Clostridium difficile is a Gram-positive anaerobe that can cause diarrhea, pseudomembranous colitis and, sometimes, death. Commonly found in hospital inpatients or nursing-home residents, C. difficile is a major, community-acquired infection seen worldwide. Current detection methods include polymerase chain reaction (PCR), enzyme immunoassays (EIAs), cultures and cytotoxicity assays. The current gold standard is a type of cytotoxicity assay where the addition of fecal filtrate to a human cell-line culture allows the researcher to observe cell-rounding (a sign of cell death), which indicates if the fecal sample contains C. difficile toxins.
Healthcare professionals have commonly observed the unique odor of C. difficile, as compared to other gastrointestinal distresses.1 This smell is not actually the bacteria itself but a volatile organic compound (VOC) produced by C. difficile. VOCs can be detected using gas chromatography–mass spectrometry (GC-MS); Tait et al. highlight a novel method for detecting C. difficile using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS).2
Previous research papers showed a number of VOCs associated with C. difficile infection but, due to a lack of specificity, existing methods of detection were ineffective. Tait et al. discovered that adding the enzyme substrate 3-fluoro-4-hydroxyphenylacetic acid (FHPAA) to the HS-SPME-GC-MS workflow increased the specificity of the test because of C. difficile’s unique ability to use the enzyme to release the VOC 2-fluoro-4-methylphenol. To reduce the viability of non-C. difficile bacteria, the group used alcohol shock as an initial step prior to processing. By preparing 100 fecal samples (both culture positive and culture negative) according to the adjusted protocol, the group was able to successfully utilize FHPAA along with HS-SPME-GC-MS (PolarisQ ion trap mass spectrometer, Thermo Scientific) to show an improved specificity as compared to previous research studies.
Additionally, after multivariate analysis of data compiled from C. difficile culture positive and negative fecals, the researchers found that sensitivity could be further improved when they measured 2-fluoro-4-methylphenol in combination with either isocaproic acid or p-cresol (two previously identified VOCs associated with C. difficile). Next, the group observed whether the new method could discriminate between toxin positive or toxin negative samples (a sign of more serious infection). Although toxigenic samples had a higher average concentration of 2-fluoro-4-methylphenol, no significant difference existed between the two groups.
The future detection of VOCs as a method of clinical diagnosis of C. difficile would allow doctors to non-invasively identify infected patients. This could prevent hospital or nursing home outbreaks and could also help facilities determine effective methods for containing such a community-acquired disease. As technology advances, VOC detection may evolve to a handheld device designed to alert healthcare workers to contaminated surfaces such as doorknobs or bedrails, thus saving resources—and lives.
1. Garner, C.E., et al. (2007) “Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease,” The FASEB Journal, 21(8) (pp. 1675–88), doi: 10.1096/fj.06-6927com.
2. Tait, E., et al. (2014) “Development of a novel method for detection of Clostridium difficile using HS-SPME-GC-MS,” Journal of Applied Microbiology, 116(4) (pp. 1010–19), doi: 10.1111/jam.12418.
Post Author: Rebecca Easley.