2-pentylfuran: biosynthesis by Aspergillus fumigatus in-vitro and investigation of confounders of a breath test in-vivo
A. fumigatus is the principle cause of fatal invasive pulmonary aspergillosis (IPA) in severely immunocompromised hosts and rapid treatment is compromised by the limitations in the current diagnostic tests. 2-pentylfuran (2-PF) is produced in-vitro by A. fumigatus and detectable by gas chromatography with tandem mass spectroscopy (GC-MS/MS) in the breath of patients with chronic lung disease who are colonised with this organism (sensitivity 77%, specificity 78%). The 2-PF breath test has the potential of being a rapid and reliable test for the diagnosis of IPA. The aims of this thesis are: 1) to confirm that 2-PF is produced consistently by A. fumigatus under conditions similar to the microenvironment of lung tissue, and identify a biochemical pathway for 2-PF biosynthesis, 2) to demonstrate that 2-PF is detectable in the breath of patients with IPA, 3) to identify causes of false positive breath tests, and 4) to validate a 2-PF breath test in an animal model. Using GC-MS/MS, A. fumigatus cultured on potato dextrose agar in closed vials produced higher 2-PF levels in the vegetative phase than conidiating phase (P<0.001). Both vegetative and conidiating cultures incubated under 0% and 20% oxygen flow produced 2-PF (P<0.05). To determine whether oxygenases contributed to the synthesis of 2-PF mutant strains were studied. In A. fumigatus, RNAi silenced triple ppo (precocious sexual inducer (psi) producing oxygenase) strain (RNAippoABC) produced 36% less, and the double lipoxygenase (lox) mutant (∆loxAB) produced 43% less 2-PF than the wild-type AF293 strain (p<0.05). In A. nidulans, the triple ppo mutant (∆ppoABC) produced 91% less 2-PF than the wild-type strain (p<0.001). The 2-PF breath test was repeatedly positive in two clinical cases of IPA and became negative with effective antifungal treatment. Subsequently 2-PF was found acontaminant of environmental air and glass breath collection bulbs so that Tedlar® breath collection bags were substituted. The precision and accuracy of this breath analysis method were 90.40% and 94.89%, respectively, and the empirical limit of quantitation for 2-PF was ~1.13 attograms per 2L. Foods containing 2-PF included soy products and vegetable oils. There was no detectable difference in 2-PF levels between morning and afternoon, or fasting and non-fasting breath samples in healthy subjects consuming a 2-PF poor diet. 2-PF was present in breath samples immediately after a soy milk mouth rinse (p<0.001), and intermittently after soy milk ingestion followed by a water mouth rinse. In a pilot study, a small proportion of subjects with pneumonia (consolidation) (5 of 19) or acute exacerbations of COPD (2 of 22) had quantifiable 2-PF in breath samples but this was not significantly different from controls. These findings do not exclude the possibility that 2-PF is produced by an inflammatory response in-vivo. The development of a sheep model of IPA was hindered by the susceptibility of sheep to cyclophosphamide (CPA) toxicity but quantifiable 2-PF was not present in the 115 breath samples of eight normal sheep or in sheep with CPA-induced diffused alveolar damage. To our knowledge, this is the first report providing evidence that 2-PF is an endogenous metabolite of A. fumigatus and is synthesized via oxygenase pathway. A 2-PF breath test may be confounded by consumption of foods containing 2-PF but lung inflammation as a cause of elevations in 2-PF in breath has not been excluded. Development of a breath collection system that can eliminate environmental contamination with 2-PF is a key improvement necessary to optimise the performance of this breath test.
Advisor: Chambers, Steve; Scott-Thomas, Amy; Weld, Richard; Charles, Chris
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology
Publisher: University of Otago
Research Type: Thesis