Administration of the hematopoietic growth factor granulocyte-colony stimulating Factor (G-CSF) has been reported to enhance recovery from controlled cortical impact (CCI) in rodent models. G-CSF exerts actions in both the periphery (stimulation of hematopoiesis) and in the brain, where it serves as a neurotrophic factor, promoting neuronal survival and stimulating neural stem/progenitor cell proliferation in the hippocampus. In order to distinguish the direct CNS actions of G-CSF from its peripheral actions, experiments were designed to block the recruitment of peripheral monocytes to the site of the lesion produced by CCI. The selective C-C motif receptor 2 (CCR2) antagonist (RS504303) was co-administered with G-CSF for three days after CCI in a chimeric mouse previously transplanted with GFP-expressing (GFP+) blood stem-progenitor cells. Results: The drug significantly impaired infiltration of GFP+ bone marrow-derived cells to the frontal cortex and striatum without impeding recovery performance and hippocampal neurogenesis in the behavioral test, the Radial Arm Water Maze (RAWM). Administration of the CCR2 antagonist alone, without G-CSF, was effective in promoting recovery in RAWM. These results support the hypothesis that the direct action of G-CSF on neural cells, independent of its hematopoietic effects, is primarily responsible for enhanced recovery from CCI. In addition, this study confirms the importance of CCR2 and its ligand, monocyte chemotactic protein-1 (MCP-1), in mediating the inflammatory response following CCI. Granulocyte colony-stimulating factor (G-CSF) is one of several hematopoietic cytokines that regulates the production of circulating blood cells by the bone marrow. G-CSF is commonly used to treat leukopenia, but it has also been investigated in animal models of stroke. G-CSF administration was reported to reduce brain damage and improve functional outcomes ,2,3,4. GA recent study in mice examined the sub-acute response (up to two weeks after mild CCI) to a lower dose of G-CSF (100 µg/kg) given for three consecutive days after the injury . That stIn addition to its impact on hematopoietic cells, G-CSF readily passes the blood–brain barrier  and inteTo further understand the mechanisms for the beneficial effects of G-CSF in models of TBI, the present study was designed to determine the extent to which the recruitment of bone marrow-derived cells (BMDC), in particular monocytes, from the blood into the brain is responsible for enhanced recovery from TBI. The approach taken here utilized a selective chemokine receptor antagonist of monocyte chemoattractant protein-1 (MCP-1) to decrease the infiltration of monocytes into the CNS. If enhanced recovery from TBI occurs despite inhibition of monocyte recruitment to the site of injury, then the direct actions of G-CSF on neural cells can be considered a critical mechanism for enhanced functional recovery from TBI.Two weeks after CCI, the cohort of mice treated with G-CSF exhibited an increased infiltration of GFP+ (bone marrow-derived) cells into the right frontal cortex and striatum . The GFPp < 0.05) (p < 0.05). The GFP+ signal in the hippocampus was not significantly increased two weeks following CCI compared to vehicle-treated controls  consistent with earlier reports ,13. Co-a < 0.05) . When thcontrols .G-CSF treatment improved performance in the RAWM compared to vehicle-treated controls . Interesp < 0.05). DCX+ cell counts were not performed in this study because an earlier report from this laboratory showed a positive correlation between the DCX+ signal in the hippocampus and the DCX+ cell count in that structure  1Osb/J, 003291) were obtained from Jackson Laboratory . All the experiments utilized chimeric mice, prepared from C57BL/6 mice transplanted with green fluorescent protein-expressing (GFP+) bone marrow. More specifically, the procedure for bone marrow harvesting from transgenic (Tg) GFP+ mice has been previously published -spiro -2(1H)-one) was purchased from Tocris Biosci Inc. . This chemokine receptor CCR-2 and its primary ligand, monocyte chemoattractant protein-1 (MCP-1), represent a critical signaling pathway for the recruitment of peripheral blood monocytes to sites of immune-mediated inflammation, where they become inflammatory macrophages.Human recombinant G-CSF  task was employed to study the cognitive effects of G-CSF in mice that had undergone mild to moderate CCI. The RAWM is a hippocampal-dependent, spatial learning task that is not based on locomotor ability or swimming speed . The RAWMice were anesthetized with 150 mg/kg Ketamine, 15 mg/kg Xylazine and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were stored in 4% paraformaldehyde, and then transferred to 25% sucrose solution in 4% paraformaldehyde, until the brains sank to the bottom. Then brains were slowly immersed into isopentane (cooled on dry-ice), left in isopentane for 20 s, removed, placed on a small piece of aluminum foil sitting on powdered dry-ice for 1–2 min (to let the isopentane evaporate) and finally wrapped in the foil and stored at −80 °C until sectioning. Brains slices were cut 30 μm thick, in a cryostat  set to −25 °C.Selective immunostaining of astrocytes and microglia was performed with antibodies to glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule-1 (Iba-1), respectively. Iba-1 is protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. Antibodies to doublecortin (DBX) were used to label immature neurons in the dentate gyrus of the hippocampus. Brain sections were preincubated in phosphate buffered saline (PBS) containing 10% normal serum  and 0.3% Triton X-100 (Sigma) for 30 min. The sections were then transferred to a solution containing primary antibodies in 1% normal serum, 0.3% triton X-100/PBS and incubated overnight at 4 °C. The specific antibodies used in each experiment were: rabbit anti-DCX , 1:1000; 1:500; rabbit anti-Iba-1 , 1: 500, rabbit anti-GFAP , 1:50 in PBS containing 1:100 normal serum without Triton X-100. After incubation with a primary antibody, the sections were washed and incubated for one hour with Alexa Fluor 488 goat anti-mouse IgG diluted 1:400 in PBS  at room temperature. The sections were then rinsed in PBS three times and covered with a cover glass. Green fluorescence signals from the labeled cells were visualized with fluorescence microscopy using appropriate filters.Quantitation of microgliosis  and astrogliosis  was made by computerized image analysis. An estimation of hippocampal neurogenesis  was also determined by image analysis. Previous reports from our laboratory documented a strong correlation between DCX+ signal analysis and stereologic counts of hippocampal DCX+ cells .The method for quantitative image analyses has been previously described ,27. Eighp < 0.05.Neurohistologic measures, as well as measures of neurotrophic factors were expressed as mean ± SEM and statistically evaluated using two-way ANOVA and multiple t-tests (comparing vehicle vs. G-CSF) with the Holm-Sidak correction for multiple comparisons . Analysis of RAWM data utilized repeated measures analysis of variance (ANOVA) with one between-subjects factor (treatment) and one within-subjects factor . One-way ANOVAs followed by Bonferroni’s post hoc test were also used to determine which trials were different between groups when a significant overall group difference or group × trial interaction was found. All comparisons were considered significant at In conclusion, the present study supports the hypothesis that the direct action of G-CSF on neural cells, independent of its hematopoietic effects, is primarily responsible for enhanced recovery from CCI. In addition, this study confirms the importance of CCR2 and its ligand MCP-1 in mediating the inflammatory response following TBI. More importantly, the administration of a drug that blocks MCP-1 actions (without the need for co-administration of G-CSF) appears to be sufficient to enhance recovery of cognitive performance in the RAWM. Going forward, it will be important to study the mechanism by which mitigation of the inflammatory response, by interfering with chemokine signaling, results in stimulation of the hippocampal neurogenesis.


To develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma.® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts.Glioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArrayp=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the ‘signature’ for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF.miRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor “signature” consisting of nine miRNAs. The “signature” correlated with glioblastoma tumor volume (We report a CSF miRNA signature as a “liquid biopsy” diagnostic platform for glioblastoma. Glioblastoma, defined by the World Health Organization (WHO) glioma classification as grade IV astrocytoma, is the most common form of primary brain cancer in adults , 2. DiagCSF is an appealing and accessible bio-fluid for glioblastoma “liquid biopsy”. The bio-fluid lies in close proximity to tumor tissue, often bathing tumor or its associated microenvironment . The CSFExtracellular Vesicles (EVs) are cell-secreted vesicles that range 30-2000 nm in size that mediate critical biologic functions, including cellular remodeling and intracellular communication . Cancer Here, we examined miRNA profiles of the CSF EVs and “crude” CSF derived from glioblastoma patients. miRNA is an attractive biomarker platform given its stability in bio-fluids , selectiT cut-off of 35, we found that 200-400 miRNAs were detected in the glioblastoma specimens . Between 30-50% of these miRNAs were detected in the matched EV CSF , significant variation in miRNA profiles were found between CSF derived from the first three cohorts . To account for this variability, we used miRNA profiles derived from all three cohorts in our signature development. Details of the analysis can be found in We then used LASSO  to develP < 0.0001, Figure Pre-operative MRI was available for 11 of the patients in Cohort 1. We created tumor volumes based on the Agfa CD Viewer and related these to the CSF miRNA gene signature scores. A positive correlation was observed between miRNA signatures and tumor volumes Figure . GlioblaWe tested the performance of the 9-miRNA signature in a prospective manner. Since most EV miRNAs are also detected in crude CSF, we opted to validate our signature using unfractionated crude CSF. We prospectively collected and profiled cisternal CSF from an additional 22 patients (Cohort 4: 10 glioblastoma and 12 non-oncologic patients). Using the cutoff (0.4) established during the discovery process, the signature correctly identified 8/10 subjects with glioblastoma and 8/12 non-oncologic subjects, yielding a sensitivity of 80% and specificity of 67%. The AUC was 0.75  Figure .We also prospectively collected and profiled lumbar CSF from 18 glioblastoma and 20 non-oncologic patients (Cohort 5). Using the same coefficients and cutoff score, the 9 miRNA signature correctly identified 5/18 glioblastoma subjects and 19/20 non-oncologic subjects, yielding a sensitivity of 28% and specificity of 95%. The AUC was 83% . Figure . Notably13 lumbar glioblastoma CSF samples were collected as a part of Cohort 1. We had compared the performance of our miRNA signature in these samples in order to afford direct comparison to that seen in the cisternal samples. In the cohort 1 lumbar CSF samples, the 9 miRNA signature correctly identified glioblastoma subjects in 3/13 glioblastoma samples yielding a sensitivity of 23%. These results were comparable to those observed in the validation cohorts, confirming our observation that the diagnostic utility of the 9-miRNA signature is optimal when applied to cisternal CSF .miR-21  play a pIn current clinical practice, CSF sampling is not routinely performed in glioblastoma patients. The sensitivity of CSF cytology as a diagnostic tool for glioblastoma is ∼10%  and belovs. lumbar). However, even after correcting for site of collection, this variability remained. It is worthwhile noting that the CSF samples were collected in our study through a Standard Operating Procedure (SOP) and processed identically post-collection. The variability observed between these cohorts, in the context of the published literature, suggests that CSF miRNA profiles are likely influenced by physiologic factors or perturbation that was not accounted for by the SOP . As such, the robustness of the CSF miRNA signatures are largely a function of the sample size, since larger sample sizes afford a greater likelihood of minimizing the undue influence of any particular perturbation/physiology. Our study is particularly important in this context, since our study design is the only one in the literature that derived the signature through three independent cohorts, summing to 135 CSF samples. We subsequently validated our results in another 60 prospectively collected CSF. The scale of our study as well as the meticulous effort devoted to validation is notable in the reported literature of glioblastoma CSF biomarkers.There has been significant variability in the reported miRNA profiles in CSF derived from glioblastoma patients –23. We oAn important finding in this study is that the miRNA contents of cisternal and lumbar CSF differ. We found that less than half of the miRNAs detected in cisternal CSF were detected in lumbar CSF , likely EVs have been touted as platforms for diagnostic and prognostic biomarker interrogation –28. The The literature that examined altered miRNA regulation in glioblastoma has expanded over the past decade . It is nWhile our miRNA signature performed well as a diagnostic tool in cisternal CSF, opportunities for obtaining these samples are admittedly limited. Such samples can be obtained only from patients with an Ommaya reservoir or a ventriculo-peritoneal shunt. Because these procedures involve placement of an indwelling catheter that is in direct communication to cisternal CSF, serial samples can be safely acquired in this patient population. As such, clinical testing of the cisternal CSF signature is feasible in the subpopulation of glioblastoma patients with an indwelling shunt system. Moreover, serial sampling of cisternal CSF from this patient population may afford a minimally invasive platform for tracking glioblastoma disease burden. We are in the process of collecting and testing CSF from recurrent glioblastoma patients to further test the utility of our miRNA signature.In sum, our study provides a proof-of-principle study demonstrating the plausibility of CSF miRNA profiling as a “liquid biopsy” platform for glioblastoma diagnosis and provides the basis of future validation of this platform.Five cohorts of patients totaling 195 subjects provided CSF for these studies . Manufacturer's cartridges consisted of 818 TaqMan qPCR assays arranged on 384 well plates, with primers targeting 754 miRNA species, and 16 replicate wells of one negative and 3 positive RNA controls. Megaplex™ RT Primers, Human Pool A v2.1 and Megaplex™ RT Primers, Human Pool B v3.0 were used for the reverse transcription step. Megaplex™ PreAmp Primers, Human Pool A v.2.1 and Megaplex™ PreAmp Primers, Human Pool B v3.0 were used for the PreAmp step. The samples within each of the 3 discovery cohorts were assayed on the same date using the same reagents. The validation samples (cohorts 4 and 5) were assayed in two different batches on two different dates and data was combined for analysis.RNA was extracted from each sample using the miRCURY™ RNA Isolation Kit (Exiqon). Samples assayed were EV, supernatant and tissue from cohort 1; EV and supernatant from cohort 2; EV from cohort 3; and whole CSF from cohorts 4 and 5. Four microliters of RNA extract (4-20ng/μl) was used as input for microRNA profiling on the TaqManT value ≥35 were considered below the detection threshold. In tumor tissue samples, CT values for the query miRNAs were normalized using the mean of the positive controls . For the CSF samples, the positive control miRNAs were not uniformly expressed at high levels across samples. For the discovery cohorts global mean normalization was performed in which normalized CT values were calculated as the raw CT value minus the arithmetic mean of all expressed miRNAs in the sample [miRNA species with Ce sample . For thee sample  with assp-value < 0.05  and the same direction of differential expression; this test of replication has overall Type I error rate ∼1%. This candidate selection plan was pre-specified and documented.The classifier was trained using the three discovery cohorts . When both supernatant and EV miRNA was available within a cohort, we used the fraction with the higher median number of detected miRNA species for analysis within that cohort. For cohorts with low detection rates, we used both fractions with a Bonferroni correction for the two comparisons. Differentially expressed CSF miRNAs between glioblastoma and non-oncologic subjects were identified using the limma Bioconductor package , with FDCandidate miRNAs were carried forward to a multivariate model to discriminate glioblastoma from non-oncologic controls using L1-penalized logistic regression . The mod4 cells in 4 μl HBSS) were stereotactically injected into the brains of nude mice at age 6 weeks old. The coordinates were: 1.8 mm to the right of bregma and 3 mm deep from the dura. Aged-matched nude mice were used as controls. Four weeks after injection, CSF samples were collected from the cisterna magna as previously described [Dissociated glioblastoma stem cells JVJ , followed by qPCR using SsoAdvanced™ Universal Probes Supermix (Bio-Rad) and miRNA specific Taqman assay on a Bio-Rad CFX96 instrument. For the detection of miRNA from murine CSF, collected CSF was lysed directly in buffer containing 50mM Tris pH 8, 140mM NaCl, 1.5mM MgClTaqman miRNA assay for miR-10b, miR-16, and miR-21 were purchased from ThermoFisher Scientifics.


Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker’s yeast, in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae . Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.Codon optimization P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.Our data support a view that the methylotrophic yeast The online version of this article (doi:10.1186/s13104-017-2471-6) contains supplementary material, which is available to authorized users. Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), a glycosylated cytokine, plays a vital role in proliferation and differentiation of granulocytes and macrophages from bone marrow progenitor cells . hGM-CSFN- and 4 O-glycosylation sites leads to different glycoforms with molecular weights ranging from 14 to 60 kDa . Approximately 20 mL of filtered supernatant was loaded onto the column at 1 mL/min. The column was then washed with 10 column volumes of buffer B. The bound protein was subsequently eluted with buffer B  in a step-wise manner, ranging from 5, 10, 20, 40, 60 and 100% buffer B. The target fractions were identified by SDS-PAGE. The protein was eluted maximally at 10 to 20% buffer B.rhGM-CSF was purified using hydrophobic interaction chromatography. A HiPrep Phenyl Fast Flow column was equilibrated in buffer A [ in RPMI plus 10% FBS to obtain a final rhGM-CSF concentration ranging from 2 × 104 to 2 × 10−1 pg/mL. 100 µL of each combination was aliquoted into a 96-well plate and cultured for 72 h in a 5% CO2 incubator. The proliferation of TF-1 cells was measured by using a CCK-8 kit at OD 450 nm according to the manufacturer’s instructions. Crude supernatants from P. pastoris and S. cerevisiae served as controls. LU (Laboratory unit) was calculated as follows:Purified rhGM-CSF from each yeast species was added to 1 × 10in silico-optimized sequences show a high relative adaptiveness .Time-course expression showed an accumulation of rhGM-CSF Fig. . In the iae Fig. . ConsequP. pastoris was eluted maximally at 10–20% buffer phosphate. The purity was 85.2% and the recovery yield was 53.8%  (pg/mL) and laboratory unit (LU) (units/mg) were calculated from each curve as shown in Table 50 of purified rhGM-CSF increased on going from protein produced using P. pastoris and IEC, P. pastoris and HIC, S. cerevisiae and IEC to S. cerevisiae and HIC. Notably, purified rhGM-CSF from P. pastoris using IEC had higher purity than protein from P. pastoris using HIC . The discrepancy could be due to the glycosylation moiety added to rhGM-CSF [P. pastoris had an activity of 1.2x107 LU/mg. This activity is almost equal to the standard sample announced by WHO [E. coli-derived rhGM-CSF (non-glycosylated), our data show that rhGM-CSF is 1.5 times lower than that of E. coli origin (unpublished data). With the same purity, rhGM–CSF from S. cerevisiae was 327 times less active than that from P. pastoris.The activity of purified rhGM-CSF from d by WHO  and is td by WHO , our purS. cerevisiae and P. pastoris. Our results suggest that rhGM-CSF expressed in P. pastoris is more active than the same protein from S. cerevisiae. To our knowledge, the data presented here are the first head-to-head comparison of S. cerevisiae and P. pastoris as host cells for rhGM–CSF production.In this study, we have been able to compare the pipeline from gene to recombinant protein between Additional file 1. Results of fed-batch fermentation process of P. pastoris X33::hgmcsf and S. cerevisiae BY4742/pYES2-hgmcsf.Additional file 2. Results of bioactivity analysis of purified hGM-CSF derived from P. pastoris X33::hgmcsf and S. cerevisiae BY4742/pYES2-hgmcsf.


Corynebacterium striatum. The bacterium was detected from the removed central venous catheter tip too, and the patient was diagnosed with catheter-related bloodstream infection by C. striatum. However, treatment was not effective, and the patient died on day 73 of admission.A 49-year-old woman visited our emergency department because of exertional dyspnea due to severe left ventricular functional failure. It progressed to disseminated intravascular coagulation and disturbance of consciousness on day 67 of admission. Gram-positive bacilli were detected from two different blood culture samples on day 67 of admission. An API-Coryne test and sequencing (1~615 bp) of the 16S rRNA gene were performed, and the strain was identified as Corynebacteria are a group of aerobic, Gram-positive, catalase-positive, nonsporulating, generally nonmotile rods [ Corynebacteria are divided into two groups: Corynebacterium diphtheriae and nondiphtherial Corynebacteria, collectively referred to as diphtheroids. When isolated from clinical specimens, nondiphtherial Corynebacteria, such as Corynebacterium striatum, Corynebacterium amycolatum, Corynebacterium minutissimum, Corynebacterium xerosis, and Corynebacterium freneyi, were originally thought to be contaminants [ C. striatum infections including cases of bacteremia, endocarditis, meningitis, pleuropneumonia, osteomyelitis, arthritis, and intrauterine infections [ C. striatum, in a 49-year-old immunocompetent female patient which has multiple organ failures.Theile rods . The Coraminants , as thesaminants . There afections . In the A 49-year-old woman was brought to our emergency department because of exertional dyspnea due to severe left ventricular functional failure. Her vital signs were unstable; hence, she was immediately admitted to the intensive care unit (ICU). She had two comorbidities: one was diastole cardiomyopathy, and the other was complete atrioventricular block (c-AVB), already treated with a pacemaker implantation (PM).Although an implantable cardioverter defibrillator , with biventricular pacing function, was replaced with PM for severe left ventricular functional decline, on day 12 of admission, an intra-aortic balloon pump (IABP) was also inserted because of multiple organ failure. The IABP was removed on day 16 of admission. Thereafter, there was no obvious fever, signs of infection, so no antibiotics were administered. However, intermittent hemodialysis was continued due to liver failure and renal failure, and an IABP was necessary again after a worsening of cardiac function on day 66. The illness progressed to disseminated intravascular coagulation (DIC) and disturbance of consciousness on day 67 of admission. Therefore, two sets of blood samples for blood culture were collected. Gram-positive bacilli were detected in both blood culture samples; each set included aerobic and anaerobic cultures Figures . A centrSome asynergy in wall motion was detected by echocardiography, but no vegetation was seen. Initially, on day 67 of admission, tazobactam/piperacillin (TAZ/PIPC) (2.25 g every 6 hours) was prescribed. After the results from blood cultures on day 69 of admission, vancomycin (VCM) (1 g every 6 hours) was added to the therapy, while TAZ/PIPC was changed with meropenem (MEPM) (1 g every 6 hours) on day 72. At this time, another two sets of blood samples were collected and blood cultures were negative. However, the patient died on day 73 of admission. C. striatum/C. amycolatum strain was identified with a probability of 89.7%. The nucleotide sequence (1~615 bp) of the 16s rRNA gene revealed a 99.7% homology to a specific subtype, that is, C. striatum ATCC 6940 (GenBank: NZ_GG667536). The bacterium was detected from the removed central venous catheter tip too. Thus, the patient was diagnosed with a C. striatum catheter-related bloodstream infection. The C. striatum strain was susceptible to VCM, linezolid (LZD), and gentamicin (GM)  was performed. This method is based on the assessment of biochemical properties.cin (GM) .C. striatum colonizes the skin and mucous membranes of both healthy people and hospitalized patients [ C. striatum infection are hospital-acquired as wound infections and a few reports on systemic infections [ C. striatum from a sterile site, are available. However, most of these cases are represented by patients with implanted indwelling devices or who present an immunosuppression [ C. striatum regardless of patient's history.patients . The majfections , that ispression , 6. Beca C. striatum [ C. striatum may have been isolated from blood sample cultures, it is difficult to distinguish an innocuous contamination from a dangerous infection. Outbreaks caused by multidrug-resistant C. striatum have been reported in patients with prolonged hospitalization, mechanical ventilation, or use of broad-spectrum antibiotics [To our knowledge, this is the second report which found both blood cultures and cultures from a central venous catheter tip positive for the same strain ofstriatum . Since Cibiotics , 8. Corynebacterium spp., while 9% of early and 4% of late prosthetic valve endocarditis are caused by members of the genus [ Corynebacterium sp. [Approximately 0.2 to 0.4% of native valve endocarditis is caused byhe genus . Althoughe genus . In addi C. striatum from C. amycolatum. Although the biochemical properties of C. amycolatum and C. striatum are similar, only C. striatum contains mycolic acid [ C. striatum identification was confirmed by 16s rRNA gene analysis.The API-Coryne test is a method to distinguishlic acid . However C. striatum as susceptible to a wide range of antibiotics [Since most of reports classifiedibiotics , it has ibiotics . C. striatum is resistant to penicillin but sensitive to other β-lactam antibiotics and to vancomycin. In a previous report, vancomycin was recommended as empirical therapy for serious infections caused by Corynebacterium spp. [ C. striatum was susceptible to VCM in this case, the patient might have died because administration of VCM was delayed. Therefore, in this case, appropriate antibiotics could not be judged in vivo. However, the optimal antimicrobial therapy for these infections is still controversial. In vitro susceptibility tests showed that linezolid and tigecycline are active against coryneform bacteria, revealing a potential therapeutic value [ Corynebacterium spp. infections. Appropriate susceptibility tests and interpretive criteria are critically needed, in light of the growing emergence of multidrug resistance and its involvement in nosocomial infections.In general,ium spp. . Althougic value , 16 of t Corynebacterium could be isolated from a blood culture as a common contaminant, in certain case this observation could conceal a dangerous infection. Patients with a history of exposure to broad-spectrum antibiotics or immunosuppression, as well as critically ill patients with an implanted indwelling device or a central venous catheter, must be considered at high risk of severe infection for this type of bacteria and it is necessary to recognize C. striatum as an emerging nosocomial pathogen. In conclusion, we encountered a case of catheter-related bloodstream infection caused by C. striatum. Unfortunately, we could not successfully treat the patient because of her poor general condition and comorbidity.In conclusion, although


Device-related infections with this pathogen frequently require prolonged parenteral therapy. Corynebacterium striatum is an emerging multidrug-resistant bacteria. We retrospectively identified 179 isolates in a clinical database. Clinical relevance, in vitro susceptibility, and length of parenteral antimicrobial drug use were obtained from patient records. For patients with hardware- or device-associated infections, those with C. striatum infections were matched with patients infected with coagulase-negative staphylococci for case–control analysis. A total of 87 (71%) of 121 isolates were resistant to all oral antimicrobial drugs tested, including penicillin, tetracycline, clindamycin, erythromycin, and ciprofloxacin. When isolated from hardware or devices, C. striatum was pathogenic in 38 (87%) of 44 cases. Patients with hardware-associated C. striatum infections received parenteral antimicrobial drugs longer than patients with hardware-associated coagulase-negative staphylococci infections . C. striatum commonly shows resistance to antimicrobial drugs with oral bioavailability and is associated with increased use of parenteral antimicrobial drugs. Corynebacterium striatum has historically been the second most commonly isolated Corynebacterium species, after C. jeikium , C. striatum isolates were frequently susceptible to many antimicrobial drugs, including β-lactams, tetracycline, and fluoroquinolones  were included if C. striatum was isolated from a specimen submitted for bacterial culture and identified to the species level. Because we were interested in whether C. striatum would be considered pathogenic in an immunocompetent population, patients with active immunosuppression were excluded from our analysis. We excluded 34 patients with immunosuppressant use at the time of culture with microbiological growth on the basis of pharmacy records , or with a diagnosis of active malignancy or HIV/AIDS according to codes from the International Classification of Diseases, 9th Revision. Our algorithm-based method was confirmed by using a manual chart review.We used an algorithm-based query of the De-Identified Clinical Data Repository maintained by the Institute for Translational Health Sciences of the University of Washington  to identify 213 patients from the university medical center infected with Corynebacterium isolates to the species level (C. striatum in 100% (51/51) of clinical isolates tested . This kit correctly identifies 95% of C. striatum were obtained, we considered only the first isolate in our analysis.Phenotypic susceptibility testing was performed by using the E-test  and Remel blood Mueller Hinton agar (Thermo Fisher Scientific) and incubation for 24–48 h at 35°C in ambient air, according to breakpoints of the Clinical and Laboratory Standards Institute , whether the culture grew C. striatum cases compared with that for persons infected with coagulase-negative staphylococci (controls). Persons infected with coagulase-negative staphylococci were chosen as controls because isolates are frequently found in clinical samples (enabling appropriate matching), colonize the skin, and generally have low virulence, similar to C. striatum. Cases were matched to controls on the basis of age (± 5 years), site of infection, and presence or absence of hardware associated with the infection site.In a subset of patients with hardware-associated osteomyelitis or infections of implanted cardiac devices, we performed a matched case−control analysis to examine length of parenteral therapy in t-test, and nonparametric data were compared by using χ2 or Mann-Whitney U tests. A p value <0.05 was considered significant.Statistical analysis was performed by using Prism 6 . Data are reported as mean ± SE. Parametric data were compared by using the C. striatum isolated from clinical specimens. A substantial proportion  of these isolates were believed to be from clinically relevant infections. For comparison, in our laboratory system during a similar period, ≈42,000 isolates of Staphylococcus aureus and 4,800 isolates of coagulase-negative staphylococci were recovered with in vitro susceptibilities determined.We identified 179 immunocompetent adult patients infected with C. striatum is a colonizer of the skin and mucous membranes, isolates were frequently reported in sputum and skin samples (65/179) (Consistent with previous reports that (65/179) . Interpr(65/179) . IsolateEighty percent (143/179) of isolates were found in an inpatient setting. Susceptibility testing was performed for 121/179 isolates, and 72% (87/121) were resistant to all oral antimicrobial drugs tested . The perWe determined in vitro susceptibilities for several antimicrobial drugs . In geneAll patients with infections deemed to be clinically relevant were initially given vancomycin, except 1 patient with a documented allergy to vancomycin who received daptomycin. As expected, the length of time parenteral antimicrobial drugs were administered varied widely by anatomic site of infection and presence of a foreign body. Prosthetic joint infections were treated with parenteral therapy for 54 ± 7 days, and other hardware- or device-associated infections were treated for 65 ± 10 days. One patient with a ventricular assist device−associated infection who received vancomycin for 650 days was excluded from this analysis (outlier).C. striatum, we found that those infected with C. striatum had a longer course of parenteral antimicrobial drugs   than coagulase-negative staphylococci  (p = 0.02).In a subset restricted to the 38 patients with hardware (including prosthetic joint) or device-associated infections deemed clinically meaningful, we successfully matched 27 patients with control patients infected with coagulase-negative staphylococci. Eleven patients could not be matched for site of infection or age. When we compared control patients infected with coagulase-negative staphylococci with patients infected with p<0.001) . C. striC. striatum group and only 1 serious adverse event in the coagulase-negative staphylococci group (p = 0.19). The serious adverse events  were 1 drug reaction with eosinophilia and systemic symptoms syndrome, 2 acute kidney injuries with creatinine levels >3 times baseline values, and 3 absolute neutrophil counts <1,000 cells/mm3.Five serious adverse events were associated with parenteral antimicrobial drugs in the C. striatum can be a component of the skin microbiota, determination of whether microbiologic growth in a clinical sample represents an infection depends on clinical judgment. Previous investigations have described C. striatum primarily as a nosocomial pathogen, frequently in the setting of underlying malignancy or organ transplantation  with an expensive oral antimicrobial drug (linezolid)  ranging from 5 to 17 episodes/100 devices and infection rates of 0.5 to 5 infections/100 lines (C. striatum infections is linezolid. Although testing for C. striatum linezolid susceptibility is rarely performed in our clinical microbiology laboratory, to our knowledge, linezolid resistance has never been reported for corynebacteria. Nevertheless, linezolid is poorly tolerated during the long courses of treatment required for hardware- or device-associated infections and has shown a rate of adverse events leading to treatment discontinuation ranging from 34% to 80% (,One oral treatment option for multidrug-resistant C. striatum are probably circulating in the community rather than emerging under nosocomial pressure, but further studies would be needed to establish the ecologic niche of drug-resistant C. striatum strains.We demonstrated that multidrug resistance was common even in isolates that were not considered to be clinically meaningful in an outpatient setting. We hypothesize that these resistant strains of C. striatum infections in immunocompetent hosts and the large number of C. striatum case reports we reviewed. Detailed clinical information, including the treating physician’s interpretation of the sample, was linked to microbiological isolates. C. striatum will probably be recognized in more clinical settings because use of MALDI-TOF mass spectrometry enables C. striatum to be rapidly identified to the species level with a high degree of confidence without molecular techniques (Strengths of our study include a systems-wide approach to Limitations of our study include its retrospective nature and use of data from 1 health system, which restricted potential generalizability of the results. Given that our study was a retrospective study conducted over a 10-year period, we also cannot state whether the isolates are clonally related or reflect a diverse group with divergent mechanisms of drug resistance. In addition, we relied on the treating physician’s interpretation to determine the clinical relevance of an isolate. We have no other way of determining whether the isolate was causing disease, but we believe that this limitation is indicative of general clinical practice. Determining causality would require a different series of mechanistic investigations.C. striatum is an emerging multidrug-resistant pathogen. Our results highlight the need to identify corynebacteria to the species level, which is now readily performed by using MALDI-TOF mass spectrometry, and perform susceptibility testing for any isolate that is believed to be clinically meaningful. The frequent resistance of C. striatum to all easily tolerated oral antimicrobial drugs supports the need for development of new agents with good oral bioavailability and acceptable long-term safety profiles that are active against gram-positive organisms.Our study demonstrates that


Cerebrospinal fluid leakage remains a significant cause of morbidity following posterior fossa surgery, and its treatment remains a difficult problem. The aim of the study was to propose a treatment algorithm for its management.for a variety of diseases. Patients with post operative CSF leakage had either been treated initially with conservative measures including re-suturing of the wound, with CSF lumbar drainage to be employed in case the CSF leakage didn’t stop, or the initial intervention was the institution of CSF lumbar drainage simultaneously with conservative measures. VP  shunt was done in patients with gross hydrocephalus on postoperative CT brain.A retrospective, single-center study was conducted on 147 patients who underwent elective posterior fossa surgery There were 25 (17%) cases of CSF leakage, including 24 incisional CSF leaks and one case of CSF otorrhea. In eight patients with incisional CSF leakage treated initially with conservative measures including re-suturing of the wound, CSF leakage stopped in only two cases. CSF lumbar drainage instituted later on in six cases with persistent leakage stopped the CSF leakage. In fourteen patients managed initially with re-suturing of the wound and concomitant CSF lumbar drainage, CSF leakage settled in all the cases. Two patients with gross hydrocephalus on post operative CT were managed successfully with VP shunt. Re-suturing of the wound with concomitant CSF lumbar drainage was found to be significantly associated (p=0.003) with the stoppage of CSF leakage, and the settlement of meningitis (p= 0.014).CSF leaks after posterior fossa surgery should be managed with re-suturing of the wound and concomitant CSF lumbar drainage, instead of an initial trial of conservative therapy alone.Incisional  Cerebrospinal fluid (CSF) leak remains a significant source of morbidity in neurosurgery, particularly after posterior fossa surgery.1CSF leak following posterior fossa surgery remains a difficult and perplexing problem.Treatment of postoperative CSF leak was diagnosed when clear fluid was observed draining through the incision, irrespective of the development of central nervous system infection. CSF rhinorrhea or otorrhea was diagnosed when clear fluid was observed draining through the nose or ear respectively.The study was a retrospective, single-center study conducted in the Department of Neurosurgery, Unit-1, Lahore General Hospital from January 15, 2012 to September 15, 2015 for forty five months. Patients of both sexes and all age groups operated for posterior fossa pathologies were included in the study. Post-operative CSF leakage at any point in time was noted. Incisional CSF leakage the initial mode of treatment was the institution of conservative measures. These conservative measures included bed rest, head elevation, local wound care (such as pressure dressing) and suturing of incisional leaks. In case the CSF leak didn’t stop, CSF lumbar drainage was started. In the remaining patients developing incisional CSF leakage, re-suturing of the wound was accompanied by concomitant CSF lumbar drainage. A surgical re-exploration was to be employed if these non-operative measures failed to stop the CSF leakage. A VP shunt was done in patients having gross hydrocephalus on post-operative CT brain. Cases of otorrhea were also initially managed conservatively. Surgical re-exploration was to be done if the otorrhea didn’t settle.In one third of the patients developing incisional The development of meningitis in any patient at any point in time was also noted. The follow up of all the patients was of two months. The collected data was entered and analyzed using SPSS version 20. Qualitative data like gender, intervention type, CSF leakage, leakage stopped and meningitis settled are presented in the form of frequency and percentages. Mean ± S.D was used for age, time of interventions and meningitis settled. Chi-square test was applied for comparison of types of interventions and leakage stopped / meningitis settled. P-value ≤ 0.05 was considered as significant.th ventricle/cerebellar sols, twelve for foramen magnum SOLs, and six for chiari malformations. CSF leakage occurred in 7/50 (14%) cases of retrosigmoid surgeries for Cerebellopontine (CP) Angle SOLs, 12/59 (20.3%) cases of midline suboccipital craniectomies for 4th ventricle/cerebellar SOLs, 4/12 (33.3%) cases of foramen magnum SOLs, and 2/6 (33.3%) cases of chiari malformations. There were no cases of CSF leakage after microvascular decompression (MVD) for trigeminal neuralgia in 20 cases.One hundred forty seven cases with posterior fossa pathologies were operated in the Department of Neurosurgery, Unit-1, Lahore General Hospital from January 15, 2012 to September 15, 2015. Of the one hundred forty seven cases of posterior fossa surgeries, retrosigmoid suboccipital posterior fossa craniectomies were carried out in seventy cases. Fifty of these suboccipital retrosigmoid craniectomies were carried out for CP Angle SOLs (space occupying lesions), and twenty were done for microvascular decompression of trigeminal neuralgia. Midline suboccipital craniectomies were done in seventy seven cases. Fifty nine of these were done for 4Over all, CSF leakage occurred in twenty five (17%) cases. Out of these twenty five cases, there was incisional CSF leakage in twenty four cases, and there was a single case of otorrhea. The incidence is shown in Out of the twenty four cases of incisional CSF leakage, eight patients were managed initially with conservative measures including bed rest, head elevation, local wound care (such as pressure dressing) and re-suturing of the wound. In only two (25%) cases the CSF leakage settled with these conservative measures. CSF lumbar drainage was then instituted in the remaining six (75%) patients, and that lead to the cessation of CSF leakage.In fourteen patients the initial management was re-suturing of the wound along with concomitant CSF lumbar drainage. In all these fourteen cases the CSF leakage stopped. In two cases CSF leakage was accompanied by gross hydrocephalus on postoperative CT and a ventriculoperitoneal (VP) shunt was the intial intervention in these cases and the CSF leakage stopped. There was a single case of otorrhea that settled with conservative expectant management. The initial management was found to be significantly associated (p=<0.05) with the stoppage of CSF leakage in case of an incisional leak as shown in The stoppage of CSF leakage was significantly associated with the initial management (p=0.003). The details of age, timing of first and second intervention, and timing of developing meningitis are given in Four (50%) of the eight patients with incisional CSF leakage initially managed conservatively with resuturing of the wound alone also developed meningitis. Two of these patients developed meningitis before conservative measures were instituted, and two developed meningitis after conservative measures had been instituted. Although CSF leakage settled with CSF lumbar drainage, the condition of these four patients didn’t improve.Of the fourteen patients managed initially with re-suturing and simultaneous CSF lumbar drainage, eight (57%) developed meningitis. Of these eight patients, four (28.5%) had already developed meningitis before the CSF drainage was started. However, not only did the CSF leakage stop but the meningitis also settled in these patients, and they were discharged later on. In the other four (28.5%) patients meningitis developed after CSF lumbar drainage had been started. Two of these patients recovered and were discharged, while two patients didn’t recover from the meningitis although the CSF leakage stopped. The single patient suffering patient suffering from otorrhea didn’t develop meningitis.The first intervention was found to be significantly associated (p= 0.014) with the settlement of meningitis in cases of incisional CSF leakage as shown in Cerebrospinal fluid leakage remains a significant cause of morbidity after posterior fossa surgery.13Although the incidence of CSF leakage after posterior fossa surgery has been quoted to be as high as 26.7%Treating postoperative CSF leakage following posterior fossa surgery is a difficult propositon.Bayazit et al.7However, Fishman et al.In our study CSF leakage stopped in only two of the eight patients managed conservatively, and the remaining six patients required CSF diversion in the form of CSF lumbar drainage. Four of these eight patients developed meningitis and this meningitis didn’t settle later on. In the fourteen cases managed initially with re-suturing of the wound and concomitant CSF lumbar drainage, CSF leakage stopped in all the patients. Meningitis also settled in six of the eight patients that had developed meningitis in this group. The settlement of CSF leakage (p <0.05), and settlement of meningitis (p <0.05) were significantly associated with the first intervention. The results of our study lead to the conclusion that conservative measures such as re-suturing the wound alone are not successful in stopping CSF leakage after posterior fossa surgery. Infact, an initial trial of conservative therapy alone increases the morbidity associated with meningitis. Our findings are thus consistent with the findings of Fishman et alOther variables that may have influenced the risk of CSF leak, including poor wound healing  were not tracked, possibly confounding the analysis. Also, these surgical results have been pooled by three neurosurgeons at a single institution—as a result, individual variations in operative protocol unrelated to the above discussion might also potentially confound data analysis.Incisional CSF leaks after posterior fossa surgery should be managed with re-suturing of the wound and concomitant CSF lumbar drainage, instead of an initial trial of conservative therapy alone.


Cerebrospinal fluid (CSF) cytology has low sensitivity for leptomeningeal metastasis (LM); thus, new markers are needed to improve the diagnostic accuracy of LM. We measured carcinoembryonic antigen (CEA) and cytokeratin 19 fragments (CYFRA 21-1) in paired samples of CSF and serum from patients with LM and patients with nonmalignant neurological diseases (NMNDs) as controls. Receiver operating curve analysis was performed to assess their diagnostic accuracy for LM. In patients with NMNDs, CEA and CYFRA 21-1 levels in the CSF were significantly lower than the serum levels. In patients with LM, there was no significant difference between the CSF and serum CEA levels, whereas the CYFRA 21-1 levels were significantly higher in the CSF than the serum. CSF/serum quotients of CYFRA 21-1 were higher than those of CEA in patients with LM and patients with NMNDs. CSF CYFRA 21-1 and CSF/serum quotient of CYFRA 21-1 had high accuracy for differentiating LM from NMNDs that was similar to CSF CEA and CSF/serum quotient of CYFRA 21-1, whereas serum CYFRA 21-1 is of poor diagnostic value. Measurement of CSF CYFRA 21-1 should not be overlooked in patients with suspected LM, even if the serum CYFRA 21-1 level is within normal limits.  Leptomeningeal metastasis (LM) is the metastatic dissemination of malignant cells to the leptomeninges and subarachnoid space of the central nervous system. Cerebrospinal fluid (CSF) cytology remains the gold standard for the diagnosis of LM, but it has relatively low sensitivity . The faiμg/mL and 0.1 ng/mL, respectively. Measurement of a panel of TMs, including CEA and CYFRA 21-1, is a routine aspect of the CSF analysis workup for ascertaining the diagnosis of intracranial malignant metastasis in our institution and had been approved by the institutional ethics committee.CEA and CYFRA 21-1 levels in the CSF and serum were measured with an immunochemiluminescent assay on a Roche MODULAR ANALYTICS E170 analyzer. Specifically, the serum or CSF sample, biotinylated monoclonal CEA-specific or CYFRA 21-1-specific antibody, and a monoclonal CEA-specific or CYFRA 21-1-specific antibody (Elecsys CEA and CYFRA 21-1 immunochemiluminescent assay kit) labeled with a ruthenium complex were reacted to form a sandwich complex. After addition of streptavidin-coated microparticles, the complex bound to the solid phase via the interaction of biotin and streptavidin. The microparticles were then magnetically captured onto the surface of the electrode, and unbound substances were removed. Application of voltage to the electrode then induced chemiluminescent emission, which was measured by a photomultiplier. The results were determined via reference to a calibration curve that was instrument-specifically generated. The lower detection limits for CEA and CYFRA 21-1 were 0.2 The data of patients with LM were retrospectively collected among patients admitted to our hospital from 2005 to 2015. The diagnosis of LM in this study was based on at least one of the following criteria: (1) CSF cytology identified malignant cells; (2) if CSF cytology was negative for malignant cells, primary tumor and/or systematic metastasis was ascertained by pathologic evidence obtained through biopsy or tumor resection, and the clinical manifestation, neuroimaging characteristics, and results of the routine CSF analysis workup were consistent with those of LM –14. PatiFor each patient with LM, the data of a control patient with NMND were retrospectively reviewed. As of January 1, 2011, patients who met the following criteria were finally included in the control group: (1) age and sex were matched with the corresponding patient with LM; (2) a lumbar puncture had been performed; (3) CEA and CYFRA 21-1 were measured in paired samples of the CSF and serum for diagnostic purpose (the two samples had to have been collected from the patient within one week); (4) central nervous system and systematic malignant disease had been ruled out.QCEA) and CYFRA 21-1 (QCYFRA  21-1) were calculated. When CSF CEA was below the lower detection limit (0.2 μg/mL), 0.2 μg/mL was used as CSF level in calculating QCEA.   QCEA was compared with QCYFRA  21-1 in patients with LM and patients with NMNDs, respectively, by Wilcoxon signed ranks test. We used receiver operating characteristic (ROC) analysis to determine the area under the curve to assess the diagnostic accuracy of CEA and CYFRA 21-1 in CSF, CEA and CYFRA 21-1 in serum, QCEA and QCYFRA  21-1 for LM arising from systemic solid tumors. We included diagnostic accuracy measures of optimal cut-off values based on ROC analysis by maximizing the Youden index. All statistical analyses were performed using the software SPSS 19.0 .CEA and CYFRA 21-1 levels in paired samples of the CSF and serum were retrospectively evaluated in patients with LM and in control patients with NMNDs. The Wilcoxon signed ranks test was employed to compare the CSF level with the serum level. CSF/serum quotients of CEA  were included in this study. The primary tumor arose from the lung in 53 patients, stomach in three patients, and colon, liver, breast, and pleura in one patient each. The pathological type of the primary tumor was determined to be adenocarcinoma in 54 patients and mesothelioma in one patient through CSF cytology and/or pathological analysis of the primary tumor. In the remaining five patients, the CSF cytology assay only revealed malignant cells in the CSF; however, the exact pathological type could not be clarified and pathological evidence of the primary tumor was not available.P = 0.54), whereas the CSF level of CYFRA 21-1 was significantly higher than its serum level (P < 0.01)  . Two patIn patients with NMNDs, the levels of both CEA and CYFRA 21-1 in the CSF were extremely low and were significantly lower than those in the serum . Only onQCYFRA  21-1 is 1.19, QCEA and QCYFRA  21-1 were below 1.0 in all other patients with NMDs, and QCYFRA  21-1 was significantly higher than QCEA in both patients with LM and patients with NMNDs  were reported according to the control group patients with different benign pulmonary disease. The optimal cut-off value to different LM from NMNDs determined in this study was almost identical to that for NSCLC when patients with pulmonary tuberculosis were used as control group . The relQCEA and QCYFRA  21-1 were used to reflect their intrathecal synthesis and both showed high sensitivity and specificity for the diagnosis of LM. We also found that QCYFRA  21-1 was higher than QCEA in both patients with LM and patients with NMNDs. The molecular weight of CYFRA 21-1 is 30 kDa, whereas that of CEA is 180 kDa; thus, the trans-blood-brain-barrier (BBB) diffusion of CYFRA 21-1 should be more active than that of CEA. In other words, it should be easier to balance CYFRA 21-1 levels in the CSF and serum with all else being equal. So, in patients with NMNDS, higher QCYFRA  21-1 most likely results from more active trans-BBB diffusion of CYFRA 21-1 from blood to CSF. Considering that the CSF CYFRA 21-1 level was higher than the serum CYFRA 21-1 in most patient, higher QCYFRA  21-1 cannot be attributed to trans-BBB diffusion in patients with LM. Higher QCYFRA  21-1 suggested other underlying mechanisms for elevation of the CSF CYFRA 21-1 level that is strong enough to counterbalance the lowering effect from trans-BBB diffusion.Intrathecal fraction is more accurate for detecting abnormal intrathecal synthesis of biomarkers  and has One possible mechanism is that tumor cells metastasizing to the leptomeninges synthesize and secrete CYFRA 21-1 at higher levels than their counterparts in the primary tumor or systemic metastasis. That is, during the process of metastasis, tumor cells might acquire the capability or increase their inherent capability to synthesize CYFRA 21-1, without a corresponding change in the synthesis ability for CEA. According to the “seed and soil” hypothesis, metastatic colonization of a given organ is not due to chance, but is influenced by a specific affinity of certain tumor cells (the “seed”) for the milieu of certain organs (the “soil”) . CentralClearance of TMs also affects their levels in the body fluid compartment. Clearance of TMs in the CSF is mainly determined by their speed of diffusion across the BBB to the blood. These markers might have a similar clearance speed from the CSF, as they are all large molecules that cannot easily cross the BBB. However, in actuality, the half-life of different TMs in the blood varies greatly. In patients with malignant lung neoplasms, the half-life of CEA was reported to be 1.3 days after curative resection, whereas that of CYFRA 21-1 was found to be much faster at 0.17 days . This miμg/mL was used to calculate QCEA in patients whose CSF CEA is below the lower detection limit. This limitation resulted in artificially high values CSF CEA and QCEA. However, we believe that this limitation does not weaken the diagnostic value of CSF CEA for LM. Even artificially elevated CSF values of CEA and QCEAS in 59 NMND patients were used as controls, CSF CEA  was still demonstrated to be high sensitive and specific for LM. In other words, the actual sensitivity and specificity of CSF CEA and QCEA for LM may be even higher than the results of our study. Fourth, the LM in this group of patients consisted of different pathological types and the number of patients was relatively small. Our findings are most likely relevant for the major pathological types such as lung adenocarcinoma; however, whether or not they are applicable to the more relatively rare pathological type requires further verification.Our study has some limitations worth mentioning. First, the optimal control patients for assessing the diagnostic accuracy of TMs in the CSF for LM are those with pathologically matched solid tumors without central nervous system metastasis. Nonetheless, lumbar puncture is unnecessary in patients without suspected central nervous system metastasis and it is ethically infeasible to perform lumbar puncture just for study purpose. Second, intrathecal fraction is usually employed in evaluating biomarkers in CSF for diagnostic purpose. It reflects intrathecal synthesis of biomarker more accurately than CSF concentration by adjusting the effect of BBB dysfunction. In LM, BBB is usually damaged, elevated TM in CSF may result from trans-BBB diffusion from blood rather than intrathecal synthesis. Because of the retrospective nature of our study, intrathecal fraction could not be obtained. Nonetheless, this limitation does not weaken our findings about the diagnostic value of CSF CYFRA 21-1 for LM. CSF CYFRA 21-1 was higher than serum in most patients with LM, which rule out the possibility that elevated CSF CYFRA 21-1 solely resulted from trans-BBB diffusion from blood. Third, 0.2 Notwithstanding the above limitations, from a clinical perspective, our findings highlight the diagnostic significance of CSF CYFRA 21-1 as a marker for LM. Therefore, CYFRA 21-1 in the CSF should not be overlooked in patients with suspected LM, even if their serum CYFRA 21-1 levels are within normal limits.


Kappaphycus are farmed in tropical countries as raw material for carrageenan, which is widely used in food industry. The sea area available for farming is one limiting factor in the production of seaweeds. Though cultivation is spreading into subtropical regions, the lower seawater temperature is an important problem encountered in subtropical regions for the farming of Kappaphycus. This research of physiological response to low temperature stress will be helpful for screening Kappaphycus strains for growth in a lower temperature environment.Kappaphycus alvarezii and Kappaphycus striatum were evaluated during low temperature treatments . Compared with the controls at 26 °C, the H2O2 concentrations increased in both species when the thalli were exposed to low temperatures , but these increases were much greater in K. striatum than in K. alvarezii thalli, suggesting that K. striatum suffered more oxidative stress. The activities of some important antioxidant enzymes (e.g. superoxide dismutase and ascorbate peroxidase) and the hydroxyl free radical scavenging capacity were substantially higher at 23, 20 and 17 °C than at the control 26 °C in K. alvarezii, indicating that the antioxidant system of K. alvarezii enhanced its resistance to low temperature. However, no significant increases of antioxidant enzymes activities were observed at 20 and 17 °C in K. striatum. In addition, both the maximal efficiency of PSII photochemistry (FV/Fm) and the performance index (PIABS) decreased significantly in K. striatum at 23 °C, indicating that the photosynthetic apparatus was damaged at 23 °C. In contrast, no significant decreases of either FV/Fm or PIABS were observed in K. alvarezii at 23 °C.Responses of antioxidant systems and photosystem II (PSII) behaviors in K. alvarezii has greater tolerance to low temperature than K. striatum.It is concluded that  Kappaphycus alvarezii and Kappaphycus striatum , two important carrageenophyte species, are used as the major commercial source of κ-carrageenan, which is widely utilized as a gelling and stabilizing agent for some food products, including frozen desserts, chocolate-flavoured milk, cottage cheese dressings and soyamilk  and hydroxyl free radical (·OH), which are harmful to the plant’s photosynthetic apparatus. Collén et al. , catalase (CAT) and ascorbate peroxidase (APX)], hydroxyl free radical (·OH) scavenging capacity and the level of malondialdehyde (MDA), an important index of oxidative lesions    in a 12:12 h light:dark (L:D) cycle. They were acclimatized to their new surroundings for a day in the tank before performing the following low temperatures treatments assay.−2 s−1  with 40 L of natural seawater  collected in Li’an bay  and irradiated with a fluorescent lamp at approximately 55 μmol photons ms−1 Fig. . The samAfter treatment at each temperature, algal samples of each species were immediately froze in liquid nitrogen, labeled and stored in a freezer at −80 °C for the following assay  were ground in a mortar in liquid nitrogen and placed in 10-mL centrifuge tubes in ice. The algal powder was mixed with 3 mL of pre-cooled extraction buffer  and homogenized using an S10 homogenizer  in an ice-water bath . H2O2 was bound with molybdenic acid to form a complex, which was measured spectrophotometrically at 405 nm and the concentration of H2O2 was then calculated  based on measuring the enzyme’s ability to inhibit the photochemical reduction of tetrazolium blue. The developed blue color was measured spectrophotometrically at 550 nm  was determined by using a commercially available kit—Hydroxyl Free Radical Assay kit (Nanjing Jiancheng Bioengineering Institute) based on the deoxyribose degradation assay described by Halliwell et al. .To evaluate the extent of oxidative damage in algal cells under low temperature stress, the concentration of MDA, a marker of lipoperoxidation, was measured in the algal thalli by the method of Fraga et al. .Enzyme activities were expressed relative to protein concentrations, which were estimated by the method of Deng et al.  using boFast Chla fluorescence transients were measured with a Plant Efficiency Analyzer . Each transient obtained from the samples was analyzed according to the JIP-test Strasser . Each teV/Fm), the maximum PSII photochemical efficiency, namely the maximum quantum yield of primary photochemistry. FV/Fm = O/ABS, where TRO and ABS denote the trapped and absorbed excitation energy fluxes, respectively.o, the probability that a trapped exciton moved an electron into the electron transport chain beyond (plasto)quinone (QA−). ψo = ETO/TRO, where ETO and TRO denote the electron transport and trapped excitation energy fluxes, respectively.ψA−, namely quantum yield of electron transport. O/ABS.ABS, the performance index on an absorption basis. PIABS = (RC/ABS)·[o/(1 − ψo)], where RC/ABS, o/(1 − ψo) reflect the efficiency of the reaction centers, the light energy absorption efficiency and the accepting efficiency of the electron acceptor, respectively.PIThe following variables were calculated directly or indirectly.t test (p < 0.05).Data are presented as mean ± SD for at least three replicate measurements (the number (n) of replicates is presented in each figure caption). To determine the statistical significance between experimental groups, the data were examined using a 2O2 in both K. alvarezii and K. striatum thalli were observed at 23 °C; they decreased at 20 and 17 °C. A H2O2 concentration similar to the control (26 °C) was observed at 17 °C in K. alvarezii thalli but K. striatum thalli at 17 °C still had a higher H2O2 concentration than the control at 26 °C , but the increase was much greater in K. striatum thalli than in K. alvarezii thalli. For example, the H2O2 concentration at 23 °C was 1.9 times as much as that of the control in K. alvarezii thalli, while the H2O2 concentration at 23 °C was 3 times as much as that of the control in K. striatum thalli. These results suggest that K. striatum thalli suffered more serious oxidative stress from H2O2 than K. alvarezii thalli.The highest concentrations of HK. alvarezii and K. striatum thalli. Compared with the control, SOD activities and APX activities increased significantly in both species when algal thalli were exposed to 23 °C  , whereas those of K. striatum thalli had no significant differences at either 20 or 17 °C compared with the control. Although CAT activities did not vary significantly in the two species after exposure to low temperatures  . In addition, compared with the control, hydroxyl free radical scavenging capacity (HFRSC) had significant increases at lowered temperatures  in K. alvarezii thalli. Hydroxyl free radical scavenging capacity (HFRSC) of K. striatum thalli peaked at 23 °C (HFRSC was 2.2 times as much as the control), but dropped to the same level as the control at the lower temperatures, 20 and 17 °C  whereas that of K. alvarezii thalli still had resistance to the lower temperatures (20 and 17 °C). No significant differences of MDA were found in the two species during exposure to lower temperatures (23 and 20 °C)  in both V/Fm = K. striatum thalli at 23 °C  . When exposed to 17 °C, both K. alvarezii and K. striatum thalli had significant decreases in FV/Fm compared to the control . Because the value of FV/Fm largely reflected the physiological activity in plants, it was inferred that physiological activity in K. alvarezii might have greater tolerance to low temperature stress than that in K. striatum.Compared with the control, a significant decrease in the maximum PSII photochemical efficiency (F68) Fig. , indicatA− (ψo), both K. alvarezii and K. striatum thalli only exhibited significant decreases of ψo when the temperature dropped to 17 °C  , indicating again that K. alvarezii had stronger low temperature tolerance than K. striatum with regard to electron transport.With regard to the excitation efficiency for electron transport beyond QABS) and its three variables RC/ABS, Since the parameters of photosynthetic efficiencies only reflect the energy cascade from light absorption to electron transport and don’t indicate the specific PSII component damaged by low temperature stress, the performance index on an absorption basis . Compared with the control, there were significant decreases of PIABS in K. striatum thalli when the temperature was below 23 °C , whereas a significant decrease of PIABS was observed in K. alvarezii thalli only when the temperature was dropped to 17 °C  , were calculated. RC/ABS, o/(1 − ψo) reflect the efficiency of reaction centers, the light energy absorption efficiency and the accepting efficiency of the electron acceptor, respectively. When the temperature was dropped to 23 °C, RC/ABS decreased significantly in K. striatum thalli (the ratio RC/ABS was 0.32 at 23 °C while the control was 0.46), whereas no significant decrease of RC/ABS was observed in K. alvarezii until the temperature was decreased to 17 °C (the ratio RC/ABS was 0.45 at 17 °C while the control was 0.72) , indicating that the low temperature reduced the efficiency of the PSII antenna in both species ) in both K. alvarezii thalli and K. striatum thalli were impacted negatively at 17 °C (the ratios ψo/(1 − ψo) were 0.58 and 0.32 in K. alvarezii and K. striatum thalli, respectively, while the respective controls were 0.90 and 0.60), indicating that the acceptor sides of PSII in these two species were damaged  activities. Barros et al.  but those of K. striatum thalli didn’t. However, it is worth noting that no significant difference of CAT activity was observed during any of the low temperature treatments in either of the two species. Because CAT and APX are two important H2O2-scavenging enzymes, why doesn’t CAT respond to the increase of H2O2? Barros et al.  has a greater affinity for H2O2 than CAT. So it is surmised that concentrations of H2O2 didn’t reach the level at which CAT would play a role during these low temperature treatments. Not only H2O2, but also ·OH (a powerful oxidizing agent) can cross membranes and may oxidize a variety of compounds , indicating that K. alvarezii retained a stronger capacity for eliminating ·OH at 20 and 17 °C but K. striatum did not. Besides, the lipoperoxidation damage was only observed in K. striatum thalli   of res Fig. , indicats et al.  found thes) Fig. . From thV/Fm values decreased) in both species as a consequence of low temperature stress, indicating that photo-damage had occurred such that photosynthetic activities decreased in both species.Given that PSII is the most sensitive part of the photosynthetic apparatus, changes of the PSII state can indicate changes in physiological processes during low temperature stress  and the quantum yield of electron transport  and can reflect the states of different photosynthetic components  for the two species, the values of RC/ABS, K. striatum thalli at 23 and 20 °C while no significant difference of RC/ABS was observed in K. alvarezii thalli at these temperatures. This suggests that efficiency of PSII reaction centres in K. alvarezii thalli remained at a normal level while the efficiency of PSII reaction centers in K. striatum thalli decreased at lower temperatures (23 and 20 °C). It was inferred that PSII reaction centers of K. striatum might have weaker resistance to low temperature stress than those of K. alvarezii.PIg et al.  studied red Fig. . From ouK. alvarezii and K. striatum are important commercial red seaweeds. Strains of Kappaphycus could be selected for temperature tolerance by using the Chl fluorescence method, which is non-destructive to the algal tissue and takes only a few secs to record .κ-Carrageenan-producing


Surface electromyography (sEMG) signals reflect muscle contraction and hence, can provide information regarding a user’s movement intention. High-density sEMG systems have been proposed to measure muscle activity in small areas and to estimate complex motion using spatial patterns. However, conventional systems based on wet electrodes have several limitations. For example, the electrolyte enclosed in wet electrodes restricts spatial resolution, and these conventional bulky systems limit natural movements. In this paper, a microneedle-based high-density electrode array on a circuit integrated flexible substrate for sEMG is proposed. Microneedles allow for high spatial resolution without requiring conductive substances, and flexible substrates guarantee stable skin–electrode contact. Moreover, a compact signal processing system is integrated with the electrode array. Therefore, sEMG measurements are comfortable to the user and do not interfere with the movement. The system performance was demonstrated by testing its operation and estimating motion using a Gaussian mixture model-based, simplified 2D spatial pattern. Muscle contraction is triggered by action potentials that propagate through muscle fibers. The combination of the action potentials can be measured from the skin surface using a technique known as surface electromyography (sEMG). This technique gathers information on movement, and it is widely used in various fields such as prosthetics  and rehaA high-density sEMG system requires tiny electrodes to retrieve a high-resolution spatial distribution. Generally, wet electrodes are used for a high-density sEMG system because the conductive electrolyte enclosed in the wet electrode helps to reduce skin impedance, and assists in measuring signals from small areas by diffusing into the skin. Lapatki et al.  developeA solution for a high-density sEMG electrode is a microneedle array (MNA) electrode. The microneedles penetrate the high-impedance outermost skin layer and make direct contact with the more conductive living-skin layer. Therefore, an MNA electrode can measure low skin impedance sEMG signals for a long time without an additional medium. Furthermore, even a single microneedle can measure meaningful sEMG signals given its high sensitivity . In otheHowever, conventional MNA electrodes are not suitable for constructing a high-density sEMG system. Most MNA electrodes ,12,13 haNot only the electrode specification, but also the signal processing system is important for sEMG measurement. sEMG signals are of the order of microvolts, and they are highly susceptible to noise such as power supply interference and cable artifact. Therefore, the signal amplifier unit and processing unit should be as close as possible to the electrodes for high signal-to-noise ratio (SNR). However, conventional sEMG systems ,20,21 foIn this paper, a gold electroplated stainless steel microneedle-based high-density (MNHD) electrode array on a circuit integrated flexible substrate is proposed, as illustrated in The proposed system involves signal measurement, signal amplification, feature extraction and feature transmission. In addition, Gaussian mixture model (GMM) -based spThe rest of this paper is organized as follows. The fabrication of the MNHD electrode array on a flexible substrate and the sEMG system implementation are described in Both the hardware and software of the proposed wireless compact sEMG system were developed. A diagram of the complete system is shown in Gold electroplated stainless steel needles, having the diameter of the body as The fabrication of the array is simple and cost effective, and it is depicted in The desired needle length was The target limb of the developed electrode array in this study is the forearm flexor group; thus, the specific array size was designed as  results . Four neThe proposed system measures sEMG signals in a bipolar configuration. The muscle activation signals measured on the four needles constituting an electrode were merged. Thereafter, the differential activation between two electrodes against a reference electrode, which is placed on the bony body part such as the elbow, was measured. Raw sEMG signals are of the order of microvolts, and are thus highly susceptible to noise sources such as power supply interference and movement artifacts. Therefore, an amplifier is required to increase the SNR. The RHD2216 amplifier  was selected. Sixteen bipolar sEMG signals were sampled at The complexity of sEMG signals hinders their analysis. Hence, a signal processing module was embedded in the proposed system to extract and transmit useful features  value of an sEMG signal window) from such signals to an external device. For this stage, an appropriate processing unit was considered. An sEMG signal should be sampled and wirelessly transmitted every The software embedded in the microcontroller processes the sEMG signal and transfers data every System performance can be evaluated by considering signal acquirement and processing latency. The typical bandwidth of the sEMG signal is 5–500The sEMG system amplifies the differential amplitude between two electrodes. Therefore, the performance can be evaluated by applying known input signals  to the electrodes and measuring the amplifier output.A sine wave was simulated and generated by NI PCI-6259 , and wires were connected to one channel of the system. The frequency and amplitude of the simulated sine wave were The measured signal and the simulated signal are shown in During full signal processing and transmission processes , the execution time and its interval loop were evaluated using the microcontroller timer and an oscilloscope. Specifically, a pulse wave was generated, and its amplitude was changed in the loop interval in which mean the frequency of the pulse wave should be The experimental results are shown in The measured sEMG signals vary depending on the electrode placement. Hence, spatial patterns retrieved from different electrodes in a specific area can be used to retrieve motion information. As an example, the sEMG signal during hand-close and two-finger (index finger and middle finger) flexions were measured. The electrode array was placed on the flexor group of the left forearm and the reference electrode was place on the elbow. The motions were performed sequentially. Then, the measured signals were divided into During the hand-close motion, channel 2 exhibited higher amplitudes than channels 7, 13, and 15. During two-finger flexions, channels 2 and 7 showed a similar tendency in that the exhibited amplitudes were similar to the amplitudes during the hand-close for each channel. On the other hand, channel 13 exhibited a weak amplitude compared with the amplitude during the hand-close. By contrast, channel 15 exhibited a higher amplitude than that during the hand-close. Thus, the sEMG activation signal amplitude varies according to motion, which is affected by the electrode placement.To obtain a simplified spatial pattern, signals were discretized by a GMM, which is a clustering method. The model retrieved the probabilistic model parameters from a set of samples. One-dimensional GMM was trained using RMS signals from all channels. The simplified 2D spatial patterns during hand-close and two-finger flexion are shown in In this section, finger-flexion discrimination was conducted to demonstrate the performance of the system and GMM-based simplified 2D spatial pattern. The finger-motion-related muscles in the forearm have a complex structure. For example, flexor digitorum superficialis has four tendons connected to individual fingers. A 2D spatial pattern can provide meaningful motion information owing to its high spatial resolution.The system was placed on the flexor group of the left forearm. Signals were measured during single-finger flexion motions . Then, the measured signals were divided into These patterns were highly related to the anatomical parameters of the muscle and electrode array placement. Thus, 2D spatial pattern has high inter-subject variability and may not guarantee the performance even on a single subject when the electrode array placement is changed. In a future study, we plan to develop a muscle model-based electrode array placement recognition for robust motion identification.This paper reports on a gold electroplated stainless steel microneedle-based high-density (MNHD) electrode array on a circuit integrated flexible substrate with a compact wireless system for surface electromyography (sEMG). A stainless steel needle is rigid and robust to needle breakage; thus, the needle guarantees high-quality signal measurement in long-term use. In addition, the needle is mass producible and, variable needle parameters such as needle body diameter and needle tip shape can be easily controlled. Furthermore, an electroplated gold layer achieves a low skin-electrode impedance. The flexible substrate has sufficient flexibility to cope with skin curvature, and its circuit provides stable electrical contact and robustness to external noise. The wireless signal processing system transmits the features to an external device via Wi-Fi. Therefore, the proposed wireless system provides freedom for the user’s movement, and a natural motion estimation is available. Additionally, simple and effective GMM-based simplified pattern is proposed for motion estimation.The performance of the system was evaluated by considering signal acquirement and processing latency. In addition, finger motion recognition was demonstrated to prove the simplified pattern-based approach. The experimental results indicated that the proposed system can measure and transmit reliable sEMG signals in real time, and the GMM-based simplified pattern can be used to estimate motions.


In 2009, three years after stopping mass treatment with azithromycin, a trachoma impact survey in four health districts in the Kayes region of Mali found a prevalence of trachomatous inflammation—follicular (TF) among children aged 1 to 9 years of >5% and a trachomatous trichiasis (TT) prevalence within the general population (≥1-year-old) of <1%. As a result, the government’s national trachoma program expanded trichiasis surgery and related activities required to achieve trachoma elimination.In 2015, to assess progress towards elimination, a follow-up impact survey was conducted in the Kayes, Kéniéba, Nioro and Yélimané health districts. The survey used district level two-stage cluster random sampling methodology with 20 clusters of 30 households in each evaluation unit. Subjects were eligible for examination if they were ≥1 year. TF and TT cases were identified and confirmed by experienced ophthalmologists. In total 14,159 people were enumerated and 11,620 (82%) were examined. TF prevalence ) was 0.5% (0.3–1%) in Kayes, 0.8% (0.4–1.7%) in Kéniéba, 0.2% (0–0.9%) in Nioro and 0.3% (0.1–1%) in Yélimané. TT prevalence (95% CI) was 0.04% (0–0.25%) in Kayes, 0.29% (0.11–0.6%) in Kéniéba, 0.04% (0–0.25%) in Nioro and 0.07% (0–0.27%) in Yélimané.Eight years after stopping MDA and intensifying trichiasis surgery outreach campaigns, all four districts reached the TF elimination threshold of <5% and three of four districts reached the TT elimination threshold of <0.1%. Trachoma is a leading infectious cause of preventable blindness worldwide. The World Health Organization (WHO) recommends that endemic states implement the SAFE strategy  to achieve trachoma elimination by the year 2020. Mali has implemented this strategy from 2002. In the Kayes region in southwest Mali, the districts of Kayes, Kéniéba, Nioro and Yélimané stopped antibiotic treatment in 2006 after three rounds of mass drug administration (MDA). In 2009 an assessment was conducted and found that the prevalence of active trachoma (TF) among children aged 1–9 years and trichiasis (TT) prevalence in adults were above the elimination thresholds in these districts. An intensified effort to expand trichiasis surgery and related interventions, including community-based behavior change communication on facial cleanliness and environmental improvement activities using women’s groups, community health workers, and radio. A follow-up survey was conducted in 2015 in the four districts to assess whether one more round of mass treatment was needed. The results showed that TF prevalence in all four health districts decreased to below the 5% elimination threshold and three of four districts achieved the TT elimination threshold of <0.1%. Chlamydia trachomatis [Trachoma is an eye disease caused by infection with chomatis , 2. Tracchomatis . In 2017chomatis . Trachomchomatis . Worldwichomatis .Surveys conducted in Mali from the 1980s showed high prevalence of active trachoma in children below 10 years in many regions, often exceeding 25% , 7. NatiIn 1996, the WHO created the Alliance for the Global Elimination of Trachoma by 2020 (GET 2020) , 9. The WHO also defined a threshold for the implementation of mass drug administration (MDA) of a TF prevalence in a health district ≥10% among children aged 1–9 years –14. MaliAs recommended by the WHO 2006 guidelines , an impaIn light of these recommendations, the Mali PNSO decided to conduct an impact survey in these four health districts in 2015 before making the decision to implement another round of MDA. In this paper, we present the results of the impact survey and discuss the next steps for trachoma elimination in these four districts.2. Nearly the entire population (90.6%) of the region lives in rural areas. The region is ethnically diverse with the population composed primarily of Khassonké, Malinké and Soninké people. The climate is arid in the north and semi-arid in the south, with average annual rainfall of 516 mm in the region. The region lacks adequate access to clean water, with an average of 190 inhabitants per clean water point [The Kayes region is Mali’s largest administrative region and is located in the western part of the country, bordered on the east by the Koulikoro region, west by the Republic of Senegal, north by the Islamic Republic of Mauritania, and south by the Republic of Guinea. The population in 2015 was estimated at 2,445,000 projected from the 2009 General Population and Housing Census, Mali , with aner point . The KayTo assess TF and TT prevalence in the four districts included in the study, a cross-sectional survey was conducted using two-stage, cluster random sampling methodology , 19. BasAssuming a TF prevalence of 4%, a precision estimate of ±2%, an alpha of 5%, and a design effect of 2.7, a sample size of 996 children aged 1–9 years for each evaluation unit was calculated. An extra 12% was added to the sample size to adjust for refusals and this gave rise to an expected sample size of 1132 children aged 1–9 years per evaluation unit. The sample size for adults (≥15 years) was not calculated as all adult household members present at the selected households were to be examined for TT and other trachoma signs.Twenty clusters were selected per evaluation unit using the probability proportional to size sampling strategy, from a complete list of villages, and population, in each health district , 20. TheThe survey was implemented by teams, composed of an examiner  and a data collector. Each team was responsible for 10 clusters, with two teams per district. Teams were supervised by a senior ophthalmologist, with expertise in screening of TF and TT cases, responsible for confirming all detected cases .With the assistance of village authorities, field teams prepared a complete list of households in selected villages, broken down into segments of five households. The last segment could include four or six households. Six segments were then chosen randomly, for a total of 30 households per village . The houAll household members aged ≥1 year were examined for signs of trachoma. The eye examination was conducted using a magnifier visor (2.5x magnification) in natural light or with a flashlight. When a case of trichiasis was found, the examiner was advised to evert the eyelid to determine and record the presence or absence of conjunctival scarring. If such scarring was present, or if the eyelid could not be everted, this was considered a TT case. Information was recorded on whether surgery was offered to individuals with TT in order to estimate the number of TT cases unknown to the health system . The WHOIn addition to looking for signs of trachoma, examiners also checked for discharge from the eyes and/or nose to assess facial cleanliness among all children aged 1–9 years. Children aged 5–14 years were asked about school attendance. All individuals examined were asked whether they had taken azithromycin during previous MDA campaigns; examiners used containers of azithromycin tablets as visual aids. One adult in each household was questioned about the location of the water source used for daily household tasks. Latrines in households were visually inspected to confirm use and type . GPS coordinate data were gathered for each cluster.Prior to the implementation of the surveys, the PNSO, with support from technical and financial partners, trained examiners and interviewers. The training followed the guidelines of the Global Trachoma Mapping Project (GTMP) . At the 2 were used to compare proportions between gender and signs of trachoma and between the SAFE strategy components and signs of trachoma. A 95% confidence interval (CI) was calculated for prevalence. TT backlog was calculated taking the TT prevalence and the district population into account. A Z-test was used to compare trachoma prevalence from the present study with prevalence in 2009 [Data were collected using the ONA interface on tablet computers and analyzed using Excel , Epi Info 7.2.1.0  and R software . Fisher’s exact test and Pearson χThe survey was conducted as part of routine monitoring and evaluation activities of the national program of the Ministry of Health to eliminate trachoma as a public health problem in Mali. As it was a standard public health measure and all procedures for survey and diagnosis were following the WHO guidelines, review of the protocol by the Institutional Review Board was considered not necessary and therefore not conducted. Authorization was sought, and granted, from village chiefs before surveys were conducted in the selected villages. At the household level, oral informed consent was obtained from the heads of the selected households, and any other adult, parent or caregiver, prior to beginning the survey for their participation and that of their children. In addition, assent was obtained from each child >6 years who participated. Survey participants were informed of the purpose of the trachoma examinations and their rights not to participate or to stop the examination at any time. Oral consent was documented on a survey electronic data collection tool. All children presenting signs of TF or trachomatous inflammation—intense (TI) were provided free tetracycline eye ointment and caregivers were instructed to apply it twice daily for six weeks. Those identified with TT were recorded, counselled, and offered free consultation and surgery with a trained TT surgeon.A total of 14,159 people aged ≥1 year were enumerated and 11,620 (82.1%) were examined in the selected households in the health districts of Kayes, Kéniéba, Nioro and Yélimané. Of those examined, 4,502 (38.7%) were children aged 1–9 years and 5,573 (48%) were aged 15 years and above. 2, p>0.05) ; 0.81% (95% CI: 0.41–1.59%); 0.23% (95% CI: 0.06–0.84%) and 0.31% (95% CI: 0.11–0.91%) for the districts of Kayes, Kéniéba, Nioro and Yélimané, respectively . Boys 1– p>0.05) . In cont p>0.05) .The prevalence of unknown TT cases in the population examined (≥1 year old) was less than 0.1% in three of the four surveyed districts. The prevalence was 0.04% (95% CI: 0.00–0.25%) in Kayes, 0.04% (95% CI: 0.00–0.25%) in Nioro and 0.07% (95% CI: 0.00–0.27%) in Yélimané. The TT prevalence in Kéniéba was 0.29% (95% CI: 0.11–0.60%), which is greater than the WHO elimination threshold (>0.1%). Although five (38.5%) of the 13 TT patients found were female, the difference in TT prevalence between males and females was not statically significant . TT prevalence decreased considerably from 2009 to 2015 for all districts  .The prevalence of TT in those aged ≥15 years ranged from 0.09% to 0.65% in the four districts surveyed . The preBased on the 2015 survey, the TT backlog i.e. those patients who needed surgery but had not yet received it, was recalculated from 2009 to 2015 respectively to: 3,850 to 314 in Kayes; 1,495 to 571 in Kéniéba; 360 to 113 in Nioro and 357 to 131 in Yélimané.More than 85% of children aged 1–9 years had a clean face at the time of the survey. This proportion was 92.1% in Kayes, 81.1% in Kéniéba, 88.5% in Nioro, 78.2% in Yélimané . Basic sAmong the 4,502 children (1–9 years) examined, those with clean faces had a TF prevalence of less than one-half that of children who did not have clean faces . In addition, these gold mine sites were sometimes not visited by the surgical teams because they are not official villages and thus not counted during screening. The TT surgical refusal rate was higher than 20% sometimes according the TT surgery activity reports in this district. This may explain the small drop in TT prevalence, despite the good geographical coverage of health areas for surgical campaign.Improved hygiene and sanitation play an important role in controlling the transmission of trachoma . Facial One limitation of this study was that the current survey was not powered to give an accurate estimate of TT prevalence. This underpowering may have contributed to the large spread of the 95% CI of the TT and TF prevalence. Despite that TT prevalence in three districts met the elimination threshold, it is noted that the upper bound of the 95% CI of TT prevalence in these districts was above the 0.1% elimination threshold in the population. Secondly, the result on TT in the 2015 survey was recorded differently from the 2009 survey as only those cases unknown to health systems were counted as TT cases in the result. The 2015 TT result was not fully comparable to the 2009 result; however, it did represent the field situation at the time. Based on the current data, the national program should continue with the TT surgery effort in all four districts for the remaining TT cases. For future trachoma survey to verify whether the TT prevalence in an EU has reached the elimination threshold, a properly powered TT survey may be needed.In conclusion, these results showed that the TF prevalence in children aged 1 to 9 years fell considerably between 2009 and 2015 for all four HDs without the need of one additional round of MDA. All four health districts met the TF elimination criteria and three of the four surveyed met the TT elimination threshold. Based on these results, the TT surgery campaigns will continue only in the Kéniéba health district. The PNSO also recommended that in Kayes, Nioro and Yélimané districts, TT case management should continue in fixed-site centers in hospitals and the referral health centers in order to ensure availability of surgery for remaining TT cases after elimination of trachoma as a public health problem has been achieved. The results of these surveys show that Mali is on the right track toward eliminating trachoma as a public health problem by 2018.S1 Checklist(DOC)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.