S. aureus (SA) infective endocarditis (IE) has a very high mortality, attributed to the age and comorbidities of patients, inadequate or delayed antibiotic treatment, and methicillin resistance, among other causes. The main study objective was to analyze epidemiological and clinical differences between IE by methicillin-resistant versus methicillin-susceptible SA (MRSA vs. MSSA) and to examine prognostic factors for SA endocarditis, including methicillin resistance and vancomycin minimum inhibitory concentration (MIC) values > 1 μg/mL to MRSA.Patients with SA endocarditis were consecutively and prospectively recruited from the Andalusia endocarditis cohort between 1984 and January 2017.We studied 437 patients with SA endocarditis, which was MRSA in 13.5% of cases. A greater likelihood of history of COPD , invasive procedures, or recognized infection focus in the 3 months before IE onset  and of diagnostic delay  was observed in patients with MRSA versus MSSA endocarditis.p = 0.32).The one-year mortality rate due to SA endocarditis was 44.3% and associated with decade of endocarditis onset (1985–1999) ; 2000–2009 ; active neoplasm  and sepsis . Methicillin resistance was not associated with higher IE-related mortality (49.7 vs. 43.1%; MRSA IE is associated with COPD, previous invasive procedure or recognized infection focus, and nosocomial or healthcare-related origin. Methicillin resistance does not appear to be a decisive prognostic factor for SA IE. Staphylococcus aureus (SA) is one of the most frequently involved bacteria [S. aureus (MRSA) versus methicillin-susceptible S. aureus (MSSA) [OR of 1.8]  . BesidesThe objective of this study was to determine differences in epidemiological, clinical, and prognostic variables between MRSA versus MSSA IE and to analyze the prognostic value of vancomycin MIC > 1 μg/mL for MRSA.We conducted a prospective, multicenter, longitudinal study of consecutive patients with IE hospitalized in eight hospitals of the Health Service of Andalusia (Spain) between 1984 and 2017. During this period, these hospitals prospectively enrolled a total of 2076 patients with IE from whom informed verbal consent to study participation was obtained. Ethics committee approved this procedure.S. aureus endocarditis who met eligibility criteria. Information was prospectively gathered by attending physicians on epidemiological, clinical, analytical, and prognostic  data and on medical and surgical treatments. Heart surgery was available at five of the eight hospitals, to which candidates for surgery from the other three hospitals were transferred. Patients were followed up for 12 months after IE, monitoring clinical, analytical, and microbiological results. The EuroSCORE, logistic EUROSCORE, and modified Duke Criteria were calculated for all patients and included as study variables when they became incorporated into clinical practice, with the agreement of the endocarditis study group, as well as data on new antibiotics. There were no changes during the study period in the other study parameters, i.e., clinical, epidemiological, microbiological, analytical findings, duration of antibiotic therapy, performance of surgery, adequate antibiotic treatment (according to contemporary recommendations), or mortality data.Among these patients, we prospectively enrolled in the present cohort the 437 patients with All information was treated in accordance with national legislation on personal data protection , and the study was approved by the ethics committee of the coordinating center .S. aureus IE according to modified Duke Criteria  and the non-IE-related mortality  at 1 year post-discharge.Severe sepsis was defined by ≥2 criteria of systemic inflammatory response syndrome with organ dysfunction; and Septic shock by sepsis with refractory hypotension and end-organ perfusion dysfunction despite adequate fluid resuscitation   , one forWe included 378 patients with MSSA IE and 59 with MRSA IE from 1984 through January 2017 .p = 0.048); nosocomial acquisition ; history of COPD , elevated Charlson index (3 [p = 0.006), history of previous invasive procedure or focus , and more frequent diagnostic delay . In comparison to patients with MRSA IE, higher percentages of those with MSSA IE had congenital heart disease , were in a hemodialysis program , and had infectious perivalvular involvement during IE . The remaining results are listed in Tables According to bivariate analyses, the main epidemiological and clinical differences between IE by MRSA versus MSSA were age  but no significant difference in mortality from IE at 1 year  or in the percentage of patients undergoing surgery . Among the patients with MRSA IE, there was a larger percentage for whom surgery was indicated but not performed  and a higher relapse rate  , previous invasive procedure or recognized focus of infection in the three-month period before IE onset , and a delay of ≥7 days between symptom onset and hospital admission  (Table p = 0.7). The Mortality attributable to MRSA IE in hospital or during the first 30 days post-discharge was 49.1% (29/59) of which Vancomycin AUC:CMI was available in 18 patients. The hospital mortality rate was 61.1% (11/18) in strains with MIC ≤1 μg/mL versus 38.8% (7/18) with MIC> 1 μg/mL, also a non-significant difference (p = 0.847).MIC values were determined by E-test in 37 (62.7%) of MRSA cases and by microdilution in 22 (37.3%). Vancomycin MICs for MRSA were available in 74.6% (44/59) of cases and were > 1 μg/mL in 38.6% (17/44). There were 4 relapses, observing 3 (75%) in strains with MIC > 1 μg/mL and 1 (25%) with MIC ≤1  patients died from SA endocarditis-specific mortality. The mortality rate was 56.9% (37/65) in 1985–1999; 53.8% (78/145) in 2000–2009; and 34.2% (67/196) in 2010–2017. During the one-year follow-up, 13 (2.9%) died from a cause other than IE, 6 (1.4%) died from a new IE episode due to a resistant strain, and 16 (3.7%) were lost to the follow-up. Data were available on the treatment of 378 patients (86.5%), and 342 (90%) of these received adequate initial antibiotic treatment according to the antibiogram and clinical practice guidelines.p = 0.032; 2000–2010: 42.8% (78/182) vs. 36.8% (67/224), p = 0.007; 2010–2017: 36.8% (67/182) vs 57.6% (129/224); p = 0.0001); older age ; active neoplasm ; early prosthetic IE ; mitral valve involvement ; sepsis/septic shock ; kidney failure during IE episode ; de novo heart failure ; CNS involvement ; high surgical risk . MRSA itself did not emerge as a risk factor for mortality in our cohort  Protective factors were: IE on pacemaker lead or automatic implantable cardioverter-defibrillator (AICD) ; osteoarticular spread of the infection ; heart surgery conducted when indicated without delay . According to the multiple logistic regression analysis, poor prognostic factors for SA endocarditis were: decade of endocarditis onset in 1985–1999  or 2000–2009 ; active neoplasm ; and sepsis/shock   vs. survival rate of 12.5% (28/224), S. aureus endocarditis, 13.5% were MRSA, all elderly men with multiple diseases, elevated Charlson index, and high surgical risk. Around half of the patients with MRSA had prior valve disease, which was rheumatic or degenerative in one-third of cases and of nosocomial origin or healthcare-related in the remainder. The IE predominantly involved left-sided native valves  and less frequently cardiac devices, although such cases have become increasingly frequent. The mortality rate was very high, with around half of the patients dying from IE in hospital or within 30 days of their discharge. These data are similar to previous reports associating endocarditis with high morbidity and mortality rates and linking its acquisition to health care in up to 30% of cases [SA etiology, high Charlson index score, and EuroSCORE II ≥ 9 [S. aureus and endocarditis; this review reported contradictory data on the benefits of early surgery, observing that surgery was sometimes delayed for weeks or months beyond the end of antibiotic IE treatment to correct the valve damage responsible for heart failure [In this study of patients with of cases . These tof cases . The maiE II ≥ 9 . In our  failure .The main differences between MRSA and MSSA endocarditis in our study population was the greater frequency of the former in patients with COPD and its association with a longer interval between symptom onset and hospital admission (≥ 7 days in around 60% of cases). One reason may be the higher MRSA colonization rate in patients with COPD due to their repeated contact with the health care system . The exaS. aureus bacteremia do not include cases of endocarditis. It should also be acknowledged that the prognosis of IE is influenced by numerous factors; therefore, the added prognostic value of data on methicillin resistance and vancomycin MIC may be limited. The association of methicillin resistance with higher treatment failure rate in our cohort did not reach statistical significance, possibly due to the low relapse rate after IE treatment with prolonged antimicrobial therapy and frequent removal of the infection focus (heart surgery).Previous invasive procedures and/or infectious foci were also more frequent in the patients with MRSA versus MSSA IE, with 70% of the former being of nosocomial origin. There were no significant differences between MRSA and MSSA IE in surgical intervention or mortality rates or in the receipt of adequate antibiotic therapy. Many authors have associated mortality due to SA with methicillin resistance. A recent meta-analysis of 62 studies of bacteremia  found a higher mortality risk for MRSA versus MSSA, with an OR of 2.65  [Staphylococcus coagulase-negative IE with vancomycin MIC ≥2 μg/mL [The importance of vancomycin susceptibility in methicillin-resistant and even methicillin-susceptible strains is controversial. After initial studies described worse outcomes for methicillin-resistant strains with high vancomycin MIC values , 30, var≥2 μg/mL  and even≥2 μg/mL  and bact≥2 μg/mL , 34. In ≥2 μg/mL . Likewis≥2 μg/mL .S. aureus endocarditis was very high in our cohort, despite the receipt of antibiotic treatment that accorded with antibiogram results and was recommended in available clinical practice guidelines by 90% of the patients. Active neoplasm, sepsis/shock, and decade of endocarditis onset (1985–1999 or 2000–2009 vs. 2010–2017) emerged as poor prognostic factors, but early surgery (within first 2 weeks) did not appear to influence the prognosis. A recent multicenter, longitudinal, observational study of SA IE (n = 213 cases) reported a mortality rate of 37% and identified a high Charlson index, congestive heart failure, CNS involvement, and sepsis/septic shock as risk factors [The mortality rate of  factors .p = 0.001) but not in cases of prosthetic IE  [S. aureus IE on prosthetic valve when the surgery was performed during the first 60 days of hospitalization rather than later . The authors therefore recommended that surgery be considered on a case-by-case basis, regardless of whether SA is involved [With regard to the possible beneficial effect of early surgery in SA endocarditis, a meta-analysis reported a lower mortality rate when the surgery was conducted within the first 2 weeks rather than later in cases of native IE  . Anotherinvolved .One study limitation is that our analysis considered data gathered over three decades rather than shorter time periods in order to obtain adequate statistical power. Strengths include the large patient sample and its prospective longitudinal multi-center design, involving specialist hospitals that formed a specific study group for this purpose. The results provide a reliable understanding of the current state of endocarditis in our region and may possibly be extrapolated to other regions of our country.S. aureus endocarditis has a very high mortality rate in our setting. MRSA IE is associated with COPD, previous invasive procedure or recognized infection focus, and nosocomial or healthcare-related origin. Although methicillin resistance does not appear to have a decisive influence on the mortality risk, it may increase the therapeutic failure rate among patients receiving recommended treatments.


Although microbial diversity and richness did not differ between groups, there were significant differences in terms of individual species. In particular, Lactobacillus crispatus was associated with samples from a full-term pregnancy, whereas one community state-type was associated with samples from preterm pregnancies. Furthermore, by predicting gene functions, the functional potential of the preterm microbiota was different from that of full-term equivalent. Taken together, we observed a discrete structural and functional difference in the microbial composition of the vagina in women who deliver preterm. Importance: with an estimated 15 million cases annually, spontaneous preterm birth (PTB) is the leading cause of death in infants under the age of five years. The ability to accurately identify pregnancies at risk of spontaneous PTB is therefore of utmost importance. However, no single cause is attributable. Microbial infection is a known risk factor, yet the role of vaginal microbes is poorly understood. Using high-resolution DNA-sequencing techniques, we investigate the microbial communities present in the vaginal tracts of women deemed high risk for PTB. We confirm that Lactobacillus crispatus is strongly linked to full-term pregnancies, whereas other microbial communities associate with PTB. Importantly, we show that the specific functions of the microbes present in PTB samples differs from FTB samples, highlighting the power of our sequencing approach. This information enables us to begin understanding the specific microbial traits that may be influencing PTB, beyond the presence or absence of microbial taxa.An association between the vaginal microbiota and preterm birth (PTB) has been reported in several research studies. Population shifts from high proportions of lactobacilli to mixed species communities, as seen with bacterial vaginosis, have been linked to a twofold increased risk of PTB. Despite the increasing number of studies using next-generation sequencing technologies, primarily involving 16S rRNA-based approaches, to investigate the vaginal microbiota during pregnancy, no distinct microbial signature has been associated with PTB. Shotgun metagenomic sequencing offers a powerful tool to reveal community structures and their gene functions at a far greater resolution than amplicon sequencing. In this study, we employ shotgun metagenomic sequencing to compare the vaginal microbiota of women at high risk of preterm birth ( The rate of spontaneous PTB, defined as the onset of labour and subsequent delivery before 37 weeks of gestation, is reported to be at one in ten pregnancies  worldwide3. In addition to increased mortality, prematurity is associated with significant morbidity in terms of chronic lung disease, increased rates of neurodevelopmental delay, and long-term health problems6. The economic burden through healthcare costs related to prematurity is estimated to be $26 billion in the United States alone7.Preterm birth (PTB) is the leading cause of mortality in infants under the age of 5 years8, smoking9, obesity10, stress11, age12, and previous history of obstetric complications13 as indicators of PTB. Microbial infection, specifically urinary tract infections, vaginitis, bacterial vaginosis (BV), and even periodontal disease have also been associated with an increased risk of spontaneous preterm delivery16. The composition of the microbiota of the mother, and in particular the mother’s vaginal microbiome, has more recently been linked to spontaneous PTB3. In general, the vaginal microbiota is stable throughout pregnancy with the dominant species rarely changing18; however, biogeography and ethnicity can play a considerable role in determining the apparent ‘normal’ microbiota19. A study of 90 women, mainly of African American decent, found that there was no association between the vaginal microbiota and PTB20. In contrast, DiGiulio et al.21 found that changes in the vaginal populations of Caucasian women were correlated with PTB. Further studies have provided additional evidence that race is a key factor when identifying patterns linking PTB with vaginal microbiota composition23.Several studies have investigated links between maternal health status including dietLactobacillus species have generally been associated with positive health states for the woman24. In contrast, CST-4, which is reduced in lactobacilli and contains an increased abundance of mixed species, has been associated with poorer health outcomes. These mixed species, including Gardnerella vaginalis ), Atopobium vaginae (assigned as Fannyhessea vaginae in the GTDB), Mobiluncus sp., Prevotella sp., and others, are most often associated with BV. Multiple studies have reported a twofold increased risk for PTB in women suffering from BV26. Despite this, large cohort studies have reported that CST-4 can also be present in a high percentage of the healthy population with no symptoms of BV27. Indeed, B. vaginale has also been frequently isolated from asymptomatic women28.The common means of categorizing the vaginal microbiome has been to group them into community state types (CSTs). CSTs dominated by B. vaginale29, while targeting the cpn60 gene, has shown an improvement in terms of identifying Bifidobacterium30. Shotgun metagenomics presents an opportunity to overcome many of the limitations of amplicon sequencing, with the additional benefit of understanding the functional potential of a community. An increasing number of studies have now used this approach to elucidate the vaginal microbial communities. In particular, functional distinctions among different metagenomic assembled B. vaginale isolated from the same sample31 and an ability to sub-speciate important taxa32 provide examples for the power of this approach.Determining a role for the vaginal microbiota in influencing pregnancy outcome has been limited by the ability to fully differentiate taxonomic groups. This resolution may prove important if discrete communities, species, or strains prove to represent a risk factor. In light of this, it is important to consider that the use of standard 16S rRNA targeted amplicon sequencing has been reported to under-report certain subgroups of In a cohort of women with a predisposed risk for PTB, we aimed to use shotgun metagenomics to distinguish any vaginal microbial signatures different to a low-risk control group.n = 49). Of these, 8 pregnancies ultimately were preterm (PTB) and the remaining 41 were full-term birth (FTB). In addition, for the 35 participants initially considered at risk of PTB, 7 women delivered before 37 weeks’ gestation and were grouped into the PTB group (risk_PTB). The remainder were grouped into a risk but full-term group (risk_FTB). For the 14 women at low risk of PTB, 13 delivered at term (no-risk_FTB). The single sample from this control group that delivered prior to 37 weeks was subsequently provided a distinct group of no-risk_PTB. Within the PTB samples, six deliveries were late preterm (32–37 weeks), one was a moderate preterm (31.7 weeks), and one was a preterm at 26 weeks (Table p < 0.001 Student’s t-test).A total of 57 participants with singleton pregnancy were successfully recruited over the study period, 20 at low risk of PTB and 37 with risk factors for PTB. Multiple swabs were collected from some women depending on clinic visit frequency, accounting for 89 total samples. To control for multiple sampling of some woman and different trimester timepoints, we focused our analysis on single samples for each woman in the second trimester of pregnancy . In terms of β-diversity, the risk_PTB communities were significantly dissimilar from both the no-risk_FTB and risk_FTB samples .Following quality filtering, a mean of 828,950 high-quality microbial sequencing reads were obtained per sample. The median number of observed species for either the FTB or PTB groups was 168 and 185.5, respectively Fig. , with thLactobacillus crispatus (50.86%) and Lactobacillus iners (21.27%) were dominant. Within the risk_FTB group, L. crispatus (25.83%), L. iners (14.83%), Lactobacillus gasseri (11.70%), and L. gasseri A (7.30%) were the dominant species detected. L. crispatus was never >0.03% relative abundance in the risk_PTB group. In addition, L. gasseri was only detected in two samples in the risk_PTB group, accounting for just 0.01% mean relative abundance. Although L. iners (30.82%) accounted for a greater proportion of the mean population in the risk_PTB group compared to both FTB groups, this increase was not statistically significant. Due to the large variation in both the abundance and number of species observed between samples, L. crispatus (p < 0.001), L. gasseri (p = 0.028), and Bifidobacterium breve (p = 0.036) were the only species that significantly differed between full-term and preterm groups, with a higher respective mean relative abundance , and after correcting for multiple comparisons, these q-values were below than 0.25  was assigned as multiple sub-species. The two most abundant BV-associated species across all samples were B. vaginale and B. vaginale_G; however, neither were significantly increased in either FTB or PTB samples . In addition, F. vaginae and F. vaginae_A (this database assigns A. vaginae as F. vaginae) were positively correlated with PTB samples . There was a weak association between samples from the risk_PTB groups and CST-5 (Supplementary Table p = 0.09). There were five observations of the BV-associated CST-4, three of which were from the risk_PTB group. This CST had a positive association with samples from the risk_PTB group (p = 0.02). Neither CST-3 nor CST-8 were associated with any pregnancy outcome.Resolution of the microbial species into CSTs revealed that six distinct CSTs were present across all swabs Fig. . CSTs wep-value 0.038 and 0.045, respectively). From ADONIS analysis, only L. gasseri was significantly influencing the variaton in gene function across all three categories , 0.027 (CC), and 0.013 (BP)). Stratification of the samples into the risk groupings did not show any significant differences in diversity of function . For gene functions with significant association to preterm delivery, the MF category contained the most differential features. Among these were genes involved in ‘2′ 3′-bisphosphoglycerate-independent phosphoglycerate mutase activity’, ‘receptor activity’, ‘aldose 1 epimerase activity’, ‘carboxyl or carbamoyltransferase activity’, and ‘copper exporting ATPase activity’ , biological process (BP), and molecular functions (MFs) revealed functional diversity between the risk groupings. Both Shannon and Simpson diversity indexes determined there was a significant increase in the diversity of gene functions for both BPs and metabolic functions for PTB samples compared to FTB Fig. . When saL. crispatus with full-term pregnancies. In addition, evidence is provided that CST-4 is more associated with the vaginal microbiome from a spontaneous PTB. This study also identified functional differences in the vaginal microbiome of women who subsequently deliver preterm.In summary, using a shotgun metagenomics approach, this study has confirmed the association of a vaginal microbiome dominated by B. vaginale  subtypes can be difficult with the potential to omit subtle nuances when associating vaginal communities with PTB.To date, numerous microbiome sequencing studies have been conducted with the aim of understanding the vaginal microbiota and its’ association with PTB. A limiting factor in several of these studies has been the use of targeted amplicon sequencing. In particular, it has been shown that certain key vaginal taxa can be underrepresented or not detected, depending on the gene region targeted. Of note, BV has been determined as a risk factor for PTB, yet the detection and differentiation of B. vaginale were detected with each determined to correlate differently with pregnancy outcome  had a mean relative abundance of 14%. In contrast, B. vaginale_D (9/49 occurences), B. vaginale_E (8/49 occurences), and B. vaginale_F (8/49 occurences), which significantly correlated with PTB outcome, were never >0.2% relative abundance. At such low abundance, it remains to be determined whether these species can exert a meaningful biological influence within the microbiota. Nonetheless, this difference within the B. vaginale group highlights the importance of stratifying this species when investigating associations with vaginal health and perinatal outcome. Another important member of BV-associated bacteria, F. vaginae (A. vaginae), was identified in three of the PTB samples at relatively high abundance (4.42–8.10%), yet was rare in full-term samples at a maximum relative abundance of 3% (in 3/41 samples). This species has been shown to develop strong biofilms with B. vaginale35. Previous studies identified high loads of this species as a risk for PTB37. Of interest in our study, there was an increased relative abundance of F. vaginae in the control group sample that had no perceived risk of PTB, yet delivered preterm , or had two previous LLETZ (n = 11). High-risk participants in the study were recruited from women attending the PTB clinic at The National Maternity Hospital Dublin, Ireland. Anonymized patient data were collected from patient charts by an independent researcher and pregnancy outcome data collected from patient charts and a computerized database in the National Maternity Hospital, Dublin. Low-risk controls (n = 14) were women who had previously delivered a full-term baby with no prior history of PTB or LLETZ, and were attending routine antenatal care. Inclusion criteria were women over 18 years of age and pregnant. Exclusion criteria were women currently on antibiotic treatment. Following informed consent, a high vaginal swab was taken using a speculum from the posterior vaginal fornix and external orifice of the cervix prior to transvaginal ultrasonography using a dry cotton swab and delivered to the laboratory within 24 h.This was a prospective cohort study with institutional ethics approval by the National Maternity Hospital Research Ethics Committee and maternal written consent. Women were determined to be at risk of spontaneous PTB if they had either a history of previous spontaneous PTB, and therefore at high risk of subsequent spontaneous PTB  and purified with AMPure magnetic beads (Beckman Coulter). A negative sterile water control was included during the enrichment and carried forward through subsequent sequencing steps. Finally, all samples were normalized to 0.2 ng µlLibraries of DNA were prepared according to standard Illumina protocols. Briefly, DNA was sheared by heating to 55 °C for 7 min. Paired-end indexes were added and amplification occurred for 12 cycles before samples were purified with AMPure magnetic beads. DNA was quantified by Qubit dsDNA HS assay kit and the Agilent Bioanalyser 2100 with high sensitivity DNA chips before being pooled to 2 mM. Quality of the sample pool was confirmed by quantitative PCR. A sample of sterile water was processed in parallel with the DNA during library preparation to act as a negative control (Supplementary Table https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html). TrimGalore (v 0.6.0) , a perl wrapper for Cutadapt (v. 1.18)60 and FastQC (v. 0.11.8) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), was used to remove adapter sequences and low-quality sequences using default parameters. Removal of human DNA contamination was performed by aligning all high-quality paired-end reads to the latest draft of the human genome (hg38) using Bowtie2 (v. 2.3.4)61. The resulting SAM files were converted to BAM format and filtered to keep only unmapped paired-end reads using SAMtools62. Bedtools63 was used to convert the remaining reads from BAM to FASTQ format. Taxonomic assignment of paired-end reads was performed using Kraken264 alignment against the GTDB_54k database created by Méric et al.65 (https://github.com/rrwick/Metagenomics-Index-Correction). Functional profiling was performed using the HUMAnN2 pipeline (v. 2.8.1)66. The gene families output were renormalized as copies per million reads and regrouped according to Gene Ontology terms.Raw sequencing data were base-called using Illumina’s bcl2fastq software (v 2.19) . Statistical analysis was carried out in R using the vegan package (https://cran.r-project.org/web/packages/vegan/index.html) and RVAideMemoire (https://cran.r-project.org/web/packages/RVAideMemoire/index.html). Multivariate Association with Linear Models 2  was used to determine independent associations of species and functions with metadata factors. A q-value of >0.25 was considered significant in our analysis.Data were visualized using both Graphpad Prism 6 and RStudio (R v.3.6.0). Heatmaps were generated using the ‘ComplexHeatmap’ packageFurther information on research design is available in the Supplementary InformationReporting Summary


Early gastric carcinoma is heterogeneous and can be divided into early gastric cardiac carcinoma (EGCC) and early gastric non-cardiac carcinoma (EGNCC) groups. At present, differences in clinicopathology remains obscure between EGCC and EGNCC fundus–corpus and antrum–angularis–pylorus subgroups, especially between EGCC with and without oesophageal invasion.In this study, we studied 329 consecutive early gastric carcinoma radical gastrectomies with 70 EGCCs and 259 EGNCCs.H. pylori infection rate, and lymphovascular/perineural invasion. EGCC with oesophageal invasion (n = 22), compared to EGCC without (n = 48), showed no significant differences in the H. pylori infection rate and oesophageal columnar, intestinal, or pancreatic metaplasia, except for a higher percentage of the former in size > 2 cm and tubular differentiation.Compared to the EGNCC antrum–angularis–pylorus (n = 181), but not fundus–corpus (n = 78), sub-group, EGCC showed significantly older age, lower prevalence of the grossly depressed pattern, better tumor differentiation, higher percentage of tubular/papillary adenocarcinoma, but lower frequency of mixed poorly cohesive carcinoma with tubular/papillary adenocarcinoma, and absence of lymph node metastasis (LNM) in tumors with invasion up to superficial submucosa (SM1). In contrast, pure poorly cohesive carcinoma was less frequently seen in EGCCs than in EGNCCs, but mixed poorly cohesive carcinoma with tubular/papillary adenocarcinomas was significantly more common in the EGNCC antrum–angularis–pylorus sub-group than in any other group. No significant differences were found between EGCC and EGNCC sub-groups in gender, tumor size, There exist distinct clinicopathologic differences between EGCC and EGNCC sub-groups; EGCC was indeed of gastric origin. Further investigations with larger samples are needed to validate these findings. Gastric cancer is heterogeneous in epidemiology, pathology, and pathogenesis mechanisms, and may be divided into cardiac and non-cardiac categories , 2, bothWe searched the electronic pathology database stored in the Jiangsu Province Hospital of Chinese Medicine in Nanjing, China, over the 7-year period from January 2011 to December 2017 for gastric cancer radical resection and collected 2184 consecutive cases with pathologically confirmed gastric cancer. According to the 2019 World Health Organization (WHO) diagnostic criteria , 443 EGCHelicobacter pylori (HP) infection was confirmed by microscopic identification of the bacterium on routine hematoxylin–eosin and basic fuchsin stained sections with appropriate controls.The 5th edition WHO diagnostic criteria on gastric carcinoma were followed and EGC was defined as invasive carcinoma confined to the mucosa or submucosa . EGCC reIn most Chinese patients, endoscopic mucosal GEJ and squamous-columnar epithelial junction lines overlap at the same level . Microsc2, Fisher’s exact, or Kruskal–Wallis H test. P values < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS software, version 13.0 .Differences between groups with continuous or categorical variables were statistically compared with appropriate statistical methods, such as Student t, χP < 0.01).As shown in Table P < 0.05), there were no significant variations in the elevated or flat sub-group of EGC between the two groups, despite the fact that the frequency of the elevated pattern was more commonly seen in the former (10.0%) than in the latter (7.3%). However, EGC tumors with the depressed pattern (Types 0-IIc and 0-III) were significantly less commonly seen in the EGCC group (67.1%) than in the antrum–angularis–pylorus group (81.2%) (P < 0.05), but not in the fundus–corpus sub-group of ENGCC tumors. Statistically, differences in overall tumor size and invasion depth were not significant between EGCC and ENGCC sub-groups.As shown in Table P < 0.01). As displayed in Table P < 0.05). In contrast, pure PCC , while mixed PCC with tubular/papillary adenocarcinoma was more commonly seen in the antrum–angularis–pylorus (P < 0.05), but not the fundus–corpus, sub-group of EGNCC. There were no significant differences in uncommon variants of EGC, such as micropapillary adenocarcinoma  between the EGCC and EGNCC groups. Although well differentiation in EGC was more commonly noted in EGCC (24.3%) than in EGNCC (10.0%) groups, the difference did not reach a statistically significant level. However, poor differentiation in EGC was significantly less commonly identified in EGCC (15.7%) than in EGNCC (43.6%) groups (P < 0.05). There were no significant differences between EGCC and EGNCC groups in lymphovascular invasion, perineural invasion, HP infection, and LNM.In this cohort, differences in the EGC histologic type were significantly different between EGCC group and EGNCC sub-groups P < 0.0. As dispP < 0.01). Surprisingly, no LNM was detected in 38 EGCC cases with the invasion depth up to superficial submucosa, ie, M2 + M3 + SM1; thus, the prevalence of LNM was significantly lower in EGCC than in EGNCC  groups (P < 0.05). Specifically, the percentage of LNM in EGNCC was 6.8% (4/59), 7.0% (5/71), and 18.2% (6/33) for invasion depth at M2, M3, and SM1, respectively. There was no significant difference in risk of LNM of EGC with deep submucosal invasion (SM2) between EGCC and EGNCC sub-groups.The average number of regional lymph nodes retrieved and examined per case was 21.5 (range: 2–68). As illustrated in Table , extending to an average distance of 4.16 mm (range: 1–10), primarily underneath benign oesophageal squamous epithelium. No Barrett’s oesophagus was recognized in all 22 EGCC cases. In addition, high-grade intraepithelial neoplasia was observed in cardiac mucosa adjacent to invasive carcinoma in the gastric cardia  and the overall difference in the microscopic tumor type was also statistically significant between the two sub-groups (P < 0.01), in that EGCC tumors with oesophageal invasion showed a significantly lower proportion of tubular adenocarcinoma than those without oesophageal invasion.In this study, 22  EGCC tumors showed their epicenters in the gastric cardia with a small proportion of the tumor crossing the GEJ line into the distal oesophagus  in EGCC cases with oesophageal invasion. These findings, if confirmed with larger samples in the future, may have lasting impact on the current clinical management strategy for patients with EGC, especially EGCC.EGC heterogeneity is well-known in risk factors, histopathology, molecular pathobiology, and prognosis. The percentage of EGCC in our cohort was much higher than that previously reported in Japan , 12, KorWhile HP infection has been shown to be the most important risk factor for EGC and inversely correlated to Barrett’s adenocarcinoma , we did Compared to EGNCC, EGCC tumors invaded deeper with a higher percentage of cases with submucosal invasion, as we reported recently in a multicenter study . SubmucoEGCC pathologic staging stays controversial. According to the 8th edition of the American Joint Committee on Cancer staging manual , if tumoMajor limitations of this study include the retrospective study design with no standardized lymphadenectomy so that the number of lymph nodes retrieved varied. However, the overall quality of nodal retrieval remained sound because of a high average number of lymph nodes (over 21) retrieved per case in this cohort. The other undesirable issue in this study was the small number of EGC cases in 3 sub-regions  so that differences in several clinicopathologic variables could not be effectively analyzed statistically. To overcome this short-coming, our next research plan was to expand the current investigation into a multi-center study, which is on-going, to verify the results of this single-center study.In summary, EGCC, compared to EGNCC, showed more advanced patient age, better tumor differentiation, higher percentage of tubular and papillary adenocarcinomas, but lower frequency of PCC, and lower risk of LNM. EGCC with epicenter in the gastric cardia may invade the oesophagus, but with rare intestinal metaplasia and high prevalence of HP infection in our cohort, which support the classification of EGCC as gastric, not oesophageal, carcinoma. Further investigations with larger samples in multi-center studies are urgently needed to validate our findings for a better understanding of EGCC and more appropriate patient management strategy.


Treatment of transplant-ineligible (TNE) newly diagnosed multiple myeloma (NDMM) requires a balance between disease control and maintaining quality of life (QoL). Patients value treatment-free remission periods in this incurable condition, as they are associated with better QoL. We set out to study clinical outcomes of consecutive TNE NDMM patients in routine care treated in Thames Valley Cancer Network between 2009 and 2017. The primary outcome was the evaluation of the treatment-free interval (TFI) after 1st and subsequent lines of therapy in the total cohort and in individual subgroups, according to age (≤75 vs. >75 years), and co-morbidities using Charlson Co-morbidity Index (CCI): mild: 0–2 vs. moderate: 3–4 vs. severe: ≥5). Secondary outcomes include response rates, overall survival (OS) and progression-free survival (PFS) between subgroups: according to age and according to co-morbidities. In a total cohort of 292 patients, median TFI (IQR) was longest after first-line therapy 6.9 months (1.4–16.9), reducing after second line therapy to 1.8 months (.7–6.9), and after third line therapy to 0.6 months (0.2–1.5). Median TFI followed the same trend across the different subgroups, by age  and by CCI . Overall response rate (ORR) to first line therapy for total cohort was 67%, with responses categorised as complete response (CR): 21%, very good partial response: 16%, partial response: 30%, stable disease: 18%, and progressive disease: 8%. ORR in individual subgroups by age were (≤75: 70% vs. >75: 63%), and by CCI (0–2: 65% vs. 3–4: 71% vs. ≥5: 77%). Median OS and PFS for the total cohort were , and , respectively. Patients aged >75 years showed a significant reduction in OS and PFS compared to those ≤75 years of age: OS , PFS . Median OS was significantly reduced with worsening co-morbidities: . Median PFS was significantly reduced in the severely co-morbid subgroup . This study demonstrated that first line therapy in the TNE NDMM setting resulted in the longest TFI which was modest at a median of 6.9 months, and decreased significantly following subsequent lines of therapy and across the different subgroups by age and by co-morbidities. Therapy objective should be to maximise the benefit of first line treatment. We envisage that the recent shift towards a continuous therapeutic approach will benefit TNE patients in view of improved survival data demonstrated by a number phase 3 trials. When continuous therapy is not appropriate due to patient choice or toxicities, an efficacious  but tolerable first line FDT strategy, which can maximise TFI and maintain a good QoL, remains a reasonable alternative approach. Multiple myeloma is primarily a disease of the elderly with up to 45% of new diagnoses in the UK made in patients aged 75 and over . Age-speElderly myeloma patients are typically ineligible for autologous stem cell transplant (ASCT) due to advanced age and co-morbidities. Objectives of first-line treatment in this patient population are disease control whilst maintaining quality of life (QoL), which translates into improved survival .As the myeloma treatment landscape continues to be shaped, continuous therapy has become the new standard of care. Progression-free survival (PFS) advantage of continuous lenalidomide with dexamethasone (Rd) was demonstrated in the MM015 trial and the FIRST trial , 4. In aHowever, the decision to employ a continuous first-line strategy in routine practice requires a careful account of a number of patient-related factors. At least 30% of patients are frail, due to disease-related symptoms and/or age-related decline in physical capacity in addition to co-morbidities, polypharmacy, nutritional status, and cognitive impairment . AchieviTreatment free interval (TFI) in routine practice can occur in a planned or unplanned fashion. Physicians and patients often decide to plan for a fixed duration therapy (FDT) strategy based on response achieved from therapy. In addition, upfront therapy can be discontinued in an unplanned fixed duration fashion as a result of significant toxicities leading to early treatment discontinuation. In both scenarios, TFI becomes an issue of immense importance to patients and clinicians, and can allow patients to recover from toxicities and restore a good QoL.It is, therefore, important to tailor the choice and duration of therapy to patients’ priorities and individual needs. Functional status and QoL are primary objectives in this population . Health-TFI and good QoL have been described as additional measures of efficacy which can be employed to make individualised treatment decisions. A UK cross-sectional survey of 370 myeloma patients demonstrated that being in a first TFI and experiencing a longer TFI were significantly associated with a better HRQL as assessed by various domains of the QLQ-C30, MY20 and EQ-5D .UK’s National Institute for Health and Care Excellence (NICE) recently approved lenalidomide with dexamethasone as first-line therapy in TNE NDMM patients where thalidomide is not tolerated or inappropriate . Prior tIn view of the recent shift towards continuous therapy, we looked to evaluate the TFI as an additional metric of efficacy in routine practice, after 1st and subsequent lines of therapy, in a large cohort of TNE NDMM patients. To our knowledge, there are no published data quantifying TFI exclusively in the TNE NDMM setting.We have also assessed the influence of age (<75 vs. ≥75 years) and co-morbidities (as per Charlson Co-mordibity Index: CCI) on clinical outcomes. The aim was to understand current practice and identify strategies which can increase first TFI and improve future outcomes.TNE NDMM patients are defined as those with a new diagnosis of symptomatic multiple myeloma which requires initiation of systematic first line therapy, but who are not eligible for, and did not receive a autologous stem cell transplantation (ASCT) due to age and/or co-morbidities. All patients with TNE NDMM within the UK Thames Valley Cancer network with measurable disease, as defined by International Myeloma Working Group (IMWG) guidelines, treated with at least one cycle of systemic chemotherapy between 2009 and 2017 were eligible for inclusion. Patients were excluded if they had been treated as part of a clinical trial. The study was approved locally by the Clinical Governance Committee.Data was retrospectively collected from patients’ medical and chemotherapy records including baseline demographic and clinical characteristics ), anti-myeloma treatment . All patients consented for retrospective analysis of their records at the point of treatment, and all patient records were anonymised at the point of analysis.The primary endpoint was the evaluation of treatment-free interval (TFI) following 1st and subsequent lines of therapy in the total cohort, and the following subgroups: according to age (≤75 vs. >75 years) and according to co-morbidities: (CCI: 0–2) vs. (CCI: 3–4) vs (CCI: ≥5). TFI was evaluated as the time between the last dose of one line of treatment and the first dose of a subsequent treatment. Response rates for the total cohort were also evaluated after first line therapy.Secondary outcomes include response rates, OS and PFS between subgroups: according to age (≤75 vs. >75 years) and according to CCI.OS was defined as time from initiation of first anti-myeloma treatment to death from any cause. PFS was evaluated as the time between initiation of first anti-myeloma treatment and progressive disease (based on IMWG uniform response criteria) or death. Patients who did not receive another anti-myeloma therapy were censored at the last available date the patient was known to be alive.Descriptive statistics for quantitative variables are presented as mean (standard deviation [SD]) or median (interquartile range [IQR] and/or range). Descriptive statistics for categorical variables are presented as number (%). Time-dependent variables were evaluated and presented using the Kaplan-Meier method and reported as median . Time-dependent variables were compared between the different age groups (≤75 years vs. >75) CCI co-morbidity subgroups (mild: 0–2 vs. moderate: 3–4 vs. severe: ≥5) using unstratified log-rank tests and Cox regression analyses, with proportionality of hazards evaluated using Schoenfeld residuals, and hazard ratios (HR) presented with 95% CI.A total of 292 patients were eligible for inclusion. The baseline demographic and clinical characteristics of the total cohort as well subgroups  are presented in At presentation, 48% of patients in the total cohort had an International Staging System (ISS) of III. A meaningful analysis of outcomes based on cytogenetics was not possible due to lack of data. The proportion of patients with renal impairment (serum creatinine ≥140 μmol/L) were comparable between the two age subgroups , but renal impairment was more prevalent in the severely co-morbid subgroup (CCI 0–2: 7% vs. CCI 3–4: 22% vs. CCI≥5: 48%).The older cohort (>75 years) presented with a significantly higher proportion of moderately to severely co-morbid patients compared to the younger cohort (60% vs. 39%).2 (range 0.7–5.2 mg/m2), Within the total cohort, thalidomide was the most commonly used first line therapy (61%), followed by PI-based (bortezomib) (22%), alkylator-based (10%), and lenalidomide-based (7%). Bortezomib was only approved for use as first line in the UK in 2014, which explains the smaller patient numbers compared to THAL group. The mean daily thalidomide dose was 51.4mg (range 0-133mg). The mean cumulative bortezomib dose per cycle was 4 mg/mThe mean number of treatment cycles was comparable (6 cycles) for the total cohort as well as individual subgroups according to age or according to co-morbidities. Except for only 20 patients in the total cohort who received lenalidomide until disease progression or unacceptable toxicity, the rest of patients in this large study were treated with FDT in a pre-planned fashion.Overall response rates for total cohort as well as individual subgroups are presented in In the total cohort, median (IQR) TFI was longest after first-line therapy 6.9 months  TFI in individual subgroups according to age were also longest after first line therapy, and decreased with increasing lines of therapy: ≤75 years (median decreased from 7.5 months to 1.8 then to 0.5 months), >75 years (median decreased from 6.1 months to 2.1 then to 1.2 months). rd line therapy, TFI in individual subgroups according to co-morbidities were also longest following first line therapy and decreased with increasing lines of therapy: In the mildly co-morbid group (CCI = 0–2), median TFI decreased from 4.8 months to 1.9 then to 0.4 months. In the moderately co-morbid group (CCI = 3–4), median TFI decreased from 6.9 months to 2 then to 0.5 months. In the severely co-morbid group (CCI≥5), median TFI decreased from 8.9 months to 1 month then increased to 3.2 months following 3Median OS for the total cohort was 30.2 months (95% CI: 23.8–36.9), OS was significantly longer in the ≤75 years group (49.0 months (95% CI: 31.3–61.8) compared to the >75 years group (22.4 months (95% CI: 18.6–29.6)), P<0.0001, HR: 2.08 (95% CI: 1.5–2.8), PFS was significantly longer in the ≤75 years group (9.7 months (95% CI: 8.6–12.0)) compared to the >75 years group (8.0 months (95% CI: 7.4–9.4)), P<0.01, Hazard ratio: 1.47 (95% CI: 1.1–1.8), Median OS was significantly reduced with worsening co-morbidity index: , st line: 10 months, 2nd line: 5 months, 3rd line: 3 months, 4th line: 1 month) [This large retrospective study of 292 patients suggests that TNE NDMM patients are able to achieve a good ORR (67%) in routine care, and this is despite a short median duration of treatment (4.6 months), older age and higher rates of co-morbidity. However, the median TFI in the total cohort was only 6.9 months and declined with subsequent treatment phases . These findings are consistent with a large international real-world study of 4997 myeloma patients, which suggested that median TFI and time to progression decrease with increasing lines of therapy (11 month) . Our cohWhen we examined the different subgroups, by age (≤75 years vs. > 75 years), and according to co-morbidities (CCI: 0–2 vs. 3–4 vs. ≥5), our study also demonstrated that TFI was longest after first line therapy and decreased significantly following second and subsequent lines of therapy.The cross-sectional study of 370 patients was the first UK-based myeloma study to report the impact of the first TFI on HRQoL. Overall, the results demonstrated that being in the first TFI was associated with better HRQL compared to later treatment lines, and that longer TFI were largely associated with better HRQL . In anotTFI has also been previously identified as a metric of patient experience and a valuable health outcome in the relapsed myeloma setting, as demonstrated in an additional analysis of Panorama-1 trial .Our study findings also suggest that a FDT approach for patients >75 years confers an inferior PFS and OS compared to patients ≤75 years. Our data is consistent with a number of subgroup analyses from large phase 3 trials in the TNE NDMM setting which demonstrated evidence of poorer survival outcomes for patients aged >75 years compared to their younger counterparts (≤75 years). In the FIRST trial, patients aged >75 years benefited from continuous Rd versus MPT; however, PFS and OS periods were shorter for the older versus young group  . In the Our study demonstrated that OS and PFS were significantly shorter with worsening co-morbidity . The Greek Myeloma Group showed that performance status was an independent prognostic factor influencing survival in an octogenarian myeloma cohort . A retroIn our large dataset, 50% of patients were moderately to severely co-morbid which partly explains poorer outcomes of therapy particularly in >75 years, and the short median duration of first line treatment (4.6 months).Our data demonstrated that the use of FDT with a thalidomide or a bortezomib-based regimen was associated with modest PFS and OS outcomes particularly in patients >75 years. This FDT strategy also resulted in a short median TFI. We envisage that the shift towards a continuous therapy approach will benefit TNE NDMM patients as demonstrated by survival advantage in large phase 3 trials, and should be standard of care in order to improve outcomes.FIRST trial demonstrated that median PFS in the continuous lenalidomide and dexamethasone arm (Rd) was significantly superior to 18 cycles of Rd and to FDT with melphalan in combination with prednisolone and thalidomide (MPT) . The 4-year OS rates were (continuous Rd: 59% vs. Rd18: 56% vs. MPT: 51%) . MM-015 In patients with advanced age or with co-morbidities, tolerable dosing and close monitoring are required to limit toxicities and maintain continuous therapy.If the continuous therapy option is not available, or not appropriate (due to toxicities or patient choice), an effective FDT combination containing novel agents  and with good tolerability remains a reasonable alternative approach. FIRST trial showed no overall survival benefit between continuous Rd and 18 cycles of Rd . In certain scenarios where a decision is made to employ FDT over continuous therapy, the aim besides improving PFS and OS outcomes, should be to maximise TFI which can in turn, help improve QoL.Our study is limited by its retrospective nature, lack of QoL data which can further inform of the benefits of TFI, in addition to the use of thalidomide or bortezomib-based therapies which are not reflective of the current shift towards newer therapies and a continuous strategy.In conclusion, this large real-world data study details the outcomes of treating TNE NDMM with a FDT approach. First line therapy resulted in the longest TFI which was modest at a median of 6.9 months, and decreased significantly following subsequent lines of therapy. This was demonstrable for the total cohort as well as for individual subgroups by age, and according to co-morbidities. The treatment objective for TNE NDMM should be to maximise the benefit of first line treatment. Continuous therapy has demonstrated its significantly improved survival outcomes, compared to FDT. Therefore, we envisage that the recent shift towards a continuous therapeutic approach will benefit TNE patients, but only if patients are closely monitored with a tolerable regimen. However, when continuous therapy is not appropriate due to patient choice, or toxicities leading discontinuation, an efficacious  but tolerable FDT strategy remains a reasonable alternative approach, which can produce a meaningful TFI.


ALPL variants in subjects with persistent hypophosphatasaemia and determine the associated clinical and laboratory features. For this cross-sectional study, laboratory records of 386,353 subjects were screened by measurement of ALP activity. A total of 85 (0.18%) subjects with persistent hypophosphatasaemia  were included (secondary causes previously discarded). ALPL genetic testing and a systematized questionnaire to retrieve demographic, clinical and laboratory data were performed. Descriptive analysis and logistic regression models were employed to identify the clinical and laboratory characteristics associated with ALPL variants.Hypophosphatasia (HPP) is an inborn error of metabolism characterized by low levels of serum alkaline phosphatase (ALP). Scarce evidence exists about features that should signal the potential association between hypophosphatasaemia and HPP in adults. The aim of this study is to estimate the prevalence of (47%) had a variant(s) in ALPL. With regard to clinical characteristics, the presence of an ALPL variant was significantly associated only with musculoskeletal pain . Nevertheless, a trend to present more dental abnormalities  was observed. Metatarsal stress fractures were also more frequent  in this group. Regarding laboratory features, median ALP levels were lower in subjects with ALPL variants . Interestingly, the threshold of ALP levels < 25 IU/L showed a specificity, positive predictive value and positive likelihood ratio of 97.8, 94.4% and 19.8 to detect a positive ALPL test, respectively.Forty subjects ALPL variants. Musculoskeletal pain and ALP levels < 25 IU/L are associated with this variant(s). In this scenario, ALP levels < 25 IU/L seem to be very useful to identify individuals with the presence of an ALPL variant.In subjects with persistent hypophosphatasaemia –secondary causes excluded– one out of two presented  ALPL, located on chromosome 1p36.1-p34, which encodes the tissue-nonspecific alkaline phosphatase  and two likely pathogenic variants . The substitutions p.(Gly112Arg), p., p.(Glu291Lys) and p.(Thr115_Ala116dup) were each present in three subjects; the disease-causing variants p.(Arg152Cys), p.(Asp183Asn), p.(Asp378Gly), p.(Thr166Ile), p.(Gly491Arg) and p.(Glu191Lys) in two subjects and the rest were observed in single subjects. Thirty-four subjects (85%) had missense disease-causing variants, three had duplications (7.5%) and the rest had deletions, insertions or splice site mutations . Most of the variants were located in exons 5,6 and 9, were predicted to have a damaging effect in silico pathogenicity prediction tools and were absent or existed at extremely low frequencies in gnomAD. Additional file ALPL variants and the clinical findings associated to each of them.Regarding the subjects who showed variants in nt, 2.5% /40 for aDemographic characteristics of study participants are shown in Table Clinical features of +GT and –GT groups are detailed in Table p < 0.05] although functional assessments, as measured by HAQ-DI were comparable in the two genetic groups.Data from the knee and/or pelvis X-ray was available for 11 subjects: 5 in the +GT group and 6 in the -GT group. Among them, two subjects in the former had radiographic chondrocalcinosis signs (one of them with a previous diagnosis of pyrophosphate arthropathy), contrasting with a negative previous history in the -GT group. A higher VAS was observed in the +GT group [3 (2–5) vs 1 (0–5); ALPL disease causing variants. The results are shown in Table Furthermore, regression models adjusted for possible confounders  were employed to evaluate the association between clinical manifestations and n = 9) and 11.9 (7–21.2) for those who did not report symptoms before age 18 (n = 29). In terms of initial HPP-associated symptoms, 8 subjects reported dental problems at a median age at onset of 14.51 (7.7–19.5); 18, musculoskeletal symptoms at a median age at onset of 43 (14.8–51.8) and 2 of them, kidney complications.In the +GT group, a median 19-year delay between first signs or symptoms and diagnosis was observed, being the median (IQR) age at the onset of the symptoms of 40.3 years (13.6–50.5) and at the diagnosis of 49.8 (37.2–63.3) years. Median diagnostic delay was 22.7 (19.6–34.6) for those who experienced a pediatric onset  vs 3.4 mg/dL (3.1–4.1), p < 0.05, respectively). Eleven percent (5/44) of subjects, based on the available data, presented hyperphosphoremia (> 4.5 mg/dL), most (4/44) in the +GT group. Median calcium levels were within the normal range, statistically comparable in both groups, and hypercalcemia was not observed in subjects using the available data. Levels of 24-h urinary calcium and phosphate excretion did not differ between groups.The biochemical profile of study participants is presented in Table ALPL variants based on different ALP cut-off levels  was assessed  and two individuals diagnosed with HPP experienced metatarsal fractures. The specificity and positive predictive value for musculoskeletal pain in this group of subjects were 52.3 and 41.7%, respectively and 86.4 and 50% for dental abnormalities; thus, the discriminative power did not increase  displayed a positive variant and 21 (58.3%), a negative genetic test. In addition, 12 subjects reported dental abnormalities  had HPP, which is in accordance with the study of Riancho-Zarrabeitia and co-workers [ALPL disease-causing variants (84%), most likely because a more stringent definition of hypophosphatasaemia was used. We identified four previously unreported variants based on the American College of Medical Genetics and Genomics (ACMG) classification criteria [The prevalence of persistent hypophosphatasaemia in adults of our clinical setting was 0.18%, which is in agreement with two studies, one conducted in a large rural multispecialty clinic population in the USA   and anot-workers  found a criteria . The majcriteria , 18.ALPL genetic testing is required. Nevertheless, in clinical practice, access to such testing is not always available or rapid testing is required. Therefore, it would be very useful to identify the clinical and routine biochemical characteristics of the disease and its predictive value for a proper and early diagnosis, in order to avoid erroneous therapeutic decisions.According to our results, approximately 50% of persistently low APL levels signify a different underlying etiology than HPP. Therefore, genetic confirmation by ALPL variant. In contrast, ALP levels seem to be discriminative enough for this purpose. ALP levels below 25 IU/L showed a high specificity (97.8%), positive predictive value (94.4%) and positive likelihood ratio (19.8). Consequently, in the presence of symptoms potentially related to HPP and biochemical abnormalities, specific ALP cut-off levels could help diagnostic strategies, especially when genetic testing is not available. If these results are confirmed in other populations, these key findings could prove very useful in clinical practice.Based on the results of this study, neither single nor combined clinical manifestation are useful enough for identifying the presence of an ALPL status. Previous publications also showed a high incidence (41–95%) of musculoskeletal pain in subjects with HPP [ALPL disease-causing variants. In a recent publication, metatarsal fractures reached a prevalence of 21% and were considered typical of HPP [With regards to clinical features, only the presence of musculoskeletal pain was significantly and strongly associated with a positive genetic ence 41–9% of muscwith HPP –17. Indewith HPP . Howeverl of HPP . The numALPL allele and the limitations of conventional genetic studies [On the other hand, to implement strategies that will enable an early detection of the disease is mandatory. In our study, a significant 19-year delay was observed, which is in accordance with the global HPP Registry, the largest observational study including real-world data cases . Our stu studies , 23.ALPL could be carriers. Furthermore, ALP substrates and the analysis of the dominant negative effect of the variants have not been performed due to limitations in their determination.The main weaknesses of our study are the lack of pediatric medical histories, the use of a clinical questionnaire for identifying clinical symptoms and limited data available regarding certain variables, such as radiographic records. As a limitation, the design of the study does not allow us to establish a definitive clinical diagnosis of HPP: although a detailed questionnaire was designed for the evaluation of subjects included, a complete visit and complementary tests targeted to study clinical features or laboratory abnormalities were not performed, so that some subjects with variants in ALPL genetic test, although these results need further investigation.The main strengths are the thorough analysis of medical records, which yielded a considerably large sample size for a rare disease, the integration of data across a wide range of variables, the analysis of information obtained through a detailed clinical questionnaire and the genetic analysis encompassing this large population. In addition, to the best of our knowledge, this is the first time that ALP cut-off levels are used to identify a positive ALPL disease-causing variant. It is imperative that the clinical and biochemical characteristics and its predictive value are defined if early diagnoses are to be obtained and erroneous therapeutic decisions avoided. Musculoskeletal pain and ALP levels below or equal to 25 IU/L are associated with a positive result in ALPL genetic testing. In this scenario, ALP levels below this threshold seem to be very useful for predicting the presence of ALPL disease-causing variant. If confirmed in other populations, these key findings can be useful in clinical practice. Prospective studies evaluating these findings and establishing the natural evolution of HPP will definitely enable a comprehensive determination of the disease’s complete clinical spectrum in adults.In subjects with persistent hypophosphatasaemia, one out of two individuals with primary low ALP levels, presented an This cross-sectional study was performed at La Paz University Hospital . The study adhered to the tenets of the Declaration of Helsinki and approval was obtained from its ethics committee. Each subject provided written informed consent prior to inclusion.First, 1,536,711 laboratory records including ALP serum levels values from 386,353 subjects were screened to identify abnormally low ALP results. These values were recorded in the biochemical database of the hospital from 2009 to 2015. The main criteria for inclusion were: adults aged 18 years or older with persistent hypophosphatasaemia, defined as ≥2 ALP measurements below or equal to 35 IU/L. Subjects were excluded if ≥1 ALP values were above 45 IU/L (lower limit for adults), or if clinical records revealed secondary underlying causes of hypophosphatasaemia, such as cancer or chemotherapy, adynamic renal osteodystrophy, severe hypothyroidism, Cushing’s disease, Wilson’s disease, nutritional deficiencies, hypomagnesemia, hypozincemia, myocardial infarction, sepsis, major trauma or surgery, massive transfusions and clofibrate therapy among others [Eligible subjects completed a questionnaire to report family or personal history of musculoskeletal pain, muscle weakness, fractures, premature dental loss and previous orthopedic surgery, among other clinical features classically associated with HPP. Additionally, radiographic data were retrieved from medical records. Musculoskeletal pain was considered when symptoms were recurring or chronic (> 6 months) and not when transient and muscle weakness was defined by a chronic subjective perception of a decrease in muscle strength, but no physical examination was performed. Dental abnormalities were defined as tooth shape abnormalities, structure and colour abnormalities of enamel or dentin, thin enamel, late teeth eruption or severe/recurrent cavities and early loss of permanent teeth was defined as the loss of several teeth or extraction (> 10) due to tooth abnormalities in the past, prior to the age of 50. In terms of fractures, peripheral traumatic and fragility ones were evaluated as were stress metatarsal and atypical fractures. Multiple fractures were defined as more than three. Chondrocalcinosis and calcific periarthritis were considered when the subject had a previous diagnosis or if a prior imaging test confirmed the diagnosis. Subjects were required to grade their pain on a 100 mm visual analog scale (VAS) and to complete the Health Assessment Questionnaire-Disability Index (HAQ-DI). Individuals were requested to provide a blood sample for the genetic analysis.Between 2009 and 2013, the University La Paz Hospital Laboratory utilized an Olympus 5400 analyzer (Beckman Coulter) to measure serum ALP activity. In February 2014, it switched to Siemens Healthineers (Advia 2400 chemistry system) and clinically acceptable correlation and comparison was demonstrated between the two devices . Both methods measure ALP activity by a kinetic rate method in which p-nitrophenyl phosphate (a colourless organic phosphate ester substrate) is hydrolyzed by ALP to the yellow-coloured product pnitrophenol and phosphate at pH 10,3. Enzymatic activity of ALP is directly proportional to changes in absorbance at 410 nm. The normal adult’s range is 45 to 116 IU/L.ALPL (NM_000478.4) was performed by Sanger sequencing. In silico pathogenicity prediction and control population frequency analysis were assessed using Alamut V2.6 software , Varsome (https://varsome.com/), CADD software (http://cadd.gs.washington.edu) and Silvent et al. criteria [http://gnomad.broadinstitute.org/) and the in silico tools included CADD V1.3, DANN, SIFT, Polyphen, MutationTester, Mutation assessor, FATHM and SpliceSiteFinder-like, MaxEntScan, NNSPLICE, GeneSplicer. The ALPL disease-causing variants database (http://www.se-sep.uvsq.fr/03_hypo_mutations.php) was also consulted to obtain up-to-date information about the genetic variants included in our study  and the screening of the exons and intron/exon boundaries of criteria . The allALPL variants among subjects with persistent hypophosphatasaemia and to compare clinical and laboratory characteristics between individuals with and without ALPL variants. Continuous variables were described as median (interquartile range –IQR-) and categorical variables as an absolute number and relative percentage. Comparisons between two independent groups for continuous variables were performed using the Student’s t-test for unpaired data if normally distributed, or a Mann-Whitney U test when not. Statistical significance difference between groups for categorical variables was calculated using the Chi-square test or the Fisher’s exact test, as appropriate. Logistic regression models adjusted for confounders were employed to investigate the association between clinical and laboratory characteristics and the genetic status. Finally, diagnostic utility measures  to predict the likelihood of having an ALPL variant were calculated. The level of statistical significance was set at p < 0.05. Statistical analyses were performed using the IBM SPSS Statistics 23.0 for Windows.First, descriptive analysis was employed to determine the prevalence of Additional file 1: Table S1. List of subjects displaying ALPL disease causing variants observed in the cohort according to the transcript NM_000478.4, clinical features associated and family history available.Additional file 2: Table S2. Diagnostic utility measures for each of the symptoms combined with ALP levels between 25 and 35 IU/L.


We have developed a novel in vitro approach of precision-cut adipose tissue slices from northern elephant seal (Mirounga angustirostris) as a complementary approach to whole animal models. Blubber biopsies were collected from 14 pups during early and late post-weaning fast , precision-cut into 1 mm thick slices and maintained in culture at 37°C for at least 63 h. The slices exhibited an efficient response to ß-adrenergic stimulation, even after 2 days of culture, revealing good in vitro tissue function. The response to lipolytic stimulus did not vary between regions of outer and inner blubber, but was higher at early than at late fast for inner blubber slices. At early fast, lipolysis significantly reduced leptin production. At this stage, inner blubber slices were also more efficient at producing leptin than outer blubber slices, especially in the non-lipolytic condition. This model will aid the study of adipose tissue metabolism and its response to environmental stressors in marine mammals.Adipose tissue plays key roles in energy homeostasis. Understanding its metabolism and regulation is essential to predict the impact of environmental changes on wildlife health, especially in fasting-adapted species. However,  Adipose tissue has been traditionally considered for its role in insulation and energy storage. More recently, it has been shown to be an essential endocrine organ, secreting adipokines that are involved in various crucial biological functions, such as appetite regulation, immunity, inflammation, and regulation of glucose and lipid metabolism . It is aMirounga angustirostris)  is a multi-functional organ playing a key role in metabolic homeostasis and energy balance regulation, especially in fasting adapted species such as northern elephant seals (NES – rostris) . Knowledrostris) , and morIn vitro approaches, when properly developed and executed, may provide an important and viable complement to whole animal models by minimizing the animal impact, and allowing experiments with multiple treatments under controlled conditions. Marine mammal blubber is relatively accessible to sample. In fact, for some species such as large cetaceans, it is the only tissue that is feasible to sample in living individuals  blubber  model from NES. Because of its accessibility, physiology and natural life history, the NES represents an excellent model to investigate adipose tissue metabolism . This phAll methods were carried out in accordance with relevant guidelines and regulations. All capture and handling procedures were approved by the Sonoma State University Institutional Animal Care and Use Committee (IACUC #2017-56) and were performed under the National Marine Fisheries Service Marine Mammal Permit #19108. Free ranging weaned NES pups were sampled at Año Nuevo State Reserve, CA, United States , from February to May 2018. Four early weaned pups were sampled for PCATS method setup. Afterward, six early  and eight late  weaned pups were sampled for lipolysis experiments. Pup anesthesia was initiated with an intramuscular injection of Telazol (1 mL/100 kg of estimated body mass)  and then maintained with an intravenous injection of Ketamine (as needed) (Ketaset). Full-length blubber biopsies were taken in the lateral pelvis area with 6-mm biopsy punches . Samples were rinsed in phosphate buffered saline  before being kept in sterile sampling medium at around 25°C as recommended for adipose tissue explants , in orde2) on a shaker for 3 h. This first 3-h incubation period was added in order to collect and remove cellular components resulting from damaged cells at the edges of the slices. After 3 h of incubation, media were removed and replaced by new media, differing in their composition as described below. Shaking was used throughout the culture to keep a homogeneous solution, facilitate gas exchange  was compared to the LDH activity present in the slices at the end of the experiment.Tissue viability was assessed during two different cultures (two different pups) for the three conditions investigated  by measuring the release of lactate dehydrogenase (LDH) in the culture medium and compare it to the LDH present in the tissues, as reviewed in T = 0 h which corresponds to the start of the lipolytic treatments, after the 3 h of incubation to collect damaged cells contents, T = 15, 39, 63 h). Glycerol was measured using a glycerol assay kit  and Perkin Elmer Victor X3 spectrophotometer. Concentrations were calculated as μmoles per g of tissue.Lipolysis was monitored by quantifying glycerol in culture media at four different times  previously validated for NES .Statistical analyses were performed using JMP PRO 13.0 software.10 to achieve normality. T = 0 h was not taken into account. Variance homogeneity was tested using Levene’s test. Differences between treatments within a particular time or over time within a particular treatment were assessed with linear mixed models (with the pup as a random variable and the time or treatment as fixed variables) and a post hoc Tukey’s test.Distribution normality was tested using Shapiro-Wilk’s test and, when necessary, data were transformed in log10 to achieve normality. Variance homogeneity was tested using Levene’s test. Afterward, a Student t-test (paired data for comparisons between inner and outer blubber and non-paired data for comparisons between early and late fast or between sexes) was applied to compare the means (comparison by treatment).Distribution normality was tested using Shapiro-Wilk’s test and, when necessary, data were transformed in logp < 0.05 for all analyses.The level of statistical significance was set at n = 111). Histological sections were conducted on inner blubber slices from late fasting pups after 63 h of culture. Round adipocytes could be observed, with their nucleus pushed to the side of the cell , two conditions of ß-adrenergic-induced lipolysis  with either high (4.2 g/L) or low (0.9 g/L) glucose concentrations in the medium (“Lipolysis high glucose” LHG and “Lipolysis low glucose” LLG), and a last condition in which the slices were in NL medium between 0 and 39 h and then in LLG medium from 39 to 63 h (NL LLG). Cell viability (measurement of lactate dehydrogenase release in culture medium) was maintained during the 15 h-period as cell death estimates were only 3.2 ± 3.8% for the LLG condition, 2.9 ± 3.0% for LHG condition and 0.8 ± 0.4% for NL condition during that period. Between p < 0.05), whereas it remained constant in the NL condition (T = 15 h to T = 63 h (p < 0.05). Within the two lipolytic conditions, there was no significant difference of glycerol release under low versus high glucose concentration conditions in the medium. In both low and high glucose lipolytic conditions, the highest rate of glycerol release was observed between T = 0–15 h, with progressively slower rates of release between T = 15–39 h and T = 39–63 h (p < 0.05)  .p < 0.05), whereas it did not change for the other conditions  did not vary with sex or blubber depth (inner or outer blubber), either at early or late fast (data not shown). In inner blubber, the total release of glycerol was significantly higher at early as compared to late fast in the LLG condition (nditions . In outenditions .p < 0.05) (p < 0.05), the latter of which did not differ from one another (p < 0.05) but not the one in the LLG condition. At late fast, the difference of leptin production between the NL and the lipolytic conditions (factor 1.5–2) was not significant (inner or outer blubber).Leptin release over time in culture media was analyzed in inner and outer blubber and at early and late fast in the NL, LLG and LHG conditions. Cumulative leptin release in culture media increased significantly over time in NL, LLG and LHG conditions, especially during the first 39 h of culture ( < 0.05) . At earl another . The sitp < 0.05)  did not vary with sex or fasting stage (data not shown). In contrast, the quantity of leptin released by inner blubber slices tended to be higher than the amount released by outer blubber slices. This difference was however only significant for the NL condition at early fast, where a 50% drop of leptin production could be observed between inner and outer blubber, and for the LLG condition at late fast ( < 0.05) .14C glucose into lipids , hormone sensitive lipase (HSL) and monoglyceride lipase (MGL) . Among oin vitro lipolysis protocols (in vivo in NES pups (34.8 to 94.1 pg/mL)   . Insulin1 pg/mL) . NES exh1 pg/mL) , the higNES pups . The lev (1–2 h)  and/or w (1–2 h) . The sti (1–2 h) .in vitro culture of adipose tissue explants of marine mammals were conducted using gray seal blubber  may explain why they exhibited the same response. ant load , 2015. IIn vivo, circulating free fatty acid and glycerol levels of NES increase over the fast while the lipolytic rate appears to remain constant  between feeding and fasting periods of their life cycle .CaD conceived the study and designed field sampling, tissue culture and related experiments with input from DC, LP, ClD, and J-FR. CaD and MV carried out sample collection, tissue cultures and associated biochemical tests. DC supervised sample collection and leptin analyses. CR performed leptin assays. LP and GT conducted data analyses. Tissue culture experiments were carried out in DS’s lab, with all the access to cell culture facilities. CaD and DC were involved in funding acquisition. CaD drafted the manuscript with input from LP and MV, DC, J-FR, DS, YL and GT edited, revised and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Individuals with panic disorder frequently face ongoing symptoms, suboptimal treatment adherence, and increased relapse rates. Although mobile health interventions have shown promise in improving treatment outcomes for numerous mental health conditions, their effectiveness, specifically for panic disorder, has yet to be determined.This study investigates the effects of a mobile-aided case management program on symptom reduction and quality of care among individuals with panic disorder.This 3-year cohort study enrolled 138 participants diagnosed with panic disorder. One hundred and eight participants joined the mobile-aided case management group and 30 in the treatment-as-usual group. Data were collected at baseline, 3-month, 6-month, and 12-month treatment checkpoints using self-report questionnaires, in-depth interviews, direct observation, and medical record analysis.p = 0.008) and state anxiety (p = 0.016) more than the control group at 6 months. Participants who underwent case management experienced enhanced control over panic symptoms, heightened self-awareness, and elevated interpersonal support.During the maintenance treatment phase, the mobile-assisted case management group decreased both panic severity (The mobile-aided case management is beneficial in managing panic disorder, especially maintenance treatment. Panic aCase management is a collaborative approach to coordinating and providing care for individuals with complex needs. It involves assessing, planning, implementing, and evaluating care plans and resources to ensure optimal health outcomes , 3. CaseThe treatment-as-usual (TAU) approach for panic disorder typically consists of pharmacotherapy , 12 and Patients with panic disorder sometimes struggle to consistently follow their prescribed treatment plan, resulting in insufficient adherence and impacting overall effectiveness. In previous studies, the unremitted rate of panic disorder is around 42–46% , 22, andg = 0.38, 95% CI: 0.24–0.52, p < 0.001)  . However< 0.001) .We hypothesized case management brings less recurrent rate or better recovery in anxiety and depressive disorder. In this report, we sought to demonstrate the efficacy of combining mobile-aided service with case management for panic disorder.2.The design of this entire study is initially a prospective cohort study and development plan for the panic prediction model . However2.1.This study enrolled 108 participants from psychiatric clinics at a community hospital between June 15, 2020, and March 22, 2023, to receive mobile-aided case management (MCM) augmented with regular care. The eligibility criteria included being an adult aged 18 or older and having a Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5)  and Mobile-aided Case Management (MCM) with TAU. The aim was to compare the efficacy of these methods in managing panic disorder symptoms. We invited all participants to the MCM-TAU group initially. Those who did not want contact with a case manager, but agreed to regular surveys and clinic follow-ups, formed the TAU group. Due to the limited number of available case managers, participants unable to access this resource also joined the TAU group. To ensure various experiences and detect meaningful differences, we strategically increased participant numbers in the MCM + TAU group.Treatment as Usual (TAU): TAU refers to the standard care provided to patients with panic disorder, typically including a combination of pharmacotherapy and cognitive behavioral therapy. The pharmacological treatment includes escitalopram, sertraline, and venlafaxine as first-line treatment and low dosage augmented use of benzodiazepines.Mobile-aided Case Management (MCM) with TAU: MCM involves integrating mobile technology, such as smartphones and wearable devices, to supplement TAU. This approach provides additional support and patient monitoring through mobile applications and remote contact with healthcare professionals. In this study, MCM was implemented alongside TAU, with patients receiving pharmacotherapy, CBT, and mobile-aided case management services. The MCM component consisted of regular contact with case managers via telephone and a dedicated mobile application. The contact frequency in the MCM group was determined based on each patient’s needs and symptom severity, with a minimum of one contact per week. This contact aimed to monitor the patient’s progress, provide support, and address any concerns or questions about their treatment plan. The wearable devices are smartwatches that collect patients’ daily physical activities, sleep, and heart rate during the study period; we presented the wearable outcome in the previously published article . The curIn treating panic disorder, psychiatrists guide patients through specific self-guided CBT techniques. They help patients understand the disorder’s nature, suggest tracking panic episodes using journals or apps, and teach deep breathing techniques for symptom relief. Exposure therapy is introduced by gradually facing trigger situations. Patients learn to challenge cognitive distortions and remain calm during anxiety peaks. Additionally, psychiatrists recommend having an emergency plan, like deep breathing or contacting someone during sudden attacks.In summary, this study sought to compare the efficacy of TAU and MCM with TAU in managing panic disorder symptoms. Both approaches included pharmacotherapy and self-guided CBT, while the MCM group received additional support through mobile technology and remote case management services.2.4.We investigated the effects between groups using a mixed-methods  approach. For both groups, quantitatively, we examined changes in panic disorder severity , stCase managers assist individuals with panic disorder to stay committed to their treatment plan, ensure they have access to essential services, and support them in preventing relapses. In the MCM group, the contact frequency varies based on each patient’s needs and symptom severity, but at least one contact per week via apps or telephone. This regular contact aims to oversee the patient’s progress, provide support, and address any treatment-related concerns or questions. Additional contacts are tailored based on the case manager’s understanding of the patient. Typically, an extra contact becomes necessary if the weekly PDSS Question One score exceeds 1, indicating that a recurrent panic attack occurred in the previous week, or if the weekly PDSS sum score (ranging from 0–28) surpasses 10, indicating a slight illness. However, the frequency of additional contacts is a joint decision between the case manager and the patient, factoring in the patient’s needs and personal situation. At times, increased life stress or a depressed mood might also necessitate more frequent contact.The practical interventions from case managers include: (1) If a case’s PDSS sum exceeds 10 or PDSS Question One exceeds 1 in the previous week, case managers contact patients using mobile apps or phones to ascertain recent panic attack details: frequency, intensity, and specific somatic symptoms. They can also monitor these symptoms through the app interface presented in The anticipated impact on patients includes: (1) Interactions with case managers offer patients interpersonal support, a crucial factor in mental health management. (2) These interactions make patients more aware of their condition and the dynamics of a panic attack. As a result, they prioritize psychiatric care over frequenting varied internal medicine clinics or emergency departments in search of physiological causes. (3) In terms of medication, patients recognize the significance of drugs, commonly selective serotonin-reuptake inhibitors, and opt for sustained use. (4) Patients learn to relax effectively. Skillful relaxation, before or during a panic attack, or proactive stress management (like ensuring less demanding work schedules for those with stress-induced panic attacks) diminishes the likelihood and severity of subsequent attacks.The participants for the qualitative interviews were selected using a purposive sampling technique. This approach allowed for picking individuals who had completed at least three-month treatments and could provide rich, detailed, and relevant information about their treatment experiences and the support they received. We conducted a semi-structured interview with a guide ; please 2.5.z-test with the numpy  between the MCM-TAU and TAU groups. We did not use the crossectional mean score of both groups because each participant’s initial severity differed at enrollment. Later, we employed a he numpy  and sciphe numpy  modules For qualitative data, our team used thematic analysis for in-depth interviews, direct observation, and medical chart reviews with Python Natural Language Toolkit (NLTK)  library,3.3.1.p < 0.05) exist in covariate-adjusted PDSS change at six and 12 months, STAI-S, BAI, and BDI change at 3 and 6 months.n = 108 vs. n = 30), we used Levene’s Test  = 0.026, p = 0.871], age , GAD , and AG . Only depression displayed a variance concern . Nonetheless, these findings support the use of RMANCOVA. The relevant code was provided in the modified Given the disparity in sample sizes . At 12 months, MCM + TAU’s mean improvement was 6.6 (SD: 1.1) which is better than TAU’s mean improvement of 5.5 (SD: 0.9), and the difference between them was 1.1 .For PDSS at 6 months, the mean improvement for MCM + TAU was −7.5 (SD: 2.2) compared to TAU’s mean improvement of 5.9 (SD: 1.5), resulting in a difference of 1.6 . At 6 months, the mean improvement for MCM + TAU was −17.4 (SD: 0.7) whereas TAU’s was −15.6 (SD: 1.3), yielding a difference of 1.8 .For STAI-S at 3 months, MCM + TAU showed a mean improvement of −12.0 (SD: 1.4) compared to TAU’s mean improvement of −9.7 (SD: 1.7), a difference of 2.3 . At 6 months, the difference widened with MCM + TAU’s mean improvement of −12.8 (SD: 8.1) compared to TAU’s mean improvement of −4.8 (SD: 7.2), a staggering difference of 8.0 .For BAI  at 3 months, MCM + TAU improved by a mean of −8.9 (SD: 2.7), while TAU’s mean improvement was −6.6 (SD: 1.8), with a significant difference of 2.3 . At 6 months, the mean difference increased slightly with MCM + TAU improving by −5.4 (SD: 0.9) while TAU improved by −4.2 (SD: 0.9), resulting in a difference of 1.2 . However, Levene’s Test for depression at baseline indicates a variance concern  that could suggest bias. Thus, this improvement should be interpreted with caution.Finally, for BDI at 3 months, the mean improvement for MCM + TAU was −3.0 (SD: 0.4) against TAU’s − 2.0 (SD: 0.5), a difference of 1.0 (3.2.p = 0.028). For emergency department visits, none have been to the emergency department due to panic attacks in the MCM + TAU group; However, 3 of 10 participants in the TAU group visited the emergency once due to panic attacks. They acknowledged their emergency visit because they were unsure if this was a panic symptom, especially chest pain, and difficulty breathing.Given the appropriate methodologies for smaller sample sizes, distinct patterns emerge from the MCM + TAU and TAU groups. We demonstrated the results of remission status and emergency department visits at 3 months by Fisher’s exact test in A thematic analysis was conducted using the Python NLTK library and NVivo on the qualitative data from semi-structured interviews. This process helped identify frequently occurring words and phrases, allowing us to pinpoint critical themes and sentiments in the participants’ responses. The identified themes from thematic analysis are interpersonal support, reassurance, self-recognition, care coordination, and medical adherence. From in-depth interviews, 18 of the 36 participants from the MCM + TAU group reported high satisfaction with interpersonal support from the case managers through face-to-face or telephone interviews. Twenty participants reported they could manage panic symptoms and felt reassured and improved self-recognition with regular case management. Both groups were satisfied with care coordination. However, the MCM + TAU group reported more access to medical professionals and increased motivation to adhere to treatment plans because of an increased understanding of the panic disorder. Twenty-one of the thirty participants said they were motivated to keep the maintenance phase of treatment despite being remitted from panic symptoms.Each theme was supported by substantial evidence from participant responses. For instance, the theme of Interpersonal support was evident in a response such as: “The support from my case manager has been instrumental in my recovery. Our regular phone check-ins make me feel like I am not alone in dealing with this.” Similarly, the theme of reassurance emerged from comments like: “Having regular case management sessions made me feel more secure about handling my panic symptoms.” The process also illuminated a strong presence of Self-recognition in statements like: “With the treatment, I’ve become more aware of what triggers my panic attacks and how to manage them effectively.” Care coordination was echoed in sentiments like: “The coordination between my case manager and the doctor has been seamless. I’ve been able to get the help I need when I need it.” Lastly, Medical adherence responses such as: “Understanding my disorder and the purpose behind each part of the treatment plan has motivated me to adhere to it.”4.4.1.p = 0.008) and state anxiety (p = 0.016) over TAU at 6 months. The effect for panic disorder severity remains significant at 12 months (p = 0.035). They also felt reassurance and interpersonal support, more understanding of panic disorders, less emergency need, and higher satisfaction with the care. However, the two groups have no significant difference in trait anxiety change at 3 and 6 months.In summary, case management is a practical approach to treating panic disorder, and mobile-aided interventions can be helpful tools for case managers in remotely evaluating patients. Patients with mobile-aided case management show significant improvement in panic disorder severity  in several domains: significant panic and state-anxiety symptoms reduction in the maintenance treatment phase, self-recognition of symptom control, increased adherence to medication, and reduced emergency visits due to panic attacks. Further robust studies should be conducted for confirmation.tsaichanhen@gmail.com.The datasets presented in this article are not readily available because the dataset contains identifiable patients’ private information. Requests to access the datasets should be directed to C-hT, The studies involving humans were approved by En Chu Kong Hospital research plan (ECKH D11101). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.C-hT designed the study and completed the data analysis and writing. MC supported the data analysis and information engineering. FL directed and supervised the project. All authors contributed to the article and approved the submitted version.