The aim was to evaluate the patients with chronic kidney disease stage 5 (CKD 5) and their prospective renal transplant donors with regard to their renal replacement choices, and to assess the medical and non-medical factors that affect living-related renal donor selection. Over 24 months, consecutive patients with CKD 5 and their relatives were interviewed at presentation. Reasons for the choice of modality were analyzed; the prospective recipients and their donors were again interviewed separately and the medical and nonmedical factors that affected the donor selection were determined. A total of 1257 patients were enrolled. Conservative therapy, maintenance dialysis, and renal transplantation were chosen by 513 (40.8%), 320 (25.5%), and 424 (33.7%) patients, respectively. Only socioeconomic status affected the modality chosen. The age, gender, and donor availability did not emerge as significant factors. Patients or donors were likely to withdraw from transplant evaluation due to the absence of a voluntary donor, presence of a male donor, coercion not to donate, and the absence of reimbursement. The commonest cause of rejection of a donor was blood group incompatibility (45.8%), followed by diabetes mellitus (DM) or risk of DM (24%), renal disease (5.9%), hypertension (5.5%), and persistent cross-match positivity (5.1%). To improve donation rates, the donor’s spouse should be involved in the early stages of donor evaluation, financial support for the recipient has to be improved, and the apprehensions about complications of nephrectomy among the donors need to be alleyed. Male donors are at increased risk of leaving the program in the evaluation phase. Chronic kidney disease is a worldwide public health problem with an increasing incidence and prevalence. An increasing number of patients are treated with renal replacement therapy (RRT) – dialysis or transplantation. The annual incidence of end-stage kidney disease (ESRD) has doubled over the past decade to reach about 135 per million in Europe and a similar rate is seen in USA. It is expected to continue to rise at an annual rate of around 5–8%. In India, the annual incidence is 34–240 per million population.[Increasing elderly population and the diabetic epidemic worldwide are largely responsible for the increasing incidence of ESRD. The number of people with diabetes worldwide (currently about 154 million) is predicted to double within the next 20 years. This is In India, nearly 18,000–20,000 patients (10% of new ESRD cases) get RRT. In 2004,The social factors and the perception of the complications of donation by the donor, family members, or even the recipient can affect the act of voluntary donation. In India, the Human Organ Transplantation Act of 1994 and its amendments discourages unrelated transplant due to ethical reasons and to avoid exploitation of the financially disadvantaged people. Data from India on the factors that determine the donor selection are lacking. This study was an effort to explore the medical and nonmedical factors that affect the donor selection.The study was a prospective study on consecutive patients with chronic kidney disease stage 5 (CKD 5) who presented to the nephrology services of Christian Medical College, Vellore. The renal replacement options chosen by the patients and the medical and nonmedical factors that determine the living kidney donor selection were studied.Subjects with newly diagnosed CKD 5 from December 2006 to November 2008 were recruited. The diagnosis was based on clinical evaluation, imaging studies including ultrasound, and estimated glomerular filtration rate (GFR) calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation. Patients diagnosed as CKD or where chronicity was doubtful prior to the study period were excluded.The patients and the relatives were interviewed with regard to the planned renal replacement options. The prospective recipients and their prospective donors were interviewed separately. The medical and nonmedical factors that could possibly have a bearing on the selection and rejection or withdrawal of a donor were analyzed.t-tests for continuous variables, and logistic regression) were done using the SPSS version 14  software package.Descriptive statistics and tests of significance  had chosen conservative treatment. Poor finances influenced the decision in 466 (96.1%) of the 485 patients otherwise fit for transplantation. No suitable donor was available in 16.A similar trend was seen among the 320 (25.5%) patients who chose long-term dialysis as RRT. Here 217 (93.5%) of the 232 patients were fit to be a renal transplant recipient, and the decision was influenced by poor finances.Of the 1257 patients with CKD stage 5, 424 (33.7%) chose renal transplant as their modality of treatment and of these 382 (90.1%) came for recipient evaluation. Seventy-three (19.1%) of the 382 patients did not have any donor and that contributed to 35.3% (73/207) of the prospective recipients who left our transplant program during subsequent evaluation due to various reasons.We noted a trend toward older patients choosing maintenance dialysis as a modality of RRT and younger people choosing renal transplantation but it was not statistically significant. In addition, the number of patients choosing conservative treatment was not different across the various age groups suggesting age was not a major factor influencing the choice of therapy .Similarly, gender had no influence on the choice of therapy and the difference across the groups was not significantly different .Higher proportion of patient with better education and higher monthly family income chose renal transplant over the other modalities as shown in  and the The mean age of the recipients who came for evaluation was 38.47±13.02 years and the range was from 6 years to 65 years. The recipient population under study was predominantly male forming 74.6% of the cases. The majority of the prospective transplant recipients, i.e., 94.4%, had a level of education better than high school certificate and only one patient was illiterate .As per the modified Kuppusamy classification (urban), 18.1% ofThe commonest blood groups in both the recipient and the donor populations were O positive and B positive. The O positive blood group was seen in 50.5% of the recipients while it was seen in 34.6% of the prospective donors. As our program strongly encourages first-degree relatives to donate, most of the donors were near relatives. Mother was the donor in 21.2% of cases and brother, sister, and father were donors in 19.6%, 16.5%, and 10.4%, respectively. Spouse came forward for donation in 68 (15.2%) cases .The donor was first asked by the recipient to donate in 29.6% of cases; a first-degree relative came forward himself/herself in 28% of the cases. The physician helped with donor selection in 25.7% of the cases. In 46 (22%) of cases, there was at least one instance of an attempt to discourage donation and the donor’s spouse was the commonest identified cause in 47.8% of the cases. The commonest reason for a donor not coming forward to donate was spouse’s unwillingness. One case of donor withdrawing consent was due to medical reasons (horseshoe kidney) in the donor. Out of the 382 prospective recipients, 56.8% (217/382) were not eligible for reimbursement by governmental or nongovernmental institutions or by medical insurance .The commonest cause of donor rejection was ABO incompatibility in 45.8% followed by 24% with diabetes or risk of diabetes and renal disease in 5.9% (36/613) in prospective donors. We rejected donors if either of their parents was diabetic or if the status of diabetes was not known in parents and if more than one of the sibling was diabetic. We did consider some of the at-risk group prospective donors who were older and had no impared glucose tolerance. There was a delay (defined arbitarily as duration of more than 30 days) in getting a renal transplant surgery in 91.1% of patients. The medical reasons contributed to 29.6% of the delay. The commonest nonmedical reason was government procedures in 26.4% (101/382) .The risk factors we identified among the prospective recipients who left our program were the absence of any donor, presence of male donor, attempted coercion to donate, and absence of financial support by the governmental or nongovernmental institutions either fully or partly .Chronic kidney disease has become an important public health problem. Though life style modification, prevention and early detection of kidney disease prolong life with reasonable quality, the ever-widening gap between the numbers of people waiting for renal transplantation is all too familiar. In our country, our perception of the patient’s choice of the modality of RRT is that it is largely dependent on the means of financial support for transplantation followed by donor factors. We evaluated if that was really the case.per se is more in males. In a study in a French urban area, the male:female ratio was 2:1 after the age of 20 years onward.[We observed that age did not have a significant influence on the modality chosen. The males constituted 77.2% of the total population. This was comparable to the 69.6% as per the Indian CKD registry. Though is onward. Hospitals onward.There was no significant difference in the male:female ratio in the three modalities of therapy chosen by the patient suggesting that age and gender were not the major factors involved in choosing a particular modality of RRT. Even in countries where the treatment is state sponsored, males outnumber females. Some of the gender difference in prevalence may be due to risk factors shared by cardiovascular disease and end-stage renal failure, both of which are commoner in men.There was a significant difference in the monthly family income, education status, and socioeconomic status of the three treatment groups with higher income group and patients with better education opting for transplant and dialysis compared to the group that chose conservative treatment. In our study, only 7.6% were fully sponsored and 5.5% were partly sponsored by governmental or nongovernmental organizations for the treatment expenses. In 2004, poor Indians spent 40% of their income on health care; the rich spent about 2.4%. Studies We did not evaluated the “health-related literacy” as a part of our study with instruments like Short Test of Functional Health Literacy in Adults (STOHFLA) which would have affected the decision-making process. However, the majority of the prospective transplant recipients, i.e., 94.4%, had a level of education better than high school certificate and only one patient was illiterate.As per the modified Kuppusamy classification (urban), 18.1% ofThe number of patients who chose conservative treatment, dialytic treatment, and renal transplant were 40.8%, 25.5%, and 33.7%, respectively, as against the Indian CKD registry data of 75.1%, 22.5%, and 2.5%, respectively. Most of et al. from a state-funded hospital from the state of Tamil Nadu, India, had reported that two-thirds of their donor population was females and this had only marginally changed over the last 10 years.[For the 172 patients who either had a transplant or were in the final stages of evaluation, 70.9% were males. Females were the donors for 59.8% of the recipients. Muthusethupathi 10 years. Interest10 years.15The donors volunteered themselves for transplant evaluation in 28% of cases and in the rest, it was requested by the recipient or suggested by the recipient’s physician. Of all the donors who came for evaluation, 46 (22%) reported at least one instance of an attempt to discourage donation and donor’s spouse was the commonest (47.8%) identified cause. Similarly, the commonest cause for some of the possible donors declining to even come forward for donor evaluation was again the spouse refusing to give consent. And in the rest, no reason was disclosed. This emphasizes the fact that the donor’s spouse must be a part of decision making along with the donor to alley some of the fears associated with donation. In fact, despite the donors voluntarily donating the organ, 40.5% were either unsure or felt it was likely (38.7%) to have complications peroperative or have long-term morbidity like renal failure (1.8%). Imparting better education of the immediate and long-term complications with kidney donation to the general public and the prospective donors will for sure alley some of the fear about the surgery and its complications and may increase the donation rates.For the 382 prospective recipients, there were 1725 age compatible donors with 4.5 donors per recipient. Interestingly the male female ratio was approximately 1:1 among the total number of the donors . One-thiWe observed that compared to the group that successfully got a transplant or was waiting for the transplant surgery, the group of patients who left our center without renal transplant had more number of prospective recipients who did not have a donor (30%), had less female prospective donors (44.4%), their donors were more likely to be coerced not to donate (14%), and were less often sponsored (36.2%).To improve donation rates, the donor’s spouse should be taken into confidence in the early stages of donor evaluation; financial support for the recipient have to be improved; and the apprehensions about complications of the renal transplant surgery among the donors to be alleyed both for individual donors and the general public. The prospective recipients with male donors are at increased risk of leaving the program in the evaluation phase.


This new method is effective in the treatment of several intractable diseases and conditions, such as autoimmune diseases in aging, advanced malignant tumors, exposure to supralethal irradiation, multiple organ transplantation from different donors, and type 2 diabetes mellitus, for which conventional methods are ineffective. Our findings suggest that allo-HSCT + TT is preferable to conventional allo-HSCT alone or allo-HSCT + DLI. This method may become a valuable next-generation HSCT technique.Although allogeneic hematopoietic stem cell transplantation  has become a valuable strategy for some intractable diseases, a number of problems remain to be resolved. We have developed a new HSCT method, HSCT + thymus transplantation (TT) from the same donor, which induces elevated T cell function with mild graft- Howeve (GVHD) . Converse (GVHD) –11. Recee (GVHD) , 13. TheThe thymus is the organ in which T cells are differentiated and produced with induction of tolerance to the host. The thymus also regulates biological homeostasis using the cells with several cytokines and hormones . Therefo+ FoxP3− effector T cells (Teff  cells) but also CD4+ FoxP3+ regulatory T cells (Treg  cells), which prevent GVHD and autoimmunity [reg cells in HSCT + TT is lower than that in HSCT alone, but higher than that in HSCT + DLI, while the opposite is true for the percentage of Teff cells [We recently developed a method of allo-HSCT in conjunction with TT from the same donor –27. Thisimmunity , 29, proff cells . We haveFirst, we present the theory of allo-HSCT + TT . In the We examined the effects of HSCT + TT in treatment of several intractable diseases . Female We next investigated the effects of HSCT + TT in tumor-bearing mice . Mice trWe also examined the effects of HSCT + TT on leukemia . In contThe thymus atrophies with bone marrow suppression in hosts with advanced tumors, thus causing immunodeficiency, which is one of the major causes of death in such cases , 39. We Advanced age is one of the risk factors for unsuccessful BMT , 10. TheTransplantation of multiple organs from different donors is desirable in patients with several intractable diseases. Although grafting of the thymus itself can also induce tolerance , its effReduction of the intensity of the HSCT conditioning regimen will decrease the side effects for both host and donor. Therefore, we examined the effects of HSCT and TT for two conditions, that is, low-dose irradiation ), which attenuates host damage, and low numbers of BMC (injection of low numbers of BMC (ILNBMC)), which reduces the burden on the donor . BMT + DThere is increasing evidence that both autoimmune and autoinflammatory mechanisms are involved in the development of not only type 1 diabetes mellitus (T1 DM) but also type 2 DM (T2 DM) , 43. TheAs described above, allo-HSCT + TT is functionally superior to either conventional HSCT alone or HSCT + DLI for several intractable diseases and conditions. Elevation of T cell function with low GVHD facilitates engraftment of donor cells in HSCT + TT, and these allo-T cells may work more effectively and safely in treatment of several diseases compared to HSCT alone or HSCT + DLI.eff and Treg cells from the transplanted allothymus. Although the detailed mechanism is not yet clear, it suggests that thymic antigen-presenting cells such as dendritic cells (DC) and/or medullary thymic epithelial cells (mTEC) in the transplanted thymus may play important roles with the antigen [The elevation of T cell function in HSCT + TT is due to the newly developed T cells from the transplanted thymus . In addi antigen , 45–49  , 27. AltHowever, it is both ethically and technically difficult to obtain adequate thymus tissue for clinical use. In this respect, grafts could be obtained from patients with congenital heart diseases or from aborted fetuses, as utilized previously , 16. TheThe findings presented here indicate that allo-HSCT + TT is more effective against several intractable diseases compared with conventional allo-HSCT methods. This method may become a valuable strategy for the treatment of various diseases in humans.


P < 0.001 for PFS, P < 0.001 for OS, respectively). Cox regression analyses revealed that 1p/19q co-deletion and 19p polysomy were independent prognostic factors (P < 0.05). Pearson’s Chi square test demonstrated GBMO-STS exhibited lower 1p/19q co-deletion, IDH1 mutation rates than AOA or GBMO-LTS  but higher chromosome 1q, 19p polysomy rates compared with AOA or GBM . Patients with glioblastomas with oligodendroglioma component concurrent with polysomy for chromosomes 1 and 19 always confers an unfavorable prognosis which needs our extra attention in clinic.This study was designed to display the molecular genetic features of short-term survivors in glioblastomas with oligodendroglioma component (GBMO). A total of 186 patients with histological diagnosis of primary gliomas, including 11 GBMO-STS , 29 GBMO-LTS , 36 anaplastic oligoastrocytoma (AOA) and 110 glioblastoma multiforme (GBM), enrolled in the study. An evaluation form was developed and used to document molecular pathological, clinical and treatment-associated parameters between subgroups. Kaplan–Meier plots for survival showed that the median progression-free survival (PFS) and overall survival (OS) of GBMO-STS were 5.0 and 10.0 months, respectively. Intergroup comparison revealed that the GBMO-STS harbored the most dismal prognosis than those with AOA, GBMO-LTS or GBM (The online version of this article (doi:10.1007/s11060-013-1311-3) contains supplementary material, which is available to authorized users. The latest WHO classification of tumors of central nervous system (CNS) introduced an entity–AOA with necrosis was to be diagnosed as glioblastomas with oligodendroglioma component (GBMO). As a new pathology entity, the studies devoted to revealing the prognosis of GBMO were relatively limited.Increasing evidence suggested GBMO was a heterogeneous group with considerable survival variant. Several reports found that the survival of GBMO was significantly longer than glioblastoma multiforme (GBM) whose median overall survival (OS) was only 12–15 months in spite of multimodal aggressive treatment, comprising surgical resection, local radiotherapy and systemic chemotherapy . NeverthGBMO displayed OS ≤12.0 months is a relatively rare event which is worthy of further inquiry. It has not been firmly established which, if any, of the molecular genetic aberrations is important for the pathogenesis of GBMO-STS represent prognostic factors. The identification of molecular genetic markers that are associated with survival in patients with GBMO would be beneficial for its diagnostic and prognostic potential. In this regard, we report a retrospective analysis of 186 primary high-grade gliomas recruited in the Beijing Tiantan Hospital Neurosurgery Department. In addition to basic clinical data, we evaluate the clinical characteristics and screen for glioma associated genetic aberrations, i.e. 1p/19q co-deletion, IDH1 mutation, MGMT promoter methylation, PTEN, p53, Ki-67, EGFR, VEGF expression, as well as chromosome 1q, 19p polysomy.A total of 186 patients  with histological diagnosis of primary supratentorial high-grade gliomas  in Beijing Tiantan Hospital from May 2008 to May 2011 were enrolled in the study. The secondary GBM were excluded. All patients provided written informed consent for the current study and the clinical study was approved by the Medical Ethics Committee of Capital Medical University. The mean age of this cohort was 47.5 ± 12.5 years at the time of surgery. All specimens were independently re-evaluated by three experienced neuro-pathologists according to the 2007 WHO classification of the CNS tumors  were considered as being methylated.The progression-free survival (PFS) was designated as the time period from the first operation to the time of tumor recurrence or evidence of progression based on magnetic resonance imaging (MRI). Patients who were recurrence-free at last follow-up were considered as a censored event in analysis. OS was defined as the period between the first operation and death or last follow-up. Patients who were still alive at last follow-up were considered as a censored event in analysis. All the survival data were collected mainly when patients visited the clinics and during the phone interview with patients and/or their relatives.P < 0.05.SPSS 13.0  was used for statistical analysis. Pearson’s Chi square test and Fisher’s exact test were used to compare the frequencies between groups. Kaplan–Meier method was used for survival analysis. Probability value was obtained from two-sided tests, with a statistical significance of The basic clinical characteristics of these patients enrolled in the study were summarized in Table P < 0.05) . In multivariate Cox regression analysis, the presence of 1p/19q co-deletion and 19p polysomy were independent prognostic factors (P < 0.05) (Table P > 0.05).In the cohort 186 high-grade glioma, univariate analysis demonstrated that 1p/19q co-deletion, polysomy for 1q and 19p were associated with prognosis (5) Table . UnexpecP < 0.001 for PFS, P < 0.001 for OS, respectively) . Patients in subgroup 3 had the shortest survival time that the median PFS was 11.0 months and the OS was only 17.5 months. No significant difference of prognosis was observed between subgroup 1 and 4  , we could classified the 186 patients into four subgroups  which conferred different survival time. Patients in subgroup 2 exhibited the most favorable prognosis compared with subgroup 1, 3 and 4 .Fluorescence in situ hybridization (FISH) for chromosome 1p and 19q was available in 121 cases. Among them, 28 (23.1 %) had 1p/19q co-deletion . The frequencies of 1p/19q co-deletion in AOA, GBMO-STS, GBMO-LTS and GBM were 40.0, 0.0 %, 37.9 and 6.5 %, respectively. The frequency of 1p/19q co-deletion in GBMO-STS was significantly lower than those in AOA or GBMO-LTS .DNA sequencing for IDH1 was available in 137 patients. Among them, 38 out of 137 (27.7 %) had IDH1 mutation . The frequencies of IDH1 mutation in AOA, GBMO-STS, GBMO-LTS and GBM were 42.4, 0.0, 55.2 and 12.3 %, respectively. The frequency of IDH1 mutation in GBMO-STS was significantly lower than those in AOA or GBMO-LTS . Furthermore, GBMO-STS exhibited higher 19p polysomy rate than AOA or GBM   had 1q ploysomy ; 46 (37.4 %) had 19p ploysomy . 1q polysomy was slightly more frequent in GBMO-STS compared with AOA or GBM (Anaplastic oligoastrocytoma (AOA) with necrosis, formerly categorized in WHO grade III, now is regarded as GBMO (WHO grade IV) which is a heterogeneous group with considerable survival variant. Several reports dedicated to the survival analysis about GBM versus GBMO showed that no favorable prognostic value of an oligodendroglial component was found –6, althoP = 0.048), no other significant difference was observed between the four subgroups   influenced the patients’ survival, we recruited all the clinical factors in the present study. We found that except for the age at diagnosis of patients with GBM was older than those in GBMO-STS (M) Table . But theIn our cohort, we found that the GBMO-STS exhibited lower frequency of 1p/19q co-deletion than AOA or GBMO-LTS but resembled it with GBM. This finding was similar with Jiang’s report, which maintained there was no significant difference of 1p/19q co-deletion rate between GBMO and GBM . 1p/19q As elaborated by experts, IDH1 mutation was associated with a better outcome in patients with low-grade diffuse gliomas, AA, GBM and had been shown to be a powerful independent prognostic factor for prolonged survival , 20. IDHUnivariate analysis revealed that polysomy for 1q and 19p were associated with the dismal prognosis of patients with high-grade gliomas. These findings were in consistent with many previous reports. Snuderl et al.  reportedFrom the above, experts, all over the world, in regard of the prognostic value of an oligodendrogial component in glioblastomas couldn’t arrive at a consensus Table . Based oThe small sample of GBMO-STS which would weaken the conviction of this study to some extent. So we will enlarge our sample for the further inquiry in the future. It was also a limitation that the EGFR amplification information was absent in distinguishing GBMO from small cell GBM.Patients with GBMO concurrent with polysomy for chromosomes 1 and 19 always confers an unfavorable prognosis which needs our extra attention in clinic.Supplementary material 1 (TIFF 197 kb)Supplementary material 2 (DOCX 22 kb)Supplementary material 3 (DOCX 15 kb)Below is the link to the electronic supplementary material.


Radionuclide Cisternography (RNC) is of potential value in pointing out the sites of cerebrospinal fluid (CSF) leakage in patients with spontaneous intracranial hypotension (SIH). In the current report, we present two patients who underwent RNC for suspected CSF leakage. Both patients underwent magnetic resonance imaging (MRI) and RNC for evaluation. We describe a simple method to increase the detection ability of RNC for CSF leakage in patients with SIH. Spontaneous intracranial hypotension (SIH) is a condition which is caused by spontaneous spinal cerebrospinal fluid (CSF) leakage without a history of dural trauma, which results in low CSF pressure. The most common neurologic symptom caused by low CSF pressure is headache, which is often orthostatic, i.e., occurs or worsens in the upright position and improves when the patient is recumbent -5. EvideA 45-year-old woman had a sudden onset of severe headache over bilateral temporal and frontal regions for one week. Her headache worsened in the upright position and improved after lying down. Physical examination, routine blood tests and chest radiography on admission were normal. A CSF study after a lumbar puncture showed a CSF opening pressure of 3.5 cmH2O, 3 erythrocytes, 1 leukocyte, 1 lymphocyte/cumm, a protein level of 42 mg/dl  and a glucose level of 65 mg/dl. Results of all spinal fluid cultures were negative. Brain MRI showed no mass effect but pachymeningeal enhancement at the bilateral cerebral convexity with more marked enhancement at the left frontal convexity on contrast-enhanced T1 weighted study  was seenA 36-year-old woman was admitted with sudden onset of positional headache for 9 days. Her headache occurred when sitting up or standing up for 20-40 minutes and was relieved when lying down. The patient had no history of spinal trauma, including previous lumbar puncture, surgery or accident. Lumbar puncture and CSF study showed a CSF pressure of 0 mmH2O. Brain magnetic resonance imaging was negative. An RNC was performed using the same protocol mentioned above. Areas of tracer accumulation along both sides of the paraspinal areas at the upper thoracic level and lumbar level were demonstrated on 1-hour, 3-hour and 6-hour images. Immediately after the 6-hour supine imaging, the images taken in the sitting position showed clearer tracer accumulation along both sides of the paraspinal areas at the upper thoracic level and the right side of the lumbar level. Furthermore, additional sites of CSF leakage were identified at both sides of the paraspinal areas at the thoracic and lumbar levels, especially at the lumbar spine . EBP usiRNC is of potential value in diagnosing CSF leakage for SIH.5 Extra-dural accumulation of radioactivity is a direct sign of CSF leakage , 7. In aPotential technical errors during lumbar puncture may result from incomplete penetration of the subarachnoid space by the needle. The RNC images usually reveal intense and focal accumulation of radioisotope in the extra-spinal space at the lumbar level. In addition, non-visualization of the upper spinal region or slow ascent of radioisotope to the upper spinal region may also occur. In our two cases, smooth ascent of radioisotope from the lumbar area to the cerebral convexity and no obvious accumulation of radioisotope in the lumbar region were demonstrated which indicated a successful injection of the radioisotope via the lumbar puncture.Complications from lumbar puncture include herniation, headache, cranial neuropathies, nerve root irritation, low back pain, infection and bleeding. The most life-threatening complication is herniation, which may result from a large pressure gradient between the cranial and lumbar compartments. Headache is the most common complication occurring in approximately thirty-six percent of patients within 48 hours after the procedure. Headaches may be caused by CSF leakage through the puncture site at a rate that exceeds the rate of CSF production. SufficieDifferential diagnosis in the conditions causing focal areas of radionuclide concentration or retention including radionuclide leakage following lumbar puncture or neurosurgical procedures, spinal meningeal diverticula (type I and type II = Tarlov’s cyst), CSF fistula, CSF leak, subdural hematoma, subdural hygroma, myelomeningoceles, congenital anomaly of meningoceles, congenital arachnoid cysts, Sturge-Weber syndrome and Dandy-Walker cysts. RadiologTo our best knowledge, there is no similar report about usage of the upright method to increase the detection of CSF leakage in SIH. In a case reported by Caroline et al., extra-sIn conclusion, RNC is useful in diagnosing and managing SIH because it can identify the CSF leakage. We emphasize that hours in the lying position followed by the upright position may be effective in CSF leakage detection, which provides accurate diagnosis and proper treatment of SIH.


Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental and genetically heterogeneous disorder with decreased head circumference due to the abnormality in fetal brain growth. To date, nine loci and nine genes responsible for the situation have been identified. Mutations in the ASPM gene (MCPH5) is the most common cause of MCPH. The ASPM gene with 28 exons is essential for normal mitotic spindle function in embryonic neuroblasts.We have ascertained twenty-two consanguineous families with intellectual disability and different ethnic backgrounds from Iran. Ten out of twenty-two families showed primary microcephaly in clinical examination. We investigated MCPH5 locus using homozygosity mapping by microsatellite marker.Sequence analysis of exon 8 revealed a deletion of nucleotide (T) in donor site of splicing site of ASPM in one family. The remaining nine families were not linked to any of the known loci .More investigation will be needed to detect the causative defect in these familiesWe detected a novel mutation in the donor splicing site of exon 8 of the ASPM gene. This deletion mutation can alter the ASPM transcript leading to functional impairment of the gene product. Autosomal recessive primary microcephaly  is clinically characterized by an occipitofrontal head circumference (OFC) of at least three standard deviations (SDs) below the expected mean for age and sex. The disMicrocephaly is divided into primary (present at birth) and secondary microcephaly . The birClinical examinationWe evaluated 22 families with two or more intellectual disability patients referred to the Genetics Research Center, Tehran, Iran. These families were from various ethnicities and different provinces of Iran. Informed consents were taken from the family members who participated in this study. Ten out of twenty-two families showed primary microcephaly. Consanguinity was observed in 7 out of these 10 families. On examination, head circumferences were -3 to -13 SD below the population age and sexrelated mean values. All parents had normal intelligence scores and normal head circumferences .DNA Extraction and GenotypingDNA was extracted from peripheral blood lymphocytes following a standard protocol. A panel of 70 microsatellite markers was selected from the Genome Databases (http://www.gdb.org/ and http:// genome.ucsc.edu/). Population-specific allele frequencies were available for the Iranian population, because of previous studies in Iran. polymerase chain reaction (PCR) amplification of the microsatellite markers are performed. Polyacrylamide gel electrophoresis and standard silver stain protocol were used to visualize the PCR products. When the haplotype at a MCPH locus was found to be homozygous in all affected members of a family, mutation screening was initiated. If different homozygous haplotypes or heterozygous markers were found in the affected individuals, the respective locus would be excluded.Sequencing of ASPM All 28 exons, and exon/intron splice sites of the ASPM gene , of the family linked to the MCPH5 locus on chromosome 1q31, were sequenced using a set of 33 PCR primers (designed with the Primer3 software). Sequences were compared with the reference genomic and cDNA sequence (NM_018136).Clinical FindingsThe consanguineous Iranian family with primary MCPH (9000013) had two affected subjects, including two males  with ageHead circumference of the two affected individuals was 10 to 13 SD below the expected mean for sex and age and they (V: 1 and V:2) had severe intellectual disability (intelligence quotient [IQ] of 30 to 35). They were unable to read or write and did not have basic self-care skills. With the exception of intellectual disability, there were no other neurological and motor development problems. The parents had normal head circumference and normal intelligence .Genotyping and Mutation AnalysisLinkage was performed using STR markers present within the known MCPH loci. Linkage of a family to MCPH locus was based on the observation that all affected individuals had the same homozygous pattern. One out of ten families showed homozygosity at MCPH5 locus (family [9000013]).Sequence analysis of exon 8 in 2 affected individuals  and in parents  revealed a deletion of nucleotide (T) in donor site of splicing site . This deThe MCPH5 has been shown to be the most prevalent MCPH loci in Iran with a frequency of 13.3% . It was In this study, one family showed linkage to MCPH5 (ASPM). We detected a novel mutation in the donor splicing site of exon 8 of the ASPM gene. This deletion mutation can alter the ASPM transcript leading to functional impairment of the gene product. The ASPM gene with 28 exons is the human ortholog of the Drosophila melanogaster ‘abnormal spindle’ gene (asp), which is essential for normal mitotic spindle function in embryonic neuroblasts , 23 Fig. ASPM was highly expressed in fetal and adult human tissues with lower levels in adult tissues. The predicted full-length protein contains 3477 amino acids and has a calculated molecular mass of 410 kD. ASPM contains two conserved regions, ASPM N-proximal (ASNP) repeats, and C-terminal calmodulin-binding IQ motifs with variable length. Immuno-staining of cultured human cells revealed that ASPM was localized in the spindle poles during mitosis . ASPM geThe remaining nine microcephalic families were not linked to any of the known loci. These families will help to refine the mapping of the other MCPH loci or the genes which have not been identified.


In Bangladesh diarrhoeal disease and respiratory infections contribute significantly to morbidity and mortality. Handwashing with soap reduces the risk of infection; however, handwashing rates in infrastructure-restricted settings remain low. Handwashing stations – a dedicated, convenient location where both soap and water are available for handwashing – are associated with improved handwashing practices. Our aim was to identify a locally feasible and acceptable handwashing station that enabled frequent handwashing for two subsequent randomized trials testing the health effects of this behaviour.We conducted formative research in the form of household trials of improved practices in urban and rural Bangladesh. Seven candidate handwashing technologies were tested by nine to ten households each during two iterative phases. We conducted interviews with participants during an introductory visit and two to five follow up visits over two to six weeks, depending on the phase. We used the Integrated Behavioural Model for Water, Sanitation and Hygiene (IBM-WASH) to guide selection of candidate handwashing stations and data analysis. Factors presented in the IBM-WASH informed thematic coding of interview transcripts and contextualized feasibility and acceptability of specific handwashing station designs.selection of candidate designs were market availability of low cost, durable materials that were easy to replace or replenish in an infrastructure-restricted and shared environment. Water storage capacity, ease of use and maintenance, and quality of materials determined the acceptability and feasibility of specific handwashing station designs. After examining technology, psychosocial and contextual factors, we selected a handwashing system with two different water storage capacities, each with a tap, stand, basin, soapy water bottle and detergent powder for pilot testing in preparation for the subsequent randomized trials.Factors that influenced A number of contextual, psychosocial and technological factors influence use of handwashing stations at five aggregate levels, from habitual to societal. In interventions that require a handwashing station to facilitate frequent handwashing with soap, elements of the technology, such as capacity, durability and location(s) within the household are key to high feasibility and acceptability. More than one handwashing station per household may be required. IBM-WASH helped guide the research and research in-turn helped validate the framework. Poor water quality and hygiene are major contributors to the spread of diarrhoeal disease and acute respiratory infections (ARI). Estimates suggest that in 2011 there were 1.3 million ARI-related deaths and 700,000 diarrhoea-related deaths among children under the age of five  to clean my hands before food preparation because it is placed far away from where I cook. But now I wash regularly with the kitchen handwashing station before cooking., “All of my family likes the bucket handwashing station because after washing hands the waste water is stored in the bowl, and the handwashing station doesn’t get muddy underneath.”Operating at the Individual level were reactions to the handwashing station, such as attractiveness and quality. Participants testing the bottle with metal valve viewed the reclaimed 2 litre plastic bottle and metal holster as lacking durability and that the design would not withstand frequent use. For others, appearance and perceived value contributed to the acceptability of a handwashing station in the household. Both the 40-litre bucket and the kitchen bucket were brightly coloured, and installed complete with a water receptacle and a stool to place the bucket upon. Users reported that these features made these handwashing stations attractive. Some noted that the water receptacle kept the area surrounding the handwashing station clean and dry, and the stool added stability. For example, one participant saidOperating at the Interpersonal / Household level were factors influencing how a handwashing station was used and shared by households and their neighbours.bodna, or soapy water bottle when used by a large number of people, required frequent refilling and were not conducive to repeated use throughout the day. One participant said, “The size of the bottle [with pump] is small so we need to refill it frequently, but sometimes we forget.” Most participants using handwashing stations with small storage capacities acknowledged that it was not feasible to use the handwashing station at all key times—after defecation or cleaning a child’s bottom, and before food preparation or feeding children.Because handwashing station designs needed to be refilled with water and soap, the frequency of regular maintenance influenced acceptability of the designs. Handwashing technologies with smaller capacity such as the bottle with valve, In the urban field site, several participants mentioned concerns regarding shared access to a handwashing station placed next to a shared latrine and the implications this had on maintenance among sharing households. Even with the large storage capacity of the drum, one urban participant said,In the last few days, when water and soap have run out, I have managed to refill it. But our compound environment is not good. After some time the renters change, so who will take responsibility? Taking care of the soap and water is not possible for everybody. There is no good place to install the drum… [and it] can be broken. Then, quarrels arise. So, single ownership is better.bodna, 40 L bucket, and kitchen bucket said they moved the handwashing station inside at night, so that it would not be stolen. In both urban and rural sites, the large size of the soapy water bottle and the use of inexpensive detergent in the mixture was advantageous compared to bar soap because it was not attractive to steal and was less likely to be misplaced by children.Not only was use and maintenance difficult to negotiate when the handwashing station was accessible to multiple unrelated households, but theft and damage were also concerns. Several participants using the “In the last few days we are becoming habituated to hand washing, and now if we don’t wash our hands then we feel bad.” Several participants mentioned that though they had past experience with washing hands after defecation, routine use of the handwashing station increased their hygiene habits to include handwashing before cooking and eating.At the Habitual level, past handwashing experiences influenced current attitudes and practices. Many of our participants shared that they were not habituated to using soap and water for handwashing during all key times. However, some participants noted that their newly acquired experience of a designated handwashing station facilitated habit formation. One participant described how her household’s frequent use of the drum changed how they felt about handwashing, bodnas are traditionally used for anal cleansing after defecation, using it as a multipurpose handwashing station rendered this design unacceptable in both urban and rural sites. Similarly, placing other handwashing station designs too close to latrines elicited feelings of disgust when used for handwashing prior to food preparation, eating, or feeding a child. This was particularly pronounced in those cases where the handwashing station was in a location accessible to multiple households.At the Individual level, knowledge of handwashing station use, self-efficacy, and attitudes toward threats did not significantly distinguish between handwashing station designs, nor seemed to influence handwashing practice. All participants knew how to use their handwashing station. They were fully capable of using the handwashing stations and soap, indicative of self-efficacy. However, some handwashing station designs and locations were preferred or avoided due to feelings of disgust related of defecation. Because “Everybody should wash their hands regularly,” indicating that some level of hygiene was expected.At the Interpersonal / Household level, participants explained how different members of the family interacted with the handwashing stations designs. For example, mothers often described how their children learned to use the handwashing station, suggesting that handwashing was part of a parent’s nurturing role. In addition, participants in both urban and rural sites alluded to descriptive norms for handwashing. Though many lacked established handwashing routines, several participants stated, Age was an important factor in use of the handwashing station because age often indicated who was in the home and how easy a handwashing station was to use. A child’s age and developmental stage was extremely important in determining acceptability and feasibility. The drum had low acceptability for some participants because children might leave the tap running and muddy the floor. Many participants expressed concerns about children using the soapy water bottle [Phase 1] because it was too heavy for young children to lift and they preferred the modified design with a pump [Phase 2] instead.At the Interpersonal / Household level, gender roles and responsibilities of household members played a large part in determining handwashing station use and maintenance. Women came in more frequent contact with soap and water for household chores than their male counterparts, were more likely to be in charge of teaching children to wash hands, and were primarily responsible for refilling and maintaining the handwashing station. In both urban and rural sites, several participants noted that men worked and socialized outside of the home, so they did not have access to a handwashing station throughout the day, and their role in maintenance was limited.At the Community level interviews with participants revealed that the built environment shaped response to handwashing designs. Access to water had a critical impact on functionality of the handwashing station, especially in designs with small water storage capacity. In the urban site, access to water was shared and determined by electrical pumps drawing from a low pressure and intermittent municipal supply. One participant described this problem saying, “In a slum, our hands become dirty the whole day. Moreover, electricity is absent, so water is not available… Water from the bodna is finished after one person washes his or her hands.”“Our mobility inside the room was interrupted due to the installation of the handwashing station because it is congested inside the room.” This was another reason to test the smaller kitchen bucket.In the urban site, living quarters were small and densely arranged. Finding a convenient location to install a large handwashing station was difficult because living space was at a premium. One participant noted, “If it is stolen, we won’t be able to replace it because the pumper is not available.” Another participant commented on the feasibility of stocking a handwashing station with soap if not provided through the study, saying that soap was expensive and rare in their household.Lastly, urban households were more likely to be engaged in income generating activities, and have more access to urban markets than rural households. Participants in the urban site often assessed the handwashing station design they had received in terms of availability of replacement parts at the market. One participant said that the soapy water bottle with a pump was not a good option because, bodna was associated with disgust related to defecation and was deemed inappropriate for other handwashing purposes; and the drum was not very attractive, less stable, and left the area surrounding the handwashing station a muddied mess. Due to the numerous advantages that the soapy water bottle designs presented over bar soap —low cost, easy to make, and not easily stolen, —we included the soapy water bottle as the method of soap delivery for use in our final handwashing station designs.Some of the candidate handwashing station designs were eliminated due to significant factors effecting feasibility and acceptability. The bottle with valve was not durable and held small amounts of water; the The bucket handwashing stations were also incorporated into our final handwashing station designs Figure . They weThe study explored factors influencing the feasibility and acceptability of candidate handwashing station designs in two infrastructure-restricted settings in Bangladesh, using the IBM-WASH framework. An acceptable and feasible handwashing station in our case required more than the presence of soap and water. Instead, attributes specific to the handwashing station technology, such as water storage capacity, durability, and maintenance requirements; contextual factors such as consistent access to water, the physical location of the technology within a household’s daily workflow; and psychosocial factors such as disgust, all contributed to how a handwashing station facilitated or inhibited handwashing at key times.Ease of use the physical act of operating the handwashing station facilitates habit formation , and thiThe development of the IBM-WASH was concurrent with this formative research in which we were able to develop operational definitions of factors and examine interactions between dimensions of the IBM-WASH. Some of the factors related to the technology and contextual dimensions were organized in the framework based on the data in this paper . In thatRegarding contextual factors, provision of a soapy water bottle without a water storage vessel helped to convert an existing tube well or hand pump to a handwashing station where the former provided reliable water access. However, this did not address the convenience of a second station at the food preparation area. It soon became evident that more than one handwashing stations might be required. This eventuality was not originally anticipated.Psychosocial factors such as disgust towards faeces has been described in the literature as a motivator to handwash ,24. In tPhysical attributes of the handwashing station included in the IBM-WASH and other models ,24 are eAdditionally, our findings suggest that handwashing station components must coexist with the surrounding household environment. Figueroa and Kincaid discuss how attitudes toward a product can positively or negatively influence use . FurtherWe focused our analysis on factors occurring in the Habitual, Behavioural, Individual and Interpersonal/Household Levels of IBM-WASH for two reasons. First, Community and Societal/Structural factors shaping the feasibility and acceptability of handwashing station designs were already considered in selecting candidate designs. Second, the subsequent RCTs were to measure the effects of handwashing that occurs in the household, hence our formative research had the most direct relevance to the Behavioural, Individual, and Household Levels of our conceptual framework.This research focused largely on the acceptability and feasibility of different handwashing station designs. Another study is necessary to measure the effectiveness of a chosen handwashing station on actual handwashing practice. Because handwashing stations were provided free of charge, there was likely a positive bias toward handwashing hardware, and in particular to the more costly designs. Likewise, the study did not include measures for desirability or market value. However, in the urban site, there were references to perceived product value, indicating that such measures would be beneficial. Specific questions targeting factors at the Community level, such as access to markets and resources and shared community values surrounding hygiene, may have provided greater context for selecting a handwashing station design for programmatic use, beyond a randomized trial.A number of contextual, psychosocial and technological factors influence use of handwashing stations at five aggregate levels, from habitual to societal. In interventions that require a handwashing station to facilitate frequent handwashing with soap, elements of the technology, such as capacity, durability and location(s) within the household are key to high feasibility and acceptability. Access to water, convenience, and disgust towards faeces-related handwashing stations may require more than one handwashing station per household.Using constructs outlined in IBM-WASH may help identify key components of a behavioural intervention and key multi-level determinants influencing behaviour change in specific settings. Though the findings from this research were used to directly inform an intervention for subsequent research, this methodology may be used to develop successful interventions for both programmatic and research purposes.The authors declare that they have no competing interests.KRSH conducted qualitative data analysis, the synthesis and interpretation of results and was the primary author of the final manuscript. PJW provided conceptual guidance for data analysis and manuscript development. PJW, EL, LU, FN, PK, AA and SL conceived of the study design and provided support for field trials and data collection. PJW, EL, RD and KRSH contributed to the development of the IBM-WASH-structured analytical approach. AA and NCD provided detailed information on study methods and lead the qualitative field research team. All authors provided feedback and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/877/prepub


Immunohistochemical studies have revealed that cystatin C (CysC) co-localizes with amyloid-β (Αβ) in amyloid-laden vascular walls and in the senile plaque cores of amyloid. In vitro and in vivo animal studies suggest that CysC protects against neurodegeneration by inhibition of cysteine proteases, inhibition of Αβ aggregation, induction of autophagy and induction of cell division. CysC levels may be altered and may have a potential link with cerebrospinal fluid (CSF) Aβ levels in various types of dementia with characteristic amyloid deposits, such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and the atrophic form of general paresis (AF-GP). We assessed the serum and CSF levels of CysC and the CSF levels of Aβ40 and Aβ42 in patients with AD (n = 51), DLB (n = 26) and AF-GP (n = 43) and normal controls (n = 30). Using these samples, we explored the correlation between CSF CysC and CSF Aβ levels. We found that in comparison to the normal control group, both CSF CysC and CSF Aβ42 levels were significantly lower in all three dementia groups ; serum CysC levels were the same in the AD and DLB groups, and were lower in the AF-GP group (p = 0.008). The CSF CysC levels were positively correlated with both the CSF Aβ40 and Aβ42 levels in the AD, AF-GP and normal control groups . Lower CSF CysC levels might be a common feature in dementia with characteristic amyloid deposits. Our results provide evidence for the potential role of CysC involvement in Aβ metabolism and suggest that modulation of the CysC level in the brain might produce a disease-modifying effect in dementia with characteristic amyloid deposits. Cystatin C (CysC) is secreted by all human cells and found in all mammalian body fluids and tissues. It is almost completely cleared from the circulation by glomerular ultrafiltration CysC has been found to co-localize with Aβ in the senile plaque cores of amyloid plaques in brains of patients with amyloidosis AD, DLB and AF-GP share similar pathological process of neurodegeneration with amyloid deposits, oxidative stress and inflammation. Experimental All subjects gave their written informed consent. In case patients were considered not to have the capacity, written inform consent was given by the nearest relatives. The study was conducted according to the principles expressed in the Declaration of Helsinki and approved by the ethics committees at the Guangzhou Brain Hospital.A total of 150 subjects  were consecutively recruited from the ward and the outpatient clinic of the Neurology Department of the Guangzhou Brain Hospital. Both a neurologist and a psychiatrist evaluated all of the individuals. All enrolled patients fulfilled the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria for dementia. The AD patients fulfilled the NINCDS-ADRDA The CSF samples (10–12 ml) were collected in the morning by lumbar puncture. The first 2 ml of CSF was examined for cell counts, glucose and total protein. The CSF samples with erythrocyte counts less than 500 cells/µL were required for further analysis. Within 1 hour after lumbar puncture, blood samples (12–15 ml) were collected. All of the samples were centrifuged at 3000× g for 15 minutes at 4°C after collection. The CSF and serum were aliquoted into polypropylene tubes and stored at −80°C until use.The serum and CSF CysC levels were quantitatively measured by a latex immunoturbidimetric assay using CysC reagents  with an auto-analyzer  with the following instrument settings: Wavelength: 546 nm, 2 μL samples and 160 μL reagent 1 were mixed with 40 μL reagent 2. The CSF Αβ40 and Αβ42 levels were quantified using commercially available enzyme immunoassay kits . All the samples were analyzed in duplicate.−1.154×age−0.203×1.212 (if patient is black) ×0.742 ) to estimate the GFR for each participant. The serum levels of creatinine were analyzed using a standard technique.We used the Modification of Diet in Renal Disease 4 equation  13.0 statistical software. The Kolmogorov-Smirnov test was performed for all of the continuous variables to define the presence of normality. Comparisons of normally distributed data were performed using a one-way analysis of variance (ANOVA), followed by post hoc Bonferroni's test. When comparing CysC levels, analysis of covariance (ANCOVA) was also used to adjust for potential confounders for association with CysC, including age and GFR. Non-parametric analysis was performed for non-normally distributed data and categorical variable. The correlations of the measured values were examined using the Pearson correlation coefficient. A p value of less than 0.05 was considered statistically significant.There were no significant differences in education among the four groups. We found a lower percentage of female patients (p<0.001) and younger ages (p<0.001) in the AF-GP group compared to the normal control group . As expeThe CSF CysC levels were significantly lower in all three dementia groups compared to the normal control group   These diThe serum CysC levels were negatively correlated with GFR  and positively correlated with age  in the total sample. There were no correlations between CSF CysC levels and GFR  or age . CSF CysC levels were positively correlated with serum CysC levels in the total sample . We also analyzed the correlation between CSF and serum levels of CysC according to the tertiles of CSF Αβ42 levels in the total sample. A significant positive correlation was found in subjects in the lowest CSF Αβ42 tertile (CSF Αβ42 level: 108.4 pg/ml ∼490.7 pg/ml) , but not in those within the middle (CSF Αβ42 level: 490.8 pg/ml ∼847.4 pg/ml)  and highest (CSF Αβ42 level: 847.5 pg/ml ∼1495.0 pg/ml)  tertiles. CSF CysC levels were positively correlated with CSF Αβ40 and Αβ42 levels in the AD, AF-GP and normal control groups . HoweverTo explore the association between the CysC levels and the disease severity, we compared the serum and CSF CysC levels among groups defined by Clinical Dementia Rating Scale (CDR). Based on their CDR scores , patients with AD, DLB and AF-GP were divided into three groups respectively. No significant differences in serum or CSF CysC levels were observed as determined by one-way ANOVA  or by ANTo dig up biological evidence for a role of CysC in dementia with characteristic amyloid deposits, we evaluated the serum and CSF levels of CysC and the correlation of CSF CysC with CSF Aβ in patients with AD, DLB and AF-GP and normal controls. Our main finding was that the CSF CysC levels were significantly lower in all three dementia groups compared with normal controls. Moreover, positive correlations were detected between CSF CysC levels and CSF Aβ40 and Aβ42 levels in patients with AD and AF-GP, as well as in normal controls.In consistent with recent published data The serum CysC, which is cleared from the circulation by glomerular filtration, is affected by renal function Recent studies reported positive correlations of CSF CysC with CSF Aβ40 and Aβ42 in patients with AD and normal controls Our results showed that CysC levels were lower only in the CSF of patients suffering from AD and DLB, but reduced in both the CSF and serum of patients suffering from AF-GP. Amyloid deposits are present in the brain of AD and DLB, while in GP, they accumulate both in the central nervous system and the periphery. It seems that there are decreased CysC levels in the tissues, organs or systems where there is a certain amount of amyloid deposits. These findings provide further support for the notion that CysC is related to Aβ metabolism.Several protective effects of CysC on the brain that plays a role in neurodegeneration have been described in in vitro and in vivo studies, ranging from inhibition of cysteine proteases Relatively low level of serum or plasma CysC was supposed to be a marker for future risk of AD Previous studies showed that the CysC levels in CSF of AD patients Decreased CSF Αβ42 levels had been reported to be associated with disease severity Although CysC has multi-targeted neuroprotection in the brain, its levels expression in patients with AD, DLB and AF-GP might not be sufficient to maintain the long-term inhibition of Αβ deposition and adequately protect the neuronal cells from Aβ toxicity and oxidative stress. It had been demonstrated that CysC protects neuronal cells from death in a concentration-dependent manner A limitation of our study is the lack of amyloid plaque imaging in our subjects. Patients with pure DLB without any amyloid pathology may be included. However, there are strengths in this study. We analyze the CysC level in both the serum and CSF in different types of dementia with characteristic amyloid deposits. Furthermore, the causes of dementia include not only degenerative neurological diseases but also infections of the central nervous system.In summary, our results suggest that lower levels of CSF CysC are related to dementia with characteristic amyloid deposits. Decreased levels of Aβ42 are accompanied by a decrease in the CSF CysC levels in patients with AD, DLB and AF-GP, and there are positive correlations between these proteins in AD, AF-GP and normal control subjects. Our findings strengthen the evidence for the role of CysC involved in Aβ metabolism. Modulation of the CysC level may represent an alternative therapeutic strategy in dementia with characteristic amyloid deposits.


Over 1.2 million people are blind from trachomatous trichiasis (TT). Lid rotation surgery is the mainstay of treatment, but recurrence rates can be high. We investigated the outcomes (recurrence rates and other complications) of posterior lamellar tarsal rotation (PLTR) surgery, one of the two most widely practised TT procedures in endemic settings.We conducted a two-year follow-up study of 1300 participants who had PLTR surgery, conducted by one of five TT nurse surgeons. None had previously undergone TT surgery. All participants received a detailed trachoma eye examination at baseline and 6, 12, 18 and 24 months post-operatively. The study investigated the recurrence rates, other complications and factors associated with recurrence. Recurrence occurred in 207/635 (32.6%) and 108/641 (16.9%) of participants with pre-operative major (>5 trichiatic lashes) and minor (<5 lashes) TT respectively. Of the 315 recurrences, 42/315  had >5 lashes (major recurrence). Recurrence was greatest in the first six months after surgery: 172 cases (55%) occurring in this period. Recurrence was associated with major TT pre-operatively , pre-operative entropic lashes compared to misdirected/metaplastic lashes , age over 40 years  and specific surgeons (surgeon recurrence risk range: 18%–53%). Granuloma occurred in 69 (5.7%) and notching in 156 (13.0%).Risk of recurrence is high despite high volume, highly trained surgeons. However, the vast majority are minor recurrences, which may not have significant corneal or visual consequences. Inter-surgeon variation in recurrence is concerning; surgical technique, training and immediate post-operative lid position require further investigation. Trachoma is the most common infectious cause of blindness worldwide. It causes trichiasis (inturning of the eyelashes to touch the eye), which can cause visual loss. Trachomatous trichiasis (TT) affects over eight million people, 1.2 of whom live in Ethiopia – the most affected country worldwide. Surgery is the mainstay of treatment for TT. However, results of surgery in the field are often very mixed. We investigated the surgical outcomes of one of the two most widely used surgical techniques (posterior lamellar rotation), in 1300 individuals in the Amhara Region of Ethiopia. We found that recurrence occurred frequently: 315/1276 (24.7%) participants. However, recurrence was rarely severe (greater than 5 lashes): 42 participants (3.3%). Recurrence occurred much more frequently in participants who had severe pre-operative disease and with specific surgeons. The high recurrence rates and inter-surgeon variation is concerning. Further research will be required to investigate factors such as surgical technique, surgeon training and immediate post-operative lid position, in order to improve surgical outcomes. Chlamydia trachomatis. The major risk factor of blinding corneal opacification (CO) is trichiasis (TT), the in turning of the eyelashes. TT traumatises the delicate epithelium of the cornea, rendering it vulnerable to secondary infection. TT encompasses a range of eyelid and eyelash abnormalities from a few peripheral in turned lashes to the entire upper eyelid pulled inwards by scarring (entropion). TT can also occur without entropion, from metaplastic or misdirected lashes Blindness from trachoma is the end result of progressive scarring of the conjunctiva driven by Several different surgical procedures have been used to correct upper lid TT, some of which have been compared in randomized trials TT surgery can prevent or reduce progression of corneal opacity, improve vision and relieve pain Serious surgical complications are rare in TT surgery. However, complications such as granuloma and lid contour abnormalities (notching) occur relatively frequently During the course of two recently reported randomised controlled trials conducted in Ethiopia we recruited 1300 individuals with the full spectrum of TT type and severity, who received PLTR surgery with silk sutures, and were followed up for two year The National Health Research Ethics Review Committee of the Ethiopian Ministry of Science and Technology, the London School of Hygiene & Tropical Medicine Ethics Committee and the Emory University Institutional Review Board approved this study. Informed consent was taken at the time of enrolment. The research adhered to the tenets of The Declaration of Helsinki. All participants gave written informed consent to take part in the study.Two previously reported randomised controlled trials of the management of TT were conducted in Ethiopia from 2008 to 2010 Participants presented during TT surgical treatment campaigns in rural villages in the West Gojjam Zone, Amhara Regional State, which were advertised in local markets, churches and schools. Additionally, health extension workers from every sub-district (kebele) across West Gojjam were trained to recognize trichiasis. They visited each village in their kebele to identify potential participants.study eye and included in the analysis, although both eyes were treated. In both trials, participants were allocated to surgeons sequentially. Surgeons were not permitted to select specific participants and participants were not allocated according to severity.In the first trial, individuals with major TT (>5 lashes touching the eye) were randomly allocated to PLTR surgery with either silk or polyglactan (vicryl) sutures. In the second trial individuals with minor TT (<6 lashes touching) were randomly allocated to either PLTR surgery with silk sutures or repeated epilation. In individuals with bilateral TT, one eye was randomly designated  as the The group described in this report is comprised of all the individuals who were randomly allocated to the PLTR with silk suture arms of the two studies. They represent the full spectrum of TT disease (both major and minor TT) and received exactly the same surgical intervention performed by the same group of surgeons.Participants were examined immediately before surgery and again at 6, 12, 18 and 24 months. The methods used have been described in detail Surgery was performed under local anaesthesia using the technique previously described The primary outcome measure was trichiasis recurrence defined as either (1) one or more eyelashes touching the eye or (2) clinical evidence of epilation. Secondary outcome measures were surgical complications, entropion and conjunctivalisation.Data were double entered into an Access (Microsoft) database and transferred to Stata 11 . For participants who had bilateral surgery only the randomly designated ‘study eye’ was included in the analysis. The cumulative incidence of failure in each six-month block of follow-up was calculated using the Kaplan-Meier method. The associations of binary outcomes with exposures were assessed using logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI). Variables associated with the outcome on univariate analyses (p<0.2) were retained in the multivariable model. The p-values for the association between categorical variables and specific outcomes were calculated using likelihood ratios. For visual acuities of counting fingers or less, logMAR values were attributed: counting fingers: 2.0, hand movements: 2.5, perception of light: 3.0, no perception of light: 3.5.We recruited 1300 individuals with previously unoperated trichiasis in at least one eye. No individuals refused participation. Recruitment took place between March and July 2008. The follow-up assessments were conducted at the following times: 6-month, August to December 2008; 12-month, February to July 2009; 18-months, August to December 2009; 24-months, January 2010 to May 2010. The final follow-up was conducted two months ahead of schedule because of the Ethiopian general election (May 2010), during which time follow-up would not have been possible. Almost all individuals (98.1%) were reviewed on at least one occasion.Baseline characteristics are shown in Overall, recurrence occurred in 315/1276 (24.7%) study eyes. Recurrence was more frequent in participants with pre-operative major TT (32.6%) compared to minor TT (16.9%): OR 2.39, 95%CI 1.83–3.11, p<0.005 . This asOverall, there was a significant reduction in mean lash burden between baseline (4.66 lashes) and 24 months  . Amongst2: p = 0.035), as did their post-operative under-correction rates (X2 = 0.003)  . Their r= 0.003) . There wMultivariable logistic regression modelling identified increased baseline TT severity, the presence of entropic lashes (compared with misdirected or metaplastic lashes without entropion), specific surgeons (No. 2 and No. 5) and older participant age as independent risk factors for recurrence .Early  and later (seen at any subsequent follow-up) post-operative complications and their association with recurrent trichiasis are presented in 2: p = 0.495), gender (X2: p = 0.239) or younger (<41 years) age (X2: p = 0.41)Univariate and multivariable associations for developing a granuloma and lid notching are shown in Surgery successfully corrected entropion: 1148 (93.5%) participants at 12 months and 1126 (92.1%) at two years had no entropion, compared to 327 (25.2%) at baseline . SurgeryTrichiasis recurred in a quarter of study eyes by two years. Recurrence severity was very variable, ranging from a single peripheral metaplastic lash to complete entropion. However, only 13% of recurrences had more than five lashes touching they eye and there was a substantial reduction in lash burden. Therefore, although any recurrence is unsatisfactory, the likely severity of the recurrence should be considered in a balanced assessment of the risks and benefits of surgery.Over half of all recurrences occurred by six months and the risk decreased significantly for each subsequent six-month period. Higher rates of early recurrence have been reported previously. In a study from The Gambia the recurrence at four years was 41%, over three quarters of which occurred within the first six months after surgery Recurrence was twice as frequent in individuals with major TT pre-operatively. Furthermore, pre-operative entropic trichiasis (rather than misdirected or metaplastic) was an independent risk factor for recurrence. More severe pre-operative trichiasis is consistently a major risk factor for recurrent TT In our study only the PLTR procedure was used and gave recurrence rates in the middle of the reported range for this procedure: 12% to 55%, with reported follow-up periods of between 3 months and four years The surgeons in this study were selected for their surgical ability and given additional training. Their technique was intermittently observed. Nevertheless, two surgeons had significantly higher recurrence rates than the best performing surgeon. Surgeon No. 5, who had the highest recurrence rate, did operate on a higher proportion of major TT cases than the other surgeons, but remained an independent risk factor for recurrence after adjusting for baseline TT severity.Inter-surgeon variability has previously been highlighted as a concern in trachoma surgery with one study finding recurrence rates ranging from 0–83% between surgeons Notching is focal external rotation or irregularity of part of the lid usually caused by excessive suture tension. Some authors include notching within a broader category of ‘lid contour abnormalities’ Granulomas usually develop at the incision site within weeks of surgery. They require excision when they are large. In ophthalmic surgery they have been described following tarsal rotation and chalazion surgery and found to be associated with residual suture fragments, male gender and younger age This study has a number of limitations that potentially constrain the generalisation of the conclusions. It is possible that the results are better than those achieved under routine operational conditions. The five surgeons were selected for their technical skill, received additional training and supervision and performed relatively large volume surgery. They are, therefore, not truly representative of many ‘field’ TT surgeons, who typically perform few cases, have limited training and supervision Set against these limitations, this study has a number of strengths. Firstly, we report the results of a large number of operations performed in a standardised manner. Secondly, follow-up rates are high despite the inaccessibility of many participants; reducing follow-up bias. Finally, participants were representative of the spectrum of TT disease in the wider population of TT patients in Ethiopia, which remains the country with the highest prevalence of TT in the world.Recurrence rates were comparable to previous studies. Baseline disease severity and inter-surgeon variation are major determinants of recurrent disease. However, PLTR surgery successfully corrected most entropion and much of the recurrence was minor, which may not represent a significant risk for most patients. The inter-surgeon variation in recurrence rates is concerning. Further research is needed to ascertain whether recurrence can be predicted immediately after surgery, and whether it can be ameliorated.Checklist S1STROBE Checklist.(DOC)Click here for additional data file.


Given that the blood-testis barrier is more permeable than the blood–brain barrier, the use of semen to detect rare parasitic antigens/infections of the CNS in males is hypothesized. Acanthamoeba and Balamuthia mandrillaris and primary amoebic meningoencephalitis due to Naegleria fowleri has made accurate diagnosis challenging [Infections of the central nervous system (CNS) can progress rapidly and cause substantial damage or even death if they are not diagnosed promptly and treated aggressively. Given the involvement of the CNS, it is typical to use cerebrospinal fluid for the identification of infectious agents/antigens, and the host factors to suspected disease. In general, pathogens colonize mucosal surfaces, followed by invasion of the intravascular space leading to haematogenous spread and entry into the CNS at the sites of the blood–brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier at the choroid plexus. The use of CSF specimens has been of tremendous value in the diagnosis of CNS infections but the rarity of pathogens/antigens in the CSF in some of the parasitic infections such as neurocysticercosis, cerebral toxoplasmosis, granulomatous amoebic encephalitis due to llenging ,2. For sllenging -5. Physillenging . The BBBThe authors declare that they have no competing interests.Both authors contributed equally to this manuscript and both read and approved its final version.


Thermoanaerobacter tengcongensis. In this study, we revealed that the Tt-tolB gene located downstream of the Tt-rpoE1 gene encoded the cognate anti-σ factor, which could inhibit the transcription activity of Tt-RpoE1 by direct interaction with Tt-RpoE1 via its N-terminal domain. By in vitro transcription assay, the auto-regulation ability of Tt-RpoE1 was determined, and band shift assay showed that Tt-RpoE1 preferred to bind a fork-junction promoter DNA. With truncation or base-specific scanning mutations, the contribution of the nucleotides in −35 and −10 regions to interaction between Tt-RpoE1 and promoter DNA was explored. The promoter recognition pattern of Tt-RpoE1 was determined as 5′ tGTTACN16CGTC 3′, which was further confirmed by in vitro transcription assays. This result showed that the Tt-RpoE1-recognized promoter possessed a distinct −10 motif (−13CGTC−10) as the recognition determinant, which is distinguished from the −10 element recognized by σ70. Site-directed mutagenesis in Region 2.4 of Tt-RpoE1 indicated that the “D” residue of DXXR motif was responsible for recognizing the −12G nucleotide. Our results suggested that distinct −10 motif may be an efficient and general strategy used by ECF σ factors in adaptive response regulation of the related genes.Extracytoplasmic function (ECF) σ factors, the largest group of alternative σ factors, play important roles in response to environmental stresses. Tt-RpoE1 is annotated as an ECF σ factor in  This family regulates the transcription of genes involved in cell envelope functions, including periplasmic stress, heat shock response, iron transport, metal ion efflux, and alginate secretion As an essential component of RNA polymerase (RNAP), bacterial sigma (σ) factors play an important role during the initiation of transcription by specifically recognizing and binding to promoter DNA elements E. coli σE and Bacillus subtilis σWA common feature of most ECF σ factors is their regulation by a co-transcribed trans-membrane anti-σ factor. The best understood archetypes include Streptomyces coelicolor, living in a hostile and changing soil environment, has 55 ECF σ factors among a total of 65 σ factors E. coli σE by different bioinformatic analyses W, σX and σM in B. subtilisGenome sequencing has revealed numerous ECF σ factors existing in a wide variety of bacteria including many pathogens, and in many organisms, the different ECF σ factors often outnumber all other σ factors combined. For example, Thermoanaerobacter tengcongensis belonging to the phylum Firmicutes, is an anaerobic, rod shaped, and low G+C content (33%) thermophilic bacterium, which was isolated from a freshwater hot spring in China and grows between 50–80°C, with an optimum temperature of approximately 75°C T. tengcongensis revealed seven ECF σ factors , which were predicted to contribute to the adaptation of this thermophile to a high temperature environment in vitro run-off transcription analysis. In addition, we identified that the “DXXR” motif in Region 2.4 of Tt-RpoE1 is responsible for recognizing the −12G nucleotide of −10 element. Our studies indicate that a specific element (−13 CGTC −10) in the −10 region of the promoter recognized by Tt-RpoE1 distinguishes it from the σ70 promoter, and that may be a general strategy used by ECF σ factor to regulate its extracytoplasmic functions.Tt-tolB gene is located immediately downstream of the Tt-rpoE1 gene, and there are two other genes located downstream but with opposite direction (Tt-rpoE1 and the start codon of Tt-tolB (Tt-tolB contained a 23-amino acid (residues 39–61) trans-membrane domain predicted by the program SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html). Based on the features of Tt-TolB, we hypothesized that it was the potential anti-σ factor of Tt-RpoE1. As such kind of anti-σ factor usually harbours a trans-membrane domain and could tightly bind to the ECF σ factor Tt-rpoE1 into pGBKT7 to obtain a DNA-binding domain (BD) fusion construct and then cloned all the other genes of this cluster into pGADT7 to obtain activation domain (AD) fusions. As shown in lacZ reporter gene, indicating a specific interaction between Tt-RpoE1 and Tt-TolB, whereas no interaction was observed between Tt-RpoE1 and the other two gene products. In many cases of RpoE-like ECF σ factors, it was sequestered by the intracellular domain of its cognate anti-σ factors Tt-tolB into pGADT7 and found that the N-terminal domain of Tt-TolB interacted with Tt-RpoE1 specifically  . It exhibited little difference in binding efficiency to that of Tt-RpoE1 alone (data not shown), implying that high concentration of Tt-RpoE1 (5 µM) could decrease the “activation effect” of core RNAP on the binding affinity T. tengcongensis grows at an optimum temperature of approximately 75°C, we also assayed the interaction between Tt-RpoE1 and the fork-junction promoter DNA at different temperatures from 25 to 80°C. Tt-RpoE1 exhibited a similar binding affinity to promoter DNA from 25 to 55°C, indicating that it functioned very well at a wide range of temperatures (data not shown). However, when performed at 60 to 80°C, the fork-junction promoter DNA partially melted (data not shown), which is not favorable for studying the interaction between Tt-RpoE1 and the promoter DNA. Therefore, in order to mimic the Tt-RpoE1/promoter interaction in an open complex of transcription initiation in vivo, we performed the EMSA experiment at 25°C to investigate the interaction between Tt-RpoE1 and the fork-junction promoter DNA in the following experiments.We also detected the interaction between the promoter and the reconstituted holoenzyme , respectively, to the EMSA reaction with Tt-RpoE1, using the fork-junction promoter DNA (T+1/B−10) as a template. As shown in To experimentally determine the sequences of the promoter recognized by Tt-RpoE1, we analyzed the interaction between Tt-RpoE1 and different fork-junction promoter probes with truncations in the putative −10 or −35 regions. For the −35 region using T+1/B−10 as the parental probe, we cut back the double strands from −40 to different positions  was left intact and the bottom strand was cut back from +1 to different positions . As showAlthough the truncation results of the −35 and −10 regions gave clues to the location of promoter recognition by Tt-RpoE1, the contribution of each nucleotide to recognition was still unresolved. To precisely define the conserved nucleotides needed for recognition by Tt-RpoE1, we performed scanning mutagenesis by nucleotide substitutions between G-C and A-T in the −35 and −10 regions.For the −35 region using D−5/−10 as the parental probe, the nucleotides were substituted in top and bottom strands simultaneously. The EMSA results were shown in For the −10 region using the strongest binding structure (D−5/−12) as the parental probe, scanning mutations were made from −11 to −6 on the top strand, while the −13 (C/G) and −12(G/C) base pairs were substituted in both top and bottom strands. The EMSA results were shown in in vitro transcription assays to test if those important nucleotides determined by EMSA would affect the transcription activity of Tt-RpoE1. Since the structure of the complex of E. coli σE4 and its −35 element has been determined We also carried out 16CGTC 3′.Based on the results of EMSA and transcription analysis, the determinant sequence (−35 and −10 regions) for the promoter recognized by Tt-RpoE1 could be identified as 5′ tGTTACN70 (TATAAT) 70 family, Region 2 has been implicated in recognition of −10 regions The studies above showed that Tt-RpoE1 recognized a specific −10 element (−13CGTC−10) which is distinct from that recognized by σT. tengcongensis. Y2H and EMSA results showed that Tt-TolB, the cognate downstream gene product of Tt-RpoE1, interacted with Tt-RpoE1 via its N-terminal domain. Tt-TolB also inhibited the transcription of Tt-RpoE1. These results demonstrated that Tt-TolB (TTE0322) was the anti-sigma factor of Tt-RpoE1. While TTE0322 was originally annotated as Tt-TolB for containing a conserved domain of TolB, a periplasmic component of the Tol biopolymer transport system In this work, we addressed the function of Tt-RpoE1, one of seven ECF σ factors annotated in the genome of 70, ECF σ factor could bind to double-strand promoter DNA, but it preferred to bind fork-junction structure promoter  σ factors Being an auto-regulated ECF σ factor, the ECF σ factor Tt-RpoE1 was first subjected to investigate the interaction with its promoter. Different from σpromoter . With suE. coli σE4 and its −35 element. In that complex, specific protein-DNA base interactions occurred only at three positions of its 7 bp −35 element GGAACTT (underlined): −35G, −34G, and −31C, which were specifically recognized by residues R176, S172 and R171 of E. coli σE, respectively GTTAC) of Tt-RpoE1 promoter played the same roles as −35G, −34G, and −31C in the E. coli σE promoter, serving as the key nucleotides to form strong hydrogen bonds or van der Waals interactions with Tt-RpoE1. For the −33T, −32TA−31, they may be similar to the “AA” motif in the −35 element in E.coli σE promoter E4/promoter interaction. Thus substitution at any one of those nucleotides would disrupt the structure, and affected the Tt-RpoE1/promoter interaction.For the −35 region of the Tt-RpoE1 promoter, we found that −34G, −33T, −30C and −32TA−31 were functionally important for recognition by the ECF σ factor Tt-RpoE1 . Substitin vitro transcription assay  are functionally important. Mutations at these bases resulted in loss of Tt-RpoE1 binding affinity and decreasing the transcription activity. However, the “TATA” box downstream of the CGTC motif did not seem to contribute to the interaction between Tt-RpoE1 and its promoter. Taking one of the substitutions −9T to G as example, we have not detected any effect in the EMSA and on assay , indicatP. aeruginosa, the results also suggested that the “D” residue participated in discriminating −10 region contacts We have also identified residues in Tt-RpoE1 contributing to base-specific interactions in the promoter by site-directed mutagenesis employing the same strategy as Koo and his colleagues in their studies 70. This is consistent with their different functions in bacteria. The group I σ factors contain conserved melting residues  70 tolerate a great deal of promoter sequence diversity when directing the transcription of thousands of housekeeping genes. Whereas only one melting residue corresponding to “W”  recognized by group I factor σT. tengcongensis MB4T was routinely grown in modified MB medium at 75°C without shaking E. coli DH5α was used as a host for the cloning experiments, and E. coli BL21DE3 (lysS)  for overproduction of the recombinant proteins. Both E. coli strains were grown in LB medium containing the appropriate antibiotic, ampicillin  or kanamycin sulfate  if necessary. The plasmids and partial oligonucleotides used in this study were described in Tt-rpoE1 (TTE0323) and Tt-tolB (TTE0322) coding regions were amplified by PCR from genomic DNA with primers P1/P3 and P9/P10  according to the manufacturer’s protocol. Genes encoding Tt-RpoE1 (TTE0323) and the other three proteins (TTE0320-0322) including in vitro assays were performed as described previously by Huang et al with minor modifications in vitro transcription system. Typical reaction mixtures (25 µl) contained 1 µg template DNA, 2.5 pmol of E.coli core RNAP , 50 to 60 pmol of ECF σ factor Tt-RpoE1 in transcription buffer  with 0.8 mM ATP, GTP, CTP and 5 µCi [α-32P]-UTP. DNA and RNAP were preincubated at 4°C for 30 min and 37°C for 8 min to allow promoter binding. Nucleotide triphosphates (NTPs) were then added, and transcription proceeded for another 8 min. The RNA transcripts were extracted with phenol-chloroform and precipitated with ethanol. The pellet was resuspended in 10 µl of urea stop solution, heated to 95°C for 3 min, and separated by 7 M urea–6% polyacrylamide gel electrophoresis and autoradiography. For assay to detect the effect of Tt-TolB on the in vitro transcription of Tt-RpoE1, Tt-TolB was added into the reaction system at the same time with Tt-RpoE1 or after Tt-RpoE1 and RNAP incubated for 38 min with the same concentration of Tt-RpoE1 (5 µM).Run-off transcription 32P]-UTP was substituted with UTP. The primer P16  harboring plasmid p28Tt-RpoE1, p28Tt-TolB and p28Tt-TolB-C were cultivated in LB to an optimal density at 600 nm of 0.6 at 37°C and induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 24°C overnight. Then the proteins were extracted and purified as described previously To overproduce the His-tagged proteins Tt-RpoE1, Tt-TolB and Tt-TolB-C, 2, 50 mM NaCl and 1 mM EDTA, with the top strands labeled at the 5′-end with [γ-32P] ATP. The 20 µl-standard binding reaction contained: 50 mM Tris-HCl (pH 8.0), 10 mM MgAc2, 30 mM NaCl, 1 mM DTT, 5% glycerol, 30 µg/ml BSA, 0.5 mM EDTA, 1 µg poly (dI-dC), 20 fmol labeled DNA probe and the indicated amounts of appropriate proteins. After incubation at 25°C for 20 min, samples were immediately loaded on native 5% polyacrylamide gel  in 0.5× TBE buffer and electrophoresis at 150 V for 2 h. Gels were dried and exposed to Biomax radiographic film (Kodak) for autoradiography.Double-stranded or fork-junction probes were obtained by annealing reaction containing equimolar concentration of two oligonucleotides  in 50 mMTable S1Nucleotide sequences for electrophoretic mobility shift assay.(DOC)Click here for additional data file.


To understand epidemiologic patterns of meningococcal disease in Asia, we performed a retrospective molecular analysis of cerebrospinal fluid (CSF) specimens collected in prospective surveillance among children aged < 5 years of age in China, South Korea, and Vietnam.Neisseria meningitidis (Nm) ctrA gene. Multi-locus sequence typing (MLST) was performed in Nm gene amplification analysis and incidence rates for meningococcal meningitis were estimated.A total of 295 isolates and 2,302 CSFs were tested by a meningococcal species- and serogroup-specific polymerase chain reaction (PCR) assay targeting the ctrA gene), including 67 (23.6%) from China, 92 (32.4%) from Korea, and 125 (44.0%) from Vietnam. Neonates and infants aged < 6 months of age accounted for more than 50% of Nm-PCR positive CSF. Two CSF specimens from Vietnam were identified as serogroup B using MLST. In addition, 44 specimens underwent sequencing to confirm meningococcal serogroup; of these, 21 (48%) were serogroup C, 12 (27%) were serogroup X, 9 (20%) were serogroup Y and 2 (5%) were serogroup B. The incidence rates of meningococcal meningitis among children < 5 years of age was highest in Vietnam  ).Among 295 isolates tested, 10 specimens from Vietnam were confirmed as serogroup B and all were Sequence Type (ST) 1576 by MLST. Among the 2,032 CSF specimen tested, 284 (14%) were confirmed by PCR (N. meningitidis disease.These results suggest that there is a previously undetected, yet substantial burden of meningococcal meningitis among infants and young children. Standardized, sensitive and specific molecular diagnostic assays with Nm serogrouping capacity are needed throughout Asia to understand the true burden of  Neisseria meningitidis (N. meningitidis) is a gram-negative, encapsulated β-proteobacterium and the leading cause of epidemic meningitis. Globally, N. meningitidis causes an estimated 1.2 million cases and 135,000 deaths each year X and 33% Y [n = 2]), while serogroup X comprised 56% [n = 9] and serogroup Y [n = 6] comprised 38% in Korea  were less than six months of age  in this region.The burden of The authors declare that they have no competing interests.SAK and PEK carried out study design, data collection and analysis, and drafted the manuscript. DWK performed study design and conducted molecular laboratory test and drafted the manuscript. BQD, JSK and DDA participated in its design and helped to draft 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/1471-2334/12/310/prepubTable S1. Primers used for serotyping of N. meningitidis A, B, C, Y, and W135 (4)*. S2. Neisseria meningitidis identifications in CSF specimens, in China, South Korea, and Vietnam, 1999 through 2002. S3. Multi-locus sequence typing (MLST) results of 10 N. meningitidis isolates and 2 CSF specimens collected in Vietnam.Click here for file


The incidence of healthcare associated infections in a surgical ward in Shiraz has beenÂ established as 18%Â with associated costs of approx US150,000. Healthcare workers (HCWs) had acceptable levels of knowledge and attitude about hand hygiene but poor self-reported practices. We used the Theory of Planned Behavior (TPB) to identify predictors of handwashing to underpin a theory-driven intervention.Between April and September 2008, 1700 healthcare workers (HCWs) from all wards in 18 private and 10 public hospitals in Shiraz answered a self-administered survey designed in accordance with the TPB. Multiple logistic regression analysis was used to model two handwashing for patient contacts perceived to be clean and contacts perceived to contaminate hands.=0.001), peer pressure from ICPs  and attitudes about contaminated contact handwashingÂ .90% of HCWs returned a completed survey. SignificantÂ predictors for clean contact handwashing in the hospital included compliance withÂ similar community practice  and contaminated contact handwashingÂ , perception that clean contact handwashingÂ required little effort  and nursing peer pressure . SignificantÂ predictors for contaminated contact handwashing included clean contact handwashing compliance , community contaminated contact handwashing (AOR1.5 PCommunity-based handwashing practices exert strong influence on handwashing in the hospital. Given the interdependence between community and hospital handwashing a campaign to improve aware about the benefit of community handwashing may improve HCWs’ compliance.None declared.