Molecular indentification, antifungal susceptibility and virulence factors of Candida glabrata isolated from Malaysian patients / Azadeh Lotfalikhani

Azadeh, Lotfalikhani (2016) Molecular indentification, antifungal susceptibility and virulence factors of Candida glabrata isolated from Malaysian patients / Azadeh Lotfalikhani. Masters thesis, Universiti of Malaya.

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Abstract

There has been an increase in the incidence of candidiasis among hospitalized patients in many healthcare institutions in recent years. Among the Candida species, Candida glabrata has been described as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. Accurate identification of Candida spp. from clinical specimens and antifungal susceptibility testing are important to facilitate optimal antifungal therapy and patient management. Molecular techniques are excellent tools for identification of new species and strain typing of yeasts. In vitro assessment of the virulence properties of a pathogen, for example, the ability of an organism to produce hydrolytic enzymes and biofilm, may provide some insights on the pathogenesis of the infection it causes. This study aims to determine the identification and the characteristics of C. glabrata isolated from various clinical specimens in the Diagnostic Microbiology Laboratory, University of Malaya Medical Centre, from 2008-2012. A total of 185 C. glabrata isolates were identified in this study based on sugar assimilation tests. Majority of the isolates were obtained from vaginal swabs (n=142, 76.8 %), followed by blood samples (n=11, 5.9%), urine (n=14, 7.6%), respiratory secretions (n=4, 2.2%), and others (n=14, 7.6%). The yeast DNA was extracted from each isolate and subjected to testing using a singleplex PCR assay for discrimination between C. glabrata, C. bracarensis and C. nivariensis, as reported by Enache-Angoulvant et al. (2011). Four isolates obtained from a blood specimen and three vaginal swabs of two patients were identified as C. nivariensis, a rare and emerging yeast pathogen. The identity of the isolates was confirmed based on sequence analysis of the yeast internal transcribed spacer (ITS) region and D1D2 domain. The low prevalence (1.1 %) of C. nivariensis isolates is in agreement with several studies reported in other geographical regions. Based on the ITS sequence data (ranging from 619 to 624 bp) obtained from 35 randomly selected C. glabrata isolates, a total of 9 iv sequence types were identified. All C. glabrata and C. nivariensis isolates were susceptible to amphotericin B, caspofungin, fluconazole, and voriconazole. Randomly amplified polymorphic DNA (RAPD) analysis of C. glabrata isolates using two single primers [M13 and (GTG)5] identified six and five genetic clusters, respectively. C. nivariensis isolates formed a unique genetic cluster as compared to C. glabrata isolates. A total of 15 RAPD types of C. glabrata were identified in this study. Weak phospholipase activity and biofilm production was demonstrated by C. glabrata isolates investigated in this study. This study reports for the first time the identification of C. nivariensis in a Malaysian hospital. The yeast was misidentified as C. glabrata as it shared many phenotypic characteristics with C. glabrata. Despite of the genetic diversity of C. glabrata (as exhibited by sequence determination of the ITS region and RAPD analysis), all isolates are susceptible to the antifungal drugs tested in this study. Although C. glabrata is probably not as virulent as compared to C. albicans due to its low production of phospholipase and biofilm production, however; further work is necessary to validate the finding.

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