Simultaneous Detection and Identification of Aspergillus and Mucorales Species in Tissues Collected from Patients with Fungal Rhinosinusitis

Abstract

Rapid detection and differentiation of Aspergillus and Mucorales species in fungal rhinosinusitis diagnosis are desirable, since the clinical management and prognosis associated with the two taxa are fundamentally different. We describe an assay based on a combination of broad-range PCR amplification and reverse line blot hybridization (PCR/RLB) to detect and differentiate the pathogens causing fungal rhinosinusitis, which include five Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, and A. nidulans) and seven Mucorales species (Mucor heimalis, Mucor racemosus, Mucor cercinelloidea, Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, and Absidia corymbifera). The assay was validated with 98 well-characterized clinical isolates and 41 clinical tissue specimens. PCR/RLB showed high sensitivity and specificity, with 100% correct identifications of 98 clinical isolates and no cross-hybridization between the species-specific probes. Results for five control isolates, Candida albicans, Fusarium solani, Scedosporium apiospermum, Penicillium marneffei, and Exophiala verrucosa, were negative as judged by PCR/RLB. The analytical sensitivity of PCR/RLB was found to be 1.8 ؋ 10 Rhinosinusitis is a common disease with multiple manifestations and can even cause death in acute invasive cases. Fungi are considered to be major etiological agents, of which members of the order Mucorales and genus Aspergillus (order Eurotiales) are the most pathogenic (5, 10, 13). The course, therapeutic treatment, and prognosis of fungal rhinosinusitis caused by Aspergillus and Mucorales species are radically different; therefore, early diagnosis and accurate identification of pathogenic fungal species are crucial for effective treatment and clinical decision-making (21). Currently, diagnosis of fungal sinusitis still depends on histopathological examination and culture from nasal biopsy, but conventional culture-based phenotypic identification techniques often include significant delays and can fail to yield growth in clinical samples (17). In a significant number of cases, fungal culture is negative and only formalin-fixed paraffin-embedded (PE) tissue specimens are available for diagnosis of fungal infection. However, rapid diagnosis of surgical tissues is urgently needed in acute invasive infection cases. In addition, histopathological observations of fungal shape and arrangement may not be sufficient for the accurate identification of fungal species if only a limited quantity of anamorphic fungal hyphae is present. Therefore, to improve the outcome for fungal rhinosinusitis patients, the rapid and accurate detection and identification of pathogenic fungal species are needed to allow early initiation of targeted therapy.In this study, we developed an assay combining broad-range PCR amplification and reverse line blot hybridization (PCR/ RLB) to detect and differentiate Aspergillus and Mucorales pathogens in tissue specimens. The fungal pathogens tested included five Aspergillus species (Aspergillus fumigatus, A. f...

Identification of Mycoses in Developing Countries

“…Simple PCR assays are relatively easier and more straightforward than PCR-RFLP assays and differentiation of target species mainly relies on specific primers and the length of the amplified fragments. Ease of primer design, straightforward processing, and low-cost account for the wide use of this assay in the identification of a broad range of clinically relevant fungal species, including the most common pathogenic yeasts [114], cryptic Candida species [115], onychomycoses agents [116], and Aspergillus [117,118]. Multiplicity significantly affects sensitivity and specificity resulting in a higher detection limit and non-specificity in some cases [119].…”

“…Multiplicity significantly affects sensitivity and specificity resulting in a higher detection limit and non-specificity in some cases [119]. Although simple PCR does not have sufficient sensitivity to detect low numbers of target organisms and, therefore, may not provide desired results when applied directly to clinical specimens, it showed good results when used on blood cultures [120,121] or combined with reverse line blot hybridization, PCR-RLB [118]. Moreover, some of these multiplex PCR assays showed a higher degree of sensitivity when compared to phenotypic assays, such as direct microscopy and culture [116,118].…”

Frequency of fungal agents identified in sinus samples from patients with clinically suspected rhinosinusitis

“…Clinical and physical examinations are inconclusive (Vergara et al, 2005), and there are several reports indicating a low correlation between clinical criteria and endoscopic or imaging findings (Vergara et al, 2005). Culture remains the indispensable technique for suspected fungal infection, although its sensitivity may not exceed 50% (Zhao et al, 2011). Sensitivity of 32% and specificity of 94% have been described for direct examination, with positive and negative predictive values of 81.3% and 63%, respectively (Pulido Murillo and Gaona Hernández, 2007).…”

“…Mixed infections with Aspergillus and Mucorales have been described in patients with FRS and are considered to be the most pathogenic of agents (Montone et al, 2012;Ponikau et al, 1999;Zhao et al, 2011); it is noteworthy that they were identified by molecular techniques (mainly DNA amplification by PCR), suggesting the need to use other methodologies to identify the causative agents of this mycosis to species level (Zhao et al, 2011). It is thus important to know what fungal species is involved to ensure that a proper and effective antifungal treatment can be administered.…”

Rapid Detection and Identification of Aspergillus from Lower Respiratory Tract Specimens by Use of a Combined Probe–High-Resolution Melting Analysis

“…Molecular and nonmolecular assays have been developed (2,12,18,29,(44)(45)(46) to improve these limitations. As the lung is the main organ affected in most cases of IA, it seems logical to investigate Aspergillus in lower respiratory tract samples.…”