fumigatus and Aspergillus terreus, eight sputum samples were collected between 22 November 2007 to 16 February 2010, and no Scedosporium was detected by culture. Likewise, for patient 14 (sample 26), 37 samples were collected between 4 July 2007 and 29 May 2009. Mycological analysis gave strictly the same results for almost all these samples,
with an exclusive growth of Candida albicans. Mycological analysis of 21 sputum samples and three broncho-alveolar fluids collected between 3 January 2007 and 29 May 2009 from patient 10 (sample 21 in this study) revealed a chronic colonisation of the airways by C. albicans, sometimes associated with Candida dubliniensis or A. fumigatus, but Scedosporium species were never detected. In addition, www.selleckchem.com/products/azd9291.html two consecutive samples from seven CF patients were analysed. For one of these patients (patient 24), PCR-RLB yielded identical results for the two samples, with a positive signal with
the P. apiosperma-specific probe, and mycological analysis of the second sample (sample 93 collected on 15/12/2008) permitted the recovery of this fungus. This suggests a lack of sensitivity of culturing for the first sample (sample 150 collected on 16/10/2007). Discrepant results between the two successive samples were obtained for the six other patients, with a PCR-negative signal for one of the samples in three patients (patients 2, 21 and patient 29) or with positive signals with different species-specific probes between the two samples in the other three cases (patients 19, 22 and 25). Several molecular methods Selleckchem Small molecule library targeting the internal transcribed spacer (ITS) region have been described, but with insufficient resolution to differentiate all clinical species of the P. apiosperma/P. boydii complex. The fragment BT2 of the β-tubulin gene provided more information than
ITS as a target for the identification of Scedosporium species.17,22 We chose the RLB format, given the advantages of low cost and the simultaneous analysis of multiple specimens against multiple probes. The assay analyses up to 43 specimens in a single run and the membrane can be reused up to 20 times without loss of signal. The PCR-RLB assay was able to identify Clostridium perfringens alpha toxin five species except S. dehoogii. The latter species has not been proven to have clinical relevance, and thus PCR-RLB is sufficient for use in clinical diagnostics. Although a single amplification PCR round was sufficient for DNA extracted from cultures, a second PCR format maximises detection limits. Two PCR reactions might carry a higher risk of cross contamination due to the amplification of contamination from e.g., Scedosporium DNA contaminated tubes, sampling equipment (bronchoscope), PCR water or reagents; however, it made it possible to detect DNA extracted directly from clinical specimens. Fifty-nine sputum samples were analysed using methods dedicated to the selective isolation of Scedosporium species; five samples (8.5%) proved to be positive.