Mycology
Rapid and specific detection of section Fumigati and Aspergillus fumigatus in human samples using a new multiplex real-time PCR

https://doi.org/10.1016/j.diagmicrobio.2014.06.003Get rights and content

Abstract

Invasive aspergillosis is an opportunistic infection caused primarily by Aspergillus fumigatus. However, other common fungal pathogens belonging to section Fumigati are often misidentified as A. fumigatus. Thus, we have developed a multiplex real-time PCR (qPCR) assay with primers and specific TaqMan probes based on internal transcribed spacer regions or benA gene to discriminate, in less than 3 h, species of section Fumigati and, specifically, A. fumigatus. The multiplex qPCR showed a limit of detection of 20 and 50 fg of DNA for section Fumigati and A. fumigatus, respectively. Moreover, it enabled detection of a single germinated conidia. The inclusion of some PCR facilitators together with the dilution of samples makes it possible to completely avoid PCR inhibitions in all bronchoalveolar lavage (BAL) samples assayed. This technique may be a useful complementary tool in the diagnosis of invasive pulmonary aspergillosis caused by A. fumigatus using BAL fluid.

Introduction

Aspergillosis is a group of diseases caused by opportunistic species of the Aspergillus genus. These diseases range from allergies to invasive aspergillosis (IA), the most serious being bronchopulmonary allergic aspergillosis, aspergilloma, and IA, which appear in immunocompromised patients, especially those with prolonged neutropenia (Walsh et al., 2008). Among these, IA stands out, with mortality rates of greater than 50%, reaching 95% in certain clinical scenarios (Balloy and Chignard, 2009, Maschmeyer et al., 2007), and Aspergillus fumigatus is the major etiological agent, followed by Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and other species with a lower incidence. Although the spores of A. fumigatus are a small proportion of the airborne spores in hospitals (0.3%), this fungus causes approximately 90% of the systemic infections due to Aspergillus (Brakhage and Langfelder, 2002).

Nowadays, it remains relatively difficult to identify the species causing aspergillosis. Classical diagnostic methods are very unreliable, as they are based on morphological features, which depend mainly on growth conditions, and tend to require a high level of experience and/or a long period of incubation. Moreover, there is a risk of misidentifying the etiological agent. The section Fumigati that includes A. fumigatus provides a prime example of this problem: several species, such as Aspergillus lentulus, Aspergillus viridinutans, Aspergillus fumigatiaffinis, Neosartorya pseudofischeri, Neosartorya hiratsukae, and Neosartorya udagawae having been wrongly identified and reported as A. fumigatus (Balajee et al., 2005a, Balajee et al., 2005b, Balajee et al., 2006, Hong et al., 2008), illustrating how difficult it can be to identify species when morphological examination alone is used. In addition, some of these species (A. lentulus, A. viridinutans, N. pseudofischeri, and N. udagawae) have been reported to be resistant in vitro to azole antifungals itraconazole, miconazole, posaconazole, ravuconazole, and/or voriconazole (Alcazar-Fuoli et al., 2008), and although their incidence is not high, it would be useful to distinguish A. fumigatus from them.

From a clinical point of view, it is essential to detect IA and identify the species responsible as early as possible, to define specific therapeutic strategies (Rüping et al., 2008) and, consequently, improve patient outcome; diagnostic assays targeting fungal biomarkers have been developed (Cuenca-Estrella et al., 2011), and these offer the potential for new paradigms in prevention and early treatment (Almyroudis and Segal, 2009). Among them, detection of circulating galactomannan in serum or bronchoalveolar lavage (BAL) fluid (Hage et al., 2011, Pfeiffer et al., 2006, Rex, 2006) may be a useful complementary diagnostic procedure for IA. Nevertheless, in such tests, false-positive and false-negative results are problematic as well as it being impossible to identify individual species of the Aspergillus genus.

For the correct identification of Aspergillus clinical isolates, molecular identification is usually recommended. Comparative sequence analysis of the internal transcribed spacer (ITS) regions, specifically the ITS1 and ITS2 non-coding regions flanking the 5.8S rDNA, was suggested as appropriate for identifying Aspergillus to the level of subgenus/section (Balajee et al., 2007). Meanwhile, amplification followed by the sequencing of some codifying genes, such as actin, calmodulin, rodlet A, and/or β-tubulin, has been used to distinguish A. fumigatus from related species (Balajee et al., 2007, Samson et al., 2007). Other techniques, such as analysis based on random amplified polymorphic DNA (Brandt et al., 1998) or restriction fragment length polymorphism (Staab et al., 2009) and a new method based on microsphere-based Luminex assays (Landlinger et al., 2009), may allow molecular identification without sequencing. However, these methodologies are time consuming and laborious, and they require technology that is not available to all clinical mycology laboratories. On the other hand, there are currently a variety of commercial assays based on real-time PCR (qPCR) that can assist in this diagnosis. Among them, MycAssay Aspergillus (Myconostica, Cambridge, UK), Aspergillus tracer (Affigene, Bromma, Sweden), Septifast (Roche, Madrid, Spain), MycoReal Aspergillus (Ingenetix Gmb H, Vienna, Austria), and Aspergillus spp. q-PCR Alert (Nanogen, Turin, Italy) should be mentioned. None of these, however, achieve identification at the species level.

The use of multicopy sequences, such as the ITS region or benA gene (that codes the β-tubulin protein), together with different strategies to avoid PCR inhibitions, might improve the detection level of A. fumigatus DNA in human samples and at the same time distinguish it from other related species, even those in section Fumigati. Therefore, the aim of this work was to develop a multiplex qPCR that enables species of section Fumigati to be quickly and easily distinguished from other fungi and specifically identifies A. fumigatus in a single reaction, avoiding PCR inhibitions.

Section snippets

Collection of microorganisms

The following microorganisms were used: 70 strains of 40 species of the Aspergillus genus, 10 species of Mucorales, 3 species of other filamentous fungi (genera Fusarium, Penicillium, and Scedosporium), 5 species of yeast, and 4 species of bacteria (Table 1). The procedures for maintaining and harvesting the microorganisms to obtain DNA were carried out following methods described previously (Abad-Diaz-De-Cerio et al., 2013).

Human samples

Six samples of human DNA were extracted from cells obtained from the

Collection of microorganisms

The Fumigati primers and the ITS probe allowed all species and strains of section Fumigati tested to be detected, except N. pseudofischeri, which was detected using the Np-ITS probe. The benAfum probe specifically identified DNA from all A. fumigatus strains studied. Table 3 summarizes the positive results obtained from the microorganism collection by multiplex qPCR with the probes designed. All the other strains and species in the collection analyzed (included within Aspergillus genus or not)

Discussion

A. fumigatus continues to be the most common etiological agent of IA. Identification of A. fumigatus has historically been based on morphological features. However, recent studies have shown that species identification considering only morphology has limitations due to morphological variability with growth conditions. Nowadays, there are still many hospitals that use culture as the reference method for microbiological confirmation and identification despite its low sensitivity and the

Acknowledgments

This work was partly supported by the UPV/EHU (grants PES13/03, GIU12/44, and UFI11/25), and the Government of the Basque Country (grant S-PC11UN007). Jimena V. Fernandez Molina and Aize Pellon have been supported by Pre-doctoral Research Grants of the University of Basque Country (UPV/EHU). Monica Sueiro Olivares has been supported by Pre-doctoral Research Grants of the Government of the Basque Country. Technical and human support provided by Advanced Research Facilities (SGIker) of the

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