dilution and greatly improve the convenience and throughput of neutralizing antibody testing. Predictably, it will greatly facilitate the screening and evaluating anti-PRRSV therapies, as well as the mechanistic study of its replication and pathogenesis in the future. Porartervirus of the family Arteriviridae within the order Nidovirales (Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in breeding sows and respiratory distress in pigs of all ages, initially emerged in North America and Western Europe in the late 1980s . In the ovirales  Based onovirales , 6. As aovirales . Since mThe PRRSV genome is approximately 15 kb in length and contains at least 12 identified open reading frames (ORFs) with the 5′-cap and 3′-polyadenylated tail , 11. Twoin vivo, and to screen the antiviral reagents  and reverse genetic operation were employed to rescue several recombinant viruses with inserted NanoBiT fragments , 26. Nanin vivo and can be used to monitor viral dynamics in serum and tissue. Collectively, our findings revealed that the split-luciferase reporter system is a practically helpful tool for evaluating viral propagation both in vitro and in vivo that can provide a highly efficient platform for screening and evaluating anti-PRRSV therapies.In this study, the fragment of the HiBiT subunit was inserted into the PRRSV genome at different loci with two expression strategies to generate luciferase-tagged recombinant viruses. The results showed that the rescued recombinant virus displayed similar growth kinetics as the parental virus, and the infectious titers and luciferase of the recombinant viruses were stable after eight rounds of serial passages in cells. The convenience of this reporting system for screening antiviral reagents was also confirmed by testing the antiviral effect of four nucleoside analogs, because the decrease of luciferase activity was correlated with reduced infectious titers of recombinant virus, and a novel luciferase-based PRRSV neutralization assay was also developed, with higher throughput and a more convenient process compared with the conventional one. Furthermore, an animal inoculation experiment was also set to indicate that the recombinant virus was stable 12-HiBiT, pWSK-JXwn-Nsp2325-HiBiT, pWSK-JXwn-Nsp2338-HiBiT, and pWSK-JXwn-Nsp2419-HiBiT, respectively. In another full-length plasmid, the HiBiT fragment was inserted into the region between the ORF7 and the 3′-UTR, combined with a transcription regulatory sequence (TRS) of PRRSV ORF2, named pWSK-JXwn-ORF7-HiBiT . Among them, the recombinant virus RvJX-Nsp2325-HiBiT had the highest titer  is genetically stable during eight serial passages in MARC-145 cells.To investigate whether there is a correlation between infectious titers and luciferase expression, we simultaneously measured the luciferase activity of the samples used to determine the growth kinetics above. The data indicated that this recombinant virus RvJX-Nsp2325-HiBiT and treated with various concentrations of remdesivir, sofosbuvir, β-d-N4-hydroxycytidine (NHC), and ribavirin, respectively, which have been previously reported to show different levels of antiviral activity against other viruses  have the risk of reversion to virulence , the ant viruses –31. At 3To compare the efficiency of the screening system based on luciferase activity or traditional viral titer testing by IFA, the throughput and time consumed for each process were recorded and calculated. As 325-HiBiT and its parental virus RvJXwn was confirmed to be similar, shown in 325-HiBiT that the standard positive serum cannot completely neutralize , the luciferase activity value of virus neutralized with serum at different dilutions was detected, and the results show that the luciferase signal of standard serum gradually increases with the gradient dilution, and a simple linear regression can be fitted with R2 = 0.9248  around 5 to 13 dpi and had a peak temperature of nearly 42°C. Nevertheless, the rectal temperature of several pigs in the RvJX-Nsp2325-HiBiT-inoculated group rose 1 or 2 days earlier than that in the JXwn06-inoculated group, but they dropped down faster after 13 dpi . The typical HP-PRRS-related lung lesions including severe interstitial pneumonia with extensive and marked pulmonary edema, hemorrhage, and consolidation can be observed in both inoculated groups , but theted pigs . The lun325-HiBiT can grow as well as its parental virus JXwn06 at the first week postinoculation, but viral titers of HiBiT-tagged virus were lower than that of JXwn06 during the second and third weeks postinoculation, indicating it was earlier to be cleared by the host, which corresponded to the changes of ADG and rectal temperatures during that period.The viremia of inoculated animals was examined by using a microtitration infectivity assay. The data showed that RvJX-Nsp2in vivo, the collected serum and tissue samples of the inoculated pigs were submitted for luciferase activity assay. Two strategies were designed in this study: one was to directly measure the luciferase activity of the serum samples, and another one was to inoculate MARC-145 cells with collected serum samples and test luciferase activity at 36 hpi , and inguinal lymph nodes (ILNs)  , suggestCurrently, the vaccine is widely used on pig farms for PRRS prevention and control; however, the genetic variation of PRRSV limits the vaccine efficiency of cross-protection against heterological field strains. Meanwhile, the risk of reversion to virulence from its modified live vaccine raises some concerns about its utilization, so scientists and veterinarians are trying to find out some new strategies to control this disease, especially caused by variated strains. Some reagents that are able to nonspecifically inhibit the PRRSV proliferation, such as the macrolide antibiotic tylvalosin tartrate, have been practically used for treating herds with PRRS outbreak . However325-HiBiT and RvJX-ORF7-HiBiT were as high as that of RvJXwn. However, the luciferase activity of RvJX-ORF7-HiBiT was a little lower than the other four strains, which is speculated to relate with the lower transcription level of this additional ORF or some individuals of the quasispecies lost this nonessential ORF during proliferation, the mechanism is unclear but worthy of study in the future. As a result, the recombinant strain RvJX-Nsp2325-HiBiT was selected as the representative one for the rest of the study.Reporter genes, such as fluorescent and luciferase tags, have been used to visualize the intracellular distribution of viral protein, monitor infection events, or quantitatively detect viral protein expression or virus proliferation. The key point of successfully constructing the recombinant virus with reporter genes is to choose a suitable loci for reporter gene insertion or replacing viral fragments. To increase the possibility of successfully rescuing the recombinant virus and screening out the virus with the highest expression level of the reporter gene, five loci based on two construct strategies were set to insert the HiBiT fragment into the PRRSV genome. Among the nonstructural proteins of PRRSV, Nsp2 is viable and highly heterogeneous, suggesting that this region is tolerant for inserting foreign genes –35. Howe–325-HiBiT indicated that the HiBiT gene is genetically stable in vitro, and this has been further confirmed in the inoculation experiment, as the environment in vivo can provide more complicated, restricted selection pressure for the recombinant virus, including the interaction with the host immune system. Interestingly, the original sequence of aa 325 to 335 region in Nsp2 is conserved among highly pathogenic PRRSV (HP-PRRSV) strains such as JXA1 and HuN4, and there were no deletions nor mutations found in this region while JXwn06 was serial passaged to P80 in MARC-145 cells. The data demonstrated that the NanoBiT luciferase system is applicable to PRRSV, as the HiBiT recombinants are genetically stable and exhibit similar replication characteristics with its parental backbone virus both in vitro and in vivo.Stability is another prerequisite for a good viral reporter system. As mentioned above, a recombinant PRRSV carrying GFP fused with the Nsp2 protein lost the green fluorescent protein after only seven serial passages in cells, due to the accumulation of mutants or the deletions. The insertion of a large fragment of the reporter gene into a viral genome presents the risk of impairment of viral growth or genetic stability. The split-luciferase NanoBiT provides a good solution, which only needs to insert an 11-amino-acid coding region into the viral genome. The serial passaging data of RvJX-Nsp2It is noticeable that the luciferase signal of recombinant PRRSV was hard to detect in the culture supernatant before cell fragmentation or directly from the serum samples of inoculated pigs. They might be reasonable, as the reporter protein is fusion expressed with viral nonstructural protein; however, PRRSV Nsp2 has been identified as a virion-associated structural protein and exits in or on viral particles in the previous study , 37, so in vitro, but the sofosbuvir did not. Taking the advantage of reduced time-consumption and increased throughput to monitor the HiBiT-tagged PRRSV proliferation, this system will be a convenient tool for screening antiviral reagents.To evaluate the feasibility and efficiency of this HiBiT recombinant PRRSV as an unbiased high-throughput screening platform for anti-PRRSV reagents, a proof-of-concept study was performed by using four potential antiviral reagents: remdesivir, sofosbuvir, NHC, and ribavirin. Both luciferase activities and viral titers of inoculated cells were determined at 36 h postinfection, with the treatment of these reagents, and the changing trend of luciferase activity was consistent with viral titer in all treated groups, which indicated that remdesivir, NHC, and ribavirin show the various level of anti-PRRSV activity For many viruses, the NAbs are a key part of humoral immunity against viral infection, but their role in controlling PRRSV is controversial. In a primary infection, PRRSV-specific NAbs are usually detected after the viremia is already resolved. On the other hand, the passive transferred homologous NAbs were shown to prevent reproductive disease and vertical transmission to offspring , and higin vitro; to evaluate the viral proliferation, virulence, and the validity and stability of HiBiT expression in vivo, the animal inoculation test was carried out in this study. The results indicate that RvJX-Nsp2325-HiBiT-inoculated pigs showed no mortality, lighter clinical signs, shorter duration of viremia, and high fever compared with the JXwn06-inoculated group, suggesting that the replacement of an 11-amino-acid subunit in Nsp2 (aa 325 to 335) impaired PRRSV fatal virulence in pigs. Interestedly, the viral titer of both RvJX-Nsp2325-HiBiT and JXwn06 were similar during the first week postinoculation, indicating that RvJX-Nsp2325-HiBiT retained its proliferation well in vivo, showing consistency in that it displayed indistinguishable growth kinetics from the parental virus RvJXwn in both MARC-145 cells and PAMs. However, the viremia of RvJX-Nsp2325-HiBiT can be cleared more quickly by the host after the second week postinoculation, which might be due to inducing a different immune response compared with its parental strain, JXwn06. Fortunately, the luciferase activity can be directly detected from tissues or after inoculating MARC-145 cells, indicating that the HiBiT gene retains its genetic stability in the recombinant PRRSV, even during infection in vivo. Just as in early stages of inoculation in vitro, no luciferase signal can be detected from the supernatant before freeze-thawing. We prefer that the amount of HiBiT-tagged Nsp2 “leaked” from infected target cells into the serum is too low to detect, compared with a higher concentration in tissue homogenate. Even though the luciferase activity cannot be directly detected from serum, the titer of viremia can still be obtained more easily through TCID50 assay by detecting the increased luciferase activity to indicate the PRRSV infection compared with observing the IFA signal or CPE in the traditional assay. Summarily, the HiBiT-tagged virus is still a convenient tool for investigating viremia dynamics and tissue tropism in infected animals.In previous studies, most recombinant PRRSVs with reporter genes were only studied in vitro and in vivo were characterized by using one represent strain. The results demonstrated its advantages in screening antiviral reagents and NAbs. Furthermore, it will greatly facilitate the mechanistic study of PRRSV replication and pathogenesis in the future.To conclude, serial recombinant PRRSVs harboring a split-luciferase gene HiBiT were constructed and rescued in this study, and the feasibility, genetic stability, and efficiency for viral quantification both The animal experiments in this study were carried out according to the Chinese Regulations of Laboratory Animals: The Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China) and Laboratory Animal Requirements of Environment and Housing Facilities . All protocols for primary PAMs preparation and animal inoculation were approved by the Laboratory Animal Ethical Committee of China Agricultural University under approval No. AW81801202-2-1.2 atmosphere. PAMs, the target cells of PRRSV, prepared from 1-month-old SPF pigs were maintained in RPMI 1640 (Gibco) medium, containing 10% FBS and penicillin (50 U/mL) and streptomycin (50 μg/mL) as previously described (The MA104-derived monkey kidney cell line MARC-145 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and penicillin (50 U/mL) and streptomycin (50 μg/mL) at 37°C under a humid 5% COescribed . The fulescribed .To construct a luciferase-tagged recombinant virus, two strategies were designed to insert the high-affinity NanoBiT tag  into the PRRSV genome as the fusion expressed protein or an individual ORF. According to previous reports on PRRSV genomic modification and our predictive analysis, four loci in the Nsp2 coding region at aa 12 to 22, aa 325 to 335, aa 338 to 348, and aa 419 to 429 were initially selected to replace the nucleotide with NanoBiT tag by overlapping fusion PCR  (34). In2 atmosphere, the cells and supernatant were collected and freeze-thawed once; then the supernatant was harvested and serially passaged in MARC-145 cells for three passages. The rescued viruses were examined by indirect immunofluorescence assay (IFA) using the PRRSV anti-N monoclonal antibody (McAb) SDOW17 . To further verify the correctness of the HiBiT gene, the RNAs of third-passage recombinant viruses were extracted and subjected to reverse transcription (RT)-PCR, followed by sequencing. The rescued viruses were named RvJX-Nsp212-HiBiT, RvJX-Nsp2325-HiBiT, RvJX-Nsp2338-HiBiT, RvJX-Nsp2419-HiBiT, and RvJX-ORF7-HiBiT, respectively.To rescue the recombinant viruses, the full-length plasmids were respectively transfected into MARC-145 cells by using Lipofectamine LTX reagent (Thermo Fisher), according to the manufacturer’s instructions. After 4 to 5 days of incubation at 37°C under a humid 5% CO50/mL), according to the Reed-Muench method. The kinetics were analyzed based on the data from three independently repeated experiments.MARC-145 cells and primary PAMs were infected with recombinant viruses at the same multiplicity of infection (MOI) of 0.1. After 1.5 h of incubation at 37°C, unbound viruses were washed off with PBS, and then the cell cultures were supplemented with maintenance medium (DMEM for MARC-145 or RPMI 1640 for PAMs) containing 2% FBS. The viruses were cultured until 72 h postinoculation (hpi), and at 12-h intervals, the inoculated cells with their supernatant were collected for titration on MARC-145 cells by using the endpoint dilution assay, recorded as 50% tissue culture infective dose per mL  according to the manufacturer’s instructions. Briefly, the Nano-Glo HiBiT lytic reagent is reconstituted by adding LgBiT protein (1:100) and substrate (1:50) to a detergent-containing buffer and mixed with collected cell supernatant. The luminescence generated is detected by the luminometer after a 10-min reaction.325-HiBiT was serially passaged in MARC-145 cells to the eighth passages. The luciferase activity and virus titer of each passage were tested as above, and the HiBiT gene, together with its flanking sequences, was applied by RT-PCR and submitted for sequencing.To test the stability of recombinant virus with HiBiT tag in cells, the recombinant virus RvJX-Nsp250) was calculated by using the software GraphPad Prism 8.0.The cytotoxicity of remdesivir, sofosbuvir, NHC, and ribavirin was assessed by 3--2,5-diphenyl-2H-tetrazolium bromide (MTT). Briefly, MARC-145 cells were treated with each reagent at a range of concentrations (6.25 to 200 μM) and incubated for 48 h at 37°C. Dimethyl sulfoxide (DMSO) was set as the negative control. Cell viability was tested using an MTT cell proliferation and cytotoxic assay kit  following the manufacturer’s instructions. The 50% cytotoxic concentration  in the medium with remdesivir, sofosbuvir, NHC, and ribavirin at a range of concentrations , respectively. After 1.5 h of incubation, the supernatant was removed, followed by three washes with PBS; then the medium with the same concentration of reagents as in the initial incubation was added. At 36 h postinfection, the luciferase activities and viral titers of inoculated viruses were determined by using Nano-Glo HiBiT lytic detection system and endpoint dilution assay, respectively. The average time consumed for each step was recorded for further comparison with the traditional method based on viral titration.325-HiBiT or RvJXwn at the titer of 2 × 103 TCID50/mL. After 1 h of incubation at 37°C, the mixture of virus and serum was transferred to a 96-well cell culture plate (100 μL/well) seeded with monolayered MARC-145 cells. After 1 h inoculation at 37°C, the cells were washed triple times with PBS and then added 100 μL 2% FBS DMEM to each well. At 24 hpi, PRRSV-positive wells were detected by IFA or Nano-Glo HiBiT lytic detection system recorded as RLU. The NAbs titer against RvJX-Nsp2325-HiBiT or RvJXwn were calculated using the Reed-Muench method.Serum samples with a high level of neutralizing antibody against PRRSV were prepared by repeatedly inoculating the pigs with HP-PRRSV strain JXwn06 in our previous study . They we5 was set as the standard positive serum. The lowest amount of PRRSV that the serum cannot neutralize was first determined by reacting with the RvJX-Nsp2325-HiBiT, with 2-fold serially dilution from 210 to 20 TCID50/mL. The lowest titer of RvJX-Nsp2325-HiBiT that the standard positive serum cannot completely neutralize was further used for reacting with the 2-fold serially diluted standard positive serum. At 24 hpi, the neutralization effect was determined by testing the luciferase activity of each well, treated with serum at different concentrations. Then the standard curve of NAbs was built based on the value of luciferase activity and serum dilutions. And the R2 value was calculated to evaluate the quality of the standard curve. To confirm the accuracy of this method, the NAbs titers of four serum samples from PRRSV-infected pigs and one negative serum were parallelly detected by traditional virus neutralization test with serial dilution and IFA, as well as by RLU and the standard curve built with standard positive serum.One stocked PRRSV-specific pig serum with the NAbs titer of 1:2in vivo, the animal inoculation study was further performed. Fifteen 4-week-old SPF pigs, obtained from the Beijing Center for SPF Swine Breeding and Management, were confirmed to be negative for PRRSV, pseudorabies virus (PRV), classic swine fever virus (CSFV), circovirus type 2 (PCV2), and Mycoplasma hyopneumoniae infections. The pigs were randomly divided into three groups (n = 5), and each group was separately raised in the isolated rooms in the animal facility of China Agricultural University (CAU). The pig in each group was intranasally inoculated with 2 × 106 TCID50 virus (RvJX-Nsp2325-HiBiT or JXwn06) or 2 mL supernatant of MARC-145 cells as the negative control, respectively. The clinical symptoms, including respiratory disease scores and rectal temperature, were recorded daily, and the average daily weight gain (ADG) was calculated by weighting the pigs weekly.To evaluate the pathogenicity of the recombinant virus, together with the availability and stability of the HiBiT tag Necropsy, gross and microscopic pathological evaluation, and immunohistochemistry (IHC) examinations were carried out once the pigs died during the experiment, and all of the surviving pigs were euthanized and necropsied on 28 dpi . Lung tiThe blood samples were collected at 0, 1, 3, 5, 7, 14, 21, and 28 days postinoculation (dpi) to determine the viral loads in the sera of the infected animals by using a microtitration infectivity assay on MARC-145 cells. In parallel, the luciferase activity of the virus in the collected sera was measured by directly adding Nano-Glo HiBiT lytic reagent into serum samples or inoculating the MARC-145 cells with the sera for 36 h before testing the supernatant with Nano-Glo HiBiT lytic reagent. Furthermore, the tissue samples, including lung, MLNs, ILNs, liver, spleen, and kidney, were also collected after the pig died during the experiment or euthanized at the end of 28 dpi. The collected tissue samples were initially homogenized by a grinding mill with DMEM (with the ratio of 500 mg:1.5 mL), followed by freeze-thawing and centrifugation at 12,000 rpm/min for 15 min at 4°C. Then the supernatant was measured by luciferase assay as above.t test, and the asterisks indicate the statistical significance. p<0.05 was considered statistically significant, and p<0.01 or p<0.001 means extremely significant. Error bars indicate means ± standard deviation (SD).All statistical analysis in this study was performed by GraphPad Prism (version 8.0) software. Statistical significance was evaluated by two-tailed unpaired Student’s


Social isolation has been recommended as a strategy for reducing COVID-19 risk, but it may have unintended consequences for mental well-being. We explored the relationship between social isolation and symptoms of depression and anxiety in older adults during the first wave of the COVID-19 pandemic and assessed the role of cardiometabolic diseases (CMDs) in this association. Between May and September 2020, 1,190 older adults from the Swedish National Study on Aging and Care in Kungsholmen were surveyed about their behaviors and health consequences during the first wave of the COVID-19 pandemic. In total, 913 (76.7%) participants reported socially isolating at home to avoid infection during this period. Social isolation was associated with a greater likelihood of reduced mental well-being  . In joint exposure analysis, there was a significant likelihood of reduced mental well-being only among people who were socially isolating and had CMDs  . In conclusion, social isolation as a COVID-19 prevention strategy was related to reduced mental well-being in an urban sample of Swedish older adults, especially among individuals with CMDs. In early 2020, a highly infectious novel coronavirus (SARS-CoV2) began to spread across the world, leading the World Health Organization to declare COVID-19, the respiratory disease caused by the virus, an international public health emergency on January 30 and a global pandemic on March 11 .In an effort to curb the virus’ spread and reduce COVID-19 mortality, many countries implemented strict measures, including lockdowns and stay-at-home orders. In contrast, Sweden’s pandemic response emphasized voluntary adherence to recommendations from the Public Health Agency, including avoiding contact with others if one showed signs of COVID-19 symptoms, maintaining hand hygiene, and practicing social distancing . AdditioThough it is undoubtably effective for reducing the spread of infection, social isolation – that is, staying at home and minimizing in-person contact with others – may have unintended negative consequences. Before the COVID-19 pandemic, social isolation has been identified as a risk factor for poorer mental health , 7. ConsDespite increased interest in the impact of pandemic-related social isolation, no studies to our knowledge have explored this issue in relation to the growing population of older adults with cardiometabolic disease. On one hand, people with cardiometabolic diseases (CMDs) stand to benefit most from social isolation from a COVID-19 prevention perspective, given the higher COVID-19 fatality rates associated with T2D and CVD . On the Pandemics are predicted to become more intense and more frequent with continued globalization and expansion. To this end, a recent investigation estimated that the probability of a COVID-19-scale pandemic is as high as 2% in any given year . For theIn this study, using data from the Swedish National Study on Aging and Care in Kungsholmen (SNAC-K), we aimed to assess the association of social isolation with depression and anxiety during the COVID-19 pandemic, and to examine the role of CMDs in this association. We hypothesized that social isolation would have an adverse impact on mental health and that these associations may be modified by the presence of CMDs.Of 1,190 study participants , 913 (76.7%) had been socially isolating during the first wave of the COVID-19 pandemic. Compared to those who did not isolate, socially isolating participants were more likely to be older, have a lower education level, and have CMDs, but were less likely to smoke or drink heavily .Participants who had been socially isolating showed a higher likelihood of reduced mental well-being (multi- adjusted OR [95% CI]: 1.74 [1.15 – 2.65]) compared to those who were not isolating, including a higher likelihood of both depressive (2.08 [1.10 – 3.94]) and anxiety (1.82 [1.16 – 2.84]) symptoms .The presence of CMDs was not significantly associated with reduced mental well-being  compared to those without CMDs. However, in joint exposure analysis, compared to CMD-free participants who were not socially isolating, participants who were socially isolating and had CMDs had over twice the likelihood of reduced mental well-being . Similarly, there was a significantly increased likelihood (OR [95% CI]) of depressive (3.34 [1.50 – 7.45]) and anxiety (2.05 [1.13 – 3.70]) symptoms only among participants who both had CMDs and were socially isolating . There wAfter stratifying by gender, the association between self-isolation and reduced mental well-being  was statistically significant among women . Moreover, in joint exposure analysis, the OR (95% CI) of reduced mental well-being was 2.26 (1.16-4.42) among women who were isolating and had CMDs compared to those who were CMD-free and not isolating. Among men, these associations had a similar effect size, but were not statistically significant . There wIn this population-based cohort study of older adults in Stockholm during the first wave of the COVID-19 pandemic, we found that: 1) social isolation was associated with higher levels of depression and anxiety and 2) reduced mental well-being was greatest among participants who were socially isolating and had CMDs.Social isolation is a widely recognized risk factor for depression and anxiety and its effect on mental health may be particularly pronounced among older adults , 7. The Consistent with these reports, we found that social isolation was related to reduced mental well-being in the form of feelings of both depression and anxiety in an urban population of Swedish older adults. It is notable that these adverse effects of social isolation were apparent even in the context of Sweden’s comparatively relaxed COVID-19 prevention measures, where people who isolated themselves at home did so by individual choice and not because of an external mandate. Additional studies are needed to better understand the long-term impacts of COVID-19-related social isolation beyond the pandemic’s first wave.Our study takes the further step of examining the impact of cardiometabolic disease on the association between social isolation and mental well-being. Older adults with CMDs like T2D and CVD have far greater risk of COVID-19 fatality  and wereThe health consequences of social isolation for people with CMDs may be even more severe if social isolation itself impacts individuals’ ability to self-manage CMDs. Social isolation has been identified as a risk factor for coronary heart disease and stroke . FurtherA previous investigation indicated that loneliness, anxiety, and insomnia during the pandemic were particularly pronounced among women aged ≥60 years . ConsistIt is possible that the association between social isolation and reduced mental well-being was impacted by acute or time-limited anxiety deriving directly from the pandemic situation. We addressed this in sensitivity analyses stratified by participants’ self-reported levels of worry that they themselves  or a memStrengths of this study include the use of a study sample from a well-characterized population-based study, SNAC-K, and the use of a questionnaire developed by a multidisciplinary team of experts. However, some limitations should be acknowledged. First, these findings are based on self-reported information and therefore may be affected by recall bias. Second, given the pandemic restrictions, participants’ mental well-being was measured in terms of self-reported feelings of anxiety and depression via a telephone interview, rather than a formal diagnosis of anxiety or depression based on a clinician’s visit. Third, in this study, data on mental well-being was not collected in the same way before and after the onset of the COVID-19 pandemic, thus we could only examine the cross-sectional relationship between social isolation and feelings of depression and anxiety. However, considering the possible influence of pre-pandemic mental health on mental well-being during the first wave of COVID-19, we included participants’ MADRS scores from the most recent regular SNAC-K follow-up visit (conducted from 2016 to 2019). Further population-based longitudinal studies are warranted to confirm the social isolation-mental wellbeing association. Additionally, the generalizability of these findings – which reflect an affluent, highly educated, urban population – may be limited, particularly given the voluntary nature of Sweden’s COVID-19 prevention recommendations, which differed substantially from the mandates issued in most other western countries. Finally, we could not rule out the influence of potential residual confounding due to unmeasured factors, like the number of social contacts or the severity of CMDs, which may be related to both mental health and the choice to self-isolate.To the best of our knowledge, this is the first study to examine the combined impact of social isolation and CMDs on mental well-being during the COVID-19 pandemic. Pandemic-related social isolation was associated with depressive and anxiety symptoms in older adults, particularly those with cardiometabolic comorbidities. Our findings highlight the collateral damage of social isolation as a COVID-19 prevention strategy and underscore the need for mental health support for older adults during subsequent waves of the current pandemic. Furthermore, in the unfortunate but highly probable event of a future pandemic on the scale of COVID-19 , the ampThe study population was derived from SNAC-K, an ongoing population-based cohort study that includes a random sample of older adults aged ≥60 years living in central Stockholm, Sweden. Between May and September 2020, individuals who participated in the regular SNAC-K follow-up assessment in 2016-2019 were invited to participate in a structured survey administered over the telephone by trained SNAC-K staff to assess their behaviors and direct and indirect health consequences during the first wave of the COVID-19 pandemic . The telephone questionnaire was developed by the SNAC-K data collection team with input from experts in geriatric medicine, mental health, neurology, and public health. The questions contained some items from the regular SNAC-K assessments (which participants undergo every 3 or 6 years) as well as items from the WHO Europe survey tool . Before The study was approved by the Karolinska Institutet Ethical Committee and the Regional Ethical Review Board in Stockholm, Sweden. All participants provided informed and written consent.The SNAC-K protocol has been described in detail previously . Brieflyth version (ICD-10).Education level was defined as elementary, high school, or university. Smoking status was grouped into never, former, or current smokers. Alcohol consumption was categorized as no/occasional, light-to-moderate (1–14 drinks/week for men or 1–7 drinks/week for women), or heavy (>14 drinks/week for men or >7 drinks/week for women) drinking . Living In the telephone questionnaire, participants were asked to report whether or not they had been staying at home and avoiding in-person social contact to minimize their chances of being infected by the coronavirus. Social isolation status was dichotomized as isolating vs. not isolating.The telephone questionnaire included questions to assess mental well-being since the onset of the COVID-19 pandemic in Sweden in March 2020. Participants’ experience of depression during the pandemic was assessed by the question, “Have you experienced depressive symptoms since March 2020?” Participants responded using a 1 to 6 scale, with 1 indicating a “neutral mood” and 6 indicating “consistent experience of maximum depression.” Participants were coded as having experienced depressive symptoms if they scored 3  or higher. Participants’ experience of anxiety during the pandemic was ascertained by two questions: 1) “Have you experienced feelings of anxiety since March 2020?” ; and “Have you experienced anxiety symptoms since March 2020?” . Participants were coded as having experienced anxiety if they scored 3 or higher on either question. We additionally created a combined mental well-being endpoint defined as reduced mental well-being  or sound mental well-being .CMD status, defined as the presence of T2D and/or CVD , was assCharacteristics of socially isolating vs. not isolating participants were compared using Chi-square tests for categorical variables and t tests for continuous variables. Data were presented as numbers and percentages for categorical variables and as means and standard deviations for continuous variables.Odds ratios and 95% confidence intervals for the association between social isolation status and depression, anxiety, or overall reduced mental well-being were obtained from logistic regression analyses. Models were adjusted for age, gender, education, living status, smoking status, alcohol consumption, and pre-pandemic levels of depressive symptoms (i.e. MADRS score from the most recent regular SNAC-K follow-up visit).We additionally assessed the joint effect of isolation status and CMDs on mental well-being. Participants were categorized into four groups according to combined social isolation (yes vs. no) and CMD (CMD-free vs. any CMD) status. We assessed the additive interaction between social isolation status and CMDs using the attributable proportion due to interaction (AP). We examined the multiplicative interaction between social isolation status and CMDs by adding the cross-product term  into the model. To assess possible gender-specific aspects of the associations between social isolation, CMDs, and mental well-being, we repeated all analyses separately among men and women.Finally, we accounted for potential acute or time-limited anxiety deriving directly from the pandemic situation in sensitivity analyses stratified by participants’ level of worry about being affected by COVID-19 , level of worry about family members being affected by COVID-19 , and feelings of nervousness and stress  since the beginning of the pandemic.P-values were two-sided, and P-values <0.05 were considered statistically significant. Statistical analyses were performed using Stata SE 16.0 .All Supplementary Figure 1Supplementary Tables


Digital health technologies have been used to enhance adherence to TB medication, but the cost-effectiveness remains unclear.We used the real data from the study conducted from April 2014 to December 2020 in Morocco using a smart pillbox with a web-based medication monitoring system, called Medication Event Monitoring Systems (MEMS). Cost-effectiveness was evaluated using a decision analysis model including Markov model for Multi-drug resistant (MDR) TB from the health system perspective. The primary outcome was the incremental cost-effectiveness ratio (ICER) per disability adjusted life-year (DALY) averted. Two-way sensitive analysis was done for the treatment success rate between MEMS and standard of care.The average total per-patient health system costs for treating a new TB patient under MEMS versus standard of care were $398.70 and $155.70, respectively. The MEMS strategy would reduce the number of drug-susceptible TB cases by 0.17 and MDR-TB cases by 0.01 per patient over five years. The ICER of MEMS was $434/DALY averted relative to standard of care, and was most susceptible to the TB treatment success rate of both strategies followed by the managing cost of MEMS.MEMS is considered cost-effective for managing infectious active TB in Morocco. Tuberculosis (TB) is the leading cause of mortality among infectious diseases, accounting for 1.5 million deaths worldwide in 2018 . HoweverRecently, digital health technologies and management have been used to enhance adherence to TB medication. Many studies have reported that digital adherence technology (DAT) substantially improved adherence  and led In Morocco, where TB incidence in 2018 was 99/100,000 nationwide and 130/100,000 in cities like Kenitra . We prevThe study was conducted in Salé, Morocco by the Korean International Cooperation Agency (KOICA) in collaboration with Global Care International and the Ministry of Health and Welfare of Morocco from April 2014 to December 2020. Briefly, the study enrolled and provided patients a smart pillbox with a web-based medication monitoring system, called MEMS, which reminded patients to take medication at certain times . MEMS asMEMS were applied after one to two weeks of routine direct supervision for medication at the treatment site, depending on the circumstances.The system provided regular statistical monitoring results including drug adherence status according to time, region, and patient information to staffs. When patients failed to take their medication, staffs made phone calls or home visits to patients. This original study compared patients who received a MEMS (n = 206) with patients who received standard TB care (n = 141) among new infectious active TB patients in five rural health centers of Sale area. The results of this study showed that MEMS increased TB treatment success rate and decreased the lost to follow-up rate overall significantly (P<0.001).The national TB treatment guidelines of Morocco recommend two months of rifampin, isoniazid, pyrazinamide, and Ethambutol (2RHZE) followed by four months of rifampicin and isoniazid (4RH) for new smear-positive cases , 15.Direct supervision at the treatment site for two months is recommended (modified DOT) before self-administered treatment (SAT). Retreatment regimens include two months of streptomycin, rifampin, isoniazid, pyrazinamide, and Ethambutol (2SRHZE), followed by one month of RHZE and five months of RHE (1RHZE/5RHE). Multi-drug resistant (MDR) TB treatment consists of six months of intensive phase with injectable drugs in addition to levofloxacin, ethionamide, cycloserine, pyrazinamide, and ethambutol, and 18 months of continuation phase without injectable drugs before the WHO TB treatment guidelines were updated in 2018 .We used epidemiology and costing data from the KOICA project to perform a cost-effectiveness analysis from the health system perspective in a hypothetical cohort of infectious TB patients with acid-fast bacilli (AFB) smear-positive results in Morocco. We used a five-health state Markov model to evaluate two strategies for the first line treatment of drug-susceptible TB: 1) standard of care by modified DOT ; or 2) MEMS. Based on the WHO guideline , we inclThe patients lost within the initial two months of treatment were assumed to be recruited again for drug-susceptible treatment. We assumed that drug susceptibility testing did not influence treatment because all treatment regimens were considered standardized medications. We limited our analysis to 5-year time horizons to accommodate the following lengths of treatment: six months for initial treatment regimens, eight months for retreatment regimens, and 24 months for MDR-TB treatment regimens with two cycles. All analyses were performed using TreeAge 2019 R2.0 software .Epidemiological parameters were determined from literature review and real data of the KOICA project . We condHuman resource costs for each activity component were calculated based on the approximate time engaged in each activity during the operations, assessed using a workload survey. For the costs of goods, equipment, and services, we divided direct costs into capital and recurrent costs. Capital costs were annualized over its expected remaining lifetime, and discounted at a 3% annual rate. Costs were collected as Moroccan Dirhams (MAD), and were converted as 2018 US dollars ($US) using the standard inflation adjustment method, at the rate of 9.116 Moroccan Dirhams (MAD) per US$1, based on the average UN exchange reported for 2018.The primary outcome of analysis was the incremental cost-effectiveness ratio (ICER), measured in units of 2018 US$ per disability adjusted life-year (DALY) averted. For DALY calculation, the life table of Morocco and the raw data of the WHO reports were adopted . We alsoThe consent for this study was waved, as it was based on a simulated cohort of patients. This study was approved by the Institution Review Board (IRB) of Mohammed V University (IRB number: Dossier number 48/16)Under the SoC strategy, 0.48 drug-susceptible TB cases and 0.03 MDR-TB cases / per patient (first year) would occur over the 5-year time horizon . The devIn tornado diagram of ten influential factors identified in one-way sensitivity analysis , the ICEWe found that MEMS intervention in Morocco would cost $434/DALY averted for managing drug-susceptible TB patients with AFB smear-positive sputum over 5 years of time horizon, compared to SoC. The MEMS strategy would reduce the number of drug-susceptible TB by 0.17 and MDR-TB by 0.01 per patient over 5 years. The result was most sensitive to the treatment success rate of drug-susceptible TB using the MEMS strategy. In light of the GDP of Morocco , our study results support that MEMS is considered cost-effective in Morocco.The strength of our study is that we included micro-costing and measured the detailed costs of implementing MEMS and the standard care of drug-susceptible TB and MDR-TB, including staff salaries, follow-up visits, and costs for monitoring tests and drugs. The estimated total cost of MDR treatment in our study was lower than that reported from other studies . PotentiThe key factor driving a relatively low ICER for MEMS was the higher success rate of TB treatment with MEMS. There has been limited evidence of MEMS effectiveness on percentage of cure and completion as the indicators for treatment success . In a smIn addition to the TB treatment success rate, the ICER was also sensitive to the managing costs of MEMS during continuation and intensive phases. In another study in Brazil, the unit cost of TB medication monitor was $423, which was slightly higher than the unit cost per person $398.70) in our study [.70 in ouOur study has several limitations. First, MEMS only allows to monitor opening of the pillboxes electronically. Therefore, drug adherence is not guaranteed without virtual confirmation of the medication counts. Second, costs for MDR-TB were assumed based on outpatient clinics only and did not include any costs for in-patient treatment. Third, we did not include bedaquline and delamanid as MDR-TB new drugs. Fourth, actual data were used only for drug-susceptible TB, and we used the data from literature reviews to estimate the costs related to recurring TB and MDR-TB management. Lastly, because the original KOICA study that provided epidemiologic parameter for this analysis had been a non-randomized study, the input data for this cost-effective analysis could not have been fully adjusted, reflecting the difference of patients’ characteristics and behavior between the enrolled MEMS and SoC group. Therefore, considering much higher medical costs and hindrance to actual data gathering for MDR-TB as well as retreated DS-TB, a deeper investigation based on more reliable data is needed.Our study demonstrated the cost-effectiveness of MEMS among infectious active TB patients in Morocco. To inform scale-up of digital health intervention as part of TB control strategy, further cost-effectiveness studies are needed under different national tuberculosis programs and TB burdens.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Resting-state (rs) fMRI has been widely used to examine brain-wide large-scale spatiotemporal architectures, known as resting-state networks (RSNs). Recent studies have focused on the temporally evolving characteristics of RSNs, but it is unclear what temporal characteristics are reflected in the networks. To address this issue, we devised a novel method for voxel-based visualization of spatiotemporal characteristics of rs-fMRI with a time scale of tens of seconds. We first extracted clusters of dominant activity-patterns using a region-of-interest approach and then used these temporal patterns of the clusters to obtain voxel-based activation patterns related to the clusters. We found that activation patterns related to the clusters temporally evolved with a characteristic temporal structure and showed mutual temporal alternations over minutes. The voxel-based representation allowed the decoding of activation patterns of the clusters in rs-fMRI using a meta-analysis of functional activations. The activation patterns of the clusters were correlated with behavioral measures. Taken together, our analysis highlights a novel approach to examine brain activity dynamics during rest. Resting-state functional MRI (rs-fMRI) is a functional neuroimaging technique during which subjects are at rest and not engaged in any behavioral task, and it is thought to monitor intrinsic physiological signals in the brain , in multiple brain areas comprising the network. Although prior studies assessed RSFC in RSNs using the entire duration of rs-fMRI scans (static RSFC), recent studies have examined fluctuations in RSFC for timescales ranging from seconds to a few minutes, so-called time-resolved RSFC , with all data included in the S900 data release. For each subject, resting-state scans (approximately 15 min) were conducted for four functional runs with a repetition time (TR) of 0.72 s and 2-mm isotropic spatial resolution.Resting-state and task-related fMRI data were obtained from the Human Connectome Project (HCP: rs-fMRI data were preprocessed by HCP and cleaned by ICA-based X-noiseifier (FIX) from the FMRIB Software Library (FSL)  was applied  was then defined as the 1occurred . Becauseme point . The curst PC of windowed timecourses for window sizes ranging from 15 to 65 TRs. The size of the sliding window is a critical parameter in time-resolved RSFC analyses, because an excessively short window introduces spurious fluctuations  from derivatives of the six rigid-body realignment parameters were calculated over the scanning run . Voxel-based data were expressed in grayordinates space  the arbitrariness of the sign of PCs, (2) the spatial pattern of clusters, and (3) autocorrelation of BOLD timecourses. Therefore, we named these 6 groups as clusters and each SAP in a cluster as a sub-cluster. For example, Cluster 1 consists of Sub-clusters 1-1 and 1-2, and Cluster 2 consists of Sub-clusters 2-1 and 2-2. Note that we avoided the use of terms such as states and attractors because similar SAPs were obtained in phase-randomized null models (see Discussion for an interpretation of clusters).https://neurosynth.org; Yarkoni et al., To characterize the SAP maps, we decoded SAP maps based on their topographical structure . PMI was calculated as the ratio of two observation probabilities  of TS events of Clusters :To examine the temporal structure of cluster (sub-cluster) occurrences, occurrence probabilities were calculated based on the frequency of occurrence across time series. The probability was calculated for each of the 6 clusters. For each cluster, the probability associated with sub-clusters  was averaged. Then, we examined point-wise mutual information  reaching zero. Then, at each scanning frame, PMIs were calculated for all possible pairs of the 6 clusters.The two probabilities were computed by counting observations for each pair at times t + τ), by using multiple regression analysis. PMIs in the first and last 100 time points were discarded from the analysis. The lag effect was examined for τ > 100 TR to minimize bias derived from autocorrelation of PMIs  time effect reflecting monotonic change in PMI over the scanning period and (2) lag-time effect reflecting PMI change at time t and .Then, relationships between TSP maps in resting state  and taskClusters were examined in relation to the RSFC, which was calculated as pair-wise correlation coefficients based on the full timecourse of the rs-fMRI scan .To characterize TSPs in relation to psychological and behavioral characteristics, we explored cross-subject correlations between TSPs and behavioral measures. The HCP dataset includes behavioral measures [subject measures (SMs)] of individual subjects, including demographic, psychological, and behavioral measures, collected by standard procedures conducted outside the scanner. Detailed descriptions of SMs are available elsewhere  were concatenated, resulting in 6 images each involving 182,564 pixels . The images were subjected to rank-based inverse Gaussian transformation to avoid the influence of potential outlier values . st PC explained the largest fraction of variance of resting-state brain activity within a window. Timecourses of the variance explained by the 1st PC showed large temporal fluctuations . With larger window sizes, the timecourses did not show a temporally consistent pattern or monotonic changes .The large variance explained by the 1s as TSs  red dotsS window . The tim changes , suggest changes . Becauseincrease . Becausest PC  during one functional run . To identify dominant patterns of activity dynamics during rest as observed in prior studies  RSFC and TSP. If TSs involving two RSNs  in its TSP occurred in a scanning run, high temporal correlation between the two RSNs should be found. To confirm this possibility, we performed multiple regression analysis where the strengths of RSFC (13 × 12/2 = 78 combinations) were predicted by the co-occurrence of each cluster, and R-squared values of the regression were calculated. The cluster occurrence explained well the strength of static RSFC among many of the networks including sensory, visual, cingulo-opercular, auditory, ventral attention, and cerebellar networks .2 = 0.3; FPM (b): r2 = 0.2]. These results confirmed that cluster occurrence and SAPs could explain the characteristics of static RSFC.The strength of RSFC was also predicted by SAP. SAP explained well the FC strength among those networks . When thThe clusters extracted above may be a statistical artifact due to sampling variability rather than reflecting distinct brain states . Then, for all combinations of clusters, PMI was calculated for all t and τ , providing a PMI matrix (t (time effect), τ (lag effect) and a constant (zero-lag PMI) (P < 0.05) suggests that these temporal structures of cluster occurrences cannot be fully explained by autocorrelations of rs-fMRI signals.Next, we examined the temporal structure of cluster occurrence probability by calculating temporal point-wise mutual information  . The parlag PMI) . Statistlag PMI) . Note thleft). Clusters 2 and 6 showed strong negative lag effects, indicating that Clusters 6 and 2 alternated in short time intervals. However, Cluster 6 showed greater positive lag effects with other Clusters 1-5, indicating that Cluster 6 and other clusters infrequently occurred especially in a short time interval. Note that small tau effects (τ <100 TRs) and early (first 100 frames) and late (last 100 frames) scanning periods were eliminated from fitting (see Methods). This was because the current analysis focused on cluster co-occurrences in long periods, and cluster co-occurrences in short periods were derived from autocorrelation of temporally filtered rs-fMRI signals.Lag effects showed large negative effects in diagonal components left. Clleft) was derived from the frequent occurrence of Cluster 2 and 6 in the early and later periods, respectively (middle). To confirm this effect further, we performed a simulation analysis in which randomized null data sets preserved the temporal characteristics of cluster occurrence frequency of the real data   analyses between spatiotemporal characteristics during resting state and those during task states or individual characteristics. Classification of clusters (6 in the current study) also involves a certain degree of arbitrariness. Therefore, more precise mental events or states would be identified if the clusters were classified and examined in more detail. Compared to ROI-based approaches , BOLD signals used in the present study likely contain physiological  noise. Such physiological noise may have affected the event-based analysis. Nevertheless, recent studies using simultaneous optical recording of neuronal and hemodynamic signals in mice reported that many transient events of neuronal activity were indeed visible in hemodynamics  within a window, for various window sizes. For window size smaller than 25 TR, TSP time course showed monotonic change within a window, suggesting that resting-brain activity did not show sharp transitions at fast (<25 TR) time scale. In contrast, for window size larger than 35 TR, TSP time course did not show monotonic change, suggesting that the resting-brain activity showed activity change at a time scale shorter than these window sizes. Taken together, while we acknowledge a methodological limitation in sensitive dynamic range, the present data suggest that the current choice of window size was appropriate for capturing dominant FC transitions.There is a possibility that the choice of a particular window size limited the analysis of FC transitions within a limited dynamic range. For example, FC transitions occurring at a shorter time scale (<25 TR) or at a much longer time scale (>25 TR) maybe undetected by the current choice of the window size. Nevertheless, our comparison of different window size  suggestspost-hoc manner provides a helpful way to examine the nature of the resting state.We observed repeated occurrences of TSs, which may reflect physiological functions. The current clustering and voxel-based reconstruction approach are useful techniques to understand the physiological processes underlying rs-fMRI. Indeed, the current study demonstrated that spatiotemporal patterns during TS were related to those during task engagement and the behavioral/psychological characteristics of individual subjects. These results suggest a potential link between individual moments during rs-fMRI and functional processes taking place in that moment. Because it is almost impossible to keep subjects' mental state stable during rs-fMRI, our approach to cluster TS events determined in a https://www.humanconnectome.org/). Further inquiries can be directed to the corresponding authors.The data set analyzed in the study is publicly available from Human Connectome Projects (Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.YN designed the study and performed data analyses. YN, RL, TM, and KJ wrote the manuscript. All authors contributed to the article and approved the submitted version.