Eleven fungal strains were received from the clinical microbiology laboratory collection of Queen Mary Hospital and Pamela Youde Nethersole Eastern Hospital in Hong Kong from 2010-2011. The collection comprised of ten ascomycetes and one zygomycete. They were identified down to the genus level based on the morphological criteria. Internal transcribed spacer (ITS), beta-tubulin, actin and 28S gene sequencing were used for genotypic characterization. The ITS sequences of four of the strains demonstrate <3%-base difference to a single fungal species. They were species of the genus, Acremonium, Aspergillus, Cladophialophora and Ochroconis respectively. Five strains belonging to the genus, Trichophyton, Monascus, Mucor, Arthrinium and Acremonium could not be identified to the species level due to low interspecies heterogeneity. The tubulin gene was used for two of the strains. The tubulin sequence of a strain of Phaeoacremonium was identified to the species level with 0%-base difference. The ITS, partial beta-actin and 28S rDNA genes were sequenced for a strain of Exophiala. They showed a distinct cluster, mostly closely related to, but distinct from, Exophiala xenobiotica, Exophiala jeanselmei and Exophiala oligosperma. The genotypic characteristics suggest the strain to be a novel species of Exophiala. More genotypic and phenotypic characterization are required to described this strain of Exophiala. / published_or_final_version / Microbiology / Master / Master of Research in Medicine

In clinical microbiology laboratories, the conventional method for identification of pathogenic fungi is based on fungal culture and observation of fungal phenotypic characters. However, it is time-consuming, subjective and unreliable due to the long incubation period and variations in fungal colony morphology. Thus, there is a need for a rapid, objective and accurate identification of pathogenic fungal isolates. ITS regions are most commonly used targets for molecular identification of fungal pathogens because of the optimal inter- and intra-species variations and large copies in fungal genome. In this study, twenty-two clinical fungal isolates were identified using the phenotypic method and ITS sequencing. The results showed that there were only thirteen isolates identified to species level by phenotypic method, while others were only differentiated in genus level. Due to the poor differentiation based on the conventional phenotypic approach, misidentification of fungal pathogens occasionally occurred. However, ITS sequencing successfully achieved accurate species-level identification of all fungal isolates. The results were demonstrated in phylogenetic trees with high bootstrap support. In conclusion, ITS sequencing is a rapid and reliable for the identification of pathogenic fungal isolates. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Identification of pathogenic fungi isolated from clinical specimens in clinical microbiology laboratories is primarily based on observing fungal phenotypic structures under the microscope and performing biochemical tests for fungal cultures. This conventional method is very time-consuming and labor-dependent. It usually requires several weeks for the fungi to grow sufficiently on culture media, and the identification processes on fungal phenotypic structure rely very much on experienced staff. Therefore, a more accurate and rapid method for pathogenic fungal identification is necessary for clinical laboratories to get abreast of modern development. Gene sequencing and phylogenetic analysis targeting the internal transcribed spacer (ITS) region in the fungal genomes are the most commonly used molecular methods for fungal identification. Because of the optimal inter and intra-species variation property of the ITS region, it can act as the DNA barcode to identify fungi to the species level. In this study, 33 clinical fungal isolates were identified by both phenotypic method and ITS sequencing. The results showed that 23 isolates were successfully identified to thespecies level by both phenotypic and molecular methods. Moreover, five isolates were only identified to the genus level by phenotypic method, but they could be successfully identified to the species level by ITS sequencing. However, five isolates have not been differentiated because there were mismatched results from phenotypic and sequencing methods. It may be due to the limitation of sequencing method on some fungal species. Building up a more comprehensive database or setting up a standard platform to guide the molecular process may help improve the performance of molecular method. To conclude, molecular method is a rapid and reliable way for fungal identification because ITS region acts as the DNA barcode for pathogenic fungi. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences