The latest worldwide prevalence rate projects that over 65 million patients suffer from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), an illness with known effects on the functioning of the immune and nervous systems. We performed an extensive metabolomics analysis on the plasma of 52 female subjects, equally sampled between controls and ME/CFS patients, which delivered data for about 1750 blood compounds spanning 20 super-pathways, subdivided into 113 sub-pathways. Statistical analysis combined with pathway enrichment analysis points to a few disrupted metabolic pathways containing many unexplored compounds. The most intriguing finding concerns acyl cholines, belonging to the fatty acid metabolism sub-pathway of lipids, for which all compounds are consistently reduced in two distinct ME/CFS patient cohorts. We compiled the extremely limited knowledge about these compounds and regard them as promising in the quest to explain many of the ME/CFS symptoms. Another class of lipids with far-reaching activity on virtually all organ systems are steroids; androgenic, progestin, and corticosteroids are broadly reduced in our patient cohort. We also report on lower dipeptides and elevated sphingolipids abundance in patients compared to controls. Disturbances in the metabolism of many of these molecules can be linked to the profound organ system symptoms endured by ME/CFS patients. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a perplexing disease, silently destroying millions of lives. Indeed, the latest prevalence rate of ME/CFS estimates that there are over 65 million suffering individuals throughout the world , and 35–At least half of the 11 major organ systems in the human body appear to be disrupted in ME/CFS patients. Some established instances include the reported dysfunction of several types of immune cells ,3,4,5, aThe circulatory system is fundamental to all the other systems as it supplies them with necessary compounds while evacuating metabolic waste. The effort to characterize the blood metabolome of ME/CFS patients, with the dual goal of establishing routine diagnostic strategy and gaining further insight into the underlying cause of the disease, has been carried out by several groups on distinct populations of limited size ,19,20,21®’s mass spectrometry. The metabolic panels yielded datapoints for about 1750 blood compounds with a large emphasis on lipids (over 70%). Our analysis provides novel insights into plasma metabolomics in ME/CFS in several promising areas. Primarily, we expose a reproducible disruption in acyl choline metabolism, a class of lipids that remain largely unstudied but are theorized to have fundamental functions. Another key observation, possibly relevant to the nervous system disruptions in the ME/CFS population, involves sphingolipids, which are also widely affected in our patient cohort. We also find, for the second time, that dipeptides are altered. Finally, three steroid classes  are consistently reduced in our ME/CFS patient cohort, a disturbance that could have major repercussions on the well-being of patients.For this latest study, we provided plasma from 52 female subjects, equally sampled between controls and patients, for analysis by MetabolonThe female cohort for this study is comprised of 26 healthy controls and 26 ME/CFS patients between the age of 22 and 72 and with comparable body mass index (BMI). Further description of the patient population is detailed in Moreover, half of the patients also reported gut inflammation symptoms, and 69% of the patients reported being diagnosed with orthostatic intolerance. The Bell scores reported for the patient cohort attest of the severity of the symptoms endured by the patients, with almost all of them under the score of 50, far from the score of 100 expected of a healthy individual. Furthermore, the SF-36 aggregated summaries (PCS and MCS) for both groups, demonstrate the impairment of the patients, especially with a SF-36 PCS score at 26, well below the US general population norm of 50 .®’s standards. The partitioning is as follow: amino acids (196), carbohydrates (25), cofactors and vitamins (29), energy (10), lipids (259), nucleotides (33), partially characterized molecules (2), peptides (33), and xenobiotics (181). Across 52 subjects, there are a cumulative 39,936 datapoints, which can be further divided into 94 sub-pathways , while 117 metabolites had missing values in more than 20% of the subjects for a total of 2375 imputations (38.4%). The level of imputation is similar for each cohort.Such level of detail is crucial when handling missing data either because of the absence of a compound or present in amounts below the threshold of detection of the equipment used. For this dataset, 15.5% of values are missing (6170), similar to the 14% of imputed data for our previous study . The hanTM generated concentration data for 1007 complex lipids across 11 super-pathways and 19 sub-pathways, per Metabolon®’s nomenclature  to be significantly different between controls and patients at p < 0.05 . Only eiFold change analysisA fold change analysis between the two cohorts finds 25 metabolites to have absolute values changes with a threshold of two . Only fiMost noteworthy is the presence of several lipids belonging to the sub-pathway of fatty acid metabolism (acyl cholines). Indeed, the means of oleoylcholine, dihomo-linoleoyl-choline, linoleoylcholine, stearoylcholine, and palmitoylcholine were more than twice as low in patients compared to controls. Additionally, The other lipid reduced by over three-fold in the patient cohort is cholate, a major primary bile acid produced in the liver and known to facilitate fat absorption because of its detergent properties . All theSimilarly, pyridoxate is reduced by over three-fold in the patient cohort. The only other measured metabolite belonging to the Vitamin B6 sub-pathway is pyridoxal and it was also reduced in patients by 2.3-fold .Volcano plot analysisq-values does not yield any significantly different metabolites between controls and patients. However, when fold change is combined with a p < 0.1, five out of the ten metabolites deemed significant belong to the acyl choline pathway, namely, dihomo-linoleoyl-choline, oleoylcholine, stearoylcholine, linoleoylcholine, and palmitoylcholine  analyzed, but not all , and canA volcano plot analysis does not reveal any difference between controls and patients.In an effort to pursue biological relevance, it is crucial to interpret our datasets from a different and more comprehensive perspective. To this end, we used MetaboAnalyst Pathway Enrichment as well as ChemRICH  to further explore our data.Global metabolomics panel®, 527 had such ID, and 477 were suitable after ID standardization. As a result, 38% of the metabolites were not part of this analysis, including all of the compounds that are part of the acyl choline pathway . Out of the 768 metabolites analyzed by Metabolon pathway , for exap < 0.05, namely glutathione metabolism, steroid hormone biosynthesis, and propanoate metabolism. However, none of them are significantly enriched after multiple testing correction  and their impact score is very low, below 0.08 . Out of the 768 metabolites with data from Metabolon®, 623 were suitable for analysis. This means that 19% of the metabolites were not part of this analysis, and this time, three of the compounds classified as part of the acyl choline pathway , the Pubchem ID, the SMILES identity (Simplified Molecular Input Line Entry System), which is a line notation for both entering and representing molecules, and finally  pathway  were incp = 0.025, although with a q-value of 1  and adipoylcarnitine (C6-DC)] which are more abundant in patients compared to controls.None of the other 85 cluster names are significantly different between controls and patients. However, with TMComplex lipid panelAs stated previously, 392 of the lipids have an HMDB ID, which was used to gather the required fields to run the ChemRICH analysis. In the end, 347 were included in the pathway enrichment analysis, only 34% of the dataset. None of the 26 monacylglycerols and none of the 518 triacylglycerols had an HMDB ID and are therefore not included in this analysis.p = 0.0004, shows three out of nine compounds that are increased in ME/CFS patients compared to controls, namely: CER (18:0), CER (18:1), and CER (24:1)  and SM (18:1) (Two clusters are significantly different out of 20 cluster names . The firR (24:1) . The firM (18:1) . Sphingo®’s nomenclature, all five metabolites discussed here are part of the super-pathway sphingolipids, which were first discovered in brain extracts in the 1870s, and have a severe impact on neural tissue when imbalance occurs [According to Metabolone occurs .Sub-pathway-based enrichment analysis® was performed using the ChemRICH concept. This analysis combined both complete datasets to look for difference in patterns between our control and patient cohorts, according to sub-pathway classification, p-values and fold changes. Six of the seven significantly (p < 0.05) impacted metabolite clusters were not classified as drugs and all are displayed in p-values were considered, but for the first time, three remain significant after correction (q < 0.05), and the first five if we apply a q < 0.15 . Similar to androgenic steroids, both of the altered corticosteroids are decreased in patients compared to controls.To circumvent the limitations described above, a sub-pathway-based analysis according to the sub-pathways provided by Metabolonq < 0.15 . The tre® , with no differences to report.One striking observation is that no broad difference is observed when comparing the control cohort to the patient cohort. This is, by itself, a result worth noting that we will discuss later. Nevertheless, it is also vital to reflect on the methods used and their limitations, especially in light of the numerous variables ignored (from 19 to 66%) by both MetaboAnalyst and ChemRICH. Although the ChemRICH sub-pathway-based analysis provides valuable and statistically sound insight into our dataset, we opted to design simple but equally comprehensive calculations to analyze our datasets with less stringent statistical rigor in order to enhance our ability to fully understand the metabolomics status of ME/CFS patients. Therefore, we decided to compute the ratio (controls/patients) of the median for each metabolite analyzed by Metaboloncs panel . The samThe most obvious alteration is acyl cholines with a ratio of 1.9 . As depip-values < 0.05, even though we initially ignored them based on multiple testing correction. Androgenic steroids are already highlighted in the ChemRICH analysis . Here again, all members of this sub-pathway are lower in ME/CFS versus controls. Seven of them are present in analysis . All 18 Dipeptides were also part of the sub-pathways with a high averaged ratio of fold change medians. Six dipeptides are in our dataset, and four of them have been mentioned throughout our data analysis in Two other sub-pathways should be mentioned: pyrimidine metabolism (cytidine containing) and the vitamin B6 metabolism as those are recurring results in the ME/CFS metabolomics literature ,20 and mFinally, one compound classified as an acyl glutamine (hexanoylglutamine) had the lowest ratio at 0.77. A higher concentration of this metabolite in patients would indicate an increase in mitochondrial fatty acid β-oxidation (FAO) and could affect energy levels in patients. Such changes have also been linked to Parkinson’s disease although not as a sole biomarker .q-values above 0.4), their p-values and fold change are comparable to the data presented in This class of compound is extremely intriguing in the context of ME/CFS. Although this is only the second time that acyl cholines have been assessed in ME/CFS, the results are reproducible between two separate cohorts. Our previous study  containsAcyl cholines are still an obscure class of compounds first described in 1911, in conjunction with blood pressure, where a remarkably extensive study found that short-chain choline esters had a depressor effect while long-chain ones tended to have a pressor effect . Those f®’s expertise, acyl cholines have recently been mentioned in a few nutrition metabolomics studies on both plasma and serum. In one case, the effect of coffee consumption on the blood metabolism of subjects was evaluated, and while six acyl cholines were found to be significantly associated with coffee intake, there were an additional 99 unrelated metabolites with the same association [Acyl cholines have been vastly neglected since the 1950s. Linoleoylcholine, oleoylcholine, palmitoylcholine, and stearoylcholine have been sparingly discussed as enhancers of drug absorption from the gastrointestinal tract while a pharmacological experiment found arachidonoylcholine to exhibit cholinomimetic activity, which stimulate the PSNS. Mostly because of Metabolonociation . As expeociation . In anotociation . Here agociation .Dipeptides are another class of compounds found to have decreased levels in our patient cohort, both by statistical test  and ChemTM dataset using both MetaboAnalyst and ChemRICH , meaning that the acyl group is attached to a larger molecule as is the case in all the acyl compounds highlighted throughout this analysis. While the acetyl group is a sub-type of acyl, no acetyl molecule was found different between controls and patients. Instead, we uncovered a class of lipids belonging to the acyl choline sub-pathway as one of the rare compounds that were consistently lower in ME/CFS patients compared to controls. This prompted us to duplicate the analysis displayed in We have very limited knowledge of both the synthesis pathway and the functions of the majority of these compounds, but they are part of the lipids super-pathway and some links can be found between them. For example, monoacylglycerols are released from diacylglycerols by various lipases, including hormone sensitive lipases which in some cases are involved in steroid production. Another example is the length of the fatty acid chain of virtually all compounds highlighted, between 16 and 28 carbons.®. For instance, 458 metabolites with an HMDB ID are common between studies, with 25% of the data from Germain and colleagues [In our previous study, blood processing was done differently, with blood tubes shipped overnight from a collecting location in New York City, compared to more rapid processing for the samples in this study . Keepinglleagues  not preslleagues .Out of the 52 female subjects who participated in this study, 26 were healthy controls, while the remaining 26 were established patients of a ME/CFS specialist in New York City (NYC), Susan Levine, M.D. All patients had a confirmed and rigorous diagnosis of ME/CFS according to the CDC criteria . HealthyBoth ME/CFS subjects and healthy controls completed the medical outcomes survey short form-36 (SF-36)  and the TM P100 tubes  at Dr. Levine’s office. Tubes were transported within two hours, at room temperature, to the NYC Cornell Weill Medical School, where plasma was produced before storage at −80 °C. Shipment of plasma to Cornell’s Ithaca campus was done on dry ice.Collection of whole blood, from an antecubital vein with subjects in the seated position, was achieved directly in 8.5 mL BD®’s facility in NC for analysis (www.metabolon.com). The two metabolomics solutions selected included the global metabolomics and the lipidomics panels, resulting in two distinct datasets provided as excel tables.Plasma samples were thawed and 400 μL of each sample was shipped frozen on dry ice to Metabolonwww.metaboanalyst.ca) for its statistical analysis and pathway analysis modules [Two online services were used to analyze the data. The first one is MetaboAnalyst ( modules . After uhttp://chemrich.fiehnlab.ucdavis.edu) for its statistical enrichment approach [www.github.com/barupal. Additional analyses were performed using Microsoft Excel.The second one is ChemRICH .We are reporting on the largest number of metabolites in the ME/CFS field to date, about 1750 plasma compounds, encompassing 20 super-pathways and 113 sub-pathways. However, we have not unequivocally identified a plasma biomarker or set of biomarkers with abundances drastically different between controls and ME/CFS patients, despite the fact that our clinical data indicates our patient cohort had a substantial level of disability . The samNevertheless, the metabolites emphasized as a result of our analysis, most specifically acyl cholines and steroids, should be considered in light of the metabolic impact even modest changes can have along with the complexity of sources that drain metabolites into the circulatory system. Indeed, the changes observed might result from a disturbance occurring in another part of the body , one whose impact may be diluted in plasma. Acquisition of samples in conditions that increase patient symptoms  could further widen the observed gap between the plasma metabolome of patients and controls. Because liquid biopsies such as plasma and serum remain the most accessible resource from human subjects, our results combined with more explorative metabolomics will help stimulate our efforts before we move toward different, yet more invasive sample collection techniques. Furthermore, set-enrichment statistics with a control for false discovery rates can be used to identify chemical groups instead of individual metabolites that can be associated with a disease or phenotype. Follow-up studies can develop new targeted analytical methods to measure those chemical groups in studies with power calculations from this discovery stage data.


The key to modern drug discovery is to find, identify and prepare drug molecular targets. However, due to the influence of throughput, precision and cost, traditional experimental methods are difficult to be widely used to infer these potential Drug-Target Interactions (DTIs). Therefore, it is urgent to develop effective computational methods to validate the interaction between drugs and target.We developed a deep learning-based model for DTIs prediction. The proteins evolutionary features are extracted via Position Specific Scoring Matrix (PSSM) and Legendre Moment (LM) and associated with drugs molecular substructure fingerprints to form feature vectors of drug-target pairs. Then we utilized the Sparse Principal Component Analysis (SPCA) to compress the features of drugs and proteins into a uniform vector space. Lastly, the deep long short-term memory (DeepLSTM) was constructed for carrying out prediction.A significant improvement in DTIs prediction performance can be observed on experimental results, with AUC of 0.9951, 0.9705, 0.9951, 0.9206, respectively, on four classes important drug-target datasets. Further experiments preliminary proves that the proposed characterization scheme has great advantage on feature expression and recognition. We also have shown that the proposed method can work well with small dataset.The results demonstration that the proposed approach has a great advantage over state-of-the-art drug-target predictor. To the best of our knowledge, this study first tests the potential of deep learning method with memory and Turing completeness in DTIs prediction. Drug targets are the foundation of drug research and development, and over the past few centuries, people have relied heavily on hundreds of drug targets currently known to detect drugs . AlthougAt present, the computational method for DTIs prediction can be classified into three categories: the ligand-based approach, the docking approach and the feature learning approach. Ligand-based methods are often used to estimate potential targets of action by calculating the chemical structural similarity of a given drug or compound to active compounds of known targets. Keiser et al.  proposedenzymes, ion channels, GPCRs and nuclear receptors. The results are exposed to give superior performance to the existing state-of-the-art algorithms for DTI prediction.In this work, we proposed deep learning-based method to identify unknown DTIs. The proposed method consists of three steps: (i) Representation for drug-target pairs. The drug molecules are encoded as fingerprint feature and the protein sequences features are obtaining by using Legendre Moments (LMs) on Position Specific Scoring Matrix (PSSM) that contains evolutionary information about protein. (ii) Feature compression and fusion. The Sparse Principal Component Analysis (SPCA) is used to decrease the features dimension and information redundancy. (iii) Prediction. The Deep Long Short-Term Memory (DeepLSTM) model is adopted for executing prediction tasks. The flow of our proposed model is represented in Fig. We collected information about the interactions between drug compounds and target proteins form KEGG , DrugBanThe ability of substructure fingerprints in characterizing drug molecules has been confirmed in some studies. Through the comprehensive analysis of previous research results, PubChem fingerprint was used to characterized each drug molecules. In this work, drugs are encoded Boolean substructure vector representing the presence or absence of corresponding substructures in a molecule. The PubChem database defines 881 chemical substructures in which each substructure is assigned to a particular location. Therefore, for a substructure appears in the drug compound, the position corresponding to the substructure in the fingerprint vector is set to 1, otherwise, and the corresponding position is set to 0. Hence, each drug was represented as an 881-dimensional vector .A with N amino acids residue can be expressed asThe position specific scoring matrix (PSSM) was firstly introduced for finding distantly related proteins. In recent years, PSSMs is widely used in proteomics and genomics research, such as prediction of DNA or RNA binding sites and membrane protein types. In this paper, PSSM is used to encode proteins and obtain evolutionary information about amino acids. The PSSM of protein Ai → j is a score that represents probability of i-th residue being mutated to j-th native amino acid and N is the length of amino acids residue of sequence A, 20 means the 20 native amino acid types. To get the PSSM for each protein sequence, the Position Specific Iterated BLAST (PSI-BLAST) . In this work, I denotes PSSM, C is the number of rows of a PSSM, V means the sum of each column of a PSSM [The invariant moments are a global statistical feature and has excellent characteristics in size invariance, rotation invariance and displacement invariance which avail to the extraction of stability features. Legendre moments (LMs), as a fast moment invariant feature extraction technology, show good performance in the application of many pattern recognition, viz., graphic analysis, target recognition, image processing, classification and prediction. Here, we use Legendre moment to further refine the evolutionary information contained in PSSM and generate feature vector. LMs are continuous orthogonal moments, which can be used to represent objects with minimal information redundancy , 22. Thef a PSSM , 24. ThewhereThe integral terms in  are comma, b = 30.As a result, using LMs on PSSM of protein sequence, we have obtained 961 features from each protein sequence by setting We got an 1842-dimensional drug target feature vector from each drug target pair by combining drug substructure fingerprint features (881-D) with protein LMs features (961-D). To economize calculating time of classifier, reduce memory consumption and remove noisy features from the original feature space, the sparse principal component analysis (SPCA) is used to integrate both features of drugs and target proteins into an organic whole, reduce the feature dimension and redundant information. Classical principal component analysis (PCA) has an obviously drawback, that is, each PC is a linear combination of all variables and the loadings are typically nonzero. Thus, when dealing with a combination of two different types of features, such as the drug and protein features produced herein, often results in unpredictable results. SPCA is an improved PCA the using lasso (elastic net) to produce principal components with sparse loadings, which overcome above problem. Finally, we gain 400-dimensional refined feature vector as the input of classifier.LSTM is a special recurrent neural network (RNN) architecture, providing more excellent performance than the traditional RNNs . In thisOne of the major differences with standard RNNs network is the LSTM architecture use memory blocks to replace the summation units. Memory blocks, as shown in Fig. We constructed DeepLSTM by stacking multiple LSTM layers , 30. Com35 different subnets during training. In the testing phase, an “mean network” strategy is adopted, which contains all of the original network connection, but their efferent weights are halved in order to make up for the fact that twice as many of them are active [Neural networks often optimized with a large number of parameters. However, there may be overfitting problems in such networks. Dropout is used for solving this problem by randomly removing units from the neural network and their connections in the train of training. The meaning of “dropout” is to extract a “sparse” network from the original network, the sparse network is composed of all the surviving units, as shown in Fig. e active , 32.FigIn this paper, we evaluate the performance of our predictor by calculating accuracy (ACC), true positive rate (TPR), specificity (SPC), positive predictive value (PPV), and Matthews’s correlation coefficient (MCC). The ACC is used to reveal the overall level of prediction. The TPR exposes the proportion of positives samples that have been correctly predicted in the test results. The SPC exposes the proportion of negatives samples that have been correctly predicted in the test results. The PPV is used to reveal the proportion of the true positive samples in the samples that were predicted to be positive. The MCC is a general measure of predictive performance for two classification problems. These performance indicators are defined as follow:FN, FP, TN, TP represents the number of false negative, false positive, true negative and true positive, respectively, and the area under the Receiver Operating Characteristic curve (AUC) is calculated used for measuring the quality of prediction [Here, ediction –35.For four datasets, we divided each dataset into: the training set; the verification set; the test set. Test sets account for one tenth of the total, the training set account for eight tenths of the remaining data, the rest are used as validation sets. We use the training set to fit a DeepLSTM prediction model, use the validation set to optimize the DeepLSTM neural network weight, use the test set to verify the model performance. Another benefit of using validation set is to prevent overfitting by early stopping: terminate model training when errors on the validation dataset no longer decrease and have an increasing trend. This trick avoids the overfitting and reduces the training cost of the model.Nadam optimizer, using dynamic learning rate with initial value of 0.002, decay of 0.004 and momentum of 0.5. The time step was set to 1 and batch size was 64. Training was stopped after a maximum of 500 iterations or early stopping if there was no new best error on the validation data.We use hyperbolic tangent activation for the cell input units and cell output units, and logistic sigmoid for the input, output and forget gate units. The input to the LSTMs and RNNs is 40-dimensional features. The output layer is a fully connected network and uses softmax function to produce probability results. In order to find the best network structure, we test the performance of DeepLSTM models with different number of layers and units on the validation data. The number of hidden layers that were trialed from 1 to 6. With respect to the number of units, these were trialed from 20 to 200 with stride s = 4. Finally, the DeepLSTM model with 4 hidden layers and 36 units was determined. The weights of the DeepLSTM were initialized using random numbers with 0 mean and standard deviation 0.1. We trained model with mean squared error and enzymes data sets, our predictor has given satisfying result of 92.92% accuracy, along with of 99.31% sensitivity, of 86.57% specificity, of 88.04% precision, of 86.75% MCC and AUC of 0.9951. The same good results also appear on other three data sets by using our method. The results achieved of our method on ion channels dataset is 91.97% accuracy, along with 93.23% sensitivity, of 90.87% specificity, of 89.95% precision, of 85.19% MCC and AUC of 0.9705. The results achieved of our method on GPCRs dataset is 91.80% accuracy, along with 83.71% sensitivity, of 100% specificity, of 100% precision, of 84.44% MCC and AUC of 0.9511. The results achieved of our method on nuclear receptors dataset is 91.11% accuracy, along with 95.24% sensitivity, of 87.50% specificity, of 86.96% precision, of 83.76% MCC and AUC of 0.9206. There is particularly noteworthy is our method achieved over 90% accuracy on nuclear receptors datasets with only 180 sample. This clearly shows that our method can provide excellent performance in the case of very small training samples. This is a huge advantage that will be clearly distinguished from other methods. The extraordinary performance comes mainly from the following three points: 1) our feature representation method can effectively extract the discriminative features from drug molecular and target protein sequence; 2) SPCA enjoys advantages in several aspects, including computational efficiency, high explained variance and an ability in identifying important variables, which compresses two different feature vectors into a unified feature space and extracts heterogeneous features; 2) The hierarchical structure enables the neural network to convert the input data into new feature space which is more conducive to complete classification tasks.Statistics of the prediction performance for the proposed models are given in Table enzymes, ion channels, GPCRs and nuclear receptors datasets by using other two prominent classifiers (Multi-layer Perceptron and Support Vector Machines). For fairness, except for the different classifiers, the other settings are completely consistent. We build multi-layer perceptron (MLP) networks, in which the number of hidden layers and neurons is the same as the DeepLSTM network. The Support Vector Machine (SVM) was available by using LIBSVM tool [To exhibit the advantage of DeepLSTM, computations were performed on SVM tool . The parenzymes data set, 91.97% in ion channels data set, 91.80% in GPCRs data set, 91.1% in nuclear receptors data set. and clearly outperform MLP  and SVM . The AUC obtained by the DeepLSTM net are 0.9951 in enzymes data set, 0.9705 in ion channels data set, 0.9951 in GPCRs data set, 0.9206 in nuclear receptors data set. However, the MLP net respectively achieve the average AUC of 0.9967, 0.9972, 0.9853 and 0.8421 in four datasets. The SVM respectively achieve the average AUC of 0.9686, 0.9613, 0.9230 and 0.9910 in four datasets. There are five main reasons for the proposed method to produce better results. The first one is that the hierarchical structure of the deep neural network is convert the input data to more complexity space, which is more conducive to complete classification tasks. The second one is that the design of our DeepLSTM not only avoid overfitting effectively, but also makes it possible to train a large number of different neural networks in a short period of time, which makes the network produce better performance. The third one is that the memory units of LSTM can retain more knowledge, which helps to make more accurate decisions at the prediction stage. The fourth is that the LSTM solves the gradient vanishing problem in the Back-Propagation (BP) algorithm, which is helpful to get better prediction model than MLP. The fifth is the use of the validation set helps to train more flexible models.From the results summarized in Table enzymes dataset. Focus on nuclear receptors dataset, the value obtained by our method is 10% higher than those the highest in several other methods, 21% than those the minimum in several other methods. The obviously higher AUC indicates that our scheme obviously outperforms the other compared methods. The results of comparison with other methods also confirm this fact that our method can improve the performance for drug–target interaction prediction. In fact, from the results shown in Table In this section, we compared the AUC of our proposed method with that of some state-of-the-art methods including DBSI , KBMF2K In this paper, we have developed a deep learning-based method to infer potential DTIs using compounds and proteins sequence. To evaluate the ability of our method, we compared it with several state-of-the-art approaches. The experimental results proved that this approach is significantly better than others in terms of performance. Comparing with other classifiers, we have provided initial evidence that DeepLSTM outperforms traditional machine learning system on the DTIs task. For the characterization and quantitative method of drug-target pairs, an interesting scheme was proposed by using SPCA to fuse PubChem fingerprint and protein evolutionary features obtained by the combination of PSSM and LM. Promising results were observed when the characterization method cooperates with three different classifiers, respectively. These results indicate that the proposed scheme has great advantage on feature expression and recognition. We have shown that the proposed method can work well with small dataset, which is distinguish from the predecessor’s methods and goes in its own special way. We also found that prediction quality continues to improve with increasing dataset size. This underscores the value of this model to train and apply very large datasets, and suggests that further performance gains may be had by increasing the data size. On the whole, the theoretical analysis and experimental results give strong theoretical and empirical evidences for the efficacy of using the proposed method to predict DTIs.Additional file 1: Table S1. Prediction performance of SVM-based for the enzymes datasets in term of ACC, TPR, SPC, PPV, MCC, and AUC. Table S2. Prediction performance of SVM-based for the ion channels datasets in term of ACC, TPR, SPC, PPV, MCC, and AUC. Table S3. Prediction performance of SVM-based for the GPCRs datasets in term of ACC, TPR, SPC, PPV, MCC, and AUC. Table S4. Prediction performance of SVM-based for the nuclear receptors datasets in term of ACC, TPR, SPC, PPV, MCC, and AUC.


Placenta previa describes a placenta that extends partially or completely over the internal cervical oss. Placenta previa is one of the leading causes of widespread postpartum hemorrhage and maternal mortality worldwide. Another cause of bleeding in pregnant women is Placenta accreta spectrum. Therefore, the aim of the present systematic review and meta-analysis is to determine the effect of prophylactic balloon occlusion of the internal iliac arteries in patients with placenta previa or placental accreta spectrum (PAS).2 index, and subsequently a random effects model was applied. Data analysis was performed within the Comprehensive Meta-Analysis software (version 2).In this systematic review and meta-analysis, to identify and select relevant studies, the SID, MagIran, ScienceDirect, Embase, Scopus, PubMed, Web of Science, and Google Scholar databases were searched, using the keywords of internal iliac artery balloon, placenta, previa, balloon, accreta, increta and percreta, without a lower time limit and until 2020. The heterogeneity of the studies was examined using the IP < 0.05).In the review of 29 articles with a total sample size of 1140 in the control group, and 1225 in the balloon occlusion group, the mean difference between the two groups was calculated in terms of Intraoperative blood loss index (mL) and it was derived as 3.21 ± 0.38; moreover, in 15 studies with a sample size of 887 in the control group, and 760 in the balloon occlusion group, the mean difference between the two groups in terms of gestation index (weeks) was found as 2.84 ± 0.49; and also with regards to hysterectomy balloon occlusion after prophylactic closure of the iliac artery, hysterectomy (%) balloon occlusion was calculated as 8.9 %, and this, in the hysterectomy control group (%) was obtained as 31.2 %; these differences were statistically significant and showed a positive effect of the intervention , which can be considered by midwives and obstetricians. Placenta previa is a type of placenta that extends partially or completely over the internal cervical oss . PlacentAnother cause of bleeding in pregnant women is placental adhesions. placental adhesions is an abnormal invasion of the placenta to the uterine wall and is classified as accreta, percreta, or increta depending on the degree of invasion of the myometrium , 6. The Establishing homeostasis in placenta requires the use of surgical techniques. These methods include multiple placement of hemostatic sutures in the placental bed, compression sutures of the uterus with or without intrauterine balloon tamponade, use of FLOSEAL in the placental bed, and uterin artery lrgation –13. If tCollapsed intra-arterial balloons do not completely block blood flow to the uterus, yet they lower blood pressure to the site of obstruction. By reducing the amount of bleeding, hemostatic methods can be more practical –22. ObstConsidering the importance of reducing bleeding due to placenta previa and Placenta accreta spectrum to reduce their mortality among pregnant women, and also due to the inconsistent results from research in this area, this systematic review and meta-analysis aimed to determine the effect prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum.In this meta-analysis, the SID, MagIran, ScienceDirect, Embase, Scopus, PubMed, Web of Science and Google Scholar databases were searched to identify and select relevant studies. The keywords used were: internal iliac artery balloon, placenta, previa, balloon, accreta, increta, and percreta, and all possible combinations of these words. No time constraints were considered in the search. The information of the identified articles was transferred into the EndNote bibliography management software (EndNote X8). In order to maximize the comprehensiveness of the search, the lists of references used within all identified articles were manually reviewed.Studies that examined the effect prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum, interventional studies, and studies for which a full text was available.Unrelated studies, studies without sufficient data, duplicate papers, studies with an unclear methodology.Initially, duplicate studies in various databases were excluded from this study, and only one copy was retained. Then a list of the titles of all the remaining articles was prepared, for further analysis. In the first stage, screening, the title and abstract of the remaining articles were carefully examined, and a number of articles were removed considering the inclusion and exclusion criteria. In the second stage, i.e. eligibility evaluation, the full text of the studies, remaining from the screening stage, were thoroughly examined according to the criteria, and similarly, a number of other unrelated studies were excluded. To prevent subjectivity, article review, and data extraction activities were performed by two reviewers, independently. If an article was not included, the reason for excluding it was mentioned. In cases where there was a disagreement between the two reviewers, a third person reviewed the article.In order to examine the quality of the remaining articles , a checklist appropriate to the type of study was adopted. STROBE checklists are commonly used to critique and evaluate the quality of observational studies. The checklist consists of six scales/general sections that are: title, abstract, introduction, methods, results, and discussion. Some of these scales have subscales, resulting in a total of 32 fields . In fact, these 32 fields represent different methodological aspects of a piece of research. Examples of subscales include title, problem statement, study objectives, study type, statistical population, sampling method, sample size, the definition of variables and procedures, data collection method(s), statistical analysis techniques, and findings. Accordingly, the maximum score that can be obtained during the quality evaluation phase and using the STROBE checklist is 32. By considering the score of 16 as the cut-off point , any artInformation on all final articles was extracted from a different pre-prepared checklist. This checklist includes article title, first author’s name, year of publication, sample size, place of study, maternal age (years), intraoperative blood loss (mL), blood transfusion (units), blood transfusion volume (ml), placenta type, and gestation (weeks), Hysterectomy (%).2 test was used to evaluate the homogeneity between studies, and due to the found heterogeneity in the studies, a random effects model was used to combine the studies and perform the meta-analysis. When the I2 index is less than 25 %, heterogeneity was considered as low; between 25 and 275 %, this was considered as moderate heterogeneity, and more than 75 % as high heterogeneity. P value less than 0.05 was considered significant. The Egger’s test and corresponding funnel plots were also used to examine the publication bias. Data analysis was performed within the Comprehensive Meta-Analysis software (version 2).Frequency and percentage as well as the standardized mean difference indices were used to combine the results of different studies. The IThe study selection process was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Based on the initial search in the database, 295 possible related articles were identified and transferred into the EndNote bibliography management software. Moreover, 12 further studies were added after reviewing the lists of references of the identified articles. Out of a total of 307 identified studies, 69 studies were duplicates and were therefore excluded. In the screening phase, out of the remaining 238 studies, 88 articles were removed after examining their titles and abstracts, and based on the inclusion and exclusion criteria. In the eligibility evalaution stage, out of the remaining 150 studies, 115 irrelevant articles were removed by studying the full text of the articles and in accordance with the inclusion and exclusion criteria. In the quality evaluation stage, by reading the full text of the article and based on the score obtained from the STROBE checklist, out of the remaining 35 studies, 6 low quality studies were omitted. Finally, 29 articles that were published between 2006 and August 2020 entered the analysis stage . According to the results of studies by Yu et al. (2020) [P > 0.05).The first criterion for comparison was the intraoperative blood loss index (mL). In 26 studies , 47–50 p. (2020) , Feng et. (2020)  and McGi. (2020) , no sign2 test, in the study of intraoperative blood loss index (mL), there was a heterogeneity between the control group and the case group (I2 = 97.4), thus, a random effects method was applied to amalgamate the results of all studies and approximate a final result. Based on the results of the meta-analysis, the mean difference of intraoperative blood loss (mL) in the balloon occlusion, and control groups were obtained as 1436 ± 211 and 403 ± 2503 respectively. Therefore, prophylactic internal iliac artery balloon occlusion reduces postpartum hemorrhage .Based on the results of IP < 0.05) was observed. However, in reviewing the results of studies by Yu et al. (2020) [P > 0.05). Therefore, reduction of blood transfusion index (units) in the balloon occlusion group is not definite, and varies according to the study conditions. Thus, with respect to blood transfusion index (units), it can be argued that prophylactic internal iliac artery balloon occlusion has no advantage.The second criterion is the comparison was blood transfusion index (units). In the studies of Dai et al. (2018) , Picel e. (2020) , Darwish. (2020) , Omar et. (2020) , Broekma. (2020) , Bodner . (2020)  and Feng. (2020) , no signP < 0.05).The third criterion for comparison was gestation index (weeks). In 14 studies , 47, 50 2 test, in the study of gestation index (weeks), there was heterogeneity between the control group (I2 = 99.1), thus, a random effects model was applied to combine the results of the studies and approximate a single final outcome. Based on the results of meta-analysis, mean of gestation (weeks) in the balloon occlusion and control groups were 37.0 ± 0.18 and 35.9 ± 0.17 respectively. Therefore, it can be concluded that prophylactic internal iliac artery balloon occlusion increases the gestation index (weeks). The mean difference between the two groups was 2.84 ± 0.49, which indicates the positive effect of the intervention. Figure P = 0.055) . While reviewing the results of studies by Omar et al. (2017) [P˃0.05).The third criterion for comparison was the hysterectomy index (%). In 8 studies , 47, 50 . (2017) , Jiang e. (2017) , Fan et . (2017) , Dai et . (2017) , Zhou et. (2017) , and Cho. (2017) , no sign2 test, in the study of hysterectomy index (%), there was a heterogeneity between the control group and the case group (I2 = 74.9), (I2 = 87.5), thus, a random effects method was applied to combine the results of the studies and approximate an overall. Based on the results of meta-analysis; the percentages of hysterectomy (%) in the balloon occlusion and control groups were 8.9 % and and 31.2 % respectively. Therefore, it can be concluded that prophylactic internal iliac artery balloon occlusion reduces the hysterectomy index (%), which indicates the positive effect of the intervention. The horizontal line on each square, in the hysterectomy forest plot, represents the 95 % confidence interval  (P = 0.499) , I2 = 7.5, thusThe aim of the present systematic review and meta-analysis was to determine the effect of prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum.Bleeding is still a major cause of maternal death following childbirth. In many developed countries, bleeding is the most important factor in admitting a pregnant woman to an intensive care unit (ICU). In developing countries, bleeding is the cause of almost half of all maternal mortality . PlacentAccording to the present systematic review and meta-analysis, the mean difference of the two groups in terms of intraoperative blood loss (mL) was obtained 3.21 ± 0.38, and this difference was statistically significant. This also showed a positive effect of the intervention. The most valid study in terms of sample size, was a research conducted by Wu et al. (2016) , in whicTimely transfusion of blood and blood products can save the lives of these patients. Therefore, the use of a variety of blood products, can be prevent the risk of death in 15,000 pregnancies per year, globally . Since bP < 0.05). However, in the results of 6 studies [P > 0.05). According to the results results, the decrease in blood transfusion index (units) in the balloon occlusion group is not definite and varies according to the study conditions. Therefore, in terms of Blood transfusion index (units), it can be stated that prophylactic internal iliac artery balloon occlusion has no advantage.In 7 studies , 47, 50, studies , 36, 41,Premature birth or gestational age less than 37 weeks from the last date of maternal menstruation is one of the most important indicators of health in any society, and the survival of newborns is directly related to gestational age and birth weight . The preAccording to the present systematic review and meta-analysis, the mean difference of the two groups in terms of gestation index (weeks) was 2.84 ± 0.49 which was statistically significant, and showed a positive effect of the intervention. The most valid study in terms of sample size was conducted by Wu et al. (2016) , which rHysterectomy is a major surgery that is almost always performed in cases of severe, life-threatening bleeding after vaginal delivery or cesarean section . Its preAccording to the present systematic review and meta-analysis, the percentage of hysterectomy (%) in the balloon occlusion and hysterectomy control groups were 8.9 % and 31.2 % respectively, which was statistically significant and, showed a positive effect of the intervention. The most comprehensive study in terms of sample size was conducted by Wu et al. (2016) , who repIn general, according to the present systematic review and meta-analysis; prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum has benefits, such as reduced intraoperative blood loss, reduced hysterectomy, and increased gestation (weeks). Therefore, the procedure can be applied prophylactically by obstetricians and gynecologists.One of the limitations of this study is that some samples were not based on random selection. Moreover, the non-uniform reporting of articles, and methods of implementation, lack of consistency in the data analysis, incomplete data, and the unavailability of the full text of some of the papers presented at the conferences can be mentioned.The results of this study show that prophylactic internal iliac artery balloon occlusion in patients with placenta previa or Placenta accreta spectrum has benefits such as reduced intraoperative blood loss, reduced hysterectomy and increased gestation (weeks), which can be considered by midwives and obstetricians.


There has been a major resurgence of tuberculosis (TB) and drug-resistant tuberculosis in the last few decades. Although it has been brought under control in most Western countries, it is still a major cause of death in endemic regions like India. Osteoarticular tuberculosis (OA TB) forms a small proportion of the total cases of tuberculosis. Perceptions and practices of orthopedic surgeons are entirely different in endemic and non-endemic regions around the world, due to the vast difference in exposure. Literature from endemic areas puts stress on clinico-radiological diagnosis and empirical anti-tubercular treatment (ATT). Such practices, although non-invasive, simple to implement, and economical, carry a significant risk of missing TB mimics and developing drug resistance. However, OA TB is still perceived as a “diagnostic enigma” in non-endemic regions, leading to a delay in diagnosis. Hence, a high index of suspicion, especially in a high-risk population, is needed to improve the diagnosis. Evolving drug resistance continues to thwart efforts to control the disease globally. This review article discusses the perceptions and practices in different parts of the world, with India as an example of the endemic world, and lays down priorities for overcoming the challenges of diagnosing osteoarticular TB. Tuberculosis (TB) is one of the oldest recognized diseases of mankind with significant morbidity and mortality . It is iHowever, the disease load is not uniform, with a greater prevalence in low-income countries around the world. As per the WHO, the 2018 figures are about 220 per 100,000 in African and Southeast Asian countries, whereas they are less than 30 in the United States and Europe . UnfortuOsteoarticular tuberculosis (OA TB) represents between 1% and 3% of the TB population, with variable incidence in endemic and non-endemic areas of the world. This has resulted in varying perceptions and practices in these different areas. With high load and poor infrastructure, surgeons in India have adopted the practice of initiating empirical anti-tubercular treatment (ATT) based on clinical and imaging findings. This concept of “clinico-radiological” diagnosis of OA TB in endemic areas is based on a presumably better clinical acumen due to high patient load, the easy availability of imaging modalities like magnetic resonance imaging (MRI), the shortage of laboratory facilities, and slow culture and drug sensitivity results. However, such practices increase the risk of missing TB mimics and drug-resistant TB. In contrast, surgeons in high-income countries often do not initiate investigations for mycobacterial disease, leading to a delay in diagnosis, and hence consider this as a “difficult clinical diagnosis” and a “diagnostic enigma” .Thus, the priority is to improve practices throughout the world, helping to institute a “protocol-based treatment” in endemic areas and improving “perceptions” in non-endemic areas.In 2018, an estimated 10 million people globally became infected with TB with almost half a million new cases of resistance, of which 78% were MDR TB. India bears almost 27% of the global burden of new cases as well as resistant cases. Additionally, every year, a million “missing” undiagnosed or inadequately diagnosed cases are not notified . In 2018The true incidence of OA TB is unknown and there are no epidemiological data or studies available. We can, however, only extrapolate the incidence representing 1–3% of pulmonary TB. OA TB appears to follow the trends of pulmonary TB, and a high incidence of resistance is reported in the study by Kapil et al. In a series of 686 culture-positive cases of Koch’s spine studied at a tertiary referral center, drug resistance was noted in 111 (16%) cases with 87 12.7%) being MDR .7% being.The European Union (EU) and European Economic Area (EEA) as well as the United Kingdom (UK) comprise 31 countries with a population of 517 million. The report “Tuberculosis surveillance and monitoring in Europe 2020 (2018 data)” states that, in 2018 in the EU/EEA and the UK, there were 52,862 tuberculosis patients, with a notification rate of 10.2 per 100,000 population. Of all notified TB cases, 40,625 (76.9%) were newly diagnosed and 36,047 (68.2%) were confirmed by culture or smear and nucleic acid amplification test. Of all TB cases with reported HIV status, 4.2% were co-infected with the virus. TB in prisons remains poorly reported; for the 17 EU/EEA countries reporting data, the notification rate amounted to 217 new and relapse TB cases per 100,000 inmates, that is, a relative risk of 14.7 compared to the general population in the same countries.Thirty-five per cent of all TB cases reported in the EU/EEA were of foreign origin, but a recent analysis failed to demonstrate a positive correlation between migration and incidence of TB in Europe . Of 26,8A recent Centers for Disease Control and Prevention (CDC) report from the United States shows a decrease in incidence from 9 cases per 100,000 persons in 1993 to 2.8 cases per 100,000 persons .The incidence of OA TB in non-endemic areas is unknown. OA TB should be part of differential diagnosis with a high index of suspicion in the prison population, people with foreign travel, any unusual presentation, and patients suffering from HIV.OA TB occurs primarily by hematogenous spread from a primary focus such as lung, kidney, and lymph node. OA TB is often a reactivation of an OA lesion that has been implanted at the time of initial bacteremia .Tubercle bacilli are obligate aerobes in the lungs but become facultative anaerobes in situations when there is reduced oxygen tension. Thus, bacilli are rather less active in the osteoarticular lesions than the pulmonary ones (paucibacillary)  18]..18].More than 50% of OA TB affects the spine. Thoracic vertebrae are the most commonly affected region (50%) . HematogIn the appendicular skeleton, the bacteria reach the joint space directly through the sub-synovial vessels or indirectly by eroding the epiphyseal bone. In the appendicular skeleton, the hip and the knee are the most common sites affected .Skeletal tuberculosis can affect any gender or age group in the endemic regions, but it is mostly seen during the first three decades of life and equally in both men and women .Patients generally present with joint pain, deformity, and the characteristic cold abscess, sinus, or a tubercular ulcer. Classical constitutional symptoms associated with tuberculosis like low-grade evening fever, anorexia, and weight loss are seen in less than one-third of patients and an active concomitant pulmonary infection is rarely seen . On examThe tubercular sinus has a wide mouth and thin, blue undermined margins with serosanguinous discharge. The sinus has a thick wall and can be fixed to the underlying tissue or bone  22]..22].The tubercular ulcer is slightly painful. It has thin, reddish-blue undermined edges. There is pale granulation tissue with scanty serosanguinous discharge in the floor and slight induration at the base  22]..22].The disease most commonly involves the osteoarticular region but may rarely present as osteomyelitis.The osteoarticular disease goes through five stages in its natural history .1.Stage of synovitis. Patient presents with night pain and spasm of the affected group of muscles. Affected joint has >75% of the joint movement preserved. X-ray shows rarefaction and haziness of the articular regions. This stage has excellent prognosis.2.Stage of early arthritis. Patient presents with joint pain, spasm of the surrounding muscle, and difficulty in doing some activities of daily living. Affected joint has 50–75% of the joint movement preserved. X-ray shows classical Phemister’s triad of juxta-articular osteopenia, mild joint space reduction, and peripheral osseous erosions. This stage has good prognosis with mild stiffness.3.Stage of late arthritis. Patient presents with joint pain, spasm, and difficulty in activities of daily living. Affected joint has >75% loss of range of movement of the joint. X-ray shows marked joint space reduction and joint destruction. This stage has fair prognosis with severe loss of motion.4.Stage of advanced arthritis with subluxation and dislocation. Patient presents with deformity of the involved joint. Affected joint has gross restriction of all movements. X-ray shows pathological dislocation with wandering acetabulum in the hip. This stage has poor prognosis.5.Terminal or sequelae of arthritis. Fibrous ankylosis of the joint is seen.X-rays: The commonly observed features on X-rays in spinal TB are loss of vertebral height, indistinct vertebral endplates, erosions, angular kyphosis, and paravertebral masses. Involvement of two or more contiguous vertebrae generally occurs later .In appendicular skeleton, Phemister’s triad of juxta-articular osteopenia/osteoporosis, peripheral osseous erosions, and gradual joint space narrowing suggests tuberculous arthritis but is nonspecific .OA TB can rarely present as tuberculous osteomyelitis involving long bones. When it involves flat bones of the hand or foot, medullary expansion is seen by the classic picture of “spina ventosa”.The MRI is very sensitive with respect to bone and soft tissue changes.MRI findings considered to be diagnostic of tuberculosis of the spine are a combination of marrow edema, paravertebral collection with subligamentous and epidural extension, contiguous vertebral body involvement with relative preservation of disc space, epidural involvement , intraosseous abscess, and septate paravertebral shadows  25]..25].Cord edema, myelomalacia, cord atrophy, and syringomyelia are observed in cases with neural complications. Healing or healed disease is suggested by resolution of marrow edema and collection, fatty replacement of bone marrow, resolution of cord signal intensity, and absence of contrast enhancement .In the appendicular skeleton, the most common MRI findings are synovitis, bone marrow edema and erosions, osteomyelitis with para-articular abscess, and tenosynovitis  21,27].,27.21,27The CT scan is a valuable tool when the X-rays are normal and MRI facilities are unavailable. It is particularly useful for skeletal locations not clearly visible on radiographs like craniovertebral spine, cervicodorsal spine, rib, sternum, sacroiliac joint, and posterior elements of vertebrae. The CT scan detects abscess within the vertebral canal. Varying degrees of bone destruction with presence of sequestrum is pathognomonic of TB on a CT scan . PresencPositron emission tomography/computed tomography (PET/CT) imaging allows three-dimensional localization of tracer activity to bone lesions and is useful for differentiating soft tissue infection from bone infections. It is used to assess sites of suspicious bone infection and can observe the extent of the lesion at complex anatomical sites . FluorodAn appropriate sample must be obtained for processing when OA TB or any OA infection is suspected.Cotton swab specimens from discharging sinuses should be avoided. These specimens are unreliable for isolation of mycobacteria or they may contain skin contaminants skewing culture reports .Tissue samples yield the best results ,36. TissWhile some authors favor fine-needle aspiration cytology in superficial affected joints , some doPercutaneous transpedicular biopsy is favored by most spine surgeons for obtaining a sample of suspected spinal tuberculosis .Arthroscopy provides visualization of the lesion, biopsy as well as option for surgical excision if needed.The samples (pus and tissue) for bacteriological examination are collected in a predesignated sterile leak proof container and transferred to the laboratory at the earliest to process them for pyogenic and mycobacterial smear and culture. The histopathological sample must be collected in a formalin container .TB culture is still considered as the gold standard in the diagnosis of tuberculosis. Additionally, it also helps us in assessing drug resistance.The commonly used culture media are as follows:Mycobacterium tuberculosis (M. tb). However, it has low sensitivity especially in smear-negative pulmonary TB and extrapulmonary TB. Additionally, reports are available at six to eight weeks and delay in laboratory diagnosis of TB is a major obstacle in TB control programs.(A) Lowenstein–Jensen (LJ) medium is the conventional medium. It is a solid medium requiring basic infrastructure with low cost and is highly specific for Mycobacterium tuberculosis (M. tb) complex consisting of M. tuberculosis, M. bovis, M. africanum and M. microti. However, it is more expensive and its implementation requires an advanced technical infrastructure that is not always available in resource-poor countries [(B) BACTEC Mycobacterium Growth Indicator Tube 960 (MGIT 960) is a liquid medium and is sensitive, accurate, and reproducible. It has a better yield and the culture report results are available within two to three weeks as opposed to the six to eight weeks needed for LJ medium ,42,43,44ountries .Laboratory technicians require special training in safe practices for specimen and culture handling. Risk of contamination or carryover contamination exists because of the use of a highly enriched growth media for culture .In short, MGIT is the top performer with regard to sensitivity, while LJ medium sets the current standard for specificity. Hence, international guidelines recommend that all specimens that are cultured on automated liquid systems be inoculated on solid medium as well .Mycobacterium tuberculosis: This medium consists of a lower layer of LJ medium and an upper layer of Middlebrook (MB) 7H10 with an added indicator to detect growth by color change. The isolation rate is almost double and it has a better recovery time than that of LJ, and it is less expensive than MGIT. However, its role in OA TB has not yet been proven.(C) Bilayered Medium for Rapid Isolation of Drug sensitivity studies: These should always be done whenever mycobacterial culture is positive. The importance of performing drug sensitivity studies can be judged when one reviews the literature on drug-resistant cases of OA TB from endemic areas. It reveals any one drug resistance to be around 30% and MDR to be between 9% and 25.5% .Culture is the gold standard and is the only method to assess complete drug sensitivity; however, deep inaccessible lesions, paucibacillary state, and initiation of empirical ATT pose difficulties in the accurate diagnosis of OA TB . Most InM. tb as well as resistance.Molecular Methods: These methods can detect Mycobacterium tuberculosis DNA with results available within two hours. Moreover, it can detect rifampicin (RIF) resistance, helping to start therapy immediately for MDR TB. The specificity of GeneXpert is 100% and the sensitivity is quite low for OA tuberculosis (about 60% to 70%). A negative GeneXpert cannot be used to rule out tuberculosis infection and, thus, culture remains the gold standard [GeneXpert is a novel test used to detect the presence of standard .A new diagnostic tool, GeneXpert MTB/RIF Ultra or Xpert Ultra, has improved sensitivity in the detection of TB and RIF resistance compared to GeneXpert and line probe assays (LPAs) in both pulmonary and extrapulmonary samples, but at the cost of lower specificity than the GeneXpert ,55. GeneM. tuberculosis. It is useful as a molecular diagnostic tool for screening and predicting resistance to re-treatment of pulmonary TB patients [Pyrosequencing is a rapid method for the detection of resistance to rifampicin, isoniazid, streptomycin, ethambutol, ofloxacin, and amikacin in patients . The respatients .The management flowchart for a suspected case of osteoarticular tuberculosis is presented in Drug sensitivity studies with molecular methods: It must be borne in mind that most current molecular methods detect rifampicin resistance, where a few others detect resistance to isoniazid. Resistance to other drugs may go unnoticed if only these methods are used, and the patient may receive one or two ineffective drugs .Hence, these methods must be supported by culture-based drug sensitivity tests.M. Tb infection and non-TB mycobacterial infection [Smear: Ziehl–Neelsen (ZN) stain is a rapid method to detect acid-fast bacilli (AFB). The number of bacilli needed for detection are considered to be 10,000 per slide or ml, and hence sensitivity is often low especially in biological fluids (5% to 20%) . The sennfection .Histopathology: The microscopic pathological lesion of TB is the “tubercle” characterized by central necrosis surrounded by epithelioid cells, giant cells, and mononuclear cells. Two types of lesions have been described in the literature: (a) caseating exudative type—more commonly seen in children characterized by rapid and extensive destruction of bone and cartilage with predominating caseation and cold abscess formation; and (b) proliferating type—seen in adults mostly characterized by less bone destruction and cellular proliferation predominating with minimal caseation. The tuberculous granuloma is the extreme form of the caseating type . The speAlthough many tests have been introduced for the indirect diagnosis of TB, serodiagnosis has limited utility in disease-endemic countries with high infection rates and an acquired immunodeficiency syndrome (AIDS)-prevalent population . InterfeThis is the most common method of diagnosing OA TB in India, and is based on clinical and imaging features without attempting biopsy, and hence is prone to miss TB mimics and drug-resistant cases ,71,72. HThus “astute clinicians and comprehensive diagnostic algorithms” play a vital role in clinico-radiological diagnosis . HoweverTo summarize, there is no diagnostic test which can diagnose OA TB with high sensitivity and specificity. The hierarchy of investigative modalities for diagnosis of OA TB. is shown in 1.Non-tuberculous mycobacteria (NTM). Initiating ATT would not yield desirable results in cases of NTM .2.Pyogenic arthritis3.Rheumatoid arthritis (RA). In RA, the disease is usually polyarticular, the synovium tends to be thicker and irregular, and erosions are smaller as compared to OA TB .4.Pyogenic spondylodiscitis. Presents as single segment involvement with poor soft tissue mass and early disc changes on MRI .5.Seronegative spondyloarthropathy, especially while dealing with sacroiliac lesions.The other diagnoses to be entertained are tumors and pigmented villonodular synovitis in the appendicular skeleton, herniated disc, canal stenosis, and mechanical back pain in cases of spinal TB ,79.The world TB scenario improved dramatically in the middle of the 20th century when anti-TB drugs were introduced to the world. Dr. B. Mukhopadhaya from Patna, India, while delivering the Hunterian lecture on OA TB at the Royal College of Surgeons in 1955, had concluded saying that we appear to be on the “threshold of success” . HoweverIndia reported totally drug-resistant TB (TDR-TB) in 2012 shortly after Iran ,83. AlthThe irrational prescription of ATT by family physicians could have been another cause of rising resistance. A study done among family physicians serving Dharavi in Mumbai, the largest slum in Asia and a TB hotspot, revealed that only 6 out of 106 physicians could write an appropriate prescription for first-line ATT and these 106 doctors prescribed 63 different regimens for TB . PovertyThe current situation is described by Chatterjee et al. in an eye-opening article entitled “Drug-resistant tuberculosis: is India ready for the challenge?”. The authors mentioned that resistance to fluoroquinolones is 21% in non-MDR and 36% in MDR patients and resistance to aminoglycosides such as amikacin, capreomycin, or kanamycin is 7% in new patients. In addition, resistance to ethionamide and para-aminosalicylic acid (PAS) was 11% in new patients .This increased resistance in pulmonary TB naturally translated into increased resistance in OA TB, and this has been noted in a few studies from India mainly about metropolitan cities ,41. SuchAt the other end of the spectrum, OA TB is quite rare in developed countries. The low incidence can be judged by the fact that, in a 20-year study of OA TB in children from a pediatric center in Spain, there were only 11 cases of OA TB. Of these, 9 patients were children of immigrants .1)a)Confirming that other conditions are not treated as tuberculosis ;b)Establishing that TB is not treated as other conditions .Ruling out false-positive and false-negative cases and achieving probable diagnosis of TB.2)Achieving accurate diagnosis of TB and establishing sensitivity pattern, ideally in every patient of OA tuberculosis.3)a)Ruling out false-positive cases: Empirical ATT is often erroneously initiated in NTM infections and pyogenic infections, among many others.M. tb was cultured in 189 specimens [NTM infections are often not even considered and ATT is instituted when histopathology reveals granulomatous infections. Some details of history like infection following open fractures or invasive procedures like arthroscopy or intra-articular injections would point to NTM infection . It is bpecimens .Pyogenic infections are to be ruled out in every case of suspected OA TB by smear and culture for pyogenic organisms. In a study of 93 patients referred from city public hospitals and private healthcare facilities across Mumbai on the threshold of receiving ATT for suspected OA TB, 7 out of 93 were diagnosed to be suffering from pyogenic infections . Other cb)Ruling out false negative: Confirming that TB is not treated as other conditions. Several studies from non-endemic areas or developed countries have reported a delay in diagnosis and treatment for OA TB, mainly because the condition is not high on the clinician’s differential diagnosis list. The medical community should bear in mind that immigration from disease-endemic regions has reintroduced the relatively rare tuberculosis infection.In a paper entitled “Delays in the diagnosis and treatment of bone and joint tuberculosis in the United Kingdom”, Broderick et al. described a median delay of seven months between the onset of symptoms and the referral to an appropriate center in 30 patients who were finally diagnosed as OA TB; 89% of their patients were migrants. They also noted that in 23% of the patients the initial sample was not sent for mycobacterial culture at the time of index surgery at the tertiary center, needing a second surgery .Staphylococcus aureus, he was initially treated with targeted antibiotics [Arrabal et al. described a three-year delay in diagnosis of TB of the hip joint in a 54-year-old patient in Spain, even though he had multiple discharging sinuses around the hip. Based on a culture from the sinus that grew coagulase-negative ibiotics .Thus, it must be borne in mind that there is an increased incidence of this “difficult clinical diagnosis” in developed countries  Figure .Performing diagnostics in patients who were treated with empirical ATT and not responding to ATT.4)Establishing a sensitivity pattern ideally in every patient of OA tuberculosis: The importance of establishing a sensitivity pattern can be judged by a recent publication on prevalence and patterns of drug-resistant pulmonary tuberculosis in India by Lohiya et al. . They foM. tb culture. Appropriate sample and container, early transfer to a laboratory with experience to tackle extrapulmonary specimens, and availability of liquid culture medium play an important role in improving the yield of the sample.Since OA TB is likely to follow the same trend, targeted ATT is essential to addressing the challenge of drug-resistant OA TB infection. Prerequisite to this is improving the yield of the 5)Diagnosing and Addressing Non-Respondents: After the initiation of empirical ATT based on either clinico-radiological or histopathological findings, the patients must be closely observed.If a satisfactory response is not determined in about three months, a close review is needed to find the reason for poor response. The poor response can be judged by persistent or increased swelling, development of new swelling or new lesion, persistent discharging sinus, development of new sinus, development of non-healing ulcer, neurological deterioration, or development of new neurological deficit. Agashe et al. under the aegis of the Bombay Orthopaedic Society studied 89 patients not responding to ATT at a mean duration of 9.32 months (3 months to 60 months). Surprisingly, 80.9% patients were initiated on ATT without tissue diagnosis. Of the 89 patients, 33 had resistance to at least one drug, 24 were MDR, and 3 were XDR. Noteworthy findings were varying resistance pattern, primary resistance to second-line drugs, and high number of resistant patients in the pediatric age group. On comparing the resistance pattern with another Bombay Orthopaedic Society project conducted between 2004 and 2007, the authors noted significant deterioration in the resistance pattern . AnotherOnce a probable diagnosis of OA TB is made, the cases may be investigated along the lines discussed below:This drastic change in the management of OA TB in endemic regions like India would mean a significant improvement in infrastructure, a change in mindset of orthopedic surgeons and doctors at large, and the willingness on the part of patients to undergo invasive investigations for a condition “traditionally” treated with medicines empirically.Similarly, a drastic change would be needed in the mindset of surgeons in non-endemic regions to put OA TB on a “suspect list”.1.“Complete diagnosis of OA TB”—i.e., culture-positive cases with drug sensitivity known and in whom targeted chemotherapy can be initiated.2.“Qualify for ATT”—i.e., ATT is initiated based only on clinical and imaging findings or on results of tests that do not establish a sensitivity pattern. This is an acceptable practice but only after attempts at establishing a “complete diagnosis” have failed and the surgeon strongly suspects OA TB.3.The term “clinico-radiological diagnosis” should be replaced by clinico-radiological impression as there is no consensus on clinical and/or radiological criteria for diagnosing OA TB.To summarize, with the epidemic of drug resistance in a paucibacterial disease like OA TB, the definitions must be modified. We propose them as follows:Protocol-based therapy is very difficult, particularly in cash-strapped economies, but even in progressive economies like India (6th largest GDP in the world) it would be tricky to implement it. In India, there are currently 628 GeneXpert machines and 74 National Tuberculosis Elimination Program (NTEP)-certified laboratories to perform susceptibility testing . These fIf current practices are not modified in India, it is postulated that, by 2032, the incidence of MDR may increase by 275% and an estimated 85% of these cases will be primary resistant tuberculosis .Tremendous political will is needed at the national level to tackle such a deteriorating scenario. Significantly more resources, up from the current 1.28% of GDP (2017–18) that India spends on public healthcare, are needed to improve poor infrastructure . The numAssociations and societies in endemic areas, such as the Indian Orthopaedic Association (IOA), must take rapid steps to educate its members on coming out of their comfort zone of instantaneous diagnosis obtained by non-invasive means and trying to achieve “complete diagnosis”.Similarly, orthopedic societies in developed countries must educate their surgeons on keeping OA TB in mind when dealing with a case of osteoarticular infection.The WHO will have to funnel its resources to increase and upgrade laboratories in developing nations and endemic areas.Lastly, international bodies such as the World Association Against Infection in Orthopaedics and Trauma (WAIOT) and the SICOT  can play a major role in research by connecting endemic and non-endemic countries. The high volume of patients in endemic areas and the advanced technology available in developed and non-endemic countries can help us understand more about this disease.1.TB is a tenacious disease having bounced back from the brink of defeat to a position of infecting 10 million people annually.2.The incidence of TB is disproportionately high in India, China, and some African countries.3.Drug resistance is increasing in endemic areas and, at present, at least a quarter of new TB cases are resistant to at least one drug and about 3% have MDR. Resistance to fluroquinolones, aminoglycosides, ethionamide, and PAS are also noted in new cases.4.OA TB is paucibacterial, often slowly progressive, accompanied by constitutional symptoms in about one-third to one-half of cases. Spinal TB is the most common presentation of OA TB.5.OA TB constitutes between 1% and 3% of the TB population and, hence, surgeons in endemic areas are exposed to a greater patient load of OA TB cases as compared to surgeons in non-endemic areas.6.Surgeons in endemic areas tend to “over-diagnose” TB as tissue diagnosis is hardly obtained due to the lack of infrastructure, a reportedly poor culture yield, the confidence in clinical acumen, and a belief in MRI. The initiation of empirical ATT is almost a norm in most parts of India.7.While surgeons in non-endemic areas often “under-diagnose” OA TB because pyogenic infections are always high on the list and investigations are directed accordingly, this often leads to a delay in diagnosis.8.The initiation of ATT is a fateful decision; the practice of empirical ATT, although reasonably successful in previous years, is certainly not advisable in view of rising drug resistance. Every attempt must be made to establish a drug sensitivity pattern before the initiation of ATT.9.Due to the paucibacterial nature and moderate culture yield at best, empirical ATT must be started in a proportion of cases even if the culture yield is negative, if OA TB is strongly suspected. These patients must be observed very carefully and, if a good response is not obtained after three to four months, the cause for the poor response must be investigated and addressed.10.Infrastructure development on a priority basis is the need of the hour in endemic regions and an improved perception is required in non-endemic regions.


Spontaneous intracerebral hemorrhage (sICH) is a serious stroke subtype. The effective therapies for patients with sICH are still unclear, and the role of hemostatic agents in sICH is still unclear. Although some studies have shown that hemostatic agents could benefit patients with sICH, different hemostatic drugs have different effects on patients with sICH, and which hemostatic drug has the best effect on the prevention of hematoma expansion and neurological deterioration in sICH patients remains unclear. To better understand the effects of hemostatic agents in patients with sICH, it is necessary to carry out a network meta-analysis to comprehensively compare the effects of different hemostatic agents.This protocol has been designed following the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols statement. Related studies in the following databases will be searched until September 2020: PubMed, Embase, Scopus, Web of Science, the Cochrane Library, China National Knowledge Infrastructure, VIP and Wanfang. Randomized controlled trials and nonrandomized controlled studies comparing at least 2 different hemostatic agents in sICH patients will be included. A quality assessment will be conducted with the Cochrane Collaboration tool or the Newcastle-Ottawa Scale based on the study design. The primary outcome will be the incidence of hematoma expansion, and the secondary outcome will be the functional outcome. Pairwise and network meta-analyses will be conducted using STATA V.14 . Mean ranks and the surface under the cumulative ranking curve will be used to evaluate every agent. Statistical inconsistency assessment, subgroup analysis, sensitivity analysis and publication bias assessment will be performed.According to disseminate through academic conferences, the results of this network meta-analysis are expected to publish in a peer-reviewed journal.This study will provide high quality evidence about effects of different hemostatic agents in patients with sICH.CRD42020196039. The occurrence of hematoma expansion (HE) was detected within 3 hours of symptom onset in approximately 73% of sICH patients, clinically obvious expansion was present in 35% of patients, and HE was independently related to death and adverse outcomes.–5Spontaneous intracerebral hemorrhage (sICH) is a devastating health condition, accounting for 10% to 15% of all strokes, Hence, drug  interventions to accelerate hemostasis and limit HE have been the main focus of treatment for acute sICH.In theory, early interventions to reduce hematoma volume may improve prognosis. Compared to medical management alone, surgical craniotomy to evacuate supra tentorial hematoma and reduce hematoma volume was found to reduce mortality and disability rates, but the result is not very reliable, so surgical treatment is not often used. In Sprigg et al.'s study, tranexamic acid was compared with a placebo. In another study by Zhang et al., hemocoagulase and tranexamic acid were analyzed. In addition, Piriyawat et al. explored the potential role of aminocaproic acid in the prevention of early HE after sICH, and aprotinin was analyzed in a study by Li. Some systematic reviews have also been published. In Yuan et al.'s study, rFVIIa was compared with a placebo. In another study by Huang et al., tranexamic acid and placebo were analyzed.Some studies about hemostatic agents, including aminocaproic acid, tranexamic acid, aprotinin, recombinant activated factor VII (rFVIIa), and hemocoagulase, in patients with sICH have been published previously. For example, Stephan et al randomly assigned sICH patients to receive a placebo, 20 μg of rFVIIa per kilogram of body weight, or 80 μg of rFVIIa per kilogram to compare the percentage of hematoma volume change and poor outcomes in patients from these 3 groups. To better understand hemostatic treatment of sICH patients, it is necessary to carry out a network meta-analysis to comprehensively compare the effects of different hemostatic agents.However, different hemostatic drugs have different effects on patients with sICH, and it is still unclear which hemostatic agent is most suitable for preventing HE and neurological deterioration in sICH patients. Based on its methodology, a network meta-analysis can evaluate the relative efficiency of different hemostatic drugs and rank them.22.1https://www.crd.york.ac.uk/prospero/) and the study registration number is CRD42020196039. The protocol has been reported following the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols statement .This network meta-analysis protocol has been registered on the PROSPERO website  and nonrandomized controlled studies will be included in this study. Case reports, case series and reviews will not be included in this study.2.3.3Studies comparing at least 2 different hemostatic agents among the following will be included in this study: aminocaproic acid, tranexamic acid, aprotinin, rFVIIa, and hemocoagulase.2.3.4The primary outcome will be incidence of HE. HE will be evaluated by the imaging index threshold of each study. The secondary outcome will be the functional outcome at the end of follow-up. Functional outcome will be categorized as good or poor according to the scale and threshold in each study.2.4We will conduct the literature search for the related RCTs and nonrandomized controlled studies until September 2020 in the following databases: PubMed, Embase, Scopus, Web of Science, the Cochrane Library, China National Knowledge Infrastructure, VIP and Wanfang. Restrictions on language will not be set. The detailed search strategy is provided in online .2.5Two authors (YL and JZ) will independently screen the titles and abstracts of all records after removing duplicates. Any record which does not meet the eligibility criteria will be deleted. The full-text papers of the remaining studies will be obtained and screened by 2 authors independently. Studies that meet the eligibility criteria will eventually be included. If data are used in more than 1 study, the study with the larger sample size and longer follow-up time will be included. Any controversy between the 2 authors will be addressed by another author (XY).2.6According to a predetermined extraction form, 2 authors (YL and JZ) will independently extract data from all included studies. The following information will be extracted: first author, year of publication, area, study duration, sample size, age, percentage of males, time from onset to first brain CT scan, time from first CT to follow-up CT, inclusion/exclusion criteria, details of the intervention in each group, number of patients in each group, follow-up time and outcomes in each group. We will attempt to contact authors to access data that cannot be gotten directly from the papers. Any controversy between 2 authors will be addressed by consensus, and another author (XY) will review all the data.2.7The quality of all RCTs will be evaluated with the Cochrane Collaboration tool. The quality of all nonrandomized controlled studies will be assessed by the Newcastle-Ottawa Scale. Two authors (YL and JZ) will independently conduct quality evaluations, and any controversy will be addressed by discussion with another author (XY).2.82.8.1If quantitative analysis cannot be carried out, the results will be described narratively. When quantitative analysis is feasible, we will conduct all of the following statistical analyses by STATA V.14 .2.8.2 Heterogeneity among the included studies will be assessed by the I2 statistic.If at least 2 studies provide related data, conventional pairwise meta-analyses between different interventions will be performed first using the DerSimonian–Laird method and a random effects model.2.8.3 The network geometry will indicate the interactions among all the included studies, and the contributions of direct comparisons will be shown in the contribution plot for the network. The effects of every intervention on both the incidence of HE and the functional outcome in sICH patients will be assessed using mean ranks and the surface under the cumulative ranking curve.A network meta-analysis will be conducted using a random effects model.2.8.4 and the local inconsistency will be assessed using the loop-specific method.Inconsistency between direct and indirect comparisons will be evaluated using global and local methods. The design-by-treatment model will be adopted for the global method,2.8.5We will perform subgroup analyses, if possible, according to age, sex, race, Glasgow Coma Scale score, baseline hematoma volume and hematoma location. Sensitivity analysis, by eliminating each study, will be used to test whether the results are stable.2.8.6The network funnel plot will be used to assess the potential publication bias in the network meta-analysis.2.8.7We will follow the Grading of Recommendations, Assessment, Development and Evaluation approach, which is used to rate the quality of treatment effect evaluations from network meta-analyses, to assess the evidence quality.3This study will be the first network meta-analysis that comprehensively compares different hemostatic agents in sICH patients. Nonrandomized controlled studies will be included to strengthen the statistical power of this network meta-analysis because of the limited number of related randomized controlled studies. We hope that the results of this network meta-analysis will offer more information about the safety and efficacy of different hemostatic agents in sICH patients and provide help for future clinical practice and research design. However, this network meta-analysis will still have limitations. First, studies with inferior quality that are included in this network meta-analysis may decrease the significance of the results. Moreover, the final results of this network meta-analysis may be influenced by high heterogeneity among different studies.In conclusion, this study will help to compare the effects of different hemostatic agents in patients with sICH. We hope this network meta-analysis can offer a high evidence for hemostatic treatment of sICH patients and guide future clinical practice.Conceptualization: Yujian Li, Hao Li.Data curation: Yujian Li, Huiqing Zhou, Xiang Yang.Methodology: Jun Zheng.Project administration: Xiang Yang.Writing – original draft: Yujian Li.Writing – review & editing: Hao Li.