Host biomarkers and combinatorial scores for the detection of serious and invasive bacterial infection in pediatric patients with fever without source.

BACKGROUND: Improved tools are required to detect bacterial infection in children with fever without source (FWS), especially when younger than 3 years old. The aim of the present study was to investigate the diagnostic accuracy of a host signature combining for the first time two viral-induced biomarkers, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interferon γ-induced protein-10 (IP-10), with a bacterial-induced one, C-reactive protein (CRP), to reliably predict bacterial infection in children with fever without source (FWS) and to compare its performance to routine individual biomarkers (CRP, procalcitonin (PCT), white blood cell and absolute neutrophil counts, TRAIL, and IP-10) and to the Labscore. METHODS: This was a prospective diagnostic accuracy study conducted in a single tertiary center in children aged less than 3 years old presenting with FWS. Reference standard etiology (bacterial or viral) was assigned by a panel of three independent experts. Diagnostic accuracy (AUC, sensitivity, specificity) of host individual biomarkers and combinatorial scores was evaluated in comparison to reference standard outcomes (expert panel adjudication and microbiological diagnosis). RESULTS: 241 patients were included. 68 of them (28%) were diagnosed with a bacterial infection and 5 (2%) with invasive bacterial infection (IBI). Labscore, ImmunoXpert, and CRP attained the highest AUC values for the detection of bacterial infection, respectively 0.854 (0.804-0.905), 0.827 (0.764-0.890), and 0.807 (0.744-0.869). Labscore and ImmunoXpert outperformed the other single biomarkers with higher sensitivity and/or specificity and showed comparable performance to one another although slightly reduced sensitivity in children < 90 days of age. CONCLUSION: Labscore and ImmunoXpert demonstrate high diagnostic accuracy for safely discriminating bacterial infection in children with FWS aged under and over 90 days, supporting their adoption in the assessment of febrile patients.

Host test based on tumor necrosis factor-related apoptosis-inducing ligand, interferon gamma-induced protein-10 and C-reactive protein for differentiating bacterial and viral respiratory tract infections in adults: diagnostic accuracy study.

OBJECTIVES: To assess the performance of a test (called BV), integrating the blood levels of three immune proteins into a score, to differentiate bacterial from viral infection among adults with suspected lower respiratory tract infection (LRTI). METHODS: Prospective diagnostic accuracy study, enrolling febrile adults >18 years with LRTI signs or symptoms for less than 7 days presenting to several hospitals' emergency departments in Israel. The main exclusion criterion was immunodeficiency. Reference standard diagnosis (bacterial/viral/indeterminate) was based on three experts independently reviewing comprehensive patient data including follow-up data. BV generated three results: viral infection or other nonbacterial condition (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65) and bacterial infection including co-infection (65 < score ≤ 100). BV performance was assessed against the reference standard with indeterminate reference standard and equivocal BV cases removed. RESULTS: Of 490 enrolled patients, 415 met eligibility criteria (median age 56 years, interquartile range 35). The reference standard classified 104 patients as bacterial, 210 as viral and 101 as indeterminate. BV was equivocal in 9.6% (30/314). Excluding indeterminate reference standard diagnoses and equivocal BV results, BV's sensitivity for bacterial infection was 98.1% (101/103; 95% confidence interval 95.4-100), specificity 88.4% (160/181; 83.7-93.1) and negative predictive value 98.8% (160/162; 97.1-100). DISCUSSION: BV exhibited high diagnostic performance for febrile adults with suspected LRTI among patients with reference standard diagnoses of bacterial or viral LRTI.

Automating a new host-protein assay for differentiating bacterial from viral infection to reduce operator hands-on time.

Distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse, and detrimental ramifications for the patient, the healthcare system and society. A novel ELISA-based assay that integrates the circulating levels of three host-response proteins (TRAIL, IP-10 and CRP) was developed to assist in differentiation between bacterial and viral etiologies. We developed a new protocol for measuring the host-based assay biomarkers using an automated ELISA workstation. The automated protocol was validated and was able to reduce technician hands-on time by 76%, while maintaining high analytical performance. Following automation, the assay has been incorporated into the routine workflow at a pediatric department, and is performed daily on admitted and emergency department patients. The automation protocol reduces the overall burden on the hospital laboratory performing the assay. This benefit has potential to promote adoption of the host-based assay, facilitating timely triage of febrile patients and prudent use of antibiotics.

A host-protein signature is superior to other biomarkers for differentiating between bacterial and viral disease in patients with respiratory infection and fever without source: a prospective observational study.

Bacterial and viral infections often present with similar symptoms. Etiologic misdiagnosis can alter the trajectory of patient care, including antibiotic overuse. A host-protein signature comprising tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein-10 (IP-10), and C-reactive protein (CRP) was validated recently for differentiating bacterial from viral disease. However, a focused head-to-head comparison of its diagnostic performance against other biomarker candidates for this indication was lacking in patients with respiratory infection and fever without source. We compared the signature to other biomarkers and prediction rules using specimens collected prospectively at two secondary medical centers from children and adults. Inclusion criteria included fever > 37.5 °C, symptom duration ≤ 12 days, and presentation with respiratory infection or fever without source. Comparator method was based on expert panel adjudication. Signature and biomarker cutoffs and prediction rules were predefined. Of 493 potentially eligible patients, 314 were assigned unanimous expert panel diagnosis and also had sufficient specimen volume. The resulting cohort comprised 175 (56%) viral and 139 (44%) bacterial infections. Signature sensitivity 93.5% (95% CI 89.1-97.9%), specificity 94.3% (95% CI 90.7-98.0%), or both were significantly higher (all p values < 0.01) than for CRP, procalcitonin, interleukin-6, human neutrophil lipocalin, white blood cell count, absolute neutrophil count, and prediction rules. Signature identified as viral 50/57 viral patients prescribed antibiotics, suggesting potential to reduce antibiotic overuse by 88%. The host-protein signature demonstrated superior diagnostic performance in differentiating viral from bacterial respiratory infections and fever without source. Future utility studies are warranted to validate potential to reduce antibiotic overuse.

A novel host-protein assay outperforms routine parameters for distinguishing between bacterial and viral lower respiratory tract infections.

Bacterial and viral lower respiratory tract infections (LRTIs) are often clinically indistinguishable, leading to antibiotic overuse. We compared the diagnostic accuracy of a new assay that combines 3 host-biomarkers (TRAIL, IP-10, CRP) with parameters in routine use to distinguish bacterial from viral LRTIs. Study cohort included 184 potentially eligible pediatric and adult patients. Reference standard diagnosis was based on adjudication by an expert panel following comprehensive clinical and laboratory investigation (including respiratory PCRs). Experts were blinded to assay results and assay performers were blinded to reference standard outcomes. Evaluated cohort included 88 bacterial and 36 viral patients (23 did not fulfill inclusion criteria; 37 had indeterminate reference standard outcome). Assay distinguished bacterial from viral LRTI patients with sensitivity of 0.93±0.06 and specificity of 0.91±0.09, outperforming routine parameters, including WBC, CRP and chest x-ray signs. These findings support the assay's potential to help clinicians avoid missing bacterial LRTIs or overusing antibiotics.

Validation of a Novel Assay to Distinguish Bacterial and Viral Infections.

BACKGROUND: Reliably distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse. A novel assay that integrates measurements of blood-borne host-proteins (tumor necrosis factor-related apoptosis-inducing ligand, interferon γ-induced protein-10, and C-reactive protein [CRP]) was developed to assist in differentiation between bacterial and viral disease. METHODS: We performed double-blind, multicenter assay evaluation using serum remnants collected at 5 pediatric emergency departments and 2 wards from children ≥3 months to ≤18 years without (n = 68) and with (n = 529) suspicion of acute infection. Infectious cohort inclusion criteria were fever ≥38°C and symptom duration ≤7 days. The reference standard diagnosis was based on predetermined criteria plus adjudication by experts blinded to assay results. Assay performers were blinded to the reference standard. Assay cutoffs were predefined. RESULTS: Of 529 potentially eligible patients with suspected acute infection, 100 did not fulfill infectious inclusion criteria and 68 had insufficient serum. The resulting cohort included 361 patients, with 239 viral, 68 bacterial, and 54 indeterminate reference standard diagnoses. The assay distinguished between bacterial and viral patients with 93.8% sensitivity (95% confidence interval: 87.8%-99.8%) and 89.8% specificity (85.6%-94.0%); 11.7% had an equivocal assay outcome. The assay outperformed CRP (cutoff 40 mg/L; sensitivity 88.2% [80.4%-96.1%], specificity 73.2% [67.6%-78.9%]) and procalcitonin testing (cutoff 0.5 ng/mL; sensitivity 63.1% [51.0%-75.1%], specificity 82.3% [77.1%-87.5%]). CONCLUSIONS: Double-blinded evaluation confirmed high assay performance in febrile children. Assay was significantly more accurate than CRP, procalcitonin, and routine laboratory parameters. Additional studies are warranted to support its potential to improve antimicrobial treatment decisions.

ENViz: a Cytoscape App for integrated statistical analysis and visualization of sample-matched data with multiple data types.

ENViz (Enrichment Analysis and Visualization) is a Cytoscape app that performs joint enrichment analysis of two types of sample matched datasets in the context of systematic annotations. Such datasets may be gene expression or any other high-throughput data collected in the same set of samples. The enrichment analysis is done in the context of pathway information, gene ontology or any custom annotation of the data. The results of the analysis consist of significant associations between profiled elements of one of the datasets to the annotation terms (e.g. miR-19 was associated to the cell-cycle process in breast cancer samples). The results of the enrichment analysis are visualized as an interactive Cytoscape network.

miRNA target enrichment analysis reveals directly active miRNAs in health and disease.

microRNAs (miRNAs) are short non-coding regulatory RNA molecules. The activity of a miRNA in a biological process can often be reflected in the expression program that characterizes the outcome of the activity. We introduce a computational approach that infers such activity from high-throughput data using a novel statistical methodology, called minimum-mHG (mmHG), that examines mutual enrichment in two ranked lists. Based on this methodology, we provide a user-friendly web application that supports the statistical assessment of miRNA target enrichment analysis (miTEA) in the top of a ranked list of genes or proteins. Using miTEA, we analyze several target prediction tools by examining performance on public miRNA constitutive expression data. We also apply miTEA to analyze several integrative biology data sets, including a novel matched miRNA/mRNA data set covering nine human tissue types. Our novel findings include proposed direct activity of miR-519 in placenta, a direct activity of the oncogenic miR-15 in different healthy tissue types and a direct activity of the poorly characterized miR-768 in both healthy tissue types and cancer cell lines. The miTEA web application is available at http://cbl-gorilla.cs.technion.ac.il/miTEA/.

Silencing of a large microRNA cluster on human chromosome 14q32 in melanoma: biological effects of mir-376a and mir-376c on insulin growth factor 1 receptor.

BACKGROUND: Metastatic melanoma is a devastating disease with limited therapeutic options. MicroRNAs (miRNAs) are small non coding RNA molecules with important roles in post-transcriptional gene expression regulation, whose aberrant expression has been implicated in cancer. RESULTS: We show that the expression of miRNAs from a large cluster on human chromosome 14q32 is significantly down-regulated in melanoma cell lines, benign nevi and melanoma samples relative to normal melanocytes. This miRNA cluster resides within a parentally imprinted chromosomal region known to be important in development and differentiation. In some melanoma cell lines, a chromosomal deletion or loss-of-heterozygosity was observed in the cis-acting regulatory region of this cluster. In several cell lines we were able to re-express two maternally-induced genes and several miRNAs from the cluster with a combination of de-methylating agents and histone de-acetylase inhibitors, suggesting that epigenetic modifications take part in their silencing. Stable over-expression of mir-376a and mir-376c, two miRNAs from this cluster that could be re-expressed following epigenetic manipulation, led to modest growth retardation and to a significant decrease in migration in-vitro. Bioinformatic analysis predicted that both miRNAs could potentially target the 3'UTR of IGF1R. Indeed, stable expression of mir-376a and mir-376c in melanoma cells led to a decrease in IGF1R mRNA and protein, and a luciferase reporter assay indicated that the 3'UTR of IGF1R is a target of both mir-376a and mir-376c. CONCLUSIONS: Our work is the first to show that the large miRNA cluster on chromosome 14q32 is silenced in melanoma. Our results suggest that down-regulation of mir-376a and mir-376c may contribute to IGF1R over-expression and to aberrant negative regulation of this signaling pathway in melanoma, thus promoting tumorigenesis and metastasis.

Ischemia caused by time to freezing induces systematic microRNA and mRNA responses in cancer tissue.

Time to freezing tumor tissue for RNA expression analysis will always vary to some extent. To evaluate the effect of ischemia time, tumor tissue from ten breast cancer patients was collected and aliquots of tissue were snap frozen at different time points after surgery (0, 0.5, 1, 3 and 6 h). Using miRNA and mRNA expression microarrays and statistical analysis, 56 miRNAs and 1788 mRNAs were found to be significantly altered with ischemia time up to six hours. Several of the 56 miRNAs have been reported to play a role in cancer, such as hsa-miR-663 and hsa-miR-125a-3p. Known stress response genes such as GADD45B, JUND and FOSB were among the mRNAs most significantly affected by time to freezing. A novel statistical method for identification of consistently correlated miRNA-mRNA pairs and miRNA-associated biological processes in time course data is presented. Application of this method revealed that several miRNAs, including hsa-miR-1228, hsa-miR-1225-5p and hsa-miR-574-5p, were associated through their correlation to mRNAs to biological processes such as "response to stimulus" and "stress response". These miRNAs also showed enrichment of predicted targets among either their positively or negatively correlated mRNAs. The induced miRNAs may play both direct and indirect roles in biological responses. Caution should be taken when the miRNAs and mRNAs reported to be affected by ischemia time are included in a prognostic or predictive signature.

miRNA-mRNA integrated analysis reveals roles for miRNAs in primary breast tumors.

INTRODUCTION: Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information. METHODS: We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods. RESULTS: We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process. CONCLUSION: This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

A novel translocation breakpoint within the BPTF gene is associated with a pre-malignant phenotype.

Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma--one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis.

Novel rank-based statistical methods reveal microRNAs with differential expression in multiple cancer types.

BACKGROUND: MicroRNAs (miRNAs) regulate target genes at the post-transcriptional level and play important roles in cancer pathogenesis and development. Variation amongst individuals is a significant confounding factor in miRNA (or other) expression studies. The true character of biologically or clinically meaningful differential expression can be obscured by inter-patient variation. In this study we aim to identify miRNAs with consistent differential expression in multiple tumor types using a novel data analysis approach. METHODS: Using microarrays we profiled the expression of more than 700 miRNAs in 28 matched tumor/normal samples from 8 different tumor types (breast, colon, liver, lung, lymphoma, ovary, prostate and testis). This set is unique in putting emphasis on minimizing tissue type and patient related variability using normal and tumor samples from the same patient. We develop scores for comparing miRNA expression in the above matched sample data based on a rigorous characterization of the distribution of order statistics over a discrete state set, including exact p-values. Specifically, we compute a Rank Consistency Score (RCoS) for every miRNA measured in our data. Our methods are also applicable in various other contexts. We compare our methods, as applied to matched samples, to paired t-test and to the Wilcoxon Signed Rank test. RESULTS: We identify consistent (across the cancer types measured) differentially expressed miRNAs. 41 miRNAs are under-expressed in cancer compared to normal, at FDR (False Discovery Rate) of 0.05 and 17 are over-expressed at the same FDR level. Differentially expressed miRNAs include known oncomiRs (e.g miR-96) as well as miRNAs that were not previously universally associated with cancer. Specific examples include miR-133b and miR-486-5p, which are consistently down regulated and mir-629* which is consistently up regulated in cancer, in the context of our cohort. Data is available in GEO. Software is available at: http://bioinfo.cs.technion.ac.il/people/zohar/RCoS/