title: Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.
abstract: PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.
authors.affiliation: Department for Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Springer
authors.affiliation: Division of Clinical Microbiology, University Hospital Basel, Basel, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Goldenberger
authors.affiliation: Department for Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany.
authors.forename: Friderike
authors.initials: F
authors.lastname: Schmidt
authors.affiliation: Clinic of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland.
authors.forename: Maja
authors.initials: M
authors.lastname: Weisser
authors.affiliation: Clinic of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Wehrle-Wieland
authors.affiliation: Department for Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany.
authors.forename: Hermann
authors.initials: H
authors.lastname: Einsele
authors.affiliation: Division of Clinical Microbiology, University Hospital Basel, Basel, Switzerland.
authors.forename: Reno
authors.initials: R
authors.lastname: Frei
authors.affiliation: Department for Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Löffler
mesh_terms: D000328:Adult; D000368:Aged; D005260:Female; D016681:Genome, Fungal; D006801:Humans; D008297:Male; D008875:Middle Aged; D009090:Mucorales / Q000302:isolation & purification*; D009091:Mucormycosis / Q000175:diagnosis / Q000382:microbiology*; D012329:RNA, Bacterial / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D012329:RNA, Bacterial; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S