The biological and clinical heterogeneity of neuroblastoma (NB) demands novel biomarkers and therapeutic targets in order to drive the most appropriate treatment for each patient. Hypoxia is a condition of low-oxygen tension occurring in poorly vascularized tumor tissues. In this study, we aimed to assess the role of hypoxia in the pathogenesis of NB and at developing a new clinically relevant hypoxia-based predictor of outcome. We analyzed the gene expression profiles of 1882 untreated NB primary tumors collected at diagnosis and belonging to four existing data sets. Analyses took advantage of machine learning methods. We identified NB-hop, a seven-gene hypoxia biomarker, as a predictor of NB patient prognosis, which is able to discriminate between two populations of patients with unfavorable or favorable outcome on a molecular basis. NB-hop retained its prognostic value in a multivariate model adjusted for established risk factors and was able to additionally stratify clinically relevant groups of patients. Tumors with an unfavorable NB-hop expression showed a significant association with telomerase activation and a hypoxic, immunosuppressive, poorly differentiated, and apoptosis-resistant tumor microenvironment. NB-hop defines a new population of NB patients with hypoxic tumors and unfavorable prognosis and it represents a critical factor for the stratification and treatment of NB patients. Neuroblastoma (NB) is a common extracranial solid tumor of the developing sympathetic nervous system, which accounts for roughly 5% of all diagnosed pediatric cancers . PatientEfforts to identify prognostic biomarkers from the genomic interrogation of NB tumors have been made with the aim of improving patient stratification and providing novel therapeutic targets ,2. MolecThe importance of hypoxia in conditioning the aggressiveness of tumors, including NB, is documented by an extensive literature ,15,16,17The tumor microenvironment (TME) is a heterogeneous milieu that is composed by neoplastic, stromal, endothelial, and infiltrating immune cells . The funTelomere maintenance mechanisms (TMM) are adopted by tumor cells to prevent telomere shortening and acquire immortality and they represent a malignant hallmark of several cancer cells . TelomerIn this study, we aimed at assessing the role of hypoxia in the pathogenesis of NB by analyzing the molecular mechanisms and biological effectors that are involved in NB hypoxic TME and at dissecting the prognostic value of a new hypoxia-based predictor in a large multicenter and multiplatform study. Our results show the unfavorable prognostic value of hypoxia in a large number of patients and the ability of hypoxia to additionally stratify clinically relevant groups of NB patients. Furthermore, our results reveal the deregulation of specific biological processes and pathways affecting NB TME. Analyses were carried out using the 11-probe set signature to assess the role of hypoxia in the pathogenesis of NB and to dissect the prognostic value of a new hypoxia-based predictor in a large multicenter and multiplatform study . To thesThe relative percentage of patient outcome was comparable among the four data sets. Age at diagnosis, international NB staging system (INSS) stage, MYCN status, event overall, and event-free data were available for all data sets. Patient follow-up was available for RNA-seq498, Affymetrix413, and Agilent709 data sets, but not for Agilent262, whereas telomere maintenance, ALT, documented regression, TERT rearrangement, ATRX mutation, RAS, and p53 mutation data were only available for the Agilent262 data set.The gene expression profiles of RNA-seq498, Agilent709, and Affymetrix413 data sets were integrated into a single data set to achieve a large-scale genomic data analysis. 577 patients were filtered out from the analysis either because of missing information about outcome or of follow-up shorter than five years . FurtherIt is known that the batch effect may be introduced when data sets from different gene expression platforms are integrated . Batch eThe expression profiles of 236 randomly selected tumors out of 786 (30%) served to build a classifier and the profiles of the remaining 550 tumors (70%) were used to test its prognostic value in a validation data set. The clinical and molecular characteristics of patients in the training and test sets are summarized in We have previously used a biology-driven approach in order to assess the hypoxic status of NB cells identifying a 11 probe set signature that was able to accurately predict NB patient outcome . This sip-value < 0.05; p-value < 0.05; These genes encode for proteins that are involved in metabolic response to hypoxia. Univariate analysis of overall survival in the batch-adjusted training set based on the NB-hop genes showed that high expression of PGK1, PDK1, MTFP1, and FAM162a genes was associated with a significantly higher risk of death (hazard ratio (HR) > 1 and p < 0.0001; The classifier was built from the expression of NB-hop genes and patient outcome in the training set using the LibSVM library and the leave one-out cross validation (LOOCV) technique . The prep value < 0.0001). These findings support our conclusion that the NB-hop classifier is an accurate predictor of NB patients’ outcome. Our classifier obtained a significant overall performance of 44% of Matthew’s correlation coefficient (MCC) (78% of Accuracy) in the test set . For comPatients predicted as UF by NB-hop had a clear different expression profile with respect to those predicted as F NB-hop, demonstrating that NB-hop was able to distinguish two groups of patients at the gene expression level . p < 0.0001). These data indicate that UF NB-hop tumors are more hypoxic than F NB-hop tumors. We also correlated the HIF-1a gene expression and that of each NB-hop marker by Pearson correlation. We found a significant correlation between HIF1a and NB-hop markers (p < 0.05). HIF-1a and HIF-2a are hypoxia inducible factor α-subunits that mediate the cellular response to hypoxia . We compp < 0.0001; The prognostic power of the NB-hop classifier was compared with that of established markers utilizing NB-hop classifier predictions (UF vs. F), INSS stage , age at diagnosis (age ≥ 18 months vs. < 18 months), and MYCN status (amplified vs. single copy) in the test set. UF NB-hop, advanced stages 4, age ≥ 18 months, and MYCN amplification were significantly associated with a higher risk of death or undergoing an event according to a univariate analysis . The NB-hop classifier was not able to significantly stratify patients with amplified MYCN or stage 4s tumors (p > 0.05). The NB-hop classifier significantly stratified patients with tumor stage 1, 2, 3, and 4, patients with age < 18 months, patients with age > 18 months, and patients with not amplified MYCN tumor . In the subgroup of low and intermediate-risk patients with stage 4 tumors lacking MYCN amplification and age < 18 months, NB-hop classifier identified two groups of patients with significant different EFS  . Among tPathway analysis is a well-known bioinformatic tool that is used to explore the biological processes and pathways associated with a list of differentially expressed genes using curated ontologies . The lisUp-regulated genes were mainly involved in the cellular response to hypoxia, telomere maintenance, chromatin remodeling, DNA damage response, P53 mediated cell cycle arrest, cellular senescence, cell cycle, and proliferation. Down-regulated genes were mainly involved in immune response, cell differentiation, motility, inflammation response, cell death, and angiogenesis. These results showed that patients with UF NB-hop tumor are characterized by a hypoxic, immune suppressive, poorly differentiated, and apoptosis-resistant TME. The up- or down-regulation of these genes have been previously associated with an unfavorable prognosis of NB patients, in accordance with our results. Among the up-regulated genes, several were involved in glycolysis , pH regulation (SLC16A1), and homeostasis (PDK1), indicating a metabolic reprogramming typical of hypoxic cells. In addition, a prominent set of up-regulated genes coding for proteins with a primary role in telomere maintenance , DNA damage response , P53 mediated cell cycle arrest , and proliferation . On the contrary, we demonstrated the down-regulation of genes coding for proteins that are involved in immune responses , cell differentiation , cell motility and invasion , and angiogenesis (EPAS1). We also found the down-regulation of genes coding for proteins that are involved in chromatin remodeling (ARID1A and CHD5) and of the MYC gene and the up-regulation of genes involved in cell differentiation , and coding for proteins that are involved in apoptosis and cell death (BIRC5 and TWIST1). p-value < 1.0 × 10−16). Our findings establish a connection between HIF-1a and genes reported in We performed a network analysis using STRING-DB software in order to assess the biological connection between HIF-1a and genes reported in The estimation of the abundance of immune and stromal cell populations in the TME may uncover their role in tumor development/progression . DiffereThe heat map shows a clear tendency to a lower abundance of T cells, CD8+ T cells, NK cells, cytotoxic lymphocytes, B cell lineage, monocytic lineage cells, myeloid dendritic cells, endothelial cells, and fibroblasts in patients with UF NB-hop tumors than in F NB-hop tumors, suggesting the association between an immunosuppressive TME and UF NB-hop tumors. p value < 0.05, p value < 0.05, We carried out additional analyses to provide more links between MCP-counter analysis and hypoxia. For each cell type MCP-counter defines a set of characteristic genes that we refer to as MCP-counter markers . OverlapThe presence of specific immune regulatory cell populations is associated with poor outcome in different types of cancer, including NB . Tumor iOne of the main mechanisms exploited by tumors for immune system escape is modulating immune checkpoints, inhibitory pathways that physiologically maintain self-tolerance, and limit the duration and amplitude of immune responses . We focuWe used the gene expression profiles of 262 untreated primary NB tumors (Agilent262), for the majority of which we had information on the TMM activation in addition to other patient clinical and molecular data, such as age at diagnosis, ALT activation, event overall, event-free, TERT rearrangement, INSS stage, MYCN status, p53/RAS gene mutations, and spontaneous regression, in order to evaluate the association between hypoxia and TMM in NB . Patientp < 0.00001, We, then, analyzed the distribution of TERT mRNA expression in the profiles of patients belonging to the Agilent262 test set that was grouped by NB-hop predictions. We found that TERT mRNA expression was significantly elevated in patients with UF prognosis with respect to those predicted with F prognosis . We investigated the association between hypoxia, TMM, and other available clinical and molecular prognostic factors for NB  in the Ap-value < 0.05, p-value > 0.05, On the contrary, patients with documented tumor regression were associated with a lower odd of UF NB-hop prognosis , a process that allows epithelial cells to adopt a more mesenchymal state with stem-like properties . EMT invCellular proliferation is a hallmark of cancer . A complThe abundance and composition of immune cells that infiltrate the TME is a critical determinant of tumor development, therapy efficacy, and clinical outcome in NB ,97. CytoWe decided to carry out a correlation analysis between HIF-1a and each MCP-counter marker in order to investigate this association in NB. Although network, correlation and MCP-counter analyses are based on different methodological and statistical approaches, results obtained with these methods support the association between immune suppression and hypoxic TME in NB. Because the tumor suppressing/promoting role of several immune markers remains controversial to date, we also assessed the potential suppressive role of the immune markers significantly correlated with HIF-1a. Bibliographic search evidenced that MAL, BANK1, CXCR2, and KCNJ15 genes play a tumor suppressive role in different types of cancer, including colorectal cancer, B-cell lymphoma, and renal cell carcinoma ,94,95,96The high expression of TAM-specific genes was associated with poor five-year event-free survival in not amplified MYCN tumors . In ordeImmune checkpoints are important pathways that are exploited by tumor cells to escape immune system response . We analDespite the therapeutic advances in the last years, a large number of NB patients still undergo a refractory disease . The mecp53 is a transcription factor that is involved in cellular defense against malignant transformation . HypoxiaCell growth arrest promotes cellular resistance to apoptosis, which is associated with an unfavorable prognosis . Our finAltogether, our data indicate that NB patients with hypoxic tumors are potentially more susceptible to acquire resistance to conventional chemotherapeutic drugs than patients with normoxic tumors. We believe that information derived from the assessment of the hypoxic status of the tumors may potentially be used for taking chemotherapeutic decisions in order to improve treatment response and reduce the side effects that are associated with aggressive therapies. Hypoxia is a potential therapeutic target and may offer several therapeutic opportunities in cancer treatment ,151. TheWe used the gene expression profiles of 1882 primary tumor samples from four different cohorts of NB patients ,11,12,13http://www.ncbi.nlm.nih.gov/geo; accession: gse62564) and ArrayExpress  databases. The gene expression profiles from 709 (Agilent709) and 498 (RNA-seq498) tumor samples were generated by Agilent customized 4 × 44 k oligonucleotide microarray  and IlluThe third data set cohort (Affymetrix413) consisted of 413 NB patients that belonged to six sub-cohorts whose gene expression has been profiled using Affymetrix U133 Plus 2.0 GeneChips. Eighty-eight patients were collected by the Academic Medical Center  ; twenty-Gene expression profiles from additional 262 (Agilent262) tumor samples were generated by the Agilent customized 4 × 44 k oligonucleotide microarray . Gene exThe identifiers of the different data sets were converted into the corresponding gene symbols to make them comparable to each other. Probe sets with no gene symbol annotation were excluded from the analysis because they have no multi-platform annotation. Patients that were profiled with more than one platform were considered once in order to prevent that inclusion may introduce optimistic and biased predictions. http://r2.amc.nl), GEO or the BIT-Gaslini biobank. Samples were obtained from primary tumors at the time of diagnosis. Tumor stage was defined according to the INSS stage. The data were retrieved from the R2: genomic analysis and visualization platform (The primary endpoint was the OS defined as the time (in years) from disease diagnosis to patient death or the last follow-up if the patient survived. Alive patients with follow-up less than five-years were filtered out to make our primary endpoint as homogeneous as possible across patients. The secondary outcome was the EFS defined as the time (in years) from disease diagnosis to tumor progression, relapse, or death from disease or the last follow-up if no event occurred. The COMBAT algorithm implemented in inSilicoMerging package  was usedwww.graphpad.com. Exact log-rank test was provided when log-rank p-value was >0.0001. The univariate Cox model was used to assess the prognostic value of the markers when patient follow-up was available. Multivariate analysis with a Cox proportional hazards regression model was used to assess the prognostic effect of hypoxia signature in the context of concomitant effects of other known prognostic factors . The patients with missing data were discarded from the survival analysis. The Survival R package (R 3.1.2) was used for the computation of the Cox regression models. Logistic regression analysis was carried out in order to assess the prognostic value of the markers when only event overall was available. Univariate logistic regression was computed using the bayesglm method implemented in the arm R package. Differential expression analysis considered as significant gene up- or down-regulation with fold change ≥1.5 or ≤1.5 (0.58 in log2) and p value lower than 0.05 after adjustment for multiple hypothesis testing by Benjamini–Hochberg method. We used the MCP-counter method in order to assess the abundance of different tumor-infiltrating immune and stromal cell populations [OS and EFS curves were plotted by the Kaplan-Meier method and they were compared with the log-rank test by GraphPad Prism version 6.0 for Windows, ulations . MCP-couulations . Immune ulations . BioGRIDulations . BioGRIDIn this study, we reported independent evidences of the unfavorable prognostic value of hypoxia in NB and highlighted the role of hypoxia as a condition for the development and progression of NB. We identified NB-hop as a new biomarker that is able to define a new population of patients with hypoxic tumor and unfavorable prognosis, characterized by the activation of telomere maintenance mechanisms and a deregulated hypoxic TME. We believe that hypoxia evaluation should, thus, be considered for NB patient risk stratification and treatment. A prospective study and further in vitro and in vivo assessment of the effects of hypoxia in NB initiation and progression may guide the future development of hypoxia-directed therapeutic strategies.


This study aimed to assess whether the emergency service of a major Swiss dental institution faced different demands  during a lockdown, issued to mitigate the COVID-19 pandemic, compared with the weeks before and after.α=0.05.Data of patients receiving urgent care at a university center for dental medicine  during the 6-week lockdown, pre-lockdown, and post-lockdown periods were retrospectively evaluated. Statistical analysis involved tests for equal proportions and logistic regression models. The level of significance was set at p<0.001). Among all treatments, aerosol-generating procedures dropped from 56.1% (pre-lockdown) to 21.3% during lockdown (p<0.001), while teledentistry follow-ups became more frequent (p<0.001). Patients with comorbidities sought urgent dental care less frequently during the post-lockdown period (p=0.004).The study comprised 3109 dental emergency visits in the period from February 2 to June 5, 2020. Daily caseloads increased during lockdown. Abscesses, orthodontic emergencies, and surgical follow-ups were more common during lockdown, whereas the number of dento-alveolar injuries declined (≤0.048). Urgent dental care provision involved intraoral radiographs more frequently in the pre-lockdown period compared with the following weeks (The lockdown significantly impacted the dental emergency service in terms of patients’ diagnoses, treatment needs, and the characteristics of the urgent care that was delivered.Access to essential dental care must be monitored and safeguarded throughout the COVID-19 pandemic and beyond as deferred care entails risks for serious sequelae and persons with comorbidities may change their dental care-seeking behavior. The delivery of dental care regularly involves close contact to patients, exposure to blood and saliva, and procedures that produce droplets and aerosols. Consequently, it may entail a heightened risk for cross-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) , 4.The appropriate management of dental emergencies is crucial to alleviate the burden faced by hospital emergency departments . The timTo curb the spread of SARS-CoV-2, the Swiss Federal Council gradually imposed public health measures which included restrictions for dental care services. Dental practitioners had to defer or suspend treatments that were not considered urgent in the 6-week period from March 17, 2020, to April 27, 2020. The University Center for Dental Medicine Basel (UZB), a major tertiary center for dental medicine, was responsible for delivering urgent dental care to COVID-19 patients in the northwest of Switzerland (5/26 cantons of Switzerland). Thus far, its emergency service has remained open for patients with urgent dental treatment needs throughout the COVID-19 pandemic.Until an effective treatment or a safe and effective widespread immunization program or both are available, populations remain highly vulnerable to SARS-CoV-2 infections and the resulting disease. When one wave of COVID-19 has abated, there is a substantial risk for viral reintroduction or resurgence and possible subsequent waves ought to be anticipated [The aim of this study was therefore to retrospectively evaluate the dental emergency services that were provided at the UZB during the 6-week lockdown period of wide-ranging public health measures issued by the Federal Council (16 March [starting a midnight] until 26 April 2020). Data on urgent dental care delivered in the 6-week period before and after 17 March and 26 April 2020, respectively, were used to determine if the dental emergency service faced different demands at distinct stages of the pandemic. The demand faced by dental emergency service was defined by the volume and composition of patients seeking urgent care, their treatment needs, and the treatment modalities used in the provision of urgent dental care. Given the urgency of the COVID-19 health crisis and the singularity of the federal lockdown measures, a retrospective study design was chosen. The null hypothesis was that the three time periods assessed would exhibit no difference regarding the patient population, patients’ treatment needs, and the characteristics of urgent dental care provision.The study was conducted in accordance with the regulatory requirements of the Swiss Human Research Act and Human Research Ordinance and it complied with Good Clinical Practice principles. The local Ethics Committee (EKNZ) approved this retrospective study (EKNZ 2020-01512).On their first visit, patients treated at the UZB received information and General Consent (GC) documents from the admission staff or other UZB employees. Patients gave their informed GC by signing the UZB GC form.Located in Basel, Switzerland’s third most populous city with about 179,000 inhabitants, the UZB is a major tertiary center for dental medicine. In 2019 and 2020, the UZB recorded a total of 85,800 and 72,500 dental patient consultations, respectively.During the COVID-19 pandemic, along standard hygiene practices, the UZB has implemented additional infection prevention and control practices, which are outlined in Table The three periods, pre-lockdown, lockdown and post-lockdown, each comprised 27 working days (Monday to Friday) during which urgent dental care was delivered. The timeframes of the three periods used for data retrieval were as follows: pre-lockdown, 07 February 2020 until 16 March 2020; lockdown, 17 March 2020 until 24 April 2020; and post-lockdown, 27 April 2020 until 06 June 2020. For the retrospective assessment, personal and medical data, including medical imaging data, of patients receiving urgent dental care at the UZB in the timeframe from 07 February 2020 until 05 June 2020 were used.Utilization of urgent dental care  at the UZB in the period from 07 February 2020 until 05 June 2020.Inclusion criteria were the following:Data from patients who received scheduled, routine, elective dental care were not used .Exclusion criteria were the following:With data encoding implemented from the outset, data were retrieved in a standardized manner from the individual case files of all patients who were eligible according to the inclusion/exclusion criteria. No additional data were acquired for research purposes. The dataset extracted comprised patient characteristics with epidemiological background data , characteristics of the main urgent dental health problems, and characteristics of the urgent care. The dataset generated and analyzed during the current study is available from the corresponding author on request. Table α = 0.05. All analyses were carried out with the statistical program R version 3.5.1 .The statistical analysis included descriptive statistics of the patient population receiving urgent dental care and of the treatments that were provided during the three time periods  with the corresponding overall significance tests. Logistic regression models adjusted for age and gender were performed in order to predict the binomial data structure  of the clinical parameters. Pairwise comparisons were performed by the subsequent analysis by Tukey contrasts. The level of significance was set at p=0.285). The average length of the journey from the patients’ residence to the UZB increased during lockdown compared with the periods before and after (p<0.001). The percentage of patients with underlying medical conditions decreased over the observation period: Fewer patients with one or more comorbidities sought urgent dental care during the lockdown (p=0.695) and the post-lockdown period (p=0.004). One patient with confirmed and five with suspected COVID-19 were treated during the lockdown period. Table The study included 3109 dental emergency visits of eligible patients who sought urgent dental care in the entire study period from 07 February 2020 until 05 June 2020. The demand for urgent dental care saw an increase during lockdown: The daily patient volume in the emergency service was, on average, 32.9 in the pre-lockdown period, 41.5 in the lockdown period, and 40.8 in the post-lockdown period. The gender balance of patients seeking urgent dental care was slightly skewed towards males. The patients’ mean age, 43.7 years across the three periods, showed no significant differences (p≤0.007). Dental abscesses, orthodontic emergencies, and surgical follow-ups were more common during lockdown. During lockdown, the number of dento-alveolar injuries decreased (≤0.048). The caseload of dento-alveolar injuries was highest in the weeks after lockdown. Restoration fracture and prosthodontic emergencies were a less common reason to seek urgent dental care during lockdown (≤0.013). A significant drop in patients seeking preparatory dental measures  was observed during lockdown und post-lockdown (p≤0.021). The characteristics of patients’ main dental health problems are provided in Table The percentage of patients with acute pain, the main reason for seeking urgent dental care throughout the observation period, was significantly lower during lockdown compared with the pre- and post-lockdown periods (ased ≤0.08. The cap<0.001). No shift towards a more frequent use of extraoral imaging was observed over time. Pharmacological management was more frequent during lockdown (7.4%) in comparison with the period after (4.6%) (p=0.018). During lockdown, 21.3% of all treatments involved aerosol-generating procedures, a drop of 34.8% compared with the pre-lockdown period (p<0.001). Rubber dam was more frequently used during lockdown compared with the post-lockdown period (p=0.004). Restorative and periodontal treatments  were less common in the lockdown period. The lockdown period saw an increase in abscess treatments, simple tooth extractions, surgical follow-ups, and treatments for necrotizing periodontal diseases. Telephone follow-ups, rare across the three periods, showed a slight increase during lockdown (<0.001). During the lockdown and post-lockdown period, four patients with severe fascial space infections requiring hospitalization were recorded, while no such infection was observed before lockdown (p≥0.20). Detailed characteristics of the dental care delivered are reported in Table Urgent dental care provision involved intraoral radiographs more frequently in the pre-lockdown period compared with the following weeks , offers a high level of protection for DHCP even during splatter and aerosol-generating procedures . The weaThis retrospective study has some limitations that need consideration. First, the dataset contained no information on SARS-CoV-2 infections among DHCP, and data on SARS-CoV-2 infections among patients were based on self-reports and a screening that did not involve virological testing. Consequently, this study precludes any conclusion about the impact of the precaution measures regarding cross-infections at the dental emergency service.Second, the retrospective study examined data from a single major dental institution in Switzerland. Its findings should be interpreted in the context of demographic aspects, dental public health, and the government response to the COVID-19 pandemic because these factors likely affect the demands that dental emergency services face.Third, thanks to rapid advances in COVID-19 research, the list of underlying medical conditions considered to increase the risk for severe illness from COVID-19 has seen some changes over the past months. The list of underlying medical conditions used during data extraction for this study was not exhaustive and it reflects the known risk factors at that time rather than those currently known. For example, no information on patients’ body mass index or other indicators for overweight or obesity was acquired in their standard medical history.Fourth, no data were gathered on patients’ acceptance of the special precaution measures or on characteristics of their dental health seeking prior to their visit to the UZB. Further research focusing on the perspective of patients is required to gain insight into patients’ experience of seeking and receiving dental care during health crises such as the COVID-19 pandemic. In addition, survey or focus group data are needed to assess and draw lessons from the experience of DHCP delivering dental care under such unprecedented circumstances.Within the limitations of this retrospective study, it can be concluded that the dental emergency service faced different demands at distinct phases of the COVID-19 pandemic. The nationwide lockdown significantly impacted the dental emergency service in terms of patients’ treatment needs and the characteristics of the urgent dental care that was delivered. Persons with comorbidities changed their dental care-seeking behavior during lockdown and beyond. During health crises such as the COVID-19 pandemic, which put restrictions on dental healthcare services, it is therefore important to monitor and confirm equitable access to essential dental care.


Current recommendations for diagnostic imaging for moderately to severely ill patients with suspected coronavirus disease 2019 (COVID-19) include chest radiograph (CXR). Our primary objective was to determine whether lung ultrasound (LUS) B-lines, when excluding patients with alternative etiologies for B-lines, are more sensitive for the associated diagnosis of COVID-19 than CXR.This was a retrospective cohort study of all patients who presented to a single, academic emergency department in the United States between March 20 and April 6, 2020, and received LUS, CXR, and viral testing for COVID-19 as part of their diagnostic evaluation. The primary objective was to estimate the test characteristics of both LUS B-lines and CXR for the associated diagnosis of COVID-19. Our secondary objective was to evaluate the proportion of patients with COVID-19 that have secondary LUS findings of pleural abnormalities and subpleural consolidations.We identified 43 patients who underwent both LUS and CXR and were tested for COVID-19. Of these, 27/43 (63%) tested positive. LUS was more sensitive , 71.1–97.0) for the associated diagnosis of COVID-19 than CXR . LUS and CXR specificity were 56.3%  and 75.0% , respectively (p = 0.453). Secondary LUS findings of patients with COVID-19 demonstrated 21/27 (77.8%) had pleural abnormalities and 10/27 (37%) had subpleural consolidations.Among patients who underwent LUS and CXR, LUS was found to have a higher sensitivity than CXR for the evaluation of COVID-19. This data could have important implications as an aid in the diagnostic evaluation of COVID-19, particularly where viral testing is not available or restricted. If generalizable, future directions would include defining how to incorporate LUS into clinical management and its role in screening lower-risk populations. Novel coronavirus, SARS-CoV-2, is responsible for causing the coronavirus disease 2019 (COVID-19). With an estimated case fatality rate of 1%, COVID-19 has resulted in over 305,000 deaths worldwide to date.5When compared to CXR, lung ultrasound (LUS) may offer improved diagnostic accuracy in the evaluation of patients with suspected COVID-19 pneumonia. LUS has a high sensitivity and often out-performs CXR in the diagnosis of other pulmonary infections.10LUS is a safe, readily available tool that can be employed by EPs to provide real-time clinical assessment for COVID-19. Lab testing utility is hampered by delays in results, accuracy, and availability. CXR may miss pulmonary disease, and the ACR has cautioned against routine screening with chest computed tomography (CT), citing concerns of poor specificity of ground-glass opacities for COVID-19 as well as infection control procedures necessary to decontaminate the CT scanner.Our primary aim was to determine whether detection of B-lines on LUS, among patients without alternative etiologies for their presence, is more sensitive for the diagnosis of COVID-19 than CXR. Our secondary aim was to evaluate the proportion of patients with COVID-19 that have secondary LUS findings of pleural abnormalities and subpleural consolidations.This was a retrospective, observational, cohort study of patients undergoing COVID-19 testing . This study had institutional review board approval and was conducted based on Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines and best practices for retrospective reviews.11What do we already know about this issue?Lung ultrasound (LUS) has been shown to outperform chest radiograph (CXR) in its ability to detect abnormalities with non-coronavirus disease 2019 (COVID-19) pulmonary infections.What was the research question?To determine if B-lines detected by LUS are more sensitive for the associated diagnosis of COVID-19 than an abnormal CXR.What was the major finding of the study?B-lines detected by LUS were more sensitive for the associated diagnosis of COVID-19 than an abnormal CXR.How does this improve population health?In locations where viral testing is not available or has significant delays, LUS may provide important information for the evaluation of suspected COVID-19.This investigation was performed at a large urban academic ED in the United States with >140,000 visits per year. The ED is associated with an emergency medicine residency and clinical ultrasound fellowship, and has six dedicated portable ultrasound machines . All ultrasound studies are transferred wirelessly and stored in QPATH . There was no formal education for LUS specific to COVID-19; however, all physicians have had structured training in LUS. All physicians were provided literature from a small study of 20 patients with COVID-19 that had 12 lung zones evaluated with ultrasound, which found 75% of patients had abnormal LUS findings at the posterior lung bases.All ultrasound studies completed in the ED between March 20, 2020–April 6, 2020, were reviewed for LUS imaging. We reviewed the electronic health record (EHR), EPIC  to determine whether COVID-19 testing was performed. Subjects were included for evaluation if they had a COVID-19 test performed during the index hospitalization or within two weeks of the LUS examination. At the hospital during this time period, COVID-19 testing was performed only on people with symptoms concerning for disease, and no routine screening practices were in place. However, performance of viral testing was at physician discretion, and those without viral testing were excluded from analysis. We also excluded subjects if they did not have a CXR. Lastly, based on EHR review from patient history or physician documentation, patients were excluded if they had reasons for alternative causes of B-lines , as it would not be possible to determine the etiology of the abnormal ultrasound results.All lung ultrasounds were reviewed by two expert EPs, both with clinical ultrasound fellowship training (JRP and KCM), who were blinded to COVID-19 results. When disagreements occurred, a third ultrasound fellowship-trained, blinded independent expert reviewer adjudicated (MML). LUS were scored as positive or negative after review of all images. Subjects were considered to have a positive LUS if any B-lines were detected. The reviewers further graded positive ultrasounds as having 1–2 B-lines or ≥3 B-lines.The images were subsequently evaluated for subpleural consolidations and pleural abnormalities –E. We deAfter LUS scoring and data collection, clinical data including demographics, co-morbidities, vital signs, and laboratory values, was collected from the EHR by two investigators (JRP and FS) using a standardized abstraction technique and entered into REDCap.The primary outcome measure was the sensitivity of LUS compared to CXR for the detection of COVID-19, using the RT-PCR laboratory test as the reference standard. Secondary outcome measures were the proportion of additional secondary LUS findings  detected.A sample size of 43 patients with an estimated sensitivity of 40% for CXR and 70% for LUS yields 81% power with an alpha of 0.05 assuming 70% disease prevalence. We used an estimated sensitivity of 40% based on results of CXR findings in influenza, as the referenced paper of 69% was not available at the time this study was designed.A total of 304 ultrasound studies were completed over the 18-day study period . Of thesThe sensitivity and specificity of B-lines on LUS associated with COVID-19 were 88.9%  and 56.3% , respectively. The association between CXR and COVID-19 results had a sensitivity and specificity  of 51.9%B-lines were more frequently detected in patients with COVID-19 . Of the 27 patients with confirmed COVID-19 infection, 21 had pleural abnormalities (77.8%) and 10 had subpleural consolidations (37%). Of the 16 subjects without COVID-19, three had pleural irregularities (18.8%) and two had subpleural consolidations (12.5%).There was a mean of 6.2 LUS images recorded per patient, which was not significantly different between COVID-19 results, and a median of 6 LUS images taken per patient. Images were more frequently obtained with a curvilinear probe 37/43, (86%), than the phased array probe, 6/43 (14.0%). Of the LUS studies, 8/43 (18.6%) were completed by residents or physician assistants, 4/43 (9.3%) by an ultrasound fellow, 17/43 (39.5%) by ultrasound faculty, and 14/43 (32.6%) by non-fellowship trained EPs. Of the CXRs performed, 42/43 (97.7%) were performed as portable examinations. The one 2-view CXR was a false negative.To our knowledge this is the first study to evaluate the test characteristics of LUS for COVID-19. We also are the first to compare the diagnostic performance of LUS to the more conventional use of CXR. Although preliminary, this work provides important results for the application of LUS for detection of COVID-19. This investigation offers compelling evidence that B-lines detected by LUS are more frequently associated with COVID-19 than an abnormal CXR. This finding is in line with the performance of LUS in other pulmonary disease entities.10We used RT-PCR as the reference standard for diagnosis of COVID-19. However, it is known that the test characteristics of RT-PCR are dependent on collection technique, timing in disease process, and processing technique. In our population there were two negative RT-PCR tests that were positive on repeat testing. Both patients with initially negative RT-PCR tests had positive LUS findings; thus, it is possible LUS is more sensitive than RT-PCR for COVID-19. Further research would be necessary to substantiate this theory.Our study reports a sensitivity of 52% for CXR, which is lower than the reported 69% for portable CXR. It is unknown whether the radiologists in that previous study were blinded, and it is also unclear how body mass index or other variables may have resulted in our reported lower sensitivity for CXR. It is unknown how two-view CXRs would perform for the detection of lung involvement from COVID-19, as it might outperform portable CXR. However, given the infectious nature of COVID-19 portable CXR is the recommended diagnostic test for patients with suspected COVID-19, and these results demonstrate a generally low sensitivity.Evidence that LUS is more sensitive for the associated diagnosis of COVID-19 than CXR has potential global implications. These results may be of particular importance to settings with significant delays in viral RT-PCR testing, settings in which RT-PCR testing is restricted or not available, or where CXR or CT are not accessible. Further scientific investigation could determine how LUS at the time of initial evaluation may aid the physician in counseling patients with regard to findings suggestive of COVID-19. Our investigation provides important new data for the role of LUS relative to CXR for patients being evaluated for COVID-19.Conversely, LUS did have a lower specificity than CXR. As noted, 1–2 B-lines may be non-pathologic; however, only one patient in this study was found to have 1–2 B-lines that did in fact have COVID-19. It is possible that using LUS with only one or two B-lines to direct care for patients suspected of having COVID-19 could lead to unnecessary isolation or further medical testing. Additionally, there are other etiologies for LUS B-lines, and our results will likely be most valuable when interpreted in the clinical context of the medical evaluation.Physicians should have an estimation of pretest probability when performing and interpreting diagnostic testing, and LUS for COVID-19 is no exception to this rule. In this population with a high prevalence of disease (as judged by RT-PCR results), a positive LUS was a good predictor of disease. Further work is necessary to better delineate how to incorporate these findings into screening for asymptomatic patients, diagnostic algorithms, and clinical management strategies.Since this was a retrospective study, it is unclear why physicians chose to perform both CXR and LUS. It is also unknown whether the result of either diagnostic test affected the physician’s choice to perform the other test. Additionally, the treating physician was not blinded to the patient’s history, exam, or CXR. It is possible that knowledge of these data points would change the extent to which the physician performed their LUS. Despite this, there were a similar number of images recorded for patients with and without COVID-19.Over half of the studies performed were performed by non-fellowship trained EPs. Further work is needed to validate these findings in a population of EPs without fellowship training. Identification of B-lines is a core skill of EPs; therefore, we anticipate the findings would be similar.Another limitation was the use of RT-PCR for the diagnosis of COVID-19, as it likely misses some cases. Some of the tests classified as false positive may have actually been true positives. RT-PCR was chosen as the reference standard since that is what is currently used at our, and most, institutions nationally, and viral culture is not feasible at this time. Inconclusive CXRs were scored as negative, which might favor the analysis toward LUS. This was done, in accordance with STARD guidelines, because inconclusive CXRs do not provide diagnostic guidance in real time.11We used B-lines in this study as a reliable marker for COVID-19. It is possible a comprehensive evaluation including pleural abnormalities and subpleural consolidations would improve the test characteristics of LUS. We chose to only include B-lines for our assessment as B-lines are already familiar to EPs and would be easier to implement. We included any number of B-lines (one or more) as abnormal; however, it has been reported 1–2 B-lines may not be pathologic. We selected this approach to maximize the sensitivity of LUS at the cost of specificity.This investigation provides evidence that LUS is more sensitive for the associated diagnosis of COVID-19 than CXR when excluding patients with other expected causes of B-lines. This work could have important implications where viral testing is restricted or alternative diagnostic imaging is not available. Further work may find LUS for the evaluation and care of COVID-19 patients to be of clinical benefit and may also have a role to guide testing as screening and contact tracing are expanded.


Currently, most state-of-the-art therapeutics for treating autoimmune diseases or preventing allograft rejection work either by general immunosuppression or blocking inflammatory reactions and are non-specific. Hence, these approaches cannot provide satisfactory long-term results, let alone a cure. However, in animal models the therapeutic potential of Treg expansion for inducing effective tolerance has now been demonstrated in various models of autoimmunity and allogeneic transplantation. Here, we focus on therapies for increasing the size of the Treg pool by expanding endogenous Treg numbers in vivo or by adoptive transfer of Tregs. In particular, we discuss IL-2 based approaches  for inducing Treg expansion in vivo as well as cell-based approaches  for adoptive Treg therapy. We also mention new questions arising from the first clinical studies on Treg therapy in the fields of transplantation and autoimmunity.Induction of immune tolerance is the Holy Grail in transplantation medicine and autoimmunity. Currently, patients are required to use immunosuppressive drugs for the rest of their lives, resulting in unwanted side effects and complication from global suppression of the immune response. It is well established that regulatory T cells (Tregs) are critical for the maintenance of immune tolerance towards self-antigens by several mechanisms of immune regulation, in parallel with intrathymic deletion of self-reactive T cells during ontogeny. Therefore, approaches for increasing Treg numbers or function  Furthermore, Treg cells have since been demonstrated to be impaired in a variety of autoimmune settings as manifested by a reduction in Treg cell numbers, function, or survival  after haematopoietic stem cell engraftment (HSCT), Tregs have also been prepared from umbilical cord blood  are used as the starting population for rd blood . For HSCrd blood .Do transferred Tregs need to be antigen specific? Although Tregs have to be activated to express their suppressor function, once activated their function is largely non-specific . For treSeveral reports suggest, that the superiority of antigen specific Tregs is due to specific homing and activation in lymph nodes . HoweverThe current methods for preparing polyclonal and antigen specific Tregs are considered below.+CD25+ T cells could rescue mice from organ-specific autoimmune diseases and GvHD-like wasting disease .The potency of polyclonal Tregs for tolerance induction by adoptive cell therapy was shown many years ago by Sakaguchi and co-workers who showed that injections of sorted CD4 disease . These feic HSCT . Thereaf potency . In our + CD25+ cells with cross-linked anti-CD3/CD28 antibodies and IL-2 in vitro for 1–6 weeks, with most protocols using rapamycin to prevent Teff proliferation [The ONE study  .TCR-transduced Tregs can be rapidly generated from polyclonal Tregs in large numbers. This is clearly an advantage over expanding Tregs from rare antigen-specific precursor cells, which can take up to 6 weeks of tissue culture. Moreover, TCR-transduced Tregs have been shown to migrate to the target tissue where they exert both antigen-specific and non-specific suppressive effects . As mentWith their capacity to recognize native antigen while retaining typical T cytolytic activity, CAR T cells are an invaluable tool for treating hematologic malignancies . With fuIt should be noted that, for both TCR-transduced and CAR Tregs, their production requires retroviral transduction techniques. Hence, their safety for therapy is receiving scrutiny.in vivo by various methods. This approach increases the Treg : Teff cell ratio and allows polyclonally-expanded Tregs to mediate non-specific immunosuppression. This method is simpler and less expensive than adoptive Treg therapy. For organ transplantation, both methods are generally limited to situations where the organ concerned is taken from a living donor.As an alternative to adoptive Treg therapy, Tregs can be selectively expanded in vivo induction of Tregs have been described and date back to initial studies on the capacity of anti-CD3 antibody to prolong allograft survival. Thus, treatment with anti-CD3 antibody is now known to induce Treg cell expansion while selectively depleting T effector cells, both in pre-clinical murine . As discussed below, this problem can be avoided by using IL-2/antibody complexes or IL-2 fusion proteins.Another problem with IL-2 therapy is that CD25 expression is not unique to Tregs and is also expressed at a lower level on activated T cells. Hence, even in low doses, IL-2 based therapies may cause some level of effector T cell stimulation in addition to Treg expansion.in vivo showed that injecting mice with IL-2 bound to particular IL-2 mabs, notably JES6-1, led to selective expansion of Tregs and the onset of immunosuppression  pression . The cappression . Thus, spression , 52.Despite prolonging the survival of islet allografts, IL-2 mab complexes failed to augment the survival of skin allografts as monotherapy . HoweverAs with pegylation and binding to albumin, fusing IL-2 with the Fc portion of IgG (IL-2-Fc) retards excretion and thereby considerably increases the half-life of IL-2. For low-dose IL-2 therapy, IL-2 fused to non-FcRγ binding human IgG1 has been shown to be superior to unmodified IL-2 for the induction of Tregs in a non-human primate model .Based on the binding interaction of IL-2 with IL-2 mabs, IL-2 can be mutated to selectively stimulate either CD8 T cells and NK cells or Tregs . For TreIt is now more than a decade since the first clinical trial using adoptive Treg therapy for the treatment of GvHD after allogeneic HSCT , which wvia Treg therapy will soon be available for several types of autoimmune disease. Data on long-term results are still very limited, however, and many questions remain open, including which cell product/expansion strategy is optimal in terms of cell yield/specificity/potency, and the number and frequency of Treg cell injections required for efficient tolerance induction. Hopefully, the ongoing clinical trials will resolve these questions.Results of published and ongoing clinical trials in the setting of autoimmunity are summarized elsewhere . NotablyFor solid organ transplantation, a large number of clinical trials using Treg therapy for tolerance induction are ongoing. These trials are summarized in In these studies, Treg isolation under GMP conditions was carried out with the “CliniMACS” system  which involves clinical-scale magnetic enrichment of cells (CD8 depletion followed by CD25 enrichment) in a closed and sterile system, or, outside European Union countries, by flow sorting of cells for expression of CD4, CD25, and CD127 (yielding populations of >99% purity). For preparation of antigen specific Tregs, magnetic bead isolation may be superior to flow-sorting of Tregs because contamination with residual antibodies impairs Treg expansion , perhapsThe results of these trials will clearly be awaited with great interest. The results are not easy to predict because, in contrast to mice, inducing tolerance in humans is proving to be especially difficult. This issue is discussed below.For autoimmune diseases, Treg based therapies are promising and have shown initial success for treatment of T1D, SLE and inflammatory bowel disease [, 60 and Despite the importance of Tregs, long-term tolerance to all antigens, including alloantigens, requires efficient elimination of reactive T cells in the thymus during ontogeny . In pre-These clinical data are intriguing and are in line with comparable studies in various preclinical models , 74. Ovein vivo show considerable promise  and grants from the National Health and Medical Research Council of Australia to JS.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.


Drought is a major limiting factor of crop yields. In response to drought, plants reprogram their gene expression, which ultimately regulates a multitude of biochemical and physiological processes. The timing of this reprogramming and the nature of the drought-regulated genes in different genotypes are thought to confer differential tolerance to drought stress. Sorghum is a highly drought-tolerant crop and has been increasingly used as a model cereal to identify genes that confer tolerance. Also, there is considerable natural variation in resistance to drought in different sorghum genotypes. Here, we evaluated drought resistance in four genotypes to polyethylene glycol (PEG)-induced drought stress at the seedling stage and performed transcriptome analysis in seedlings of sorghum genotypes that are either drought-resistant or drought-sensitive to identify drought-regulated changes in gene expression that are unique to drought-resistant genotypes of sorghum. Our analysis revealed that about 180 genes are differentially regulated in response to drought stress only in drought-resistant genotypes and most of these (over 70%) are up-regulated in response to drought. Among these, about 70 genes are novel with no known function and the remaining are transcription factors, signaling and stress-related proteins implicated in drought tolerance in other crops. This study revealed a set of drought-regulated genes, including many genes encoding uncharacterized proteins that are associated with drought tolerance at the seedling stage. Drought is one of the most important abiotic stresses that adversely affects plant growth and productivity (reviewed in ). It is Sorghum bicolor (L.) Moench), a C4 crop plant used for food, feed, and fiber, is one of the best-adapted cereals to water-limited environments and ranks amongst the most drought-tolerant of all crops grown in the US .,69.22,69In this study, we identified a set of genes that are differentially expressed only in resistant genotypes in response to PEG-induced drought. This set of genes encodes known transcription factors and other proteins  that are implicated in drought responses and also many novel proteins with no known function. Bioinformatics analysis indicated potential regulatory elements in these genes and cognate transcription factors that may modulate the expression of these genes. Future functional studies with the uncharacterized genes that are expressed only in the drought-resistant genotypes will further our understanding of drought tolerance in sorghum at the seedling stage and help in developing drought-resistant crops. Increased expression of rapidly induced transcription factors identified in drought-resistant genotypes in drought-sensitive genotypes through genetic engineering approaches could lead to the generation of drought-resistant cultivars.Sorghum bicolor genotypes  were obtained fromCourtney Jahn, Department of Bioagricultural Sciences and Pest Management at Colorado State University and other genotypes  were obtained from United States Department of Agriculture (USDA). Seeds of the seven cultivars were propagated in a greenhouse at 16/8 h light/dark cycle, 26 °C and light intensity 100–120 microeinstein·m−2·s−1.Seeds of To evaluate the drought tolerance of these genotypes at the seedling stage we compared the root length of seedlings grown vertically on MS and MS-containing polyethylene glycol (PEG)-8000  agar plates with water potential of –0.5 MPa according to . DroughtFor drought treatment, seeds of the selected four genotypes were surface sterilized with 20% bleach, rinsed with distilled water, and germinated on wet filter paper for 24 h. The seedlings were then transferred to culture tubes (two seedlings per tube) and allowed to grow vertically on 0.5× Hoagland nutrient solution-wetted 3 M filter paper bridge for an additional 7 days as described . On the Total RNA was extracted using miRNeasy Mini Kit  and treated with DNase I using in-column DNase I digestion as described in the user’s manual. The integrity of RNA was checked using Bioanalyzer  and RNA samples with RNA integrity number (RIN) ~9.0 or more were used for further analyses. Poly (A) mRNA enrichment and construction of stranded mRNA-seq libraries were performed using KAPA Stranded mRNA-seq kit (Cat# KR0960) . Briefly, poly (A) mRNA was captured using magnetic oligo (dT) beads and fragmented using heat and magnesium. First-strand cDNA was synthesized using random primer and the second strand was synthesized in the presence of dUTP. Adenosine was added to the 3′-end followed by adaptor ligation. The library was amplified using high fidelity, low-bias PCR. Single-end sequencing of the libraries was done at the Sequencing and Analysis Core Resource, Duke University using Illumina Hi seq 2000 .https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). To get rid of adapter sequences and other artifacts, fastx-trimmer (from FASTX-toolkit at http://hannonlab.cshl.edu/fastx_toolkit) was used to trim the first 10 and last 3 bp from each read, respectively. After filtering, the remaining reads were aligned to the Sorghum bicolor version 3 reference genome using TopHat2 [The RNA-seq reads generated by Illumina sequencing were first analyzed for quality control using FastQC . For protein-coding genes, EdgeR [p-values were applied with q-value adjustment using the Benjamini–Hochberg method. Moreover, we used the log fold change value of 2 (logFC2) as the threshold for differential gene expression. To assess replicate reproducibility (same- and cross-condition replicate correlations) we used Cufflinks  to compus, EdgeR  was usedCircos  plots wePromoters were defined as 1000 bp regions upstream of the transcription start site of the up- and down-regulated genes. Next, the DNA sequences were extracted from the identified promoter regions. For motif discovery, MEME suite  was used


There is considerable state-level variation in the incidence of dialysis-requiring acute kidney injury (AKI-D). However, little is known about reasons for this geographic variation.National cross-sectional state-level ecological study based on State Inpatient Databases (SID) and the Behavioral Risk Factor Surveillance System (BRFSS) in 2011. We analyzed 18 states and six chronic health conditions , and chronic obstructive pulmonary disease [COPD]). Associations between each of the chronic health conditions and AKI-D incidence was assessed using Pearson correlation and multiple regression adjusting for mean age, the proportion of males, and the proportion of non-Hispanic whites in each state.The state-level AKI-D incidence ranged from 190 to 1139 per million population. State-level differences in rates of hospitalization with chronic health conditions (mostly < 3-fold difference in range) were larger than the state-level differences in prevalence for each chronic health condition (mostly < 2.5-fold difference in range). A significant correlation was shown between AKI-D incidence and prevalence of diabetes, ASHD, and COPD, as well as between AKI-D incidence and rate of hospitalization with hypertension. In regression models, after adjusting for age, sex, and race, AKI-D incidence was associated with prevalence of and rates of hospitalization with five chronic health conditions--diabetes, hypertension, CKD, ASHD and COPD--and rates of hospitalization with cancer.Results from this ecological analysis suggest that state-level variation in AKI-D incidence may be influenced by state-level variations in prevalence of and rates of hospitalization with several chronic health conditions. For most of the explored chronic conditions, AKI-D correlated stronger with rates of hospitalizations with the health conditions rather than with their prevalences, suggesting that better disease management strategies that prevent hospitalizations may translate into lower incidence of AKI-D. Dialysis-requiring acute kidney injury (AKI-D) is an important cause of morbidity and mortality in hospitalized patients and is associated with a large public health burden. We have recently reported considerable geographic variation in incidence of AKI-D in the United States –3 with sWe hypothesized that geographic variation in prevalence of certain chronic health conditions predisposing either directly or indirectly to acute kidney injury may be an important contributor to the observed geographic variation in AKI-D incidence. Candidate chronic conditions include diabetes mellitus (diabetes), hypertension, chronic kidney disease (CKD), arteriosclerotic heart disease (ASHD), cancer, and chronic obstructive pulmonary disease (COPD).Furthermore, since AKI-D occurs only among hospitalized patients , we hypothesized that geographic variation in the hospitalization rates of patients with the chronic health conditions is also potentially important.To explore these associations, we analyzed data from 18 states derived from two community representative US databases—the State Inpatient Databases (SID)  and the We conducted a national, cross-sectional, ecological study among populations aged 45 years or older in the United States. We limited our study to adults aged 45 years or older because AKI-D incidence and chronic health conditions prevalence and hospitalizations are low in younger populations.Based on the existing literature, we selected a priori which chronic health conditions to study as candidates to correlate with AKI-D incidence. We included the top causes of ESRD , the top morbid conditions accompanying of ESRD (as they may act as triggers of acute on chronic kidney disease), and possible risk factors for AKI-D from previous study , 6, 7. SState-level prevalence estimates of chronic health conditions were obtained based on self-report data from the BRFSS in 2011. The BRFSS is the nation’s premier system of health-related telephone surveys that collect data at the state and local levels from US residents regarding their health-related risk behaviors, chronic health conditions, and use of preventive services , 8. ThesThe BRFSS questions and answer choices for these six chronic health conditions are listed in Supplementary Table We used the State Inpatient Databases (SID) to determine the number of AKI-D hospitalizations , 4. We tAKI-D was defined when there was both an acute renal failure diagnostic code  and a dialysis procedure code  without concurrent arteriovenous fistula creation or revision procedure codes  . Prior sDue to cost reasons, we purchased only 25 out of the 30 SID available in 2011: Arizona (AZ), Arkansas (AR), California (CA), Colorado (CO), Florida (FL), Iowa (IA), Kentucky (KY), Massachusetts (MA), Maryland (MD), Maine (ME), Michigan (MI), Mississippi (MS), North Carolina (NC), Nebraska (NE), New Jersey (NJ), New Mexico (NM), New York (NY), Nevada (NE), Oregon (OR), Rhode Island (RI), South Carolina (SC), Utah (UT), Vermont (VT), Washington (WA), and West Virginia (WV)  SID databases reported > 6 procedure codes. In none of these 19 states did more than a tenth of the hospitalizations have > 6 procedure codes In our primary analysis, to reduce ascertainment bias, we did not include SID databases with fewer than 15 diagnostic codes  and we only analyzed the first 15 diagnostic codes listed for each hospitalization  on discharge but not on admission. Our primary analysis did not include states  in which we could not tell whether ESRD was an admission diagnosis or a discharge diagnosis [or both])   and excluded all hospitalizations containing a diagnostic code for ESRD (585.6) since for five states , we could not tell whether a this diagnosis was already present on admission .We identified and compiled hospital discharges related to each chronic health condition and calculated rates using estimated state-level patient populations. We hypothesized that hospitalization rate with each chronic health condition could be influenced driven by patients with that chronic health condition who had more severe disease or had complications that needed to be treated in the hospital.Specifically, we used Clinical Classifications Software (CCS) in SID which collapses the > 14,000 standardized diagnosis codes in the International Classification of Diseases-Ninth Revision-Clinical Modification (ICD-9-CM) into a smaller number of clinically significant categories . Each onState-level AKI-D incidences were expressed as per million population (pmp) as described previously , 12, 14.We computed the chronic condition prevalence estimates from the BRFSS and the resident population from the 2011 US Census Bureau . We calcTo obtain prevalence estimates and associated confidence intervals (CIs), we used complex sample survey design specifying strata, clusters and sample weights for the combined landline telephone and cellular telephone data  .Pearson correlation coefficients were used to determine i) the state-level correlation between prevalence of each chronic health condition and AKI-D incidence, and ii) the state-level correlation between rate of hospitalization with each chronic health condition and AKI-D incidence.p-values from the linear regression models were used to gauge which chronic health condition may have had the greatest influence on AKI-D incidence. We also compared adjusted R-squared values in different models for each chronic health condition to evaluate whether differences in prevalence or rate of hospitalization with some chronic health conditions could potentially account for more of the observed state-level variation in AKI-D incidence.Linear regression analysis was performed to determine the state-level association between the prevalence of or rate of hospitalization with each chronic health condition and AKI-D incidence, adjusting for mean age, the proportion of males, and the proportion of non-Hispanic whites in each state. Size of parameter estimates and In all regression models, we checked the linearity assumption. If the relationship between an exposure of interest and the outcome appeared non-linear, we added a quadratic term into the model and displayed our results graphically.As a negative control, we examined skin cancer as a chronic health condition that we hypothesized would not have any correlation or association with AKI-D incidence. Information on skin cancer was also based on self-report in BRFSS  and verified independently by a separate analyst using SAS version 9.4 .. Estimated prevalence of the six chronic health conditions of interest varied from state to state . Specifically, rates of hospitalization with diabetes ranged from 252 to 438 per 1000 patients (1.7-fold difference), whereas that with hypertension ranged from 153 to 278 per 1000 patients (1.8-fold difference), that with CKD 360 to 1144 per 1000 patients (3.2-fold difference), that with ASHD 289 to 547 per 1000 patients (1.9-fold difference), that with cancer from 197 to 384 per 1000 patients (1.9-fold difference), and that with COPD from 213 to 473 per 1000 patients (2.2-fold difference).Note the 6-fold range difference in AKI-D incidence among states (1139/190 pmp) is more than the state-level variation in chronic health condition prevalence (mostly < 2.5-fold difference in range) or state-level variation in chronic health condition hospitalization rate (mostly < 3-fold difference in range).p = 0.01, Fig. p = 0.01, Fig. p = 0.02, Fig. p = 0.15), CKD .At the state-level, a significant correlation was shown between AKI-D incidence and prevalence of diabetes , CKD , cancer , and COPD .A significant correlation was also shown between AKI-D incidence and rate of hospitalization with hypertension . Generally, these relationships were adequately captured in a linear model. For example, for each 1 per 1000 higher rate of hospitalization with diabetes, we estimated there was a 3.0 (95% CI: 1.8–4.1) pmp higher incidence of AKI-D in the state. For cancer, states with higher hospitalization rates also had higher AKI-D incidence but the association was not linear and is more accurately shown graphically in Fig. 2 than models with disease prevalence as the main exposure   may influence geographic distribution of AKI-D.We acknowledge that firm conclusions cannot be made by ecological studies and correlation is not causation. But, we believe that it is very plausible state-level variations in these chronic health conditions are patho-physiologically relevant since these are known to be risk factors for AKI. For example, diabetes has been recognized as a risk factor for contrast-associated nephropathy and other forms of AKI-D , 20. HypWe believe our conclusions are strengthened by the findings regarding skin cancer, our negative control . We had hypothesized that state-level variation in skin cancer prevalence and hospitalization rate should not correlate or be associated with state-level AKI-D since skin-cancer is not a known AKI risk factor. We indeed observed this. Thus, this provides some reassurance about the specificity of the observed state-level associations between the 6 selected chronic health conditions and AKI-D.2 was higher in the models with hospitalization rate as the main exposure vs. the models with disease prevalence as the main exposure, which is consistent with the hypothesis that rates of hospitalization with chronic health conditions are more influential than prevalence rates in the community in explaining state-level variation in AKI-D incidence. Since it is plausible that patients with poorly controlled chronic health conditions are the ones more likely to be admitted, these results highlight the possibility that improved management of chronic health conditions could reduce AKI-D episodes. Our study potentially identify which states would garner the most benefit in terms of these prevention strategies. Overall, intensive patient education programs to improve self-management of chronic conditions and the skill and supply of non-nephrologists  may be important modifiable factors to reduce AKI-D disease burden.We believe that a potentially important observation is that state-level differences in rates of hospitalization with chronic health conditions  were larger than the state-level difference in prevalence for each chronic health condition . Furthermore, as noted, for most of the chronic conditions, the adjusted ROur study is strengthened by encompassing multiple states across the nation. To our knowledge, the BRFSS is the only comprehensive state-level dataset available regarding the chronic health conditions of interest. Previous studies reported that BRFSS data highly agree with data from other national surveys –31. As aHowever, we recognize a number of limitations. Due to limitations in the SID data, we did not have data on all 50 states. Thus our results may not generalize to the entire country. We lacked clinical data  to capture AKI-D and chronic health conditions and this may introduce biases in our estimations of the strengths of associations. We identified each health condition using diagnostic codes and so were not able to capture disease severity. We were unable to quantify any pre-existing reductions in glomerular filtration rate for those patients who developed AKI-D. There were other factors which may contribute to AKI-D incidence  which we did not have available information to adjust for. We were not able to account for any potential differences in threshold to initiate dialysis or death at home as a competing event that may vary regionally. Likely because of reliance on patient self-report, the population prevalence of CKD according to BRFSS is much lower than the population prevalence of CKD defined using biochemical markers from the National Health and Nutrition Examination Survey (NHANES) , 34. ThiThere is considerable state-level variation in the incidence of dialysis-requiring acute kidney injury (AKI-D). AKI-D incidence was associated with prevalence of and rates of hospitalization with five chronic health conditions--diabetes, hypertension, CKD, ASHD and COPD--and rates of hospitalization with cancer. We believe our results are provocative and shed light on potential reasons for the geographic variations in AKI-D incidence. Our results, if replicated by others, suggest that measures which reduce hospitalization of patients with chronic health conditions may translate into lower incidence of AKI-D.Additional file 1 Supplementary Table 1 The BRFSS questionnaires included in our study. Supplementary Table 2 State-level AKI-D incidence in 2011 for the 18 states in the primary analysis. Supplementary Table 3 State-level demographic characteristics of BRFSS respondents in 2011 for the 18 states in the primary analysis. Supplementary Table 4 State-level prevalence estimates for diabetes mellitus and rates of hospitalization with diabetes mellitus in 2011 for the 18 states in the primary analysis Supplementary Table 5 State-level prevalence estimates for hypertension and rates of hospitalization with hypertension in 2011 for the 18 states in the primary analysis. Supplementary Table 6 State-level prevalence estimates for chronic kidney disease and rates of hospitalization with chronic kidney disease in 2011 for the 18 states in the primary analysis. Supplementary Table 7 State-level prevalence estimates for arteriosclerotic heart disease and rates of hospitalization with arteriosclerotic heart disease in 2011 for the 18 states in the primary analysis. Supplementary Table 8 State-level prevalence estimates for cancer and rates of hospitalization with cancer in 2011 for the 18 states in the primary analysis. Supplementary Table 9 State-level prevalence estimates for chronic obstructive pulmonary disease and rates of hospitalization with chronic obstructive pulmonary disease in 2011 for the 18 states in the primary analysis. Supplementary Table 10 State-level prevalence estimates for skin cancer and rates of hospitalization with skin cancer in 2011 for the 18 states in the primary analysis. Supplementary Table 11 Linear regression model for AKI-D incidence and each chronic health condition for the 25 states in the sensitivity analysis. Supplementary Figure 1 Flow diagram showing selection of state for data analysis. Supplementary Figure 2 Map demonstrating the regional variation of AKI-D incidence in sensitivity analysis with 25 states. Supplementary Figure 3 Rate of hospitalization with cancer and predicted AKI-D incidence for the 25 states in the sensitivity analysis. A) Adjusted for age, % male, and % non-Hispanic White. B) Adjusted for age, % male, % non-Hispanic White, and prevalence of cancer.


Polish physicians and researchers lack an extensive and precise instrument in their native language for evaluating sexual dysfunction in individuals with neurogenic disorders. The aim of this study was to create a culturally adapted, validated, Polish language version of the Multiple Sclerosis Intimacy and Sexuality Questionnaire (MSISQ-15) for persons with multiple sclerosis (MS) and spinal cord injury (SCI).International recommendations and standardized methods for instrument validation were followed. Sexually active patients with MS and SCI completed the MSISQ-15, International Index of Erection Function , and Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire . IIEF-15 and PISQ-31 were used as reference questionnaires. Responses were collected at baseline (test) and after 2 weeks (re-test).r = − 0.487) and PISQ-31 (r = − 0.709). These correlations substantiated the high quality construct/criterion validity. An analysis of reliability presented good internal consistency  and reproducibility . There were no ceiling or floor effects.We recruited 299 Polish-speaking patients with MS or SCI. Interviews disclosed that the translated questionnaire had optimal content validity/cross-cultural adaptation. MSISQ-15 scores correlated significantly with the severity of sexual dysfunction as evaluated by IIEF-15 (The Polish version of MSISQ-15 exhibited excellent measurement properties. It is a suitable and reliable instrument to assess sexual dysfunction in MS and SCI individuals. The Polish MSISQ-15 will enhance routine clinical practice and assist research for neurogenic patients in Poland.The online version contains supplementary material available at 10.1186/s12883-021-02132-9. Sexual functioning is a part of life’s vitality. Chronic neurogenic disorders such as multiple sclerosis (MS) or spinal cord injury (SCI) may have profound, negative effects on sexual life . Both coVigorous concerted efforts of many different healthcare professionals are needed to ameliorate prospects and well-being of neurogenic patients. This goal requires thorough diagnosis and treatment of sexual dysfunction (SD) in this distinctive patient group. The European Association of Urology currently recommends the use of validated instruments to assess SD for patients with chronic neurogenic disorders because nonspecific instruments are not sufficiently sensitive . CliniciThe Multiple Sclerosis Intimacy and Sexuality Questionnaire MSISQ-15) is a well-known, acknowledged, sensitive, and specific questionnaire designed to accurately evaluate symptoms of SD in MS individuals  is a wel. The MSIThe research ethics committee of our institution approved the study. The MSISQ-15 copyright owner provided written permission to develop and distribute a Polish version of the questionnaire. All patients were apprised about the study purpose and procedures, and all provided written informed consent to participate.This was a prospective (cohort) validation study. We recruited the study population from the Department of Urology of the Jagiellonian University Medical College, Krakow, Poland. We invited all adult MS and SCI individuals who were registered in a patient database. We excluded patients who had no sexual activity during the preceding 6 months, and patients with cognitive impairment, pre-existing major chronic illnesses, dementia, mental retardation, active malignancies, or Polish language difficulties. After translation (see later), MS and SCI individuals assessed the MSISQ-15 instrument during direct discussions. Then, other MS and SCI persons were requested to complete the Polish version of the MSISQ-15, and either the International Index of Erection Function (IIEF-15) or the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-31), first as outpatients (test) then at home 2 weeks later (re-test). IIEF-15 and PISQ-31 were used as reference questionnaires. Demographic and clinical attributes were obtained from patient medical records and subjected to assessments with pre-specified outcomes that included validity  and reliability . Standardized guidelines and international recommendations for translation, adaptation, and validation of questionnaires were followed , 12.The MSISQ-15 is a self-administered instrument that investigates the effects of neurogenic symptoms on sexual activity and sexual satisfaction during a preceding 6-month period . The MSIThe IIEF-15 is a brief, reliable, and multidimensional scale for evaluation of male sexual function . We chosThe PISQ-31 is a reliable, well-known, and comprehensive questionnaire to assess sexual functioning in women with pelvic organ prolapse and/or urinary incontinence . BecauseTwo professional and avowed translators whose native language was Polish separately translated the questionnaire from English into Polish (forward-translation). The investigators compared the two translations, made slight corrections without changing the content, and settled on a provisional version. A similarly credentialed professional translator, blinded to the original English version of the questionnaire, translated the provisional version into English (back-translation). Then, a concurrence meeting produced the final Polish version of the MSISQ-15. The final version was evaluated based on semantic, idiomatic, experiential, and conceptual equivalence. Three staff urologists, one psychiatrist, one gynecologist, and one neurologist checked and agreed on the final Polish version of the questionnaire.With direct discussions, 60 patients  assessed the content validity  . First, Construct/criterion validity  was evaluated by analyzing the correlation between the Polish version of the MSISQ-15 and the reference questionnaires [11]. Notably, the score correlations of the MSISQ-15 to the IIEF-15 for men and the MSISQ-15 to PISQ-31 for women were determined by Pearson correlation coefficients .Ceiling/floor effects, analyzed to determine sufficient variance in observations, were considered to occur if > 15% of participants had the highest or lowest possible score . The ceiInternal consistency referred to intercorrelation of the questions; a parameter that verifies correlations between items within a subscale, domain, or questionnaire, i.e., whether the items gauged the identical fundamental construct. The Cronbach’s alpha was computed for the total score and for the three separate domains of the MSISQ-15 . A Cronb2,1) [Reproducibility referred to test-re-test reliability that analyzed whether the investigated instrument consistently replicated the result more than once in the same situation and population, i.e., produced repeated measurements. We ensured that there was not any type of treatment intervention with the patients between the two measurements. The reliability was computed from the intraclass correlation coefficient that confronted the initial MSISQ-15 score with the repeat MSISQ-15 score 2 weeks hence  . A coeff2,1) .p < 0.05. Data analysis was conducted with SPSS Statistics software, version 24.0 .Descriptive results for continuous data were measured as means ± standard deviations. For discrete data, we used counts and percent. Statistical significance was regarded as Following standard recommendations for instrument validation and other validation studies, initially, we sought sample sizes of 50 MS and 50 SCI persons , 11. HowBecause the European Association of Urology recommends individual validations for specific neurogenic patient groups, we validated the MSISQ-15 independently with individual data presentations for the MS and SCI patients . AlthougWe invited 328 patients to adapt and validate the questionnaire but 29 persons were excluded following the exclusion criteria. Therefore, 299 patients participated in this study. From this group, thirty MS and thirty SCI individuals were selected randomly to assess content validity/perform cross-cultural adaptation with in-person interviews. Those 60 individuals were excluded from the later steps of validation to avoid interviewer bias. After evaluation of content validity, 299 patients completed the instruments at baseline (test phase). For unknown reasons, eight patients did not return the second set of questionnaires, two refused to complete the second MSISQ-15 questionnaire, one did not fully complete the second set of questionnaires, and one died from pneumonia. Two hundred twenty-seven (117 MS and 110 SCI) persons completed the second set of questionnaires (re-test phase) on average 19.8 (standard deviation 10.3) days after the first set of questionnaires; we included these 227 individuals in the final analysis. In the re-test phase, no patient disclosed a change in sexual functions nor did any patient receive new medication/treatment for their SD.Table During direct discussions, MS and SCI individuals discerned a wording issue in the Polish translated instrument that necessitated a minor adjustment before finalizing the questionnaire . Includer = − 0.487 for the test phase and r = − 0.456 for the re-test phase; see Table r = − 0.709 for the test phase and r = − 0.688 for the re-test phase). Thus, we concluded that the construct/criterion validity was adequate.We found a significant correlation between the total scores of MSISQ-15  and the total scores of IIEF-15 for men  were all higher than 0.7 in MS and SCI patients, which indicated excellent reproducibility Table . We did This study provides the first comprehensive Polish questionnaire that evaluates sexual function in patients with neurogenic disorders. Whereas some extant instruments for this specific patient population are available in the Polish language, existing questionnaires evaluate primarily bladder problems –21. The Complex care for neurogenic individuals, particularly for MS and SCI persons, is multidimensional and multifactorial. Because neurological disorders may strongly affect sexual function, assessment of SD is an important aspect for these patients’ quality of life. Current recommendations advocate specific instruments for investigation of sexual function . SpecifiAn asset of our study was the contributions of two consistent groups of individuals with two distinct neurogenic disorders, i.e., MS and SCI, with individual and independent adaptations and validations. Individuals with MS and SCI represent non-traumatic and traumatic neurologic dysfunctions. Although the pathophysiology of these two neurogenic conditions differ, there are some similarities between the effects of neurological symptoms on sexual functioning and between treatment methods. These similarities lead to a hypothesis that some instruments may perform well in different neurogenic populations. Thus, our study is as an example of this hypothesis because it showed clearly the measurement properties of the MSISQ-15 for two types of patients and because we used discrete analyses based on current guidelines and recommendations , 11. In All patients confirmed content validity of the Polish version of the MSISQ-15 during in-person discussions. We substantiated optimal construct/criterion validity for both MS and SCI patients because the MSISQ-15 translated into Polish correlated significantly with the IIEF-15 for men and the PISQ-31 for women. Positive associations were also observed for the three specific domains of the MSISQ-15. Because of homogeneity of included patients and adequate sample size, we did not detect ceiling or floor effects . Our valHow widely applicable is the Polish version of the MSISQ-15? Koltuniuk et al. reported that more than half of MS patients were unsatisfied with their sex life, and most had significant sexual disorders . Poland Our investigation should be also assessed in the context of its limitations. We did not evaluate the responsiveness of the Polish version of the MSISQ-15. This omission was due to short follow-up that we adopted to ensure adequate test-re-test analyses with no clinical interventions between the two measurements and to minimize possible symptom changes. In addition, we acknowledge that a correlation coefficient for interconnection between IIEF-15 and MSISQ-15 was lower than a correlation coefficient between PISQ-31 and MSISQ-15. Nonetheless, moderate correlations of some MSISQ-15 components have been described , 9. ThesIn this study, following international and well-established recommendations, we successfully translated, adapted, and validated the Polish version of MSISQ-15. It is a valid, comprehensive, and reliable tool for evaluation of SD symptoms in MS and SCI patients. The instrument will promote improvement of the quality of rehabilitative intervention and assessment of the sexual lives of neurogenic patients. We recommend that clinicians and investigators use the Polish version of MSISQ-15 in clinical practice and research in Poland.Additional file 1. Polish MSISQ-15.Additional file 2. English MSISQ-15.


Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.More human deaths have been attributable to  Mycobacterium tuberculosis complex. MIRU is based on measuring the length of variable number tandem repeat (VNTR) loci in the M. tuberculosis genome, which is conceptually the same as subtyping of other bacterial genera by multiple locus VNTR analysis (MLVA). Different sets of loci can be used for analysis, and common variants include 12-locus MIRU , 15- and 24-locus MIRU (MIRU-24). The increased discrimination provided by a greater number of informative loci needs to be balanced against the cost and complexity of the test. MIRU-24 is widely regarded as a well-standardized strain typing method that provides adequate discrimination for the majority of M. tuberculosis strains.Mycobacterial Interspersed Repetitive Unit (MIRU) typing –3 has beM. tuberculosis transmission within Australia. By comparison, incidence rates of tuberculosis in England are somewhat higher but broadly similar . FASTQ files from the Ion Torrent platform, used to sequence clustered Lineage 2 strains identified in NSW, underwent quality-score-preserving error correction using muffinec . Mapping of error-corrected reads against this reference sequence was performed using the mem algorithm of bwa  and were excluded from further analysis. The remaining SNP variants for each sample were patched into the reference genome using vcf2fasta [https://github.com/vcflib/vcflib].FASTQ files corresponding to 110 eligible isolates from the Merker  collectimuffinec . FASTQ fnec  is associated with descent from more ancestral strains; in other words, for a leaf with high MPD the branches leading toward it are long with few near neighbors. Nearest taxon distance (NTD) is a property of leaves and refers to the total substitution distance between a leaf and its nearest neighbor. Calculation of these distances from the mrbayes tree was performed using DendroPy  of <10 substitutions was arbitrarily chosen as consistent with recent transmission; this is roughly equivalent to 14–32 years of bacterial evolution  based on recent estimates of the ar clock , 42–44, bfc error correction. Four of 21 isolates from the NSW MRL were excluded from further analysis, one due to insufficient number of reads after muffinec error correction and three that represented likely laboratory cross-contamination. Of the 245 isolates, 118 (48%) were from Lineage 2, 35 (14%) from Lineage 3 and 92 (38%) from Lineage 4. From a total of 10,965 SNPs, 115 (1%) were excluded from analysis as potentially homoplasious (in genes where resistance-associated SNPs are believed to occur); 37 of these 115 SNPs were synonymous and 58 were present in more than one isolate. The median number of isolates possessing one of these 115 SNPs was two, suggesting that the impact of their exclusion on the phylogeny is minimal and that few of these 115 SNPs were truly homoplasious.In total, we assessed the MIRU-24 profiles and SNP-based phylogeny of 245 isolates; 17 were derived from two MIRU-24 clusters identified in NSW Australia, 132 from the Walker collection of United Kingdom community MIRU-24 clusters, and 96 from a global collection of Lineage 2 strains, as shown in The Simpson diversity index for individual MIRU-24 loci ranged from 0.008 to 0.77 . The Simpson index for the complete MIRU-24 was 0.96. In this dataset from lineages 2, 3, and 4, four loci could account for almost all of the total MIRU-24 diversity . Other data transformations could preserve comparable diversity . The Bayesian inference phylogeny, with tips color-coded according to membership of the 10 largest MIRU-24 clusters (containing six or more members), is shown as One 8-member and one 9-member MIRU cluster among the 17 NSW isolates were identified using an identical MIRU-24 match as the cluster definition. From the Merker collection, one 17-member and one 11-member cluster were identified. In the Walker collection, there were one 27-member, one 17-member, one 16-member, one 13-member, one 7-member, and one 6-member clusters. The remaining 114 isolates were MIRU-24 singletons (68 isolates), pairs (22 isolates), or members of MIRU-24 clusters with three to five members (24 isolates). Extending a MIRU-24 cluster to include isolates with up to one mismatched locus produced a cluster with 50 strains; including up to two mismatched loci  producedThe distance tree revealed 138 isolates that were <10 substitutions from their nearest taxon, consistent with a recent shared ancestor shown in . These wOut of 138 isolates that were members of a SNP-cluster, MIRU-24 profiles were identical within each SNP-cluster in all but seven instances  was 95% , with seven instances where SNP-clustered isolates were misclassified by MIRU-24 see . The truThe Simpson diversity index for MIRU remained reassuringly high (0.96). However, by MIRU-24, 177 isolates were clustered, vs. 138 isolates by the permissive criterion of substitution distance <10. This represents a reduction in potential secondary cases from 149 isolates in 28 clusters with MIRU, to 108 isolates in 30 clusters with SNP typing (of which 13 isolates in seven SNP clusters had the “wrong” MIRU-24). In other words, 32% (48/149) of potential secondary cases by MIRU-24 were “false positives,” using a permissive standard for recent transmission.R package phytools , a density plot of reconstructed total number of repeats at the root node, and a histogram of total repeats by lineage for the 245 isolates at the tips.One possible selective pressure acting on repeat loci is genome reduction due to gene loss, as observed for M. tuberculosis isolates of modern lineages using genome-wide phylogeny shows evidence of extensive homoplasy of repeat number at MIRU loci. Comparison of repeat loci with genomic deletions has demonstrated fewer repeats per locus in lineages that separated later from the common ancestor of modern M. tuberculosis , whilst more ancient lineages  have a higher average number of repeats per locus . In contrast, the Merker data is intended to be globally representative of Lineage 2 strains. However, it derives primarily from a culture collection at a single major reference laboratory, and the high proportion of MDR strains within the collection is typical of culturing bias in resource-limited settings. Other datasets  captureAnother limitation of this analysis is the handling of potential errors in ascertainment of repeat number at MIRU loci. MIRU-24 data employed in this study were generated by reference laboratories, and have been published previously, so are likely to match or exceed real-world quality of MIRU data. The reproducibility of MIRU-24 is good (87–100%) –49, but M. tuberculosis clades.Our data suggest that the number of repeats at specific MIRU loci evolve at random, without evidence of selective pressure. Homoplasy of repeat numbers reduces the utility of MIRU-24. In these data, MIRU-24 showed poor specificity and modest positive and negative predictive values for the detection of recent transmission, using a permissive SNP threshold. Unless supported by independent evidence , clusters detected using MIRU-24 will often be polyphyletic and unhelpful for detecting recent transmission, especially in settings with incomplete sampling, limited local transmission, or high rates of imported disease. Inclusion of additional hypervariable repeat loci  could heM. tuberculosis lineages, arising through stochastic processes without natural selection. This homoplasy contributed to relatively poor specificity and low positive predictive values for the detection of recent transmission. These findings strengthen the case for transition away from MIRU-based to WGS-based methods of typing, which display much higher discrimination and rarer homoplasy, and provide important drug resistance information.In conclusion, we found frequent homoplasy of MIRU repeats in modern The datasets generated for this study can be found in the European Nucleotide Archive, PRJEB11778, PRJEB7281, and PRJEB2221.The studies involving human participants were reviewed and approved by Human Research Ethics Committee University of Sydney. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AO, BM, and VS developed the concepts for this analysis. UG collected data and initiated the analysis of NSW MRL MIRU-24 clusters. UG, PJ, NM, and QW performed laboratory procedures including MIRU-24 and whole genome sequencing for the NSW MRL isolates. GAH-C provided expertise regarding whole genome sequencing and bioinformatic analysis. AO performed bioinformatic and statistical analyses. AO and BM drafted the initial manuscript. BM and VS supervised the research. All authors participated in manuscript revision.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.