The diagnosis and treatment of invasive mould infections in haematology patients
Histological appearances of many moulds overlap and there is a need for a method to allow identification in tissue specimens. Two methods for extracting fungal DNA from wax tissue sections, based on the TaKaRa DEXPAT TM kit and QIAampÒ DNA Mini Kit, were optimised and compared. DNA was amplified by PCR using pan-fungal primers, and detected by Southern blot hybridisation with a probe specific for Aspergillus fumigatus and A. flavus. The TaKaRa DEXPAT TM kit based method, with additional steps using lyticase and ethanol precipitation, was superior and was used to test sequential wax tissue specimens from 56 patients with IFI. PCR products not hybridising with the probe were identified by sequencing. The species was confirmed in all tissue culture positive cases (23 A. fumigatus or A. flavus, one Chaetomium globosum and one Scedosporium apiospermum). Of culture negative cases, a diagnosis of A. fumigatus or A. flavus was established in 25 and emerging moulds in two (one probable Alternaria species and one unidentified). Overall, emerging moulds were identified in 4 cases (7%) with a trend toward a temporal increase in these infections. This method provides a valuable diagnostic tool for both patient management and future antifungal and epidemiological trials. Reasons for therapeutic failure in IFI are unclear. Amphoterican B susceptibility of isolates cultured from tissue biopsies from patients who had received a median of 12 days therapy, were tested using a method based on the NCCLS M38-A broth microdilution method. The difficulty in treating IFI does not appear to be due to susceptibility of the isolates, but may be due to poor penetration of antifungal agents into infected tissue.