P = .67) and previous systemic treatment (P = .16). Interestingly, but with the caveat of the limited number of patients included in this series, primary extragastric disease had a superior PFS compared with gastric lymphoma (P = .04) and also depth of response, i.e. complete or partial response versus stable disease was associated with significantly prolonged PFS (P = .01). We documented four patients (8%) with pronounced improvement of response during follow‐up including three patients initially rated as partial remission and finally achieving complete remission at 12 to 32 months. This highlights the potential of delayed responses to LEN treatment. Estimated overall survival at 5 years was excellent at 92%. These “real‐world” data confirm long‐term activity of LEN in MALT lymphoma.Based on results of two pilot trials, lenalidomide (LEN) was found to be active and safe as monotherapy and showed an increased response rate of 80% in combination with rituximab (R) for patients with mucosa‐associated lymphoid tissue (MALT) lymphoma. While initial results were promising, there are currently no data on long‐term outcome, and larger international phase II/III trials on LEN for indolent lymphoma lack specific subgroup analyses. Thus, we have systematically analyzed 50 patients treated with LEN‐based therapy  at the Medical University of Vienna 2009 to 2019 and investigated long‐term outcome and relapse patterns. At a follow‐up of more than 5 years (median 68 months), 54% of patients are free of relapse, and estimated median progression‐free survival (PFS) was 72 months (95%CI 49‐96). There was no difference in PFS according to stage of disease, i.e. localized versus disseminated disease ( Considering above discussed caveats in the current trial landscape of MALT lymphoma, the objective of this article is to present here consistent “real‐world” follow‐up data on response, relapse patterns, and safety with a median follow‐up of more than 5 years.2For the current investigation, we have retrospectively analyzed patients treated with LEN‐based therapy for histologically verified MALT lymphoma at the Medical University of Vienna, Clinical Division of Oncology, between 2009 and 2019. All diagnoses were made by a reference hematopathologist according to the definition of MALT lymphoma as outlined in the WHO classification for tumors of hematopoietic and lymphoid tissues.2.12 day 1 plus LEN 20 mg daily day 1 to 21 in a 28‐day cycle (maximum of eight cycles). Response was evaluated after cycles 3, 6, and 8 if applicable, and outcome was classified for best response based on either radiological or GELA histological criteriaTreatment regimens applied included LEN‐monotherapy, i.e. 25 mg daily from day 1 to 21 in a 28‐day cycle (maximum of six cycles) or R‐LEN consisting of R at 375 mg/m2.2Data of interest for this analysis included basic patient characteristics, i.e. sex, age at treatment start, primary localization of MALT lymphoma, stage of disease, presence of risk factors/disease relevant comorbidities including lactate dehydrogenase (LDH) levels, beta‐2 microglobulin (B2M), presence of an autoimmune disorder and HP status, and histological features, i.e. documentation of plasmacytic differentiation. Furthermore, MALT‐IPI scores as recently published were retrospectively assessed.2.3P‐values less than 0.05 were considered statistically significant (two‐sided).Statistical analysis was performed using IBM statistics for Mac OS version 25.0 . Metric data were described using median and range or interquartile range (IQR), respectively. For categorical data, absolute frequencies and percentages are presented. Associations of binary variables were calculated by use of Fisher's exact test. Overall survival (OS) and PFS estimations were plotted by Kaplan Meier method, and differences between groups were compared using log‐rank test. 3We could identify a total of 50 patients who received LEN‐based treatment for histologically verified MALT lymphoma at our institution. Median age at treatment start was 67 years ; 62% of patients were female and 38% male. At initial diagnosis, 32% of patients had primary gastric involvement, while the majority presented with primary extragastric MALT lymphoma (68%). The most common extragastric manifestation were the ocular adnexa 30%, 15/50), followed by MALT lymphoma of the lung  and parotid gland , and one patient each had primary of the breast, colon, skin, liver, and small intestine. Furthermore, five patients (10%) had multiple organ involvement at first diagnosis. In terms of Ann Arbor stage at LEN‐treatment start, 66% were rated as localized disease (stage IE or IIE) and 34% had disseminated disease (stage IIIE or IV). A history of autoimmune disease was present in 24% of patients. Risk stratification according to the MALT‐IPI scoring system resulted in 26% low risk, 62% intermediate risk, and 12% high risk patients. See Table 5/50, folThe median time from initial diagnosis to treatment start with LEN‐based treatment was 5.9 months , and while extragastric patients were directly eligible for LEN‐treatment, patients with gastric MALT lymphoma and presence of HP had been pretreated with adequate antibiotic eradication therapy. However, we also included two patients (4%) with HP‐negative gastric MALT lymphoma as assessed by histology and serology, receiving up‐front LEN‐based therapy. Analyzed for the entire cohort, LEN was applied as first‐line treatment in 52% (26/50), while the remaining patients had been pretreated including a total of 28% (14/50) who had received prior systemic treatment for MALT lymphoma, ie, chemotherapy and/or immunotherapy.P = .731 and P = .136, respectively). There was no difference of frequency in response in terms of primary localization  or disease dissemination status .In terms of treatment schedule and initial response rates as published previously, 32% (16/50) of patients were treated within the LEN monotherapy trial and 68% (34/50) with the combination of R‐LEN. Initial ORR for the entire cohort was 72%, with 48% achieving a CR, 24% a PR and 22% SD as best outcome, while 6% (three patients) progressed during treatment. In the entire cohort, median time to documentation of best response was 5.1 months , and remarkably four patients (8%) improved their outcome during prolonged follow‐up and converted to a better classification of response after the last restaging within the study protocol, including three patients with delayed CR at 12 to 32 months after LEN‐start and one converting from SD to PR after 11 months. Thus, the final outcome according to best response documented during follow‐up was 74% ORR, 54% CRs, and 20% SDs. ORR and CR rates were higher for the combination group being 77% versus 69%, and 62% versus 38%; however, none of these differences reached statistical significance , a total of 46% 23/50) of patients have relapsed with a median estimated PFS of 72.3 months , MALT‐IPI scores (P = .186), or presence of an autoimmune disease (P = .279), but patients with primary extragastric MALT lymphoma at initial diagnosis had statistically a longer estimated PFS than patients with gastric origin , and in view of the limited number of cases included in this series. As expected, there was a trend toward worse PFS in patients who had received prior treatment but without reaching statistical significance (P = .083). Prior immuno‐/chemotherapy did not significantly influence PFS (P = .155).There was no difference in PFS in terms of dissemination, i.e. localized versus disseminated disease  are currently alive without evidence of lymphoma, 38% (19/50) are alive with residual MALT lymphoma, and six patients (12%) have died including two (4%) lymphoma‐related deaths: one patient with heavily pretreated disseminated disease and one with transformation to refractory diffuse large B‐cell lymphoma (DLBCL). In addition to the later patient, another patient showed histological transformation for a total transformation rate of 4%. PCR‐based clonality analysis, however, revealed that only one of these DLBCLs was clonally related to the primary MALT lymphoma , while the second showed no alignment of immunoglobulin‐rearrangement pointing to a clonally unrelated, secondary manifestation of DLBCL.4Application of LEN has become a widely used approach in various lymphoid malignancies including follicular lymphoma in combination with rituximab, as well as in mantle cell lymphoma.Activity of LEN‐based therapy was high, with an ORR of 74% (including 54% CR rate) and 20% SD as best outcome, while 6% (three patients) progressed during treatment. Whereas ORR and particularly CR rates were higher for the combination group , these differences were not statistically significant. Importantly, a total of four patients showed delayed responses including three patients initially rated as PR converting to a CR at 12 to 32 months, highlighting the potential of late remissions following immunomodulatory treatment in such an indolent disease, a phenomenon that has been reported not only for LEN but also thalidomide in MALT lymphoma patients before.P = .01), and in addition PFS following CR was also longer over PR (P = .05). This suggests an influence of depth of remission on PFS following LEN‐based therapy, while currently there is no clear‐cut influence of response quality in patients with gastric MALT lymphoma following HP‐eradication as judged from two series in the literature.In terms of long‐term outcome, at a median follow‐up time of 68 months, 54% of patients are free of progression, and the estimated PFS was 72 months in our study. Interestingly, our data suggest a statistically superior PFS for patients achieving a response to LEN‐based therapy over patients with SD only  for our patient collective is comparable to a recently published analysis for R‐LEN in patients with MZL from the MD Anderson, who reported on 30 patients with median follow‐up of 75 months.The authors certify that they have no conflicts of interest to disclose in the subject matter discussed in this paper.Design of the study: B.K., M.R. Collection of data: B.K., W.L., O.N., M.E.M., I.S.K., M.R. Interpretation of data: B.K., W.L., O.N., M.E.M., I.S.K., M.R. Manuscript design and writing: B.K., M.R. Manuscript approval and final editing: all authors.


Cachexia is an important extra‐pulmonary manifestation of chronic obstructive pulmonary disease (COPD) presenting as unintentional weight loss and altered body composition. Previous studies have focused on the relative importance of body composition compared with body mass rather than the relative importance of dynamic compared with static measures. We aimed to determine the prevalence of cachexia and pre‐cachexia phenotypes in COPD and examine the associations between cachexia and its component features with all‐cause mortality.We enrolled 1755 consecutive outpatients with stable COPD from two London centres between 2012 and 2017, stratified according to European Respiratory Society Task Force defined cachexia [unintentional weight loss >5% and low fat‐free mass index (FFMI)], pre‐cachexia (weight loss >5% but preserved FFMI), or no cachexia. The primary outcome was all‐cause mortality. We calculated hazard ratios (HRs) using Cox proportional hazards regression for cachexia classifications  and component features (weight loss and FFMI) and mortality, adjusting for age, sex, body mass index, and disease‐specific prognostic markers.P < 0.001). There were 313 (17.8%) deaths over a median (interquartile range) follow‐up duration 1089 (547–1704) days. Both cachexia  and pre‐cachexia  were associated with increased mortality. In multivariable analysis, the unintentional weight loss feature of cachexia was independently associated with mortality , whereas low FFMI was not . Sensitivity analyses using body mass index‐specific, age‐specific, and gender‐specific low FFMI values found consistent findings.The prevalence of cachexia was 4.6% [95% confidence interval (CI): 3.6–5.6] and pre‐cachexia 1.6% (95% CI: 1.0–2.2). Prevalence was similar across sexes but increased with worsening Global Initiative for Chronic Obstructive Pulmonary Disease spirometric stage and Medical Research Council dyspnoea score (all Despite the low prevalence of cachexia and pre‐cachexia, both confer increased mortality risk in COPD, driven by the unintentional weight loss component. Our data suggest that low FFMI without concurrent weight loss may not confer the poor prognosis as previously reported for this group. Weight loss should be regularly monitored in practice and may represent an important target in COPD management. We propose the incorporation of weight monitoring into national and international COPD guidance. Chronic obstructive pulmonary disease (COPD) is a major cause of global morbidity and mortality.2 plus ≥3 of five markers of metabolic disturbance  to diagnose cachexia.Cachexia is a complex syndrome that encompasses multiple manifestations of COPD. The prominent and dynamic feature of cachexia is unintentional weight loss, driven by a variable combination of reduced food intake and metabolic disturbance.2 in males and <15kg/m2 in females'.The European Respiratory Society (ERS) Task Force on nutritional assessment and therapy in COPD recently provided expert‐derived pragmatic definitions for different metabolic phenotypes, using practical assessment modalities that can be more readily applied in practice.A prospective validation of these practical definitions is lacking in COPD. Moreover, whilst previous studies in COPD have demonstrated the additional prognostic importance of baseline body composition compared with BMI alone,We therefore aimed to determine the prevalence of cachexia and pre‐cachexia in patients with stable COPD, to characterize the cachexia phenotype, and to examine the associations between cachexia and its component features with mortality. We hypothesized that mortality risk would be increased in the presence of cachexia as compared with the presence of unintentional weight loss or low FFMI alone.1) to forced vital capacity of less than 0.7 and a physician diagnosis of COPD consistent with the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) guidelines.For this prospective cohort study, a consecutive series of patients attending outpatient respiratory, pulmonary rehabilitation, and community assessment clinics in northwest London, UK, were approached between January 2012 and May 2017. Eligible patients were aged 40 years or above, with a ratio of forced expiratory volume in 1 s (FEV2 in kg/m2). Using the ERS Task Force on nutritional assessment in COPD definitions,2 for men and <15 kg/m2 for women were classified as having cachexia. Patients reporting unintentional weight loss of >5% over the preceding 6 months but with a preserved FFMI were classified as having pre‐cachexia. As per usual local practice, those classified as having pre‐cachexia or cachexia were provided with an information leafletUnintentional weight loss was assessed using current weight  and a structured clinical history following British Association For Parenteral And Enteral Nutrition (BAPEN) guidance.1 using spirometry, functional exercise capacity using the incremental shuttle walk (ISW) test,All‐cause mortality, our primary outcome, was assessed over a 6‐year observation window (2012–17). Deaths were recorded up to 29 August 2017 using data retrieved from the UK National Health Service ‘spine', maintained by the NHS Care Records Service. Age, sex, and ethnic origin were recorded from medical records. Other outcomes assessed at study enrolment were FEVχ2 for trend. Baseline clinical characteristics were compared across groups through one‐way analysis of variance or Kruskal–Wallis tests with χ2, independent t‐test, Mann–Whitney U‐tests for pairwise comparisons.The recruitment target was 1700 participants based on the precision to which cachexia prevalence could be estimated: ±2.5% with a large sample normal approximation (nQuery Advisor V.6.0) and previous studies that identified modified mortality risk from altered body composition parameters. The prevalence of cachexia, with 95% confidence interval (95% CI) calculated using Wilson's method, was determined overall and compared across sex, age, GOLD spirometric stage, and MRC dyspnoea score categories using 1 per cent predicted, MRC dyspnoea score, and ISW distance as reported predictors of mortality in COPD. After checking for collinearity (r < 0.75), all variables significantly associated with mortality (P < 0.10) were considered in a multivariate model. Hazard ratios (HRs) with 95% CI were estimated. Analyses were performed using SPSS , and P values less than 0.05 were considered significant.Survival was plotted using Kaplan–Meier curves, and the log–rank test was used to compare survival curves according to cachexia classifications  and component features . After censoring and proportionality assumptions had been satisfied, associations with mortality were investigated using Cox proportional hazards regression models. In univariate separate models, either cachexia classifications or component features were entered as independent variables along with the covariates age, sex, smoking status, Charlson co‐morbidity index, exacerbations in the previous year, BMI category, FEV1 48.6 (19.5) % predicted, and BMI 27.8 (6.7) kg/m2 (TableP for trend <0.001 each).Of the 2250 consecutive patients approached, 1755 (78%) were enrolled. Participants had a mean (standard deviation) age of 70 (10) years, FEV1% predicted and exercise capacity, more exacerbations in the previous year (P < 0.001 each), and a higher level of respiratory‐related disability (P = 0.005) as compared with patients with no cachexia. The groups did not differ statistically with respect to sex distribution, age, smoking history, long‐term oxygen therapy use, or health‐related quality of life  over median (interquartile range) follow‐up duration 1089 (547–1704) days. All‐cause mortality at the end of the first, second, and third years of study follow‐up was 5.3%, 10.3%, and 15.7%, respectively.P < 0.001). In contrast, no difference in survival was observed between groups with baseline cachexia and pre‐cachexia (P = 0.699)   Table. In multP = 0.214). Furthermore, in patients with low FFMI, survival was significantly reduced in patients with unintentional weight loss compared with those with no weight loss (P < 0.001)   Table. In mult2] Table.1% predicted, co‐morbidity burden, and ISW distance ] after adjusting for age, sex, FEVThis large prospective cohort study found a prevalence of 4.6% and 1.6% for cachexia and pre‐cachexia in outpatients with stable COPD. Patients with these phenotypes had a poorer exercise capacity and more severe respiratory disability as compared with those with no cachexia, including those with a low FFMI alone. Both cachexia and pre‐cachexia were associated with increased mortality risk. However, contrary to our hypothesis, low FFMI did not add prognostic value to unintentional weight loss alone. Our findings underscore the prognostic importance of unintentional weight loss in COPD.per se has consistently been associated with mortality in COPD; indeed, BMI is a component of the BODE index, the best known prognostic index in COPD.Low body weight In line with previous studies, we found reduced FFMI to be associated with increased mortality risk when considered in isolation.All cachexia definitions stress the dynamic nature of the syndrome and require a change in weight over time.There are potential limitations to our study. First, the prevalence of cachexia in our cohort was lower than previously reported. This is most likely explained by previous studies focusing on cohorts with more advanced COPD, the application of different cachexia criteria, and the increase in body size with obesity. We acknowledge that our population consisted primarily of stable, Caucasian (93%) outpatients, and our findings need to be corroborated in other settings and countries. We hypothesize that the ERS Task Force cut‐offs for FFMI may over‐estimate or under‐estimate the prevalence of cachexia in different ethnic populations. Cachexia may also be more prevalent in specific settings, such as in care or nursing homes or acute hospitals. Second, we assessed unintentional weight loss through clinical history as serial measurements of weight were often not recorded in patient care records. Multiple measures of weight and body composition would have extended our study findings and allowed us to examine the impact of different trajectories on clinical outcome, and we recommended longitudinal assessment in future studies. However, we followed a consensus‐recommended systematic process, and patient‐reported weight history is reliableGiven the prognostic significance of weight loss, we propose that assessing weight change should be an essential part of the clinical assessment (both through history and examination) of the patient with COPD and incorporated explicitly into international guidance.There is significant interest in understanding the mechanisms underlying low muscle mass in patients with COPD.In summary, although the prevalence of cachexia and pre‐cachexia in outpatients with stable COPD is low, these phenotypes are associated with important clinical manifestations including poorer exercise tolerance and greater respiratory disability. Unintentional weight loss is independently associated with mortality risk in COPD and regular monitoring of weight should be an essential part of the clinical assessment of the patient with COPD. Our data suggest that low FFMI without concurrent weight loss may not confer the poor prognosis as previously reported for this group.W.D‐C.M. and M.M. did the concept and design of the study. Acquisition of the data was made by H.Y.K., C.N., S.J., S.P., R.B., and S.K. Analysis and interpretation of the data were carried out by H.Y.K., M.M., M.I.P., P.C., and W.D‐C.M. Drafting of the manuscript was performed by H.Y.K., M.M., M.I.P., P.C., and W.D.‐C.M. All authors revised the manuscript critically for important intellectual content and approved the final manuscript.The authors declare no competing interests.Journal of Cachexia, Sarcopenia, and Muscle.The authors certify that they comply with the ethical guidelines for authorship and publishing of the The recruitment of the cohorts was supported by a National Institute for Health Research (NIHR) Clinician Scientist Award, a Medical Research Council (UK) New Investigator Research Grant, and a NIHR Clinical Trials Fellowship awarded to W.D.‐C.M. H.Y.K. was supported by a European Respiratory Society Short Term Research Fellowship. C.M.M. was supported by a NIHR Doctoral Research Fellowship and a NIHR Clinical Trials Fellowship. S.E.J. and R.E.B. are supported by NIHR Doctoral Research Fellowships. M.M. is supported by a NIHR Career Development Fellowship (CDF‐2017‐10‐009) and the NIHR Collaboration for Leadership in Applied Health Research and Care for South London and Cicely Saunders International. W.D.‐C.M. was part funded by the NIHR Collaboration for Leadership in Applied Health Research and Care Northwest London.Table S1. Baseline characteristics of patients with COPD without cachexia (n = 1646) classified according to the presence of low or preserved fat free mass indexTable S2. Cox proportional hazard models for all‐cause mortality in patients with COPD according to cachexia constituents using BMI‐, age‐ and gender‐specific low fat‐free mass index values.Click here for additional data file.


Lymphocytosis is relatively common in cats, but few studies describe lymphocyte populations or the clinical course associated with different immunophenotypic expansions.We hypothesized that cats frequently develop non‐neoplastic lymphocytosis and that different neoplastic immunophenotypes have variable prognoses. We aimed to characterize the lymphocyte expansions in a large population of cats with lymphocytosis and to assess clinical presentation and outcome in a subset.Three cohorts of cats older than 1 year with lymphocytosis (>6000/μL) were examined to define immunophenotypic categories (n = 146), evaluate outcome (n = 94), and determine prevalence of immunophenotypes (n = 350).Retrospective study of cats with blood submitted for flow cytometry. Medical records (n = 94) were reviewed for clinical data, treatment, and survival information.Five major immunophenotypic categories were identified: B cell, heterogeneous (≥2 lineages expanded), CD4+ T cell, CD4−CD8− (double negative [DN]) T cell, and CD5‐low‐expressing T cell. B‐cell and heterogeneous phenotypes were more consistent with a non‐neoplastic process, having polyclonal antigen receptor gene rearrangements, younger age at presentation, lower lymphocyte counts, and prolonged survival. The neoplastic phenotypes, CD4+ T cell, DN T cell, and CD5 low T cell, had different median survival times . Among CD4+ T‐cell cases, cats with abdominal lymphadenopathy, intestinal involvement, or both and females had shorter survival. Among 350 cats with lymphocytosis, CD4+ T‐cell lymphocytosis was most common, followed by heterogeneous and B‐cell phenotypes.Neoplastic CD4+ T‐cell lymphocytosis is common in cats and has a prolonged clinical course compared to aberrant T‐cell phenotypes. Cats with heterogeneous and B‐cell lymphocyte expansions commonly have non‐neoplastic disease. CLLchronic lymphocytic leukemiaCSU‐CIColorado State University‐Clinical ImmunologyDNdouble negativeFeLVfeline leukemia virusIGimmunoglobulinIGH‐VDJimmunoglobulin heavy chain variable (V)‐diversity (D)‐joining (J) gene rearrangementIMHAimmune‐mediated hemolytic anemiaIQRinterquartile rangeLGLlarge granular lymphocyticMSTmedian survival timePARRPCR for antigen receptor rearrangementsPBMCperipheral blood mononuclear cellsPEphysical examinationTRGT‐cell receptor gamma1Lymphocytosis is relatively common in cats, but few studies have examined its flow cytometric features. Lymphocytosis may be attributed to non‐neoplastic or neoplastic processes. Our goals were to determine how frequently lymphocytosis in cats is neoplastic, and among cases of neoplastic lymphocytosis, the utility of flow cytometry to characterize clinically relevant phenotypes.Cytauxzoon felis, Toxoplasma gondii, Aelurostrongylus abstrusus, Leishmania infantum, Mycoplasma haemofelis, Bartonella henselae, feline leukemia virus (FeLV), and feline immunodeficiency virus.Few studies have described non‐neoplastic expansions of peripheral lymphocytes in cats, which may be attributed to physiologic or inflammatory processes. Absolute lymphocytosis has been documented with a number of infections, including Lymphocytosis attributed to a neoplastic process may be seen in cases of leukemia or cases of lymphoma with circulating neoplastic lymphocytes. Clinical findings and outcome have been reported for 18 cats with clinically defined chronic lymphocytic leukemia (CLL).Our experience is that cats present more commonly with ≥2 expanded subsets of peripheral lymphocytes than do dogs. How often these cases are non‐neoplastic in nature has not been determined, and in cases where the cells are neoplastic, the prognostic value of immunophenotyping is unclear. We performed a retrospective study to examine the range of phenotypes in cats presenting with lymphocytosis without any a priori definitions of disease. We examined clonality, clinical presentation, and outcome on a subset of cases.22.1A retrospective study was performed using feline blood samples submitted to the Colorado State University‐Clinical Immunology (CSU‐CI) laboratory for immunophenotyping by flow cytometry. All cats included in the study were older than 1 year of age and had >6000 lymphocytes/μL . Three cohorts of patients were examined for different components of this study: (i) definition cohort; (ii) outcome cohort; and (iii) concordance cohort.2.1.1An initial study population of 146 cats was examined to define immunophenotypic categories. This population included all cats that had both flow cytometry and PARR performed on blood samples submitted between October 24, 2014, and November 16, 2017. In some cases (25%), concurrent flow cytometry and PARR were requested by the veterinarian at the time of submission, but in many cases, PARR (75%) was performed after an equivocal flow cytometry result.2.1.2A separate study population of 94 cats with outcome data was examined, which included cats that had immunophenotyping on blood performed between July 12, 2006, and April 20, 2010. Veterinary clinics were contacted for medical records and clinical data were collected by reviewing records. All cats included in this cohort had persistent or recurrent lymphocytosis as described above identified on ≥2 separate CBCs.2.1.3A third study population of 350 cats was examined to determine the prevalence of immunophenotypes in a larger cohort of animals and to determine whether the outcome cohort was representative of a larger population. This population included all cats that had blood samples submitted for flow cytometry between January 1, 2017, and December 1, 2017.2.2For all samples, signalment, physical examination (PE) findings, and laboratory findings were provided by the submitting veterinarian on the standard CSU‐CI laboratory submission form. Complete blood count data from the time of submission were used to identify hematologic abnormalities. Anemia was defined as a hematocrit <28%, thrombocytopenia by a platelet total <200 000/μL with no clumps noted, and neutropenia as <2000 neutrophils/μL.Additional information was gathered for the 94 cats in the outcome cohort. Medical records were provided by the submitting veterinarians, which represented non‐referral and referral clinics distributed across 24 states. Clinical signs and concurrent diseases were abstracted from the medical history. Lymph nodes, spleen, liver, and intestine were considered abnormal if enlargement, masses, or thickening were identified by palpation, ultrasound examination, or radiology. Overall survival was calculated from the time of flow cytometry submission, because this date was available for all cases, to the time of death due to any cause. In many cases, the exact cause of death was not known. Overall survival additionally was calculated from the date of onset of lymphocytosis, but this information was not available for all cases. Cats alive at the time of data collection or lost to follow‐up were censored at the last date of contact.2.3Flow cytometry was performed on blood samples as detailed in the Supporting Information, using a panel consisting of antibodies recognizing canine CD21, canine CD18, and feline CD5, CD4, and CD8. Total cell counts were determined for CD4+ T cells, CD8+ T cells, total CD5+ T cells, and CD21+ B cells by multiplying the percentage of each subset within the gated lymphocyte population by the total lymphocyte count on the CBC. Subset cell counts for each case were compared to internally generated reference intervals to identify subset expansions. Our laboratory reference interval upper limits for feline blood are 3300 CD4+ T cells/μL, 1800 CD8+ T cells/μL, 5400 CD5+ T cells/μL, and 1700 CD21+ B cells/μL. The percentage of CD4−CD8− T cells was calculated by determining the percentage of CD5+ T cells that did not express CD4 or CD8 antigens. Among 10 control cats with normal peripheral lymphocyte counts and no histologic or flow cytometric evidence of lymphoproliferative disease, the median percentage of CD4−CD8− T cells was 22.6% , suggesting minor populations of CD4−CD8− T cells are not uncommon in healthy cats.2.4Polymerase chain reaction for antigen receptor rearrangements was performed on DNA extracted from blood samples, as previously described,We previously determined that our IGH‐VDJ PARR reaction only detected 50% of feline B‐cell neoplasms, but targeting additional IG gene rearrangements increased B‐cell PARR sensitivity from 50% to 87%.2.5P‐value <.05 was considered significant.For all cases in the outcome cohort, continuous  and categorical variables were summarized and descriptive statistics calculated. Cats in each immunophenotypic category were compared. For continuous variables, an omnibus Kruskal‐Wallis test was calculated; if significant, Dunn's test subsequently was calculated. For categorical variables, Fisher's exact test was used with subsequent pairwise tests if significant. Individuals with missing or unknown data were excluded from analysis. Log‐rank tests were used to compare survival of the immunophenotypic categories from the time of flow cytometry, adjusting for multiple comparisons. Survival also was calculated using the date of lymphocytosis onset as a sensitivity analysis. Among CD4+ T‐cell cases, a log‐rank test was used to determine whether age, sex, hematologic variables, PE findings, and treatment affected survival time. Multivariable survival analysis was not performed because of inadequate sample size. Continuous risk factors were categorized into groups above and below the median for survival analysis. Treatment protocols were variable across cases, and thus treatment was categorized as (i) none; (ii) PO: corticosteroid, chlorambucil, or both; (iii) injectable/multi‐agent chemotherapy: single‐agent injectable chemotherapy, or multi‐agent chemotherapy. Corticosteroid treatment included prednisone or prednisolone, and in many cases, it was not clear which of these 2 drugs was used. Corticosteroid only and corticosteroid/chlorambucil combination treatment were combined for analysis because there was no difference in outcome between these groups. Phenotype distribution, age, sex, hematologic variables, and PE findings were compared between the outcome and concordance cohorts using Fisher's exact and Kruskal‐Wallis tests. All statistical analysis was performed in R version 3.3.2, and a 33.1One hundred forty‐six cases (definition cohort) with flow cytometry and PARR results were reviewed to define phenotypic categories. Cases with sole subset expansions of CD21+ lymphocytes were classified as B cell, of CD8+ T cells as CD8+ T cell, and of CD4+ T cells as CD4+ T cell. When ≥2 of the subsets were expanded, the case was classified as heterogeneous. Cases in which CD4−CD8−CD5+ T cells made up ≥50% of all CD5+ T cells were classified as double negative (DN) T cell. Cases with low surface expression of the CD5 antigen by flow cytometry were classified as CD5 low T cell. Expression of CD5 was considered low if the fluorescence intensity of the entire T‐cell population was contained in the second decade on a log scale, rather than the third decade , DN T‐cell cases (71%), and CD5 low T‐cell cases (86%) had clonal TRG rearrangements, indicating a neoplastic process Figure . Half ofAll 146 cats in the definition cohort had IG PARR with IGH‐VDJ primers. All CD4+ T‐cell cases, DN T‐cell cases, and CD5 low T‐cell cases had polyclonal IG rearrangements. One B‐cell case and 1 heterogeneous case had clonal IG rearrangements with IGH‐VDJ primers. We performed expanded PARR using additional IG primer sets in 92 of 98 B‐cell and heterogeneous cases where DNA was still available. The 2 clonal IG cases originally detected remained clonal, and 1 new clonal B‐cell case was detected with kappa deleting element primers. Overall, only 2 of 46 B‐cell cases and 1 of 52 heterogeneous cases had a clonal IG rearrangement . Six of 7 clonal cases were >7 years old, whereas 17 of 19 non‐clonal cases were <7 years old, suggesting age may be useful in predicting clonality for these cases. All the normal cats used to generate reference intervals had a CD4:CD21 ratio < 1.8. All the cases diagnosed with CD4+ T‐cell lymphocytosis in our study had a CD4:CD21 ratio >4. Second, cases in which ≥50% of the CD5+ T cells expressed neither CD4 nor CD8 antigens were classified as DN T cell. Ten of 14 DN T‐cell cases (71%) had a clonal TRG  cases with heterogeneous expansions, where the magnitude of the CD4+ T‐cell expansion was substantially larger than the B‐cell expansion and (ii) cases with DN T‐cell expansions of small magnitude. In these 2 types of cases, clonality results were used to help refine classification criteria to better segregate non‐neoplastic from neoplastic phenotypes. First, a substantial number of cases n = 31) had dual expansions of CD4+ T cells and CD21+ B cells. A clonal TRG rearrangement was detected in all cases where the CD4:CD21 lymphocyte ratio was >4 . Fifty‐three percent of all cases were female, and 47% were male. There was no significant difference in sex between the groups. Breed was reported for 93 cats with 14 breeds represented. Most cats across all groups were domestic shorthair, followed by domestic longhair; remaining breeds were represented in small numbers.Overall median age for cats in the outcome cohort was 11.5 years . The median age for heterogeneous and B‐cell cases was 5.0 and 8.8 years, respectively, whereas the median age was >12.0 years for each of the other groups , and heterogeneous cases had significantly lower counts than DN and CD5 low T‐cell cases . Anemia was present in 30% of cases and generally was mild, with a median hematocrit in anemic patients of 24% . Thrombocytopenia was present in 26% of cases. There was no significant difference in anemia or thrombocytopenia among groups. Neutropenia was rare , and the total neutrophil count was not significantly different among groups. Hypercalcemia was rare, affecting 1 B‐cell case and 1 CD4+ T‐cell case. Hyperglobulinemia also was rare, affecting 2 heterogeneous cases and 1 DN T‐cell case, and serum protein electrophoresis was not performed.The median lymphocyte count at the time of flow diagnosis for heterogeneous and B‐cell cases was 16 000/μL and 11 100/μL, respectively Figure B. MedianP = .03). Additionally, B‐cell cases (13% affected) had significantly less vomiting or diarrhea reported compared to CD4+ T‐cell cases , DN T‐cell cases , and CD5 low T‐cell cases . Five cats had gastrointestinal histology performed: 3 CD4+ T‐cell cats were diagnosed with intestinal lymphoma, 1 heterogeneous case had lymphoplasmacytic and eosinophilic enteritis, and 1 CD8+ T‐cell case had lymphocytic gastritis with associated helicobacter organisms.Incidence of peripheral lymphadenopathy, hepatic abnormalities, splenic abnormalities, and abdominal lymphadenopathy was not significantly different among immunophenotypic groups. Intestinal thickening or masses were detected in 39% of cases, and B‐cell cases had significantly less intestinal involvement (none affected) than did all other categories (C felis and 1 had a M haemofelis infection recognized after flow cytometry diagnosis. One heterogeneous case had systemic cryptococcosis. Three cats in this B‐cell/heterogeneous group were diagnosed with IMHA. Other diseases affecting this group included hyperthyroidism (n = 2), pancreatitis (n = 2), urinary disease (n = 3), asthma or allergic disease (n = 2), inflammatory bowel disease (n = 2), and non‐lymphoid neoplasia (n = 1). A small subset of cases had mild neutrophilia, suggesting that a catecholamine stress response may be a differential diagnosis in some cats. Only 2 of the B‐cell cases in this cohort were diagnosed with lymphoma and both were renal‐based. Renal disease, hyperthyroidism, inflammatory bowel disease, and pancreatitis also were reported sporadically in the other immunophenotypes.Concurrent diseases were evaluated for B‐cell and heterogeneous cases. Two B‐cell cases and 1 heterogeneous case were positive for FeLV. One B‐cell case was infected with 3.4P < .01 for all comparisons; Figure P = .01), CD8+ T‐cell (P = .04), and CD4+ T‐cell (P = .03) cases. The B‐cell cases  and heterogeneous cases  had prolonged survival. The CD4+ T‐cell cases had prolonged survival , which was not significantly different from heterogeneous or B‐cell cases. The CD8+ T‐cell cases had a MST of 1217 days , but this group only contained 5 cats and 3 of 5 were lost to follow‐up whereas 1 cat died of complications associated with diabetes mellitus. We performed a sensitivity analysis to determine if using the first date of lymphocytosis rather than the date of flow cytometry diagnosis altered our results; our conclusions did not change, except that an MST was now achieved for the B‐cell group (1196 days), and DN T‐cell cases had significantly shorter survival than did the B‐cell cases (P = .05).Overall survival from the time of flow diagnosis was examined for all cases in the outcome cohort. Low CD5 expression predicted poor outcome, with CD5 low T‐cell cases having a median survival time (MST) of 27.5 days , which was significantly shorter than heterogeneous, B‐cell, CD8+ T‐cell, and CD4+ T‐cell cases (P = .02). There was no difference in survival between patients receiving corticosteroid alone (n = 6) versus corticosteroid and chlorambucil (n = 17), and so these patients were combined in the PO treatment group. Patients receiving combination treatment  had shorter survival than did those that received PO treatment alone  or no treatment . The CD4+ T‐cell patients with abdominal lymphadenopathy, intestinal abnormalities, or both had inferior outcome  compared to patients without abnormalities detected .Factors were assessed for association with survival in the CD4+ T‐cell group because of the size of this cohort and are summarized in Table 3.5P = .007). Anemia was more common among CD4+ T‐cell cases in the outcome cohort (28% cases affected) compared to the concordance cohort , but anemia incidence was not significantly different for other phenotype groups. These findings suggest that the outcome cohort of 94 cats is representative of a larger population.A concordance cohort of 350 patients was examined to determine whether the composition of the outcome cohort of 94 patients was representative of a larger population. Phenotype distribution, age, and hematologic variables were compared between cohorts. In the large concordance cohort, CD4+ T cell was the most common phenotype , followed by heterogeneous , B cell 19%, n = 67), CD5 low T cell , DN T cell , and CD8+ T cell  predominantly polyclonal TRG and IG genes, (ii) younger age at diagnosis, (iii) lower presenting lymphocyte counts, and (iv) prolonged survival. Together, these 2 phenotypes accounted for 42% of the cases in the larger cohort of 350 cats. Expansions of multiple lymphocyte subsets are not typical of true lymphoid neoplasms, and thus it is not surprising that the majority of heterogeneous cases (71%) had polyclonal PARR results. However, it was surprising to see the extent to which cats can have a sole expansion of polyclonal B cells. A relatively pure expansion of 1 subset typically is suggestive of a neoplastic process, but most (89%) of the B‐cell cases were polyclonal and only 2 of these cases had clonal IG rearrangements. We recently have demonstrated that the PARR assay used in our laboratory is 87% sensitive for detecting clonal B‐cell populations. The paucity of clonal IG rearrangements suggests that neoplastic B‐cell lymphocytosis is rare in cats, which corroborates the findings of a previous study.C felis, FeLV, and cryptococcosis. Three cases  had apparent immune‐mediated destruction of erythrocytes; these patients were mycoplasma‐negative and the anemia responded to immunosuppression. Interestingly, these cats often maintained the lymphocytosis even while receiving corticosteroids. A review of the literature indicates that, across multiple studies, lymphocytosis has been reported in 15%‐52% of cases of primary IMHA in cats.Many cats with B‐cell or heterogeneous lymphocyte expansions in the outcome cohort had a variety of infectious and inflammatory diseases. The magnitude of the lymphocytosis was striking, in some cases reaching 28 000 lymphocytes/μL. Evidence from experimental Cytauxzoon and Aleurostrongylus infections corroborate these findings where lymphocyte counts up to 31 630 and 36 600 lymphocytes/μL, respectively, were reported.The propensity for cats to develop non‐neoplastic lymphocytosis created a diagnostic dilemma in some cats with true clonal expansions of T cells. In the definition cohort of 146 cats, 31 cats had expansions of both B cells and CD4+ T cells, and 12 of these 31 cats had clonal TRG rearrangements. We attempted to account for the potential for a non‐neoplastic B‐cell lymphocytosis to accompany a primary clonal T‐cell process by using the ratio of CD4+ T cells:B cells in peripheral blood. Based on our PARR findings, a cutoff of 4:1 helps with this discrimination yet is still imperfect. All cats with a CD4+ T‐cell:B‐cell ratio > 4 (5/5) had clonal TRG rearrangements, whereas 27% of cats with a ratio < 4 (7/26) that were classified as heterogeneous expansions had clonal TRG rearrangements. We felt a conservative cutoff threshold would minimize false‐positive interpretations of neoplasia in a clinical setting. These data suggest that even with an underlying T‐cell neoplasm, cats can mount a secondary non‐neoplastic B‐cell lymphocytosis, and PARR is strongly recommended in these cases to ultimately confirm clonality.The most common phenotype in our study, CD4+ T‐cell lymphocytosis, had an indolent clinical course consistent with previous reports.The proportion of cats in our study with intestinal involvement was notable. Forty to 57% of heterogeneous, CD4+ T‐cell, DN T‐cell, and CD5 low T‐cell cases had intestinal abnormalities detected by palpation or imaging. Among cases with intestinal histopathology performed, 1 heterogeneous case was diagnosed as inflammatory bowel disease and 3 CD4+ T‐cell cases were diagnosed as intestinal lymphoma. These findings suggest that cats with intestinal abnormalities and concurrent lymphocytosis may have non‐neoplastic or neoplastic intestinal diseases. One study describing both intestinal lymphoma and chronic enteritis also reported increased lymphocyte counts in cats from both categories.Our results indicate that cats presenting with aberrant phenotypes, in this case increased DN T cells or low CD5 expression, had significantly shorter survival times, but the number of cases was small. The MST of 27.5 days in the CD5 low T‐cell group was particularly notable. Interestingly, in a study of LGL lymphoma with peripheral lymphocytosis, 15 of 21 cases were described as CD5− and had variable expression of CD8 and CD4 antigens.A notable feature of our case series is the relative extent to which the DN T‐cell population can expand in cats with both neoplastic and non‐neoplastic conditions. Using the antibodies in our flow cytometry panel, we routinely can detect small numbers of circulating CD5+CD4/8− cells in normal cats (up to 1000 cells/μL). Because the simple presence of this phenotype could not be used to classify a sample as neoplastic, we utilized the definition cohort to delineate a cutoff of >50% DN T cells. Similar to the ratio of CD4+ T cells:B cells, this cutoff was chosen to conservatively classify cases as neoplastic. Polymerase chain reaction for antigen receptor rearrangements remains a necessary clinical follow‐up test in any cat with substantially expanded DN T cells. In a small number of PARR‐negative B‐cell and heterogeneous cases, DN cells accounted for as many as 49% of all T cells, although the absolute numbers of DN T cells remained relatively modest, demonstrating the extent to which the proportion of non‐neoplastic DN T cells may be expanded. Neoplastic changes resulting in the loss of surface molecule expression are relatively common in lymphomas and leukemias,In summary, our study highlights some of the challenges in characterizing lymphocytosis in cats, especially given the propensity for dual expansions of lymphocyte subsets. Although flow cytometry alone was diagnostic in many cases, a subset still required PARR to distinguish a non‐neoplastic from neoplastic process. We found that cats frequently have heterogeneous lymphocyte and sole B‐cell expansions in the blood, which are predominantly non‐neoplastic in nature. Among the neoplastic phenotypes identified, CD4+ T‐cell, DN T‐cell, and CD5 low T‐cell cases had prolonged, intermediate, and short MSTs, respectively. Flow cytometry is a useful tool to differentiate these T‐cell phenotypes with highly variable clinical courses.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.This retrospective study used materials submitted for diagnostic purposes and information in medical records.Authors declare human ethics approval was not needed for this study.AppendixS1: Supporting Information1Click here for additional data file.


Mitral regurgitation (MR) has a severe impact on hemodynamics and induces severe structural changes in the left atrium. Atrial remodeling is known to alter excitability and conduction in the atrium facilitating atrial fibrillation and atrial flutter. PMVR is a feasible and highly effective procedure to reduce MR in high‐risk patients, which are likely to suffer from atrial rhythm disturbances. So far, electroanatomical changes after PMVR have not been studied.In the current study, we investigated changes in surface electrocardiograms (ECGs) of patients undergoing PMVR for reduction of MR.We evaluated 104 surface ECGs from patients in sinus rhythm undergoing PMVR. P wave duration, P wave amplitude, PR interval, QRS duration, QRS axis, and QT interval were evaluated before and after PMVR and at follow‐up.P < .05, respectively).We found no changes in QRS duration, QRS axis, and QT interval after successful PMVR. However, P wave duration, amplitude, and PR interval were significantly decreased after reduction of MR through PMVR (The data we provide offers insight into changes in atrial conduction after reduction of MR using PMVR in patients with sinus rhythm. Thus, we performed an extensive analysis of patients undergoing PMVR to establish whether atrial unloading induces changes in electrocardiogram (ECG) morphology.22.1Of 233 patients undergoing PMVR using the MitraClip system  with MR grade 2+ to 4, patients were included in the study with ECG in sinus rhythm at baseline and within the first 2 days after PMVR and/or at the follow‐up visit to evaluate P wave characteristics  were evaluated by standard evaluation methods, using Philips CX 50 or iE 33 cardiovascular ultrasound machines with transthoracic and transesophageal probes . Severity of MR was determined according to the guidelines of the European Association of Echocardiography at baseline and at follow‐up.2.4t test was used to compare means, and P < .05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism  and SPSS . Categorical variables are expressed as absolute numbers or as percentage, continuous variables as mean ± SEM, or mean ± SD were specified. Paired 33.1The baseline characteristics of the patient cohort are summarized in Table 3.2P < .0001). At follow‐up, no significant change compared to post‐PMVR was apparent , as shown in Figure P < .001). At follow‐up, no significant change of amplitude was found , as illustrated in Figure At baseline, patients undergoing PMVR showed a mean P wave duration of 113.8 ± 2 ms before the intervention. In the ECG control shortly after the intervention, a reduction of the P wave duration to 103.3 ± 2 could be observed (3.3P < .05). Similar to the other atrial parameters, the mean PR interval did not change significantly during follow‐up compared to post‐PMVR .The QT interval was unchanged after the procedure and did not change during the follow‐up period (421.3 ± 5.1 ms vs 424.1 ± 5.6 ms [P > .05] vs ‐16.9° ± 8 [P > 0.05]).Furthermore, we found no shift in the QRS axis after PMVR and, similarly to the aforementioned ventricular parameters illustrated in Figure 4PMVR is now a widely used approach to reduce MR in patients unsuitable for surgical repair of the mitral valve. With MR being a well‐established risk factor for severe left atrial remodeling, PMVR might be a tool to reduce atrial strain and have a beneficial effect on atrial conduction.In this study, we describe for the first time distinct changes of atrial conduction found in surface ECGs of patients undergoing PMVR: (a) after PMVR, P wave duration PR interval, and P wave amplitude showed a strong decrease compared to baseline, (b) at follow‐up, P wave duration, PR interval, and P wave amplitude remained unchanged compared to post‐PMVR values, (c) QRS duration, QRS axis, and QT interval were not affected by PMVR.In our cohort, patients undergoing PMVR experienced a significant reduction of MR, illustrated in Figure Atrial enlargement has been found to be an independent predictor for onset of AF after mitral valve repair, with atrial remodeling being the major culprit of electrical instability and susceptibility for AF.With a reduction of MR through PMVR, a similar effect could be observed in our patient cohort, resulting in a reduction of P wave amplitude and duration immediately after the procedure. As the shortening of P wave duration and P wave amplitude, as well as the decrease in PR interval was apparent immediately after the reduction of MR, changes in atrial strain resulting in cellular excitability could be the primary underlying mechanism. Several ion channels have been found to be susceptible to atrial stretch in a fashion that alters susceptibility to atrial rhythm disturbances.We did not see changes of atrial conduction during the follow‐up period, which could be due to the fact that PMVR is usually performed in patients with a protracted history of myocardial and cardiovascular disease promoting atrial remodeling. Furthermore, the follow‐up period might have been too short to see long‐term effects of atrial reverse remodeling after initial unloading of the left atrium has equilibrated. Interestingly, we did not observe significant changes in ventricular conduction, although PMVR increases the effective left ventricular stroke volume through reduction of MR.5This study retrospectively evaluated 104 patients undergoing PMVR due to severe MR regarding changes in atrial conduction. Out of 233 patients undergoing PMVR, 104 patients were in sinus rhythm upon ECG evaluation, which was necessary to determine atrial parameters. The mean follow‐up duration of 6.95 months is a possible limitation of the study, as long‐term remodeling effects on the one hand and gradual increase of MR after PMVR on the other could affect outcome and secondary comorbidities, especially AF. Although there is strong evidence that prolonged P wave duration over 120 ms results in a higher risk of AF recurrence,In the current study, we provide data illustrating a decrease of P wave duration and amplitude, as well as a reduction of PR interval through reduction of MR using PMVR.


Background Standard treatment for Glanzmann thrombasthenia (GT), a severe inherited bleeding disorder, is platelet transfusion. Recombinant activated factor VII (rFVIIa) is reported to be effective in GT with platelet antibodies and/or refractoriness to platelet transfusions.Methods We evaluated rFVIIa effectiveness and safety for the treatment and prevention of surgical and nonsurgical bleeding in children <18 years old, with or without platelet antibodies and/or refractoriness, as reported in the GT Registry (GTR). Data were used from the GTR, an international, multicenter, observational, postmarketing study of rFVIIa that prospectively collected data on the treatment and outcomes of bleeds in patients with GT. Only patients with a diagnosis of congenital GT were included in the registry.Results Between 2007 and 2011, 27 children were treated for 44 surgical procedures ; nonsurgical bleeds occurred in 104 patients . The effectiveness of treatment for minor procedures, major procedures, nonsurgical bleeds was 6/6, 1/1, and 75/84 for rFVIIa, 6/6, 2/2, and 64/76 for rFVIIa + antifibrinolytics (AF), 11/12, 1/1, and 162/214 for platelets ± AF, and 5/6, 0/3, and 33/45 for rFVIIa + platelets ± AF. In all, 25 adverse events were reported in children; no thromboembolic events were reported.Conclusion For all patients, regardless of platelet antibody or refractoriness status, rFVIIa, administered with or without platelets (± AF), provided effective hemostasis with a low frequency of adverse events in surgical, as well as nonsurgical, bleeding in patients with GT.clinicaltrials.gov identifier: NCT01476423. Glanzmann thrombasthenia (GT) is an autosomal platelet function disorder caused by a quantitative or qualitative defect of the platelet membrane glycoprotein IIb–IIIa (integrin αIIbβ3) complex.The standard treatment for GT is platelet transfusion (PT), which carries the risk of development of antibodies (AB) to αIIbβ3 and/or human leukocyte antigen (HLA). Possible alternatives to PT include the administration of antifibrinolytics (AF), local hemostatic agents, and bone marrow transplantation. In addition, recombinant activated factor VII (rFVIIa) can be used for the treatment and prevention of bleeding episodes, and for surgery, in patients with GT and AB, as well as past or present platelet refractoriness. Nevertheless, this off-label use of rFVIIa in patients with GT who have bleeds is increasing, mainly because of its convenience and to avoid alloimmunization to platelets. Data from an international survey of patients with GT who have bleeding episodes or hemorrhagic surgery complications supported a preliminary suggestion of an optimal rFVIIa dosing regimen for the treatment of moderate or severe bleeding episodes .As the prevalence of GT is so low, data are not readily available for different subgroups of patients. Particularly rare are data on the treatment of GT in children, for both surgical and nonsurgical bleeding; though case studies are available, no large pediatric studies have been conducted. The GT Registry (GTR) was an international, multicenter, observational registry that focused on both adults and children. As such, it is one of the first efforts to provide pediatricians with comprehensive information on the effectiveness and safety of rFVIIa in children. This article summarizes new GTR data and aims to evaluate rFVIIa effectiveness and safety as a treatment for young patients with GT. Treatment results for all GTR patients have been published previously.The GTR was an international, multicenter, observational study on the effectiveness and safety of rFVIIa in patients with GT , prospectively collecting data on the treatment and outcomes of bleeds. Data were entered into the GTR from 2007 until its closure in 2011, using a customized, Web-based collecting tool. Treatment was based on local clinical practice rather than a set protocol, and performed in accordance with general data protection laws and local country requirements for conducting observational studies.The analyses focused on patients <18 years old, and only a diagnosis of congenital GT was included in the GTR. Patients with acquired thrombasthenic states caused by autoimmune disorders or medications were excluded. Refractoriness and the presence of AB were coded initially and assessed periodically as deemed necessary by the investigator. Because tests for AB may not have been available at all centers, AB may also have been present in patients classified as having refractoriness only. For GT definitions, as well as key terms regarding bleeding, surgery, and definitions of effectiveness , seeThe GTR was conducted in accordance with the Declaration of Helsinki and Guidelines for Good Pharmacoepidemiology Practices. Each participating center complied with local regulations. Ethical and/or regulatory approval was obtained before data entry into the registry, as required. Signed informed consent to participate was obtained from all parents/legal guardians of the patients.The effectiveness analysis was based on all children in the GTR and treatment-allocated bleeds for which the efficacy endpoint was known. All children and bleeding episodes were included in the safety analysis. The effectiveness data were summarized using numerical variables , while categorical variables were summarized as numbers and percentages. No formal statistical comparisons were performed. No subdivision into age groups was performed because patient numbers would be too low to yield meaningful results.Details of recruitment into the GTR and the safety and effectiveness datasets for all surgical and nonsurgical bleeds have been reported previously for adults and children.The clinical and demographic characteristics of children who participated in the GTR and underwent surgery are provided inOf the 599 admissions for nonsurgical bleeds in children, 145 (24.2%) were classified as severe and 454 (75.8%) as moderate, while 423 bleeds (70.6%) were spontaneous and 176 (29.4%) were classified as being posttraumatic , followed by nasal procedures . Most minor procedures  were trData on rFVIIa use were available for 18 minor procedures treated with rFVIIa, rFVIIa + AF, or rFVIIa + PT ± AF; 100% of treatments were rated effective for rFVIIa (6/6) and rFVIIa + AF (6/6), and 83.3% (5/6) of treatments were rated effective for rFVIIa + PT ± AF . For alTreatment with PT ± AF was used in 12 minor procedures, with 91.7% of treatments overall being rated effective . This tOne of the 36 minor procedures was rated “ineffective”  using PT ± AF.n = 3; 37.5% each). Major procedures were treated most frequently with rFVIIa + PT ± AF . All major procedures occurred in the no AB/no refractoriness group. Effectiveness ratings for the different treatments for major procedures  are provided inOf the eight major surgical procedures performed, gastrointestinal and circumcision procedures were most common  and rFVIIa + AF (2/2); 0% (0/3) of treatments were rated effective for rFVIIa + PT ± AF . For alThe effectiveness outcome for major procedures treated with platelets (PT ± AF and rFVIIa + PT ± AF) was available for four cases . Of then = 2) or ineffective (n = 1) in patients treated with rFVIIa + PT ± AF with no AB/refractoriness. No postsurgical bleeding was reported following rFVIIa and/or platelet treatment for major procedures  was reported in the GTR, which was an incidence of pyrexia in a male child without platelet AB or refractoriness, who was treated with rFVIIa + PT + AF (major surgery). It was considered unlikely that this AE was related to rFVIIa, and the patient recovered completely.Regardless of bleeding severity, the PT ± AF combination was the most commonly used treatment . Of theInvestigators reported that, for the 195/590 bleeds that occurred in patients with a history of antiplatelet AB and platelet refractoriness, AB only, or refractoriness only, PT ± AF was used in 93/195 , rFVIIa + AF in 39/195 , AF alone in 33/195 , rFVIIa + PT ± AF in 19/195 , and rFVIIa alone in 11/195  bleeds. The use of rFVIIa, alone  or together with AF , to treat bleeds was most frequent in patients with a history of refractoriness alone, while AF was mainly used to treat bleeds that occurred in patients with no history of AB and/or platelet refractoriness  .The number of rFVIIa doses, cumulative dose, and overall duration of treatment were greater in patients with severe bleeds than in those with moderate bleeds. They were also greater in patients with a history of AB and/or refractoriness to platelets versus patients without such medical history .Overall, treatment was judged as effective see. For 73Ineffective treatment was documented in 13 cases , comprising three severe bleeds and 10 moderate bleeds. Of these, seven had a history of AB/refractoriness. Regarding the initial treatment employed, 8 of the 13 cases were receiving AF, 3 were receiving PT ± AF, and 2 were receiving rFVIIa + AF.For 557 bleeds where data on rebleeding were available, 36 rebleeds in 17 patients were registered, comprising 15/136 (11.0%) severe bleeds, 21/421 (5.0%) moderate bleeds, 15/380 (3.9%) bleeds in patients without AB or refractoriness, and 21/177 (11.9%) bleeds in patients with a history of AB and/or refractoriness to platelets. Rebleeding occurred in 20/204 (9.8%) bleeds treated with PT ± AF, 9/42 (21.4%) treated with rFVIIa + PT ± AF, 1/78 (1.3%) treated with rFVIIa alone, and 3/69 (4.3%) treated with rFVIIa + AF.For the nonsurgical bleeding results presented in this article, 24 AEs were reported  in surgical patients with AB and refractoriness, in surgical patients with major procedures, in nonsurgical patients with refractoriness only, and in nonsurgical patients with posttraumatic bleeding. The numbers receiving rFVIIa ± AF were similar in both surgical patients without AB/refractoriness and those with minor procedures overall. In all other subgroups, rFVIIa, alone or with AF, was used less often than platelets. The GTR results further indicate that rFVIIa has a good safety profile in patients with GT.Assessment of rFVIIa dose and dosing schedule in this registry suggests that, in patients without AB or refractoriness, rFVIIa 90 to 120 μg/kg given at approximately 2 to 4-hour intervals (median: 2–3 hours) for ≥3 doses  could be used, with the first dose given immediately preoperatively in surgical patients. As reported by Poon et al,A median rFVIIa dose of 90 μg/kg was used most frequently in nonsurgical patients with AB and/or refractoriness, and in surgical patients with neither of these . The median dose interval was 3 hours, except in surgical patients with AB and/or refractoriness (for whom it was 2 hours). For severe bleeds, the number of doses reported was understandably higher than for moderate bleeds. In addition, severe bleeds were also associated with longer treatment duration and higher cumulative doses. Based on these results, rFVIIa at ≥90 μg/kg at intervals of ≤3 hours should be used, at least at the beginning of treatment. The subsequent number of doses required would need to be determined by the clinical situation and dosing should be continued until hemostasis is assured.PT was generally effective in covering surgical procedures and nonsurgical bleeds in patients, both with and without historic report of platelet AB and/or refractoriness. An exception is the much lower response rate for nonsurgical bleeds in children with AB and refractoriness 30/57, 52.6%) when compared with adults with AB and refractoriness 25/28, 89.3%).7, 52.6% , 89.3%.A key question is whether combined use of platelets and rFVIIa (rFVIIa + PT ± AF) has an advantage over rFVIIa  or PT ± AF.The relative proportion of AF use for bleeds (both moderate and severe), as well as for surgical prophylaxis (both minor and major), was similar in both children and adults.The successful use of AF without platelets or rFVIIa was reported in 3/7 (42.9%) surgical procedures, and in 147/171 86.0%) nonsurgical bleeding episodes. As noted previously for the GTR,.0% nonsuA potential concern with the use of rFVIIa in patients with GT is whether rFVIIa is thrombogenic. The pediatric data reported here suggest that all systemic hemostatic agents used had a good safety profile in surgical procedures for these patients. Among the 599 nonsurgical bleeding episodes in 104 patients and 44 procedures performed in 27 patients, no thromboembolic events were reported. Thus, rFVIIa appears to be a valid first-line treatment option for nonsurgical bleeds in pediatric patients with GT who are awaiting HLA-compatible platelet concentrates or for concentrates prepared from single-donor apheresis. Indeed, rFVIIa has been licensed for use in GT when platelets are not readily available.As previously reported,The rarity of GT also hinders the performance of formal clinical trials; pediatric data reported here represent the largest dataset available in the literature, including those in other databases.In summary, this post hoc analysis suggests that, when managing children with GT and surgical or nonsurgical bleeding episodes, rFVIIa, PT, and AF are valid first-line treatment options as they appear effective and safe. Given the selection bias in choice of treatment and adaption of treatment to clinical response, treatment strategies cannot be compared in observational studies. For first-line treatment of nonsurgical bleeds with AF, we suggest introducing other treatment options if AF proves insufficiently effective. For major surgical procedures, AF should only be used as a first-line therapy when rFVIIa or platelet concentrates are available as a backup treatment option. In cases of severe bleeding, a combined treatment with AF, rFVIIa, and platelet concentrates may be necessary. The available observations suggest that treatment with platelets may be attempted when other agents are ineffective or not available—even in patients with a history of AB and/or refractoriness. As there were no relevant differences between children and adults regarding effectiveness and safety of the studied treatment options , the treatment of children should not differ notably from that of adults.


Spatiotemporally continuous global river discharge estimates across the full spectrum of stream orders are vital to a range of hydrologic applications, yet they remain poorly constrained. Here we present a carefully designed modeling effort  to estimate global river discharge at very high resolutions. The precipitation forcing is from a recently published 0.1° global product that optimally merged gauge‐, reanalysis‐, and satellite‐based data. To constrain runoff simulations, we use a set of machine learning‐derived, global runoff characteristics maps  for grid‐by‐grid model calibration and bias correction. To support spaceborne discharge studies, the river flowlines are defined at their true geometry and location as much as possible—approximately 2.94 million vector flowlines (median length 6.8 km) and unit catchments are derived from a high‐accuracy global digital elevation model at 3‐arcsec resolution (~90 m), which serves as the underlying hydrography for river routing. Our 35‐year daily and monthly model simulations are evaluated against over 14,000 gauges globally. Among them, 35% (64%) have a percentage bias within  Thirty‐five‐year global reconstruction of river flows at unprecedented 2.94 million reaches extracted from 90‐m high‐accuracy DEMNovel bias correction (“sparse CDF matching”) is conducted grid‐by‐grid against machine learning‐derived global runoff characteristics mapsEvaluation against >14,000 gauges shows data robustness for hydrologic applications and utility in spaceborne discharge estimation efforts A range of state‐of‐the‐art global data sets and novel modeling practices are developed and synthesized to achieve the optimal model performance section . The 35‐The paper is organized as follows: The models, data, and newly developed methods of this study are described in section 22.1For the precipitation forcing, a recently published 0.1° and 3‐hourly precipitation data set that optimally merges a range of gauge‐, reanalysis‐, and satellite‐based precipitation with full global coverage . More specifically, Beck et al.  independently, using the baseflow index, climatology runoff (QMEAN), and runoff percentiles (Q10 and Q90) as the reference. Three sensitive VIC parameters controlling the generation of surface and subsurface flow are selected for calibration (see Text bi), thickness of soil layer 2 (thick2), and fraction of the maximum velocity of base flow at which nonlinear base flow begins (Ds). The Shuffled Complex Evolution (SCE‐UA) algorithm is employed to find the optimal parameter set for each grid cell. The objective function for each grid cell was as follows:o represents GSCD values and subscript m represents values calculated from the model simulation. wi represents the weighting factor to indicate the importance of each runoff characteristics during calibration, and it is set to {0.6, 0.2, 0.1, 0.1} for the four characteristics in this study. More details of this calibration method will be illustrated in a companion paper. The spatially distributed global VIC parameters after calibration are shown in Figures For runoff simulation, the Variable Infiltration Capacity (VIC) LSM  to be estimated for each vector river flowline: while x is a dimensionless weighting factor less sensitive in Muskingum and is set to 0.3 globally, k is a flood wave travel time parameter that can play a key role in the routing performance. To reasonably estimate k for the 2.94 million river reaches used for routing, the following steps are undertaken. Those include (1) running RAPID with QMEAN and arbitrarily set k and x to generate Qclim for all river reaches, (2) using Qclim to estimate river width (w) and depth (d) following a long‐established power law equation recently used by Andreadis et al. (c) with w, d, channel slope, and Manning's roughness assumed 0.035, similar to Allen, David, et al. (L) and c to calculate flood wave travel time (L/c) to obtain k for each river reach. To map the VIC gridded runoff . The spin‐up time for VIC and RAPID are both five years, and the internal time step are both 3‐hourly; the modeled discharge is averaged to daily and monthly values for final model evaluation  section  to deter section  is adopt section . The RAPsections  to 3.3.2.2Qc maps also delivered by Beck et al. (Qc). Our assumption is that these Qc maps trained from ML can potentially offer useful information on runoff signatures beyond our limited knowledge of model processes and parameters, which is in line with the increasing recognition that considers ML as a powerful approach to understand hydrology in ungauged basins , we propose a new BC approach that corrects the VIC runoff biases referenced against nine k et al. . The prok et al. , except R99,m, R95,m, R90,m, R80,m, R50,m, R20,m, R10,m, R5,m, and R1,m (blue dots). The corresponding nine runoff characteristics, denoted as R99,o, R95,o, R90,o, R80,o, R50,o, R20,o, R10,o, R5,o, R1,o, respectively, are used as reference points for adjustment (red dots). To use the sparse reference information, the ratio correction factor Rm) greater than https://github.com/peironglinlin/GlobalHydro/.At each VIC grid cell Figure , the modequation  at all a2.3https://hydrosheds.org/images/inpages/HydroBASINS_TechDoc_v1c.pdf) are used to roughly divide the MERIT Hydro basins, because HydroBASINS jointly considers topographic controls and groundwater connections for its basin delineation  with Princeton high‐performance computing to deal with the huge demand for computer memory. The resulting buffered data is then trimmed within Level‐2 basin boundaries by (1) locating the most downstream reaches within the HydroBASINS boundaries, (2) back‐tracing their contributing areas, and (3) redefining Level‐2 boundaries. Globally, there is less than 1% difference between MERIT Hydro and HydroBASINS Level‐2 basin boundaries on average; the final basin boundary is redefined based on MERIT Hydro. The vectorized hydrography (flowlines and unit catchments) within separated basin boundaries are referred as MERIT Basins v0.1 for future use. The 61 level‐2 MERIT Basins are further merged into nine Level‐1 continents  for RAPID routing. See http://hydrology.princeton.edu/data/mpan/MERIT_Basins/ for more details and access of the MERIT Basins shapefiles derived in this study. It is worth noting that the median (mean) reach length is 6.8 km (9.2 km) and the unit catchment size is 36.8 km2 (45.6 km2). Although the routing algorithm adopted here (Muskingum) is of relatively low complexity, the underlying data infrastructure represents significantly improved accuracy and resolution for channel representation compared to existing global river modeling studies , which covers >17,000 gauges globally Figure . Not surhttp://sedac.ciesin.columbia.edu/data/set/grand-v1-dams-rev01) and the HydroLAKES polygon data set  in the model evaluation. This is in contrast to previous global studies that mainly performed their evaluation for large drainage basins /Qo¯×100%), RV, and KGE skill metrics. Overall, the model shows better performance in humid regions than dry regions. The monthly statistics (right column) are better than the daily KGE scores (left column)—the difference is mainly a result of the CC difference, suggesting daily flow dynamics are less well captured than monthly flows. This is partly related to the difficulty for the forcing to capture the dynamic daily precipitation variability, and partly due to the use of the simple Muskingum routing method that cannot fully resolve daily flow variations. Solving the full Saint‐Venant equations may improve the simulation of the daily flow fluctuations, but the performance will still depend on the channel geometry parameterizations required by the hydrodynamic equations. For the purpose of providing a priori information for SWOT, however, we consider the relatively simple routing scheme sufficient because the monthly and climatological flow is of the primary focus in this study. Over majority global land areas, CC is in the range of 0.6 to 1 (monthly) and 0.4 to 0.8 (daily) except for a few complex‐terrain areas such as Chile, the Alps, and the central to western United States. Such a result is generally better than those when evaluating ten state‐of‐the‐art hydrologic models for discharge modeling . This suggests the VIC LSM and the ML‐derived Qc maps provide similar mean/median runoff estimates over these areas, which implies it is generally less challenging to understand the hydrology there. Note that the BC approach is not only effective in addressing systematic model biases such as Point A through Point E but also able to adjust different runoff percentiles by lowering high flows and increasing low flows . The log‐linear assumption , which is a place needing refinements in the future. The fact that the model is not representing transmission loss in dry regions can also partly explain the stubborn bias. In addition, water management activities such as diversions and irrigations are neither parameterized by the model nor implicitly incorporated in the Qc maps , the most pronounced challenge for the model simulation lies in the dry region runoff prediction  utilize a newly developed 0.1Qc maps used for BC and model calibration contain some “observational information” from a small subset of the final evaluation gauges, and they are assumed as the reference despite these maps are not free from errors . Second, although our goal is to estimate river flows across the full spectrum of stream orders and this goal is better approached compared to previous global modeling studies, many headwater streams important for freshwater biogeochemical cycling (Benstead & Leigh, Supporting Information S1Click here for additional data file.


Salmonella is one of the most important foodborne pathogens, causing outbreaks of human salmonellosis worldwide. Owing to large scales of consumption markets, pork and poultry that contaminated by Salmonella could pose a tremendous threat to public health. The aim of this study was to investigate the contamination of Salmonella from chicken, pork and the environment in slaughtering and retail processes in Chongqing, China.Salmonella isolates were recovered from 1112 samples collected from pork, chicken and the environment. Compared with the isolation rate of samples from chicken (9.50%) and the environment (6.23%), samples from pork had a significant higher isolation rate (44.00%). The isolation rates in slaughterhouses (10.76%) and in supermarkets (10.07%) showed no statistical difference. Thirty different serotypes were identified among all the isolates. S. Derby (n = 26), S. London (n = 16) and S. Rissen (n = 12) were the dominant serotypes. Antimicrobial susceptibility testing revealed that 73.04% isolates were resistant to tetracycline, followed by 66.96% to ampicillin and 59.13% to doxycycline. More than half (50.43%) of the isolates were multidrug resistant (MDR), and most of the MDR isolates were from supermarkets. Multilocus sequence typing results showed 24 out of 115 isolates were ST40, which was the most prevalent. Furthermore, isolates from supermarkets had 20 different sequence types while isolates from slaughterhouses only had 8 different sequence types.A total of 115 Salmonella was more frequently isolated in pork production chain than that in chicken. Compared with isolates from slaughterhouses, isolates from supermarkets had more MDR profiles and represented a wider range of serotypes and sequence types, indicating that the retail process had more diverse sources of Salmonella contamination than that of slaughtering process.Our study highlighted that  Salmonella, a foodborne pathogen, causes diarrhoeal diseases even death in both humans and animals [Salmonella in slaughtering and retail processes of chicken and pork could be a potential pathway to threat public health. animals ; it can  animals . Accordi animals , 4. In CSalmonella is a global issue. Large amount of Salmonellae resistant to extended-spectrum-β-lactams (ESBLs) and fluoroquinolones, which are important in treating salmonellosis [Salmonella, especially multidrug-resistant Salmonella, has been a menace to food safety and human health.Antibiotics are widely used to improve human and animal health, and also are commonly incorporated into animal feed to improve growth rate and feed efficiency in many countries , 6 A prenellosis . Drug-reSalmonella can help to enrich our knowledge of its regularity of dissemination. Serotyping presents a well-established methodology for typing of Salmonella [Salmonella with its transmission dynamics, including pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), whole-genome sequencing (WGS), and multilocus sequence typing (MLST) [Salmonella strains has been uploaded by users in the MLST database [Typing methods used to investigate the characterization of lmonella . To dateg (MLST) –13. Compdatabase , which bSalmonella isolated from food-producing animals is severe worldwide [Salmonella isolates could be recovered from farms, slaughterhouses and retail markets [Salmonella isolates could transmitted from slaughterhouses to retail markets in pig production chain [Salmonella contamination inpork and chicken as well as their slaughtering and retail chains. Therefore, the intention of this study was to compare the antimicrobial resistance, and genetic relationship of Salmonella isolates recovered from the environment, chicken and pork at abattoirs and supermarkets located in Chongqing, China.Contamination and antimicrobial resistance of orldwide , particuorldwide –18. Furt markets , 20 and on chain . HoweverSalmonella isolates were recovered from 1112 samples collected from slaughterhouses and supermarkets, the isolation rates form pork, chicken and the environment were 44.00, 9.50 and 6.23%, respectively. Samples from pork had significant higher isolation rates than that from chicken and the environment in both slaughterhouses and supermarkets , London (n = 16), and Rissen (n = 12) were the most commonly observed serotypes in this study. Isolates from slaughterhouses had 10 serotypes  isolates were resistant to at least one antibiotic and 50.43% (58/115) were MDR. For samples from both slaughterhouses and supermarkets, isolates showed resistance to tetracycline  was the highest, followed by 66.96% (77/115) to ampicillin and 59.13% (68/115) to doxycycline  were identified among 108 isolates, while 7 isolates with failed serotyping results also defected in MLST were excluded for further analysis , followed by ST155 (n = 19) and ST469 (n = 17). Some Salmonella isolates presented similar sequence types that belonged to the same serovar. For example, all ST40 isolates were S. Derby, all S. Rissen isolates belonged to a single cluster (ST469) and 15 out of 16 S. London isolates were ST155. As for the relationship between MLST and antibiotic resistance, we found that ST17 and ST155 were resistant to a wide range of antibiotics in this study, especially that 16 out of 18 ST155 isolates were MDR strains  than that in chicken (9.50%). Other studies also showed that Salmonella contamination rates in pork varies from 30 to 70% at retail markets [Salmonella-colonizing activity at retail markets. In general, all of the differences attributed to collection seasons, amounts of samples and types, isolation methods and management.The overall isolation rate of province  and Yangprovince , but cloprovince  and Germprovince . It shou markets , 24 and  markets , indicatS. Derby, generally detected in pork, could cause salmonellosis in many countries [S. Rissen was generally considered to be transported through pig products in European countries [S. Derby and S. Rissen were isolated from pork, chicken and the environment, and most of S. Derby isolates were detected in the environment. S. Derby was the dominant serotype, which was similar to other studies [S. Typhimurium and S. Enteritidis are the main serotypes causing acute human infection [S. Enteritidis or S. Typhimurium, which had potential threats to public health. In addition, more categories of serotypes and sequence types were detected in isolates from supermarkets than that from slaughterhouses, demonstrating the various and abundant sources of contamination in retail process. Some STs in this study related to specific serovars, for instance S. London with ST155, ST469 with S. Rissen, and ST40 with S. Derby. Our results supported the conjecture that multilocus sequence typing could be an alternative method for serotyping in the future [ountries , and S. ountries . In this studies , 17. S. nfection , and in e future .Salmonella contamination occurred at supermarkets was much more frequently. The sanitation control strategies in supermarkets need to be carried out in this area to improve the safety of animal products.In this study, most of the isolates showed resistance to tetracycline and ampicillin, which was similar to a previous study . The higSalmonella occurred in pork made it become a potential reservoir for human infection to some extent. In addition, although the isolation rate at supermarkets was close to that at slaughterhouses, isolates from supermarkets presented a high frequency of MDR profiles and a wider range of serotypes and sequence types; these results indicated that Salmonella isolates from supermarkets were more threatening and their sources were much more complicated than that from slaughterhouses. Therefore, strict hygiene method and HACCP management in retail process should be taken into consideration to prevent foodborne infection caused by Salmonella.In summary, the results of this study indicated that the contamination of n = 30), knives (n = 30), floor (n = 30), feces (n = 15), apparatus (n = 30), containers (n = 30), tables (n = 30), carcasses (n = 30), and blood (n = 20); retail environment including chopping boards (n = 75), ice (n = 75), knives (n = 75), floor (n = 75), containers (n = 75), wash water (n = 75), and tables (n = 75). All collected samples were stored in an icebox and transported to a laboratory within 2 h of collection for immediate processing and then held in a refrigerator at 4 °C.Convenience sampling was carried out in 7 slaughterhouses and 5 supermarkets in 12 districts of Chongqing, China. From March to October in 2015, a total of 1112 samples were isolated. Chicken and pork were unpacked fresh meat; slaughtering environment including wash water  and incubated overnight at 37 °C. 0.2 mL of each pre-enriched suspensions were added into 2 ml of Rappaport-Vassiliadis enrichment Broth (RVB) and 2 ml of Tetrathionate broth (TTB) respectively, then incubated at 42 °C for 24 h. One loopful of each RVB and TTB culture was then streaked onto Xylose Lysine Tergitol 4 (XLT-4) agar plates, which were incubated at 37 °C for 24 to 48 h. Among suspected colonies, only one was picked up from a plate and confirmed by specific gene through Polymerase Chain Reaction (PCR) of r Scheme .Salmonella isolates to 13 categories of antimicrobials : ampicillin (AMP 10 μg), cefoperazone (CFP 75 μg), piperacillin (PRL 100 μg), tetracycline (TE 30 μg), ceftazidime (CAZ 30 μg), doxycycline (DOX 30 μg), ceftriaxone (CRO 30 μg), minocycline (MH 30 μg), norfloxacin (NOR 10 μg), sulfamethoxazole (SXT 1.25 μg), ofloxacin (OFX 5 μg), chloramphenicol (C 30 μg) and ciprofloxacin (CIP 5 μg). Escherichia coli ATCC 25922 was invoked as the control organism. According to the CLSI, the isolates were considered to be susceptible, intermediate, or resistant. Salmonella isolates resistant to three or more antimicrobial classes were defined as MDR isolates.The standard Kirby-Bauer disk diffusion method recommended by the Clinical and Laboratory Standards Institute  was carried out to test antimicrobial susceptibility of the Salmonella were described online [thrA, purE, sucA, hisD, aroC, hemD, and dnaN. PCR products were purified and sequenced by Sanger method, and the alleles and STs were assigned according to the MLST scheme [Protocols used for MLST of d online . Seven hT scheme . The unwT scheme .P < 0.05) differences in this study.All statistical analyses were done using SPSS 20.0 , and the chi-squared test was applied to assess any statistically significant (