Factor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS.To investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG).In plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers.p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients.Higher FXII:Ag levels (In MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression. Multiple sclerosis (MS) is a chronic autoimmune disorder, characterized by immune-mediated inflammation and multifocal demyelinated lesions within the central nervous system (CNS) , 2. GrowAmong coagulation factors, a key role seems accomplished by factor XII (FXII), the initiator of the “old” contact (intrinsic) coagulation pathway , which cRecently, histological analysis of CNS tissue from MS patients identified FXII light chain depositions nearby dendritic cells (DCs) . The basIn EAE, FXII depletion had protective effects, reducing susceptibility to CNS inflammation, delaying disease onset, decreasing disease severity and production of T helper 17 (Th17) cells . In miceper se” is not involved in MS. Nevertheless, the FXII levels in plasma are usually assessed by a procoagulant assay (FXII:c). In fact, FXII:c has been found significantly increased in patients with relapsing-remitting MS (RR-MS) and secondary progressive MS (SP-MS) compared with healthy donors. Additionally, enhanced FXII:c was associated with relapses and shorter relapse-free period, independently from immune-modulatory therapy  .Inclusion and exclusion criteria for RAGTIME study were previously reported . The selThe healthy subjects (HS) group was represented by healthy volunteers, who were never diagnosed with MS, neurological disorder, other chronic inflammatory disease, and cardiovascular disease.p < 0.001, Student’s t-test, Table p = 0.138, Fisher’s exact test). The total number of patients under disease-modifying treatments (DMTs) at blood sampling was 12 (5 patients under DMTs and 7 patients under both DMTs and symptomatic treatments), as detailed in Table All subjects were of Caucasian origin. Patients were not under treatment with anticoagulant drugs. Written informed consent was obtained from all subjects, and the study was approved by the Ethical Committee of the S. Anna University-Hospital, Ferrara, Italy. The demographic and clinical characteristics of the study populations, which included 74 MS patients  and 49 HS, are summarized in Table g for 15 min and 11,000 g for 5 min). Aliquots were stored at −80°C until use.Venous peripheral blood samples from both MS patients and HS were collected into sodium citrate tubes. Patients enrolled in the RAGTIME study provided blood sampling at four time point: (T0) baseline point, prior to the first rehabilitative session; (T1) intermediate point, after six training sessions; (T2) end of treatment, 12 completed rehabilitative sessions, 1 month after T0; (T3) follow-up, after 3 months from the end of training program. Plasma samples were obtained after two consecutive centrifugations of blood samples, at room temperature  in plasma samples was assessed by an activated partial thromboplastin time (aPTT)-based assay . Activity and coagulation times were recorded by the ACLTOP 700 instrument . The inter-assay coefficients of variation assessed over multiple runs was 2.1%.Plasma FXII antigen (FXII:Ag) concentrations were determined using a sandwich enzyme-linked immunosorbent assay kit , following the manufacturer’s instructions. The assay uses a polyclonal capture antibody for FXII and a mouse primary monoclonal antibody raised against the heavy chain of FXII as detection antibody. The plasma samples were tested with a dilution of 1:3,000. The results were expressed as relative units in percentage generated from concentration values normalized to a pool of normal plasma loaded in all plates. The inter-assay coefficient of variation for plasma measurements was 2.6%.2 and thrombin fluorogenic substrate were 2.5 mM and 250 µM, respectively. The fluorescence was measured overtime in a fluorometer (Fluoroskan Ascent BioMed) and the amount of the generated thrombin was calculated using a normal pooled human plasma (Hyphen BioMed) as a standard. As negative control of contact activation, the FXII inhibitor “corn trypsin inhibitor” was added to the normal pooled plasma in each assay condition (single/double trigger).Thrombin generation in plasma samples was evaluated by the addition of a specific thrombin fluorogenic substrate  , 27. PlaSpecific parameters of TG-lag time, time to peak (TTP), peak height, and endogenous thrombin potential (ETP) (area under the curve) were obtained by a nonlinear regression analysis of the first derivative of relative fluorescence units using the software version 6.01 .® SPSS® Statistics version 24 software  and figures were produced by GraphPad Prism version 6.01 .All statistical analyses were performed using IBMt-test was used to compare age between total MS and HI groups.The Shapiro–Wilk test was used to test for normality of continuous variables. The Fisher’s exact test was used to compare differences in categorical variables and Student’s p-value, pairwise comparisons were Bonferroni corrected for multiple testing .Comparisons of MS vs. HS and of males vs. females of FXII:c, FXII:Ag, and FXII:c/FXII:Ag ratio (FXII:ratio) were conducted with the ANCOVA test using age as covariate. Comparisons for FXII levels among clinical subgroups were performed with ANCOVA test using age as covariate and, in case of a significant p-value, pairwise comparisons were Bonferroni corrected for multiple testing .To assess whether FXII levels were significantly different among patients receiving DMTs, symptomatic treatments, or none current treatment, one-way ANOVA was used and, in case of a significant p-value, pairwise comparisons were Bonferroni corrected . Student’s t-test was used to compare TG parameters of MS patients with those of HS, while paired Student’s t-test was used to assess differences in TG after NA addition in MS and HS.Pearson’s test was used to assess correlation over time for FXII:c and FXII:Ag. ANOVA for repeated measures was used to test FXII:c, FXII:Ag, and FXII:ratio across the four time points and, in case of a significant p = 0.003) and FXII:ratio (p < 0.001) but not in FXII:c (p = 0.421). No differences within clinical subgroups  were detected for FXII:c (p = 0.296), FXII:Ag (p = 0.248), and FXII:ratio (p = 0.765). Comparison between male and female within MS and HS groups, after age adjustment, did not reveal difference in FXII:c , FXII:Ag , and showed a trend in difference in FXII:ratio in HS (p = 0.045), but not in MS (p = 0.11).Factor XII coagulant activity (FXII:c), FXII protein concentration (FXII:Ag), and their ratio, providing quantitative information about the FXII activity in relation to the amount of circulating protein, were evaluated in plasma of MS patients and of HS and summarized in Table p = 0.98), FXII:Ag (p = 0.81), and FXII:ratio (p = 0.97).The potential modulation of FXII levels by treatments was investigated. Patients under both DMTs and symptomatic treatments Table  were catA large portion of patients under study were characterized by a small range of EDSS (6–6.5), which does not favor the investigation of the relation between EDSS and FXII levels.p = 0.031, Table p = 0.004; q = 0.023) and T0–T3 . The potential influence on FXII:c over time variations of MS phenotype or drug treatments was investigated. Differences were not detected within each clinical MS group , as well as within drug treatment groups .Factor XII:c, FXII:Ag, and FXII:ratio levels were also investigated over four time points Table  in 49 MSp = 0.596) and FXII:ratio (p = 0.151) (Table No differences over time were detected for FXII:Ag (1) Table .r2 = 0.90; T1–T2, r2 = 0.82; T2–T3, r2 = 0.75; p < 0.001) as well as FXII:Ag levels .Noteworthy, analysis of correlations among time points for each FXII parameter Table  showed tThe decreased FXII:ratio values in MS patients prompted us to investigate potential variation of FXII specific activity through a global plasma assay (TG), which describes all phases of coagulation process and the integrated amount of generated thrombin . In partTo magnify FXII-related differences in TG, 10 patients’ plasma, obtained at T0 , were selected for the lowest FXII:ratio (≤0.93), virtually undetectable in HS, and compared with 10 HS plasma with the highest FXII:ratio (≥1.4), which on the other hand was rare in MS patients.p = 0.042).Thrombin generation curves and parameters are reported in Figure p = 0.006; 750 ± 109 vs. 706 ± 91, p = 0.014, respectively) and in HS . After the double induction, the increase in thrombin peak height and ETP differed between patients and HS. Particularly, the ETP value increased only in MS patients , whereas the peak height was significantly increased only in HS .The TG triggered, in the same experiment, with the addition of NA, produced a clear decrease in main time parameters both in MS patients and in HS. As compared with the single ellagic acid trigger, both lag time and TTP were shorter in MS patients  and TTP was around 100 s longer . To note, three out of four PP-MS patients showed the most prolonged time parameters . Aimed at improving knowledge about the FXII role in the disease, we provided quantitative information about the FXII procoagulant activity in relation to the amount of circulating protein, by evaluating their ratio. This analysis indicated a significantly lower FXII:ratio in MS patients. This novel finding prompted us to investigate in selected groups of MS patients and HS the intrinsic pathway by TG, which provides kinetic information and potentially mechanistic interpretation of differences. Coherently with the decreased FXII:ratio, we report in MS patients a trend of lower amounts of thrombin potential, ETP, a stable and highly affordable parameter.We introduced in this study a modified TG assay with double activation, obtained by the addition of NA, which has been recognized among true physiological activators of the contact pathway , 32.Interestingly, NAs released from dead and dying cells may induce an autoimmune response by activating specific sensing receptors , thus reAdditional trigger by NA shortened time parameters less in MS patients as compared with HS. Overall, the lower FXII:ratio and longer TG time parameters suggested that in part of MS patients (i) FXII could be less active per antigen unit and (ii) FXII response to contact activation and its support to the intrinsic coagulation pathway could be reduced. Interestingly, it has been recently reported that in TG, triggered by extrinsic activation, time parameters were shorter in MS patients , which dIn light of the increased FXII protein levels and decreased activation that we report, pharmacological inhibition of FXII, proposed as a potentially new approach to MS treatment, needs deep investigation.The low number of patients under DMTs and the extremely heterogeneous DMTs did not permit us a productive analysis of FXII levels in relation to DMTs. Nevertheless, these study features enabled us to obtain FXII-related values reasonably independent from drugs, like interferon that is known to heavily influence gene expression in several tissues. These values could better reflect the “biological” relation between FXII and (untreated) disease. On the other hand, the investigation of DMTs effects on FXII-related variables in a properly designed study would provide a comprehensive picture of this poorly defined field.In conclusion, our study points toward FXII-related differences between MS patients and HS, with the limitation of the small sample size. Multiple specific and global coagulation assays could help stratification of patients to better define FXII contribution to disease phenotype and progression.This study was approved by the Ethics Committee of Ferrara province with approval number 101-2012. Written informed consent was obtained from all participants.NZ, MB, GM, and FB conceived the study design and wrote the manuscript; NZ, RV, and PS collected plasma samples and evaluated pre analytical variables; NZ and MB set up ELISA and aPTT; NZ performed ELISA, aPTT, and analyzed data; RM set up and performed aPTT; MB performed thrombin generation and analyzed data; SS, FM, PZ, and NB designed and supervised the rehabilitation study, recruited patients, and performed their clinical evaluation; SS and EM collected and analyzed instrumental and clinical data for patients classification. All authors critically evaluated the manuscript.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.


Clostridium difficile infection (CDI) is the leading cause of infectious nosocomial diarrhoea but the economic costs of CDI on healthcare systems in the US remain uncertain.We conducted a systematic search for published studies investigating the direct medical cost associated with CDI hospital management in the past 10 years (2005–2015) and included 42 studies to the final data analysis to estimate the financial impact of CDI in the US. We also conducted a meta-analysis of all costs using Monte Carlo simulation.The average cost for CDI case management and average CDI-attributable costs per case were $42,316  and $21,448  in 2015 US dollars. Hospital-onset CDI-attributable cost per case was $34,157 , which was 1.5 times the cost of community-onset CDI . The average and incremental length of stay (LOS) for CDI inpatient treatment were 11.1 (90 % CI: 8.7–13.6) and 9.7 (90 % CI: 9.6–9.8) days respectively. Total annual CDI-attributable cost in the US is estimated US$6.3 (Range: $1.9–$7.0) billion. Total annual CDI hospital management required nearly 2.4 million days of inpatient stay.This review indicates that CDI places a significant financial burden on the US healthcare system. This review adds strong evidence to aid policy-making on adequate resource allocation to CDI prevention and treatment in the US. Future studies should focus on recurrent CDI, CDI in long-term care facilities and persons with comorbidities and indirect cost from a societal perspective. Health-economic studies for CDI preventive intervention are needed.The online version of this article (doi:10.1186/s12879-016-1786-6) contains supplementary material, which is available to authorized users. Clostridium difficile is the leading cause of infectious nosocomial diarrhoea in the United States (US) [C. difficile infection (CDI) are increasing [tes (US)  and the creasing . CDI is creasing –5. Up tocreasing –8, patiecreasing , 10 or gcreasing , 11, patcreasing , patientCDI represents a significant economic burden on US healthcare systems. Infected patients have an increased length of hospital stay compared to uninfected patients, besides there are significant costs associated with treating recurrent infections. A few systematic reviews of cost-of-illness studies on CDI cost are available –21. ThesThe aim of the current study is to conduct a systematic review and meta-analysis of currently available data to identify and quantify the financial burden attributable to CDI, and to further estimate the total economic burden of CDI hospital management in the US.English-language databases with online search tools were searched for to offer maximum coverage of the relevant literature: Medline (via the Ovid interface 1946 to July 2015); EMBASE (via the Ovid interface 1980 to July 2015); The Centre for Review and Dissemination Library ; The Cochrane Library (via the Wiley Online Library) and Health Technology Assessment Database (1989 to July 2015).We supplemented our data by searching relevant published reports from: National epidemiological agencies, Google search for grey literature and hand searched the reference lists of the included studies. The general search headings identified were: Clostridium difficile, economic, costs, cost analysis, health care costs, length of stay, hospitalization. Examples of the strategy for Medline and EMBASE are listed in Additional file All studies that reported novel direct medical cost and/or indirect costs related to CDI management were included. Review articles, comments, editorials, letters, studies of outbreaks, case reports, posters and articles reporting results from economic modelling of a single treatment measure  were excluded in the final analysis. All relevant publications from January 2005 to July 2015 were included in the search. We included the following healthcare settings: hospitals, long-term care facilities and community. Geographical scope covered the US. We did not apply any language restriction. Our predefined inclusion and exclusion criteria are shown in Additional file Two reviewers  independently selected the included articles and extracted data. After combining their results, any discrepancies were solved by discussion with HN and MK.The primary outcomes were CDI-related costs  and CDI-attributable costs . For studies with control groups (e.g. matched patients without CDI), the CDI-attributable cost extracted was either the cost provided by the articles or calculated by reviewers using the CDI-related cost minus the treatment cost of control groups. The secondary outcome was resource utilization associated with CDI, i.e. CDI-related length of stay (LOS) in hospital and CDI-attributable LOS. The study characteristics of each article were extracted. These included basic publication information, study design, statistical methods, economic data reporting characteristics and population information.Matched data > Unmatched data.Adjusted model results > Unadjusted model results.Regression model results > Calculated difference.Total cost/charges > Subgroup cost/charge .Median (Interquantile Range: IQR) > Mean .When multiple cost data were presented in a study, we included only one cost estimate for each population subgroup as per the priority below:All costs/charges data were inflated to 2015 US$ equivalent prices adjusted for the Consumer Price Index. If the price year was not reported, it was assumed to be the last year of the data collection period. In cases where charges were reported without cost-to-charge given, costs were estimated using a cost-to-charge ratio of 0.60, which is commonly used value in US health economic studies .We carried out meta-analysis for cost studies following a Monte Carlo simulation approach, as reported by Jha et al  and ZimlWe estimated the national financial impact of CDI on the US healthcare system, by determining the potential boundaries. The higher boundary was the total number of CDI cases in the US in 2011 extracted from Lessa et al , while tWe extracted the total number of CDI patients and CDI-attributable costs from previous studies  and reviThe quality of the studies included was assessed mainly based on the complexity of the statistical method Fig. . All stuThe search strategy identified 2671 references from databases. Seven additional references were identified through other sources. After screening the titles, abstracts and relevant full texts Fig. , a totaln = 27) used national level databases, with 17 used National Independent Sample (NIS) database and the remaining 10 studies extracted data from various national databases. Fifteen studies were conducted at state level, of which 6 studies only collected data in single hospital. All studies reported cost in hospital level of care, no articles identified in LTCF and community. Nearly all identified references were retrospective hospital database studies (n = 40) and only 1 study was a prospective observational study [The characteristics of the 42 included studies –68 are sal study  and anotal study .Table 1On = 15) investigated economic outcomes in all age inpatients. Three studies reported cost data in children less than 20 years old. The mean/median age of the CDI patient groups ranged from 47.4 to 73.0 years. Other studies investigated complicated CDI in high-risk patient groups, such as those with major surgery (n = 16), inflammatory bowel diseases (n = 2), liver or renal disease (n = 4), elderly (n = 2) and ICU patients (n = 1). There was 1 study each in non-surgical inpatients, sepsis inpatients and patients with prolonged acute mechanical ventilation. There was 1 study focusing only on recurrent CDI in the general population.Most studies  from administrative datasets using the International Classifications of diseases, Ninth, Clinical Modification, ICD-9-CM 008.45. Clinical diagnosis was also used in two studies.The methods to identify CDI varied according to the type of CDI that was assessed in the study. CDI cases were identified either with laboratory test, i.e. positive n = 23) or both CDI types (n = 17). Only four studies investigated CO-CDI only. However, subgroup data of CO-CDI is also available in studies that reported both CDI types.CDI was classified in three types: Community-onset CDI (CO-CDI) requiring hospitalization, Hospital-onset CDI (HO-CDI) complicating other diseases, or both CDI Table . Most ofThe mean CDI-attributable costs per case of CO-CDI were $20,085 , lower than HO-CDI $34,149 . HO-CDI showed a wider range within which the additional cost for CDI in the general population ranged from $6893 to $90,202 and in high risk groups ranged from $7332 in congestive heart failure patients to $122,318 in renal impairment patients. The mean CDI-attributable LOS was 5.7 days (Range: 2.1–33.4) for CO-CDI, 7.8 (Range:2.3–21.6) days for HO-CDI, and 13.6 (Range: 2.2–16) days for both groups. Cost data and LOS for individual studies are presented in Tables Using a Monte Carlo simulation, we generated point estimates and 90 % CI for both cost and LOS; the meta-analysis results are shown in Table Similar patterns were observed in LOS data. The total CDI-related LOS was 11.1 days (90 % CI: 8.7–13.6) and CDI-attributable LOS was 9.7 (90 % CI: 9.6–9.8). The HO-CDI patients had longer CDI-attributable LOS 9.7 days (90 % CI: 9.7–9.7) than CO-CDI patients 5.7 days (90 % CI: 4.1–7.3).The total burden of healthcare facility CDI in US was estimated 293,300  cases per year . The totA summary of the quality assessment for statistical methods in included studies is shown in Additional file We systematically reviewed 42 published cost studies of CDI case management in the past 10 years (2005–2015) and found a significant financial burden associated with CDI in the US. The total CDI-attributable cost was US$6.3 billion, which is higher than previously reported (range US$1.1–4.8 billion) , 16, 17.This review facilitated a meta-analysis of a large number of cost studies for costs related to CDI management and provided an uncertainty range. Zimlichman et al  applied Furthermore, we evaluated hospital onset CDI and community onset CDI separately. We found that CDI complicating hospitalization cost more than CDI requiring hospitalization and the former had longer attributable hospital stay. Therefore, other factors, such as comorbidity, may contribute to infections and increase the difficulty of CDI treatment.We estimated that the total cost attributable to CDI management in the US was nearly US$6.3 (Range: $1.9–$7.0) billion, which is similar to Dubberke and Olsen’s estimates at $4.8 billion , but sigIn this study, we only assessed the quality of study emphasizing statistical methods and did not use the modified economic evaluation guideline as other COI systematic reviews. Cost and LOS estimation of healthcare-associated infections has the potential to be misleading if the confounders such as patients’ comorbidities or daily severity of illness were not properly controlled for. Using either the matching design or multivariable regression analysis allows to control known confounders and may, in part, address selection bias . We founOur systematic review has some limitations. First, all included studies reported direct medical costs from hospital perspective, therefore indirect cost to patients and society and costs of additional care after hospital discharge, have not been captured. No studies reported indirect cost (productivity loss due to work day losses) of patients or care-givers, and we failed to identify studies assessing cost of CDI in long-term care facilities, where about 9 % of CDI patients were discharged to for an average of 24 days of after-care. This would result in an additional US$141 million burden on the healthcare system and society due to LTCF transfers [Effective prevention can reduce the burden of diseases. Strategies have been promoted such as appropriate use of antimicrobials, use of contact precautions and protective personal equipment to care for infected patients, effective cleaning and disinfection of equipment and the environment, and early recognition of disease as primary prophylaxis . As CDI More cost of illness studies for recurrent CDI, or in LTCF, and indirect cost from a societal perspective are needed in the future. We would also recommend that published studies report their methods and include point estimates with uncertainty range. Further economic studies for CDI preventive interventions are needed.This review indicates that CDI places a significant financial burden on the US healthcare system. In addition, our findings suggest that the economic burden of CDI is greater than previously reported in the US. This review provides strong evidence to aid policy-making on adequate resource allocation to CDI prevention and treatment in US.


Neisseria gonorrhoeae, particularly to third-generation cephalosporins, has been reported in many countries. We examined the susceptibility  of 434 N. gonorrhoeae isolates collected from 107 female and 327 male patients in Stuttgart, south-west Germany, between 2004 and 2015. During the study period, high proportions of isolates were resistant to ciprofloxacin (70.3%), tetracycline  and penicillin (25.6%). The proportion of isolates resistant to azithromycin was low (5.5%) but tended to increase (p = 0.08). No resistance and stable minimum inhibitory concentrations were found for cefixime, ceftriaxone, and spectinomycin. High-level resistance was found for ciprofloxacin (39.6%) and tetracycline (20.0%) but not for azithromycin; 16.3% of the isolates produced betalactamase. Thus, cephalosporins can still be used for the treatment of gonorrhoea in the study area. To avoid further increasing resistance to azithromycin, its usage should be limited to patients allergic to cephalosporins, or  to patients for whom no susceptibility testing could be performed or those co-infected with chlamydiae. Increasing antimicrobial resistance of  Neisseria gonorrhoeae, have been reported in many parts of the world including Europe, even against the third-generation cephalosporins, cefixime and ceftriaxone [Numbers of gonorrhoea cases have increased, and the World Health Organization has estimated 106 million new cases in adults worldwide for 2008, which was 21% higher than numbers for 2005 [triaxone . For thitriaxone ,5.N. gonorrhoeae, as part of the European Surveillance of Sexually Transmitted Infections Project [The European Gonococcal Antimicrobial Surveillance Programme (EURO-GASP) was established by 12 European countries in 2004 in response to the emerging antimicrobial resistance of  Project . In 2016 Project ,8, but tN. gonorrhoeae isolates are not subject to reporting in Germany, data regarding current antimicrobial susceptibility and its development over time are scarce. Only three individual studies have addressed this issue in the past 10 years. Abraham et al. analysed 50 isolates collected between 2001 and 2010 in Dresden, Saxony [N. gonorrhoeae isolates collected in 2004/2005 in southern Germany have been reported [N. gonorrhoeae isolates. Data from EURO-GASP, however, have provided evidence for the presence of cephalosporin-resistant N. gonorrhoeae isolates in Germany, too [N. gonorrhoeae isolate acquired his infection in Munich, south Germany [As cases of gonorrhoea or antimicrobial resistance patterns of , Saxony , while Hreported . None ofany, too ,8; in fa Germany .N. gonorrhoeae in Germany and elucidate possible changes in antimicrobial resistance occurring over time, we analysed the susceptibility patterns of all N. gonorrhoeae isolates identified and tested in our laboratory between 2004 and 2015 (n = 434). Since age and sex have been identified as risk factors for harbouring antimicrobial-resistant N. gonorrhoeae isolates [To gain more information on the antimicrobial susceptibility of isolates ,14, we aN. gonorrhoeae is not mandatorily notifiable, the number of isolates tested may correspond to 30–70% of all isolates detected in that area within the period of time indicated. The samples were mainly submitted by private practitioners . For 284 (65.4%) patients, further samples (swabs or urine) had been submitted for the detection of Chlamydia trachomatis by PCR. Based on these laboratory results, 36 (12.7%) patients were co-infected with C. trachomatis while 248 (87.3%) were C. trachomatis-negative.Between July 2004 and March 2015, 434 bacterial isolates were grown from swabs obtained from patients aged 16 years and over living in the greater Stuttgart area (a radius of around 50 km from Stuttgart city centre). Although difficult to estimate because N. gonorrhoeae by using the API or the Vitek 2 system . Isolates were subjected immediately to antimicrobial susceptibility testing and subsequently stored at –70 °C for future analyses.Gram-negative, oxidase-positive diplococci were identified biochemically as 2 for 20–24 hours. Betalactamase production was assessed by using nitrocefin disks (BD Diagnostic Systems). The ATCC N. gonorrhoeae strain 49226 was used as an internal control. Susceptibility was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints [Briefly, bacterial inoculums were prepared in sterile saline at 0.5 McFarland standard and tested against the antibiotics indicated by using the Etest system  on Mueller-Hinton chocolate agar, except for testing of azithromycin, for which a chocolate GC II agar with IsoVitaleX was used  . Plates akpoints ,16.N. gonorhoeae isolates over time, data also were analysed for two different periods: July 2004 to December 2009 and January 2010 to March 2015, as well as for two-year periods. Differences were considered statistically significant at p < 0.05. Trends were defined as p-values between 0.05 and 0.1.Data were statistically analysed by using GraphPad Prism version 6.0a for Mac OS X. Data on antibiotic resistance, patients’ sex, or specialisation of referring practitioners were analysed using the two-tailed Fisher’s exact test. Data regarding age were analysed using the Mann–Whitney U-test. To elucidate potential changes in the antibiotic susceptibility in The median age of the patients was 33 years (range: 16–76), 107 (24.7%) were female and 327 (75.3%) were male. Analysis of antimicrobial susceptibility of the isolates to standard antibiotics revealed high proportions of intermediate or resistant isolates for penicillin (90.6%), ciprofloxacin (70.5%), and tetracycline (68.9%) with lower proportions for azithromycin 26.5%; . No resiHigh-level plasmid-mediated resistance to tetracycline (≥ 16 mg/L) was seen in 87 (20.0%) isolates whereas 172 (39.6%) isolates expressed high-level resistance to ciprofloxacin (≥ 4 mg/L). Seventy of 430 (16.3%) for which data on betalactamase production was available were betalactamase positive and thus expressed high-level plasmid-mediated resistance to penicillin. High-level resistance to azithromycin (≥ 256 mg/L) was not detected.N. gonorrhoeae isolates from female patients (n = 107) were more resistant to penicillin  and ciprofloxacin  than isolates from male patients (n = 327). There was no difference regarding resistance to azithromycin or tetracycline (data not shown). Patients older than 25 years (n = 317) and those aged 25 years or younger (n = 117) did not differ regarding resistance to penicillin (p = 0.536), azithromycin (p = 0.481), ciprofloxacin (p = 0.478) or tetracycline (p = 0.450).Demographic data of the two subpopulations, patients whose isolates were analysed in 2004–2009 and those whose isolates were analysed 2010–2015, were comparable . ApplyinAnalysis of the individual MICs of the seven antibiotics tested revealed some minor changes for most antibiotics . In contN. gonorrhoeae isolates within the study period, cefixime resistance has been reported for many European countries including Germany [Although we were unable to detect cephalosporin-resistant  Germany . Thus, o Germany ,10, cann Germany . In factN. gonorrhoeae isolates with this or lower MICs have been described as the cause of infections that did not resolve under cefixime standard therapy [The EUCAST breakpoint of cefixime may be too high, as  therapy . ConsideN. gonorrhoeae isolates and a concomitant decline in isolates expressing decreased susceptibility have been shown for Scotland from 2004 to 2007 [We detected azithromycin resistance in 5.5% of the isolates and none of the isolates expressed high-level resistance; both findings correspond to the data recently published by Horn et al. . In cont to 2007  from whe to 2007 . Only si to 2007 , Sweden  to 2007  and Irel to 2007 . To prevN. gonorrhoeae has been shown to decline after cessation of ciprofloxacin usage [N. gonorrhoeae isolates from Japan [The highest resistance rate was observed for ciprofloxacin (70.3%). This is in accordance with the data published by Horn et al.  while rein usage . It remaom Japan .Tetracycline resistance also was high (as previously observed by others ), and inWe did not detect spectinomycin resistance. This might be due to its long-lasting non-usage, since its usage leads to increasing resistance rates . Thus, tN. gonorrhoeae isolates from MSM are more resistant to antimicrobials than those collected from men who have sex with women [N. gonorrhoeae isolates than other patients [N. gonorrhoeae of MSM for the period 2009–2011, and highest MICs for isolates from men who have sex with women [In contrast to previous publications ,14, we dth women ,33. Likepatients ,34. In cth women . FurtherN. gonorrhoeae isolates in south-west Germany may be less dramatic than in other parts of Germany or other European countries. High resistance rates to some antimicrobials and changes of susceptibility over time, however, call for a more stringent monitoring system, e.g. the obligation of every laboratory to report all N. gonorrhoeae isolates together with their susceptibility testing results.In conclusion, the current resistance situation of


Muskoxen are increasingly exposed to multiple stressors that may impact their health and fitness. We measured stress hormones in their qiviut (wooly undercoat), and found differences across seasons, years and between sexes. Qiviut cortisol is a promising tool for guiding muskox conservation in a rapidly changing Arctic. Ovibos moschatus) are increasingly subject to multiple new stressors associated with unprecedented climate change and increased anthropogenic activities across much of their range. Hair may provide a measurement of stress hormones (glucocorticoids) over periods of weeks to months. We developed a reliable method to quantify cortisol in the qiviut (wooly undercoat) of muskoxen using liquid chromatography coupled to tandem mass spectrometry. We then applied this technique to determine the natural variability in qiviut cortisol levels among 150 wild muskoxen, and to assess differences between sexes, seasons and years of collection. Qiviut samples were collected from the rump of adult muskoxen by subsistence and sport hunters in seven different locations in Nunavut and the Northwest Territories between 2013 and 2016. Results showed a high inter-individual variability in qiviut cortisol concentrations, with levels ranging from 3.5 to 48.9 pg/mg (median 11.7 pg/mg). Qiviut cortisol levels were significantly higher in males than females, and varied seasonally , and by year (levels significantly increased from 2013 to 2015). These differences may reflect distinct environmental conditions and the diverse stressors experienced, as well as physiological and/or behavioural characteristics. Quantification of qiviut cortisol may serve as a valuable tool for monitoring health and informing conservation and management efforts.Muskoxen ( Ovibos moschatus), large herbivores that reside at Arctic and subarctic latitudes, may be especially vulnerable to ongoing changes in the Arctic  are increasingly recognized to be associated with chronic stress (chronic implying the stress occurs over long periods of time such as weeks to months), and may in turn lead to reduced health, fitness, and survival in free-ranging wildlife , chronicUrsus maritimus) (Passer domesticus) (Canis lupus) (Ursus thibetanus) (Ursus arctos) , and long guard hairs that are produced continuously over several years and form the characteristic ‘skirt’ of muskoxen  develop a reliable technique for cortisol quantification in the qiviut of muskoxen using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), (ii) determine the natural variability in qiviut cortisol among wild muskoxen and (iii) assess the relationship between qiviut cortisol levels and sex, season and year of collection.Adult muskoxen harvested by subsistence and sport hunters were sampled from January 2013 to August 2016 in the Canadian Arctic near the communities of Paulatuk, Sachs Harbour and Ulukhaktok in the Northwest Territories (NWT), Kugluktuk and Cambridge Bay in Nunavut (NU), and away from communities on the Kent Peninsula and Lady Franklin Point (NU) , individual subsistence and sport hunts , and the qiviut marketing pathway (Ulukhaktok). These are regular activities in the communities and no animals were culled specifically for our study. Samples were obtained under Animal Care and Use Permit #AC13-0121, the Wildlife Research Permit #2013-035, 2014-053, 2015-068 and 2016-058 for Nunavut, and the Wildlife Research Permit #WL500098, WL500158 and WL500257 for the Northwest Territories.The timing of sample collection was directly linked to the traditional muskox harvesting seasons and samples were classified to different seasons  based on their collection date. For the purposes of this study, samples collected between January and early April were considered winter samples, those collected late July through August were classified as summer, and those collected October through to mid-December as fall Figure . Qiviut Hunters were asked to record the gender of the harvested muskoxen on a provided form. When this information was left blank or marked ‘unknown’, a skin sample was tested to determine the gender of the animal using laboratory genetic analyses that were performed within the Kyle Laboratory at Trent University, Peterborough, Ontario, Canada. DNA from each sample was extracted using the Qiagen DNAeasy Blood & Tissue Kit  following the recommended protocol supplied by the manufacturer. Gender was determined by amplification of the SRY and ZFX genes using the primers SRY-Y53-3C, SRY-Y53-3D , ZFX-P2-n = 4). For the samples from Ulukhaktok, hides were dried under ambient environmental winter conditions in the community, and then shipped to Calgary, Alberta, Canada from where they were exported for qiviut processing. In Calgary, hide samples were collected from the rump area, as per the hunter protocol. All samples were stored at −20°C until laboratory processing.Hunters were directed to sample the qiviut when skinning the animals by cutting a piece of hide measuring approximately 10 × 10 cm from the rump, near, but lateral to the base of the tail. The location of sample collection was standardized as hair cortisol levels may vary depending on the body region .Qiviut cortisol was quantified using LC–MS/MS. The use of LC–MS/MS eliminates potential issues of antibody cross-reactivity that are associated with enzyme linked immunosorbent assays (ELISAs) and has become the preferred method of hair steroid analysis in human clinical research . It was The goal in sample preparation for LC–MS/MS is to provide as clean a steroid extraction as possible. For this reason, sample preparation differed from methods using ELISAs  added to 2 l of cold tap water and gently stirred for 3 min. The soap solution was then chilled on ice and 20 ml was added to each of the Falcon tubes containing the qiviut. The qiviut was washed by vortexing for 30 s, then the soapy water was decanted and the qiviut rinsed thoroughly with cold tap water, and patted dry in a clean paper towel. As a final rinse, the qiviut was next transferred to a new Falcon tube, and 20 ml of isopropyl alcohol (IPA) pre-chilled to −20°C, was added and mixed by gentle inversion for 10 s, then decanted to waste. Qiviut was air-dried in paper towel for at least 24 h before extraction.The qiviut samples were re-weighed before being placed in 13 × 100 mm borosilicate glass test tubes, and submerged under 6 ml of methanol pre-chilled to −20°C. A 100 μl spike of bio-identical, deuterated internal standard  in water/methanol  was added, with calibrators  and three quality control pools  spiked at the same time. Samples were extracted for 20 h at 4°C in upright tubes (i.e. no vortexing or spinning). Cold supernatant was pipetted off the hair, transferred into new culture tubes, dried at 40°C under nitrogen (Techne® Sample Concentrator), and then stored, capped, at 4°C until reconstitution.Each dry sample was reconstituted with 200 μl of water/methanol , and vortexed for 30 s. The total volume was transferred to a 600 μl microcentrifuge tube and centrifuged at 14 000 rpm for 20 min at 4°C. One hundred and fifty microlitres of supernatant were immediately transferred into LC autosampler vials for subsequent analysis.r2 = 0.94, P < 0.001), and there was no significant difference between the mean qiviut cortisol levels obtained with or without SPE .Solid phase extraction (SPE) was not used as, during method development, we found a high correlation between the qiviut cortisol levels from twelve paired samples, whose analysis differed only by the addition or not of a SPE step , DP = declustering potential , EP = entrance potential , CE = collision energy  and CXP = collision cell exit potential .All samples were analysed by using an Agilent 1200 binary liquid chromatography (LC) system coupled to an AB SCIEX QTRAP® 5500 tandem mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in positive mode. LC separation was performed on an Agilent Poroshell 120 C18 column  at 45°C. The mobile phase A was water/methanol  and the mobile phase B was methanol/IPA . The 8.5 min gradient was 20–40% B (0–1.0 min), 40–60% B (1.0–5.0 min), 60–100% B (5.0–5.5 min), 100% B (5.5–6.5 min), 100–20% B (6.5–7.0 min) and held at 20% B (7.0–8.5 min). The flow rate was 0.6 ml/min and the injection volume was 20 μl. Nitrogen was used as the source, nebulizer and collision gas . Mass resolution in Q1 and Q3 was set to unit resolution. Three transitions were monitored, the cortisol quantifier (–1), and qualifier (–2), and the deuterated cortisol IS quantifier transitions. Specific MRM (Multiple Reaction Monitoring) conditions were: x weighted linear regression) covered the range from 0.25 through 250 ng/ml with r2 ≥ 0.99 in each run. The intra-assay variability was on average 8.3% and the inter-assay variability was 13.5%. Each cortisol result was divided by qiviut mass to obtain pg/mg. Duplicate quantitation was repeated on a second run, with new hair samples, whenever the coefficient of variation (CV), calculated as (standard deviation/mean) × 100 was ≥15%. The average from the two new replicates was then used for statistical analyses.Cortisol peak integration was performed using Analyst 1.5.1 software (AB SCIEX). Sample quantitation used the area ratio between the analyte peak and the matched internal standard peak. Calibration curves , year , season of collection , and interactions between year and season, sex and season, and sex and year. In all models, we included a random effect for location of sampling to account for possible differences in qiviut cortisol among locations due to, for example, slight differences in hunter methodology or intrinsic differences in qiviut cortisol among locations linked to environmental  or other location specific conditions. Qiviut cortisol levels were log-transformed to satisfy the linear model assumptions.R2) for mixed-effects models were calculated using the MuMIn library  as the ratio of (sample size)/(number of parameters) was small . To avoi library .Table 2Qiviut was sampled from 150 adult muskoxen between January 2013 and August 2016  times higher in males than in females. Season also had a significant effect, with qiviut cortisol levels in fall and winter respectively 1.57 (95% CI = 1.26–1.95) and 1.70 (95% CI = 1.37–2.13) times higher on average than that in summer. Levels in fall and winter were not significantly different. Regarding differences between years, qiviut cortisol levels were on average 1.28 times (95% CI = 1.03–1.58), 1.67 (95% CI = 1.33–2.10) and 1.60 (95% CI = 1.21–2.10) times higher in 2014, 2015 and 2016, respectively, compared to 2013. Estimated qiviut cortisol values according to sex, season and year can be found in the σ2location = 0.0015) compared to the residual variation (σ2resid = 0.16), but the conditional modes for the levels of the random effect at each location suggest slight differences in cortisol among locations that are consistent with the local muskox population trends, with qiviut cortisol concentrations that tend to be higher in declining populations on Banks and Victoria islands . By contrast, in ELISAs, four-parameter logistic calibration curves will have quantitation errors outside of the linear portion of the curve. Therefore, when using LC–MS/MS, even when cortisol is present at a very low concentration, the detection of a signal corresponding to this hormone can be attributed to it with high confidence . HoweverWe found a high variability in qiviut cortisol levels among individual muskoxen, with levels ranging from 3.5 to 48.9 pg/mg, corresponding to an almost 14-fold variation. This wide range of values is consistent with other studies, where approximately 1.5 to 40-fold variations are reported depending on the study, number of animals included and species of interest Table  and our Particularly high qiviut cortisol concentrations, greater than 28 pg/mg, were measured in four individuals. Although all samples were washed the same way, the potential remains for cryptic blood to persist on the hair surface and be extracted. Since blood concentrations of cortisol are considerably higher than hair concentrations, blood contamination could substantially increase measured results. However, these exceptionally high ‘outlier’ hides were re-tested and similar results attained, suggesting that these are real values, and not a result of undetected, local blood contamination. Moreover, these results are consistent with other studies where individuals with higher cortisol levels are commonly identified  were significantly lower in the winter than in all the other seasons (Cervus canadensis) in South Dakota, USA, FGM concentrations were at their lowest in winter and peaked in the summer, conceivably due to increased human disturbance, high temperatures, or normal seasonal metabolic patterns (Cervus elaphus) in Austria, with a significant effect of snow and minimum ambient temperature (Loxodonta africana) presumably due to less nutritious and more sparsely distributed sources of food, and an increased competition between individuals (Equus quagga) and springboks  during the dry season in relation to decreased food and water availability . For example, faecal glucocorticoid metabolite (FGM) levels in bighorn sheep (Rangifer tarandus) and reindeer (R. tarandus) by significantly reducing their time spent foraging, and by augmenting their energy expenditure through increased movements and time spent standing  and lacstanding . Despitestanding . This maPudu puda) during the rutting season (Brachyteles arachnoids hypoxanthus) (Sapajus apella) . Muskoxen react to snowmobile activity  and in tW = 65, P = 0.98) , one of the muskox’s closest taxonomic relatives, described higher FGM levels in males (Bison bison) (Diceros bicornis) and white (Ceratotherium simum) rhinoceros , exposure to pathogens, or a combination of these and other factors that could have affected both long and shorter-term stress levels. During this time period, the average annual surface air temperatures in Arctic Tundra region of Canada from 2013 to 2015 were 1°C, 1.1°C and 1.3°C above the 1961–1990 reference period, with average summer air temperatures 0.8°C, 1.1°C and 1.2°C, above the reference period (data from Environment Canada). These warm temperatures, and other associated ecological changes, may in part be contributing to increasing stress levels.All the samples collected from 2013 to winter 2015 were analysed during the summer of 2015, whereas the samples collected onwards were analysed during the subsequent summer. Possible hormone degradation during storage may, therefore, have had a confounding effect on the yearly differences we observed. However, multiple studies, including a small pilot study by our team, have shown that cortisol is highly stable over long periods of time in hair at room temperature and in other matrices  at subzero temperatures (Chlorocebus pygerythrus) hair measured using four different immunoassays , DRG International ELISA , Salimetrics ELISA  and IBL luminescence immunoassay (LIA) ) with ones measured using LC–MS/MS (P < 0.0001), but hair cortisol concentrations were 2.5 to 20 times higher when measured using immunoassays than with LC–MS/MS. The Salimetrics ELISA and IBL LIA gave the closest results to LC–MS/MS with concentrations only approximately 2.5 times higher  to leach inside the hair shaft, thereby confounding our results . Its conTamias striatus) (Lynx canadensis) (Bos taurus) (Additional research is needed to provide evidence that qiviut cortisol levels reflect adrenal activity, which will enable us to link high qiviut cortisol levels with stress. ACTH challenges involving repeated weekly injections over several weeks have been used to validate that the measurement of cortisol in hair reflects adrenal changes in several species of domestic or captive wild animals, including eastern chipmunks (Finally, the determination of the qiviut growth cycle was based on the observation of captive animals in Saskatoon, Saskatchewan, Canada. The exact timing of the qiviut growth cycle in wild muskoxen in the Arctic remains unknown, but is unlikely to differ substantially as qiviut is shed at a similar time . Qiviut The Arctic ecosystem is currently experiencing one of the greatest rates of climate and ecological change in the world . These dSupplementary DataClick here for additional data file.


Determination of free testosterone, FSH, TNF-α, and IL1-β in plasma was performed. Plasma-free testosterone was significantly decreased, while plasma FSH, TNF-α, IL-1β, and testicular mitochondrial NAD+ were significantly increased in I/R group compared to control group. These parameters were reversed in Gingko biloba treated I/R group compared to I/R group. I/R caused marked testicular damage and increased APAF-1 in the apoptotic cells which were reversed by Ginkgo biloba treatment. It could be concluded that I/R caused subfertility induced by apoptosis and oxidative stress manifested by the elevated testicular mitochondrial NAD+, which is considered a new possible mechanism. Also, testicular injury could be reduced by Gingko biloba administration alone.Testicular torsion, a surgical emergency, could affect the endocrine and exocrine testicular functions. This study demonstrates histopathological and physiological effects of testicular ischemia/perfusion (I/R) injury and the possible protective effects of Ginkgo biloba treatment. Fifty adult male Wistar rats, 180–200 gm, were randomly divided into sham-operated, Gingko biloba supplemented, ischemia only, I/R, and Gingko biloba treated I/R groups. Overnight fasted rats were anaesthetized by Pentobarbital; I/R was performed by left testis 720° rotation in I/R and treated I/R groups. Orchiectomy was performed for histopathological studies and detection of mitochondrial NAD Testicular torsion, an abnormal twisting of spermatic cord or testicular rotation, represents a surgical emergency affecting mainly adolescents, probably following trauma to the scrotum, or may occur after strenuous exercise . It coulKoksal et al.  reportedMoreover, apoptosis, a physiologic process that entails the programmed cell death indicating tissue injury, is essential during normal spermatogenesis . HoweverLi et al.  mentioneLater, Kanter  reported+), and apoptotic changes of I/R on the testis and the possible protective effects of Ginkgo biloba extract regarding testicular structure and exocrine and endocrine functions after unilateral testicular torsion/detorsion.All previous studies in rat model of unilateral testes torsion/detorsion focused on the Ginkgo biloba extract effect on the antioxidant enzyme levels. The present work comprehensively investigated the morphometric parameters, mitochondrial function .Drugs. Ginkgo biloba was obtained from El Amriya Pharmaceutical Industries ; its concentration was 40 mg/ml.Fifty adult male Wistar rats, weighing 180–200 grams, were purchased from the Animal Farm, Helwan, Cairo, Egypt, and housed in animal cages (5 rats/cage) with suitable ventilation, temperature of 22–25°C, 12 hours light dark cycle, and free access to food and water, ad libitum, in the Animal House, Medical Research Unit, Faculty of Medicine, Ain Shams University. Rats were allowed to adapt to the new environment for 7 days prior to experimental procedures to decrease the possible discomfort. Unnecessary pain or stress was avoided and animal manipulation was performed with maximal care and hygiene. All rats received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared according to guidelines of animal use of the Ethical committee of Ain Shams University.Group I: main control group (n = 20) which were further subdivided intoGroup Ia: sham-operated group (negative control) (n = 10): they were exposed to the procedure of ischemia/reperfusion without induction of torsion/detorsion (the left testicular artery and vein were not occluded by clamping); then orchiectomy was performed.Group Ib: Ginkgo biloba supplemented group (positive control) (n = 10): they received Gingko biloba extract 50 mg/kg by gavage as a single dose  and 0.5 M ethanol), 100 μL of the tissue extract neutralized, and 20 μL of alcohol dehydrogenase. The reaction was initiated by enzyme addition and change of absorbance at 340 nm was recorded by a spectrophotometer.Mitochondria isolation was from testicular tissues which were stored frozen at −80°C till the day of NADA. Saleh . HydrolyDetermination of Free Testosterone Level in Plasma. Determination of free testosterone level in plasma was done by using ELISA kits supplied by ABCAM, Egypt, while plasma FSH level was determined by IRMA technique according to Santner et al. [r et al. . Determination of Plasma TNF-α. Plasma TNF-α was determined by using the RayBio® Rat TNF-α ELISA kit .Determination of Plasma IL1-β. Plasma IL1-β was determined by using ELISA kit (MABTECH® Rat), Egypt.(1) Hematoxylin and Eosin (H&E) Stain. Specimens from left testes of rats were immediately fixed into Bouin's fixative. Tissues were processed for preparation of paraffin blocks to get paraffin sections (5 μm in thickness), which were stained by conventional Hematoxylin and Eosin (H&E) stain [E) stain . The tes(2) Immunohistochemical Study (Apaf-1 Staining). The paraffin sections were used for detection of the Apoptosis protease-activating factor 1 (Apaf-1) expression. Immunostaining was performed with a Leica Bond-Max automatic immunostainer  [IL, USA) . Apaf-1,IL, USA) . When apIL, USA) . CellulaIL, USA) .(3) Preparation of Specimens for the Semithin Sections Toluidine Blue Stain. Testes were immediately cut into cubes (1 mm in diameter) and fixed overnight in 2.5% phosphate-buffered glutaraldehyde (pH 7.3) at 4°C. Fixation in 1% buffered osmium tetroxide for 1-2 h was followed by dehydration in ascending grades of ethyl alcohol and clearance in propylene oxide and finally embedded in fresh Epon capsules. Semithin sections, 1 μm in thickness, were cut with a glass knife and stained with toluidine blue and then examined by an Olympus light microscope [croscope . (4) Sperm Abnormality Test by Nigrosin and Eosin Stain. The sperm abnormality was studied as described by Wyrobek et al. [k et al.  as follo(5) Computerized Morphometric Analysis Quantitative Morphometric Measurements. Sections were examined by using Leica DM2500 microscope with built-in camera . All images were digitally acquired using an image analyser Leica Q win V.3 program  installed on a computer in Histology Department, Faculty of Medicine, Ain Shams University. Five different nonoverlapping fields from five different stained sections of five different rats were examined in each group for measuring each of the following:Mean seminiferous tubule diameter (MSTD) measured in micrometers.Number of the primary spermatocytes per cross section of a seminiferous tubule.Number of the rounded spermatids per cross section of a seminiferous tubule.The rounded spermatids/primary spermatocytes ratio.Number of the interstitial cells of Leydig cells per space.All measurements were performed at high power field of magnification (×400); all data were collected, revised, and subjected for statistical analysis.P ≤ 0.05.All results in the present study were expressed as mean ± SE of the mean. Statistical Package for the Social Sciences  program, version 20.0, was used to compare significance between each of the two groups. One-way ANOVA for difference between means of different groups was performed on results obtained in the study. Differences were considered significant when This study was approved by the Ethics Committee of Faculty of Medicine, Ain Shams University.P < 0.005, P < 0.01, resp.); however, it was nonsignificantly changed in both Ginkgo biloba treated I/R and Ginkgo biloba supplemented groups compared to negative control group. Compared to I/R group, Ginkgo biloba treated I/R group showed significant increase in plasma-free testosterone level , while it was insignificantly changed in ischemia only group.As shown in P < 0.02); however nonsignificant changes were observed in plasma FSH in ischemia only, Gingko biloba treated I/R, and Gingko biloba supplemented groups compared to the negative control group. Also, compared to I/R group, plasma FSH were insignificantly changed in ischemia only and Gingko biloba treated I/R groups, as shown in Plasma FSH level was significantly elevated in I/R group compared to negative control group ; however, insignificant changes were observed in mitochondrial NAD+ in Ginkgo biloba supplemented and Gingko biloba treated I/R groups compared to negative control group. In addition, it was significantly reduced in ischemia only group and Ginkgo biloba treated I/R group compared to I/R group , as shown in Oxidative stress was studied by assessment of mitochondrial NADα was significantly increased only in I/R group compared to the negative control group ; however, insignificant changes were present in ischemia only, Gingko biloba treated I/R, and Gingko biloba supplemented groups compared to the negative control group. Compared to I/R group, plasma TNF-α was significantly reduced in ischemia only and Gingko biloba treated I/R groups , as shown in β was significantly elevated in I/R group compared to the negative control group ; however, it was insignificantly changed in ischemia only, Gingko biloba treated I/R, and Gingko biloba supplemented groups compared to the negative control group. Plasma IL-1β was significantly decreased in ischemia only and Gingko biloba treated I/R groups compared to I/R group , as shown in Regarding inflammatory and apoptotic factors, plasma TNF-Histological examination of the testes of control rats (negative and Ginkgo biloba supplemented) groups (groups Ia and Ib) showed the same structure; therefore figures for the negative control group are representative for both.H&E and toluidine blue stained testicular sections of the control group showed normal pattern of testicular structure, enclosed by fibrous connective tissue capsule and fibrous septa dividing the gland into lobules composed of multiple rounded seminiferous tubules with regular outlines, lined by 4–6 layers of germinal epithelium at different stages of spermatogenesis. The flagella of mature sperms, which had whorly appearance, were observed filling the lumens of the seminiferous tubules. The interstitial spaces of the tubules contained Leydig cells and some blood capillaries. The lining epithelium consists of germinal and Sertoli cells, which appeared pyramidal in shape and resting on the basement membrane. The germinal epithelium consists of spermatogonia resting on the basement membrane, large primary spermatocytes, early or rounded spermatids with acrosomal caps, and late or elongated spermatids Figures  and 4.In ischemia only group, mild to moderate degeneration of the germinal epithelial lining of the seminiferous tubules was observed, in addition to interstitial edema, congestion of the interstitial blood vessels, and separation of the germinal epithelium with pyknotic nuclei of some Leydig cells that are embedded in acidophilic vacuolated exudates, some seminiferous tubules filled with an acidophilic exudates Figures  and 6.On the other hand, the I/R group showed moderate to severe degeneration and necrosis of the spermatogonia, as well as dead spermatids and interstitial edema. Within the tubules, desquamated epithelium, cellular debris, and fragments were, also, observed in addition to more prominent separation between germinative cells in the seminiferous tubules Figures  and 8. IApaf-1 immunohistochemical stained sections of the control group revealed weak reaction of spermatogenic cells , whereasThe control group showed normal sperm morphology with prominent hook shape head and single long tail ; in ischP < 0.001 for each) compared to the control group, while nonsignificant changes were observed in Ginkgo biloba supplemented and Ginkgo biloba treated I/R groups compared to control group. However, the MSTD was significantly increased in Ginkgo biloba treated I/R group compared to I/R group accompanied with insignificant change in ischemia only group when compared to I/R group, as shown in Regarding the mean seminiferous tubular diameter (MSTD) for testes, as shown in P < 0.002, <0.001, and <0.005, resp.). Although only the parameter of the mean of round spermatids number/HPF was significantly increased in ischemia only group compared to I/R group (P < 0.05), the remaining parameters of spermatogenesis were insignificantly changed in ischemia only group compared to I/R group, as shown in Regarding parameters of spermatogenesis such as primary spermatocytes/HPF, the mean number of round spermatids/HPF, and the mean round spermatids/primary spermatocytes ratio/HPF, they were all decreased in both ischemia only and I/R groups compared to control group (<0.001 for each), while there were nonsignificant changes in Ginkgo biloba supplemented and Ginkgo biloba treated I/R groups compared to the control group. However, they were significantly elevated in Ginkgo biloba treated I/R group compared to I/R group (P < 0.001 for each), whereas it was insignificantly changed in Ginkgo biloba supplemented and Ginkgo biloba treated I/R groups compared to the control group. However the mean number of Leydig cells/space was significantly increased in Ginkgo biloba treated I/R group compared to I/R group (P < 0.001).As shown in The ischemia/reperfusion was performed in this study by twisting the spermatic cord 720 degree, and a microvascular clamp was applied on it. Turner et al.  showed tIn the current study, the plasma-free testosterone level was reduced in ischemia only and I/R groups compared to control group. This is in accordance with Turner et al. , who fouDuring the ischemic phase of I/R process, a hypoxic condition resulted from disrupted blood flow to testicular tissues leading to reduced tissue ATP production and increased calcium influx into the intracellular compartment elevating superoxide generator enzyme, in addition to chemotactic factors stimulation, thereby, migrating polymorphonuclear leukocytes to the ischemic region, which could generate superoxide radicals after reperfusion . DespiteThe decline in free testosterone level could be due to decreased Leydig cell count as observed in ischemia only and I/R groups, which is in accordance with Bergh et al.  and Creaβ. Turner et al. [The damaged Leydig cell together with decreased plasma level of free testosterone observed in the present study in I/R group could be attributed to apoptosis induced by I/R injury as evidenced by elevated IL1-r et al.  reportedIn addition, FSH level was significantly elevated in I/R group compared to control group; however, it was insignificantly changed in ischemia only group compared to the negative control group. This rise could be attributed to the defective spermatogenesis in I/R group. In the present study, spermatogenesis was disrupted in ischemia only group and more affected in I/R group in the form of pyknosis and degeneration of the spermatogonia together with their separation from the basement membrane which were similar to Bergh et al. . GenescàIn the testicular tissue, mean seminiferous tubule diameter (MSTD) is used to assess histopathological damage based on the evaluation of progressive degeneration of the germinal epithelium . In the Also, the primary spermatocytes count, the number of rounded spermatids, and their ratio were decreased in ischemia only group and extensively in I/R group. Somwaru et al.  found thCongested testicular capsule was detected, in this study, in ischemia and I/R groups, which might be due to capillary dilatation and postcapillary venules slowing blood flow which became lodged with red blood corpuscles, as reported by Wheater et al. . The preHowever, in the present study, there was no apparent affection of Sertoli cells. Turner and Miller  reportedβ and TNF-α as apoptotic factors and inflammatory mediators were significantly elevated in I/R group compared to the control group, however, they were significantly decreased in ischemia only group compared to I/R group. Similarly, Minutoli et al. [α expression and a marked damage in both testes. Thus, I/R could increase the proinflammatory cytokines TNF-α and IL-1β, suggesting a role for these cytokines as early mediators of injury in testis. TNF-α and/or IL-1β could recruit neutrophils in parenchymal testis veins [The disrupted spermatogenesis, in the present study, could be due to apoptosis and inflammation. As plasma levels of IL1-i et al.  found this veins , triggeris veins .αis expressed as a transmembrane protein in pachytene spermatocytes, round spermatids, and testicular macrophages [αfrom the cell surface by shedding of the extracellular domain could be of value in the molecular I/R activated cascade. Also, the metalloproteases could have paracrine and autocrine signalling by controlling the ectodomain shedding of TNF-α [α, expressed in germ cells, and its activity is necessary to induce apoptosis in physiological conditions [αlevel was found in semen samples from infertile patients compared to control subjects, suggesting its role in male subfertility specifically in I/R induced testicular changes [α as a primary response to hypoxia and oxidative stress [TNF-rophages . The relof TNF-α , such asnditions . Higher  changes . Thus, te stress .β and TNF-α compared to I/R group and caused improved histological disruption of spermatogenesis observed in ischemia only and I/R groups. These findings agree with Serrano-García et al. [+ in Ginkgo biloba treated I/R group. Also, Ginkgo biloba contains proanthocyanidins and ginkgolic acid which promotes vasodilation [Moreover, Ginkgo biloba treated I/R group showed significantly decreased plasma levels of IL1-a et al. , who repa et al. , which wdilation ; thus Gidilation  found thErol et al.  evaluateκB and IL1-β, which is responsible for the testicular atrophy, decreased blood flow, and impaired spermatogenesis as observed in this study. The preserved histological and less affected spermatogenesis found in Ginkgo biloba treated I/R group could be explained partially by significant decrease in IL1-β compared to I/R group. Similarly, Akgül et al. [Moreover, Altavilla et al.  reportedl et al.  found de+.In addition to I/R -induced apoptotic changes, the oxidative stress could be a contributing mechanism. Lysiak et al.  reported+ in generating a state of oxidative stress in experimental I/R in testis. Mitochondrial NAD+ was significantly elevated in I/R group compared to control group and to lesser extent in ischemia only group. Also, it was significantly decreased in ischemia only group compared to I/R group. This finding assures that I/R injury could cause more harmful effects of oxidative stress than that which occurs in ischemia only.This study provides the novel finding of the role of mitochondrial NADTherefore, oxidative stress could disrupt the steroidogenic capacity of Leydig cells  in additThe link between the oxidative stress and apoptosis was explained by Shiraishi et al. , who rep+ in I/R group could explain the disrupted spermatogenesis observed in this group. Reyes et al. [Kroemer et al.  mentiones et al.  reporteds et al.  mentione+ in I/R group could explain the observed decrease in free testosterone in I/R group. Turner et al. [+ and decreased free testosterone level in plasma observed in the present study.Also, the finding of increased mitochondrial NADr et al.  reportedr et al. , stimular et al. , which ir et al. . This cor et al. , explain+ level was significantly reduced in Gingko biloba treated I/R group compared to the I/R group. Akgül et al. [While mitochondrial NADl et al.  found th+ and apoptotic factors, IL1-β and TNF-α, could result in the state of subfertility induced by ischemia/reperfusion. In addition, the mitochondrial NAD+ could be a mechanism of Ginkgo biloba in ameliorating I/R induced subfertility.In conclusion, this study demonstrates that ischemia/reperfusion adversely damages testicular tissue and significantly reduces sperm production through inducing oxidative stress and apoptosis, while Ginkgo biloba treatment effectively attenuated these changes. Therefore, the aforementioned decline in plasma-free testosterone level together with increased plasma FSH level caused by elevated mitochondrial NAD


The most efficient approach to diagnose malignant pleural effusions (MPEs) is still controversial and uncertain. This study aimed to evaluate the utility of a combined approach using ultrasound (US)-guided cutting-needle biopsy (CNB) and standard pleural biopsy (SPB) for diagnosing MPE.Pleural effusions were collected from 172 patients for biochemical and microbiological analyses. US-guided CNB and SPB were performed in the same operation sequentially to obtain specimens for histological analysis.p < 0.05). Significant pain (eight patients), moderate haemoptysis (two patients) and chest wall haematomas (two patients) were observed following CNB, while syncope (four patients) and a slight pneumothorax (four patients) were observed following SPB.US-guided CNB and SPB procedures provided adequate material for histological analysis in 90.7 and 93.0% of cases, respectively, while a combination of the 2 techniques was in 96.5% of cases. The sensitivity, specificity, positive-predictive value (PPV), negative-predictive value (NPV) and diagnostic accuracy of US-guided CNB versus SPB were: 51.2 vs 63.4%, 100 vs 100%, 100 vs 100%, 64.9 vs 72.2% and 74.4 vs 81.3%, respectively. When CNB was combined with SPB, the corresponding values were 88.6, 100, 100, 88.6 and 93.9%, respectively. Whereas sensitivity, NPV and diagnostic accuracy were not significantly different between CNB and SPB, the combination of CNB and SPB significantly improved the sensitivity, NPV and diagnostic accuracy versus each technique alone  contains supplementary material, which is available to authorized users. Pleural effusions are a common clinical problem with more than 50 recognised causes . In the The definitive diagnosis of pleural malignancy depends upon histological proof obtained via pleural biopsy. SPB, US-CNB and thoracoscopy are techniques commonly utilised for the acquisition of pleural tissue –15. ThorTo our knowledge, no prospective studies have been undertaken to assess the utility of a combination of US-guided CNB and SPB performed sequentially in the same setting and by the same operator. Consequently, in this prospective study, we evaluated the value of a combination of US-guided CNB and SPB for diagnosis of MPEs.We conducted a prospective, non-randomised study at a dedicated respiratory centre .A total of 172 consecutive patients with pleural effusions who were treated at our institution between January 2013 and December 2014 were included in the study. The inclusion criteria for enrolment of patients were: (1) undiagnosed and untreated pleural effusion; (2) unilateral transudate as suggested by clinical images but unresolved upon treatment of the cause; and (3) age greater than 18 years. Exclusion criteria included: (1) bilateral pleural effusions; (2) minimal or small effusions; (3) insufficient bleeding diathesis for pleural aspiration and biopsy; and (4) an inability of the patient to provide written informed consent.All patients underwent initial conventional US scans  without previous removal of pleural fluid. US was performed using splenic echotexture as an in vivo reference. The patients were in a sitting, prone, supine or lateral decubitus position when US was performed. They were divided into two groups: those exhibiting a maximum thickening of more than 3 mm, and those exhibiting a maximum thickening of less than 3 mm. The presence of effusion was confirmed by standard means, and the amount of effusion was documented as either minimal, small, moderate, or large . All zonPrior to pleural biopsy, pleural effusions were collected from all subjects for biochemical and microbiological analyses, including pH, total protein, lactate dehydrogenases (LDH) and adenosine deaminase (ADA) levels. The biopsy procedures were performed under real-time visualisation using a 16-gauge spring-loaded automated cutting needle  after thoracentesis. The cutting needle was inserted through the guiding channel and then introduced into the margin of the pleural area. At least four specimens were obtained from each patient, fixed in formaldehyde solution and transferred to the Pathology Department for histological examination and immunohistochemical analyses. One specimen was placed in a sterile tube and sent for mycobacterial culture. Figures Following US-guided CNB, SPB (Abrams’ biopsy) was performed at another site. For moderate effusions, the biopsies were obtained from the site exhibiting the maximum effusion as determined by US. In the case of large effusions, the puncture site was chosen to be as low as possible but not within 25 mm of the diaphragm. To acquire a sufficient number of specimens, we created one small incision (3–5 mm) on the skin; when necessary, the parietal pleura was pressed to establish complete contact with the biopsy needle. Collection of biopsy specimens with SPB was similar to that with US-guided CNB. In patients with large effusions, an indwelling pleural catheter was inserted after conducting all biopsy procedures to manage the subsequent steps for a definitive diagnosis. A routine follow-up chest x-ray was obtained within 24 h following the biopsy procedures to assess any possible complications.All procedures were performed in a dedicated respiratory unit by an experienced physician (JW), and all US procedures were performed by the same experienced sonographer (XZ). The US patterns were evaluated by two observers (JW and XZ). Disputes regarding pleural areas were discussed until a consensus was reached. An experienced lung pathologist evaluated the biopsy specimens.A definitive diagnosis of pleural malignancy [true-positive (TP)] was made by histopathological analysis of the biopsy specimens, clinical follow-up and surgery, while a benign diagnosis [true-negative (TN)] was made if: (1) the benign histological diagnosis was based on a precise aetiology; (2) the pleural effusion subsequently disappeared; or (3) follow-up chest radiographs or computed tomography (CT) scans showed a small amount of pleural effusion that remained stable for at least 12 months. Patients with a benign histology were observed for 12 months to minimise the risk of potential false-negative (FN) results.We combined the two biopsy methods for each patient and recognised a TP result if the two methods together or each of the methods individually showed a TP result. The patients’ clinical features, the characteristics of the parietal pleura, pathology reports on the biopsy specimens, the results of the cultured specimens, the definitive diagnoses, and clinical outcomes were all recorded.2 test was used to compare the adequacy of biopsy specimens, diagnostic sensitivity, NPV and the diagnostic accuracy. Significance for all statistical analyses was set at p < 0.05.Data were reported as the number and percentage of qualitative variables. Enumerated data were presented as means ± standard deviation (SD). Categorical variables were analysed and statistical analysis was performed using SPSS® version 16.0 . The primary endpoint was the sensitivity of each biopsy method (US-guided CNB or SPB) and the combination of the two methods for detection of pleural malignancy. Secondary endpoints were other elements of the decision matrix . A χOf the 172 patients who were enrolled in this study, 20 had undergone a non-diagnostic pleural aspiration before visiting our institute, but none had previously undergone pleural biopsy procedures. Table The definitive diagnosis in 90 of the 172 enrolled patients (52.3%) was pleural malignancy, while 82 (47.7%) had non-malignant disease as confirmed by the clinical follow-up (Table p = 0.577). When US-guided CNB was combined with SPB, adequate specimens were obtained in 166 patients (96.5%) using one or both techniques, but the number of specimens obtained was not significantly different from those obtained using US-guided CNB or SPB alone (p = 0.119 or 0.304).Adequate pleural biopsy specimens for histological analysis were obtained in 156 patients (90.7%) with US-guided CNB and 160 (93.0%) with SPB. The difference between the two techniques was not statistically significant (p < 0.05), but there was no significant difference in sensitivity between US-guided CNB and SPB (p = 0.147). Significant differences in the NPV and diagnostic accuracy were also observed between the combination and the individual techniques alone (p < 0.05).The sensitivities of US-guided CNB, SPB and a combination of the two techniques for diagnosis of pleural malignancy were 51.2, 63.4 and 88.6%, respectively (Table p > 0.05). However, in the group with pleural thickening <3 mm, diagnostic accuracy was significantly greater with SPB than with US-guided CNB (p < 0.05).We also evaluated whether pleural thickness affected the diagnostic accuracy of the two biopsy methods. In patients with pleural thickening ≥3 mm, the diagnostic accuracy with US-guided CNB and SPB were 84.2 and 82.5%, respectively, and the difference between the two techniques was not statistically significant (p < 0.05), but with SPB, there was no statistically significant difference the two pleural thickening groups (p > 0.05). The findings of this analysis are shown in Table With US-guided CNB, the diagnostic accuracy was significantly greater in patients with pleural thickening ≥3 mm in comparison with those with pleural thickening <3 mm . In 2014, Hallifax et al [p = 0.577), while the combination of both techniques provided adequate specimens in 96.5% of cases; however, the latter result was not significantly superior to the two techniques alone (p = 0.119 or 0.304).The definitive diagnosis of pleural diseases, particularly malignancy, depends upon histological analysis of tissue obtained via pleural biopsy. Adequate pleural tissues, which are crucial for a definitive diagnosis, can be obtained by SPB, thoracoscopy or CNB under the guidance of CT or US. Thoracoscopy allows direct visualisation of the pleura and biopsy from abnormal sites . In a stax et al . reporteCurrent guidelines on the investigation of pleural effusions emphasise the use of a diagnostic algorithm or recommend the use of a stepwise approach , 23–25. SPB was described more than 50 years ago and became the most widely utilised method for blind biopsy . This prp < 0.05).To overcome these limitations, a combination of CNB and SPB was used in this study. All procedures were performed sequentially by an experienced operator (JW) according to standardised guidelines. Several possible factors could be responsible for the diagnostic advantage of the combination in comparison with the individual techniques. During SPB, an incision in the skin in the direction of the chosen intercostal space above the lower rib was made, especially in overweight/obese patients. During the biopsy procedure, the assistant pressed the skin between the ribs, which allowed the distal tip of the needle to have sufficient contact with the pleura. A limitation of SPB is the blindness of the procedure, although an experienced operator can obtain adequate tissues for a histological diagnosis. We found that the number of adequate specimens was higher with SPB than with CNB , and, as previously reported , 32–36, p < 0.05), and significantly lower than with SPB (p < 0.05). A possible reason for this finding may be that the diagnostic accuracy is affected by pleural thickening. When a US-guided pleural biopsy is performed in patients with minor pleural thickening, there may be a lower probability of obtaining adequate specimens. However, SPB may be capable of acquiring a larger number of adequate samples.For CNB, we performed the procedures using relatively supradiaphragmatic biopsy sites or the most thickened pleural sites. Pleural malignancy is characteristically patchy and preferentially basal, or is found on the diaphragm and results in focal involvement . In addiBoth biopsy procedures were well tolerated in the patients we studied, and no serious complications were observed. However, we concerned with that although four significant pain and two moderate haemoptysis required no intervention, there were about 3.5% complications  required further intervention following CNB. Following SPB, four patients 2.3%) suffered from a slight pneumothorax, but which recovered spontaneously. Though neither procedure was abandoned for the complication, management must be to improve to avoid it, such as, better preparation for reducing syncope or significant pain, skilled procedures for avoiding haemoptysis, chest wall haematoma or pneumothorax. Reported complication rates of SPB or image-guide CNB vary widely , 37, 38..3% suffeUse of a combination of US-guided CNB and SPB afforded a high sensitivity to diagnose MPEs, it is a convenient and safe approach.