Michael Cauchi

Electronic-Nose Technology Using Sputum Samples in Diagnosis of Patients with Tuberculosis

ABSTRACT We investigated the potential of two different electronic noses (EN; code named “Rob” and “Walter”) to differentiate between sputum headspace samples from tuberculosis (TB) patients and non-TB patients. Only samples from Ziehl-Neelsen stain (ZN)- and Mycobacterium tuberculosis culture-positive (TBPOS) sputum samples and ZN- and culture-negative (TBNEG) samples were used for headspace analysis; with EN Rob, we used 284 samples from TB suspects (56 TBPOS and 228 TBNEG samples), and with EN Walter, we used 323 samples from TB suspects (80 TBPOS and 243 TBNEG samples). The best results were obtained using advanced data extraction and linear discriminant function analysis, resulting in a sensitivity of 68%, a specificity of 69%, and an accuracy of 69% for EN Rob; for EN Walter, the results were 75%, 67%, and 69%, respectively. Further research is still required to improve the sensitivity and specificity by choosing more selective sensors and type of sampling technique.

Analysis of volatile organic compounds of bacterial origin in chronic gastrointestinal diseases

Background: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. Methods: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn’s disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography–mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. Results: Crohns disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. Conclusions: The abundance of a number of VOCs in feces differs markedly between Crohns disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

Diversity and distribution of sulphate‐reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohns disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.

Assessment of Temporal Dose-Toxicity Relationship of Fumed Silica Nanoparticle in Human Lung A549 Cells by Conventional Cytotoxicity and 1H-NMR-Based Extracellular Metabonomic Assays

As nanoparticles could form aggregates in biological systems, the dynamics of their dispersity drives the temporal effect of nanoparticles in vitro. To test this hypothesis, the fumed silica nanoparticles (SiNPs) that have primary sizes of 7-14 nm and form aggregates in culture medium were selected for toxicity study in human lung A549 cells. The dispersity of SiNPs was analyzed by dynamic light scattering and transmission of electron microscopy. Cytotoxicity assays including mitochondrial activity, intracellular level of reactive oxygen species (ROS), and membrane damage together with the ¹H-NMR-based extracellular metabonomic assay were conducted to determine the temporal dose-effect relationship of SiNPs. In cell culture medium, SiNPs dispersed well initially at 25-100 μg/ml; however, they sedimented rapidly in a concentration-dependent manner. SiNPs caused a dose-dependent increase of intracellular ROS and cell membrane damage at 4 h and a loss of cell viability after 48 h. SiNPs also induced an elevation of extracellular glucose, lactate, phenylalanine, histidine, and tyrosine levels in a time- and concentration-dependent manner. The dose-effect patterns at 4 h were different from that at 12 and 24 h as assessed by both cytotoxicity and metabonomic assays. Both fitted better with polynomial regression than linear regression, implying multimode action of SiNPs at different concentrations. The early NP-cell interaction and the late sedimentation could be attributable to the temporal effects of SiNPs. The extracellular ¹H-NMR-based metabonomics demonstrated a potential as a robust nondestructive tool for monitoring the temporal effect of NPs that tend to aggregate in nature.

Use of the Analysis of the Volatile Faecal Metabolome in Screening for Colorectal Cancer

Diagnosis of colorectal cancer is an invasive and expensive colonoscopy, which is usually carried out after a positive screening test. Unfortunately, existing screening tests lack specificity and sensitivity, hence many unnecessary colonoscopies are performed. Here we report on a potential new screening test for colorectal cancer based on the analysis of volatile organic compounds (VOCs) in the headspace of faecal samples. Faecal samples were obtained from subjects who had a positive faecal occult blood sample (FOBT). Subjects subsequently had colonoscopies performed to classify them into low risk (non-cancer) and high risk (colorectal cancer) groups. Volatile organic compounds were analysed by selected ion flow tube mass spectrometry (SIFT-MS) and then data were analysed using both univariate and multivariate statistical methods. Ions most likely from hydrogen sulphide, dimethyl sulphide and dimethyl disulphide are statistically significantly higher in samples from high risk rather than low risk subjects. Results using multivariate methods show that the test gives a correct classification of 75% with 78% specificity and 72% sensitivity on FOBT positive samples, offering a potentially effective alternative to FOBT.

Discerning Data Analysis Methods to Clarify Agonistic/Antagonistic Actions on the Ion Flux Assay of Ligand-Gated Ionotropic Glutamate Receptor on Engineered Post-Synapse Model Cells

Cell-based experiments provide the efficacy of chemicals through the biological function. The authors have described post-synapse model cell-based assay based on qualified analysis for neural drug discoveries. However, in general, cell-based assays often include data fluctuation. This may be a barrier preventing the performance for various practical purposes. In this study, we tried discerning data analysis for clarify the chemical action to the ionotoropic glutamate receptor (GluR), whereby an ion-flux assay of post-synapse model cells is performed and are analyzed based on a chemometrics approach. The dynamic behavior of the GluR of post-synapse model cell was assayed with multivariate data analysis methods namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). By using HCA, we can identify and remove the non-responding samples. By using PCA, the effect of chemicals on the dynamic behavior of ion flux through GluR can be recognized clearly; as either agonist or antagonist. As shown in the results, the GluR-based assay by post-synapse model cell with data analysis methods provide a sodium influx profile which is derived by an agonists or antagonists application. By employing the data analysis methods, PCA and HCA, it is possible to develop a smart cellular biosensing system for qualified analysis.

Enteral feeding reduces metabolic activity of the intestinal microbiome in Crohn's disease: an observational study.

Background/Objectives:Enteral feeding will induce remission in as many as 80–90% of compliant patients with active Crohn’s disease (CD), but its method of action remains uncertain. This study was designed to examine its effects on the colonic microbiome.Methods/Subjects:Healthy volunteers and patients with CD followed a regimen confined to enteral feeds alone for 1 or 2 weeks, respectively. Chemicals excreted on breath or in faeces were characterised at the start and at the end of the feeding period by gas chromatography/mass spectrometry.Results:One week of feeding in healthy volunteers caused significant changes in stool colour and deterioration in breath odour, together with increased excretion of phenol and indoles on the breath. Feeding for 2 weeks in patients with CD produced significant improvements in symptoms and a decrease in the concentration of C-reactive protein. The faecal concentrations of microbial products, including short-chain fatty acids (SCFAs), and potentially toxic substances, including 1-propanol, 1-butanol and the methyl and ethyl esters of SCFAs, showed significant falls.Conclusions:A significant change occurs in the production of microbial metabolites after enteral feeding in both healthy volunteers and patients with CD. Many of those detected in CD are toxic and may feasibly lead to the immunological attack on the gut microbiota, which is characteristic of inflammatory bowel disease. The reduction in the production of such metabolites after enteral feeding may be the reason for its effectiveness in CD.

Optical Assay for Biotechnology and Clinical Diagnosis

In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

MMpred: functional miRNA – mRNA interaction analyses by miRNA expression prediction

BackgroundMicroRNA (miRNA) directed gene repression is an important mechanism of posttranscriptional regulation. Comprehensive analyses of how microRNA influence biological processes requires paired miRNA-mRNA expression datasets. However, a review of both GEO and ArrayExpress repositories revealed few such datasets, which was in stark contrast to the large number of messenger RNA (mRNA) only datasets. It is of interest that numerous primary miRNAs (precursors of microRNA) are known to be co-expressed with coding genes (host genes).ResultsWe developed a miRNA-mRNA interaction analyses pipeline. The proposed solution is based on two miRNA expression prediction methods – a scaling function and a linear model. Additionally, miRNA-mRNA anti-correlation analyses are used to determine the most probable miRNA gene targets (i.e. the differentially expressed genes under the influence of up- or down-regulated microRNA). Both the consistency and accuracy of the prediction method is ensured by the application of stringent statistical methods. Finally, the predicted targets are subjected to functional enrichment analyses including GO, KEGG and DO, to better understand the predicted interactions.ConclusionsThe MMpred pipeline requires only mRNA expression data as input and is independent of third party miRNA target prediction methods. The method passed extensive numerical validation based on the binding energy between the mature miRNA and 3’ UTR region of the target gene. We report that MMpred is capable of generating results similar to that obtained using paired datasets. For the reported test cases we generated consistent output and predicted biological relationships that will help formulate further testable hypotheses.

Comparison of GC-MS, HPLC-MS and SIFT-MS in conjunction with multivariate classification for the diagnosis of Crohn's disease in urine

The developed world has seen an alarming increase in the incidence of gastrointestinal diseases, among the most common of which is Crohns disease (CD) in the young. The current “gold standard” techniques for diagnosis are often costly, time consuming, inefficient, invasive, and offer poor sensitivities and specificities. This paper compares the performances of three hyphenated instrumental techniques that have been suggested as rapid methods for the non-invasive diagnosis of CD from urine. These techniques are gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and selected ion flow tube mass spectrometry (SIFT-MS). Each of these techniques is followed by multivariate classification to provide a diagnosis based on the acquired data. The most promising results for potentially diagnosing CD was via HPLC-MS. An overall classification accuracy of 73% (74% specificity; 73% sensitivity) was achieved for differentiating CD from healthy controls, statistically significant at 95% confidence.