Genetic identification and detection of human pathogenic Rhizopus species, a major mucormycosis agent, by multiplex PCR based on internal transcribed spacer region of rRNA gene.
BACKGROUND
Mucormycosis is an invasive opportunistic infection caused by fungi belonging to the order Mucorales. Due to the lack of laboratory tests, the diagnosis of mucormycosis is notoriously difficult. Added with its rapid progression as well as the debilitated state of the patients who contract the disease, mortality is extremely high.


OBJECTIVE
The goal of this study was to genetically identify human pathogenic Rhizopus species, a major mucormycosis agent, by the internal transcribed spacer (ITS) region of rRNA gene.


METHODS
Primers were designed to identify five Rhizopus species known to cause human disease by multiplex PCR. PCR was done not only with test strains and clinical isolates, but also with clinical samples from cutaneous mucormycosis patients. Sporangiospore morphology was observed by scanning electron microscopy to confirm the correlation of phenotypic and genotypic features.


RESULTS
Multiplex PCR identified five Rhizopus species including Rhizopus oryzae, where R. azygosporus could only be distinguished from R. microsporus by certain polymorphisms that were present in its sequence. When this multiplex PCR was applied to clinical samples from three mucormycosis patients (paraffin sections from all and sera from one patient), Rhizopus DNA corresponding to the isolated pathogens were specifically detected.


CONCLUSIONS
While fungal DNA detection from clinical samples is a rigorously studied area, this is the first report to genetically identify and detect Rhizopus species from human mucormycosis specimens. This may expand the possibility of this multiplex PCR system not only to identify isolated fungi, but also as a screening method for visceral mucormycosis.
Department of Dermatology, Keio University, School of Medicine, Japan. nagao18@sc.itc.keio.ac.jp
Keisuke
K
Nagao
Takayuki
T
Ota
Akiko
A
Tanikawa
Yujiro
Y
Takae
Tetsuya
T
Mori
Shun-ichi
S
Udagawa
Takeji
T
Nishikawa
D000293:Adolescent; D004271:DNA, Fungal / Q000032:analysis; D021903:DNA, Ribosomal Spacer; D020459:Genes, rRNA; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D009091:Mucormycosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012233:Rhizopus / Q000235:genetics / Q000302:isolation & purification*; D013172:Spores, Fungal / Q000648:ultrastructure
D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer