Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Aspergillus monitoring project in a large educational hospital using molecular assay

Background: It is important to find reliable and accessible methods for the diagnosis and identification of fungal species causing hospital acquired infections. Our main objective was using a rapid and accessible molecular method for the monitoring of Aspergillus infections and identification of causing agents in the level of species. Material and Methods: The study subjects were primarily clinical specimens collected from suspected HAI patients with clinical symptoms after hospitalization. Also some environmental specimens were collected from air and instruments of health care facilities for the investigation of Aspergillus sources in a university hospital of UMSU, Urmia. All specimens were transported to Medical Mycology Center for the detection and identification of Aspergillus species using morphological methods. Also molecular method, PCR-RFLP using single restriction enzyme as a rapid and available method was performed to investigate environmental sources of Aspergillus infections. Results: Total of 110 clinical fungal isolates included Candida and Aspergillus species and some other opportunistic fungi. Among the clinical Aspergillus findings, Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.6%) were the most frequent species respectively and also Aspergillus niger (43.7%), Aspergillus flavus (41.8%), Aspergillus fumigatus (14.7%) were isolated as the most frequent species from environmental sources. Conclusion: Because of accessibility, speed and high sensitivity of diagnosis, the PCR-RFLP was very useful for the identification of medically important Aspergillus species and epidemiological approaches