Objective
Morphological identification of moulds using traditional culture methods depends largely on the development of characteristic fruiting structures. However, a proportion of moulds do not display such features despite much efforts in attempting to encourage sporulation using 
various laboratory methods. In addition, clinical significance of these non-sporulating moulds is difficult to determine, especially when they were recovered from non-sterile sites and the identity is unknown. The objective of this study is to examine the utility of using molecular sequencing in identification of non-sporulating moulds and their significance when isolated from patients with chronic rhinosinusitis. 

Methods
Five moulds were isolated in 2017 from tissue and pus obtained from the nasal cavity or maxillary sinus in patients with chronic rhinosinusitis. They were isolated were subcultured onto Potato Dextrose Agar at 30oC were observed for sporulation for 3 weeks. They could not be identified due to the lack of distinctive features at the end of incubation period. These moulds were sent for further workup using molecular sequencing of. Briefly, DNA were extracted and PCR was performed using primers targeting ITS, D1/D1 region of the 28s ribosomal subunit and, where necessary, beta-tubulin gene. Amplified products were purified, sequenced and compared with available sequences in NCBI.

Results
All 5 non-sporulating moulds were successfully identified to species level using molecular sequencing. Three of 5 non-sporulating moulds were identified as Aspergillus fumigatus complex using sequences from ITS, D1/D2 region and beta-tubulin gene. None of the three displayed morphological features that were typical of Aspergillus species; white woolly growth with septated hyaline hyphae were observed. These isolates also failed to grow at 42oC, which is another feature typical of A. fumigatus complex. In two of the cases where tissue samples were sent for histological examination, presence of mycetoma was reported, supporting the significance of A. fumigatus complex isolated.

The remaining two moulds were identified as Schizophyllum commune using sequences from ITS and D1/D2 regions. Of note, only one case presented clamp connections, spicules and basidiospores from direct staining of specimen with lactophenol cotton blue wet mount. Basidiocarps were only observed from the culture plates after 4 weeks of exposure to light and dark conditions. Histology showed fungal elements with eosinophilic collections. Otherwise, only hyaline and septated thin hyphae were seen from white woolly moulds for both cases. No pronounced odour was observed from the non-sporulating S. commune cultures.

Conclusion 
Identification of non-sporulating moulds using phenotypic characteristics has always been challenging for most mycology laboratory. In certain clinical scenarios, failure to fully identify the moulds may result in misdiagnosis and could potentially affect management. In this study, molecular sequencing has enabled the laboratory to successfully identify all non-sporulating moulds isolated from patients with chronic sinusitis. Coupled with histological findings, molecular sequencing has assisted the laboratory in determining the significance of such moulds and differentiating them from other fungal colonisers.

Molecular Identification of Non-Sporulating Moulds Isolated from Patients with Fungal Sinusitis