However, it is not known if these approaches can be used for the comparison of metatranscriptome datasets and which dissimilarity measures perform the best.The comparison of samples, or beta diversity, is one of the essential problems in ecological studies. Next generation sequencing (NGS) technologies make it possible to obtain large amounts of metagenomic and metatranscriptomic short read sequences across many microbial communities. De novo assembly of the short reads can be especially challenging because the number of genomes and their sequences are generally unknown and the coverage of each genome can be very low, where the traditional alignment-based sequence comparison methods cannot be used. Alignment-free approaches based on k-tuple frequencies to real metatranscriptomic datasets from pyrosequencing 454 and Illumina sequencing platforms to evaluate their effectiveness for the clustering of metatranscriptomic samples, including three S2 and three classical http://code.google.com/p/d2-tools/.We applied several beta diversity measures based on k-tuple based sequence signature measures can effectively reveal major groups and gradient variation among metatranscriptomic samples from NGS reads. The The  The comparison of microbial communities is crucial for understanding how environment factors affect the composition and the function of the communities k-tuple  sequence signature of a community is defined as the frequency of k-tuples among the reads from the community. Previous studies have shown that k-tuple frequencies are similar across different regions of the same genome, but differ between genomes k-tuple method. The sequencing result of one microbial community is represented by a frequency vector of k-tuples, dependent only on the sequencing data, free from sequence alignments and reference genome information. Therefore, k-tuple based methods could potentially be very useful for the comparison of short reads data from NGS of microbial communities.The k-tuple sequence signature, each NGS data from a genome is represented by the k-tuple frequency vector whose elements are the number of occurrences of every k-tuple. With the k-tuple vectors, measurements of the dissimilarity between two genomic sequences were studied from different points of views, goals and applications. For two genomic sequences, the un-centered inner product of the two sequence signatures, k-tuple vectors S2Hao in this paper). However, the above measures were developed for evaluating the distance between two long sequences (such as genomic sequences). For better performance applying to NGS data, With the k-tuple sequence signatures have been applied to compare microbial communities based on metagenomic data k-tuple based dissimilarity measures to compare metagenomic samples by clustering them into different groups as well as recovering environmental gradients affecting microbial samples, aiming to evaluate the performances of these measures on comparing metagenomic samples. They found that k-tuple sequence signatures can successfully reveal major group and gradient relationships among metagenomic samples from NGS reads without alignment to reference databases and the The k-tuple frequencies are similar across different regions of the same genome, but differ between genomes k-tuple sequence signature measures to 99 metatranscriptomic and 16 metagenomic datasets from 13 communities/projects, among which 92 datasets from 12 communities were generated by the pyrosequencing 454 platform and 7 datasets from 1 community were generated by the Illumina Genome Analyzer IIx platform. The processing follows the same steps with our previous work counting k-tuple vectors of each dataset, calculating signature measures between dataset pair and then clustering according to the dissimilarity matrix. We conducted a series of computational experiments to study the effectiveness of the 16 k-tuple based sequence signature measures in clustering metatranscriptomic or mixture of metagenomic and metatranscriptomic datasets, identifying gradient relationships of microbial community samples, clustering ability when sequencing depth is low and the effect of sequencing errors on their performance. We also investigated the effects of various tuple sizes and the order of Markov model for the background genome sequences. We also developed a software pipeline to implement the processing procedures, which is more efficient in calculating, more comprehensive in function and more convenient to use compared to d2Meta for calculating the three d2-type measures in previous work With the development of experimental and sequencing techniques, many metatranscriptomic datasets have been generated to explore the expressed genes and their abundances in microbial communities k-tuple occurrences within the reads. This representation makes the direct comparison of two sequence datasets, for example, two metatranscriptomic sequencing datasets, feasible. The comparison is free from alignment of the reads to reference sequences, which are often incomplete or unavailable. Therefore, in our paper, the sequence signature represented by k-tuple frequency is applied to compare metatranscriptomic datasets.The sequence signature of a NGS data set counts the number of k-tuple frequencies. For metagenomic or metatranscriptomic sequencing data, with four possible alphabet k possible tuples of length k in all reads. The numbers of occurrences in the whole sample for the k-tuples form the k-tuple frequency vector of the sample.Without alignment to genome/transcriptome, the information of the reads’ strand direction cannot be obtained. Hence, we take both a read and its complement into consideration when counting k-tuple frequency vector represents the absolute number of occurrences of each k-tuple. For the distance between two datasets, proper normalization or centralization of the sequence signature is required. Therefore, there are various dissimilarity measures with different normalizations. In this paper, we studied various dissimilarity measures based on k-tuple frequency vectors, including three Hao in the paper) S2The k-tuple frequency vectors from two NGS sequencing datasets k-tuples.Let The  dissimilarity measureThe  and dissimilarity measuresk-tuple under a probability model (Markov model of order = 0–3) for the sequences. The ranges of For formulas (2) and (3), Ma), Euclidean (Eu) and Chebyshev (Ch) dissimilarity measures are classical The Manhattan (The Hao dissimilarity measure is from Hao in our paper to simplify the notation. Hao is defined as follows:k−2)-th order Markov chain.where The S2 dissimilarity measure is a relative entropy based dissimilarity measure developed by Tianming Wang’s group S2 is defined as:i-th k-tuple under an r-th order Markov chain for For formula (8), S2 and Hao dissimilarity measures. Under Markov model k-tuple r-tuple occurrences. The value of Markov models of different orders were used to model the background sequences for the Symmetric difference was originally defined to compare two sets. Robinson used symmetric difference as a criterion to evaluate the consistence between two trees A. Actually, the symmetric difference is the number of nodes that are in one tree and not in the other one. Compared with Parsimony score http://evolution.genetics.washington.edu/phylip.html).UPGMA (Unweighted Pair Group Method with Arithmetic Mean) PCoA  N<n, to optimize the function Spearman’s Ranking Correlation Coefficient (SRCC) assesses how well the relationship between two variables can be described using a monotonic function. If there are no repeated data values, a perfect SRCC of +1 or −1 occurs when each of the variables is a perfect monotone function of the other. In our study, SRCC is applied to evaluate the relationship between the gradient variable and the first principal coordinate of different measures. The SRCC is calculated by the function ‘cor’ from the R toolbox ‘stats’ of R.k-tuple vectors, calculate probabilities of k-tuples under various orders of the Markov models and calculate the dissimilarity matrices under various dissimilarity measures Hao, S2, Ma, Eu and Ch. The tool package can be downloaded from http://code.google.com/p/d2-tools/. Compared with d2Meta, the tool to implement the same processing steps for metagenomic comparison d2Tools has the following improvements: d2Meta only computes three d2-type  in fasta format. There are a total of 2,830,286 reads  and the read length is 164±102(SD) nt. It takes about 4 hours and 1.45 GB memory to finish the pipeline for 2–10 tuple sizes and 4 samples serially for all measures. The tuple sizes can be implemented simultaneously with simple shell command, which will speed up the running time of the pipeline. The running time of calculating the k-tuple frequency vector depends on the size of input datasets and the tuple size. The running memory mainly depends on the tuple size.We tested the http://camera.calit2.net/) and NCBI SRA (http://www.ncbi.nlm.nih.gov/sra) databases. Ninety two metatranscriptomic and 16 metagenomic datasets from 12 communities were generated by the pyrosequencing 454 platform and 7 datasets from one community were generated by the Illumina Genome Analyzer IIx. There are a total of 16,453,499 reads with length 198±92 nt in the 92 metatranscriptomic datasets and 6,031,133 reads with length 443±212 nt in the 16 metagenomic datasets from the 454 sequencing platform. There are 11,447,063 reads with 76 nt in the 7 metatranscriptomic datasets from the Illumina platform. In the previous study of metagenomic comparison We downloaded 99 metatranscriptomic and 16 metagenomic datasets from 13 communities/projects in CAMERA , north Atlantic (West English Channel), foot of north Atlantic (Sapelo Island) and East Pacific ocean . So the 19 samples can be clustered into 4 groups distinctively. Furthermore, Dataset 4 (Georgia_May) contains 2 control samples, 2 PUT-amended samples and 2 SPD-amended samples. Therefore, within the same community, the samples from the same condition should merge first. The reference clustering tree for the 19 samples from the 4 communities is shown in S2, Eu and Ch can achieve this optimum score with appropriate choices of tuple size k. However, the range of the values k that yields the optimal results is different. For k is between 6–9 with the order of the Markov chain not significantly affecting the results. For k between 6–10 for all orders of the Markov chain. Thus, the results are robust with respect to the length of k-tuples and the order of Markov chain. We observed that big ranges of settings under Hao yield the optimal value. For In k and dissimilarity measures are shown in nd order and 3rd order Markov model. For 1% sampling rate, the optimal average score is increased to about 14, and all measures cannot achieve the results as good as that using the complete data. When sampling rate drops to 0.1%, the Hao deteriorates significantly when the tuple size increases. This trend is more obvious with the decrease of sampling rate, that is, when the sequencing depth is low. It might be caused by Hao’s attributions of the high number of parameters that need to be estimated to fit a Markov model of order k−2. Ma can achieve pretty good symmetric difference scores when the size k are between 8 to 10 at all sampling rates, which may attribute to its summing up the difference between two communities for all the 4k k-tuples, which can reduce the bias from inefficient coverage when the sequencing depth is low. Other measures, such as Ch, Eu, and S2, do not perform well in all cases. It can be seen from To evaluate the effect of sequence depth on the performance of the different dissimilarity measures, we randomly sample 10%, 1% and 0.1% of the original reads from the 19 metatranscriptomic datasets. The read numbers are shown in Table S1 in k = 6, and the simplest 0-th order Markov model. The clustering result is shown in For the outstanding performance of The gene expression levels in microbial communities may be controlled by a gradient such as ocean depth, temperature, and pH levels. In order to see the performance of the different dissimilarity measures in recovering the gradient relationships of the microbial communities, we used eight metatranscriptomic samples from 25 m, 75 m, 125 m and 500 m depth (two samples for each depth) of North Pacific Subtropical Gyre (NPSG) in ALOHA stations  are merged first, which is not reasonable based on basic biological knowledge. Therefore, the We next used the dissimilarity measures to cluster metagenomic and metatranscriptomic samples. Our objective is to see if metagenomic samples and metatranscriptomic samples separate into two groups. The samples from collection depth of 25 m, 75 m, 125 m and 500 m (two samples for each depth) of North Pacific Subtropical Gyre (NPSG) in ALOHA stations , shown on k = 4, the clustering in metatranscriptomic dataset are still correct, while when the tuple size increases from 4 to 6, the clustering results become worse.The eight metatranscriptomic and eight metagenomic datasets from four depths are also randomly sampled with 10%, 1% and 0.1% rates to evaluate the performance of the different dissimilarity measures when the sequencing depth is low. The read numbers are shown in Table S10 in We also analyze eight metagenomic and eight metatranscriptomic samples from the Western English Channel sequenced with pyrosequencing 454 platform Seven metatranscriptomic datasets collected from cecum and colon tissues of four mice were sequenced with the Ilumina Genome Analyzer IIx (GaIIx) platform and they were downloaded from NCBI SRA. The description can be found in Dataset13 of k-tuple frequency vectors and the 16 dissimilarity measures, the dissimilarity between each sample-pair is obtained. With the dissimilarity matrix, hierarchical clustering is implemented with UPGMA. The symmetric differences between the clustering results and reference cluster under various measures, tuple size and order of Markov models are calculated as shown in According to the two tissues the datasets coming from, the seven samples can be clustered into two distinct groups, as shown in S2, Hao and and Ma can achieve this optimum score with appropriate choices of tuple size k. However, the range of the values k that yields the optimal results is different. For k with higher orders of Markov chain. For S2, the optimal results are obtained when k is between 6–8 with the order of the Markov chain playing no roles. For Hao and Ma, the optimal results are obtained for k between 6–10. One of the optimal clustering trees with k = 4 and 0-th order Markov model under In k and dissimilarity measures are shown in k value as the complete dataset. However, for Hao, althougth they can still obtain the optimal results, the range of optimal k value become smaller compared with the complete datasets. The results show that compared with other measures, Ma deteriorates slightly with the decrease of sampling rate, which can obtain the optimal clustering results with slightly narrower range of tuple size k. However, the performances of other measures, including Hao, Ma and S2, become worse when coverage decreases, and except the Ma, other measures cannot obtain the optimal clustering results, as shown on Table S14 in To evaluate the effect of sequence depth on the measures’ performance with Illumina datasets, we randomly sample 1%, 0.1% and 0.01% of the original reads from the seven metatranscriptomic datasets. The reason we sampled with 10% sampling rate for 454, but not for Illumina, is due to the high sequencing depth of Illumina data. The read numbers are shown in Table S11 in Ma keeps the same symmetric differences as the original data. Many more of the symmetric differences, 3–7 among 9 values, for Hao, Eu and Ch, are changed.The 19 metatranscriptomic samples of 4 communities having distinct geographic locations used in Experiment 1 are used to study the effect of sequencing errors on the performance of sequencing signature measures. According to sequencing error features of the 454 platform If a measure can obtain optimal clustering results based on the original data, it keeps the performance on imported-error data. If the measure cannot obtain the optimal results with the original data, the measure will not achieve the best performance either with added errors. Therefore, the relative performances of the dissimilarity measures are not affected by the sequencing error rates for the current sequencing technologies.k-tuple based sequence signature methods to compare microbial metatranscriptomic samples with NGS reads data. We studied three k = 2–10 and different Markov background models, were compared on the basis of five experiments of real metatranscriptomic datasets from global marine communities, with the objectives to explore their performance on clustering metatranscriptomic sequencing data from different communities generated by pryosequencing 454 and Illumina platforms, identifying gradient variance of metatranscriptomic datasets, clustering characteristics when metagenomic and metatranscriptomic datasets co-exists and robustness under sequencing errors.In this study, we evaluated the performance of k, k-tuple dissimilarity measures can reflect the gradient tendency, and k = 2–10, and the performance of different dissimilarity measures varies with different tuple size. The order of background Markov model does not affect the performance significantly.For geographically well separated communities, all the measures can classify the big groups correctly. With the complete data, for certain range of tuple size Hao’s attributions of the high number of parameters that need to be estimated to fit a Markov model of order k−2 leads to the poor performance under low sequencing depth. Ch considers the maximum difference between the tuple frequencies for the samples only and does not make full use of the information from all the tuples. On the other hand, Ma sums up the difference between two communities for all the k-tuples, which can reduce the bias from low coverage when sequencing depth is low. The normalization of the tuple counts by their corresponding expectations plays an important role in the superior performance of Our results indicate that Hao dissimilarity measure performs reasonably well when sequence depth is high and the tuple size is relatively low. One explanation is that it compares the numbers of occurrences of k-tuples with their corresponding expectations based on the k−2 order of Markov chain, which may not be accurate especially when the sequence depth is low and the tuple size is high. Ch considers the maximum difference between the tuple frequencies of the samples only and does not make full use of the information from all the tuples. On the other hand, Ma sums up the differences of all the k-tuple frequencies between two communities, which can reduce the bias from inefficient coverage when the sequencing depth is low. The normalization of the tuple counts by their expectations plays an important role in the superior performance of The performance of different dissimilarity measures varies with the tuple size. We show that k-tuple frequencies are similar across different regions of the same genome, but differ between genomes. However, in metatranscriptomic data, the genes within a genome can have different expression levels and the intron and inter-genetic region sequences are removed, while in metagenomic data, all the genomic regions are the same. At the same time, RNA the different characteristics from DNA, such as degradation, stability, easiness to be broken and alternative splicing, etc., which bring the different preferences and bias distributions to the sequencing process. Therefore, under the situation that: the expression abundance information is imported, the sequences of intron and inter-genic regions are taken out, and different sequencing preference and bias are introduced, whether the alignment-free approaches are valid is a critical question for their further applications to the metatranscriptomic datasets.The study design in this paper is similar to that in Jiang et al. k-tuple based sequence signature methods such as Similar conclusions about the relative performance of the different dissimilarity measures on clustering microbial metagenomic and/or metatranscriptomic communities were obtained. Our conclusion about the applicability of alignment-free statistics, in particular Supplement S1Ch, Hao and Eu. Table S1. Sampling reads number for Experiment 1. Table S2. The symmetric difference between the reference and clustering trees for Experiment 1 (10% sampling). Table S3. The symmetric difference between the reference and clustering trees for Experiment 1 (1% sampling). Table S4. The symmetric difference between the reference and clustering trees for Experiment 1 (0.1% sampling. Table S5. Sampling reads number for Experiment 2. Table S6. The GOF (times 100) by first principle coordinate of PCoA of Experiment 2 (10% sampling). Table S7. The GOF (times 100) by first principle coordinate of PCoA of Experiment 2 (1% samping). Table S8. The GOF (times 100) by first principle coordinate of PCoA of Experiment 2 (0.1% sampling). Table S9. The SRCC between the first principle coordinate and depth for Experiment 2 (10% sampling). Table S10. Sampling reads number for Experiment 3. Table S11. The number of sampling reads for Experiment 4. Table S12. The symmetric difference between the reference and clustering trees in Experiment 4 (1% sampling). Table S13. The symmetric difference between the reference and clustering trees in Experiment 4 (0.1% sampling). Table S14. The symmetric difference between the reference and clustering trees in Experiment 4 (0.01% sampling).Figure S1. The clustering results of both metagenomic and metatranscriptomic datasets in Experiment 3 based on the dissimilarity measures (DOC)Click here for additional data file.


The present study summarizes our experience in treating a patient with a suspected granulocyte colony-stimulating factor (G-CSF)-producing squamous cell carcinoma (SCC) of the lower gingiva, which is a rather rare entity.A 56-year-old woman underwent surgical excision of palate leukoplakia in 1996. In 2009, however, a leukoplakic superficial tumor was detected in the lower left gingiva, for which the patient underwent gingivectomy. This was subsequently diagnosed as SCC. The patient also underwent superselective arterial injection chemotherapy combined with radiotherapy, after local recurrence was observed. The patient was subsequently found to have bone metastasis. After chemotherapy combined with radiotherapy, the patient underwent segmental resection of the lower left jaw, left supraomohyoid neck dissection, and lower jaw reconstruction using titanium plates. Resection of the left femoral tumor and left total knee replacement were also performed. Computed tomography scan performed 1 month after the surgeries revealed multiple lung, liver, spine, and subcutaneous metastases. The patient also exhibited a sudden increase in her white blood cell (WBC) count and a fever that could not be alleviated, despite treatment with antibacterial drugs. A G-CSF-producing tumor was therefore suspected. Serum G-CSF level was high at 250 pg/ml.3/ml and her general condition suddenly deteriorated, and she died as a result of multiple organ failure. A final diagnosis of G-CSF-producing SCC of the lower gingiva was made based on the patient's clinical course.The patient's WBC count increased to 32 × 10 Solid malignancies are sometimes accompanied by leukocytosis and other leukemoid symptoms, the mechanism of which is attributed to the production of granulocyte colony-stimulating factor (G-CSF) by the tumor cells themselves ,2. The rThe patient was a woman in her 56 years old who had initially consulted our hospital in November 1996, at the onset of leukoplakia. The patient’s major complaint was the appearance of leukoplakic lesions corresponding to left side first and second molar equivalent to palate gingiva. The patient had been previously diagnosed with uterine polyps. Her family history was unremarkable. The patient was examined at our department in late November 1996, following a referral from her local dentist, who detected white lesions on the left palate during dental treatment in mid-November of the same year. The patient was diagnosed with leukoplakia, and the palatal leukoplakia was resected through an excisional biopsy in December of the same year. The patient was subsequently followed up on an outpatient basis. In May 2006, white lesions were observed on the left buccal mucosa and the patient was diagnosed with dysplasia. The condition was treated with another surgical resection, and the patient was followed up as an outpatient. In December 2008, a leukoplakic superficial tumor appeared at left side lower canine, the first premolar equivalency department buccal marginal gingiva.The patient had moderate physique and was well-nourished, and no swelling of the cervical lymph nodes was observed. Leukoderma and erosion were observed at left side lower canine, the first premolar equivalency department buccal marginal gingiva. Figure  upper.Panoramic radiography findings did not reveal any resorption of the lower jaw. Computed tomography (CT) findings did not indicate any clear destruction of the lower left jaw or lymph node enlargement, which could be suggestive of metastasis to the neck.The clinical diagnosis for the patient was tumor of lower left gingiva. In January 2009, the patient underwent an excisional biopsy, with resection of the lower left gingiva tumor. Pathological analysis revealed well-differentiated SCC of more than 20 mm (pT2). No swelling of the cervical lymph nodes was observed. Hemorrhagic leukoderma and erosion were observed at left side lower canine, the first premolar equivalent to palate gingiva. Figure  lower. P3/ml. Her general condition suddenly worsened, resulting in death due to multiple organ failure in February 2010; a month since the G-CSF producing tumor was diagnosed. Patient survived for 13 months from her time of tumor diagnosis till her death and her period of survival was uneventful.On relapse, the patient was clinically diagnosed with a lower left gingiva tumor. The tumor recurred in the lower left gingiva (rT4aN0M0) in July 2009. Systemic chemotherapy was performed with 15 mg bleomycin (BLM). Beginning in August 2009, the patient underwent selective arterial injection chemotherapy, combined with radiotherapy. The chemotherapy regimen consisted of 100 mg cisplatin (CDDP), administered in 4 doses: 30 mg to the lingual artery, 40 mg to the facial artery, 10 mg to the inferior alveolar artery, and 20 mg to the occipital artery. The radiotherapy regimen using Linac consisted of a total of 64 Gray (Gy) irradiation, administered bilaterally in 32 fractions, including levels I and IIa. The patient then complained of pain in the left femur. An imaging study indicated that the tumor had metastasized to the left femur Figure  and 4B; Pathological examination on relapse revealed well-differentiated SCC. Immunostaining of the resected specimen in January 2009 confirmed the presence of G-CSF-positive tumor cells. Staining of biopsy tissue in July of the same year revealed the G-CSF-positive tumor cells to be more pronounced Figure .It is known that solid malignancies are rarely accompanied by leukocytosis and other leukemoid symptoms, the mechanism of which is attributed to the production of G-CSF by the tumor cells themselves ,2. Inflain vitro colony formation assay, measurement of G-CSF activity after transplanting tumors into nude mice [Diagnosis of G-CSF-producing tumors previously required the use of cumbersome methods to demonstrate G-CSF production by tumor cells. Such methods included the ude mice , and detude mice . Currentude mice .3/ml with neutrophil count of 93 %, and an elevated serum G-CSF level of 250 pg/ml . G-CSF-positive tumor cells were identified using immunohistochemical staining. Thus, the patient fulfilled 3 of the 4 above-mentioned criteria, although normalization of granulocyte count and G-CSF level following tumor removal could not be determined. However, comparison of the G-CSF-positive tumor cells in an immunohistochemically stained specimen, surgically removed in January 2009, with those of the biopsy tissue specimen obtained in July of the same year, indicated a marked increase in the number of positive cells of the latter specimen. This finding, combined with the onset of leukocytosis after surgery, suggested a marked proliferation of the patient's G-CSF-producing tumor cells after resection of the tumor, leading to a diagnosis of G-CSF-producing SCC of the lower gum.Asano et al. proposed the following 4 criteria for diagnosing G-CSF-producing tumors: (1) marked granulocytosis, mainly consisting of mature neutrophils, (2) elevated serum and urine G-CSF levels, (3) normalization of granulocyte count and G-CSF level following tumor removal, and (4) demonstration of increased G-CSF in the tumor [G-CSF-producing tumors are considered to have a poor prognosis, due to the effects of G-CSF on proliferating tumor cells and enhancement of metastasis. G-CSF may therefore accelerate the clinical progression of the disease . The patG-CSF acts on the bone marrow to increase the WBC count, but does not directly trigger inflammatory responses, such as fever and elevated CRP. However, previous studies have also reported findings of inflammation, inferring that a separate cytokine is simultaneously produced within the G-CSF-producing tumor, in conjunction with the inflammatory response. A previous report speculated that interleukin 6 (IL-6) could be the cytokine responsible for these effects . The repThe present paper summarized our experience in treating a patient with G-CSF-producing SCC of the lower gum. The patient was diagnosed with a G-CSF-producing tumor on the basis of leukocytosis, elevated serum G-CSF, and G-CSF-positive tumor cells, immunohistochemically detected by anti-G-CSF antibodies, as well as clinical findings.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent can be furnished upon demand.G-CSF: Granulocyte colony-stimulating factor; SCC: Squamous cell carcinoma; PET/CT: Positron emission tomography/computed tomography; CT: Computer tomography; WBC: White blood cell; BLM: Bleomycin; CDDP: Cisplatin; Gy: Gray; UFT: Tegafur-uracil; CDGP: Nedaplatin; DTX: Docetaxel; CRP: C-reactive protein; EIA: Enzyme immunoassay; IL-6: Interleukin 6; FDG: Flurodeoxyglucose.The authors declare that they have no competing interests.AM designed research and performed research. TY, KN, RS, TK, and NS performed research. HH designed research and performed research. All authors read and approved the final manuscript.


The prevalence of obesity and its related comorbidities, such as fatty liver, in children is increasing worldwide mostly due to changes in diet and life-style. Many serological markers have been suggested for screening of fatty liver but investigations for finding more reliable factors are still in progress.This study aimed to investigate the correlation between the level of retinol binding protein-4 (RBP4) in the serum and sonographic grading of fatty-liver severity in obese Iranian children.This case-control, double-blind study involved 51 obese children aged between five and 17 years as the case group. In addition, 35 healthy lean children with no liver problems were selected as the control group. Plasma RBP4 (using an ELISA), serum triglycerides (TG), low-density-lipoproteins (LDL), high-density-lipoproteins (HDL), total-cholesterol (Chol), and body mass index (BMI) were measured. Grading the severity of the fatty liver condition was done by an expert radiologist in the case group.RBP4 levels in obese children (19 482.9 ± 3 302.2 pg/ml) were higher than those found in the lean control group (14 295.68 ± 2 381.3 pg/ml) (P < 0.05). In the obese patients, RBP4 levels showed a significant correlation with the grade of fatty liver and BMI (P < 0.05).It was found that the level of RBP4 had a strong correlation with the severity of fatty liver. Therefore, RBP4 may be considered as a useful, noninvasive predictive biomarker of intrahepatic lipid content in obese children prior to using radiological investigations. In particular, abdominal sonography, for the evaluation of intrahepatic lipid content in obese patients, as the sensitivity of a sonography is decreased due to the increased thickness of the abdominal wall as a result of fat deposits. The prevalence of obesity in children is increasing in many countries due to changes in diet and life-style. In fact, it is associated with a number of morbidities, such as insulin resistance, dyslipidemia, hypertension, and non-alcoholic fatty liver disease (NAFLD) , 2. ReceSonography of the liver, which is non-invasive, more practicable, and less expensive, having a sensitivity of 89% and specificity of 93% in detecting steatosis in the liver, has been found to be a good screening method for the evaluation and grading of fatty liver among different imaging and interventional diagnostic methods , 8. TherIn this longitudinal, case-control, double-blind study, 51 obese children aged between 5 to 17 years (10.57 ± 3.04), who were referred to the pediatric clinics of Shiraz University of Medical Sciences between June 2011 and March 2012, were selected as the case group. In this group, 35.5% of the subjects were male and 64.5% were female . In addiThe study data were analyzed using SPSS statistical software (v. 15) . Descriptive data were presented as mean ± standard deviation (SD). Comparison of RBP4 levels between the patients and the controls was done using a Student’s t-test. In addition, the correlations were evaluated by the Pearson method .The level of RBP4 was significantly higher in the case group (19 482.9 ± 3 302.2 pg/ml) compared to the control group (14 295.68 ± 2 381.3 pg/ml) (P < 0.001). Moreover, the level of RBP4 was 17 053.47 ± 2 199 pg/ml in obese children without sonographic evidence of fatty liver, 20 550 ± 1 990 pg/ml in grade 1 fatty liver, and 24 880 ± 1 998 pg/ml in grade 2 fatty liver . The greThis study revealed that the level of serum RBP4, a transport protein for vitamin A, was higher in obese children than that of the lean ones. In the obese children, RBP4 showed a significant correlation with fatty liver grading. Many published studies have also indicated higher levels of plasma RBP4 in obese adults compared to the lean ones -15. In a


Low-density lipoprotein (LDL) is an important risk factor for coronary artery disease. However, its localization in human coronary plaques is not well understood. The present study was performed to visualize LDL in human coronary artery wall.(1) The fluorescence characteristic of LDL was investigated by color fluorescent microscopy (CFM) with excitation at 470-nm and emission at 515-nm using Nile blue dye (NB) as a biomarker. (2) Native LDL in 40 normal segments, 42 white plaques and 35 yellow plaques (20 with necrotic core) of human coronary arteries was investigated by color fluorescent angioscopy (CFA) and CFM.(1) NB elicited a brown, golden and red fluorescence characteristic of LDL, apolipoprotein B-100, and lysophosphatidylcholine/triglyceride, respectively. (2) The % incidence of LDL in normal segments, white, and yellow plaques was 25, 38 and 14 by CFA and 42, 42 and 14 by CFM scan of their luminal surface, respectively, indicating lower incidence (p<0.05) of LDL in yellow plaques than white plaques, and no significant differences in detection sensitivity between CFA and CFM. By CFM transected surface scan, LDL deposited more frequently and more diffusely in white plaques and yellow plaques without necrotic core (NC) than normal segments and yellow plaques with NC. LDL was localized to fibrous cap in yellow plaques with NC. Co-deposition of LDL with other lipid components was observed frequently in white plaques and yellow plaques without NC.(1) Taken into consideration of the well-known process of coronary plaque growth, the results of the present study suggest that LDL begins to deposit before plaque formation; increasingly deposits with plaque growth, often co-depositing with other lipid components; and disappears after necrotic core formation. (2) CFA is feasible for visualization of LDL in human coronary artery wall. Low-density lipoprotein (LDL) is a pro-atherogenic agent that is converted by oxidation to oxidized low-density lipoprotein (oxLDL), which plays an important role in the initiation, progression and destabilization of atherosclerotic plaques LDL is itself a clinically proven risk factor for ischemic vascular events; coronary heart disease is associated with higher serum levels of LDL Labeled-LDL has been used for trafficking LDL movement in fish, animal and humans Although invasive, color fluorescent angioscopy (CFA) is a high-resolution imaging technique that enables percutaneous two-dimensional imaging of the vascular wall from within. We have succeeded in imaging the native lipid components, such as oxLDL, and lysophosphatidylcholine (LPC), of human coronary plaques by using CFA in vitro and/or in vivo, but have not attempted imaging of LDL because of the lack of an appropriate, biocompatible marker However, we recently discovered that Nile blue dye (NB), which is used as an electromechanical biosensor of DNA The color fluorescence of LDL and other major substances that constitute atherosclerotic plaques  [23] wasThe intensity of fluorescence was categorized as strong, weak or absent when the exposure time required for imaging was taken to be within 1 s, >1 and −5 M (the maximum concentration that does not precipitate) at 37°C and then mixed with each of the major substances in atherosclerotic plaques. The evoked fluorescence was photographed at ×40.In the present study, NB  was diluted in distilled water to a concentration of 1015 The fluorescence-excitation unit  comprises a mercury-xenon lamp and 7 sets of bandpass filter (BPF) discs, exchangeable by rotation to select the desired wavelength of light ranging from 330 to 800 nm.The CFA system we used consists of a fluorescence-excitation unit, an angioscope, a fluorescence-emission unit and a camera.The angioscope  consists of a 2.5-F fiberscope that contains 6000 quartz fibers for the image guide and 300 quartz fibers for the light guide. This fiberscope is incorporated in a 5-F guiding balloon catheter and is steerable along a 0.014-inch guide wire, which enables observation of a coronary segment of up to 7 cm in length with a single saline flush. The angioscope has been approved for clinical use by the Japanese Ministry of Health and Labor, supported by National Insurance, on a commercial basis in Japan The fluorescence-emission unit (developed in collaboration with Olympus Co.) comprises 7 sets of dichroic membranes (DM) and band absorption filters (BAF) and is connected to a 3CCD digital camera . The obtained images are displayed on a computer screen through a camera controller .To observe the vascular lumen, the light and image guides are connected to the excitation and emission units, respectively. After selecting the desired BPF and BAF, the light is irradiated through the BPF and the light guide toward the target. The evoked fluorescence is received by the digital camera through the DM and BAF for successive two-dimensional imaging at an adequate time interval from 0.01 to 5 s. The intensity of the fluorescence images was categorized as for CFM.−6 M This CFA system can visualize fluorescence of a target located within 200- µm of the luminal surface In the present study, conventional angioscopy was used to classify coronary plaques and normal segments because this imaging technology enables diagnosis of macroscopic pathological changes of the vascular wall from within, and therefore has been widely employed clinically for classification of coronary plaques and detection of vulnerable plaques in vivo Plaque is defined as a nonmobile, protuberant or lining mass that is clearly demarcated from the adjacent normal wall and the shape, location and color of which do not alter under the influence of a saline solution flush. Plaque is further classified as white or yellow based on its surface color. A normal segment is defined as milky-white and smooth-surfaced without any protrusions Images of the plaque obtained by conventional angioscopy were classified as white or yellow using an AquaCosmos image analyzer , which sets a window on an appropriate portion of an image and separates the color within the window into three primary colors, namely red, green and blue. Plaque was defined as “white” when the intensity ratio of red : green : blue was 1.0∶ 0.9∼1.1∶ 0.9∼1.1, respectively, and as “yellow” when it was 1.0∶ 0.8∼1.2∶ 0.3∼0.6, respectively The study was carried out with the approval of the Ethical Committees of the Japan Foundation for Cardiovascular Research, Ethical Committee of Toho University and Ethical Committee of Funabashi-Futawa Hospital. and after. obtaining written informed consent from the families concerned.Twenty-nine coronary arteries  were detached from 10 cadavers .A Y-connector was introduced into the proximal portion of each coronary artery for perfusion with saline solution at a rate of 10 mL/min and then the angioscope was introduced through the connector into the artery for observation. Initially, conventional angioscopy was carried out to detect plaque and because the light from the angioscope’s tip was visible through the coronary wall, the site of the targeted plaque could be confirmed.As a result, 77 plaques (42 white plaques and 35 yellow plaques) and 40 normal segments were confirmed.After conventional angioscopy, the CFA system was set up, a BPF of 470±20-nm and BAF of 515-nm were selected and a control image was obtained under saline perfusion. After ceasing the perfusion, 0.5 mL of 2% NB solution was injected into the perfusion circuit and 5 min later the perfusion of saline solution was restarted and the target portion was observed again.Quantitative measurement of a target is difficult for CFA because the target was imaged as a fish eye image through a lens attached on the angioscope tip. Deposition pattern was therefore arbitrarily classified by CFA as spotty , which stain lipids red, calcium black, and collagen fibers and smooth muscles blue 2 formulae and were compared among different deposition sites, patterns and plaque morphology. A value of p<0.05 was considered to be statistically significant.The data obtained were tested by χLDL did not exhibit autofluorescence, but exhibited a brown fluorescence in the presence of NB . This flThe % incidence of LDL deposition in normal segments, white plaques and yellow plaques were 25, 42 and 40 by CFA, 38, 42 and 42 by CFM luminal surface scan, and 14, 14, and 26 by CFM transected surface scan, respectively. The % incidence examined by CFM luminal and transected surface of normal segments was significantly higher that of CFA, and the reason for the difference was shown below. On the contrary, that in white plaques and yellow plaques was not different between CFA and CFM. By both CFA and CFM, the % incidence of LDL deposition in yellow plaques was lower than that of white plaques and normal segments .By CFM luminal surface scan, LDL deposited alone or co-deposited separately or in overlapping pattern with ApoB-100 and “R” . DepositDiffuse deposition was more frequent than spotty deposition in white plaques, and this deposition pattern alone was observed in yellow plaques .LDL deposited in superficial layer, deep layer or in diffusely in both layers, -F–H, sigLDL deposited alone or with ApoB-100 in normal segments and white plaques but not in yellow plaques. In yellow plaques, LDL deposited with “R”, or with ApoB-100 and “R”  and 6. TDeposition of LDL without deposition of other lipids that are stained with Oil Red O, such as cholesterol and cholesteryl esters, was observed in normal segments, whereas in yellow plaques, the lipids deposited in all preparations but LDL did not necessarily co-localize with these lipid components .In the present study, NB, a low-molecular-weight dye, evoked a brown fluorescence in the presence of LDL. Thus, we were able to visualize LDL in a given plaque, enabling analysis of the different deposition patterns of LDL in human coronary plaques and their relationship to plaque morphology and deposition of other lipid components. The mechanisms by which NB evoked fluorescence by LDL are not known, but one possibility is that, as is the case of Evans blue dye with oxLDL Because none of the other major substances in plaque exhibited a brown fluorescence color, it is believed that it is characteristic of just LDL, and that NB can be used as a biomarker of LDL.In this study, LDL was frequently deposited in normal segments with a thick intima, white plaques and yellow plaques without NC, more diffusely in white plaques and often co-depositing with ApoB-100 or other lipid components that exhibited red fluorescent color such as LPC, TG and/or ApoE-2 in white plaques and yellow plaques without NC and in normal segments with a thick intima, but infrequently in yellow plaques with NC.LDL contains ApoB-100 in its cortex. In the present study, brown fluorescence of LDL and golden fluorescence of ApoB-100 distributed separately, indicating that golden fluorescence was not exhibited by the ApoB-100 that was contained in LDL but by free ApoB-100.NB stains the damaged cells blue under sun light, irrespective of necrotic or apoptotic, as in case of Evans blue dye It is well known that white plaques are formed by intimal thickening caused mainly due to proliferation of collagen fibers; the white plaques grow into yellow plaques by accumulating lipids in themselves; acquire NC; and finally become vulnerable to disruption Except LDL deposits with a small size and those located in deep layer, almost all LDL deposits that were visualized by luminal or transected surface scan by CFM were also detected by CFA in the present study. Although limited to significantly large sized (patchy or diffuse type) LDL deposits located in superficial layer, CFA is feasible for molecular imaging of native LDL in coronary artery wall in clinical situations.The clinical safety of NB is unproven, unlike Evans blue dye, which is used as a biomarker of oxLDL NB excited a brown fluorescence that was characteristic of LDL, which frequently deposited in white plaques and yellow plaques without NC and also in normal segment with thick intima, but rarely in yellow plaques with NC. (2) LDL often co-localized either separately or overlapping with ApoB-100, LPC, TG and/or ApoE-2. (3) Taken into consideration on the well known process of coronary plaque growth, it is conceivable that LDL begins to deposit before plaque formation, increasingly deposits with plaque growth, and then disappears after NC formation. (4) CFA is feasible for molecular imaging of native LDL deposits with a significantly large size and located in superficial layer of human coronary artery wall in vitro and in vivo.


The objective of this study was to determine the ability of various parameters commonly used for the diagnosis of acute meningitis to differentiate between bacterial and viral meningitis, in adult patients with a negative direct cerebrospinal fluid (CSF) examination.This was a prospective study, started in 1997, including all patients admitted to the emergency unit with acute meningitis and a negative direct CSF examination. Serum and CSF samples were taken immediately on admission. The patients were divided into two groups according to the type of meningitis: bacterial  or viral . The CSF parameters investigated were cytology, protein, glucose, and lactate; the serum parameters evaluated were C-reactive protein and procalcitonin. CSF/serum glucose and lactate ratios were also assessed.Of the 254 patients with meningitis with a negative direct CSF examination, 35 had BM and 181, VM. The most highly discriminative parameters for the differential diagnosis of BM proved to be CSF lactate, with a sensitivity of 94%, a specificity of 92%, a negative predictive value of 99%, a positive predictive value of 82% at a diagnostic cut-off level of 3.8 mmol/L (area under the curve (AUC), 0.96; 95% confidence interval (CI), 0.95 to 1), and serum procalcitonin, with a sensitivity of 95%, a specificity of 100%, a negative predictive value of 100%, and a positive predictive value of 97% at a diagnostic cut-off level of 0.28 ng/ml .Serum procalcitonin and CSF lactate concentrations appear to be the most highly discriminative parameters for the differential diagnosis of BM and VM. The emergency therapeutic management of meningitis involves rapid identification of the bacterial or viral nature of this disease, so that antibiotic treatment can be started without delay , even thThe immediate identification of bacterial meningitis (BM) is based on direct examination of the cerebrospinal fluid (CSF) or the detection of bacterial antigens in the CSF. These tests have a low sensitivity, and in 30% to 50% of cases, they do not contribute to differential diagnosis -9.et al., [A model for predicting the bacterial origin of meningitis was proposed by Hoen et al., -13, but The preliminary results of our study suggested that serum procalcitonin and CSF lactate levels were better markers than were those classically used with regard to differentiating between BM and viral meningitis (VM) in patients with meningitis and a negative direct CSF examination . The objThis was a prospective study, including all adult patients admitted to the emergency unit with suspected meningitis from January 1997 onward. Patients included in this study provided written informed consent. However, and in accord with the French law, this study did not required ethical approval because of the observational nature of study. This study received the agreement of the French Data Protection Authority.3 in the CSF, were eligible for inclusion in the study. Exclusion criteria comprised the presence of bacteria in the CSF evidenced by direct examination and/or detection of bacterial antigens in the CSF, antibiotic treatment before admission (more than two successive doses of the prescribed antibiotic), the presence of another focus of infection in addition to meningitis, and meningitis finally assumed to be of bacterial origin, despite the absence of microbiologic documentation, and treated with antibiotics during the patient's hospitalization.Lumbar puncture was performed immediately on the patient's admission to the emergency unit. Patients with meningitis, defined by a leukocyte count > 5 per mmBlood tests , lactate, procalcitonin (PCT), electrolytes, blood cultures), and CSF analyses  were performed on the admission of the patient, before the start of any antibiotic treatment. The limits of detection were 0.07 ng/ml for PCT , 4 mg/L for CRP  and 1.25 mmol/L for lactate . The CSF/serum glucose and lactate ratios also were calculated. Between 1997 and 1999, neurotropic viruses were identified on the basis of two serologic examinations separated by an interval of 15 days and/or detection of the viral genome by using a polymerase chain reaction (PCR) amplification technique. After 1999, only PCR tests were used for virus identification in the CSF.BM was diagnosed on the basis of a positive bacterial culture of CSF. The diagnosis of viral meningitis or meningoencephalitis was confirmed in the absence of any bacteria detectable by direct CSF examination or in bacterial cultures, and a positive viral serology or PCR test; in the absence of proven viral etiology, meningitis was considered to be viral if a cure was achieved without any antibiotic treatment apart from antiviral therapy. In view of their practically identical cytologic and chemical characteristics, VM and viral meningoencephalitis (VME) were combined in a single VM group. The confirmed diagnosis was that recorded on the patient's discharge from hospital. From 1997 onward, all patients with meningitis were contacted between 28 and 30 days after their discharge from hospital to obtain information on the following items: need for readmission to hospital, new antibiotic prescription, and persistence or recurrence of headache.P < 0.05. The discriminative power of the various parameters studied was determined by means of receiver operating characteristic (ROC) curves. The diagnostic cut-off level for differentiation of BM and VM was defined according to the maximum value of the Youden index. The ROC curves were compared by using Hanley's method [To determine the value of the different parameters for the differential diagnosis of BM and VM, the patients were divided into two groups: those with BM (group I) and those with VM (group II). The results were expressed as the mean ± standard deviation. Mean values were compared by using the Mann-Whitney nonparametric test, the threshold of statistical significance being set at s method .Between January 1997 and December 2009, 97 patients with BM and 218 patients with VM (183 with VM and 35 with VME) were admitted to the emergency unit of the Saint-Etienne University Hospital.3; protein, from 3.8 to 8.3 g/L; glucose, from 1 to 2.8 mmol/L; and lactate, from 5 to 9.2 mmol/L. These four patients received antibiotic treatment for 18 to 22 days and were cured with no sequelae. A CSF fistula was diagnosed in one of the four patients.Among the 97 patients with BM, 62 cases were excluded: 42 had a positive direct CSF examination, 16 had an additional focus of infection (pulmonary (eight), in the ear, nose, and throat region (three), ascitic (two), intra-abdominal (two), one had a spondylitis with discitis), and four patients had meningitis assumed to be of bacterial origin in the absence of microbiologic documentation. In the CSF samples taken from these four patients, the polynuclear neutrophil count ranged from 964 to 1,480/mmStreptococcus pneumoniae (14), Listeria monocytogenes (6), Neisseria meningitidis (5), Streptococcus sp. (4), Haemophilus influenzae (2), Staphylococcus aureus (2), and other species (2). A single bacterial species was identified in the blood cultures of 14 patients.Thirty-five patients with BM and a negative direct CSF examination were included in this study. The following bacteria were identified in CSF cultures: Paramyxovirus (2). In eight of these patients, admitted prior to 1999, virus identification was based on serology alone.Among the 218 patients with VM, the virus was identified in 85 patients (39%): enterovirus (53), herpes virus (25), varicella zoster virus (five), and The main clinical characteristics of the patients are presented in Table Listeria monocytogenes and Haemophilus influenzae, respectively) had low PCT values: 0.20 and 0.28 ng/ml. The CSF samples taken from these two patients showed a neutrophil count < 100/mm3 and normal glucose levels (> 3 mmol/L), but the other CSF parameters studied allowed confirmation of the bacterial origin of meningitis according to the diagnostic cut-off levels specified in this article. In all the other patients with BM, PCT levels were > 0.93 ng/ml.The mean values of the serum parameters studied are shown in Table 3, whereas > 10% of the patients in the VM group had a neutrophil count > 500/mm3. CSF glucose levels were not depressed in 25% of the patients with BM, but lower-than-normal CSF glucose levels were seen in 5% of patients with VM. CSF protein levels were elevated in > 25% of patients with VM and were normal in 5% of patients with BM.The mean values of the CSF parameters studied and the CSF/serum glucose and lactate levels are presented in Table P = 0.1). In contrast, statistically significant differences were seen between PCT and CRP (P = 0.018), between PCT and CSF protein (P = 0.02), and between PCT and the other parameters.The sensitivity, specificity, and positive and negative predictive values of the various parameters studied are presented in Table Finally, among the 183 patients with VM, 31 (17%) received antibiotic treatment for a period ranging from 8 to 24 hours on the assumption that their meningitis was of bacterial origin. All patients with BM received empirical antibiotic treatment in the emergency unit.These results confirm the value of serum PCT and CSF lactate assays for the differentiation of bacterial and viral meningitis in adult patients with a negative direct CSF examination in the emergency unit.Clinical signs have been shown to have a low sensitivity and specificity for the diagnosis of meningitis and are of no value for differentiating between meningitis of bacterial and viral origins . In the Few published studies have focused on the value of these parameters for the differential diagnosis of BM in patients with a negative direct CSF examination, so it is difficult to compare their results.3, compared with 1,520/mm3 in the CSF of 134 patients with BM and a positive direct CSF examination; the difference between these groups was statistically significant. Ray et al. [3 in 18 patients with BM and a negative direct CSF examination. In this study, neutrophil count had a sensitivity of 78% and a specificity of 75% for the differential diagnosis of BM. In our study, this parameter was among those most poorly discriminating between BM and VM, with a sensitivity of 80% and a specificity of 85% at a diagnostic cut-off level of 118/mm3.Spanos  describey et al.  reportedLow CSF glucose levels (2 to 2.5 mmol/L) and a low CSF/serum glucose ratio (on the order of 0.30 to 0.40) have been classically described as a feature of BM -22. SpanElevated CSF protein levels are also seen in patients with BM, mean values ranging from 1 to 2.5 g/L ,17,21,23Although several studies have demonstrated the value of determining lactate levels in the CSF for the differential diagnosis of BM, none of these focused specifically on the diagnostic value of this parameter in adult patients with BM and a negative direct CSF examination ,23-27. FThe value of serum levels of C-reactive protein, a protein characteristic of the acute phase of inflammation, in differentiating BM and VM, was investigated in several published studies, as well as in a meta-analysis (including 14 studies in which serum levels of C-reactive protein were measured) ,26,33-35et al. [Mycobacterium tuberculosis (one), Borrelia burgdorferi (one), Staphylococcus aureus (one), Haemophilus influenzae (one), and Streptococcus pneumoniae (one). The sensitivity of this parameter for the differential diagnosis of BM was 69%, and the specificity was 100% at a diagnostic cut-off level of 0.5 ng/ml. In another study on 12 patients with BM, reported by Hoffmann et al. [et al. [Listeria monocytogenes. More recently, Ray et al. [PCT is another marker that has been evaluated with regard to its usefulness in distinguishing between infections of bacterial and viral origin. Serum levels of this marker increase within 2 hours after stimulation of inflammatory processes, as shown in healthy volunteers receiving an injection of lipopolysaccharides . The firet al. , among 1n et al. , PCT was [et al. , on the y et al.  publisheThe population selected for this study may be criticized. All patients with meningitis and a direct examination of CSF were included. We could have excluded patients whose CSF appeared turbid macroscopically, but this criterion may be subjective.The causative virus was identified in only 39% of the patients with VM. Improvements in PCR techniques should make it possible to increase this rate in the future. Among the patients classified as having VM in our study, none received a prescription for an antibiotic within 30 days after discharge from hospital, 11 experienced persistent headaches for 5 days or more, before these resolved spontaneously, and two were readmitted to hospital (for post-lumbar puncture syndrome).The number of patients included was too small, relative to the number of parameters investigated, for us to construct a model based on multivariate analysis to confirm the diagnostic relevance of the parameters described.The various published scores and models were not tested in our study, as these were most often based on heterogeneous populations, particularly with regard to the age of the patients and the etiology of meningitis, sometimes retrospectively ,36,43,44Although PCT appeared to be one of the most highly discriminative markers with regard to the differentiation of BM and VM, we did not study the impact of serum PCT level on the prescription or nonprescription of antibiotics. Antibiotic treatment was prescribed in the emergency unit to 31 patients in the VM group, even though all these patients had a serum PCT level < 0.12 ng/ml. In the pediatric context, a study focusing on the impact of serum PCT level on the duration of antibiotic treatment was performed during an epidemic of meningitis caused by enterovirus . The treFinally, we have not yet evaluated the impact of the results of real-time PCR testing for enterovirus on the prescription of antibiotics in patients with meningitis and a negative direct CSF examination.This study confirms the value of determining serum PCT and CSF lactate concentrations for the differential diagnosis of bacterial and viral meningitis, in the absence of microbiologic pointers at the time of the patient's emergency admission to hospital.It would be interesting to study the impact of evaluating these two parameters on the prescription of antibiotics in patients admitted to the emergency unit with meningitis with clear CSF associated with a normal neurologic examination and a negative direct CSF examination.• Identification of bacterial meningitis on direct examination had low sensitivity• Identification of bacterial meningitis with classic biomarkers is insufficient• Models for predicting the acute bacterial origin of meningitis are not easy to use• Cerebrospinal fluid lactate and procalcitonin are easy to determine• Cerebrospinal fluid lactate and procalcitonin are the best markers for differentiating between bacterial and viral meningitisBM, bacterial meningitis; CRP, C-reactive protein; CSF, cerebrospinal fluid; PCR, polymerase chain reaction; PCT, procalcitonin; ROC, receiver operating characteristic; SD, standard deviation; VM, viral meningitis.The authors declare that they have no competing interests.AV, OM, and FZ conceived the study and designed the trial. AV, OM, and AB supervised the conduct of the trial and the data collection. MB, ND, JB, and BP undertook recruitment of patients and managed the data, including quality control. SG provided statistical analysis; FZ and JCB drafted the manuscript. All authors contributed substantially to its revision and approved the final manuscript.


This research has established a method for using single channel surface electromyogram (sEMG) recorded from the forearm to identify individual finger flexion. The technique uses the volume conduction properties of the tissues and uses the magnitude and density of the singularities in the signal as a measure of strength of the muscle activity.flexor digitorum superficialis muscle during four different finger flexions. Based on the volume conduction properties of the tissues, sEMG was decomposed into wavelet maxima and grouped into four groups based on their magnitude. The mean magnitude and the density of each group were the inputs to the twin support vector machines (TSVM). The algorithm was tested on 11 able-bodied and one trans-radial amputated volunteer to determine the accuracy, sensitivity and specificity. The system was also tested to determine inter-experimental variations and variations due to difference in the electrode location.SEMG was recorded from the Accuracy and sensitivity of identification of finger actions from single channel sEMG signal was 93% and 94% for able-bodied and 81% and 84% for trans-radial amputated respectively, and there was only a small inter-experimental variation.Volume conduction properties based sEMG analysis provides a suitable basis for identifying finger flexions from single channel sEMG. The reported system requires supervised training and automatic classification. Surface electromyogram (sEMG) is the non-invasive recording of the electrical activity of the muscle. It is closely related to muscle contraction and an indicator of the associated actions. For an amputee, sEMG of the residual muscles becomes an obvious choice for natural control of the prosthetic hand. This requires the classification of sEMG signals to identify the desired finger movements and obtain the command for controlling the prosthetic hand. Some of the earlier attempts to identify finger actions from sEMG were based on an estimate of the amplitude  and the Researchers have reported success in the use of multiple channels sEMG recording for controlling the prosthetic hand -10. TenoMany researchers have worked to decompose the sEMG signal into constituent MUAPs ,24. PelvThis paper reports an alternate method to estimate the relative strength of contraction of different muscles. It is based on the transmission properties of action potentials in body tissues -28 and oEleven healthy subjects  years; Mean weight = 68.7 (±3.56) kg; and Mean height = 169.3 (±6.2) cm) volunteered to participate in this study. The healthy participants exclusion criterion was; (i) no history of myo or neuropathology, and (ii) no evident abnormal motion restriction. The technique was tested on one amputee participant  who volunteered to take part in this study. Amputee volunteer had trans-radial one-third proximal amputation of the right forearm (with 9 cm long stump).Bipolar electrodes  were placed on the forearm (FDS) muscle for the healthy participants Figure a in accoExperiments were repeated twice for the able-bodied volunteers, with electrodes placed on the proximal end in the first experiment- EXP1, and on the distal end for the second experiment – EXP2  sEMG acquisition system was used to record the signal. The system gain was 1000, CMRR was 92 dB, and bandwidth was 20–450 Hz, with 12 dB/ octave roll-off. The input impedance of the system was 115 Pico-farad in parallel with 1 K-ohm. The sampling rate of the system was 1024 samples/ second for each channel and the resolution were 16 bits/ sample. Prior to the placement of electrodes, the skin of the participant was prepared by shaving (if required) and exfoliation to remove dead skin. Skin was cleaned with 70% v/v alcohol swab to remove any oil or dust from the skin surface.Experiments were conducted where the sEMG was recorded while the participants performed four sets of generic finger flexion actions Figure  labelled• Background activity: All fingers resting.• Class 1: Flexion of little (pinkie) finger• Class 2: Flexion of ring finger• Class 3: Flexion of middle finger• Class 4: Flexion of index fingerThese generic actions were selected for the following reasons:• these actions will allow the user to control individual fingers in the recently available advanced robotic/ prosthetic hand ,10,19 an• these actions can be used for communication and control commands that can be used for several different devices and applications.The participants performed the flexion without any resistance and as was convenient and easily reproducible by them. SEMG was recorded through the experiment. The examiner gave on-screen and oral commands to the participant to perform the action without any fixed order of the fingers. Each flexion was maintained for about 7–8 seconds and was repeated twelve times. The experiments were repeated twice with changed electrode location.The experimental protocol was approved by the RMIT University Human Ethics Committee and Alfred Health Ethics Committee and performed in accordance with the Declaration of Helsinki 1975, as revised in 2004. Prior to the recording, the participants were encouraged to familiarize themselves with the experimental protocol and with the equipment. For the experiment with the amputee, bilateral action training modality was performed . The ampAs a first step, the signal corresponding to class 1 for each subject on each day was normalised to the root mean square (RMS) of the recording of the same subject. The next step was the removal of the background activity from the signal Figure . This beThe pilot analysis of the recordings showed large inter-experimental variations in the spectrum of the background activity, making stationary spectral filtering unsuitable because of the variations in the noise characteristics . For thiThe first step of the proposed method required the identification of the temporal location of the MUAPs. For this purpose, sEMG signal was decomposed using wavelet transform  and the maxima were identified based on the change of sign .Wf can be described mathematically as follows , eThe system requires supervised training, while the classification phase is suitable for being automated without manual supervision. The technique could be applied for prosthetic hand control or for human computer interface.In this study, we have investigated and reported a novel method to estimate the relative strength of contraction of different muscles to identify individual finger flexions. This method is based on the volume transmission properties of action potentials in body tissues , leadingWavelet maxima detection is a suitable technique to identify the local peaks in the sEMG. Identifying the range of the amplitude of peaks using clustering techniques using the wavelet maxima density (WMD) is an indicator of the muscle activity of different muscles. This technique uses WMD as the set of features that can be classified using twin support vector machine (TSVM) to determine the associated finger flexion. The advantage of the TSVM over other classifier techniques is that it is suitable for unbalanced data sets.The results show that using WMD, single channel EMG is suitable for accurately identifying individual finger flexion. This technique was tested for able bodied and trans-radial amputee subject and also for variations in the placement of the electrodes. This work has demonstrated that it is possible to accurately identify the intent of individual finger flexion using single channel EMG recorded from the surface of the stump of a transradial amputee patient.The authors declare that they have no competing interests.DKK has designed the experiment, discussed and developed the underlying concepts for the technique and finalised the manuscript. SPA has also designed and conducted the experiments on the amputee subject, performed data analysis and written part of the manuscript. VPS has conducted the experiment with able bodies and developed the wavelet based signal processing technique. All authors read and approved the final manuscript.


A 77-year-old woman visited our institution complaining of general fatigue. Chest radiography revealed masses in the upper and middle lung fields. Pathological findings for an endoscopic biopsy specimen revealed squamous cell carcinoma. High-grade fever developed and blood analyses revealed sustained elevated white blood cell count and C-reactive protein levels. Cytokine production by tumor cells was suspected; both serum granulocyte colony-stimulating factor  and interleukin-6  levels were high. Immunohistochemical examination of biopsy specimens showed positive staining with antigranulocyte colony-stimulating factor and anti-interleukin-6 monoclonal antibodies. Diagnosis of a tumor that produced granulocyte colony-stimulating factor and interleukin-6 was established. The patient was administered best supportive therapy since she was not eligible for surgical treatment because of her poor respiratory function. She died from interstitial pneumonia exacerbation two months after this diagnosis. We present a female with squamous cell carcinoma of the lung that produced granulocyte colony-stimulating factor and interleukin-6. Granulocyte colony-stimulating factor (G-CSF) is a cytokine associated with granulocytosis . In receA 77-year-old woman with a history of interstitial pneumonia and uveitis visited our institution complaining of general fatigue. Chest radiography revealed a mass in the upper lung fields , and she2 saturation was 96% in room air. She had a history of smoking 15 cigarettes per day for 55 years but no history of alcohol consumption. On initial clinical examination, her weight was of 50.8 kg, height was 158 cm, body mass index was 20.3 kg/m2, and BSA was 150 m2. Mild anemia was revealed in the palpebral conjunctiva. No abnormal murmur could be heard on auscultation although dry rales were heard in both lungs. Her blood pressure was 130/71 mmHg, heart rate was 106 beats/min, body temperature was 36.8°C, and O2/μL; normal: 39–94 × 102/μL) with 81% neutrophils, mild anemia , elevated platelet count , mild renal dysfunction , mild hyponatremia , mild hypercalcium , and markedly elevated C-reactive protein level . The tumor marker squamous cell carcinoma antigen was also elevated . Laboratory test results demonstrated a markedly elevated white blood cell count (266 × 10Chest computed tomography (CT) revealed interstitial shadows in both lungs. Chest CT also showed a 5.2 cm irregular mass with cavity and speculated margin in the right S3 as well as a 7.6 cm irregular mass with cavity and air bronchogram sign, which were adjacent to the pleural walls Figures . A few nAfter admission , high-grThe cause of fever and elevated inflammatory reaction levels was suspected as cytokine production by tumor cells. Therefore, serum G-CSF and IL-6 levels were measured, which showed that both serum G-CSF  and IL-6  levels were high. Immunohistochemical examination of biopsy specimens showed positive staining with anti-G-CSF monoclonal and anti-IL-6 monoclonal antibodies Figures . TherefoAlthough surgical treatment was considered, it was not indicated because her respiratory function was poor because of interstitial pneumonia. Chemotherapy was recommended, but she declined. Therefore, she was administered best supportive therapy. She was discharged 19 days after her admission, after which she visited our institution regularly. However, she was transferred by ambulance because of dyspnea, and she died of hypoxemia due to interstitial pneumonia exacerbation two months after diagnosis of lung cancer.Colony-stimulating factor (CSF) is one that promotes colony formation by leukocytes and other cells. G-CSF promotes granulocyte colony formation and is produced by macrophages, fibroblastic cells, and vascular endothelial cell. G-CSF also modulates differentiation and proliferation of granulocytes. In healthy individuals, G-CSF is present at very low levels in the blood circulation, and there is no correlation between circadian changes in serum G-CSF levels or leukocyte and granulocyte counts. Lung cancer is known to be accompanied paraneoplastic syndromes such as leucocytosis . In 1966In 1977, Asano et al. [in vitro colony formation, measuring G-CSF levels after transplanting tumor cells into nude mice [In the past, evidence of G-CSF production in tumor tissues required methods showing ude mice , or deteude mice ; therefoTakahashi et al.  reportedCurrently, there is no clear definition for IL-6-producing tumors. Fujino et al.  providedHistologically, G-CSF-producing lung cancer comprises giant cell carcinoma, poorly differentiated adenocarcinoma, and squamous cell carcinoma. Moreover, G-CSF-producing lung cancer has poor prognosis with low-grade tumors and has the potential to invade into vessels and metastasize to lymph nodes. Even in resected cases, two-year mortality is as high as 15% . Prognosis of G-CSF-producing tumors is poor, mortality over one year is as high as 12%, and the mean survival time is five months . It is uMany G-CSF-producing tumors are suggested to be biologically highly malignant, poorly differentiated, and highly invasive to local lesions and vessels for the following reasons: (1) autocrine tumor growth because of the emergence of G-CSF receptors on tumor cells, (2) promotion of tumor growth and spread by proliferating neutrophils because of G-CSF, and (3) the inhibition of cellular immunity mediated by macrophages or natural killer cells by G-CSF. These mechanisms are presumably associated with tumor growth and spread .Tumors that secrete autocrine growth factors show rapid progression. Moreover, the spread of tumor cells is closely associated with granulocyte macrophage colony-stimulating factor (GM-CSF), which is a known growth factor for macrophages and granulocytes. G-CSF-producing tumors probably produce cytokines other than G-CSF; thus, establishing a method to measure several cytokines simultaneously is desirable. G-CSF- and IL-6-producing lung cancer is comparatively rare because we could find only 5 reports concerning G-CSF- and IL-6-producing lung cancer in PubMed –21. If tWhen leukocytosis without evidence of infection is found in solid cancer including lung cancer, the serum G-CSF-level should be measured to examine whether tumor cells secrete G-CSF. When G-CSF-producing tumor is diagnosed, the administration of antibiotics is unnecessary. If tumor can be resected, surgical treatment should be considered. Inoue et al.  reportedWe encountered a case of lung squamous cell carcinoma that produced both G-CSF and IL-6. When encountering patients with malignant tumors who present with elevated white blood counts and C-reactive proteins levels, serum G-CSF and IL-6 measurements and immunostaining tumor cells with anti-G-CSF and anti-IL-6 monoclonal antibodies should be considered for diagnosis of G-CSF- and IL-6-producing tumors.


Stroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined.Cortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3–4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals.All tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner.SCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke. Stroke is a cerebrovascular disease with the highest incidence occurring in those over the age of 60 . Stroke Treatment for stroke is not well developed. Recombinant tissue plasminogen activator (rtPA) is the only US Food and Drug Administration-approved drug for treatment of ischemic stroke patients in the acute phase . This thStem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as hematopoietic growth factors two decades ago based on their effectiveness in supporting the survival and growth for hematopoietic stem cells or hematopoietic progenitor cells (HSCs/HPCs) ,6. SCF iAll the procedures in this study have been approved by the Institutional Animal Care and Use Committee and have been carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals in the United States.Male C57BL mice at age of 16–19 months old and male spontaneously hypertensive rats (SHRs) at age of 11–13 months old were used in this study. These ages were chosen based on the different average lifespan of the two species: SHRs is approximately 18 months , while tThe procedure for producing cortical brain ischemia in C67BL mice has been described elsewhere . BrieflyRecombinant mouse or recombinant rat SCF  and recombinant human G-CSF  were subcutaneously injected for 5 days beginning at 3–4 months after induction of cortical brain ischemia. Six doses were tested in chronic stroke of aged mice: SCF (200 μg/kg/day) + G-CSF (50 μg/kg/day) (S+G 200/50), SCF (100 μg/kg/day) + G-CSF (25 μg/kg/day) (S+G 100/25), SCF (50 μg/kg/day) + G-CSF (25 μg/kg/day) (S+G 50/25), SCF (20μg/kg/day) + G-CSF (10 μg/kg/day) (S+G 20/10), SCF (10μg/kg/day) + G-CSF (5 μg/kg/day) (S+G 10/5), and SCF (5 μg/kg/day) + G-CSF (2.5 μg/kg/day) (S+G 5/2.5). Five mice were used for each group of tested doses. In addition, five doses were examined in aged SHRs of chronic stroke: SCF (100 μg/kg/day) + G-CSF (25 μg/kg/day) (S+G 100/25), SCF (50 μg/kg/day) + G-CSF (25 μg/kg/day) (S+G 50/25), SCF (20μg/kg/day) + G-CSF (10 μg/kg/day) (S+G 20/10), SCF (10μg/kg/day) + G-CSF (5 μg/kg/day) (S+G 10/5), and SCF (5 μg/kg/day) + G-CSF (2.5 μg/kg/day) (S+G 5/2.5). Three to eight SHRs were assigned to each group of tested doses.6 cells were analyzed with a flow cytometer .Four hours after the final injection of the 5-day treatment of SCF+G-CSF or saline, mice were anesthetized with Avertin  , and blood was collected from the hearts. Blood cells were incubated with APC-conjugate anti-mouse CD117 antibody  or an equal amount of isotypematched APC antibodies  on ice for 30 min. Red blood cells were then lysed with a FACS lysis buffer  and washed with phosphate buffered saline containing 0.5% fetal bovine serum. Thereafter, 5 x10Blood samples were collected from the heart of mice or from the tail vein of SHRs 4h after the final injection of the 5-day treatment of SCF+G-CSF or saline and then sent to the Clinical Lab of our Medical Center for biochemical tests of liver and kidney functions. The liver enzymes including aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALK phos), and gamma glutamyl transpeptidase (GGT) were used for testing liver function. Blood urea nitrogen (BUN) and serum creatinine were used for the kidney function test.Four hours after the final injection of the 5-day treatment in mice or 14 weeks after the final treatment in SHRs, animals were anesthetized with Avertin  , or Methohexital Sodium  , and the livers and kidneys were then removed. The weight of livers and kidneys was measured and corrected with the body weight. The livers and kidneys were then quickly cut into small pieces (2–3 mm thick) on ice. The liver and kidney samples were fixed in ice-cold 4% buffered-formaldehyde for 3 days and processed for paraffin embedding. Paraffin embedded sections (5μm thick) of liver and kidney were stained with hematoxylin and eosin (H&E). The H&E-stained sections were sent to the pathology, and drug-induced acute or chronic damage in the liver and kidney was examined in a blinded manner.Limb placement test ,23,24 waOne-way analysis of variance (ANOVA) was used for analyzing the data of liver and kidney functions. Kruskal-Wallis nonparametric analysis was used for determining the statistical difference of limb placement-test data. Multiple comparison-induced errors were adjusted with Bonferroni correction. P < 0.05 was considered statistically significant. Data were presented as mean±SE.Aged mice with experimental cortical stroke were randomly divided into 7 groups at 3–4 months after induction of cortical infarction: a saline control group and 6 groups with different tested doses.To determine whether the treatment of SCF+G-CSF in aged chronic stroke mice has any toxic effects, the function and pathology of liver and kidney were examined because both liver and kidney are the most sensitive organs to drug toxicity. We observed that the levels of neither the liver function-related enzymes nor the kidney function-related chemicals were significantly increased by any dose of SCF+G-CSF Figure . In addi+ bone marrow stem cells were increased in a dose dependent manner. In addition, a significant increase in the population of CD117+ bone marrow stem cells in the blood was seen only at the three higher doses   Figure , indicatMost stroke patients have a long history of hypertension. As mentioned earlier, stroke has the highest incidence in the aged population. To consider the clinical relevance, we utilized the aged spontaneously hypertensive rats (SHRs) to make cortical infarcts (stroke). Using such a model, the safety and effectiveness of selected 5 doses of SCF+G-CSF in treatment of chronic stroke were examined.First of all, we determined the toxicity of each dose through functional and pathological examinations for the liver and kidney. The experimental design was the same as stated earlier in the study of aged mice. We observed the same results as in the aged mice that none of the tested doses caused an increase in liver function-related enzymes or in kidney function-related chemicals Figure , indicatIn our early study, we demonstrated that SCF+G-CSF treatment in chronic stroke induced a stable and long-lasting functional improvement in SHRs at relatively young age . Can thiIn this study we have examined several doses of SCF+G-CSF in chronic stroke in the aged mice and SHRs and demonstrated that SCF+G-CSF treatment in chronic stroke of the aged has no toxic effects. In addition, we have also noted a dose-dependent therapeutic effect of SCF+G-CSF on chronic stroke in the aged animal models.Stroke highly attacks aged population. About two-thirds of strokes occur in elderly people over age 65 in the United States . It has Convincing evidence has supported that SCF in combination with G-CSF can synergistically mobilize HSCs/HPCs from bone marrow to the blood, resulting in a robust elevation of HSCs/HPCs in the blood stream in humans, rodents, dogs and nonhuman primates . One of Stroke model produced in aged SHRs provides a clinical-relevant animal model of stroke because hypertension is the most important risk factor for stroke in humans . ChronicIt is worth noting that SCF+G-CSF would be easily to be translated into clinical trials, as this therapy has been proven safe and effective to mobilize HSCs/HPCs for bone marrow transplantation in cancer patients after chemotherapy -38.In summary, the present study has demonstrated that the combination of two hematopoietic growth factors, SCF and G-CSF, is a safe and effective treatment for chronic stroke in the aged condition. This observation provides new knowledge to assist in developing a new therapeutic strategy for chronic stroke.ALK: Alkaline phosphatase; phos ALT: Alanine transaminase; AST: Aspartate transaminase; BUN: Blood urea nitrogen; GGT: Gamma glutamyl transpeptidase; G-CSF: Granulocyte-colony stimulating factor; SCF: Stem cell factor; SHR: Spontaneously hypertensive rat.The authors declare no competing interests.CSP and MEG-T performed experiment. XG examined the pathology of livers and kidneys, CSP and LRZ analyzed data, and LRZ designed the experiment and prepared the manuscript. All authors read and approved the final manuscript.


Tuberculosis kills five lakh patients in India every year, out of which 7-12 % are with meningeal involvement. Delay in its diagnosis and in initiation of treatment results in poor prognosis and sequlae in up to 25% of cases. The aim of the present study is to look for a simple, rapid, cost effective, non-invasive and fairly specific test in differentiating tubercular etiology from other causes.Forty patients between the age of 6 - 24 months attending hospital with symptoms and signs of meningitis were selected and divided into two groups: tubercular and non-tubercular, depending upon the accepted criteria. CSF was drawn and ADA estimated.Out of 19 tubercular patients, 18 had CSF ADA at or above the cutoff value while one had below. Out of 21 non-tuberculous patients, two had ADA levels at or above the cutoff value while 19 had below this value. Results of our study indicate that ADA level estimation in CSF is not only of considerable value in the diagnosis of TBM, CSF ADA level 10 U/L as a cutoff value exhibited 94.73% sensitivity and 90.47% specificity in differentiating tuberculous from non-tuberculous meningitis; it also has 90.00% positive predictive value and 95.00% negative predictive value.It can be concluded that ADA estimation in CSF is not only simple, inexpensive and rapid but also fairly specific method for making a diagnosis of tuberculous etiology in TBM, especially when there is a dilemma of differentiating the tuberculous etiology from non-tuberculous ones. For this reason ADA estimation in TBM may find a place as a routine investigation.Cerebrospinal fluid; Adenosine deaminase; Tuberculous meningitis Tuberculous meningitis (TBM) is an endemic disease in developing countries , more soThe newer methods for diagnosing tubercular disease is based on pheno- and genotypic methods. For the detection of acid fast bacilli (AFB) in a smear, light microscopy is a common, rapid and specific method and is used the world over with a detection rate of 30-40% . SensitiAdenosine deaminase (ADA) is an enzyme in the purine salvage pathway that catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine respectively with the release of ammonia. It plays important role in differentiating lymphoid cells and is present in abundance in active T-lymphocytes whose concentration is inversely proportional to the degree of differentiation . Its levADA is released by T cells during cell mediated immune response (CMI) to the tubercle bacilli. ADA is now being recognized as a marker of cell mediated immunity particularly as a marker of T lymphocyte activation. Adenosine deaminase levels (ADA) have also been considered by several researchers to differentiate tubercular disease from non-tubercular .Different modalities to support the diagnosis in cases of TBM have to be considered because the cytological and biochemical analysis of cerebro-spinal fluid (CSF) have a considerable overlap. As few studies have been conducted to evaluate the role of CSF ADA levels for the diagnosis of TBM, we tried to estimate ADA levels in CSF in TBM and to find out its role as a sensitive, accurate, rapid, and affordable diagnostic tool that will work in resource-limited settings in confirming the tubercular etiology in cases of meningitis.Routine procedures of history taking, examination and routine investigations were followed. Forty patients between the age of six months to two years having symptoms and signs of meningitis, admitted in the Pediatrics ward from July 2009 to June 2010 at the Subharti Medical College, Meerut, India were included in the present study.Presence of first or more than one of the following criteria was adopted to label a case as tuberculous:Bacteriological proof of presence of Mycobacterium tuberculosis.Biopsy showing caseating granulomas.Clinico- radiological findings consistent with TB.Definite clinical and radiological improvement in one month after specific anti-tubercular treatment.History of contact with current disease and positive reaction (> 20 mm induration) to 5 tuberculin unit (TU) purified protein derivative (PPD).Lumbar puncture was done in each case and at least 2 ml of CSF was collected in a sterile vial. Hemorrhagic CSF was excluded from the study. This CSF was subjected to biochemical and microscopic examination. ADA activity was estimated in all these patients by the method of Guisti  and was Out of 40 patients, 28 children were male and 12 patients were female . NineteeOut of these 19 tuberculous patients, 18 patients were found to be having CSF ADA at or above the cutoff value while only one had below cutoff value. Of the 21 patients labeled non-tuberculous, two patients were found to be having CSF ADA at or above the cutoff value while 19 had values below the cutoff .In tuberculous group ADA activity in CSF ranged between 9.2 to 110 U/L with a median of 22, mean SD as 27.1684 (ADA ≥ 10) and 22.7660 (ADA < 10). While in non-tuberculous group ADA activity ranged between 2 to 10.5 U/L with a median of 6, mean SD as 6.0619 (ADA ≥ 10) and 2.5714 (ADA < 10).On comparison of the values of CSF ADA in the two groups, t cal is 4.0173 and the difference in these two values was found highly significant (p < 0.01) .Demonstration of AFB in CSF, CSF culture, CSF cytology, and genomic amplification are the various means to confirm the etiology of TBM but visualization of AFB in direct smears or in cultures of CSF is usually difficult  and in mRoutine CSF laboratory findings may not be helpful to differentiate tuberculous etiology in meningitis from other causes. The aim is to look for a simple and rapid test that can help in the diagnosis of TBM and differentiate it from other causes with fairly good accuracy.ADA has been considered as a marker of cell-mediated immunity and its activity has been observed in various infections including TBM. Considering that both humoral and cell-mediated immunity play an important role in TBM infection, it has been suggested that ADA activity in CSF may help differentiate TBM from non-TBM infectious meningitis.CSF - ADA estimation was reported to be useful in diagnosing TBM and to differentiate TBM from normal subject or patients with other neurological disorders . CSF - AIn the present study CSF - ADA level 10 U/L as a cut-off value differentiate tuberculous from non-tuberculous meningitis. We have observed a highly statistically significant difference in the CSF - ADA levels of meningitis due to tuberculosis and non-tuberculous etiology (P < .01) . ResultsLevels of ADA in CSF of adult patients of TBM have been evaluated in earlier studies . Raised Gupta et al  observedThe mean ADA levels in CSF of TBM cases of pediatric age groups have been reported to be ranging between 11.6-13.7 U/L in earlier study . A levelKashyap et al take cutoff value of 11.39 U/L and has obtained sensitivity of 82% and specificity as 83% in TBM cases . Rana etIn the present study, median ADA levels in CSF were significantly high in TBM patients as compared to those with other etiologies. Ribera et al have also demonstrated similar finding but his study was in TBM patients of adult age group .In conclusion, we found the sensitivity of the test to be 94.73%; specificity 90.47%, positive predictive value is 90.00 % and negative predictive value 95.00%, and so it can be concluded that ADA estimation in CSF is not only simple, inexpensive and rapid but also fairly specific method for making a diagnosis of tuberculous etiology in TBM, especially when there is a dilemma of differentiating the tuberculous etiology from non-tuberculous. For this reason CSF ADA estimation in TBM may find a place as a routine investigation.