Clostridium difficile isolates among patients with UC at an inflammatory bowel disease clinic in Iran.Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that is widely identified worldwide. This study aimed to investigate the phenotypic characterization and molecular typing of C.difficile infection (CDI). C. difficile isolates were characterized based on their toxin profile and antimicrobial resistance pattern. Multi-locus sequence typing analysis (MLST) and PCR ribotyping were performed to define the genetic relationships between different lineages of toxigenic strains.In this cross-sectional study, conducted from April 2015 to December 2015, 85 UC patients were assessed for C. difficile isolates was 31.8% (27/85) in patients, of those 15 patients (17.6%) had CDI. Three different sequence types (STs) identified based on MLST among the toxigenic isolates, that is ST54 (33.3%), ST2 (53.3%), and ST37 (13.6%).The prevalence of C. difficile strains were divided into four different PCR-ribotypes . The most common ribotype was 014 accounting for 48.3% (7/15) of all strains. The strains isolated during the first episode and recurrence of CDI usually belonged to PCR ribotype 014 (ST2). A high rate of CDI recurrence  experienced in UC patients. Colonization of the gastrointestinal tract with non-toxigenic C. difficile strains was frequent among patients with mild disease.C. difficile isolates were susceptible to metronidazole, and vancomycin, 86 and 67% of isolates were resistant to clindamycin and erythromycin respectively. There was no correlation between the toxin type and antibiotic resistance (p > 0.05).All Overall CDI is rather prevalent in UC patients. All patients with CDI experienced moderate to severe disease and exposed to different antimicrobial and anti-inflammatory agents. Close monitoring and appropriate management including early detection and fast treatment of CDI will improve UC outcomes.The online version of this article (10.1186/s12879-019-3965-8) contains supplementary material, which is available to authorized users. Clostridium difficile, a gram-positive anaerobic bacterium, is the leading cause of pseudomembranous colitis, nosocomial and antimicrobial-associated diarrhea [C. difficile involves colonization of the intestinal tract and toxin production. Toxin A encoded by tcdA, and toxin B encoded by tcdB, are the most important recognized virulence factors, while another toxin, CDT (encoded by cdtA/cdtB) is present in a subset of toxigenic strains [diarrhea , 2. Incr strains , 4.C. difficile colonization. Hypervirulent clones such as ribotype 027 and to a lesser degree ribotype 078 have been involved in severe nosocomial outbreaks of CDI [C. difficile has also been developed to study clonal relations of the bacterial populations [C. difficile have focused on human isolates, animal and food strains [Various molecular typing methods have been applied to characterize outbreaks and describe endemic CDI and s of CDI , 4. Multulations , 6. ReceClostridium difficile infection (CDI) when compared with healthy population or individuals with Crohn’s disease [Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that is widely identified worldwide. The clinical outcome of UC is highly variable from mild to an aggressive disease that may require a colectomy . Determi disease , 10. CDI disease , 11. The disease , 13. Pot disease . In addi disease . There i disease .C.difficile isolates in UC patients.Data on the prevalence of CDI in UC patients have come from developed countries. Since limited information is available on the occurrence of CDI in UC patients in Iran or other Middle Eastern countries, we carried out this study to investigate the prevalence of CDI in patients with UC and, to characterize C. difficile detection were recorded. Diagnosis of UC was based on clinical signs and symptoms combined with disease activity, histologic, endoscopic, and radiological results according to the Porto criteria and Truelove-Witts activity index [This was a cross-sectional study conducted on ulcerative colitis patients referred to the Isfahan University of Medical Sciences inflammatory bowel disease clinics in Isfahan, Iran between April 2015 and December 2015. Patients between 18 and 65 years old with a documented diagnosis of ulcerative colitis were included. Patients with chronic obstructive pulmonary disease, severe liver dysfunction, end-stage renal disease, malignancy, and immunodeficiency syndromes were excluded. The demographic information and potential risk factors of patients such as age, gender, previous surgery, all drugs used by patients and antibiotic treatment within 8 weeks before the time of ty index . The modty index .Clostridium difficile infection most commonly defines as the presence of C. difficile toxin in the context of characteristic clinical manifestations including diarrhea and abdominal pain in contrast with C. difficile colonization in healthy individuals. Because of UC and CDI symptoms overlap we described CDI as three or more daily bowel movements for a period of at least 48 h in the setting of previously quiescent UC disease [ disease .CDI recurrence was described based on looser bowel movement numbers or developed new signs of severe colitis that last for more than two days .tcdA and or tcdB positive isolates) treated with metronidazole for 2 weeks. Stools specimens were screened for the presence C. difficile. Stools specimens were analyzed for the presence of other enteropathogenic organisms .A total of 170 fecal samples were collected from 85 UC (2 samples per patient) ranging in age from 20 to 65 years. The first set of samples were collected on May 2015 and the second set was taken about 2–3 months later. Stool samples collected in sterile collectors and immediately transferred into the laboratory of Infectious Diseases and Tropical Medicine Research Centre, Isfahan, Iran and preserved at − 70 °C during the analysis period. Patients with UC were treated with anti-inflammatory and steroids drugs in accordance with clinical protocols. Patients with CDI  broth culture. The cultures were incubated in an anaerobic jar in an atmosphere composed of 86% N2, 7% H2, and 7% CO2 at 37 °C for 48 h by using an Anoxomat system . One mL of enriched broth was mixed thoroughly with an equal volume of 95% alcohol and held at room temperature for 30 min. The tubes were centrifugated at 2500 g for 5 min and the alcohol supernatant was decanted. The pellets were inoculated by a swab onto the C. difficile moxalactam norfloxacin agar (CDMN) and incubated anaerobically for 48 h at 37C°. Negative cultures remained in the incubator for up to 7 days. The colonies characterized with 2–3 mm in diameter, p-cresol odor, ultraviolet fluorescence (365 nm), typical Gram stain morphology, positive malachite green for spore and positive biochemical reactions such as L-proline aminopeptidase test  were identified as C. difficile and stored at 4 °C [Selective, enrichment culture was performed . Briefly at 4 °C .C. difficile strains grown in BHI broth were centrifuged and cells were treated with lysozyme (50 mg/ml) and resuspended in TE . Guanidium thiocyanate and sarkosyl were added to the mixture for protein denaturation [tcdA and tcdB), binary toxins  and triose phosphate isomerase (tpi) [tcdA, tcd B), 5 pM of primers (tpi), 1 U Taq polymerase  and 100 ng of DNA. Amplification was carried out in a touchdown protocol [C. difficile ribotype 027 was used as positive control for molecular and microbiological analysis. C. perfringens 450 MTCC  served as the negative control [DNA extraction was performed using the modified Pitcher et al., procedure (1989). Briefly, Cultures of turation , 26. Allse (tpi) , 27. Mulprotocol , 24, 27. control .http://www.eucast.org). Strains were deemed susceptible or resistant to the test antibiotic according to documented pharmacological breakpoint values [The following antimicrobial susceptibility disks were used for antimicrobial susceptibility test metronidazole (5 μg), vancomycin 30 μg), clindamycin (2 μg), moxifloxacin (5 μg), fusidic acid (10 μg), erythromycin (15 μg), rifampicin (5 μg), . All tests were performed on Brucella Blood Agar containing vitamin K1 (1μg/mL), haemin (5 μg/mL) and 5% defibrinated sheep red blood cells . The zon μg, clinadk, atpA, dxr, glyA, recA, sodA, and tpi) was performed as described by Griffiths et al., 2010 [Multilocus sequence typing (MLST) with seven housekeeping genes  were entered into the multivariate model. Selection of variables in the multivariate model was based on a stepwise procedure. We estimated Odds Ratios (ORs) and 95% confidence intervals for each of clinical factors using logistic regression models. All probabilities were two-tailed and a p-value of < 0.05 defined statistically significant. Statistical analysis was performed using the statistical software SPSS, version 16.Data were presented as count and percentage. A first univariate logistic regression model was fitted on each independent variable, and a multivariate regression model with adjustment for the effects of other covariates was used. Variables that were significant in univariate models (C. difficile isolates (31.8%) were recovered from their stool specimens, including 15 (17.6%) with CDI (toxigenic isolates carried one or both tcdA and tcdB genes) and 70 (82.4%) with non-CDI. The toxigenic isolates detected in both stools samples of patients with CDI. None of the specimens were positive for toxin A alone or binary toxin. Salmonella spp, Shigella spp, E. coli, and Campylobacter jejuni were not detected. The use of antibiotic was identified in 74 (87%) patients in the 8 weeks prior to CDI diagnosis.  patterns. The most common ribotype was 014 accounting for 48.3% (7/15) of all toxigenic C.difficile isolates followed by the ribotypes 012  and 017  respectively. Three different sequence types (STs) identified based on MLST among the toxigenic isolates, ST54 , ST2 , and ST37 . Strains isolated during the first episode and recurrence of CDI belonged to PCR ribotype 014 (ST2) . Two out of 12 patients (17%) of studied patients were recognized re-infection by an identical strain of C. difficile (ribotype 014/ST2). Colonization of the gastrointestinal tract with non-toxigenic C. difficile strains  was frequent  among patients with mild disease.Toxigenic C. difficile strains were susceptible to metronidazole (range 23–45 mm) and vancomycin (range 19–28 mm). Only one isolate from a patient aged 60 with severe disease was resistant to fusidic acid, while the remaining isolates  were susceptible to it (range 19–28 mm). Twenty four out of 27 patients (89%) were susceptible to moxifloxacin (range 6–28 mm) and rifampin (range 23–45 mm) while, 23 out of 27 isolates (86%) were resistant to clindamycin, 18 out of 27 isolates (67%) to erythromycin (median range 6–18 mm). There was no correlation between the toxin type and antibiotic resistance (p > 0.05).All C. difficile were more than 6 times more likely to develop CDI than those who didn’t .The stepwise multivariate logistic regression model revealed that patients who used steroids 8 weeks prior to testing for The study findings also revealed that patients with a history of previous surgery, anti-inflammatory treatment, and complicated UC were more likely to develop CDI Table . We did C. difficile strains are associated with worsening disease in UC patients. Recent decade studies have been shown a steady increase in CDI prevalence especially in UC patients [Toxin-producing patients , 23, 31.patients , 32. Recpatients . Other rpatients . SeveralC. difficile may be described either by the endogenous persistence of a C. difficile strain (relapse) or by contamination of a new strain from the environment (re-infection) [C. difficile (ribotype 014/ST2). In other similar studies that performed in hospitalized patients, the rate of re-infection with identical strains was between 38 and 56% [We found a significant relationship between the presence of CDI and steroid treatment, previous surgery, the severity of UC and anti-inflammatory treatment Table . Previoufection) . About 1 and 56% , 37.C. difficile strains in our study showed susceptibility to vancomycin and metronidazole. Recurrence CDI has been observed in patients that had taken metronidazole. Other similar studies showed that patients with an IBD flare and concurrent CDI treated with vancomycin had successful treatment and vancomycin is the first-choice therapy for moderate to severe CDI [cdtA/cdtB genes in any isolate and MLST analysis. We have previously reported ribotype 078 as a common strain in both humans and meat in Iran so the absence of this strain in the current study was surprising. [−B+ strains . Recent studies have reported an increasing number of infections due to A−B+ strains especially ST37 in Asia although such strains do not produce a binary toxin [All identified vere CDI , 23. Mosvere CDI , 39. Theprising. , 40. Mosry toxin .Fig. 1AnC. difficile strains identified in a significantly high rate of 90% of UC patients and about 55% of healthy subjects [C. difficile intestinal carriage of the much lower rate in 12 patients (14.2%) of studied patients. Its relatedness to disease pathogenesis and severity in a contaminated environment justifies further investigations.In the previous report from Southern India, nontoxigenic subjects . We founC. difficile strains and lack of a healthy control group to be compared with the UC patients. This is the first study provides information about different aspects of molecular epidemiology, clinical characteristics, and antibiotic resistance profiles of circulating C. difficile strains among Iranian patients with ulcerative colitis. Further research and clinical studies with a larger population should be performed to evaluate the epidemiology of C. difficile in this high-risk group.There are several limitations to our study including small sample size, which have led to underestimating the true prevalence and diversity of circulating Overall, CDI is rather prevalent in UC. All patients with CDI experienced moderate to severe disease and exposed to different antimicrobial and anti-inflammatory agents. Close monitoring and appropriate management including early detection and fast treatment of CDI will improve UC outcomes.Additional file 1:C.difficile, PCR- ribotyping fingerprints with the primers 16S–23S. Similarity coefficients are included in the top bar; Dendrogram is color-coded according to sequence types (STs) and toxin types. The similarity was calculated using the Dice coefficient and UPGMA clustering. (DOCX 951 kb)Normalized dendrogram of the detected isolates of


The brain-derived neurotrophic factor (BDNF) is a neuronal growth factor essential for normal cardiac contraction and relaxation. Alterations in BDNF signaling are related to the development of cardiovascular disease. Whether BDNF is related to subclinical cardiac remodeling is unclear. We related BDNF with echocardiographic parameters and NTproBNP in a large population-based cohort . Transthoracic echocardiography was performed on all subjects and BDNF was measured by ELISA. Study participants with severe kidney dysfunction, previous myocardial infarction, and LV ejection fraction <40% were excluded. Linear regression models were adjusted for age, sex, lean mass, fat mass, current smoking, systolic blood pressure and depression. Low BDNF was associated with high NTproBNP. A 10,000 pg/ml lower BDNF was related with a 2.5 g higher  LV mass, 0.01 cm posterior wall thickness  and 0.02 E/A ratio . Here we show that low BDNF levels are related with adverse cardiac remodeling and higher levels of NTproBNP. Further research is warranted to assess if BDNF may be used to monitor neuronal-cardiac damage during CVD progression. BDNF also plays a pivotal role in non-neuronal cells. For example, BDNF is expressed in smooth muscle2 and endothelial cells3. BDNF deficiency during gestation results in murine endothelial cell apoptosis, missing intramyocardial blood vessels, microvascular leakage, thinning cardiac chambers and depressed cardiac contractility3. Constitutive BDNF signaling is required for physiological murine cardiac contraction and relaxation4. This effect of BDNF was shown to be independent of and in addition to β-adrenergic signaling where BDNF acted directly on Ca2+ cycling in a calmodulin-dependent protein kinase II-dependent manner.The brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors which has been attributed to a plethora of functions including the preservation of neuronal cell viability and function as well as the prevention of neuronal degradation during stress5. In patients after myocardial infarction BDNF is related to inflammation and platelet activation6. At the same time low plasma BDNF was associated with future coronary events and mortality in 885 patients with angina pectoris7. In addition, BDNF levels are also reduced in heart failure patients and are inversely correlated with BNP8. Hence, a role of BDNF in cardiac remodeling is very likely.BDNF plays a role in the progression of human cardiovascular disease. For example, this neurotrophin promotes atherogenesis and plaque instability via the activation of NAD(P)H oxidaseOverall, previous research suggests a link between BDNF, cardiac function and cardiovascular disease. We tried to improve our understanding of this relationship by assessing the role of BDNF on left ventricular cardiac remodeling and function using data from a population based cohort from northeast Germany. We further explored the association between BDNF and the established heart failure marker N-terminal pro b-type natriuretic peptide (NTproBNP).2. A total of 38% were non-, 34% ex- and 28% current smokers. The prevalence of diabetes mellitus, hypertension and metabolic syndrome was 7%, 40% and 23%, respectively. The estimated glomerular filtration rate (eGFR) as an index of kidney function was within the normal range (median: 99 ml/min/1.72 mm2). All population characteristics and echocardiographic parameters are listed in Table Median age of the study population was 48 years (range 37 to 60) and 45% were male. Median BMI was 26.7 kg/mWe found sex specific associations between BDNF and NTproBNP Fig. . SpecifiThese results are presented in Fig. The results are presented in Fig. 9. Hence, our results extend the current knowledge by providing evidence for a putative role of BDNF not just in heart failure but also during subclinical cardiac dysfunction and remodeling prior to manifest cardiac disease. Cardiac hypertrophy is accompanied by increased sympathetic activation leading to an upregulation of the renin-angiotensin-aldosterone system (RAAS)10. Brain RAAS and chronically overactive RAAS interact through positive biofeedback to synergistically maintain the diseased condition10. BDNF might be an internal mechanism to counteract the adverse effects of acute RAAS activation after myocardial infarction11. Although, in a general population rather than in a clinical setting, this study contributes to the emerging evidence featuring BDNF as a biomarker for cardio-neuronal damage during early and subclinical cardiac remodeling.Using data from a large population based study we demonstrate that low BDNF is related with higher levels of the heart failure biomarker NTproBNP and with adverse left ventricular cardiac remodeling. Recent clinical trials reported lower levels of BDNF in heart failure patients12. Low BDNF in the heart as well as a knock-down of the BDNF-specific receptor, TrkB, resulted in increased fibrosis and lower ejection fraction. Moreover, intraperitoneal injection of recombinant BDNF rescued the cardiac phenotype. In patients with ischemic heart disease or acute coronary syndrome, BDNF levels are significantly lower compared to healthy controls14. In patients with unstable angina, circulating BDNF is lower when compared to stable angina and a non-coronary artery disease group5. These findings suggest circulating BDNF as a useful biological marker for heart disease. However, plasma BDNF, in addition, significantly positively correlates with multiple risk factors for metabolic syndrome and cardiovascular dysfunction 15. Thus, BDNF may lack the required specificity for a cardiac specific biomarker but might have the potential to improve our understanding of the triangular relationship between metabolic risk factors, neuronal damage and cardiac dysfunction.Two weeks after myocardial infarction murine BDNF levels increased in brain and plasma, but not in the heart16. BDNF also prevents the TNFα induced endothelial barrier dysfunction, while TNFα can reduce BDNF expression17. Since the role of cadherins18 and TNFα21 in left atrial remodeling is well established, low levels of BDNF may increase cadherins and TNFα which in turn modulate left atrial remodeling and thereby increase AF risk. Interestingly, in the Framingham Heart Study no relation between BDNF and AF development over a 10 year time period was found22. The non-linear relationship between BDNF and left atrial remodeling may seem counterintuitive when compared to the other results which suggest that only low BDNF is associated with more severe adverse cardiac remodeling. However, in the Baltimore Longitudinal Study of Ageing peripheral BDNF was positively associated with several cardiovascular risk factors including body mass index and diastolic blood pressure15. Further, BDNF significantly increased the extent of myocardial injury in older rat hearts23. Based on the current literature one can only speculate whether BDNF has a protective role in response to the underlying cardiac pathophysiology or if this neurotrophin contributes to disease progression. We also previously reported a U-shaped relationship between BDNF and waist-to-hip ratio as a marker of visceral adiposity24. Hence, the directionality and causality between BDNF and cardiovascular disease is still undetermined and may be related to signal cross-talk. For example, BDNF and TNFα may influence each other. Future studies need to evaluate the relationship between BDNF and left atrial remodeling in specific clinical settings.An interesting and unexpected finding was the non-linear relationship between left atrial diameter and BDNF. High and low BDNF levels were related with an enlarged left atrium, while a smaller diameter was seen in subjects with BDNF levels around 20,000 pg/ml. A large left atrium is generally considered a key risk factor for atrial fibrillation (AF) and diastolic dysfunction. In mice BDNF decreases VE-cadherin cleavage to reduce atherosclerosis and promotes vascular integrity through Ets1-mediated VE-cadherin expression25. Low BDNF levels have been reported in depressive patients26. Although, in our population based cohort BDNF was not associated with depression24. The “neurotrophic hypothesis” for depression even postulates that a stress-induced decrease in BDNF initiates depression28. As mentioned above, BDNF is also related to cardiovascular disease. In a large population-based cohort low BDNF was associated with higher mortality and incidences of cardiovascular events29. Since BDNF is related to depression and cardiovascular disease, one may speculate that this neurotrophin influences both conditions30, potentially through inflammation31 since patients with depression32 and acute coronary syndrome33 are characterized by increased inflammation. This hypothesis is additionally supported by the fact that inflammation can decrease BDNF34. Nonetheless, we used major depressive symptoms as a confounder in our analysis and BDNF was still significantly associated with adverse cardiac remodeling and NTproBNP. Hence, low BDNF may be associated with higher cardiac risk independent of depression.Depression and cardiovascular disorders often appear togetherThe findings of our analysis need to be interpreted in the context of several limitations. First, we only included Europeans living in rural areas in our sample. Hence, we do not know if our findings are true for other ethnicities, children or elderly age-groups. Second, we only had access to cross-sectional data from one time point. Thus, we do not know whether a decrease in circulating BDNF over time is associated with adverse left ventricular structure and function. Third, although we used a directed acyclic graph to identify metabolic, cardiovascular and psychiatric confounders for our multivariable models, we cannot exclude the possibility of further residual confounding. Fourth, we only used two questions to determine whether subjects ever experienced a major depressive disorder. This may be less sufficient to account for the complexity of depressive disorders. Nonetheless, strengths of our study are the population-based setting, the large number of study participants, the use of standardized data collection methods, the capacity to perform adjustment for a variety of clinical, behavioral and psychiatric risk factors and the availability of a standardized echocardiographic examination from a large number of participants.In summary, this study revealed that subjects with low BDNF have higher NTproBNP levels and adverse early and subclinical left ventricular remodeling. Further, we provided evidence of a non-linear relationship between BDNF and left atrial alterations. We showed that these findings are independent of depressive symptoms. Taken together our results might support the notion that BDNF could be a marker for neuronal-cardiac damage during the early phases of cardiac remodeling.35. The data used in the presented analysis were derived from SHIP-Trend, a cohort initiated ten years after SHIP-0 in the same region36. In brief, from the total population of West Pomerania, a rural area in the northeastern part of Germany, a two-stage stratified cluster sample of 8,016 adults between the ages of 20–79 years was drawn. In total, 4,420 individuals participated in the study (response of 50.1%). Data used in this analysis is based on data collected during the baseline examination of SHIP-TREND which took place between 2008 and 2011. We have previously used SHIP-TREND to assess the relation between BDNF and BMI24. The study was approved by the ethics committee of the University of Greifswald, complies with the Declaration of Helsinki and all study participants gave written informed consent. SHIP data are publically available for scientific and quality control purposes. Data usage can be applied for via www.community-medicine.de.The Study of Health in Pomerania (SHIP) is a prospective population-based cohort of adults from West Pomerania, a north-eastern region in Germany of approximately 220,000 inhabitants. The first sample (SHIP-0) was surveyed between 1997 and 2001 using a stratified cluster-random sample of 7,008 individuals. The net sample (without migrated or deceased persons) included 6,265 eligible individuals. A total of 4,308  subjects participated (response: 68.8%) in SHIP-02), previous myocardial infarction, left ventricular ejection fraction <40%, atrial fibrillation, extreme values for BDNF and NTproBNP  and missing data were excluded. The total sample size was 2,976 subjects.For the present analysis individuals with severely impaired renal function (estimated glomerular filtration rate [eGFR] <30 mL/min/1.73 m37. Body mass index (BMI) was calculated by dividing weight (kg) by height (cm) to the square. Diabetic patients were identified based on the self-reported use of antidiabetic medication  code: A10) in the last 7 days or a glycosylated hemoglobin level >6.5%. Blood pressure (BP) was assessed after a 5 min resting period in sitting position. Systolic and diastolic BP were measured three times, with three minutes rest in between, on the right arm using a digital blood pressure monitor . The average of the second and third reading was used. Hypertensive patients were identified by either self-reported antihypertensive medication  or a systolic BP above 140 mmHg and/or a diastolic value of more than 90 mmHg. Lean mass and fat mass were measured by bioelectrical impedance analysis (BIA) using a multifrequency Nutriguard-M device  and the NUTRI4 software  in participants without pacemakers. The electrodes were placed on hand and wrist as well as ankle and foot. The test frequency was measured at 5, 50 and 100 kHz following the manufacturer’s instructions38. Body mass index (BMI) was calculated by dividing weight (kg) by height (cm) to the square. Diabetic patients were identified based on the self-reported use of antidiabetic medication  code: A10) in the last 7 days or a glycosylated hemoglobin level >6.5%. Blood pressure (BP) was assessed after a 5 min resting period in sitting position. Systolic and diastolic BP were measured three times, with three minutes rest in between, on the right arm using a digital blood pressure monitor . The average of the second and third reading was used. Hypertensive patients were identified by either self-reported antihypertensive medication  or a systolic BP above 140 mmHg and/or a diastolic value of more than 90 mmHg. Metabolic syndrome was defined by any three or more of the five components proposed by ATP III39 and updated with minor modifications by the American Heart Association and the National Heart, Lung, and Blood Institute40 and were modified for the use of nonfasting blood samples41: (1) abdominal obesity, waist circumference ≥104 cm in men and ≥88 cm in women; (2) elevated triglycerides, >2.0 mmol/l or lipid medication (ATC code C10ab); (3) low HDL cholesterol, <1.03 mmol/l in men; (4) high blood pressure, >130/85 mmHg or antihypertensive medication (ATC code C02); (5) high blood glucose, >8.0 mmol/or diabetes medication (ATC code A10). The electrodes were placed on hand and wrist as well as ankle and foot. The test frequency was measured at 5, 50 and 100 kHz following the manufacturer’s instructions38. Alcohol consumption (in grams per day) was derived from a beverage-specific quantity-frequency index.Trained and certified staff used standardized computer-assisted interviews to ask the patients about their age, sex, years of schooling and smoking habits. Smoking was classified as current smoker, nonsmoker or former smoker. All participants underwent an extensive standardized medical examination. Anthropometric measurements included height and weight based on recommendations of the World Health Organization (WHO)44. The screening questions for depressive disorders comprised the following two items: “Feelings of sadness or depressed mood for a period of at least 2 weeks” and “Lack of interest, tiredness, or loss of energy for a period of at least 2 weeks”.Major depressive disorder (MDD) and recurrent MDD were diagnosed according to DSM-IV using the Munich-Composite International Diagnostic Interview (M-CIDI)et al. (eGFR = 186 × (plasma creatinine concentration × 0.0113118)−1.154 × age−0.203) multiplied by 0.742 for female subjects and expressed as mL/min/1.73 m45. BDNF and NTproBNP were measured according to the manufacturer’s recommendations using an ELISA  and Dimension Vista , respectively.A non-fasting venous blood sample was drawn from all subjects in supine position (between 7 am and 4 pm). The eGFR was calculated according to Levey 46. Measurements of LV end-diastolic and end-systolic diameter  and septal as well as posterior wall thickness  were performed according to the guidelines of the American Society of Echocardiography47. LV mass (LVM) was calculated according to the formula: LVM (g) = 0.8 × (1.04 × ((LVDD + SWT + PWT)3 - LVDD3)) + 0.6 g as described by Devereux and Reichek49. LVM was indexed (LVMI) for body surface area (BSA) according to Duboi (BSA = 0.20247 × height (m)0.725 × weight (kg)0.425)50, which linearizes the relations between LVM and height and identifies the impact of obesity. LV wall thickness (WT), relative wall thickness (RWT), LV ejection fraction (EF) and fractional shortening (FS) were calculated following the formulas below according to the guidelines of the American Society of Echocardiography51. Transmitral pulsed-wave Doppler was used to record early (E) and late (A) wave ventricular filling velocities. Certification examinations for inter-observer variations revealed an agreement of >90%46.Two-dimensional, M-Mode and Doppler echocardiography were performed using the Vivid-I system  as described in detail elsewhereth/75th percentile. Nominal data are expressed as percentages. Differences between groups were calculated using Kruskal-Wallis (continuous variables) and χ2 test , respectively. A linear regression adjusted for sex, age, fat mass, lean mass, systolic blood pressure, current smoking and depression was fitted to relate BDNF the echocardiographic parameters of interest. Furthermore, restricted cubic splines52 were used to detect possible nonlinear dependencies of BDNF on the investigated echocardiographic parameters. Three knots were pre-specified, located at the 5th, 50th, and 95th percentiles52, resulting in one component of the spline function. Potential sex specific associations between BDNF and echocardiographic parameters were assessed by adding the appropriate interaction term into the model. If this term was significant the analysis was stratified by sex. A p < 0.05 was considered statistically significant. All statistical analyses were performed in SAS 9.4 . All parameters are reported as median and 25th as well as 75th percentile unless indicated otherwise. All results of the linear regression analysis are given as a 10,000 ng/ml decrease in BDNF.Continuous data are expressed as median and 25All participants provided written informed consent and SHIP was approved by the Ethics Committee of the University Medicine Greifswald.


To conclude, symptomatic thyroid dysfunctions rate in well-treated HIV-infected patients is low. Age and gender are crucial elements in the onset of thyroid abnormalities, together with T CD4+ cell nadir. Interestingly, medullary thyroid cancer seems to be much more frequent in HIV-infected patients compared to the general population.Thyroid diseases (TDs) have been widely associated with HIV infection. However, data about TDs prevalence and distribution are controversial, and few published studies are available. The aim of our study was to assess prevalence and risk factors of symptomatic thyroid disturbances, including thyroid cancers, in a large cohort of HIV-infected patients. A retrospective cohort study was performed at the Department of Infectious and Tropical Diseases of the University of Brescia, Italy, in the period 2005–2017. We identified all HIV-positive patients with a diagnosis of symptomatic TD in the electronic database of our Department (HIVeDB); we also operated a record-linkage between our data and the Health Protection Agency database (HPADB) of Brescia Province. Multivariate logistic regression analysis was used to determine risk factors associated with TDs onset; an incidence rate analysis was also performed. During the study period, 6343 HIV-infected patients have been followed at our Department; 123 received a diagnosis of symptomatic TD (1.94% of the entire cohort). In the TDs group, almost half of patients were females , mean age was 47.15 years (SD: 11.56). At TD diagnosis, mean T CD4+ cell count was 491 cell/uL and most patients showed undetectable HIV-RNA . Among them, 81 patients were found to have hypothyroidism (63 with Hashimoto’s thyroiditis), 21 hyperthyroidism (17 suffered from Graves’ disease), while 11 subjects were diagnosed with a primitive thyroid cancer. Papillary thyroid cancer was the most frequent histotype , followed by medullary  and follicular thyroid cancer . Male gender was a protective factor for TDs development, especially for hypothyroidism (p < 0.001); age emerged as a variable associated with both hypothyroidism (p = 0.03) and thyroid cancer (p = 0.03), while CD4+ cell nadir <200 cell/mm In particular, thyroid diseases (TDs) have been widely associated with HIV infection since the start of the epidemic, long before the beginning of the combination antiretroviral therapy (cART) era6.Various alterations in endocrine homeostasis were described in HIV-infected patients13 and no apparent increased prevalence of TDs in this population17. In well-treated HIV-positive individuals, subclinical hypothyroidism18 and isolated low free thyroxine (FT4) levels19 are the most common described alterations. Anyway, although it is estimated that around one third of PLWH may present biochemical alterations of thyroid function, only 1–3% develops an overt illness9.However, there are controversial data concerning thyroid dysfunctions in people living with HIV (PLWH), as previous studies reported both a higher than expected prevalence of TDs6. Furthermore, several opportunistic infections as from Cytomegalovirus (CMV), Mycobacterium avium, Cryptococcus neoformans, Pneumocystis jirovecii (previously known as P. carinii), Aspergillus spp, Mycobacterium tuberculosis as well as AIDS-defining neoplasms  can occasionally involve thyroid gland, rarely resulting in thyroid dysfunction20. Antiretroviral therapy can also be implicated in TDs. Stavudine has been linked to lower FT4 levels and hypothyroidism19, whereas the role of other regimens, as integrase (INIs) and protease inhibitors (PIs), in impairing thyroid function has yet to be fully investigated. Other drugs, as phenytoin, carbamazepine and lithium, that are frequently used among PLWH for psychiatric disorders, can also modify thyroid hormone levels.Many factors could be implicated in the pathogenesis of thyroid disturbances in PLWH. Firstly, HIV itself seems to play a crucial role, determining different thyroid alterations over the course of the disease22.Moreover, the improvement of host immune response induced by cART could trigger autoimmune disorders, leading to the appearance of aberrant thyroid autoantibodies. Thus, late after the introduction of antiretroviral therapy, approximately 2% of PLWH develops Graves’ disease24. Furthermore, interferon therapy can also promote autoimmune reactions in HCV-infected patients, especially targeting thyroid gland25.Lastly, Hepatitis C virus (HCV) coinfection is frequently observed in PLWH, due to similar transmission routes. HCV has been associated with different autoimmune conditions, including thyroid disorders, although the actual causative mechanisms have not been entirely determinedTo date, few published papers regarding thyroid dysfunctions in HIV-positive patients are available and thyroid alterations prevalence and distribution in PLWH still remain controversial, since previous studies show sample, definitions and outcomes heterogeneity. Moreover, to our knowledge, no studies focusing only on symptomatic thyroid dysfunctions have been performed and data about thyroid primitive tumors in HIV-infected patients are completely lacking.Thus, the aims of this study were to assess the prevalence of symptomatic thyroid dysfunctions, including primitive thyroid cancers, in a large cohort of HIV-infected patients in the modern cART era and to evaluate their laboratory and clinical characteristics. Finally, we also determined the risk factors associated with the occurrence of symptomatic thyroid abnormalities in this cohort.We performed a retrospective cohort study including all HIV-infected patients aged more than 18 years old and receiving cART in charge at the Department of Infectious and Tropical Diseases, ASST Spedali Civili and University of Brescia, Italy. The period of follow-up ranged from January 2005 to December 2017.We considered as symptomatic TDs all thyroid disorders needing medical or surgical treatment and all thyroid primitive tumors. Symptomatic thyroid dysfunctions were subsequently classified in three groups as follows: hypothyroidisms, hyperthyroidisms and thyroid cancers (TCs).We identified all HIV-positive patients with a symptomatic TD in the electronic database used for clinical management at our Department (HIVeDB); we also performed a record-linkage between HIV-infected patients followed by our Department and the Health Protection Agency database (HPADB) of Brescia Province, which tracks all services provided by the National Health Service. In this way, we captured all subjects with HIV infection who received in-hospital services and medical treatment for hypo-/hyperthyroidism or had a medical certificate with an ICD9-CM code for TD (including thyroid cancers). Uncertain TD cases were confirmed with phone-call to patients.Patients’ demographic, epidemiological, clinical and laboratory  data were recorded in an electronic file. Information about coinfections, eventual comorbidities and medication history (including cART) were also collected. In particular, the following parameters were recorded at TD diagnosis and at the last available visit performed at our Clinic: HIV-RNA (cut-off of 37 copies/ml) and lymphocyte T CD4+ and CD8+ cell counts.a priori, considering scientific evidence and convenience to retrieve data.The outcomes of the research were TDs, considered in general and as specified diagnosis-related groups . They were chosen arbitrarily Continuous variables were reported as mean with standard deviation (±SD), categorical variables as frequency with percentages. We used chi-squared tests and Fisher exact tests for dichotomous variables comparison and t-test or Wilcoxon rank-sum test for group means comparison .3), gender, age at HIV test (per 10-years older) and HCV coinfection. All the OR estimates were reported together with their 95% confidence intervals (95% CIs) and the p value.Odds ratios (ORs) were computed through multivariate logistic regression. Our model computed ORs for all outcomes, considering CD4+ nadir  using periods of three years, from 2005 to 2016. Analysis were stratified by gender. Subjects with less than 30 days of follow up were excluded from the incidence analysis but kept in all the others. We excluded prevalent cases. For this analysis we did not include incident cases during 2017 for getting homogeneous periods.Moreover, two sensitivity analyses excluding patients with positive HCV antibodies and patients with an AIDS-defining diagnosis were conducted for both the incidence and the prevalence of TDs.All statistical tests were two-sided at the level of significance p < 0.05. Analyses were performed using Stata software version 14.0 .This study was conducted according to the Declaration of Helsinki and the principles of Good Clinical Practice (GCP). As this study had a retrospective design and was based on routinely collected data, patients’ informed consent was not required according to the Italian law . Moreover, for this study we used the general authorization of the Italian Guarantor for the use of retrospective demographical and clinical data, which have been treated according to present laws.From 2005 to 2017, 6343 HIV-infected patients have been followed at our Department with at least one visit; 71.68% was represented by males, mean age at the end of follow-up was 47.30 ± 11.90 years. HCV/HIV coinfection was present in 39.08% of the cohort. A total of 123 patients showed a symptomatic TD in our HIVeDB during the study period . All detected subjects were confirmed by the HPADB and no additional cases were identified using this source.3 and 71.54% (n = 88) experienced a previous AIDS-defining event. Mean time from HIV to TD diagnosis was 11.04 years (SD: 9.60); at TD diagnosis, mean T CD4+ cell count was 491 cell/uL (range 1–1556 cell/uL) and most patients had undetectable HIV-RNA . The logistic regression analysis showed that TDs were negatively associated with male gender  and positively associated with age at HIV test per 10-year increase . At the end of follow-up, all patients with TD had serum thyroid stimulating hormone (TSH), serum triiodothyronine (T3) and serum thyroxine (T4) levels within the reference ranges (data not shown). Overall, 99 patients (80.49%) received levothyroxine, 17 (13.82%) were cured with methimazole, 3 (2.44%) took iodine and selenium supplements. Furthermore, 20 patients (16.26%) had a history of surgical thyroidectomy. Figure All patients were known to be HIV-infected when they developed thyroid dysfunction; mean age at TD diagnosis was 46.6 years SD: 11.5). Characteristics of the study population at baseline and at the end of follow-up are summarized in Table 1.5. CharHypothyroidism was the most prevalent TD in our cohort ; the majority of patients  had a Hashimoto’s thyroiditis. Mean age was 45.80 ± 11.49 years; slightly more than half was represented by males  and overall 74.07% of subjects presented at least one comorbidity. Concerning HIV status, 22 patients (27.16%) experienced a previous AIDS-defining event, with a mean time from HIV acquisition to TD diagnosis of 11.23 years (SD: 10.20). At TD diagnosis all patients with hypothyroidism were on stable cART, the majority with a PI-containing regimen . At the end of follow-up, all patients with hypothyroidism received a levothyroxine-based treatment. At the multivariate regression logistic analysis, hypothyroidism was negatively associated with male gender  while age at HIV test per 10-year increase was borderline positively associated .3 (SD: 169.30). At TD diagnosis the totality of patients with hyperthyroidism received cART, 57.14% (n =  512) with a PI-based regimen. At the end of study, all patients were treated for their thyroid dysfunction , while 5 underwent thyroidectomy. At the multivariate regression logistic analysis, only T CD4+ cell nadir <200 cell/mm3 was independently associated with hyperthyroidism symptomatic disorders .The 18.5% (21/113) of patients with TD showed a symptomatic hyperthyroidism; the majority of them  suffered from Graves’ disease. Males represented 52.38% of this group (n = 11); mean age was 42.52 years (SD: 12.58). 76.19% of patients presented at least one comorbidity. Mean time from HIV to TD diagnosis was 12.06 years (SD: 8.14), mean T CD4+ nadir was 105.74 cell/mm3). All subjects underwent thyroidectomy and all received a substitution therapy with sodic levothyroxine. One of the patients with papillary thyroid cancer had a metastatic disease involving lungs and bones; he finally died, after six years of chemotherapies and radiotherapies. TC diagnosis was made at 47 years old; patient remained in good viro-immunological conditions until the exitus . To date, the rest of patients diagnosed with TC are in good conditions. At the multivariate logistic regression analysis TC diagnosis was independently associated only with age at HIV test per 10-year old increase .Eleven subjects were diagnosed with TC during the study period. Papillary thyroid cancer was the most frequent TC , followed by medullary  and follicular thyroid cancer . The histotype was not recorded for one patient. In this group, mean age was 48.64 years (SD: 6.30) and patients were equally divided by gender . Almost all subjects  presented at least one comorbidity , while 36.36% (n = 4) were coinfected with HCV. TC was meanly diagnosed after 8.69 years from HIV diagnosis (SD: 9.88) and 100% of patients was on stable cART at that time . We observed 96 TDs incident cases with an overall crude incidence rate of 2.2 per 1000 persons (95% CI 1.8–2.7); the crude incidence rate was 1.6 per 1000 persons (95% CI 1.2–2.2) for males and 3.8 per 1000 persons (95% CI 2.9–5.1) for females. Demographics characteristics and TDs incidence rates for the entire follow-up considered (2005–2016) and for each 3-year period are shown in Table Temporal trend was also analyzed by Poisson regression as incident relative risk (IRR). For each 3-year period TDs incidence increased of 25%, with an IRR of 1.25 , mainly attributed to an incidence increase in females .As expected, at the sensitivity analyses excluding subjects with positive HCV antibodies and with an AIDS-defining diagnosis we found only few differences for both the incidence and the prevalence of TDs due to the small number of total patients with symptomatic thyroid disorders considered (data not shown).Here we report that TDs, including cancers, are a rare event in PLWH, with double incidence for females compared to males. Thus, in our cohort, only 1.94% of HIV-infected patients were diagnosed with thyroid disturbances during the follow-up period: 1.28% showed symptomatic hypothyroidism, 0.33% symptomatic hyperthyroidism, while thyroid cancer was documented in 0.17% of the considered population.29, since they included also subclinical TDs in their analysis. If we compare the sole prevalence of overt hypo and hyperthyroidism in HIV-positive individuals, our data are similar to those found by other Authors, although their studies were performed in much smaller cohorts32. Moreover, in our cohort all patients were on stable cART at TD diagnosis, confirming that well treated PLWH are not at an increased risk of thyroid dysfunction, as previous described17.Other studies reported a significative higher prevalence of TDs in PLWH, ranging from 16% to 33.1%33, besides the influence of age, sex and environmental factors34. For these reasons and considering the high prevalence of subclinical thyroid dysfunctions in HIV-infected subjects, we analyzed only symptomatic diseases.However, it is not easy to make comparisons among distinct epidemiologic studies, due to the diversity of populations and end-points analyzed. Differences involve disease definition and severity , selection criteria, different reference ranges and laboratory techniques used to measure serum thyroid hormone levels35, a large-population based study36 evaluated the presence of autoimmune diseases among 5186 HIV-infected patients, finding only 1 case of Hashimoto’s thyroiditis and 2 cases of Graves’ disease. Thus, these TDs seem to be more frequent in our cohort, showing a prevalence comparable to international general population values34.The two major autoimmune TDs, Hashimoto’s thyroiditis and Graves’ disease, were the most frequent pathologies of thyroid gland in our cohort. Although small researches reported a prevalence of Hashimoto thyroiditis up to 2.6% in HIV-positive patients34. The biological explanation for this gender difference is not entirely clarified, but the X chromosome inactivation could significantly contribute to the high incidence of autoimmune TDs in females37; moreover, it seems that maternal immune responses against fetal antigens can trigger autoimmune processes38. In our cohort of PLWH, male gender was confirmed as a protective factor for TDs onset, especially for hypothyroidism.Autoimmune TDs are much common among women than in men, with a female to male ratio ranging from 5:1 to 10:1 in general population39. In our study, age at HIV test emerged as a variable associated with both hypothyroidism and TCs. Notably, we also focused on temporal trends, finding an increase in TDs incidence for each 3-year period from 2005 to 2016, attributable to the ageing of our cohort. Thus, increasing age and non-AIDS related comorbidities , together with chronic inflammation, immunosenescence and polypharmacy may play a role in TDs occurrence and progression.TDs development is also influenced by ageing in general population3 was independently associated with symptomatic hyperthyroidism. In this group we reported the most remarkable CD4+ T cell increase from nadir to TD diagnosis (mean CD4+ increase: 437.54 ± 306.13), supporting the previously described hypothesis of Graves’ disease as a manifestation of delayed immune reconstitution inflammatory syndrome (IRIS)22 and suggesting that some CD4+ T lymphocytes subsets may affect its occurrence40.However, immune restoration is also probably implicated in TDs development. As a matter of fact, we found that a T CD4+ cell nadir <200 cell/mm29.Finally, we did not find any correlation between HCV coinfection and thyroid abnormalities, although some Authors highlighted the role of both HBV and HCV in increasing the probability of thyroid dysfunction, especially hypothyroidism43. To our knowledge, our study is the first one to evaluate TCs prevalence and associated risk factors in a population of HIV-infected patients. According to the latest Italian general population tumor report44, the risk to develop a TC is much higher in females compared to males; furthermore, although TC incidence is increasing, the overall mortality remains extremely low. In our study, TC seems to be a very rare condition, with a lower prevalence than the one reported for general population . In this population of PLWH, patients diagnosed with TC were equally divided by gender and age was the only risk factor associated with thyroid tumor development. Italian data44 show that, from 2010 to 2014, 82% of TCs was represented by papillary thyroid cancer, followed by follicular (7%), medullary (4%) and anaplastic (1%) histotypes. As expected, in our cohort of PLWH, the most frequent TC was represented by papillary thyroid cancer. Interestingly, medullary thyroid cancer was diagnosed in almost 20% of patients considered in our study, with a percentage significantly higher compared to the Italian general population. Although medullary thyroid cancer development was associated with mutations of RET tyrosine kinase, no information is available about a possible interaction between HIV and thyroid tissue, as described for EBV45. However, due to our limited sample size, more researches are needed in order to confirm our results and to evaluate the real distribution of TCs in a population of HIV-infected individuals. A recent study46, performed using data from  > 6 million US patients from the National Cancer Data Base (NCDB), showed that PLWH were more likely to be diagnosed with advanced-stage of TCs and to experience a higher mortality after TC diagnosis compared to general population. At TC diagnosis, all individuals considered in our cohort were on stable cART, the majority with a satisfying viro-immunological profile; moreover, at the end of follow-up, TC was controlled in all patients except one, who died.In the post-HAART period, PLWH reported an increasing risk of non-AIDS-defining cancers that it is higher compared to uninfected controlsOur study should be interpreted within its limitations, firstly the retrospective monocentric design, which does not permit a more comprehensive description of HIV and immune system dynamicity. Secondly, although every TD diagnosis was accurately verified, there could be some bias in estimating the prevalence of thyroid disturbances in the population considered. Finally, all participants are from the same geographical area and it is reasonable to assume a similar iodine intake; however, our results may not apply to regions with different iodine consumption.The strengths of this study include the large population size, the enrolment of an unselected group of HIV-infected patients and an accurate retrieval of data on TDs thanks to the use of two different sources, the clinic database and the HPADB of Brescia Province.47 and that PLWH are actually ageing. Periodic screening with measurement of TSH levels could be implemented, in order to provide a rapid treatment and to minimize thyroid-related complications.To conclude, our study is the first to analyze the prevalence of exclusively symptomatic TDs  in a large cohort of HIV-infected patients, confirming age and gender as crucial elements in the onset of thyroid abnormalities and highlighting the role of T CD4+ cell nadir as additional factor. Despite the high prevalence of thyroid dysfunction described in PLWH, symptomatic TDs remain a rare event. However, in the next decades, we will probably face a progressive increase of this comorbidity, considering that TDs  are dramatically more prevalent after the age of 40Moreover, we found an interesting much more elevated rate of medullary thyroid cancer in PLWH compared to the general population; however, the distribution of different histotypes and their extensive characterizations are yet to be fully described in this category of patients.


Chronic kidney disease (CKD) is a common comorbidity of chronic obstructive pulmonary disease (COPD). Although high hemoglobin (Hb) is detrimental to CKD patients, its relationship with poor outcomes in the COPD population has not been reported. This study aimed to investigate the relationship between high Hb and in-hospital mortality and to explore reference Hb intervals in patients with COPD and CKD.2, respectively. The association between Hb level  and in-hospital mortality was analyzed in different multivariable logistic regression models by CKD stratification.This retrospective study was multicenter population-based. A total of 47,209 patients who presented with COPD between January 2012 and December 2016 were included. The average Hb level during hospitalization was used as the Hb level. CKD and advanced CKD were defined as estimated glomerular filtration rates < 60 and < 30 ml/min/1.73 mThe Hb level was decreased in the CKD subgroup. In the non-CKD group, a higher Hb level was not associated with an increased risk of in-hospital death. However, the Hb level and mortality showed a U-shaped relationship in the CKD group. After adjusting for age and Charlson Comorbidity Index, multivariable regression analysis showed that an Hb level > 17 g/dL was associated with an increased risk of death in the CKD group with an odds ratio (OR) of 2.085 . Hb > 14 g/dL was related to an increased risk of death in advanced CKD patients ).High Hb is associated with an increased risk of in-hospital death in COPD patients with CKD, especially among those with advanced CKD. In this group of patients, attention should be paid to those with high Hb levels.The online version of this article (10.1186/s12890-019-0933-4) contains supplementary material, which is available to authorized users. Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation, inflammation and lung remodeling. In 2020, COPD is projected to rank fifth worldwide in terms of disease burden and third in terms of mortality .Hemoglobin (Hb) abnormalities, including anemia and polycythemia, are common in the COPD population –4. As hyThe association of polycythemia and adverse outcomes is not well understood in patients with COPD. Several previous studies indicated a neutral or protective role of polycythemia , 6, 7. IHowever, the potential detrimental effects of polycythemia have been implicated in several other chronic conditions, one of which is chronic kidney disease (CKD). Serial randomized controlled trials (RCTs) and meta-analyses have demonstrated the risks of relatively higher Hb concentrations. These increased risks include stroke, hypertension, vascular access thrombosis, cardiovascular events, end-stage renal disease and death, and the cutoff values of high Hb did not exceed those of polycythemia –16. BaseCKD is a common comorbidity in the COPD population , 8, 19. Electronic data of COPD patients from the 13 hospitals included in the database of the China Collaborative Study on Acute Kidney Injury  were retrospectively analyzed. This dataset includes all hospitalized COPD patients with or without AKI. All patients were admitted to the hospital between January 2012 and December 2016, and the electronic data consisted of demographic information, diagnostic determination, and results of serum creatinine and Hb tests. The creatinine values from the centers were calibrated by the Clinical Laboratory Center of Guangdong General Hospital.Medical records from 78,036 admissions with COPD suitable for the purpose of this study were screened. COPD and the acute phase were judged according to clinical diagnostic coding. Twelve hospitals were tertiary hospitals, and 1 was a secondary hospital  < 60 ml/min/1.73 midelines . In-hospidelines . Conside2) and an advanced CKD group (eGFR < 30 ml/min/1.73 m2).The enrolled patients were divided into a CKD group and a non-CKD group. To analyze the influence of the severity of CKD on Hb, the CKD group was further divided into an early CKD group  and 95% confidential intervals (95% CIs) were calculated. Age and CCI were included as variables. All statistical analyses were performed using SPSS 24.0. A two-tailed P value < 0.05 was considered to indicate a significant difference.Measurement data are presented as the means ± standard deviation or medians (Q25-Q75). Independent t-tests were used for comparison between groups. Numeration data are presented as percentages (%), and Of the included 47,209 patients with COPD, 6808 14.4%) patients also had CKD. Patients in the CKD group were older and had more complications  than those in the non-CKD group  of 13.4  g/dL. Furthermore, Hb distribution frequencies were plotted based on intervals of 1 g/dL according to clinical judgment and preliminary analysis .A total of 640 patients (1.4%) died during their hospital stays. The death rates of the non-CKD group and CKD group were 1.0 and 3.3%, respectively, showing a significant difference   and 4.579 , respectively  and CCI scores as the covariates. The outcomes of the multivariable analysis were similar to those of the univariate analysis. For the non-CKD group, although mortality increased when the Hb level was below the 12–13 g/dL interval , a higher Hb level above this interval was not significantly associated with increased mortality Fig. a. For thThe results of the present study of the hospitalized COPD population are in accordance with those of other reports showing the effect of CKD on anemia  and the A high Hb concentration is double-edged. It not only is considered an adaptive physiological response to hypoxemia in the COPD population  but alsoNo previous studies have reported a clinical threat of a high Hb level in COPD patients , 7, 31. The exploration of the optimal upper Hb target in the CKD population has gone through a long journey. In early studies, exploring the optimal Hb target was difficult due to the considerable heterogeneity in the criterion for high Hb . Only inThis study has the following limitations. First, the CCS-AKI databank suffers from a lack of information regarding erythropoietin- and COPD-related medicine and hematologic testing indices except for Hb, as the initial designed endpoint of the databank was renal events. Therefore, the potential influence of these factors on mortality failed to be corrected for. Second, because the data were derived from retrospective electronic medical records, supplemental oxygen use, pulmonary function and blood gas analysis indices were not available, and the severity of hypoxia and patient conditions were not evaluated. Thus, the impact of a high Hb level could not be assessed independent of the severity of COPD. In addition, approximately 40% of subjects were excluded due to incorrect or incomplete medical records Fig. , which mIn conclusion, high Hb was associated with an increased risk of in-hospital death in the COPD population with CKD, especially those COPD patients with advanced CKD. The management of erythrocyte abnormalities in COPD should not be limited to anemia, but due attention should be given to CKD stratification and increased Hb levels.Additional file 1:Table S1. Information on the enrolled hospitals. Figure S1. Comparison of the Hb levels between males and females in the COPD population complicated with CKD. Figure S2. In-hospital mortality at different Hb levels using uncombined data in the advanced CKD group. The subgroups of the advanced CKD group with Hb levels > 14 g/dL were not merged. In the advanced CKD group, the mortalities of the subgroups with Hb levels of 13–14 g/dL, 14–15 g/dL, 15–16 g/dL, 16–17 g/dL and higher than 17 g/dL were 9.4, 4.3, 12.9, 15.4, and 16.0%, respectively. Points a, b and c indicate the lowest mortalities of the non-CKD group (0.5%), the early CKD group (1.7%), and the advanced CKD group (2.8%), respectively. Figure S3. Association between low or high Hb levels and in-hospital death using uncombined data in the advanced CKD group. The subgroups with Hb levels > 14 g/dL were not merged. Compared with the reference interval (12–13 g/dL), Hb levels of 15–16 g/dL, 16–17 g/dL and > 17 g/dL significantly increased patient mortality, with ORs of 5.831 , 7.417  and 8.781 , respectively. Bars represent the ORs and 95% confidence intervals. (DOC 1185 kb)


Previous studies have found that introduced plants commonly experience more favorable microbial interactions in their non-native range, suggesting that changes to the microbiome could be an important contributor to invasion success. Little is known about microbiome variation across native and invading populations, however, and the potential sources of more favorable interactions are undescribed. Here, we report one of the first microbiome comparisons of plants from multiple native and invading populations, in the noxious weed yellow starthistle. We identify clear differences in composition and diversity of microbiome bacteria. Our findings raise new questions about the sources of these differences, and we outline the next generation of research that will be required to connect microbiome variation to its potential role in plant invasions. Centaurea solstitialis) has been shown to experience more favorable microbial interactions in its invasions of the Americas, but the microbiome that must contribute to this variation in interactions is unknown. We sequenced amplicons of 16S rRNA genes to characterize bacterial community compositions in the phyllosphere, ectorhizosphere, and endorhizosphere of yellow starthistle plants from seven invading populations in California, USA, and eight native populations in Europe. We tested for the differentiation of microbiomes by geography, plant compartment, and plant genotype. Bacterial communities differed significantly between native and invading plants within plant compartments, with consistently lower diversity in the microbiome of invading plants. The diversity of bacteria in roots was positively correlated with plant genotype diversity within both ranges, but this relationship did not explain microbiome differences between ranges. Our results reveal that these invading plants are experiencing either a simplified microbial environment or simplified microbial interactions as a result of the dominance of a few taxa within their microbiome. Our findings highlight several alternative hypotheses for the sources of variation that we observe in invader microbiomes and the potential for altered bacterial interactions to facilitate invasion success.Invasive species could benefit from being introduced to locations with more favorable species interactions, including the loss of enemies, the gain of mutualists, or the simplification of complex interaction networks. Microbiomes are an important source of species interactions with strong fitness effects on multicellular organisms, and these interactions are known to vary across regions. The highly invasive plant yellow starthistle (IMPORTANCE Previous studies have found that introduced plants commonly experience more favorable microbial interactions in their non-native range, suggesting that changes to the microbiome could be an important contributor to invasion success. Little is known about microbiome variation across native and invading populations, however, and the potential sources of more favorable interactions are undescribed. Here, we report one of the first microbiome comparisons of plants from multiple native and invading populations, in the noxious weed yellow starthistle. We identify clear differences in composition and diversity of microbiome bacteria. Our findings raise new questions about the sources of these differences, and we outline the next generation of research that will be required to connect microbiome variation to its potential role in plant invasions. Humans continue to transport plant species around the globe, and increasing numbers of these translocations result in the invasive expansion of non-native species into recipient communities –4. While–8–12–Microbial communities have emerged as particularly likely candidates for facilitating invasions. Although many interactions between plants and microbes can be beneficial, soil microbial communities often appear to have negative net effects on plant fitness which may become more negative over time, e.g., via plant-soil feedbacks , 19–21. 19–22–Invasive plant species have provided some of the best evidence to date that microbial interactions can be locally evolved and can vary considerably over geographic regions . Introdu29–32–Centaurea solstitialis). Yellow starthistle is native to a wide region of Eurasia and was introduced from Western Europe to South America in the 1600s and North America in the 1800s as a contaminant of alfalfa seed  and roots (ectorhizosphere and endorhizosphere) of yellow starthistle plants in both the California invasion and native regions in Europe. Previous experiments with fungicide treatments have shown that plant-soil interactions between yellow starthistle and fungi in California are more negative (less favorable) than those in the native range, inconsistent with a role for fungi in beneficial species interactions in this invasion . Here, w47–We sampled fifteen populations of yellow starthistle for their microbiomes: seven sampling sites across the invasion of California, six sites in Western Europe (native source region), and two in Eastern Europe  to block amplification of plant chloroplast and mitochondrial 16S , includiSequencing yielded 9,672,898 read pairs, of which 6,217,852 remained after merging and quality control; these were 253 bp in length after removing adapter and primer sequences. The numbers of raw read counts per sample ranged from 16 to 306,200 with a median of 21,964. Analysis of the merged and processed reads resulted in 4,014 operational taxonomic units (OTUs), of which 60 were identified as plastid or mitochondrial and 428 were unidentifiable (11%). Of the remaining 3,526, 206 were identified to species (6%), 1,084 to genus (27%), and 2,229 to family (56%) levels. A total of 103 OTUs (3%) were identified as members of the 49 genera with known plant pathogens in the FAPROTAX v.1.1 database , as well as control (blank) samples. Vertical red lines indicate thresholds for rarefaction where relevant. Download Copyright © 2019 Lu-Irving et al.2019Lu-Irving et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00088-19.7TABLE S2Table S2, DOCX file, 0.05 MB.Comparison of host chloroplast blocking levels achieved by different PNA clamps in pooled endorhizosphere samples from two populations. Download Copyright © 2019 Lu-Irving et al.2019Lu-Irving et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Rarefaction levels  were 18,000 reads per sample for phyllosphere, 17,000 for ectorhizosphere, and 5,000 for endorhizosphere samples . These l10.1128/mSphere.00088-19.2FIG S2FIG S2, EPS file, 0.8 MB.NMDS plot of bacterial OTU composition in unrarefied control, phyllosphere, leaf endosphere, ectorhizosphere, and endorhizosphere samples . Download Copyright © 2019 Lu-Irving et al.2019Lu-Irving et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.001) (P = 0.001) (P = 0.001). Native and invaded range phyllosphere samples differed with marginal significance (P = 0.05). Clustering analyses within the phyllosphere and ectorhizosphere compartments consistently grouped invaded range samples together, as well as samples from the source region in Western Europe  . Within = 0.001)  and ectorope see . Native 10.1128/mSphere.00088-19.3FIG S3FIG S3, EPS file, 0.7 MB.Yellow starthistle populations clustered according to Bray-Curtis dissimilarity between rarefied, square root-transformed pooled samples from three plant compartments. Download Copyright © 2019 Lu-Irving et al.2019Lu-Irving et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes (Bacillus (Firmicutes), Chryseobacterium (Bacteroidetes), and the Proteobacteria taxa Erwinia, Pseudomonas, and Xanthomonadaceae  for plants from invaded range sites in all compartments, with the exception of richness in the phyllosphere (P = 0.20) but were significantly lower in evenness (P = 0.005) and were marginally lower in Hill diversity (P = 0.05) than native range plants. Similarly, the ectorhizosphere of invaders was not significantly different in richness (P = 0.41) but was significantly lower in both evenness (P = 0.03) and diversity (P = 0.01). In contrast, endorhizosphere samples pooled by site did not differ significantly in evenness (P = 0.13) but were marginally significantly lower in richness (P = 0.06) and diversity (P = 0.08). Nested analysis of variance (ANOVA) of individual endorhizosphere samples indicated strongly significant reductions in richness, evenness, and diversity in invading plants  , evenness (J), and diversity (elosphere  and 5. W< 0.001) . For indPseudomonas and Erwinia were among the most common pathogen-containing genera encountered in each plant compartment in both ranges. The phyllosphere also included a high frequency of Janthinobacterium, with a relative increase in Serratia in the invaded range. In the ectorhizosphere, Serratia was common in both ranges, but invading plants showed a large relative decline in Erwinia and increase in Pseudomonas. In the endorhizosphere, invading plants harbored less Pseudomonas and more Bacillus and Streptomyces than native plants (see P = 0.44) or ectorhizosphere (P = 0.48). For the endorhizosphere samples pooled by site, significantly lower diversity was indicated in the invaded range (P = 0.04). For individual endorhizosphere samples, nested ANOVA also indicated significantly lower diversity in the invaded range (P < 0.0001), and no significant differences among populations within regions.To explore the potential for these patterns to capture plant-pathogen interaction in particular, we filtered the rarefied data sets for genera containing known plant pathogens in the FAPROTAX database, which resulted in 49 phyllosphere OTUs, 69 ectorhizosphere OTUs, and 88 endorhizosphere OTUs. While pathogen-containing genera are likely to include both pathogenic and nonpathogenic strains , we expeants see . The div10.1128/mSphere.00088-19.5FIG S5FIG S5, EPS file, 1.2 MB.Proportion of rarefied read counts assigned to pathogen-containing genera in all samples according to plant compartment and range. Download Copyright © 2019 Lu-Irving et al.2019Lu-Irving et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H′) for each plant compartment using linear models with fixed effects of plant genetic diversity, region (native versus invaded), and the interaction between these two effects. The model was significant for endorhizosphere samples pooled by site [F = 5.89; P = 0.02; P = 0.03) and region . The interaction between these two effects was not significant (P = 0.71) and was removed from the final model. This same pattern was marginally significant when using only OTUs from pathogen-containing genera in the endorhizosphere samples . Similar linear models did not identify significant effects of plant genetic diversity when predicting the median diversity of individual plant endorhizosphere samples (P = 0.74) or diversity in the phyllosphere (P = 0.35). There was a marginally significant positive effect of plant genetic diversity on diversity in the ectorhizosphere (P = 0.08) in addition to the effect of region (P = 0.002).Finally, we examined the influence of plant genotype on microbial composition. Our geographic regions correspond to genetically differentiated subpopulations, and within these regions, our study sites are also known to vary in plant genetic diversity . Using e = 0.41) , with siIntroduced plants will encounter a variety of novel species interactions as they establish across biogeographic regions. For many plants, severe invasions are associated with more favorable interactions with soil microbial communities , 44. We Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, which are also characteristic of plant-associated bacterial communities surveyed to date  are known to vary at continental scales , 57, 67.73–Importantly, yellow starthistle’s invasion might also cause changes to its microbial environment. Species invasions have been shown to alter microbial composition over short timescales –78, thou–82–Finally, our invading plant populations might be experiencing disease outbreaks, resulting in a high abundance of a few strains and lower diversity of the microbiota as a whole. We think this is less likely, however. Disease outbreaks in plants tend to be local in scale, with microbial communities varying over the spatial scale of meters . Our invTrifolium introduced to New Zealand, despite more favorable soil interactions in the invasions  from North America . Gundal America , 93. For America . Reinhar America  focused lleagues , 96 explOur study is the first to find consistent differences in the microbiomes of native and invading plants which coincide with documented fitness differences in plant interactions with soil communities . Among tWe have previously argued that yellow starthistle has benefitted from the historical loss of plant competitors in California . Disturb99–In conclusion, we found consistent differences between native and invading yellow starthistle plants in their bacterial microbiomes. These differences were robust to additional variation associated with plant compartment and the diversity of plant genotypes. Invaded range microbiomes differed in composition across major taxonomic groups and harbored a lower diversity of bacteria, including reduced evenness on the surfaces of leaves and roots and reduced richness of root endophytes. We suggest that bacteria could be the source of more favorable microbial interactions that have been previously observed in this invasion. Our findings also raise questions about (i) whether lower bacterial diversity is a feature of the invaded environment or whether it is caused by the invasion itself, and (ii) how specific differences in the microbial community affect plant fitness. These questions highlight the need for additional studies that compare microbial communities (including bacteria) associated with native and invading populations, that couple microbial community identification with plant-soil feedback and fitness experiments, and that examine the interaction of environment, plant diversity, and plant density on microbial communities and their fitness effects on plants.Centaurea solstitialis L., Asteraceae) is an obligately outcrossing annual plant, diploid throughout its range (Yellow starthistle (ts range . Plants ts range . The invts range  36)..CentaureAt each location in June-July 2015, plants were sampled every meter (or to the nearest meter mark) along a 25-m transect, to yield microbial samples from 25 individual plants per population. Individuals in rosette or early bolting stages were preferentially selected. In one population (HU29), low plant density yielded 20 individuals along the 25-m transect. Using sterile technique, plants were manually pulled and each individual sampled using modified versions of protocols by Lundberg et al.  and Lebe2PO4, 61.6 mM Na2HPO4, 0.1% Tween 20). Tubes were shaken by hand for 1 min (timed). Leaf and root samples were then removed and stored on ice in separate tubes  until further processing. Wash samples were stored on ice during transport and then refrigerated at 4°C. Phyllosphere and ectorhizosphere washes were pooled across all (20 or 25) plants at a location and then centrifuged at 2,200 × g at 4°C for 15 min. Supernatants were discarded, and pellets were air-dried and stored at −20°C until DNA extraction.One to three basal nonsenescent leaves were collected from each plant, as well as the upper 2 to 5 cm of the taproot, together with accompanying lateral roots (excess soil was brushed or shaken off). Leaf and root samples were placed in individual 50-ml tubes containing 25 ml of sterile wash solution  for 2 min. Leaves were then rinsed in distilled water, patted dry using a Kimwipe, and sealed in individual sterile surgical envelopes (Fisherbrand 01-812-50). Envelopes were kept in silica gel desiccant until leaf tissue was completely dry and then stored at room temperature until DNA extraction.Roots were further washed by shaking in 10 ml of wash solution until visible residual soil was removed. Washed roots were stored and dried as described above for leaves.At each collection site, a tube of sterile wash solution was left uncapped while sampling plants. Disinfected tools were periodically agitated in the blank wash tube before sterilization and use for the next sample collection. For each population, rinse water and wipes used to process tissue samples were represented in controls by rinsing and wiping flame-sterilized forceps and then agitating the forceps in the blank wash tube. Controls were stored and processed in the same manner as for phyllosphere and ectorhizosphere samples.2) and root tissue  were collected from each individual sample per location and pooled prior to extraction. Control (blank) samples were collected for each batch of extractions by swabbing tools and surfaces and then extracting DNA from the swab head.Extractions were carried out using sterile technique in a laminar flow hood. For pooled tissue extractions, equal sections of leaf tissue . Phyllosphere and ectorhizosphere DNA was extracted from up to 0.25 g of wash pellets according to the standard kit protocol. Leaf and root tissues were ground to powder or sawdust consistency in liquid nitrogen using sterile mortars and pestles. Leaf and root DNA was extracted from 20 mg (leaf) or 100 mg (root) of ground tissue with the following modification to the standard protocol: tissue was incubated at 65°C for 10 min in extraction buffer and then vortexed for 1 min, followed by a second 10-min incubation . Control DNA was extracted by placing whole swab heads directly into extraction tubes. Extracted DNA was eluted in PCR-grade water and stored at −20°C pending library preparation.To remove secondary compounds inhibiting PCR, DNA extracted from root and leaf tissue (together with corresponding blanks) was purified using a ZR-96 genomic DNA cleanup kit (Zymo Research). All DNA concentrations were quantified using a Qubit fluorometer high-sensitivity assay for double-stranded DNA (Invitrogen) and standardized to equimolar amounts.Library preparation followed a dual-index approach  using a Centaurea diffusa, Helianthus annuus, and Lactuca sativa). We found a single nucleotide mismatch between the Asteraceae chloroplast 16S and the plastid PNA sequences, and designed an alternative plastid PNA specific to the Asteraceae sequence (5′-GGCTCAACTCTGGACAG-3′) (Peptide nucleic acid clamps (PNAs) were included in both PCR steps of library preparation to block amplification of plant chloroplast and mitochondrial 16S as recommended by Lundberg et al. . Clamp sACAG-3′) . All samACAG-3′) . To gaugACAG-3′) .https://github.com/lh3/seqtk) and a custom script. The UPARSE pipeline  for samples from native and invaded ranges within each plant compartment. Prior to performing the PCA, we performed Hellinger’s transformation to minimize the influence of OTUs with low counts or many zeros –114. We –H′ , evenness (Pielou’s J ), and thH′  of the SH′  and compPRJNA494717. Sampling locations and herbarium accession numbers of plant specimens are given in Sequence data are available under NCBI SRA accession number