Development of Three Multiplex Real-Time PCR Assays for Rapid Detection of Invasive Fungal Pathogens
Abstract
Abstract
Invasive fungal infections (IFI) are severe diseases that significantly increase morbidity and mortality in patients with immune deficiency, haematological disorders, and chronic respiratory disease. Detection of fungal pathogens has traditionally relied on methods with suboptimal sensitivity and specificity, such as fungal culture, macroscopic and microscopic, and histopathological techniques. In this context, multiplex real-time polymerase chain reaction (PCR) can be an ideal diagnostic test, as it performs multiple panels that rapidly detect the most causative agents of IFI.
To our knowledge, there is no ongoing attempt to produce a multiplex real-time PCR alternative covering an appropriate diversity of fungal genera and species (nine genera and three species) involved in IFI. This work presents the first phase of the development of three multiplex, real-time PCR assays for the detection of the most causative agents involved in IFI.
Three multiplex assays panels were designed to cover fungi associated with different diseases. Namely, a panel for fungi associated with immunocompromised patients using designed genus-specific primers for Aspergillus, Mucorales, and Cryptococcus, as well as a species-specific primer for Pneumocystis jiroveci; a panel for fungi associated with fungemia using designed genus-specific primers for Candida, Fusarium, and Saccharomyces, as well as a species-specific primer for Candida auris; and a panel for fungi associated with chronic respiratory diseases using designed genus-specific primers for Rasomsonia, Scedosporium, and Exophiala and a species-specific primer for Histoplasma capsulatum.
Primer sets and TaqMan probes were successfully designed from the aligned sequences of each target. The results showed a perfect match for each primers-probe set to their target sequence. Only one primer-probe set of Candida showed a single nucleotide variation. The results demonstrated that all the designed primer-probe sets had an identity coverage of 98% or greater for each target, which suggested a high specificity in identifying the respective genus or species.
The standard curves showed that each multiplex real-time PCR assay successfully detected all fungal target templates (n = 12) with an amplification efficiency between 75% and 104%. Moreover, in the multiplex format, the primer-probe sets displayed an excellent limit of detection (LOD) at 100 copies/reaction for 11 target templates, suggesting high sensitivity. Out of 12 templates tested, only Pneumocystis jirovecii showed an LOD of 1000 copies/reaction.
Reaching to a fully developed, optimised, and validated three multiplex PCR assays was beyond the timescale of this research project, and the late delivery of primers, probes, and Gblocks was a major limitation for completion of the project plan. Technical challenges included the propagation of uncertainty in the dilution series, causing low template concentration and amplification curves that were not equally spaced.
Future work will require extensive optimisations, evaluations, and validations accomplished through rigorous techniques and planning. However, the preliminary results were promising and encouraged the continuation of this initiative, which may produce a rapid, exhaustive, and precise method for IFI diagnosis, something that is urgently needed in the clinical setting.