Introduction: Coagulation assessment is often missing in microvascular surgery. We aimed at evaluating the predictive value of thromboelastometry for free flap thrombosis in microvascular surgery patients.Materials and Methods: We enrolled 103 adult patients with traumatic injuries scheduled for microvascular free flap surgery into a prospective observational study. Thirty-six patients with recent trauma underwent surgery within 30 days (ES group), and were compared with 67 trauma patients who underwent surgery later than 30 days  after the injury. Rotational thromboelastometry (RTE) was performed before surgery. Functional fibrinogen to platelet ratio (FPR) ≥ 42 was selected as the main hypercoagulability index. Free flap thrombosis was set as primary outcome. Thrombotic risk factors and duration of surgery related to free flap thrombosis were secondary outcomes. Statistical significance p < 0.05; not significant NS.Results: Six patients (16.7%) in the ES group and 10 (14.9%) in the LS group had free flap thrombosis (NS). In the entire cohort, free flap thrombosis rate increased in the presence of thrombogenic comorbidities  and prolonged surgery times . Although hypercoagulability occurred more frequently in the ES group (44.4%) than in the LS group , it was not associated with higher free flap thrombosis rate. In ES group patients with surgery times > 240 min, the risk of free flap thrombosis increased  with 93.3% sensitivity and 86.7% specificity . In contrast, in LS patients hypercoagulability increased the odds of free flap thrombosis . Moreover, a positive correlation was found between FPR ≥ 42 and free flap thrombosis rate . In the LS group, the presence of thrombogenic comorbidities correlated with free flap thrombosis rate .Conclusions: In LS patients with thrombogenic comorbidities, thromboelastometry supports the detection of hypercoagulability and predicts free flap thrombosis risk. In ES patients, postoperative hypercoagulability did not predict free flap thrombosis. Prolonged surgery time should be considered as a risk factor. Microvascular free flap surgery allows closure of various tissue defects in reconstructive microsurgery. Since the introduction of free tissue transfer, advancements in microsurgical techniques have increased flap survival rates –3. HowevIn trauma patients, a time-dependent hemostatic response initially increases the risk of bleeding due to hemodilution or consumption coagulopathy in combination with a rise in fibrinolytic activity . SubsequIn recent years, rotational thromboelastometry (RTE) has become an extensively used method of monitoring coagulant activity in major bleeding after trauma –26, cardWe enrolled 103 subsequent adult patients with traumatic injuries scheduled to undergo microvascular free flap surgery at the Latvian Center of Reconstructive and Microsurgery, Riga, Latvia into a prospective observational study. We compared two groups of patients according to the time that had elapsed from the traumatic event until the day of surgery. Thirty-six patients, who were operated within a 30-day period after trauma, during the primary hospital stay, were allocated to an early surgery group (ES group), and the remaining 67 patients were referred to a late surgery group (LS group) that was operated later than 30 days after the injurious trauma due to late complications, such as infections or scars. In those cases, tissue defects were closed secondary in the late posttraumatic period.The Ethics Committee of Riga Stradinš University approved the study protocol (Nr.1/26-11-16) and the informed consent form. All adult patients with traumatic injuries who underwent microvascular free flap surgery during the study period were asked to participate in the study. We excluded patients below 18 years of age and patients with ongoing oncological treatment or history of confirmed thrombophilia or pregnancy. Surgery was performed as a consequence of acute or chronic traumatic injuries. All patients gave their prior written informed consent.General anesthesia (GA) was provided for all enrolled patients according to the guidelines for free flap transfer surgery. After induction with fentanyl  1.5–2 μg/kg, and propofol  1–2 mg/kg intravenously (iv), GA was maintained with sevoflurane  0.8–1.2 MAC, and fentanyl 1–1.5 μg/kg/h. We administered cisatracurium  0.15 mg/kg iv for tracheal intubation, followed by a continuous infusion of 1–2 μg/kg/min for muscle relaxation. Crystalloid infusion  of 3.5 to 6.0 ml/kg iv per hour during the 24-h perioperative period was standardized and controlled for adequacy guided by monitoring of a urine output of 1–2 ml/kg/h. To avoid hypothermia, we measured central  and peripheral (axillary probe) temperatures (°C) of the patients, maintaining core temperatures above 35°C and a difference between central and peripheral measurements (Δt) of <2°C. We assessed patients' volume status by evaluating urine output and Δt combined, in addition to hemodynamic parameters. The decision to insert a line for central venous pressure monitoring was guided by the patient's cardiovascular status, anticipated blood loss and need of vasoactive substances.We performed peripheral blocks under ultrasound guidance when indicated. We applied brachial plexus block with axillary approach for upper extremity surgery, femoral nerve and popliteal blocks for lower extremity postoperative analgesia. Paracetamol  1.0 g four times daily in combination with non-steroidal anti-inflammatory drugs two times daily were used for postoperative multimodal analgesia. For those without a peripheral nerve block, we administered a continuous intravenous infusion of fentanyl 0.5–1 mcg/kg/h.Postoperatively, tightly followed hemodynamic monitoring, fluid management and oxygen supply was provided in the post-anesthesia care unit. According to hospital guidelines, postoperative thrombotic prophylaxis was provided with enoxaparin 40 mg  once daily from the 1st postoperative day for all included patients. Hemoglobin and hematocrit levels were optimized  in anemic patients with allogenic hemotransfusions before surgery. During and after surgery, patients with clinical symptoms of excessive blood loss or those with Hb <7 g/dl received blood product transfusions.During the study, the same team of specialists trained in microsurgery performed all microvascular flap surgeries. The surgeon monitored the microvascular flap for 3–5 days postoperatively. Clinical assessment of flap color and temperature, tissue turgor and capillary refill were evaluated as flap perfusion criteria. In all cases of free flap thrombosis, urgent surgical re-exploration was performed in order to salvage the transferred free flap. The venue of thrombosis  was assured by direct visualization during revision of the anastomosis.2 for recalcification. As activators, we added 20 μl of recombinant tissue factor for EXTEM test, 20 μl of partial thromboplastin phospholipid from rabbit brain for INTEM test, and 20 μl cytochalasin D for FIBTEM test. For all three test assays, we recorded the following parameters of RTE: clotting time ; clot formation time , MCF-maximum clot firmness  (FIBTEM) by MCFINTEM, as expressed by a numerical ratio.Preoperatively, a blood sample was drawn for RTE  assessment of the coagulation status. For each RTE test, we used 300 μl of citrated blood with 20 μl of 0.2 mol/L CaCl9–25 mm) . Hyperco9–25 mm) . To calc9/L) was determined by using Sysmex XE-5000 .In parallel, we performed standard coagulation measurements. Prothrombin index , activated partial thromboplastin time  and fibrinogen plasma levels  were analyzed in citrated plasma using the STA-R COMPACT . Platelet count . We assessed the data distribution with Shapiro-Wilk test because the sample size was small. Continuous variables were presented as mean ± standard deviation (SD) or median with interquartile range (IQR), as appropriate, and categorical variables as percentages (%). Quantitative (continuous) data were non-normally distributed (except for age of patients). Therefore, we used non-parametric Kruskal-Wallis H test to compare differences between quantitative data. Cross tabulation was used to compare free flap thrombosis (yes/no), time to operation  and demographics, clinical data and RTE variables. To assess statistical differences between the abovementioned data, we used Pearson Chi Square test and if small sample size (<5 cases) Fisher's exact test. Correlations between free flap thrombosis factors were performed with non-parametric Spearman rho test. ROC curve analysis was performed to state the diagnostic value of FPR for free flap thrombosis with threshold level of ≥ 42. We employed ROC curve analysis to find the thresholds for sensitivity and specificity of FPR, and duration of surgery and furthermore, to find the threshold of operation time at which the risk of thrombosis is high. Linear regression was performed to find adjusted predicted values of thrombosis for the length of operation. We also performed binary regression analysis in the entire population and in subgroups of early (ES group) and late surgery (LS group) patients to find significant free flap thrombosis factors. Statistical significance was reached at alpha level <0.05.p < 0.001) and thrombogenic comorbidities (p = 0.029) appeared significantly more often in the LS group, whereas posttraumatic hyperfibrinogenemia with fibrinogen plasma levels > 4 g/L were registered more frequently in the ES group (p < 0.001). There were no significant differences in smoking history, thrombocytosis or surgery time between the groups. Interestingly, the ES group patients underwent mainly lower extremity reconstruction (83.3%). The most frequent locations of myocutaneus free flap surgeries were on the scapula and in the parascapular area (21.3%), as well as in the anterolateral area of the thigh (12.6%) and the latissimus dorsi muscle area (13.5%). Fibula transposition was the most frequent bone tissue type (9.7%).Demographic data did not differ significantly (NS) between the groups . As expep < 0.001), and higher fibrinogen plasma concentration (p = 0.001), that is characteristic for the acute posttraumatic period.As shown in INTEM, p = 0.013).Assessing preoperative RTE parameters, significant shortening of clot formation time (CFT) and increase in maximum clot firmness (MCF) were detected in the ES group. In contrast, we found no differences between the groups with regard to plasma coagulation, as evidenced by clotting time (except CT Free flap thrombosis developed within the first 24 h after surgery in 16 patients (15.5 %) without significant intergroup difference .p < 0.001), as demonstrated in p = 0.009) in the LS group patients, as depicted in r = 0.362; p = 0.003), MCF EXTEM , and MCF FIBTEM .Hypercoagulability detected by RTE was seen in totally 24 patients, and more often in the ES group (Although 44.4% of the patients in the ES group had hypercoagulability (FPR ≥ 42), it did not affect the rate of free flap thrombosis.p = 0.014). The latter patients were more often presented in LS group. Additionally, multifactorial analysis showed that combination of hypercoagulability in RTE and time to surgery increases risk for free flap thrombosis  in the whole population.By assessing the possible influence of thrombogenic risk factors and duration of surgery on free flap thrombosis rate in all the enrolled patients, we found that those with previous thrombogenic comorbidities had a 4-fold increased odds of free flap thrombosis , as a marked risk factor for free flap thrombosis, a history of thrombogenic co-morbidities demonstrated an influence on flap thrombosis rate ; p = 0.05), as depicted in p = 0.026). In addition, linear regression analysis showed that surgery time from 240 up to 300 min increased the risk of free flap thrombosis by 34%.Duration of surgery appears to be a main predisposing factor for free flap thrombosis in the ES group. Prolonged duration of surgery demonstrated a tendency to promote development of postoperative thrombosis  should be considered as a major risk factor in these patients.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Riga Stradins University Ethics Commitee. The patients/participants provided their written informed consent to participate in this study.BM, JS, and AO conceived the study. JS was responsible for data collection and informed the patients. IV, JS, and MM performed data analysis and JS made interpretation of data. JS, AO, and LB drafted and revised the manuscript. All authors read and approved the final manuscript, contributed substantially to the study design, analysis and interpretation of data, and drafting or revising the article.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.


Risk factors affecting the prognosis of acute respiratory distress syndrome (ARDS) in adults were investigated. The aim was to identify new predictors for ARDS patient prognosis, including those with clinical, pathophysiological, and atypical immunodeficiency.ARDS patients were retrospectively included. The patients were grouped and analysed according to different oxygenation index grades and prognosis, and factors influencing prognosis and survival were examined. Adolescent patients, patients with typical immunodeficiency and patients who died within 24 h after being diagnosed with ARDS were excluded. The predictive value for mortality was determined by Cox proportional hazard analysis.P = 0.016), Sequential Organ Failure Assessment (SOFA) score (P = 0.027), and PaO2/FiO2 (P = 0.000), worsened from mild to severe ARDS cases. Compared with survivors, non-survivors were significantly older and had higher APACHE II and SOFA scores. Moreover, significantly lower lymphocyte/neutrophil ratios and leukocyte counts were found among non-survivors than survivors . A moderate positive correlation between the lymphocyte/neutrophil and PaO2/FiO2 ratios (P = 0.023) was observed. In predicting 100-day survival in patients with ARDS, the area under the curve (AUC) for the lymphocyte/neutrophil ratio was significantly higher than those for the PaO2/FiO2 ratio alone, body mass index (BMI) alone, and the lymphocyte count alone . Age (per log10 years), BMI < 24, SOFA score, leukocyte count, and the lymphocyte/neutrophil ratio were independent predictors of 28-day mortality in ARDS patients. Additionally, ARDS patients with a lymphocyte/neutrophil ratio < 0.0537 had increased 28-day mortality rates (P = 0.0283). Old age affected both 28-day and 100-day mortality rates .In total, 201 patients who fulfilled the Berlin definition of ARDS were included. The severity of critical illness on the day of enrolment, as measured by the Acute Physiology and Chronic Health Evaluation (APACHE) II score (10 years), BMI < 24, SOFA score, lymphocytes, and the lymphocyte/neutrophil ratio were independent predictors of 100-day mortality in patients with ARDS. The lymphocyte/neutrophil ratio may represent a potential molecular marker to evaluate atypical immunosuppression or impairment in patients with ARDS.Age (per log Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory disease with a high mortality rate in critically ill patients , 2. AlthNonetheless, clinical and animal studies have shown that the activation of multiple inflammatory cells and the release of inflammatory mediators play important roles in the development and outcome of ARDS . The invThis aim of our study was to identify a convenient and easy-to-use molecular biomarker to detect and evaluate the status of patients with atypical immunosuppression in ARDS patients.This retrospective observational cohort study was conducted with ARDS patients hospitalized in the critical care centre of a university-based tertiary care hospital  in Hunan, China, from January 2011 to August 2018. Institutional approval was provided by the Second Xiangya Hospital of Central South University Biomedical Research Ethics Committee . Written informed consent was waived because of the retrospective observational design. All patient data were anonymously recorded to ensure confidentiality.2/FiO2 ratio, patients were categorized as mild , moderate , and severe  groups. In addition, 201 patients were included and divided into a survivor group (n = 80) and a non-survivor group (n = 121) according to the final clinical results.Patients admitted to the critical care centre with a diagnosis of ARDS based on the 2012 Berlin definition  were incDemographic and baseline characteristics such as age, sex, body mass index (BMI), ARDS risk factors, severity of illness upon admission to the ICU  II score) , and theThe white blood cell count, C-reactive protein (CRP) level and procalcitonin (PCT) level were measured. Serum levels of haemoglobin, albumin, immunoglobulins , and complement components   was determined by enzyme-linked immunosorbent assay (ELISAs) in accordance with the manufacturer’s instructions. To analyse T-lymphocytes, cell staining kit  was used to detect CD4 + CD8 + CD3 + cells according to the manufacturer’s protocol. Briefly, peripheral blood mononuclear cells (PBMCs) were incubated with a mixture of luciferin isothiocyanate anti-CD4 and apc anti-CD8 at 4 °C for 30 min. Facscalibur flow cytometry  and CellQuest software  were used for flow cytometry analysis. A homotype control was used to ensure antibody specificity , 18.P-values adjusted by Bonferroni were used for multiple groups of comparison. Classification data number (percentage) aggregation, and Chi-square or Fisher’s exact test. Spearman’s rank correlation was adopted to determine correlations among variables. Area under the curve operating characteristic (ROC) was used to evaluate the prognostic value of the subject properties, diagnostic and test parameters. The cut-off point was obtained by determining the optimal den index (sensitivity+specificity-1). We used Kaplan-Meier plots and log-rank tests to compare the survival rate of each group. To calculate an independent predictor of mortality of 100 days, with a stepwise binary logistic regression variables for the regression values of P < 0.05 . The odds ratio (OR), P-value and 95% CI were used to represent results. All tests were double-tailed, and P < 0.05 was considered statistically significant. All analyses were performeded using IBM SPSS 22.0 and MedCalc v.11.0.We used the Kolmogorov-Smirnov test to assess distribution normality. The continuous variables were reported as the mean ± SD or median (IQR). An independent samples t-test was used to evaluate normally distributed data, and the Mann-Whitney test was employed to evaluate non-normally distributed data when comparing two groups. In multi-group comparisons, one-way ANOVA and Kruskal-Wallis test were used to analyze the normal and non-normal distribution data respectively, and the P = 0.016), SOFA score (P = 0.027), and PaO2/FiO2 (P = 0.000), it can be seen that the severity of critical illness on the day of enrolment worsened from mild to severe ARDS, as shown in Tables 2/FiO2 ratios than did non-survivors.A total of 201 patients meeting the Berlin definition of ARDS from January 2011 to August 2018 were included in this study. Characteristics at enrolment and outcomes of the study population are shown in Table P = 0.025). Moreover, the lymphocyte/neutrophil ratio decreased progressively with increasing ARDS severity (P = 0.001). Among non-survivors, a significantly lower lymphocyte/neutrophil ratio was found compared with that of survivors (P = 0.008)  . The PCT level of patients with severe ARDS was higher than that of patients with mild ARDS (P = 0.002). Interestingly, the lymphocyte count decreased as the severity of ARDS increased (P = 0.025) (Table P = 0.004), and had higher leukocyte and neutrophil counts  and lower BMI, lymphocyte counts and lymphocyte/neutrophil ratios  . Moreover, the levels of immunoglobulin IgE and complement C3 in non-survivors were lower than in survivors  (Table P = 0.026) , though the levels of both peripheral blood B-lymphocyte cells and CD8+ cells were similar in the three groups , as was the level of peripheral blood CD8+ cells , the lymphocyte count and the PaO2/FiO2 ratio , the immunoglobulin IgE level and the PaO2/FiO2 ratio , the C3 level and BMI , the T-CD8+ lymphocyte count and the lymphocyte count , the B-lymphocyte count and BMI , and the B-lymphocyte cell count and lymphocyte count .We noted that in all ARDS patients, the lymphocyte/neutrophil ratio was moderately negatively correlated with age , the AUC for BMI alone  or the AUC for the lymphocyte count alone   and the lymphocyte/neutrophil ratio in combination with the PaO2/FiO2 ratio  , BMI alone (P = 0.0001), and lymphocyte count alone (P = 0.0067), and the AUC for the lymphocyte/neutrophil ratio in combination with the PaO2/FiO2 ratio was significantly higher than those for the PaO2/FiO2 ratio alone (P = 0.0014), BMI alone (P = 0.0001), and lymphocytes alone (P = 0.0162).The area under the ROC curve (AUC) for the lymphocyte/neutrophil ratio for the prediction of 100-day survival in ARDS patients was 0.721 (95% CI 0.653 to 0.782) and was significantly higher than the AUC for the PaO9/L) was used to predict the survival of patients with ARDS. The sensitivity was 87.5%, and the specificity was 81.0%. The positive likelihood ratio was 4.61, and the negative likelihood ratio was 0.15.A cut-off value of the lymphocyte/neutrocyte ratio of > 0.0537 was used to predict the survival of ARDS patients. The sensitivity was 83.8%, and the specificity was 80.2%. The positive likelihood ratio was 4.23, and the negative likelihood ratio was 0.20. Moreover, a leukocyte count cut-off of > 0.415 (1010 years) , BMI < 24 , SOFA score , leukocyte count< 0.415 (109/L) , and lymphocyte/neutrophil ratio  were independent predictors of 100-day mortality in ARDS patients. Moreover, ARDS patients with a lymphocyte/neutrophil ratio < 0.0537 had a higher 28-day mortality rate than did those with a lymphocyte/neutrophil ratio > 0.0537 , BMI < 24, the SOFA score (per point) and the lymphocyte/neutrophil ratio were independent risk factors for predicting 100-day mortality in ARDS patients. Another discovery was that the lymphocyte/neutrophil ratio and age were related to ICU mortality and hospital mortality. We also found associations between the baseline lymphocyte/neutrophil ratio and age, the SOFA score, the APACHE II score, the PaO2/FiO2 ratio, and the severity of ARDS according to the Berlin classification. The lymphocyte/neutrophil ratio may help predict prognosis for ARDS patients with a high immunologic risk. Our study is a longitudinal clinical outcome study of ARDS patients, and the results demonstrate the predictive significance of the lymphocyte/neutrophil ratio.In this study, we found associations between age, BMI, the SOFA score, and the lymphocyte/neutrophil ratio at ICU admission and clinical outcomes in patients with ARDS. Age , BMI < 24, the SOFA score, the lymphocyte count, and the lymphocyte/neutrophil ratio to be independent predictors of 100-day mortality in patients with ARDS. We also observed a moderate negative correlations between the lymphocyte/neutrophil ratio and age, the SOFA score, and the APACHE II score, and a significant mild positive correlation between the lymphocyte count and BMI was also found. However, we detected a moderate positive correlation between the lymphocyte/neutrophil ratio and the PaO2/FiO2 ratio in all patients with ARDS. The AUC was greatest for the lymphocyte/neutrophil ratio combined with the lymphocyte count for the prediction of 100-day survival in ARDS. In addition, the 28-day mortality rate of ARDS patients with a lymphocyte/neutrophil ratio < 0.0537 was higher than that with a ratio of lymphocyte/neutrophil ≥0.0537. The lymphocyte/neutrophil ratio was also revealed to be a strong and independent predictor of prognosis in ARDS patients, particularly in those with atypical immunodeficiency.We found age (per log


There is a link between hyperglycemia and mechanical functions of muscle. However, existing evidence of the association between hyperglycemia and weaker muscle strength is limited and inconsistent. We examined whether glycemic status was associated with relative grip strength (RGS) in older Chinese.In 2008–2012, 9180 participants (2516 men and 6664 women) from the Guangzhou Biobank Cohort Study had fasting and 2-h post-load glucose measured. Glycemic status was categorized as normoglycaemia, prediabetes  and diabetes. RGS was assessed using a Jamar Hydraulic Hand Dynamometer divided by body mass index. General linear model was used to assess the association of glycemic status with RGS.P for trend < 0.001). The association of glycemic status with RGS was non-significant in women. Among the normoglycaemic group, no association was found between fasting glucose and RGS in men, whereas a significantly inverse association was found in women, with adjusted β for RGS per mmol/l increase in fasting glucose being − 0.05 to − 0.04 .After adjusting for age, smoking status, alcohol use, physical activity, health status, body fat percentage and waist circumference, in men, hyperglycemia was associated with a lower RGS, with the RGS being 1.38  = 1.34, 1.42) in normoglycaemia, 1.35  in prediabetes, 1.33  in newly diagnosed diabetes and 1.32  in known diabetes (Higher fasting glucose was associated with reduced grip strength in a dose-response manner, and the association was significant even in women with normoglycaemia. Our findings suggest that lowering glucose across the whole range might be important in preserving muscle strength, especially in aging women. Low muscle strength is an indicator for frailty  which is1c but sex differences or fasting glucose and 2hPG were not reported [Diabetes mellitus (DM) is a major chronic disease with severe complications. The number of DM patients has reached 463 million globally in 2019 and is estimated to increase to 700 million in 2045 , 9. Diabreported . We therAll participants were from GBCS, which is an on-going three-way collaborative project of the Guangzhou 12th Hospital and the Universities of Hong Kong, China and Birmingham, United Kingdom. Details of GBCS have been reported elsewhere . Brieflyn = 15), 9180 participants were included in the analyses. Face-to-face interviews [In this paper, we used data from participants who returned for the second examination during March 2008 to December 2012. Age range of the participants was from 53 to 98 years, with the mean age being 68 and 65 years for men and women, respectively. Within age-group, it has been shown that participants had similar levels of diabetes and hypertension to the nationally representative samples of urban Chinese –17. In 9terviews , educatiterviews , lifestyterviews  and physterviews ), healthterviews , waist cterviews  and BMI terviews ) may be All participants were required to fast for at least 10 h from the night before blood taking in the morning. Fasting glucose were measured by Shimadzu CL-8000 Clinical Chemistry Analyzer . Two-hour post-load glucose (2hPG) was measured after 75-g oral glucose administration in all participants except for those with self-reported physician diagnosis of diabetes or with glucose-lowering treatment. Participants were categorized into six groups, including (1) normoglycaemia, (2) impaired fasting glucose (IFG) only, (3) impaired glucose tolerance (IGT) only, (4) IFG and IGT, (5) newly diagnosed T2DM and (6) known T2DM . Althougr = 0.98) inter-rater reliability [max) was calculated by AGS divided by BMI. Average relative grip strength (RGS mean) was calculated by the average grip strength in both hands divided by BMI. Relative grip strength in left- or right- hand (RGS left / RGS right) was calculated by the average grip strength in left- or right- hand divided by BMI [Grip strength was assessed using a Jamar Hydraulic Hand Dynamometer in a standing position. Grip strength of each hand was tested two times and the average value was calculated, expressed as kilograms. The maximal reading of the average grip strength in right and left hands was used as the absolute grip strength (AGS). Measurements of grip strength using Jamar dynamometer showed good to excellent test-retest reproducibility (r > 0.80)  and exceiability . Maximumd by BMI , 32.2 test and one-way analysis of variance (ANOVA) were used to compare categorical and continuous variables respectively. The Bonferroni’s test was used to control the family-wise error rate. General linear models were used to assess the association of glycemic status and quartiles of fasting glucose in normoglycaemic group with grip strength, giving adjusted regression coefficient (β) and 95% confidence intervals (CIs). We also tested for interaction between sex and glycaemia groups. As significant interactions were observed on the associations with RGS mean and RGS right (P for sex interaction from 0.03 to 0.92), all analyses were done on total participants and by sex. Matrix diagram of the measures of grip strength were plotted, with the RGS max showing the highest correlation coefficient than other measures of grip strength . There were also significant differences in physical activity, education and health status in women and smoking status in men with poorer status in T2DM group (P from < 0.001 to 0.002).Table max declined from normoglycaemia to prediabetes , then to known/newly diagnosed T2DM groups . The associations of glycemic stages with grip strength were different in men and women in terms of RGS mean, RGS left, RGS right and AGS. Although the associations of glycemic status with these measures of grip strength  were not statistically significant in women (P for trend from 0.07 to 0.17), significant inverse associations were observed in men (P for trend from 0.01 to 0.03) as well as total participants (P for trend from 0.005 to 0.03).Table P for trend from 0.002 to 0.03). In women, the adjusted β (95% CI) was − 0.04  for RGS max, − 0.04  for RGS mean, − 0.05  for RGS left, − 0.04  for RGS right and − 0.98  for AGS. However, no association of fasting glucose with grip strength was found in men, when fasting glucose was analyzed as quartiles or continuous (P for trend from 0.67 to 0.88). As the associations did not vary by sex (P for sex interaction from 0.13 to 0.92), we also conducted analysis in total participants and found consistently inverse associations between fasting glucose and all measures of grip strength (P for trend from 0.006 to 0.049). No association between 2hPG and measures of grip strength in normoglycaemic group was found (Supplementary Table Table max after full adjustment (P < 0.001 for trend), with the RGS max (95% CI) being 0.88  in the highest decile and 0.95  in the lowest. In men, there was similar trend between fasting glucose and RGS max but the association was not statistically significant (P = 0.22)  , glycaemia (fasting glucose and 2hPG) and grip strength. However, our study had several limitations. First, as the associations were cross-sectional, whether the association between glycemic status and grip strength is causal is uncertain. Second, there are some factors that may influence the instantaneous strength tests and thus residual confounding could not be completely ruled out, although a wide range of potential confounding factors were adjusted for and the results remained after the adjustment. Third, as all participants were older people who might have relatively lower grip strength, the results may not be applicable to other age groups.In conclusion, higher fasting glucose was associated with reduced grip strength in a dose-response manner in men from normoglycaemia to prediabetes then to T2DM, and the association was significant even in women with normoglycaemia. Our findings suggest that lowering glucose across the whole range might be important in preserving muscle strength, especially in aging women. Further studies on the mechanisms and trials are warranted.Additional file 1. Supplementary Table 1. Characteristics by quintiles of relative grip strength max in 2498 men and 6638 women in Guangzhou Biobank Cohort Study. SD = standard deviation; Relative grip strength max, maximal of the average of the right or the left grip strength divided by body mass index (BMI)Additional file 2 Supplementary Table 2. Grip strength by post-load glucose  in 1178 men and 3080 women with normoglycaemia. Results were shown as mean , except for numbers. Relative grip strength max, maximal of the average of the right or the left grip strength divided by body mass index (BMI); Relative grip strength mean, the mean of the average of both the right and the left grip strength divided by BMI; Relative grip strength left, the average of the left grip strength divided by BMI; Relative grip strength right, the average of the right grip strength divided by BMI; Absolute grip strength, maximal of the average of the right or the left grip strength. †: Adjusted for age, education, smoking status, alcohol use, physical activity, body fat percentage and waist circumference. ††: Adjusted for age, education, smoking status, alcohol use, physical activity, body fat percentage and waist circumference and BMI. $: Adjusted for age, sex, education, smoking status, alcohol use, physical activity, body fat percentage and waist circumference. $$: Adjusted for age, sex, education, smoking status, alcohol use, physical activity, body fat percentage and waist circumference and BMI. #: P value for sex interaction with post-load glucose in terms of relative grip strength mean and relative grip strength right was 0.04 and 0.03 respectively. **: P < 0.01; ***: P < 0.001Additional file 3. Supplementary Figure 1. The matrix diagram of the measures of grip strengthAdditional file 4. Supplementary Figure 2. Association between 2hPG  and RGS max in participants without T2DM. All the means (95% CIs) were adjusted for age, education, smoking status, alcohol use, physical activity, body fat percentage and waist circumference


Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of  Leishmania major strain to be used as a resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development. Here, the authors describe the isolation, characterization, and GMP manufacture of a clinical  Leishmania. One billion people are at risk of infection across 98 countries worldwide, with over 1.5 million new cases and 20,000–40,000 deaths reported each year2. The leishmaniases are vector-borne diseases, each parasite species having co-evolved for transmission by one or more species of phlebotomine sand fly4. Disease manifestation is intimately linked to the species of infecting parasite6 and may be evident as self-healing lesions restricted to the site of skin transmission , lesions which spread from an initial skin lesion to involve the mucosae  or which spread uncontrolled across the body , or as a potentially fatal systemic disease involving major organs such as the spleen, liver and bone marrow 5. In addition, patients recovering from VL following chemotherapy often develop a chronic skin condition  that can sustain community transmission of VL8. Collectively, the tegumentary forms of leishmaniasis account for approximately two-thirds of the global disease burden, while VL accounts for most reported deaths6. In addition to the impact of primary disease, recent studies have also emphasized the importance of considering the long term sequelae of leishmaniasis, notably those associated with stigmatization, when evaluating global burden of these diseases11.The leishmaniases represent a group of diseases caused by infection with various species of the parasitic protozoan 15). Four vaccines for canine visceral leishmaniasis have reached the market, though with remaining questions about the extent of clinical versus parasitological protection that they provide16. To date, no human vaccines have achieved licensure17. Often cited barriers to vaccine development include limited investment, with only $3.7 M of new R&D funding globally in 201818, an excess of candidate antigens and delivery systems19, the questionable predictive capacity of pre-clinical animal models21, lack of good correlates of protection and a defined target product profile22, the costs and challenge of large-scale efficacy studies in disease endemic countries coupled with the high prevalence of asymptomatic infections24 and a fragmented pipeline for translational research25. As has been found with other diseases33, the incorporation of a controlled human infection model (CHIM) into the vaccine R&D pipeline can overcome many of these issues.The leishmaniases are widely regarded as vaccine-preventable diseases based on disease natural history, epidemiological data and studies in experimental models of leishmaniasis , so avoiding the stigmatization associated with CL scars (reviewed in ref. 34). More defined experimental studies were conducted sporadically through the 20th century, culminating in a WHO-sponsored evaluation of the potential for human challenge as a tool to evaluate vaccines for leishmaniasis, conducted in Iran in 200535. This study employed a L. major strain that had been produced at GMP and used for previous leishmanization studies. Results from this study demonstrated a take rate of 86% in previously non-exposed volunteers. Lesions were <3 cm diameter and ulcerated in 74% of cases. All lesions self-healed without treatment between 75 and 285 days after inoculation. In a limited re-challenge study using the same strain, 0/11 volunteers receiving leishmanization developed a lesion, compared to 5/5 in non-leishmanized controls35. Poor viability of the challenge agent and limited funding opportunities curtailed this programme before it could be developed further and to date, no defined vaccines have been tested using this approach.Artificial human infection (“leishmanization”) with 36 and in PKDL patients  and is currently in Phase IIb as a therapeutic in Sudanese PKDL patients. A live genetically attenuated L. donovani centrin−/− parasite has shown efficacy in pre-clinical models39 and a L. major centrin−/− 40 is soon to enter GMP production. An adjuvanted recombinant polyprotein vaccine (LEISH-F3/GLA-SE) has been progressed to Phase I41 and a newer derivative (LEISH-F3+/GLA-SE) evaluated in pre-clinical models21. RNA-based vaccines are also in development42. In addition, new knowledge regarding the integral nature of sand fly transmission to Leishmania infectivity has emerged in recent years45, underpinning the observation that vaccines inducing protection in mice when infected via needle inoculation fail to protect against sand fly-transmitted infection46; hence the need to incorporate vector transmission as part of a CHIM.With the advent of new candidate vaccines in or approaching the clinic, there is renewed imperative to develop a CHIM for leishmaniasis. An adenoviral-vectored vaccine (ChAd63-KH) was found to be safe and immunogenic in healthy volunteersL. major challenge agent.The pathway for development of a CHIM for sand fly-transmitted leishmaniasis requires three enabling activities: the identification of an appropriate challenge agent, optimization of sand fly transmission studies in humans, and patient and public involvement (PPI). Here, we describe completion of the first of these steps, namely the isolation, characterization and GMP production of a new Leishmania major is endemic in Israel and cases are often associated with travelers visiting areas of high transmission48. Two individuals from non-endemic areas of central Israel that had self-referred to Sheba Hospital in early 2019 after developing lesions subsequent to visiting the endemic region of Negev  on two occasions ~4 weeks apart. Her lesion fully resolved with minimal scarring by 3 months post treatment onset . Parasites were minimally cultured in GMP grade media to retain infectivity and multiple vials frozen at P1 as a seed bank and screened negative for mycoplasma. The seed stock was redistributed under dry ice using a commercial shipping agent.Parasites were isolated from slit skin preparations and diagnosis of L. major MRC-01 and L. major MRC-02 has been deposited at GenBank. A phylogeny tree was developed using all available whole-genome L. major sequences from around the world with L. major Friedlin strain from Israel used as reference . Compared to early culture parasites (pre-infection), the SNP fingerprint of the BALB/c passaged parasites (post-infection) was nearly identical  and L. major MRC-02  parasites  have been demonstrated in various Leishmania species: LRV1 in L. (Viannia) braziliensis and L. (V.) guyanensis, and LRV2 in L. aethiopica, L. infantum, L. major and L. tropica. RNA was isolated from early passage L. major MRC-01 and L. major MRC-02, and tested for LRV2 by RT-PCR49. L. aethiopica LRC-L494, previously shown to contain LRV2, was used as a positive control. Both L. major strains were negative for LRV2 , a standard drug used for the treatment of CL51, we used susceptible BALB/c mice infected subcutaneously in the rump with ~106 purified metacyclic promastigotes  favour use of this species.To more precisely quantify parasite load at each morphological stage, exact numbers of procyclic and metacyclic forms in infected sand fly females were counted using a Burker chamber. The differences between P. duboscqi given a single infectious blood meal by membrane feeding. As before, both L. major strains produced 100% infection rates in sand fly females on day 3 PBM, with >80% late-stage infections on day 6 and day 15 PBM. At day 3 PBM, 14% and 43%, respectively, of females infected with L. major MRC-01 and L. major MRC-02 had parasites located at the stomodeal valve. By day 15 PBM, thoracic midguts were filled with high numbers of parasites and the stomodeal valve was colonized in all the females infected with both strains, though heavier infections developed in sand flies infected with L. major MRC-02  under conditions identical to that for proposed GMP manufacture. Given the data above, we limited these experiments to P. duboscqi females infected by L. major MRC-01 or L. major MRC-02 were allowed to feed on anaesthetized BALB/c mice on day 15 post BM. Immediately post feeding, six ear samples per each strain were taken for determination of transmitted parasite number using qPCR. Positivity rates were 5/6 and 6/6 for L. major MRC-01 and L. major MRC-02, respectively, and numbers of parasites per ear varied from 0 to 7240 and from 92 to 5670, respectively, for the two strains  bitten by L. major MRC-01 infected flies  despite the absence of lesions  and comprises identity (PCR), resuscitation (indicating growth), sterility, endotoxin and pH. We estimate conservatively that in a sand fly CHIM, after retention for stability studies, this clinical lot will be sufficient for challenge of at least 1200 volunteers. Seed stocks are available for further GMP runs as required.A GMP clinical lot of 53. We have therefore sought to develop a new CHIM, based on best practices derived from other models. Three questions influenced our approach to developing a challenge agent, namely which (i) parasite species, (ii) challenge route and (iii) manufacturing standard?Controlled human challenge is increasingly viewed as being on the critical path for vaccine development, allowing an early demonstration of efficacy and in combination with appropriately designed Phase I trials, rapid selection/de-selection of candidate vaccinesL. major, the causative agent of Old World CL lends itself to development as a human challenge agent on a number of counts. First, unlike other species causing CL, e.g. L. tropica, L. mexicana or L. (Viannia) braziliensis and L. (V.) guyanensis, where systemic or metastatic spread is commonly documented, lesion development following L. major infection is usually localised to the site of sand fly transmission and is most commonly self-healing5. Numerous barriers to developing a CHIM model of CL using existing L. major strains were identified, including limited information on the provenance of parasites held in depositories or in use in research laboratories. Although CHIM studies per se are not under formal regulatory control in the UK, informal advice from the MHRA emphasised the importance of understanding the clinical history of the challenge agent donor, donor status with regard other human infectious agents (e.g. HIV) and the need to ensure absence of contact with bovine sera potentially contaminated with agents known to cause transmissible spongiform encephalopathies. In the case of Leishmania, passage history also represents an additional, but often poorly defined, variable that governs infectivity55. Hence, two fresh strains were obtained from donors with documented clinical histories and for which we could ensure complete traceability of parasite culture history.Addressing the first question, Leishmania parasites. While virulence factors/pathways have been identified in various species, including gp63, lipophosphoglycan, exosome production, proteases and many others56, how these vary across species or strains and associate with different clinical presentations is poorly defined57. Symbiotic leishmaniaviruses have been associated with enhanced host type I interferon responses and contribute to the metastatic potential in L. Viannia species58. Although Leishmaniavirus has been detected at lower frequency in Old World L. major strains60, there is no conclusive data to suggest an involvement in pathogenesis and treatment failure. In Iranian cases, L. major infection was not influenced by the presence or absence of LRV261. In any event, both L. major MRC-01 and L. major MRC-02 were demonstrated to be negative for LRV2.We conducted whole-genome sequencing to establish baseline characteristics and to assess genetic changes occurring after passage in animals. The two parasite strains we examined were genetically distinct, but no features were identified that directly pertain to their potential value as a CHIM agent, given that relatively little is known about the genetic nature of virulence in 62. Parasite development in two species of sand fly, both natural vectors of L. major confirmed full life cycle completion, including transmission to mice. The latter experiments also pertain directly the second question, route of challenge agent delivery. Vector transmission has been performed for other CHIMs63, and though this approach introduces potential confounding factors such as variability in infectious dose and “take rate” compared to needle injection, this needs to be weighed against the added value of such a model. For leishmaniasis, the body of evidence indicating a synergistic role of sand fly salivary components and parasite secretory products in promoting infectivity65 coupled with the value of sand fly challenge in identifying vaccine candidates66 together make a compelling case to proceed with a natural challenge model. The results of a study to optimise a human biting protocol using uninfected sand flies  and the results of our public involvement activities related to this study will be reported elsewhere.The classical BALB/c mouse model was used to evaluate in vivo infectivity and drug sensitivity to paromomycin, an often used drug for the treatment of CLFinally, we considered whether manufacture should be to GMP or GMP-like, as proposed by others. As it is not possible to generate sand flies that are “GMP”, it might have been argued that GMP-like would be sufficient for our purposes. However, given the limited additional costs of producing a clinical lot to GMP and that this leaves open the possibility for direct needle challenge or for any future change in the regulatory framework, we chose to adopt full GMP production of the clinical lot.L major MRC-02 as the challenge agent for GMP production based on an assessment of risk vs reproducibility for participants enrolled in future CHIM studies. The greater consistency in take rate by both needle and sand fly transmission and more rapid lesion development of L. major MRC-02 compared to L. major MRC-01 has an important bearing on the conduct and management of clinical studies as well as significantly decreasing the number of participants required and the duration of clinical monitoring. For example, in a simple two-arm (placebo vs. vaccine) trial with 90% power to detect a dichotomous outcome (lesion vs. no lesion) at a p value of 0.05 and with vaccine efficacy of 60%, a CHIM with 95% take rate would require 24 participants. In comparison, if the take rate was only 60%, the same study would require 74 participants. As additional parasitological and clinical data become available from our early studies using this CHIM model (see below), alternative end points to lesion development may also become valuable as indicators of vaccine efficacy in humans. For example, reducing parasite burden in the skin might impact transmission competence67 and/or reduced lesion size or rate of evolution may have some quality of life benefit9. The GMP clinical parasite bank we have generated is supported by a comprehensive data package (as described here) and should be sufficient to conduct CHIM studies in >1200 individuals by sand fly transmission. While manufacture was not of the scale associated with development of for example virally-vectored vaccines, it balances yield with the desire to limit in vitro parasite expansion and should serve as a key resource for several years to come.We selected L major was chosen as the challenge agent as this represents the species with most limited clinical severity. While the use of a L major CHIM would clearly inform the development of prophylactic vaccines against Old World CL, the degree to which this data could be extrapolated to protection against other species, for example L. donovani a causative agent of VL is untested currently. Epidemiological data has suggested68 and experimental evidence supports71 some degree of cross-protection between parasites causing CL and VL, including following vaccination. It is reasonable to suggest therefore that successful protection following vaccination in a L. major CHIM would provide highly encouraging albeit not definitive evidence to support the development of vaccines against VL or other forms of CL. Additional limitations of the current study are that it is not possible to accurately predict how infectivity in mice will translate to infectivity in humans and that it is not possible to quantify parasite load in infected sand flies prior to use in a transmission study. To mitigate against these factors, in addition to monitoring parasite load in replicate cohorts of flies alongside those used for human infections studies, we have proposed an adaptive clinical trial design as outlined elsewhere . Following ethical approval, we will initially evaluate the frequency of parasitologically-confirmed lesions (take rate), rate of lesion development and parasite load in six volunteers following exposure to five P. dubosqi infected with L. major MRC-02. As required, the adaptive design allows for variation in the sand fly species, the number of infected sand flies and the biting time. Robust monitoring and early lesion excision will further mitigate against the development of severe disease. The aim of our initial studies will be to optimise the CHIM model and inform the design of subsequent clinical trials employing controlled human infection as a measure of vaccine efficacy. These studies will also provide for detailed mechanistic insights into the early evolution of a primary CL lesion.This study has some limitations. All human studies were conducted in accord with the Declaration of Helsinki. Ethical approval was obtained from the Helsinki Committees of Hebrew University (0400-18-SOR) and The Chaim Sheba Medical Centre (5658-18-SMC) and the University of York Dept. of Biology Ethics Committee. Informed consent was obtained from patients with PCR-confirmed leishmaniasis for parasite isolation and subsequent use of these parasites in the development of a human challenge model. Animals were maintained and handled at Charles University and the University of York in accordance with institutional guidelines and national legislation  Act 1986). All the experiments were approved by (i) the Committee on the Ethics of Laboratory Experiments of the Charles University in Prague and were performed under permit from the Ministry of Education, Youth and Sports of the Czech Republic (MSMT-28321/2018-6), and (ii) The University of York Animal Welfare and Ethics Review Board and performed under Home Office license (PPL P49487014).L. major MHOM/IL/2019/MRC-01 and L. major MHOM/IL/2019/MRC-02 (herein referred to as L. major MRC-01 and L. major MRC-02), were obtained from patient lesions by culturing in 1 ml Schneider’s Drosophila medium  containing 20% foetal bovine serum  at 26 °C. Antibiotics were not included in the medium. The cultures were positive for promastigotes after 9 and 13 days, respectively. The parasites were further expanded and cryopreserved after one and two passages. For freezing, parasites  were suspended in Schneider’s Drosophila medium containing 30% foetal bovine serum and 6.5% DMSO and transferred to a Mr. Frosty box at −80 °C.Adult specific pathogen-free BALB/c mice between 8 and 12 weeks old were used in all experiments reported here and were obtained from either AnLab s.r.o (Prague) or Charles River UK (York). Mice were maintained in individually ventilated cages with food and water ad libitum and a 12 h light/12 h dark photoperiod in rooms maintained at 56% humidity, 20–21 °C. Two new strains, 72. Briefly, Illumina paired reads were aligned to the reference L.major Friedlin reference genome sequence obtained from TriTrypDB73 using the Burrows-Wheeler Aligner (version 0.7.17)74, file formats transformed using samtools (version 1.10), and variant calling was done with VarScan2 (version 2.4.3)75 to generate VCF files. Per sample candidate SNP were called by VarScan2 with a minimum coverage of 0.4× mean genome coverage, a minimum alternate allele frequency of 20% (read/read), a minimum average base quality of 15 across the reads and a 90% significance threshold. For phylogeny generation, additional sequences obtained from GenBank from whole-genome sequencing projects of L. major were also processed and aligned along with L. major MRC-01 and L. major MRC-02 strains. Polymorphisms and copy number variant were plotted using circos76 and inspected manually using the Integrative Genomics Viewer77. Homozygous SNP comparisons of L. major MRC-01 and L. major MRC-02 to the L. major Friedlin strain were analysed and reported.DNA from promastigote cultures was extracted using a DNeasy column according to manufacturer’s instruction (Qiagen). PCR-free library preparation (Lucigen) and NextSeq 500 sequencing (Illumina) was performed at Genome Quebec. Raw reads were processed as describedL. major strains were tested for the presence of LRV2 by RT-PCR using the primers LRV F-HR (5′-tgt aac cca cat aaa cag tgt gc-3′) and LRV R-HR (5′-att tca tcc agc ttg act ggg-3′)78. RNA was purified from L. major MRC-01, L. major MRC-02 or L. aethiopica (MHOM/ET/1985/LRC-L494) using the TRI reagent (Sigma-Aldrich) according to the manufacturer’s instructions. The latter strain was used as a positive control for LRV2. cDNA was synthesis using the Transcriptor Universal cDNA Master Kit (Sigma-Aldrich) with random hexamer primers. Each PCR reaction (25 μl) contained 5 μl cDNA, 10 μM each primer and 10 μl master mix ; and were carried out as follows: Initial denaturation 95 °C for 2 min, 35 cycles at 95 °C for 20 s, annealing at 55 °C for 40 s, extension at 72 °C for 40 s and final extension at 72 °C for 5 min. Amplicons were analysed on 1.5% agarose gels.6 metacyclic promastigotes, selected from stationary phase cultures using PNA agglutination50. Lesion development was monitored every 2–3 days until patency and daily thereafter. Measurements were performed in two directions using a dial caliper and the mean (8 mm) and maximum single (10 mm) diameter used to evaluate when mice had reached their clinical endpoint. Lesions were collected post mortem, amastigotes allowed to transform into promastigotes (P0) and then frozen as a stock culture (P1) for subsequent sequence analysis (as above). For drug treatments, mice reaching a pre-determined cut-off of 4 mm were randomized (n = 9–10 per group) to receive either saline or paromomycin . Treated mice were killed at day 10 post treatment for evaluation of parasite load by qPCR for kinetoplastid DNA (see below).BALB/c mice were infected s.c. in the shaved rump with 100 µl saline containing 10P. duboscqi and P. papatasi  were maintained in the insectary of the Department of Parasitology, Charles University in Prague, under standard conditions 79. The sand fly colonies have been screened by RT-PCR and found to be negative for Phleboviruses  and Flaviviruses (targeting a conserved region of the NS5 gene). Only female sand flies are used in experiments.The colonies of 6 promastigotes/ml. Sand fly females (5–9 days old) were infected by feeding through a ethanol-sterilized chick-skin membrane (BIOPHARM) on the promastigote-containing suspension. Engorged sand flies were separated and maintained under the same conditions as the colony. On day 3, 6 and 15 post bloodmeal (PBM) sample sand flies were dissected and digestive tracts examined by light microscopy. Five locations for Leishmania infection were distinguished: endoperitrophic space (ES), abdominal midgut (AMG), thoracic midgut (TMG), cardia (CA) and the stomodeal valve (SV). Parasite loads were estimated by two methods: (i) infections were qualitatively assessed in situ as light (<100 parasites per gut), moderate (100–1000 parasites per gut) and heavy (>1000 parasites per gut)80; (ii) infections were quantitatively assessed by transferring each gut into 100 μl of 0.01% formaldehyde solution, followed by homogenization and counting using a Burker chamber. Leishmania with flagellar length <2 times body length were scored as procyclic forms and those with flagellar length >2 times body length as metacyclic forms81.Promastigotes from log-phase cultures (day 3–4 in culture) were washed twice in saline and resuspended in heat-inactivated rabbit blood at a concentration of 1 × 10For transmission experiments, BALB/c mice were anaesthetized with ketamin and xylazine (62 mg and 25 mg/kg). Sand flies infected for 15 days (as above) were placed into small plastic tubes covered with the fine mesh  and the tubes were held on the ear pinnae of anaesthetized mice for 1 h. Engorged sand fly females were immediately dissected for microscopical determination of infection status (as described above). One group of mice was euthanized immediately post transmission and a second group of mice was followed for a period of 6 weeks p.i.82. One microlitre of DNA was used per individual reaction. PCR amplifications were performed in triplicates using the following conditions: 3 min at 98 °C followed by 40 repetitive cycles: 10 s at 98 °C and 25 s at 61 °C. PCR water was used as a negative control. A series of 10-fold dilutions of L. major promastigote DNA, ranging from 5 × 103 to 5 × 10−2 parasites per PCR reaction was used to prepare a standard curve. Quantitative results were expressed by interpolation with a standard curve. To monitor non-specific products or primer dimers, a melting analysis was performed from 70 to 95 °C at the end of each run, with a slope of 0.5 °C/c, and 5 s at each temperature.Sand fly-exposed ear pinnae were dissected and stored at −20 °C. Extraction of total DNA was performed using a DNA tissue isolation kit  according to the manufacturer’s instructions. Lesions from needle challenge were dissected and stored at −80 °C. Extraction of total DNA was performed using DNeasy tissue isolation kit (Qiagen) according to the manufacturer’s instruction. Parasite quantification by quantitative PCR (qPCR) was performed in a Bio-Rad iCycler & iQ Real-Time PCR Systems using the SYBR Green detection method . Primers targeting 116 bp long kinetoplast minicircle DNA sequence (forward primer (13A): 5′-GTGGGGGAGGGGCGTTCT-3′ and reverse primer (13B): 5′-ATTTTACACCAACCCCCAGTT-3′) were usedt test or ANOVA) depending on data distribution. Time to event analysis was conducted using the log-rank (Mantel-Cox) test.Data are plotted using violin plots and mean, median, 95% CI and ranges are shown as appropriate. Where shown, error bars represent standard deviation of the mean. All statistical analysis was performed with the statistical software package SPSS version 23 or with GraphPad Prism 8 for macOS (v8.4.2). Normality was evaluated using the D’Agostino–Pearson test and differences in parasite numbers in mice and sand fly tissues were tested by non-parametric  or parametric tests (student’s Further information on research design is available in the Supplementary Information FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryPeer Review File


Idiopathic central precocious puberty (ICPP) is supposed to be non-existent in a context of testicular destruction that is typically present in Klinefelter syndrome (KS). Herein, we describe a rare case of ICPP in a Klinefelter patient  with 2 maternal X chromosomes. Moreover, we highlight the differences in gonadotropin levels in comparison to males with ICPP and a normal karyotype.An 8 years old boy with a history of cryptorchidism was evaluated for precocious puberty (Tanner staging: P2/G3). Both testes measured 25x35mm. His hormonal profile confirmed a central origin of precocious puberty with high serum testosterone (4.3 ng/ml), luteinizing hormone [LH (3.5 UI/l)] and follicle stimulating hormone [FSH (7.7 UI/l)] levels. Luteinizing hormone-releasing hormone (LHRH) test amplified LH and FSH secretion to 24 and 14 UI/l respectively. Brain magnetic resonance imaging (MRI) was normal. No MKRN3 mutation was detected. He was treated for ICPP for two years. During puberty, he suffered from hypergonadotropic hypogonadism leading to the diagnosis of KS . Chromosomal analysis by fluorescent multiplex polymerase chain reaction (PCR) using X chromosome microsatellite markers identified 2 maternal X chromosomes.Analysing 8 cases of KS developing ICPP (our reported case and 7 other published cases) revealed that these KS patients with ICPP have higher LH and FSH levels during ICPP episode than in ICPP patients with a normal karyotype . Furthermore, their response to gonadotropin-releasing hormone (GnRH) stimulation is characterized by excessive LH and FSH secretion .“overrepresentation of ICPP in KS. Thus, karyotype analysis could be considered for boys suffering from ICPP, especially if testicular size is smaller or gonadotropins are significantly elevated.ICPP in boys is extremely rare. The pathophysiology of ICPP in KS is unknown. However, maternal X supplementary chromosome and early testicular destruction may play a significant role in the initiation of ICPP, in part explaining the relative The online version contains supplementary material available at 10.1186/s12610-020-00117-1. Puberty in boys is initiated when gonadotropin-releasing hormone (GnRH) secretion is amplified, inducing the secretion of gonadotropins. Pulsatile secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) is accompanied by enlarged testicular volume, testosterone secretion and initiation of spermatogenesis . The ageWe describe a case of ICPP in a boy who, a few years later, developed hypogonadism in his adulthood secondary to KS . In addition to our patient, we analyze 7 other cases of ICPP in KS patients previously reported in the literature. Among patients with ICPP, those who are also suffering from KS have higher levels of gonadotropins and an exaggerated response to GnRH stimulation compared to those with a normal karyotype. This comparison highlights the possibility of a primitive testicular factor, such as precocious germ cell and Leydig cell dysfunction, participating in the induction of ICPP in KS.A boy, born in 1994, was evaluated for suspicion of precocious puberty. At 8 years old, his height (150 cm) exceeded the mean for age by 3 standard deviations with a growth rate of 10 cm per year. He developed puberty signs (Tanner staging: P2/G3). Both testes measured 25x35mm, the penis was enlarged and increased in length. Bone age was advanced (10 years according to modified Sempé’s method). His past history was marked by cryptorchidism, operated at age of 1 year. No facial dysmorphia, behavioural abnormalities or mental retardation were noticed.His hormonal profile confirmed a central origin of precocious puberty with high serum testosterone (4.3 ng/ml) for age, and detectable LH (3.5 UI/l) and FSH (7.7 UI/l) .Luteinizing hormone-releasing hormone (LHRH) test amplified LH and FSH secretion to 24 and 14 UI/l respectively  to study short tandem repeats polymorphisms .http://projects.tcag.ca/variation). Microsatellite analysis in X chromosome showed two X chromosomes of maternal origin. Furthermore, no mutation was found in the coding regions of MKRN3 gene.Peripheral blood cytogenetic analysis of our patient revealed 47 chromosomes, with an extra X chromosome in all analyzed metaphases, confirming KS. Array-comparative genomic hybridization (CGH) excluded the presence of any other genomic imbalance, except for copy number variations (CNV) previously described in the normal population according to the Database of Genomic Variants  and those with a normal karyotype reported in the literature . Comparr et al.  comparedr et al.  or 47,XXIn our patient, despite the precocious puberty, target height was attained. Similarly, in other reported cases of ICPP with KS, the height was attained in all patients, with (3 patients including our case) , 10 or woverrepresentation” of ICPP in Klinefelter patients. The risk is estimated to be 1/370 KS patients by the Health Register of Belgium [Indeed, around 450 boys with ICPP under the age of 9 years have been described in the published cohorts to date, accounting for about 8% of all central precocious puberty cases before the age of 9 year and 10% before the age of 10 years  [MECP2 (responsible for Rett syndrome) [Secondly, in our patient, both X chromosomes were of maternal origin. None of the reported cases studied the origin of the supplementary X chromosome. It is worth noting that the supplementary X chromosome is equally inherited from paternal and maternal origin. When the origin of this supplementary X chromosome is maternal, it is hypothesized that some deleterious genes could escape the natural X chromosome inactivation , 44. Thengenita)  and MECPyndrome) . On the yndrome) . Pubertyyndrome) , and is yndrome) . Indeed,yndrome) . NeverthDespite a number of interesting studies investigating early onset of puberty in patients with KS, the genetic basis of idiopathic central precocious puberty in both sexes remains elusive. Further studies using molecular genetics could help understanding the mechanisms of puberty initiation, timing and progression. Specifically, identifying novel X-linked genes in boys with precocious puberty will be instrumental to shed light on the mechanisms underlying puberty initiation.ICPP is extremely rare in boys. ICPP patients with KS have higher gonadotropin levels than those with a normal karyotype. This suggests a peripheral or “testicular driver” that amplifies gonadotropin secretion through a positive feedback loop, which in turn triggers the initiation of central PP in such patients. Furthermore, maternal supplementary X chromosome in KS might play a role in the timing of puberty initiation. In boys with ICPP, long term endocrinological follow up until definitive puberty and karyotype analysis are important, specially if gonadotropin levels are significantly elevated or testicular volume is reduced.Additional file 1.Additional file 2: Supplementary Table 1. Incidence of central precocious puberty (ICPP) according to sex. ICPP: Idiopathic central precocious puberty. CPP: Central precocious puberty. ICPP in boys accounts for 10% of the aetiologies of CPP. Data represent number of patients or percentage (%).


Methods: We queried a multicenter longitudinal database that included 995 preterm infants (<32 weeks gestation) and included 73 cases of NEC. Dried blood spot samples were obtained on day of life 1, 7, 28, and 42. Metabolite data from each time point included 72 amino acid (AA) and acylcarnitine (AC) measures. Nutrition data were averaged at each of the same time points. Odds ratios and 95% confidence intervals were calculated using samples obtained prior to NEC diagnosis and adjusted for potential confounding variables. Nutritional and metabolic data were plotted longitudinally to determine relationship to NEC onset. Results: Day 1 analyte levels of alanine, phenylalanine, free carnitine, C16, arginine, C14:1/C16, and citrulline/phenylalanine were associated with the subsequent development of NEC. Over time, differences in individual analyte levels associated with NEC onset shifted from predominantly AAs at birth to predominantly ACs by day 42. Subjects who developed NEC received significantly lower weight-adjusted total calories (p < 0.001) overall, a trend that emerged by day of life 7 (p = 0.020), and persisted until day of life 28 (p < 0.001) and 42 (p < 0.001). Conclusion: Premature infants demonstrate metabolic differences at birth. Metabolite abnormalities progress in parallel to significant differences in nutritional delivery signifying metabolic dysfunction in premature newborns prior to NEC onset. These observations provide new insights to potential contributing pathophysiology of NEC and opportunity for clinical care-based prevention.Necrotizing Enterocolitis (NEC) is associated with prematurity, enteral feedings, and enteral dysbiosis. Accordingly, we hypothesized that along with nutritional variability, metabolic dysfunction would be associated with NEC onset.  Necrotizing enterocolitis (NEC) is an acquired disease of premature neonates with a multifactorial pathophysiology that is likely influenced by both intrinsic biological factors  and extrinsic variables including exposures inherent to medical care .Recent evidence has demonstrated that fatty acid and protein metabolism differs significantly between premature and term infants ,2,3. MetNewborn screening (NBS), which includes measurements of amino acids (AAs) and acylcarnitines (ACs)—intermediates of protein and fatty acid metabolism respectively—was initially implemented for the early identification of inborn errors of metabolism. Our group has previously demonstrated an association between levels of specific ACs measured on NBS at birth and the subsequent development of NEC in preterm infants.  We thereWe performed a case-control study derived from querying a longitudinal multicenter (23 sites in 17 states) database collected by the Pediatrix-Obstetrix Center for Research, Education and Quality across their network of neonatal intensive care units (NICUs) from April, 2009 to September, 2012 . These dn = 8), 8–28 (n = 48), 29–42 (n = 9), and >42 (n = 8). Control infants were selected from the database based on the following criteria: (1) alive at the end of the study and (2) no history of gastrointestinal disease (n = 814).Data on intestinal disease were captured in a structured field . NEC was determined to be present if an infant had at least one of the following clinical signs: bilious gastric aspirate or emesis, abdominal distention, or blood in stool without evidence of a rectal fissure; and had one or more of the following radiographic findings: pneumatosis intestinalis, hepatobiliary gas, or pneumoperitoneum. Infants with a diagnosis of NEC-medical who subsequently were treated with surgery (had both diagnoses) were assigned to the NEC-surgical group. Of 76 cases of NEC, three exited the study early due to being transferred and were excluded from analysis. A total of 73 infants with NEC were included as cases. Cases of NEC were further sub-grouped based on day of diagnosis: post-delivery day 1–7 (® (Hospira), Premasol® (Baxter) and TrophAmine® (Braun). The utilization of these varied insignificantly among the case and control cohorts. Qualitatively, those amino acids that were identified as significantly different between the cases and controls  for each of the essential and non-essential AAs reported as statistically different post adjustment in Detailed demographic and clinical data were collected for each case and control infant. Standardized fluid management and nutritional guidelines were utilized at each site. In brief, the total fluid infusion rate was set between 90 and 120 mL/kg/day. Glucose infusions were set to start immediately in critically ill premature neonates and infused at a rate of up to 8 mg/kg/min. Amino acid supplementation was started between 0.5 and 3.0 g/kg/day as early as possible and advanced by 0.5–1 g/kg/day to a maximum of 3.5 g/kg/day. Amino acid supplementation decreased to 1–2 g/kg/day once feedings reached 80–100 mL/kg/day. There were three amino acid formulations in use across the collection sites including, Aminosyn® 10%(Baxter) was utilized throughout and quantitative differences in utilization did not emerge until D28 and reflects an increase use of IL in the cases to balance the deficiency in enteral calories achieved. Low rate, small volume (<10 mL/kg/day) enteral feedings were begun within the first week of life. Feedings could be advanced at a rate between 10 and 30 mL/kg/day. Full feedings were defined as 130 mL/kg/day with consistent weight gain of 20 to 30 g/kg/day. Human breast milk was the preferred form of nutrition. Supplements such as human milk fortifier (HMF) and multi chain triglycerides (MCT) could be added to feedings as per site preference by the time that feedings had reached 100 mL/kg/day. Nutritional data were averaged over the following time intervals: day of birth (day 1), 2–7, 8–28, and 29–42. All infants were followed until discharge from hospital.Lipids emulsions were initiated with parental nutrition at a rate of 1 g/kg/day and advanced by 0.5 g/kg/day to a maximum of 3.5 g/kg/day. Qualitatively, soy based IntralipidPatient samples were collected within 24 h after birth (day 1), on approximately day 7 (7–8), on day 28 (27–29), and on day 42 (41–43) post-delivery or at discharge, whichever came first . Dried bt-test for quantitative variables and Fisher’s exact test for categorical variables.Patient characteristics were compared between case infants with NEC and control infants using Odds ratios (OR) and 95% confidence intervals (CIs) were calculated for all NEC cases using day 1 analyte levels, as well as at each time point of sampling for each sub-group. For the day 7, day 21, and day 42 time point analyses, only samples obtained prior to NEC diagnosis were used for the subjects in the NEC group. Thus, the analysis at each time-point represents only those differences between cases and controls that emerged prior to a diagnosis of NEC. This same approach was used for subsequent pre-NEC longitudinal analyses. Patient characteristic variables that were found to be significantly different in univariate analysis  were con2 (Equation (1)) for each metabolite across the four time points in terms of the fold change of the ith metabolite at the jth time point ixj,, average fold change at the jth time point μj, and the standard deviation of each time point σj (Equation (2)):To identify those metabolic changes that were associated with the development of NEC over time, we first derived each metabolite’s fold-changes between cases and controls at each time point respectively . Second, we obtained the test statistic χ2 (df = 4) < 5.62 × 10−4 after Bonferroni correction.The significant metabolites were defined as those having p-values of χt-tests in a post-hoc analysis. The individual time point analysis was also conducted using only information obtained prior to NEC diagnosis, as detailed above. The proportion of enteral feedings given as human breast milk was compared at each time point using a one-tailed t-test. Weight gain velocity was calculated for each of the time points using Equation (3):t represents the day of delivery at a beginning of a study time interval  and t + 1 represents the day of delivery at the end of a study time interval  [Loess curves were plotted over time for the nutrition data. Each nutrition variable was compared between NEC cases and their controls for each time point  using one-tailed ivery 7) ,17.R. Results were reported as significant if the p-value was <0.05.All statistical analyses were performed using Patient characteristics are summarized in After adjusting for potential confounders, those metabolites that were found to have a statistically significant association with developing NEC are summarized in p < 0.001) and citrulline/phenylalanine ratio (Cit/Phe) (p < 0.001); and two significantly up-regulated metabolites, phenylalanine/tyrosine ratio (Phe/Tyr) (p < 0.001) and octanoylcarnitine/decanoylcarnitine ratio (C8/C10) (p < 0.001), in the NEC cohort compared to the controls . Newborns who developed NEC also received significantly lower weight-adjusted total calories over the first six weeks of life , Panel ASpecific time point analyses demonstrated that infants who developed NEC began receiving significantly lower weight-adjusted total calories by post-delivery day 7 and this difference persisted at day 28 —at whichp ≤ 0.001) and took longer to achieve full enteral feeds . The proportion of enteral feedings (mL/kg/day) given as human breast milk (mL/kg/day) was not significantly different at any time point: day 1 , day 7 , day 28 , and day 42 . Despite receiving significantly less overall weight-adjusted calories than control newborns, case newborns had similar weight gain velocities (in g/kg/day) for each time interval: day 1–7: −5.99 vs. −5.10, p = 0.34; Day 7–28: 21.1 vs. 20.0, p = 0.89; Day 28–42: 18.1 vs. 19.8 p = 0.055.Newborns who later developed NEC were started on enteral feeds later  and less enteral feeds over the course of observation. It is essential to emphasize that the described differences in metabolism and nutrition were present prior to any clinical suspicion or diagnosis of disease according to stringent criteria: the requirement for surgical confirmation or radiographic evidence of definitive NEC . Whether the progressive metabolic differences led to enteral feeding intolerance requiring dependence on TPN or differences in nutritional delivery resulted in changes in nutrient metabolic processing remains unknown yet represents an important focus open to additional study.The pattern of specific metabolite differences is also revealing. At the early time points in the analyses , the differences involved predominantly AAs, whereas at the later time points , the differences were predominantly ACs. It is common clinical practice in the care of preterm infants to start IV AAs on day 1–2 of life and delay administration of IV lipids for several days—practices that may in part explain the predominance of NEC-associated AAs at the early time points. It is important to note, however, that the first newborn sample (day 1) was obtained within 24 h of life for both cases and controls. Both groups of newborns were receiving approximately the same volume of IV nutrients (AAs included) and total weight adjusted calories at the time of the first and second metabolic screen. This suggests that both groups would have been subject to nearly equivalent nutritional exposures and yet significant differences were observed. Thus, these observations support the hypothesis of inherent increased metabolic susceptibility in premature infants at birth as previously reported and built upon with the findings reported here .Several AAs that were associated with NEC in this study merit additional consideration given the interesting potential pathophysiological implications. Citrulline and arginine, both non-essential AAs, have received scrutiny in the NEC literature due to the relationship to nitric oxide (NO) synthesis—a reaction in which arginine is converted to NO and L-citrulline by nitric oxide synthase (NOS). In contrast to our findings of higher levels of arginine on days 1 and 7, several small cohort studies demonstrated reduced serum levels of arginine in infants who developed NEC ,19. It hPhenylalanine is an essential AA that is used predominately as a precursor for tyrosine synthesis, a reaction medicated by phenylalanine hydroxylase using 5,6,7,8-tetrahydrobiopterin (BH4) as a cofactor. BH4 is depleted under conditions of oxidative stress preventing the catalyzation of phenylalanine to tyrosine . The obsReduced postnatal weight gain has been associated with increased risk of NEC, bronchopulmonary dysplasia, late onset sepsis, and unfavorable neurodevelopmental outcomes ,24. LoweA limitation of this study is the lack of more detailed nutrition data. Nutrition data were averaged over each of the time intervals preventing the evaluation of daily caloric intake on the risk on developing NEC and likely resulting in an attenuation of estimated risks. Future studies will be needed to evaluate in more depth the role of daily caloric and macronutrient delivery on disease outcomes and serum metabolites. Further, given the findings that suggest a link between BH4 levels and NEC, additional nutritional variables and metabolites, including specific vitamins and minerals, should be the subject of future studies. Finally, additional studies to determine the cause or consequence for significant differences in enteral feeding and associated metabolic changes that occur with time prior to NEC diagnosis are needed.The most premature newborns, and those that are destined to develop NEC during early postnatal life, appear to be metabolically different or impaired beginning at birth. This is reflected in the significantly different metabolic patterns measured in the first metabolic screen prior to the subsequent influence of exogenous nutrition supplementation. Newborns destined to develop NEC appear to have clinical signs of early enteral feeding intolerance as reflected in the significant difference in enteral feedings , and consequent need for greater proportion of total calories given parenterally. Given the pattern of accumulating AA and AC differences longitudinally in the NEC cases suggests the need for clinicians to be exquisitely cognizant of feeding intolerance in at risk newborns and the possible metabolic derangements imparted through the increased reliance on TPN to maintain total calories. Currently, clinicians do not have the tools to fully understand the potential detrimental impact on overall clinical outcomes reflected in these metabolic abnormalities described herein. Since there was no difference in growth velocity between the NEC cases and controls, additional study is needed to determine qualifying measures of effective nutritional support beyond anthropometrics that are sensitive to these more subtle biochemical changes relative to the balance of enteral feeding and IV macronutrients and should include measures of potential biologic benefit and harm.


To compare the clinical characteristics of acute lower respiratory tract infections (ALRTIs) caused by respiratory syncytial virus (RSV) and human rhinovirus (HRV) and to explore the relationship between the development of recurrent wheezing/asthma and RSV/ HRV infections in infancy.Retrospective study was conducted to compare the clinical characteristics of acute lower respiratory tract infections (ALRTIs). Hospitalized patients with ALRTIs from March 2007 to December 2016 were screened. Single RSV cases (s-RSV), single HRV cases (s-HRV), and cases who had co-infection with the two viruses were enrolled. Follow-up was performed to determine whether either specific respiratory virus infection was related to subsequent development of recurrent wheezing/asthma.P = 0.021), they experienced the most serious condition (P < 0.001) and respiratory failure (P < 0.001), they also required highest demand of oxygen therapy (P < 0.001). And in s-RSV group, the incidence of development of recurrent wheezing was significantly higher in subgroup with the family history of wheezing than that without (P < 0.001).The s-RSV children were the youngest (The s-RSV cases suffered from the worst severity of illness, respiratory failure and required highest demand of oxygen therapy. Recurrent wheezing was more common in s-RSV group with family history of wheezing than those without. Pneumovirus genus in the Paramyxoviridae family. Nearly all children experience at least one RSV infection by the age of 2 years, and in the globle, more than 60% of acute lower respiratory tract infections (ALRTIs) in young children and 80% in infants less than 1 year old are caused by RSV [Enterovirus genus in the Picornaviridae family. HRV is also a commonly detected virus in nasal/throat swabs from young children who have been hospitalized for wheezing and from infants hospitalized for pneumonia [Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are the most common viral pathogens causing respiratory tract infection in infants and young children. RSV is a member of the neumonia . Althougneumonia , 5, therThe relationship between RSV or HRV ALRTIs in infants with the subsequent development of wheezing/asthma in childhood is still controversial. Some studies found that RSV infection in childhood increased the risk of persistent wheezing and asthma , 8. A reAs mentioned above, the co-infection rate of RSV and HRV was very high in ALRTIs in infants . WhetherFrom March 2007 to December 2016, children who were diagnosed with ALRTIs and hospitalized at Beijing Children’s Hospital were screened for this study. ALRTIs were defined by the presence of signs and symptoms of respiratory tract infection  . The incNasopharyngeal aspirates or throat swab specimens were collected from each patient within 24 h of hospitalization. All samples were stored at − 80 °C until testing.2 <  with or without PaCO2 > 45 mmHg. Heart failure would be diagnosed if meeting 4 of the following 6 criteria: (1) respiratory rate accelerated suddenly (> 60 beats/min); (2) heart rate elevated suddenly (> 180 beats/min); (3) dysphoria and cyanosis; (4) gallop rhythm, jugular venous distention and muffled heart sounds; (5) liver enlargement; (6) oliguria or anuria, and edema. Increase of creatine kinase isoenzyme (CK-MB) > 25 U/L or/and changing of ST-T in electrocardiogram (ECG) indicate myocardial damage. Aspartate aminotransferase (AST) > 40 U/L or/and alanine aminotransferase (ALT) > 40 U/L reflect liver dysfunction.Age, gender, symptoms, signs, exam results, diagnoses, complications, the need for oxygen and family history of wheezing were examined Table , and allTotal nucleic acids (DNA and RNA) were extracted from 200 μl nasopharyngeal aspirates or throat swab specimens using the Nucli Senseasy MAG™ automated extraction system  according to the manufacturer’s instructions and eluted with 60 μl elution buffer.The presence of common respiratory viral agents, including RSV, parainfluenza virus (PIV) type 1–4, 2009 H1N1 influenza virus (2009H1N1), H3 subtype influenza virus(H3), seasonal H1 subtype influenza virus(H1), influenza B virus (Flu B), human rhinovirus/enterovirus (HRV/HEV), human coronavirus , human metapneumovirus (HMPV), human bocavirus (HBoV), and human adenovirus (HAdV), were determined using multiplex RT-PCR, single RT-PCR, or PCR assays as described previously , 19. BlaTelephone follow-up was conducted to assess the subsequent development of wheezing episode and asthma in childhood at June, 2017. The questionnaire of follow-up is shown in the supplementary materials .P value < 0.05 was considered statistically significant.Continuous variables are expressed as means ± standard deviations (SD) or medians. For categorical variables, the relative numbers of patients in each category were calculated. Differences between groups were assessed using Pearson’s Chi square test or Fisher’s exact test for categorical variables and one-way analysis of variance (ANOVA) and independent-samples test for continuous variables. Games-Howell test was used for the data that do not meet the hypothesis of variance homogeneity. All analyses were performed using SPSS software, version 20.0 . All calculations were made in a two-tailed manner, and From March 2007 to December 2016, a total of 4348 children were screened for this study, and 319 children were finally enrolled, including 199 s-RSV, 47 s-HRV and 73 co-infection cases through the selection process , 22.2% (s-HRV) and 41% (co-infection), while the rate of asthma was 7.5% (s-RSV), 11.1% (s-HRV) and 12.8% (co-infection). But whether the family history was positive or not, there was no significant difference in recurrent wheezing/asthma among the three groups (Table P < 0.001, Table P = 1.000, Table P > 0.05, Table Each group was further classified into two subgroups by family history of wheezing. Only in s-RSV group, the incidence of recurrent wheezing was significantly higher in the subgroup with family history of wheezing than the subgroup without family history of wheezing (In infants and young children, ALRTIs are most frequently caused by respiratory viruses, such as RSV and HRV, both of which play a significant role in pediatric respiratory tract infections , 21. Co-It’s controversial about the role of co-infection of viruses in the severity of disease. Martin et al. found that comparing with children infected with a single virus, children with mixed viruses infection were less frequently admitted to the inpatient ward or to the intensive care unit and less likely to require supplemental oxygen or hospital stays longer than 3 days . NascimeHowever, other studies had different results. Calvo et al. reported that patients with mixed viruses infection had more frequent fever, longer hospital stays, and more antibiotic treatment than patients infected with RSV alone . Aberle A large number of studies have identified a link between RSV/HRV infection and an increased likelihood to develop either recurrent wheezing or asthma. Previous studies showed that with RSV infection, the risk of developing recurrent wheezing was from 28.1 to 30% , 33, andWe found that there were no significant differences in the development of asthma among subgroups of the three groups with or without family history of wheezing. And only s-RSV group with positive family history had a tend to have recurrent wheezing than those without. This result is consistent with that of the following studies. A Japanese study found no connection between RSV-based ALRTIs in infants and childhood asthma . Kusel, However, some studies supported that RSV or HRV infection in early childhood was associated with chronic airway diseases, especially in the subsequent development of wheezing and asthma. From some population-based birth cohort studies, RSV bronchiolitis was found to be associated with subsequent wheezing and asthma, especially in children with family histories of atopy and asthma , 39. AnoThe severity of disease in s-RSV cases was higher than that of s-HRV or Co-infection groups, with a higher demand for nasal catheter oxygen inhalation and NCPAP and higher risk for respiratory failure. No matter with or without family history of wheezing, there are no significant differences in the incidence of subsequent development of recurrent wheezing and asthma among s-RSV, s-HRV and co-infection groups. But patients with family history of wheezing are subject to have recurrent wheezing after ALRTIs caused by RSV in infancy than those without the family history.Additional file 1: Supplement 1. Clinical Scoring System. Supplement 2. Follow-up questionnaire for recurrent wheezing and asthma.