17 In general, duplex PCR amplification of BT2 yielded clear MK-2206 price Scedosporium-specific bands. Although the closely related species P. desertorum was also amplified, it gave a signal exclusively with the group-specific probe PS_P on the blot. This assay was found positive in five of six

clinically relevant Scedosporium species. Non-specific signals were found for S. dehoogii strains when probes of P. apiosperma, P. boydii, and P. minutispora were applied. No other cross-reactions with non-target Scedosporium species or other clinically relevant fungi were observed. The detection limit of the PCR-RLB method was found to be 50 cells μl−1 or 0.2 pg genomic DNA. Fifty-nine sputum samples, comprising five culture-positive samples and 54 culture-negative samples, were analysed by PCR-RLB hybridisation assay (Table 1). Twenty-two of the samples proved to be negative by PCR-RLB. The PCR-RLB hybridisation assay permitted the detection of members of the P. apiosperma/P. boydii complex in 32 of 52 patients (61.5%). Pseudallescheria

apiosperma was detected in 20 samples, while P. boydii and S. aurantiacum were detected in 17 and eight samples, respectively. Only two samples were found positive for S. prolificans and P. minutispora, respectively. RG7204 concentration Eight samples contained two distinct species or three species simultaneously. Figure 1 shows a typical result of PCR-RLB for some sputum samples and for a number of Scedosporium reference strains. Four of the five Scedosporium culture-positive samples proved also to be positive with PCR-RLB hybridisation assay. All isolates of the P. boydii/P. apiosperma complex were identified morphologically,

except one strain Forskolin in vitro recovered from sample 10 which was identified as S. aurantiacum and confirmed by sequencing the ITS1-ITS2 (99% identity with NCBI sequence AJ889599 from S. aurantiacum strain IHEM 144-458) and BT2 region (100% identity with the NCBI sequence AJ888441 from S. aurantiacum strain IHEM 15-458); this last sample gave a positive signal by PCR-RLB hybridisation exclusively with the S. aurantiacum-specific probe. Considering all analysed samples, PCR-RLB yielded more positive results than culturing (47 vs. 5, respectively). Among the 54 Pseudallescheria/Scedosporium culture-negative samples analysed, 21 were also found negative by PCR-RLB. Twenty-six DNA extracts gave a positive signal with one species-specific probe, while six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. Antifungal treatment (mostly with the azoles itraconazole or voriconazole) during the months preceding the sampling took place in seven of the patients. However, for the remaining Pseudallescheria/Scedosporium culture-negative samples producing discrepant results (26 samples), the patients did not receive any antifungal treatment preceding the sampling date and Scedosporium species were never detected by culture in previous or later sputum samples.