Development of a novel method for detection ofClostridium difficileusingHS‐SPME‐GC‐MS

Abstract

Aims: A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culturenegative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). Methods and Results:The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl. difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl. difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl. difficile within 18 h with a sensitivity and specificity of 83Á1 and 100%, respectively. Conclusions: It is recommended that this new approach could be used alongside conventional methods for Cl. difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. Significance and Impact of the Study: The ability to identify Cl. difficilepositive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.

Application of Fecal Volatile Organic Compound Analysis in Clinical Practice: Current State and Future Perspectives

“…Addition of this substrate resulted in a high sensitivity and specificity (Table 2). In absence of 2-fluoro-4-methylphenol, discrimination was not possible between C. difficile positive and negative samples [20]. In addition, three other studies demonstrated that, by means of GC-MS, C. difficile positive fecal samples could be distinguished from controls based on their VOC profiles.…”

“…However, results of culture are commonly obtained after at least 48 h, while rapid diagnosis is necessary to apply appropriate therapeutic interventions, such as prescription of targeted antibiotics and isolation of patients [18]. Most studies on application of fecal VOC analysis in infectious disease have focused on Clostridium difficile [19][20][21][22][23]. Other studies include detection of Campylobacter jejuni, cholera, giardiasis and rotavirus [21,[23][24][25][26].…”

SPME as a promising tool in translational medicine and drug discovery: From bench to bedside

“…So far, most of the reports involving SPME studies of bacteria have considered the diagnostic value of the method e.g. identification/confirmation of given bacteria in biological materials [68][69][70]. Recently, studies of E. coli employing a high throughput thin film SPME system focused on assessment of metabolome changes in bacteria influenced by the natural antibacterial agent cinnamaldehyde [71].…”

Detection of exogenous VOCs as a novel in vitro diagnostic technique for the detection of pathogenic bacteria

“…3-fluoro-4-hydroxyphenylacetic acid, resulted in the production of the VOC 2-fluoro-4-methylphenol, thus facilitating differentiation of the production of p-cresol by other species. In a blind study of 100 stool samples, 100 % of C. difficile positive stool samples could be correctly identified by this methodology [27].…”

“…Another study, by the same group, involved the development of a novel VOC assay for the detection of Clostridium difficile in stool samples by inclusion of VOC liberating enzyme substrates and selective antibiotics in a liquid growth medium [27], in conjunction with a sample pre-treatment step to eliminate competing microflora. By subjecting the stool samples to alcohol (ethanol) shock the large number of microflora present in a stool sample were killed leaving only spores of C. difficile.…”