Masahiro Kawaguchi

Simple PCR-Based DNA Microarray System To Identify Human Pathogenic Fungi in Skin

ABSTRACT Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

Bioaugmentation of TCE-Contaminated Soil with Inducer-Free Microbes

Environmental pollution is a world-wide problem since these pollutants cause serious disease not only for human beings but also for other plants and animals. Trichlo-roethylene (TCE) is a typical pollutant in soil and groundwater, and its concentration is strictly regulated since many people are using groundwater as drinking water. TCE is very stable in the environment, and soil vapor extraction (SVE) is frequently used for the removal of TCE. However, the removal efficiency in SVE decreases with low concentrations of TCE. Charcoal adsorption and photocatalytic degradation are other approaches to detoxify soil and groundwater, but in situ treatment is needed for cost-effective remediation. Bioremediation for TCE-contaminated soil is an attractive approach for in situ treatment, and many microbes which can decompose TCE have been reported.