Ismadi, Yasmin Khairani Muhammad
(2024)
Development of a multiplex real-time pcr assay for detection of fungal pathogens in invasive mycoses.
PhD thesis, Universiti Sains Malaysia.
Abstract
Invasive mycoses pose significant challenges to immunocompromised
individuals, often resulting in severe morbidity and mortality. Current diagnostic
methods are hindered by delays in identification and initiation of appropriate
antifungal therapy, stemming from the limitations of conventional techniques. This
study undertakes a thorough investigation into fungal DNA extraction protocols and
the development of a multiplex real-time PCR assay for rapid identification of
invasive fungal pathogens. This research aims to compare fungal DNA extraction
methods and develop a multiplex real-time PCR assay capable of simultaneously
identifying multiple fungal species commonly associated with invasive mycoses.
Species-specific primer design and assay optimization target Aspergillus fumigatus,
Aspergillus terreus, Candida albicans, and Candida glabrata. Results reveal certain
extraction methods exhibiting superior sensitivity and time efficiency, with
implications for clinical use. The developed multiplex real-time PCR assay
demonstrates promising accuracy in identifying fungal pathogens. Analytical
specificity testing, which involved detecting 65 isolates including various fungal
strains alongside other reference strains, yielded a 100% identification rate for target
organisms. Performance evaluation of the multiplex PCR assay using spiked blood
samples showed high specificity and sensitivity, both at 100%, with positive
predictive value (PPV) and negative predictive value (NPV) also at 100%. Overall,
this research contributes to advancing laboratory diagnosis and management of invasive fungal infections. By addressing challenges in fungal DNA extraction and
creating a high-performance multiplex PCR assay, this study lays the groundwork
for more efficient and reliable diagnostic approaches, thereby enhancing patient care
and treatment outcomes. Further validation studies on real clinical samples are
recommended to confirm the applicability of these methodologies in the current
clinical settings.
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