TLR9 polymorphisms between clinical severity variables in MM survivors.Genetic variation in immune response genes is associated with susceptibility and severity of infectious diseases. Toll-like receptor (TLR) 9 polymorphisms are associated with susceptibility to develop meningococcal meningitis (MM). The aim of this study is to compare genotype distributions of two TLR9 -1237 and TLR9 +2848 polymorphisms and compared these between thirteen clinical variables associated with prognostic factors predicting adverse outcome of bacterial meningitis in children.We used DNA samples of a cohort of 390 children who survived MM. Next, we determined the genotype frequencies of TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for Neisseria (N.) meningitidis  0.5. 95% confidence interval (CI) 0.3 – 0.9). The TLR9 +2848 AA mutant was associated with a decreased incidence of a positive blood culture for N. meningitidis . Cerebrospinal fluid (CSF) leukocytes per μL were higher in patients carrying the TLR9 -1237 TC or CC genotypes compared to carriers of the TT wild type (WT)  4987 versus 955, IQR 3938). CSF blood/glucose ratios were lower in TLR9 -1237 TC or CC carriers than in carriers of the TT WT . CSF leukocytes/μL were higher in patients carrying the TLR9 +2848 AA mutant compared to carriers of GG or GA .The We identified TLR9 genotypes associated with protection against meningococcemia and enhanced local inflammatory responses inside the central nervous system, important steps in MM pathogenesis and defense. Neisseria (N.) meningitidis is a common causing pathogen of BM, both in the Western world as in developing countries. The clinical course of meningococcal meningitis (MM) is highly diverse and depends both on pathogen characteristics as on the individual immune response of the affected patient. Host-bacteria interactions are crucial in defense against MM [N. meningitidis may lead to bacterial colonization in one patient and to fatal MM or meningococcal septic shock in the other. Survivors of BM have a high risk to develop neurological sequelae, ranging from subtle learning and behavioral disorders to deafness, paresis, and severe encephalopathy [TLR9 +2848 SNP is associated with a decreased susceptibility to MM [TLR9 -1237 C variant allele creates a potential nuclear factor kappa B (NFκB) binding site that increases the transcriptional activity of TLR9 and enhances cellular production of pro-inflammatory cytokines [The susceptibility, severity and prognosis of infectious diseases depend on the ability of the host immune system to respond to pathogens. Genetic variation of immune response genes is associated with susceptibility to and severity of infectious diseases . Bacteriainst MM . Acquisialopathy -7. Innatalopathy . TLR9 isalopathy . TLR9 isalopathy . In a prty to MM . Recent ytokines .TLR9 -1237 and TLR9 +2848 single nucleotide polymorphisms (SNPs) between thirteen clinical severity variables in order to identify patients at risk for severe disease and sequelae.The purpose of this study is to compare the genotype distributions of N. meningitidis or meningococcal antigens in the CSF. A total of 560 children were asked to participate in the study and to return a sterile swab after collecting their buccal DNA, of whom 390 patients (70%) returned a buccal swab and informed consent form. Patients were diagnosed between January 1990 and December 1995 and this cohort was previously described in detail by Koomen et al.[The study population consists of 390 Dutch Caucasian children who survived MM. These patients were identified by the Dutch Reference Laboratory for Bacterial Meningitis. The diagnosis of MM was based on a positive cerebrospinal fluid (CSF) culture with patients 0% returnen et al.. Data foChildren with ‘complex onset’ of meningitis , relapsing meningitis, or meningitis in the neonatal period were excluded.The Medical Ethical Committee of the VU University Medical Center, Amsterdam, The Netherlands approved this study.TLR9 -1237 (rs5743836) and TLR9 +2848 (rs352140) SNPs were analyzed in buccal DNA by TaqMan analysis using the standard TaqMan protocol. The AbiPrism® 7000 Sequence Detection System  was used to obtain data. Primers and probes we used have been described previously [TLR9 SNPs were chosen based on a study by Lazarus et al. In three ethnic groups they found 20 TLR9 SNPs. A set of four frequent TLR9 SNPs  accounted for more than 75% of all chromosomes in all three populations. Genotyping of both TLR9 -1237 T > C and TLR9 +2848 G > A allows all four locus haplotypes to be distinguished [nguished .We performed a literature search to identify severity variables. We used clinical variables: duration of clinical illness before admission, rectal temperature, convulsions, level of consciousness at admission, ICU admission, main clinical diagnosis at discharge (meningitis or meningitis with sepsis), and post meningitis hearing loss. Convulsions were defined as convulsions reported before or at admission or during hospitalization. Post meningitis hearing loss was defined as > 25 dB perceptive hearing loss that was not present before meningitis occurred. Laboratory variables at admission included: blood culture results, CSF leukocyte numbers, CSF/blood glucose ratios, CSF protein concentrations, blood leukocyte numbers, and C-reactive protein (CRP) concentrations in serum. The selected variables were in accordance with a recent systematic review summarizing the evidence regarding prognostic factors predicting death or sequelae after BM in children . We distTLR9 -1237 and +2848 genotypes. For statistical analysis, SPSS for Windows 17.0 and Graphpad Prism 5 were used. Genotype distributions were checked for deviations of the Hardy-Weinberg equilibrium (HWE). Recessive and dominant models were used to model the relations between genotype distributions and clinical variables. Histograms were used to assess normality of the clinical variables. T-tests, Mann–Whitney U tests, and χ2 test or Fisher’s exact tests were used where appropriate. Outliers (< 4%) were excluded by the Grubbs’ test (p < 0.01) before continuous testing. P values < 0.05 were considered statistically significant.Within selected severity groups, we compared the distribution of Table TLR9 -1237 and TLR9 +2848 respectively. Figure TLR9 -1237 TC or CC genotypes compared to carriers of the TT WT genotype  4987 versus median 955, IQR 3938).Continuous variables i.e. duration of clinical illness, rectal temperature, CSF leukocyte numbers, CSF/blood glucose ratios, CSF protein concentrations, blood leukocytes, and serum CRP concentrations were compared between carriers of wild type (WT) alleles and mutant alleles in MM patients for TLR9 -1237 TC or CC carriers than in carriers of the TT WT   .CSF/blood glucose ratios were significantly lower in No significant differences in genotype distributions of −1237 and +2848 SNPs were found for the other continuous variables (data not shown).TLR9 -1237 TC or CC mutant carriers were compared to the TT WT carriers. Significant associations are shown in Table TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for N. meningitidis  0.5, 95% confidence interval (CI) 0.3 – 0.9). These genotypes were also associated with CSF leukocyte levels > 1000 per μL , and with a CSF/blood glucose ratio ≤ 0.4 .Dichotomous variables, i.e. blood culture, convulsions in patient history, level of consciousness at admission, ICU admission, sepsis and hearing loss and the dichotomized continuous variables (as described above) were compared between WT carriers and mutant carriers. TLR9 -1237 genotype distribution were observed between the groups classified by the other severity variables (data not shown).No significant differences in TLR9 +2848 mutant carriers were compared with WT carriers. Significant associations are shown in Table TLR9 +2848 AA was associated with a decreased incidence of a positive blood culture . The TLR9 +2848 AA mutant was also significantly more present in children with > 600 and > 1000 leukocytes per μL .TLR9 +2848 genotype distribution were observed between the groups classified by the other severity variables (data not shown).No significant differences for TLR9 haplotype I was very significantly associated with blood cultures positive for N. meningitidis . Haplotype I was also significantly associated with decreased CSF leukocytes . No significant differences in haplotype distribution were observed for other severity variables (data not shown).Table TLR9 -1237 and +2848 SNPs are associated with severity variables in a cohort of MM survivors. In order to assess the biological consequence of this statistical association we focus on the essential steps in BM pathogenesis and the recognition of N. meningitidis by TLR9. Meningococci colonize the nasopharynx and may penetrate the mucosal barrier of the upper respiratory tract by transcellular passage of epithelial cells [N. meningitidis leads to activation of phagocytosing cells via TLR9 [We demonstrate that the al cells . During al cells . After pal cells . Survivavia TLR9 . Upon suvia TLR9 ,25.TLR9 SNPs with protection against meningococcemia, a prerequisite for meningeal invasion, and with elevated CSF leukocyte levels during MM. A decreased incidence of positive blood cultures in children carrying TLR9 -1237 C allele and TLR9 +2848 AA genotypes may represent a reduction in the occurrence of secondary bacteremia due to more pronounced host immune response in the CSF. A possible mechanistic explanation and biological consequence is increased NFκB binding to the TLR9 promotor region, leading to increased transcriptional regulation of TLR9[in silico regulatory SNP detection method as described by McIntyre et al.[TLR9 -1237 “C” variant was associated with significantly increased binding of NFκB, avian reticuloendotheliosis viral oncogene homolog A (RelA) and signal transducer and activator of transcription (STAT3) to the TLR9 -1237 C variant  harbouring the TC genotype show higher expression of both TLR9 and IL-6 and increased B-cell proliferation in response to CpG [TLR9-1237-C allele with cerebral malaria, indicating that enhanced TLR9 mediated immune responses are also relevant inside the CNS [We associated n of TLR9. We confre et al.: The TLRe to CpG . AnotherTLR9 KO mice displayed reduced survival and elevated levels of bacteremia compared to WT mice [TLR9 polymorphisms with prevention of bacteremia and higher levels of leukocytes in the CSF. The link between systemic inflammation and pleocytosis was previously studied. Intravenous injection of LPS prior to intracisternal LPS injection in rabbits led to impaired pleocytosis, reduced levels of TNF-α, and impaired leukocyte influx into the CNS reflecting an impaired inflammatory response in the CNS [In a mouse meningococcal bacteremia model the role of TLR9 in preventing bacteremia was also confirmed.  WT mice . We asso the CNS .TLR9 SNPs with bacteremia, no association was found with clinical sepsis. We propose that TLR9 -1237 and +2848 polymorphisms have a beneficial effect on preventing bacteremia and increase the leukocyte influx in the CNS, reflecting an enhanced immune response inside the CNS.Clinical studies show that the most severely affected patients with MM or septic shock with a rapidly evolving septic shock associated with high mortality have significant lower levels of pleocytosis . Althoug. We concluded that TLR9 polymorphisms have a small but possibly important contribution to warrant balance between beneficiary and injurious effects of inflammation in the CNS. For exact consequences of these SNPs, future studies focussing on TLR9 SNPs and long term consequences of BM should be performed.Although CNS inflammation is necessary to guarantee sterility of the CNS, its injurious properties are also evident. An adequate but balanced inflammatory response inside the CNS is essential in limiting adverse outcome of diseaseThis study does have certain aspects that limit the interpretations. Survivors of MM were retrospectively included in our study. DNA from children with fatal meningitis were not a focus of our study because including survivors of MM allows us to obtain detailed information on follow-up and long term consequences of the disease which are of particular clinical relevance. In addition, the number of patients with fatal meningitis is very low in The Netherlands. The effect of treatment of BM was not incorporated in this study. This effect may not be of great influence since comparable protocols for treatment of BM were used nationwide in the period these children have been admitted to Dutch hospitals, and timing of treatment is divided equally between groups and independent of genotype distribution.Multidisciplinary efforts are needed in order to bundle and translate genetic studies into beneficial interventions  enforced by the field called Public Health Genomics .TLR9 haplotype I and N. meningitidis). Although the multiple testing corrections reduce the number of statistically significant associations of the exploratory study, the trends that remain give indications for potential roles of the studied variables in the severity of bacterial meninigitis.We have performed several statistical analyses and therefore also performed corrections for multiple testing. Using the rough false discovery rate (rFDR) would shift the threshold for statistical significance from <0.05 to <0.026, reducing the number of significant associations described in this paper. Using the more conservative Holm-Bonferroni corrections, shifts the threshold to <0.002, resulting in one remaining significant association (TLR9 SNPs, functional consequences and long term effects in the CNS are needed to reveal the exact mechanism causing the differences in clinical course of MM and to obtain genetic traits which can be used for patient profiling and management of meningitis patients.More association and functional studies on TLR9 -1237 and TLR9 +2824 mutant genotypes were associated with protection against meningococcemia, an essential prerequisite for meningeal invasion. We also associated these SNPs with enhanced local inflammatory responses inside the CNS. Carriers of the TLR9 + 2848 mutant genotype have a lower chance of developing meningitis when colonized with N. meningitidis. When they eventually do so, they will have more efficient bacterial killing inside the CNS but may also result in a higher chance of developing neurological sequelae.In this study, All authors declare to have no competing interests of any kind regarding this study.in silico analysis, and critically revised the manuscript. AMvF and SAM designed and supervised the study and critically revised the manuscript. All authors have read and approved the final version of the manuscript.GTJvW and MSS collected the patient data and samples, performed the genotype and statistical analysis and wrote the manuscript. SO supervised the genotype and statistical analysis, performed the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/112/prepub


The lipocalin family proteins, including lipocalin-2 and retinol-binding protein 4 (RBP4), are adipokines closely associated with obesity-related metabolic disorders. In this study, we evaluated the association of serum lipocalin-2 and RBP4 with intima-media thickness (IMT) and subclinical atherosclerosis in type 2 diabetic patients.P = 0.002, P<0.001, respectively]. Moreover, positive correlations were observed between carotid IMT and lipocalin-2  or RBP4 , femoral IMT and lipocalin-2 , as well as between iliac IMT and RBP4 . Multiple logistic regression analysis further demonstrated that these two adipokines were independent risk factors for subclinical atherosclerosis in type 2 diabetes.Serum levels of lipocalin-2 and RBP4 were measured in 284 type 2 diabetic patients. Subclinical atherosclerosis was assessed by IMT at carotid, femoral and iliac arteries with ultrasound. Patients with subclinical atherosclerosis showed significantly higher circulating concentrations of lipocalin-2 and RBP4 when compared to those without [112.9 (86.4 to 202.1) µg/L versus 77.2(55.0–150.4) µg/L, 37.1(32.3–40.8) mg/L versus 23.2(20.1–29.2) mg/L, respectively; Circulating levels of lipocalin-2 and RBP4 are positively correlated with carotid IMT and subclinical atherosclerosis in type 2 diabetes, which suggests a potential role of these two lipid-binding chaperones in the pathogenesis of vascular complications of diabetes. Atherosclerotic disease is the primary cause of myocardial infarction, stroke, and peripheral vascular disease, which remains as the leading cause of death worldwide Diabetes is one of the most important risk factors for atherosclerosis Type 2 diabetes is commonly accompanied by the abnormal production of adipokines The above findings indicate a potential involvement of these two lipocalin proteins in cardiovascular diseases. However, the regulatory roles of circulating lipocalin-2 and RBP4 in type 2 diabetic patients with or without subclinical atherosclerosis have not been reported yet. In this study, we assessed serum lipocalin-2 and RBP4 concentrations in patients with newly diagnosed type 2 diabetes in relation to subclinical atherosclerosis, as measured by carotid, femoral and iliac arteries.This study was performed with the approval of the Institutional Review Board (IRB) of the Second Xiangya Hospital, Central South University. The written informed consent was received from every subject.2; (4) no ketosis within the first 6 months after diagnosis; (5) no renal dysfunction; (6) 4-week wash out if treated with insulin sensitizers; (7) no application of antihypertensive drugs or lipid-lowering drugs within recent 2 months.Total 284 type 2 diabetic patients were enrolled in the Diabetes Center at the Second Xiangya Hospital, Central South University from the Chinese National Tenth Five Tackling Key Project which was a multicenter study to evaluate the diabetic vasculopathy in newly-diagnosed type 2 diabetes. Diabetes mellitus was diagnosed according to the American Diabetes Association criteria Hypertension was diagnosed when systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg or on antihypertensive medication. Dyslipidemia was defined as having one or more of the following criteria: (1) fasting triglycerides (TG) ≥ 1.7 mmol/L; (2) HDL-cholesterol (HDL-C) < 1.30 mmol/L in female and < 1.0 mmol/L in male; (3) LDL-cholesterol (LDL-C) ≥ 3.4 mmol/L; and/or (4) already on lipid-lowering drug according to United States Adult Treatment Panel III 2 (kg/m2). Waist circumference was measured midway between the lower rib margin and iliac crest. Blood pressure measurements were taken on the right arm with the subject seated for at least 10 min. Systolic and diastolic phase blood pressures were recorded to the nearest 2 mmHg.A complete physical examination was performed on each patient. Height was measured to the nearest 0.5 cm and weight to the nearest 0.1 kg. Patients were measured in socks, stockings or bare feet and light street clothing. BMI (body mass index) was calculated as weight/heightwww.antibody.hku.hk) as previously reported After overnight fasting, a venous blood specimen was collected in the morning (around 8:00 am) for analysis of various biochemical parameters as follows. FPG (fasting plasma glucose) and 2hPG  were measured by hexokinase method on a Hitachi 7170 analyzer . Serum cholesterol and TG levels were determined enzymatically on the Hitachi 7170 analyzer (Boehringer Mannheim). Serum insulin was assessed by chemiluminescence on a Bayer 180SE Automated Chemiluminescence Systems . Insulin resistance was estimated with the homeostasis model assessment index (HOMA-IR), calculated as fasting glucose (in millimoles per liter) times fasting insulin  divided by 22.5 Examinations for subclinical atherosclerosis were performed. High-resolution B-mode ultrasound  was used to measure the intima-media thickness (IMT) of the target arteries . Linear transducers with frequency of 10 to 12 MHz were used. An anterolateral approach was used to longitudinally capture the right common carotid, femoral and common iliac arteries. The best image was selected to show the far wall intimal–lumen interface as a continuous straight line. Three determinations of IMT were made at 1 cm proximal to the bulb and at the site of greatest thickness. The values at each site were averaged, and the averaged IMT was used as the representative value for each individual. Plaque was defined as IMT ≥1.3 mm or a focal protrusion into the lumen with a thickness of at least 50% more than adjacent intima-media complex. Subclinical atherosclerosis was defined as IMT>1.0 mm and/or with plaque on one or more of the three arteries  but without clinical manifestations 2 tests for categorical variables and independent-samples T test or univariate general linear model for continuous variables as indicated. Multiple logistic regression analysis was done to determine independent factors of subclinical atherosclerosis, and included variables which were significantly different between patients with and without subclinical atherosclerosis and were biologically likely to be related with atherosclerotic status. Two-sided P values <0.05 were considered significant.All statistical analyses were performed with Statistical Package for Social Science Version 16.0 . Data were expressed as mean ± SD or median with interquartile range as appropriate. Data that were not normally distributed, as determined using Kolmogorov-Smirnov test, were naturally logarithmically transformed before analysis. Correlations between lipocalin-2 or RBP4 and biochemical variables were analyzed with Pearson correlation. Comparisons between groups were performed using χP = 0.002, P<0.001, respectively].The anthropometric and biochemical characteristics of the type 2 diabetic patients are summarized in P = 0.018) or RBP4 , femoral IMT and lipocalin-2 , as well as between iliac IMT and RBP4 .Serum lipocalin-2 levels correlated positively with BMI, waist circumference, TG and fasting insulin. Serum RBP4 levels correlated positively with systolic blood pressure . In the P = 0.033), together with age (P<0.001), BMI (P = 0.001) and FPG (P = 0.016). Similarly, if lipocalin-2 was replaced by RBP4 in the model, RBP4 was independently associated with subclinical atherosclerosis in type 2 diabetes . If FPG was replaced by 2hPG in both models, lipocalin-2 or RBP4 remained as a significant factor associated with subclinical atherosclerosis in type 2 diabetes . When waist circumference replaced BMI in all models, the results were similar . The significant association between lipocalin-2 or RBP4 and subclinical atherosclerosis remained unchanged after the HOMA-IR was taken into consideration when performing regression .Using a multiple logistic regression model, independent significant risk factors for subclinical atherosclerosis were identified, including age, sex, BMI, smoking status, FPG, fasting insulin, presence of hypertension, presence of dyslipidemia and lipocalin-2 or RBP4 . Serum lAtherosclerosis is a systemic disease affecting multiple territories in the arterial wall. Subclinical atherosclerosis, as classified by 3 vessel beds involved , is used to diagnose generalized atherosclerosis and considered as a better predictor for cardiovascular events The mechanisms by which these two lipocalin proteins mediate the progression from type 2 diabetes to atherosclerosis are unknown. We propose that they might serve as a relay between inflammation and lipid metabolism in the transition from type 2 diabetes to atherosclerosis. It is now generally believed that diabetes evokes inflammation, vasoconstriction and thrombosis that collectively contribute to atherosclerosis The pro-inflammatory features of lipocalin-2 and RBP4 have been highlighted by several studies. Expression of lipocalin-2 is induced in both acute and chronic inflammation Hyperlipidemia is another risk factor for atherosclerosis. Notably, lipocalins possess lipid-binding properties Consistent with previous findings Our study has several limitations. Firstly, due to the relatively small sample size and the cross-sectional design, the predictive value of lipocalin-2 or RBP4 needs to be confirmed in a larger cohort. Secondly, ultrasound can assess only the structural changes in arteries, but not the vascular inflammatory status or qualitative assessment. Therefore, fluorodeoxyglucose (FDG) positron emission tomography (PET) and virtual histology-IVUS may provide more precise data on the vascular inflammation and plaque components.In conclusion, our study suggests that serum lipocalin-2 or RBP4 levels reflect subclinical atherosclerosis in adults with newly diagnosed type 2 diabetes. These findings support the roles of these two lipid-binding chaperones in the pathogenesis of vascular complications of diabetes, and provide possible perspective for using lipocalin-2 and RBP4 as biomarkers for early detection of high-risk individuals developing cardiovascular diseases and potential therapeutic targets for atherosclerosis.


Significant progress is being made in the fight against blinding trachoma. However, according to estimates, there are over 7.3 million people with trachomatous trichiasis (TT) in the world.Trachomatous trichiasis occurs when in-turned eyelashes scrape the cornea. Not only is this incredibly painful, it also causes damage to the cornea; sufferers will become irreversibly blind unless it is corrected surgically.Providing access to quality surgical services, based in the community, is critical if we are to reach the target of eliminating blindness due to trachoma.Since the formation of the Global Alliance for the Elimination of Blinding Trachoma (GET2020) in 1997, there have been advances in trichiasis management on many fronts. However, surgical numbers remain quite low.The Global Trichiasis Scientific Meeting was held in Tanzania earlier this year to consider evidence from various sources about how best to manage trichiasis. The meeting was attended by scientists, national ministry of health programme managers, and NGO personnel from ten countries affected by trachoma. Here are selected conclusions from the meeting that are particularly relevant to eye care professionals actively working in the field:It is both necessary and possible to improve trichiasis surgery. Programmes and surgeons should follow the techniques outlined in the WHO manual “Final Assessment of Trichiasis Surgeons”The bilamellar tarsal rotation procedure can produce excellent results.Specific guidelines for managing recurrent TT are limited or lacking in most settings. Programmes are encouraged to develop locally appropriate guidelines for the management of recurrent TT.Evidence indicates there is no difference in the TT recurrence rates between silk and synthetic absorbable sutures.Definite indications for surgery include: having any central lashes, having peripheral lashes that touch the cornea, or surgery requested by the patient with TT. There is consensus that even a few lashes touching the peripheral globe  should be managed with an appropriate intervention.Even minor peripheral TT can progress and cause corneal damage; epilation can be considered but this must be accompanied with appropriate counselling and follow-up.Pre-operative TT should be stratified and recorded as: minor: 0–4 lashes, major: 5–20 lashes, and severe: 20+ lashes. This will make follow-up easier.Post-operative TT can be subdivided into early or late, and into clinically significant and non-significant. Clinically significant post-operative TT includes: any central lashes, peripheral lashes that touch the cornea, and TT identified as significant by the patient. Ideally, initial post-operative follow-up should take place between six and twelve weeks, and before six months.The WHO Final Assessment of Trichiasis Surgeons manual should be adopted by all programmes and incorporated at all levels of training.There is programmatic evidence that the trainees being put forward for TT surgery training are not always the most suitable candidates. Programmes should adopt and follow clear criteria for trainee selection.There isa need fora training of trainers manual. Where possible, training curricula should be standardised at the national level to ensure that different training programmes have equivalent outcomes.Supportive supervision should be built into the programmes from the beginning. Surgery team leaders should be selected from amongst active TT surgeons and receive additional training and resources to ensure they are empowered to do their job.Surgeons should maintain a surgical register of all patients. Auditing of clinical outcomes should be part of ongoing supervision. There should be a first year audit after training, including a review of patient cards and productivity, and some review of patient outcomes and observation of surgery.Static service delivery alone will not solve the problem; outreach  is needed to address the backlog.Evidence suggests that in well-planned, well-organised, and effectively managed programmes, surgeons can each operate on 20 eyes per day.Decisions on who to train as TT surgeons will be country specific. A significant proportion of general health workers who are trained as trichiasis surgeons later stop performing operations; this requires attention from the country's ministry of health.Findings in multiple settings suggest that programmes to encourage people to come for surgery should have an understanding of the barriers that a community faces. In some settings, where use of services is lower among women, gender-focused strategies are needed.There is sufficient evidence that bringing the service to the patient increases uptake; however, even with community-based trichiasis surgery, it is unlikely that a programme will achieve 100% coverage and interventions are needed to manage those who refuse surgery.Activities to address some of the challenges are underway; these will be reported in future articles in this series. The meeting was sponsored by the Fred Hollows Foundation and Lions SightFirst.


The number of circulating tumor cells (CTCs) in the peripheral blood of metastatic breast cancer patients is now an established prognostic marker. While the central nervous system (CNS) is a common site of metastasis in breast cancer, the standard marker for disease progression in this setting is cerebrospinal fluid (CSF) cytology. However, the significance of CSF cytology is unclear, requires large sample size, is insensitive and subjective, and sometimes yields equivocal results. Here, we report the detection of breast cancer cells in CSF using molecular markers by adapting the CellSearch system (Veridex). We used this platform to isolate and enumerate breast cancer cells in CSF of breast cancer patients with central nervous system metastases. The number of CSF tumor cells correlated with tumor response to chemotherapy and were dynamically associated with disease burden. This CSF tumor cell detection method provides a semi-automated molecular analysis that vastly improves the sensitivity, reliability, objectivity, and accuracy of detecting CSF tumor cells compared to CSF cytology. CSF tumor cells may serve as a marker of disease progression and early-stage brain metastasis in breast cancer and potentiate further molecular analysis to elucidate the biology and significance of tumor cells in the CSF. In the modern era of oncology, cancer-related mortality is associated with metastatic spread rather than the voraciousness of the primary tumor . Tumor sThe cerebrospinal fluid (CSF) is an important, unique, and poorly understood compartment of the central nervous system (CNS). CSF often acts as a biologic sump for neurons and glia and is continuously produced and recycled much like blood or lymph, though never filtered. Cells from solid tumors can infiltrate the CSF by several mechanisms including blood-brain barrier penetration by circulating cells, directly through tumor extension along Vichow-Robin spaces, or through patterned secondary structures of Scherer , 11. TumThe prognosis and therapeutic stratification of patients with CNS metastases is currently based on the integration of histopathologic data, the appearance and severity of neurologic symptoms, and magnetic resonance imaging (MRI) of the neuroaxis . To over+. We found that human glioblastoma cells do not express EpCAM contrary to breast cancer cells as expected .To characterize the recovery rate and linearity of recovery, we spiked cultured breast cancer cells at varying cell numbers and determined the recovery rate at these various cell counts Figure . We founr = -.66)  Figure . There w) Figure , though ) Figure . Subject) Figure . While tResponse to chemotherapy was evident in three patients that were followed over time in this pilot study Figure . Subject4 CSFTCs and exhibited facial weakness and vertical diplopia. These symptoms were resolved approximately two months after treatment and was accompanied by a near complete disappearance of CSFTCs. Subject 3 also harbored >104 CSFTCs and suffered from severe vomiting, lethargy, and headaches. Following treatment this patient had a CSFTC count that declined significantly to 267 CSFTCs but still suffered from headaches, though other symptoms resolved. The clinical outcome of leptomeningeal or parenchymal CNS metastases varies greatly. Patients with an excess of 10,000 CSFTCs had cranial nerve deficits or mental status decline. Two patients showed an improvement in KPS after chemotherapy initiation and a concomitant decline in CSFTCs. Our findings from this small pilot study suggest a possible predictive role for CSFTCs and their quantification to serve as a reliable marker that reflects disease burden dynamically unlike CSF cytology and provides information regarding the magnitude of disease burden.In addition to its general correlation with KPS, changes in CSFTC number tended to coincide with clinical deficits. Prior to treatment initiation, subject 2 had >10Gadolinium-enhanced MRI allows for the sensitive visualization of macroscopic changes with the brain, spine or leptomeninges. We found an inverse correlation with CSFTC count and the presence or enhancement pattern seen on such imaging. It should be noted that though neuroimaging is an indicator of CNS disease, it relies on the breakdown of the blood-brain barrier rather than a direct sign of response. Multi-modality imaging is an ever-advancing technology but is not sufficient to make judgments about cranial and spinal metastases. CSFTCs represent a novel marker of CNS disease that detects cancer at the single cell level and provides critical information to clinicians that attempt to combine cytology, imaging and neurologic examination as part and parcel of a treatment algorithm. While this study serves as a proof-of-principle and shows promising significance, a larger cohort of patients with follow up studies will be required to concretely establish the prognostic significance of CSFTC enumeration and the ability of therapeutic agents to eliminate these cells.The biology of metastases and their relationship to the microenvironment of the CNS remains unclear , 16. TheAccurate, early diagnosis and appropriate treatment decisions are likely to yield a better patient outcome and underscores the importance of accurately monitoring common secondary sites by a highly sensitive detection method. The ability to detect single cells at a metastatic site such as the brain may allow for therapeutic intervention that could prevent or destroy metastatic disease an early stage . Delays A number of techniques have been developed for the isolation of CTCs in peripheral blood since the first attempts in the late 1800s . InvestiThe prognostic relevance of CSFTCs is correlated here with the clinical course of the patient and the CSFTC count. The biological significance of finding up to several thousand CSFTCs in a patient sample remains unclear though it is suggestive of and correlates with high disease burden. CNS metastases are often strategically located in close proximity to ventricular surfaces or CSF cisterns. However, natural circulation of CSF can be impaired, which may cause “loculations” of malignant CSF, thus lowering the diagnostic yield of lumbar puncture .Our lab is currently pursuing post-isolation characterization of tumor cells isolated from peripheral blood as well as CSF . There iHuman SKBR3 breast cancer cells were obtained from ATCC and cultured in McCoy's 5A medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin and streptomycin. Human SF767 glioma cells were a kind gift from Akiva Mintz (Wake Forest University) and were cultured in RPMI under the same conditions. For Western blot analysis, cells were harvested by cell scraping, centrifuged, and lysed on ice for 2 hours. Lysates were harvested and the protein concentration was determined using the BioRad protein assay. Samples were electrophoresed on 4-12% Bis-Tris gels, transferred to PVDF, and blocked in 10% non-fat milk in TBST. Membranes were incubated with EpCAM  at 1:500 or Ran at 1:10,000 (BD biosciences) antibodies overnight at 4°C. Membranes were rinsed in TBST, incubated with an appropriate HRP-conjugated secondary antibody, and visualized using ECL-Plus and X-Ray film.Tumor cells were harvested from log-phase growth by trypsinization and enumerated using a Cellometer (Nexcelom Biosciences) in triplicate. The appropriate volume of cell suspension was then added to a tube of CSF previously cleared of tumor cell contaminants by centrifugation. This CSF was then subjected to the standard CSFTC detection procedure described below.The main inclusion criteria were newly discovered breast cancer with metastatic disease involving the CNS as confirmed with radiologic or cytologic findings and the commencement of intrathecal chemotherapy. All subjects provided informed consent for testing of their CSF as approved by the Institutional Review Board at the Penn State Hershey Medical Center. All subjects were diagnosed with primary breast cancer prior to enrollment into this study and had undergone neurosurgical intervention in terms of placement of a ventricular access device. All patients had involvement of the CNS, with combinations of parenchymal or leptomeningeal metastases. The ventricular access device provided a means to obtain CSF for testing. Prior to the onset of treatment, patients underwent thorough neurologic examination to identify any deficits and were stratified based on KPS. CSF was obtained every 2 to 3 weeks for each patient and often coincided with intrathecal chemotherapy deposition. The neuro-oncologist providing treatment and evaluating the patients was blinded to the CSFTC analysis.7 cells per 6.5mL sample of blood. Samples were analyzed within 3 days using the standard CellSearch protocol and the CTC Epithelial Cell Kit (Veridex). In brief, the CellSearch system qualifies a cell as a CTC if it has an evident nucleus by DAPI staining and is EpCAM+, cytokeratin+, and CD45-. Analysis and enumeration of CTCs was conducted by a blinded, certified assay operator.9mL of CSF was centrifuged at 500g for 10 minutes, the supernatant was removed, and the pellet was resuspended in 1mL of PBS. The suspension was then spiked into 6.5mL of reconstituted normal human blood. This blood was obtained from leukocyte filters that were generated from blood drives as approved by the Institutional Review Board at Penn State Hershey Medical Center. 6.5mL of filtered blood was removed from the leukocyte trap, placed into a CellSave (Veridex) tube, and inverted 8 times. The leukocyte filter was rinsed with PBS and back-flushed to obtain leukocytes that were enumerated and spiked at 10Bivariate correlation calculated by Pearson's correlation.


The specific retention volumes of nonpolar and polar probes that were chromatographed on these ordered mesoporous silica adsorbents were evaluated under infinite dilution conditions. Two methods were employed to calculate the standard free energy of adsorption, ΔGads, of each chromatographed probe on the basis its specific retention volume. These ΔGads values were then employed to estimate the van der Waals contribution and the specific contribution of the free surface energy for each MCM41. DN values  and AN* values  were employed to determine the values of parameters that characterize the ability of the MCM41s to act as electron acceptors (parameter: KA) and donors (parameter: KD). Considering the different compositions of the probes, each of which has different acceptor–donor properties, a new chromatographic test to supplement the Grob test is suggested.Three ordered mesoporous siliceous (OMS) materials known as MCM41s—unmodified MCM-41C16 (“C16”), and two MCM41s with different surface functionalities: MCM-41C16-SH (“C16-SH”) and MCM-41C16-NH The walls of the mesopores are made of amorphous silica , the solution became a lyogel, before finally taking on the consistency of porridge after mixing. The resulting adsorbent was denoted “C16.”Synthesis of the tested MCMs was carried out as follows. Approximately 15.34 g of the surfactant C3 of tetraethoxysilane and ca. 0.7 cm3 of one of the following modifiers were added:i.2H5)3Si–C3H6–SH in the synthesis of MCM41–C16–SH (denoted “C16-SH”)(OCii.2H5)3Si–C3H6–NH2 in the synthesis of MCM41–C16–NH2 (denoted “C16-NH2”).i.n-alkanes: C5–C8—0 D and cyclohexane—0.3 DNeutral: ii. sp3 hybridization and dipole moments , and benzene—0 DAcidic: dichloromethane, trichloromethane and tetrachloromethane, which possessiii.Basic: acetonitrile—3.2 D, tetrahydrofuran—1.75 D, diethyl ether—1.3 Div.Amphoteric: acetone—2.9 D, ethyl acetate—1.7 DThe following substances (categorized below according to their interactions with the surfaces of the MCM41s) were employed as probes:GadsSP values by plotting the ΔGads values of the n-alkanes and the other injected probes as a function of physicochemical parameters .As discussed in the “criptors  for all ocument}  for the Elemental analysis of the surface layers of the MCM41s was performed using a Quantax 200 spectrometer with an EDX XFlash 4010 detector . The results of this elemental analysis are collated in Table 3/min.Chromatographic measurements of the aforementioned probes were undertaken using a Unicam  type 610 gas chromatograph fitted with an on-line Unicam 4880 chromatography data handling system, which was switched on when a sample was injected. The instrument was equipped with a flame ionization detector. The injector and detector temperatures were 396 and 398 K, respectively. Elution peaks of the probes were acquired at 343–393 K and a sampling rate of 10 Hz, employing helium flowing at a rate of 20 ± 0.1 cmm1 [2 adsorbent at 100 °C and the profile of the Weibull function is depicted in Fig. n-alkanes as the column temperature is increased are depicted in Fig. Each acquired elution peak was fitted by the Weibull function in order to eliminate small distortions and obtain plausible retention data for the center of gravity of the peak . A comp3/min. The amount of probe injected onto the column ranged from 0.05 to 0.005 μl of vapor  and liquid (for diethyl ether and ethyl acetate).MCM41 samples that had been degassed at 383 K were placed in 65 × 0.4 cm I.D. glass columns each containing an adsorbent bed 10–12 cm in length. The part of the column that was unoccupied by the MCM was filled with glass beads with a mesh size of 80. The filled column was mounted in the chromatograph thermostat and heated at 423 K for 24 h in a helium stream flowing at a rate of 40 cmtm, can be calculated from the dependencetiRis the retention time of Ar, Kr, Ne, or XeMiis the atomic mass of the aforementioned gasU(z)is the Lennard–Jones 10-4 potential functiona and bare the coefficientsThe important part of these chromatographic tests was the determination of the retention volume of unadsorbed probe: the holdup. The gas holdup time, tm values has already been presented in detail [The justification for this method of calculating the n detail .tRcg, corresponding to infinitesimally low coverage of the MCM41 surface with probe molecules were estimated on the basis of the first statistic moment, m1 , based on the relation presented in the “Asymmetric elution peaks were obtained for all tested systems, and the retention times of these depended on the amount of probe injected onto the column. The retention times, Speak is the peak area, Fo is the carrier gas flow rate as measured by a soap flowmeter at the column temperature, ma is the mass of the probe injected onto the column, and q is the chart speed) was employed to produce a calibration plot.To check whether the raw detector responses changed linearly with probe concentration in the range employed, the function The specific retention volumes were calculated using the retention times determined for infinite dilution of the probe, and for 1 g of the MCM41 in the column, by means of the relation presented in the “d-electron cloud vacancies in the silicon atoms  to have only a minor effect on the values of the KA and KD parameters for different MCM41s. Therefore, we calculated the values of the KA and KD parameters for all of the polar probes used, and in the second case we omitted acetone. The results obtained are collated in Table KA and KD values.The silanol groups and siloxane bridges have a decisive influence on the acceptor–donor properties of the MCM41 tested. Unfortunately, information on the chromatographic testing of the acceptor–donor properties of siliceous materials is scarce in the literature. Therefore, it is difficult to compare them, because the values of the Upon analyzing the results collated in Table In our tests we injected the molecular probes and polar probes  separately, but the Grob test uses a mixture of molecular probes and polar probes, which must be eluted far enough from each other that mutual interactions are avoided as much as possible. Nevertheless, our attempts are not being remanded, in contrast to existing tests that are used to check the quality of chromatographic fillings.n-alkanes  are neutral and should elute intact, although partial retention of the alkanes may occur on very inert, nonpolar columns.The n-alkanes.The esters  are amphoteric, so they behave in a similar way to The alcohols 1-octanol and butane-2,3-diol are hard bases according to the classification of hard and soft acids and bases , so in tAccording to the Grob test, a primary monoalcohol is appropriate for testing a column filled with a phase that has no hydroxyl groups in its structure, such as silicones. Diols are eluted at the start of the chromatogram from columns filled with nonpolar silicone phases. However, carboxylic acids are adsorbed only slightly more strongly than 1-octanol on column fillings without any basic sites, provided that the column filling is not so strongly acidic that it causes tailing of the alcohols.Acceptor–donor interactions are gauged based on the retention of 2,6-dimethylaniline, 2,6-dimethylphenol, dicyclohexylamine, and 2-ethylhexanoic acid. The Grob test suggests that primary amines form weaker hydrogen bonds than primary alcohols, so they elute faster than primary alcohols from column fillings with acidic sites.Polar probes with sterically hindered groups are preferred in tests because they allow retention due to hydrogen bonding to be avoided. This usually arises from silanol groups or an oxidized stationary phase, or sometimes sample residues from previous injections.Dicyclohexylamine and 2-ethylhexanoic acid provide much more stringent tests of acceptor–donor properties than aniline or phenol because their functional groups are so sterically hindered that only acceptor–donor effects are seen in such systems.n-Nonanal is used to assess the capacity of the column to retain saturated aldehydes. This type of adsorption is independent of the amount of hydrogen bonding present.As presented earlier, the acceptor–donor properties of column fillings represent just one of many sets of information required from chromatographic tests; however, it is necessary to emphasize that they are the most important. Therefore, when employing the Grob test, it is important to realize that acceptor–donor adsorption cannot be interpreted on the basis of well-shaped peaks, because the results are always ambiguous.In this context, it is necessary to add that London dispersion forces, Keesom dipole–dipole forces, and Debye dipole-induced dipole forces are equally additive, and that hydrogen bonds are only dipole interactions.Lowering the number of probes employed will have a decisive influence on the results obtained.The chromatographic test performed using our set of probes will, undoubtedly, characterize the acceptor–donor properties of the column filling, and it should be performed as a complementary test to the Grob test. Since the presence of more than one polar probe exerts a decisive influence on the values of the parameters characterizing the acceptor–donor properties of MCM41s, we believe that our idea should be taken into account when testing the quality of MCM41s as both column fillings and adsorbents anchored to glass capillary columns.In the case of probe elution at infinite dilution, the resulting peak usually appears on top of a noisy baseline. Therefore, the description of the peak shape depends greatly on the number of points employed to mathematically model it. The mathematical method employed to describe raw chromatographic peaks allows more plausible retention time data to be obtained for all of the probes used, in an independent and repeatable way.GadsSP values into account, making it is possible to characterize the acceptor–donor properties of MCM41 surfaces.The IGC method employed here gives more plausible results that take ΔKA, KD, and KA/KD values and the EDX results complement one another very well and enable a deeper explanation of the acceptor–donor properties of the MCM41s when used as column fillings. Good correlation between the EDX results and the IGC ones was obtained for the acceptor–donor properties. This suggests that the specific probe–MCM41 adsorbent interactions are strongly dependent on the elemental compositions of the outermost surface layers and the bulk of the adsorbent.The sets of the It is necessary to formulate a set of probes for these tests that will allow unambiguous interpretation of the acceptor–donor properties.We hope that presenting our opinions about testing the quality of chromatographic fillings as we have done here will aid chromatographists in their attempts to solve other problems.


A high level of cerebrospinal fluid (CSF) neopterin is a marker of central nervous system inflammatory-immune mediated processes. We aimed to assess data from 606 neuropediatric patients, describing the clinical and biochemical features of those neurological disorders presenting CSF neopterin values above a new cut-off value that was defined in our laboratory.To establish the new CSF neopterin cut-off value, we studied two groups of patients: Group 1 comprised 68 patients with meningoencephalitis, and Group 2 comprised 52 children with a confirmed peripheral infection and no central nervous system involvement. We studied 606 CSF samples from neuropediatric patients who were classified into 3 groups: genetic diagnosis (A), acquired/unknown etiologic neurologic diseases (B) and inflammatory-immune mediated processes (C).The CSF neopterin cut-off value was 61 nmol/L. Out of 606 cases, 56 presented a CSF neopterin level above this value. Group C had significantly higher CSF neopterin, protein and leukocyte values than the other groups. Sixteen of twenty-three patients in this group had a CSF neopterin level above the cut-off, whereas three and seven patients presented increased leukocyte and protein values, respectively. A significant association was found among CSF neopterin, proteins and leukocytes in the 606 patients. White matter disturbances were associated with high CSF neopterin concentrations.Although children with inflammatory-immune mediated processes presented higher CSF neopterin values, patients with other neurological disorders also showed increased CSF neopterin concentrations. These results stress the importance of CSF neopterin analysis for the identification of inflammatory-immune mediated processes. White blood cell (WBC), glucose and protein levels in the CSF were analyzed for each sample . We excluded those samples with elevated WBC counts attributable to contamination with blood after a corrective formula was applied We previously established reference values for CSF neopterin concentrations in a cohort of 116 pediatric patients in whom viral or bacterial meningitis, encephalitis and other neurological conditions of non-metabolic origin were ruled out Between January 2001 and April 2012, 852 CSF samples were analyzed in the Sant Joan de Déu Hospital Laboratory. The samples were collected from patients afflicted by several neurological disorders for which a diagnostic lumbar puncture was indicated. Of the initial group of samples, 242 were excluded for one of the following reasons: traumatic (hematic) puncture and inadequate conditions for sample collection and preservation, such as the lack of rostral-caudal gradient sampling, inappropriate temperature or lack of light protection. Finally, 606 samples  were included in the study and were classified into three different clinical groups: A) patients with confirmed genetic diseases (n = 160), including inborn errors of metabolism of simple and complex molecules, mitochondrial diseases and neurogenetic disorders; B) patients with acquired neurologic disorders n = 423), including ischemic and hemorrhagic injuries, neurological disorders with motor disturbances, mental retardation plus other neurological signs and psychiatric conditions, leukodystrophy and malformations, epilepsy and epileptic encephalopathies; and C) patients with inflammatory or immune-mediated diseases (n = 23), including Aicardi-Goutières syndrome, acute disseminated encephalomyelitis, Guillain Barré syndrome, anti-NMDA (N-methyl D-aspartate) receptor encephalitis, chronic infantile neurological, cutaneous and articular syndrome, histiocytosis, myasthenia gravis syndrome and brain parasitosis. Detailed information about the diagnoses of the entire cohort is available upon request. Their main clinical and CSF biochemical data are presented in , includi2. Both total pterins were analyzed by reverse phase high performance liquid chromatography with fluorescence detection following a previously reported procedure 3 for WBCs.In the 606 patients, screening laboratory tests for the diagnosis of metabolic and genetic diseases  were performed when indicated, using standard procedures. CSF samples were collected by lumbar puncture as previously reported 3), white matter disturbances or the presence of epilepsy, we applied the Chi-square test and computed the Odds ratio for each group. Statistical significance was defined as p<0.05.All analyses were performed using the SPSS 19.0 software. To establish the new CSF neopterin cut-off, COR curve analysis was applied. The Kolomogorov-Smirnow test was applied to assess the data distribution. Because the data did not follow a Gaussian distribution, different non-parametric tests were applied. The Mann-Whitney U test was used to test for the statistical significance of different CSF variables in Groups 1 and 2. In the entire sample (n = 606), the Spearman correlation test was applied to search for correlations between the CSF neopterin level and the other variables studied in the CSF  and age. To compare the CSF neopterin level with the other CSF variables among the different clinical groups, the Kruskal-Wallis test was applied. To describe whether high CSF neopterin values were associated with a significantly higher risk of having altered CSF proteins (>40 mg/dl), altered CSF leucocytes  and 2  was created. The best discrimination was provided by the CSF neopterin value of 61 nmol/L  with a sensitivity of 91.3% and specificity of 88.5% . Table 1Neurologic cohort of patients: In the 606 patients, a significant positive correlation was observed for CSF neopterin with CSF proteins  and leukocytes  but not with CSF glucose, CSF biopterin and patient age. Given this last finding, a unique age group of patients was considered for subsequent analysis.3), high CSF proteins (>40 mg/dL) and high CSF neopterin concentrations (>61 nmol/L). In all groups, the proportion of patients with high CSF neopterin levels was higher than that of high CSF WBC. Of these 606 patients, 56 presented with increased CSF neopterin values according to the 61 nmol/L cut-off value , CSF proteins  and CSF leukocytes  compared with the other groups. Moreover, in this group, up to 69.6% of the patients (16 out of 23) had CSF neopterin >61 nmol/l. In contrast, among these 16 patients, three had increased WBC, and seven presented increased proteins , white matter disturbances or the presentation of epilepsy. The odds ratio showed a significant association between high CSF neopterin and high CSF proteins in the entire patient cohort  and increased WBC count . The risk of having alterations in white matter with CSF neopterin >61 nmol/L was only observed in group B . Conversely no increased risk of having epilepsy with CSF neopterin above 61 nmol/l was observed in any group. Additionally, there was no significant risk of having high CSF proteins, high CSF leukocytes, white matter disturbances or epilepsy when the CSF neopterin level was below 61 nmol/L.Finally, we explored whether increased CSF neopterin (>61 nmol/L) could be related to a significantly higher risk of having high CSF proteins (>40 mg/dl), high CSF WBC . In contrast, if the former upper limit of CSF neopterin (34 nmol/L) would have been used, 87 out of 606 patients would have shown neopterin values above the normal range. Using this approach, the number of patients seems excessive because most of them belonged to clinical categories with no evidence of immune-mediated processes, such as neurometabolic diseases (data not shown). Nevertheless, only one case belonging to the inflammatory-immune mediated group presented a CSF neopterin value between 34 and 61 nmol/L. In all likelihood, with the application of this new cut-off value, a better discrimination between inflammatory-immune mediated conditions and other neurological conditions will be achieved. This discrimination may be critical for etiological diagnosis and therapeutic options because treatment strategies may be completely different in these categories.Only a previous study about CSF neopterin values in neurological patients was published Concerning the neurological patients with genetic diseases, ten cases showed CSF neopterin levels moderately above 61 nmol/L . This subtle elevation most likely reflected inflammatory events associated with CNS damage Regarding the patients with acquired neurologic disorders or with unknown etiology, although the percentage of patients with high CSF neopterin was quite similar to that of group A (7.1% versus 6.2%), some patients showed values in the range of inflammatory-immune-mediated diseases . In thesWhite matter abnormalities on MRI were associated with CSF neopterin levels above 61 nmol/L in the group B patients. These results may be explained because neopterin is also produced intrathecally by different glial brain cells, acting as a possible link among inflammation, neurodegeneration and regeneration Concerning patients with neopterin levels below 61 nmol/L (n = 550), nine patients showed increased WBC, and 66 showed increased proteins. Furthermore, in groups B and C, there was a clear difference between the percentage of patients with high CSF neopterin and those with high CSF WBC , supportThis report is the first to establish a new CSF neopterin cut-off value to detect inflammatory and immune-mediated neurological disorders. This new value was higher than the upper-limit of the reference intervals previously established. Although the group of patients who more frequently present high CSF neopterin values was that with immune-inflammatory processes, patients with other neurological disorders had CSF neopterin values above the new cut-off value in a noticeable percentage of cases. Therefore, this value was not only useful to detect the presence of an immune-mediated process but also because the possibility of an anti-inflammatory treatment may arise.


Dear Sir,Polyorchidism is a rare congenital anomaly with most common occurrence of three testes (triorchidism). Polyorchidism is more common on left side. To date, in children less than 50 cases are reported. The majority of patients are asymptomatic or present with painless inguinoscrotal masses, undescended testis, and rarely, torsion of the supernumerary testis. In children the appropriate management of polyorchidism remains unclear. With the advances in imaging techniques the former practice of removing the supernumerary testes has changed. The conservative surveillance of polyorchidism in cases with a normal radiological appearance and negative tumor-markers is an accepted practice especially in the pediatric population.[3] We have encountered a child with right sided triorchidism who was managed by preservation of both the testes.A 3-year-old boy was admitted with right sided inguinal swelling for 7 days. Parents had noticed that both the testis were descended into the scrotum since birth but the right sided scrotum was empty since the child had developed the inguinal swelling. On examination, he was in obvious discomfort. Local examination of inguinoscrotal area revealed a normally descended left testis and an irreducible swelling in the right inguinal area. The right scrotum was empty but it was well developed. Ultrasonography of the swelling revealed echogenic shadows consistent with intestines and testis. After the routine haematological investigations, patient was operated with a suspicion of an obstructed inguinal hernia. At operation, after reduction of hernia contents it was found that there were two testes on the right side which were drained by a common vas deferens . Herniotomy was performed and both the testes were placed into the ipsilateral scrotum by creating subdartos pouch. The patient is well at follow up.The majority of supernumerary testes are located in the scrotal region (66%) followed by inguinal (23%), and abdominal (9%) positions.[1] The exact etiology of polyorchidism is unknown, however, an anomalous appropriation of cells in the genital ridge before 8 weeks of gestation, duplication or transverse versus longitudinal division of the urogenital ridge, incomplete degeneration of mesonephrons and development of peritoneal bands are few theories about its genesis.Leung described the first anatomical classification which was based on the possible embryological variations: Type I: Supernumerary testis lacks an epididymis or vas deferens and has got no attachment to the usual testis ; Type II: The supernumerary testis drains into epididymis of usual testis and they share a common vas deferens ; Type III: The supernumerary testis has its own epididymis and both epididymis of ipsilateral testes drain into a common vas deferens ; Type IV: Complete duplication of testis, epididymis and vas .[4] In this patient the right sided polyorchidism was of Type III.The incidence of testicular malignancy in polyorchidism is between 5.7–7% and was found only in a non-scrotal  supernumerary testis. Management of polyorchidism has been subjected to much debate. With recent improvements in imaging techniques such as ultrasound and MRI, most cases of polyorchidism can be diagnosed and followed up accurately without any need for surgical exploration or histological examination.[3] However, if the patient is symptomatic or there is a suspicion for malignancy surgical exploration, biopsy and orchidectomy are recommended when the intraoperative frozen section is positive for malignancy.[5] But there is no consensus in the literature regarding the management of polyorchidism especially in situations like the present case, where the supernumerary testes is found incidentally while operating on a obstructed hernia or an undescended testis. In such a case the orchidectomy of the supernumerary testes is advocated only when it is atrophic, separated from the normal testis or without connection to the vas deferens.[6] Moreover it has been seen on imaging during follow up of such cases that accessory testes may have reproductive potential.[6] On the basis of the available literature and taking into the above mentioned criteria, it is appropriate to preserve the supernumerary testis by performing scrotal orchidopexy as also done in the present case.Source of Support: NilConflict of Interest: None declared


Limited data exist on the impact of living kidney donation on the donor-recipient relationship. Purpose of this study was to explore motivations to donate or accept a (living donor) kidney, whether expected relationship changes influence decision making and whether relationship changes are actually experienced.We conducted 6 focus groups in 47 of 114 invited individuals (41%), asking retrospectively about motivations and decision making around transplantation. We used qualitative and quantitative methods to analyze the focus group transcripts.Most deceased donor kidney recipients had a potential living donor available which they refused or did not want. They mostly waited for a deceased donor because of concern for the donor’s health (75%). They more often expected negative relationship changes than living donor kidney recipients  who also expected positive changes. Living donor kidney recipients mostly accepted the kidney to improve their own quality of life (47%). Donors mostly donated a kidney because transplantation would make the recipient less dependent (25%). After transplantation both positive and negative relationship changes are experienced.Expected relationship changes and concerns about the donor’s health lead some kidney patients to wait for a deceased donor, despite having a potential living donor available. Further research is needed to assess whether this concerns a selected group. Due to a shortage of organ donors, kidney transplantation using living donors is increasingly performed in the Netherlands. Quality of life of most living donors is better than or equal to the general population ,2 and reResearch regarding the effect of living kidney donation on the donor-recipient relationship has mostly been performed from the donor’s perspective -8. AlthoThe recipient’s perspective has received less attention. Recipients may possibly feel they owe the donor something, hesitate to accept a kidney the donor may need later in life, or are afraid to disappoint the donor if the kidney would not function properly ,8. The rSimilar cognitions and fears about possible relationship changes may influence the recipient’s decision making when accepting a kidney offer. For example, it is unknown whether deceased donor kidney recipients have deliberately chosen for a deceased donor instead of approaching a living donor. Previous research among kidney patients on the waiting list showed that the most often mentioned first reaction in favour of deceased donor kidney transplantation was the unwillingness to burden a loved one, indicating fear of a decline in the donor’s health status . In addiPurpose of the present study therefore was to explore motivations to donate or accept a living donor kidney or to pursue deceased donor kidney transplantation, and to assess whether expected relationship changes influence decision making and whether such relationship changes are actually experienced.All donors and kidney recipients operated in the period 1997–2008 at the Leiden University Medical Center (LUMC) and who were still alive, were informed about the PARTNER study . They were asked to respond if interested in focus group participation regarding possible relationship changes. Exclusion criteria were: insufficient command of the Dutch language, living abroad, severe psychological disorder (requiring treatment), anonymous donors and their recipients, donors / recipients from the crossover program, and recipients who lost their graft to create homogeneous groups. Based on the literature  and expeOf the total population (n = 1097), 979 individuals were eligible to participate and a sample of 114 individuals was invited representative for the total study population with respect to age, gender and time since transplantation as the views of individuals may differ between e.g. younger and older individuals or by time passed since transplantation. These variables were available in the hospital information system and could thus be used for sampling. The focus groups were conducted separately for donors, living and deceased donor kidney recipients. The goal was to recruit equal numbers across the three groups with at least 6 participants and a maximum of 10 participants for each focus group to ensure everyone’s participation in the discussion. The study was approved by the LUMC Medical Ethics Committee.In June and July 2009, we conducted six focus group sessions lasting 1.5 to 2 h. Focus groups are a useful method to provide in-depth information and explore cognitions and motivations of underlying behavior ,12. We uIndividuals who consented to join the focus groups received a letter with the topics that would be discussed. Each topic contained specific questions and participants were asked to formulate their answers before the focus group sessions. During the sessions all participants wrote down their answers on post-its and posted these on a central board. This stimulated the discussion and enabled a quantitative assessment of how many participants made comments on a specific topic. The moderator ensured that the participants were prompted on all topics. The 2 observers took notes from the discussion, to ensure that the statements made on the post-its were correctly interpreted and classified into an appropriate category (see below). A quantitative count was added to support our qualitative analysis and to provide insight in the representativeness of the statements ,16. DuriThe I-change model was used as a theoretical framework -19. The All focus group sessions were audio taped and transcribed in full. We analysed the transcripts using theory-based analysis, in which the text is organized according to pre-existing theoretical categories . The traWe counted the number of participants who made comments fitting a specific category to support our qualitative analysis and to provide insight in the representativeness of the statements ,16. If pA p-value smaller than 0.05 was considered significant in all analyses. Values between 0.05 and 0.10 were considered as a trend towards significance.http://www.eurotransplant.nl).Of the 114 invited individuals, 60 (53%) consented to join the focus groups and 47 (41%) attended, representative for the total population with respect to age, gender and time since transplantation  were afraid of imbalance in the donor-recipient relationship Table . One recTo decide on kidney donation or acceptance, people need to be aware that living donor kidney transplantation is possible, and thus should have received information. All participants were aware that living donor kidney transplantation was possible, and had received the necessary information Table  and 4. OEfficacy, attitude and social pressure are motivational factors that may determine a person’s intention to donate or accept a kidney.Several donors were often available, similarly in living and deceased donor kidney recipients Table . The offDonors mostly offered a kidney motivated by the progression of the recipients’ disease and/or the prospect of dialysis in the (near) future (45%). However, they also stressed their personal benefits in donating a kidney; they hoped the transplantation would make the recipient less dependent and thus could participate more in household activities and family life Table .As expected, living donor kidney recipients mostly accepted the kidney motivated by the expected improvement of their quality of life  Tables  and 4.Deceased donor kidney recipients on the other hand, often waited for a deceased donor kidney because of feelings of obligation towards the donor (83%) Table . AnotherIn all groups, a minority experienced some social pressure. 10% of the donors experienced social pressure by family members Table  and 25% About one-third of both donors and recipients reported no change in their relationship after transplantation Table . Those wAn unanticipated finding in all focus groups was that both donors 35%) and recipients (47%) stressed that relationship changes also involved other people   . NeverthOur study is, to our knowledge, the first that explored which factors influence patients in their decision making regarding living or deceased donor kidney transplantation with both qualitative and quantitative methods. This combination of methods enables us to conclude that certain types of motivations, expectations and fears seem more common than others. At the same time we could show the inter-individual variation in the precise motivation regarding living kidney transplantation. Previous studies showed that the main motivating factors for donors are the stress and anxiety of subjecting ‘a loved one’ to prolonged dialysis therapy and the emotional and physical deterioration associated with long-term dialysis therapy ,21. BineLiving and deceased donor kidney recipients also differed in their expectations regarding donor-recipient relationship changes. Most previous studies regarding the effect of living kidney donation on the donor-recipient relationship have been performed from the donor’s perspective ,8. Some Surprisingly, we found that deceased donor kidney recipients often had a potential donor available, but that this offer was refused. These recipients chose to wait for a deceased donor kidney, because of their concern for the living donor’s health and expected negative relationship changes without any positive expectations. It is important to identify these kidney patients to address these issues and to take away any unjustified fears. This may be achieved by discussing expectations regarding changes in the relationship and their health status, as an element of standard care. If these issues are only discussed when brought up, things may be left unsaid so that the potential donor or recipient is not aware of these fears or expectations. They should at least feel reassured they can discuss their fears and doubts regarding living donor kidney transplantation; stories of previous recipients may help in this situation, as well as evidence on how many donors or recipients have actually experienced such changes. By making it part of the standard set of questions, it becomes clear that these issues are just as important as questions on medical issues and need to be considered. In this way, they are prepared what might happen after transplantation and are supported in their decision making.Most previous studies report that only a very small percentage of donors perceived external pressure to donate a kidney, with estimates in the range of 5–10% ,32,33. TWhether these results can be generalized to other centers will probably depend on differences in cultural values, health care policies and waiting list systems. For instance, Martinez-Alarcón et al. have shown that the general attitude towards living versus deceased donor kidney transplantation is different in Spain, where the majority of patients prefers to wait for a deceased donor, most likely explained by the shorter waiting time in Spain compared to the Netherlands . AnotherIn conclusion, fear of donor-recipient relationship changes and concerns about the donor’s health seem more substantial in deceased donor kidney recipients, resulting in a decision to wait for a deceased donor despite having a potential living donor available. Further research is needed to assess whether this concerns a particular group of recipients and whether it is possible to eliminate their fears or take it into account in their decision making. If confirmed, information prior to living donor kidney transplantation should address expectations regarding potential relationship changes.The authors declare that they have no competing interests.IBG analyzed data and wrote the first draft of the manuscript; KS guided the focus groups, participated in study design and interpretation of the data; SD, PJMB, AMS and AGB helped with interpretation of the data and writing the manuscript; PJM conceived the study and its design, participated in the statistical analyses and interpretation of the data, and writing the manuscript. All authors have read and approved the final manuscript.This study was funded by the Dutch Kidney Foundation, program Patient Care.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/13/103/prepub


Our objectives were to examine US hospital laboratory troponin reports to determine whether either the 99th percentile or the 10% CV level were clearly indicated, and whether nonconcordance with these guidelines was a potential barrier to detecting clinically important microscopic or ‘microsize’ myocardial infarctions (MIs). To confirm past reports of the clinical importance of microsize MIs, we also contrasted in-hospital, 28-day and 1-year mortality among those with microsize and nonmicrosize MI.International guidelines recommend that the decision threshold for troponin should be the 99th percentile or 10% CV value were clearly identified. All cases were expert adjudicated for presence of MI. Peak troponin values were used to classify MIs as microsize MI  and nonmicrosize MI.In the REasons for Geographic And Racial Differences in Stroke national prospective cohort study , 1029 participants were hospitalized for acute coronary syndrome (ACS) between 2003–2009. For each case, we recorded all thresholds of abnormal troponin on the laboratory report and whether the 99th percentile or the 10% CV level; 52% of reports indicated an indeterminate range, a practice that is no longer recommended. There were 183 microsize MIs and 353 nonmicrosize MIs. In-hospital mortality tended to be lower in the microsize than in the nonmicrosize MI group , but 28-day and 1-year mortality were similar .Participants were hospitalized at 649 acute care US hospitals, only 2% of whose lab reports clearly identified the 99Current practices in many US hospitals created barriers to the clinical recognition of microsize MI, which was common and clinically important in our study. Improved hospital troponin reporting is warranted. Tropoividuals -4,10-14,ividuals .th percentile. Troponin levels in normal reference populations are very close to zero, and recent advances in biomarker assays permit detection of levels as low as 0.006 μg/L [th percentile of a normal reference population or the level at which the assay achieves acceptable precision, defined by a coefficient of variation (CV) of 10% or better [A problem acknowledged by the Universal Definition and other preceding guidelines is that some assays may not be reliable at levels as low as the 99006 μg/L . However006 μg/L . Therefor better ,10-14. Ath percentile or 10% CV level must be clearly identified on hospital laboratory troponin reports and labeled as the decision threshold. However, many hospitals have for years reported an “indeterminate range,” which was useful in interpreting troponin relative to creatine kinase (CK)-MB fractions when troponin was first introduced. The upper limit of this indeterminate range was the World Health Organization (WHO) cut-point corresponding to abnormal CK-MB, and many reports continue to provide interpretive guidelines recommending that troponin values above this WHO cut-point represent a high likelihood of MI. Clinicians interpreting such reports today may be challenged to identify the more current, lower guideline-recommended decision threshold. Furthermore, as ST elevation MI is declining while non-ST elevation MI remains common [In order to apply this recommendation, the 99s common ,17, tropWe studied US hospital laboratory troponin practices in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study, a large national prospective cohort study. We also examined mortality risks associated with very low, or ‘microsize,’ troponin peak MIs relative to MIs with higher peak troponin levels to both confirm the risks that have been described in other populations and to highlight the potential clinical relevance of hospital troponin reporting practices.The REGARDS study is prospectively following 30,239 individuals for cardiovascular events and mortality to better understand regional and racial influences on stroke and MI mortality. Details of the study are described elsewhere . Brieflyth percentile for healthy adults or the 10% CV level for the assay utilized and whether either was clearly identified as the decision threshold for abnormal troponin. We also examined whether an indeterminate range was provided, a practice that is no longer recommended [We included the first hospitalization for ACS for each participant occurring between 2003 and 2009. ACS was defined as an urgent presentation of signs and symptoms suggestive of acute coronary ischemia resulting in hospitalization. For each case, we examined the hospital laboratory troponin report for documentation of the 99th percentile or 10% CV value if clearly identified on the report, or if not clearly identified, twice the lowest listed upper limit of normal (ULN). MIs were adjudicated as definite, probable, or possible, and only definite or probable MI events were included in this study as MI events. The highest and lowest levels of troponin and upper limits of normal were also recorded.Expert adjudicators classified cases as ACS without MI or ACS with MI following approaches used in most large epidemiology studies . For MI,After adjudication, cases were further classified into microsize MI or usual MI based on peak troponin levels. Microsize MI was defined as an adjudicated MI with peak troponin no more than five times the lowest listed ULN, and usual MIs were all other adjudicated MIs. We examined clinical characteristics of individuals and in-hospital, 28-day and 1-year age, sex, and race-adjusted mortality in each of the three ACS groups of no MI, microsize MI and usual MI.We examined differences in participant characteristics among the three ACS groups, no MI, microsize MI, and usual MI. Available characteristics included sociodemographics  and medical conditions . Physiologic variables included body mass index, low-density lipoprotein cholesterol (LDL-C) levels, high density lipoprotein cholesterol (HDL-C) levels, triglyceride levels, and creatinine levels.After conducting a descriptive analysis, we constructed a logistic regression model to examine associations with microsize MI relative to usual MI, entering the above clinical characteristics into a multivariable model that included all cases of MI. Mortality for ACS, microsize MI, and usual MI was calculated by dividing the number of deaths in the period among those at risk. Specifically, in-hospital mortality was the number of deaths prior to discharge among those hospitalized; 28-day mortality was the number of deaths at 28 days among those surviving to discharge; and 1-year mortality was the number of deaths at 365 days among those surviving to 28 days. These proportions were adjusted for age, race, and sex and tested for statistically significant differences. All analyses were carried out using SAS version 9.1, Cary, North Carolina.No ULN for troponin was provided in 35 laboratory reports, and these hospitals were excluded, resulting in the inclusion of 649 different hospitals located in 490 US cities in 45 states. The type of troponin was troponin-I in 76% of hospitals and troponin-T in 7%; the type of troponin was not specified in 17% of reports.th percentile or the 10% CV level, but only one hospital advised clinicians that the 99th percentile should be used as the decision threshold for defining myocardial necrosis. A single ULN was provided in 48% of hospitals without specification of whether it was the 99th percentile or the 10% CV level. More than one ULN was provided in the remaining hospitals. When more than one threshold was reported, it was often in the context of ranges accompanied by interpretive language . In 13% of the hospitals, there were four or more such ranges.Sixteen (2%) hospitals specifically identified either the 99th percentile values and 10% CV levels  website for 99th percentile and 10% CV values can be approximated using the IFCC table even when no manufacturer is specified on the laboratory report; the decision threshold during the observation period should have been 0.03 [Because only a single manufacturer (Roche) distributed troponin-T assays during the study period, the 99een 0.03 ,19. HoweAdditional evidence that some hospital laboratories were providing thresholds well above the recommended decision thresholds came from 56 reports in which the ULN provided was higher than the 10% CV level of any assay reported by the IFCC ,19. ThesOf the 1,029 cases of ACS included in the analysis, 493 were classified as ACS without MI, 183 as microsize MIs, and 353 as usual MIs , and in-hospital mortality was statistically different for those with ACS without MI compared to both microsize MI or usual MI Figure . Howeverth percentile or 10% CV level, and more than half of hospitals identified an indeterminate range classifying MI based on a higher threshold than the guideline-recommended approach [We found major deviations from guideline recommendations for troponin laboratory reporting in our study. Laboratory reports only rarely identified a 99approach ,20. In tAlthough our study was not able to examine the proportion of microsize MIs missed clinically by treating physicians as a result of hospital reporting practices, it is likely that some of these events were in fact not treated as MI. The potential misclassification of microsize MIs as “normal” has significant long-term prognostic implications. The higher long-term mortality risk we observed for those suffering microsize MIs was similar to past reports in which the in-hospital risks were low, but by one month and one year were similar to those with usual MIs ,22. ThisA recent report from Scotland illustrates the clinical importance of detecting microsize MIs . InvestiThese findings together with our results suggest that improved reporting of hospital troponin results in the US is warranted; the US now has a system of public accountability for the quality of health care that provides a ready infrastructure to enact rapid change. A quality indicator assessing whether the recommended decision threshold is clearly identified on laboratory reports and assessing whether indeterminate ranges are used could be added rapidly to standards for accreditation by the Joint Commission or to the Centers for Medicare and Medicaid Services’ pay-for-performance programs, such as the Premier Hospital Quality Incentive Demonstration ,28.Our study included a large proportion of black participants, which has not been the case in previous reports stemming from non-US samples . Our finth percentile or 10% CV value at each hospital at the time of the event. Like other large epidemiology studies, we had to rely on clinical laboratories from many hospitals to classify MI and, given the variations we described, it is possible that, while state-of-the art and rigorous, the adjudication process under-detected some microsize MIs. This challenge would bias our results towards finding less difference in mortality rates than may actually exist; thus, the mortality risks we describe may underestimate the true risks. Our team of experts only classified events as MI if there was a clear rising and/or falling pattern of troponin, and perennial low level elevations seen in conditions such as heart failure or renal failure would therefore not have been classified as MI; however, it is possible that some misclassification could have occurred. Unfortunately, this study was not designed to detect differences in clinical management in microsize and usual MIs, but recent reports suggest that this may be an important issue [Our study’s limitations include our inability to examine how many cases classified as microsize MI by our rigorous adjudication methods were also recognized as MI by the treating physicians. We also did not have available the 99nt issue . Future th percentiles or 10% CV levels in their troponin reports as recommended by national and international guidelines, complicating the detection of microsize MIs. This may result in misclassification of some individuals with MI, preventing optimal clinical management. Our study confirms the excess risks associated with microsize MI described in other populations, emphasizing the importance of recognizing these cases clinically. Prompt attention to improving the quality of hospital laboratory reporting of patient troponin results is warranted.Many US hospital laboratories did not report 99ACS: Acute coronary syndrome; CHD: Coronary heart disease; CK: Creatine kinase; CV: Coefficient of variation; ECG: Electrocardiogram; HDL-C: High density lipoprotein cholesterol; IFCC: International Federation of Clinical Chemists; LDL-C: Low-density lipoprotein cholesterol; MI: Myocardial infarction; REGARDS: REasons for Geographic And Racial Differences in Stroke; ULN: Upper limit of normal; WHO: World Health OrganizationThe authors declare that they have no competing interests.MS conceived of the study, participated in its design, adjudicated endpoints, and drafted the manuscript. GP participated in the study design, carried out the analyses and helped to draft the manuscript. CB participated in the study design, collected data, and helped to draft the manuscript. JH adjudicated endpoints and helped with critical redaction of the manuscript. TM conceived of the study, participated in its design, adjudicated endpoints, and critically revised the manuscript. SG adjudicated endpoints and helped with critical redaction of the manuscript. DG conceived of the study, participated in its design and critically redacted the manuscript. RP conceived of the study, participated in its design and critically redacted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/162/prepubCommercially available troponin assays obtained from the International Federation of Clinical Chemists, 2006-2008 15,17.Click here for file