The importance of appropriate processing and direct microscopic examination for the timely diagnosis and management of invasive infections caused by filamentous fungi

The gold standard for diagnosis of invasive fungal infections caused by filamentous fungi remains the visualization of fungal elements in fluids, and biopsy/tissue collected from a normally sterile body site. Parallel recovery of viable fungus from the sample subsequently permits antifungal susceptibility testing of the individual isolate. Central to both processes is the appropriate processing of tissue specimens to avoid damaging fungal elements and optimize viable organism recovery. Historically, mycologists have proposed that homogenization (grinding or bead-beating) of tissue should be avoided in cases of suspected fungal infection as it likely damages hyphae, instead preferring to chop tissue into small portions (dicing) for direct microscopic examination and culture. Here, we have compared the two processes directly on material from clinical patient cases of mucoromycosis and invasive aspergillosis. Representative portions of fresh biopsy samples were processed in parallel either by chopping (dicing) in the mycology reference laboratory or by bead-beating in the adjoining general microbiology laboratory. Aliquots of the samples were then cultured under identical conditions and subjected to direct microscopic examination. The results demonstrated that tissue homogenization significantly reduced (i) organism recovery rates in cases of both mucoromycosis and invasive aspergillosis and (ii) the number of fungal elements detectable upon direct microscopic examination. To our knowledge, this is the first study to directly compare these alternative processing methods and despite only employing a limited number of samples the data presented here, provide support for the perceived mycological wisdom that homogenization of tissue samples should be avoided when filamentous fungal infections are suspected.