Metabolomic Phenotyping of Gliomas: What Can We Get with Simplified Protocol for Intact Tissue Analysis?

Glioblastoma multiforme is one of the most malignant neoplasms among humans in their third and fourth decades of life, which is evidenced by short patient survival times and rapid tumor-cell proliferation after radiation and chemotherapy. At present, the diagnosis of gliomas and decisions related to therapeutic strategies are based on genetic testing and histological analysis of the tumor, with molecular biomarkers still being sought to complement the diagnostic panel. This work aims to enable the metabolomic characterization of cancer tissue and the discovery of potential biomarkers via high-resolution mass spectrometry coupled to liquid chromatography and a solvent-free sampling protocol that uses a microprobe to extract metabolites directly from intact tumors. The metabolomic analyses were performed independently from genetic and histological testing and at a later time. Despite the small cohort analyzed in this study, the results indicated that the proposed method is able to identify metabolites associated with different malignancy grades of glioma, as well as IDH and 1p19q codeletion mutations. A comparison of the constellation of identified metabolites and the results of standard tests indicated the validity of using the characterization of one comprehensive tumor phenotype as a reflection of all diagnostically meaningful information. Due to its simplicity, the proposed analytical approach was verified as being compatible with a surgical environment and applicable for large-scale studies.

Identification of missed viruses by metagenomic sequencing of clinical respiratory samples from Kenya

Pneumonia remains a major cause of mortality and morbidity. Most molecular diagnoses of viruses rely on polymerase chain reaction (PCR) assays that however can fail due to primer mismatch. We investigated the performance of routine virus diagnostics in Kilifi, Kenya, using random-primed viral next generation sequencing (viral NGS) on respiratory samples which tested negative for the common viral respiratory pathogens by a local standard diagnostic panel. Among 95 hospitalised pneumonia patients and 95 household-cohort individuals, analysis of viral NGS identified at least one respiratory-associated virus in 35 (37%) and 23 (24%) samples, respectively. The majority (66%; 42/64) belonged to the Picornaviridae family. The NGS data analysis identified a number of viruses that were missed by the diagnostic panel (rhinovirus, human metapneumovirus, respiratory syncytial virus and parainfluenza virus), and these failures could be attributed to PCR primer/probe binding site mismatches. Unexpected viruses identified included parvovirus B19, enterovirus D68, coxsackievirus A16 and A24 and rubella virus. The regular application of such viral NGS could help evaluate assay performance, identify molecular causes of missed diagnoses and reveal gaps in the respiratory virus set used for local screening assays. The results can provide actionable information to improve the local pneumonia diagnostics and reveal locally important viral pathogens.

Metabolic Stress Index Including Mitochondrial Biomarker for Noninvasive Diagnosis of Hepatic Steatosis

Background Mitochondrial dysfunction with oxidative stress contributes to nonalcoholic fatty liver disease (NAFLD) progression. We investigated the steatosis predictive efficacy of a novel non-invasive diagnostic panel using metabolic stress biomarkers. Methods Altogether, 343 subjects who underwent magnetic resonance imaging-based liver examinations from a population-based general cohort, and 41 patients enrolled in a biopsy-evaluated NAFLD cohort, participated in the development and validation groups, respectively. Serologic stress biomarkers were quantitated by enzyme-linked immunosorbent assay. Results Multivariate regression showed that waist-to-hip ratio, fibroblast growth factor (FGF) 21, FGF19, adiponectin-to-leptin ratio, insulin, albumin, triglyceride, total-cholesterol, and alanine-aminotransferase were independent predictors of steatosis (rank-ordered by Wald). The area under receiver-operator characteristics curve [AUROC (95%CI)] of the metabolic stress index for steatosis (MSI-S) was 0.886 (0.85‒0.92) and 0.825 (0.69‒0.96) in development and validation groups, respectively. MSI-S had higher diagnostic accuracy (78.1%‒81.1%) than other steatosis indices. MSI-S notably differentiated steatosis severities, while other indices showed less discrimination. Conclusion MSI-S, as a novel non-invasive index, based on mitochondrial stress biomarker FGF21 effectively predicted steatosis. Furthermore, MSI-S may increase the population that could be excluded from further evaluation, reducing unnecessary invasive investigations more effectively than other indices.

Surveillance and correlation of SARS-CoV-2 viral RNA, antigen, virus isolation, and self-reported symptoms in a longitudinal study with daily sampling

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of FDA emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, i.e., the presence of replicable virus. As the number of tests conducted increased, persistent SARS-CoV-2 RNA positivity by RT-PCR in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR (qRT-PCR) assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike (S), nucleocapsid (N), membrane (M), envelope (E), and ORF8. The absolute copy number of each RNA target was determined in longitudinal specimens from a household transmission study. Calculated viral RNA levels over the 14-day follow up period were compared with antigen testing and self-reported symptoms to characterize the clinical and molecular dynamics of infection and infer predictive values of these qRT-PCR assays relative to culture isolation. When detection of sgS RNA was added to the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, we found a qRT-PCR positive result was 98% predictive of a positive culture (negative predictive value was 94%). Our findings suggest sgRNA presence correlates with active infection, may help identify individuals shedding culturable virus, and that similar multiplex assays can be adapted to current and future variants.

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the “bulk” manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3’ end of the N3 probe and the 3’ end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the “bulk” material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

Serum metabolomics reveals dysregulation and diagnostic potential of oxylipins in tumor-induced osteomalacia

Context Excessive production of fibroblast growth factor 23 (FGF23) by tumor was considered as the main pathogenesis in tumor-induced osteomalacia (TIO). Despite its importance to comprehensive understanding of pathogenesis and diagnosis, the regulation of systemic metabolism in TIO remains unclear. Objectives We aimed to systematically characterize the metabolome alteration associated with TIO. Methods By means of liquid chromatography-tandem mass spectrometry (LC-MS) based metabolomics, we analyzed the metabolic profile from 96 serum samples (32 initial diagnosis TIO patients, pairwise samples after tumor resection and 32 matched healthy control subjects). In order to screen and evaluate potential biomarkers, statistical analyses, pathway enrichment and receiver operating characteristic (ROC) were performed. Results Metabolomic profiling revealed distinct alterations between TIO and HC cohort. Differential metabolites were screened and conducted to functional clustering and annotation. Significantly enriched pathway was found involved in arachidonic acid metabolism. A combination of 5 oxylipins, 4-HDoHE, leukotriene B4, 5-HETE, 17-HETE and 9,10,13-TriHOME, demonstrated a high sensitivity and specificity panel for TIO prediction screened by random forest (RF) algorithm (AUC=0.951, 95% confidence interval, CI 0.827-1). Supported vector machine (SVM) model and partial least-squares (PLS) model were conducted to validate the predictive capabilities of the diagnostic panel. Conclusions Metabolite profiling of TIO altered significant compared with HC. A high sensitivity and specificity panel with 5 oxylipins were tested as diagnostic predictor. For the first time, we provide the global profile of metabolomes and identify potential diagnostic biomarkers of TIO. The present work may offer novel insights into the pathogenesis of TIO.

From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners’ patients

Objective To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010–2012). Methods Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. Results The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34–80.06) and norovirus GII (aOR 3.10; CI 1.62–5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43–6.54)), adenovirus non-group F (aOR 1.20; CI 0.75–1.91), bocavirus (aOR 0.85; CI 0.05–13.64), enterovirus (aOR 0.83; CI 0.50–1.37), human parechovirus (aOR 1.61; CI 0.54–4.77) and sapovirus (aOR 1.15; CI 0.67–1.98). Though adenovirus group F (aOR 6.37; CI 0.80–50.92) and norovirus GI (aOR 2.22, CI: 0.79–6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. Conclusions In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Panel of genetic markers for predicting the risk of developing dry eye disease of various etiologies

Introduction. World statistics indicate an increase in patients, including young people, suffering from dry eye disease (DED). Along with exogenous factors, the development of DED depends on a genetic predisposition. Changes in the expression of genes PTPN22, TRIM21, directly or indirectly affecting the T-cell link of immunity, leads to overproduction of cytokines and, as a consequence, damage to the ocular surface.This study aimed to design a diagnostic panel of genetic markers to determine the risk for DED of various etiologies development.Materials and methods. The study included 154 patients with autoimmune diseases with and without established DED. With a diagnosis of rheumatoid arthritis (RA) n = 79 and primary Sjogren’s syndrome (PSS) n = 75. The control group consisted of 100 people without ophthalmic diseases, 31 patients with exogenous DED. In this study, we use melting curve analysis to confirm the results of the association analysis for polymorphic markers in genes.Results. The prognostic value of the predisposing genotypes of the TRIM21 gene of the markers rs915956 and rs7947461 with the risk of DED in the presence of RA (p ≤ 0.001), the marker rs4144331 at the tendency level (p ≤ 0.1) was determined. The risk of developing DES against the background of PSS is associated with the presence of the predisposing genotypes of the TRIM21 genes, the rs4144331 marker, and the PTPN22 rs33996649 marker (p ≤ 0.001). The association of polymorphic markers of the TRIM21 rs7947461 gene and the PTPN22 gene of the rs33996649 marker (p ≤ 0.01) with the risk of developing exogenous DED was established.Conclusions. The predisposing genotypes were identified and the associations of polymorphic markers of the TRIM21, PTPN22 genes were established. A diagnostic panel of genetic markers has been created to predict DED of various etiologies.