Abstract
The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 103 CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.Electronic supplementary materialThe online version of this article (doi:10.1007/s11046-014-9756-2) contains supplementary material, which is available to authorized users.
Highlights
Systemic fungal infections with high morbidity and mortality rates in immunocompromised patients are growing
Identification of the causative pathogen is a fundamental step for appropriate treatment of infectious diseases, and early initiation of antifungal therapy is crucial for reducing the mortality rate in infected patients
Among the 319 probes, six universal probes for fungi were designed, so that the array would give a positive signal at universal probe even if fungal species in the tested sample was not listed on the Table 1
Summary
Systemic fungal infections with high morbidity and mortality rates in immunocompromised patients are growing. Despite efforts by many researchers, early and rapid diagnosis of systemic fungal infection remains limited Conventional diagnostic procedures, such as cultivation of fungi from clinical samples, are time-consuming and suffer from low sensitivity. Other methods, such as PCR and serological tests, have been established for rapid and sensitive detection of fungi from clinical samples [2, 3]. These methods are difficult to use to identify a variety of fungal species at a time. To facilitate the diagnosis of fungal infectious disease, we established a rapid and specific DNA microarray system for identifying a variety of causative fungal species simultaneously. Exponential DNA amplification of the target region is proceeded under the isothermal condition
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