Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae.

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022.

Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021-2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017-2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

Evaluation of a Liquid Media MALDI-TOF MS Protocol for the Identification of Dermatophytes Isolated from Tinea capitis Infections.

One of the most common types of tinea is the superficial infection of the hair and scalp area known as tinea capitis. It is responsible for frequent outbreaks in nurseries and schools and represents a global health problem. Correct identification of the infection agent is essential in the determination of the infection source, epidemiological course, and treatment initiation. The conventional identification methods (direct exam, culture, DNA sequencing) are time-consuming, require experienced staff, are time-consuming, and the latter is expensive for routine identifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining new ground for routine identification of filamentous fungi. The main advantages of MALDI-TOF MS are its rapid and accurate identification capability, relatively low cost, and easy integration into the laboratory routine. Its accuracy heavily depends on the quality of the reference spectra database. Identification of clinical isolates with MALDI-TOF MS protocol requires a sub-culturing step to ensure reliable identification. It can take days to weeks before fungal growth appears on solid medium. In this study, a unique MALDI-TOF MS protocol using liquid cultures of dermatophyte species was developed in order to shorten the turnaround time for the culture and identification of clinical isolates. Material and Method A standard MALDI-TOF MS protocol was adapted for liquid instead of solid cultures. Three different databases were tested. Results Using the liquid media MALDI-TOF MS protocol, a global rate of 62% correct identification (RCI) was obtained, compared with 87% for the protocol based on solid cultures. Trichophyton tonsurans was not correctly identified in all isolates using liquid cultures, with 88% of the isolates misidentified as Trichophyton interdigitale. The turnaround time for primary isolates for the solid and liquid protocols were respectively 11.7 and 11.6 days (no significant difference between both methods (p = 0.96)). Conclusions The newly designed liquid MALDI-TOF MS protocol did not lead to a significantly shorter turnaround time for the identification of dermatophytes isolated from tinea capitis infections. The turnaround time for the method with primary isolates was not significantly lower, and the rate of correct identification decreased remarkably, which emphasizes the need for a sub-culturing step. Using different database did not lead to improvement in turnaround time or rate of correct identification. This study highlights the importance of the medium and the reference database when performing MALDI-TOF MS.

MALDI-TOF Mass Spectrometry Online Identification of Trichophyton indotineae Using the MSI-2 Application.

Trichophyton indotineae is an emerging pathogen which recently spread from India to Europe and that is more prone than other species of the Trichophyton mentagrophytes complex to show resistance to terbinafine, resulting in the necessity of rapid identification. Here, we improved the online MSI-2 MALDI-TOF identification tool in order to identify T. indotineae. By multiplying the culture conditions (2 culture media and 6 stages of growth) prior to protein extractions for both test isolates and reference strains, we added 142 references corresponding to 12 strains inside the T. mentagrophytes complex in the online MSI-2 database, of which 3 are T. indotineae strains. The resulting database was tested with 1566 spectra of 67 isolates from the T. mentagrophytes complex, including 16 T. indotineae isolates. Using the newly improved MSI-2 database, we increased the identification rate of T. indotineae from 5% to 96%, with a sensitivity of 99.6%. We also identified specific peaks (6834/6845 daltons and 10,634/10,680 daltons) allowing for the distinction of T. indotineae from the other species of the complex. Our improved version of the MSI-2 application allows for the identification of T. indotineae. This will improve the epidemiological knowledge of the spread of this species throughout the world and will help to improve patient care.

Implementation of MALDI-TOF Mass Spectrometry to Identify Fungi From the Indoor Environment as an Added Value to the Classical Morphology-Based Identification Tool.

Introduction: During the last decades, molds in the indoor environment have raised concern regarding their potential adverse health effects. The genera Aspergillus, Cladosporium, Penicillium, Alternaria, and yeasts, the most common fungi found indoors, include species with high allergenic and toxigenic potentials. Identification of these molds is generally performed by microscopy. This method has, however, some limitations as it requires mycologists with high expertise while identification is often limited to the genus level. Therefore, it is necessary to seek for fast and accurate tools, such as Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS), enabling an identification to the species level and guiding general practitioners in their search for the underlying cause of a health problem. Methods: In this study, 149 fungal air and dust isolates from 43 dwellings in Brussels were taken in collaboration with Brussels Environment RCIB/CRIPI and identified by both microscopy and MALDI-TOF MS in Sciensano's Indoor Mycology laboratory. Spectra obtained via MALDI-TOF MS were compared with data available in an in-house created reference database containing over 1,700 strains from the BCCM/IHEM fungal collection. Results: A total of 149 isolates including 18 yeasts and 131 filamentous fungi were analyzed. Microscopic analysis indicated 18 yeast species and allowed identification of 79 isolates (53%) to the genus level. Only 36 isolates (24%) could be identified to the species complex level. Fifteen molds (10%) could not be identified, and one was indicated as sterile mycelia. No isolate was identified to species level. MALDI-TOF MS analysis identified 137 (92%) of the 149 isolates with a logscore > 1.7. Of these 137 isolates, 129 (87%) were identified to the species level (logscore > 2.0). For only 8 isolates (5%), identification was limited to the genus/section level (1.7 < logscore <2.0), and 12 isolates (8%) could not be identified. Conclusion: A comparison of results obtained with both methods indicates an increased precision in identifications with MALDI-TOF MS analysis for 92% of the isolates, emphasizing its highly added value to the standard microscopic analysis in routine practice. In addition, MALDI-TOF MS also enables to assess the accuracy of microscopic identifications.

Investigations upon the Improvement of Dermatophyte Identification Using an Online Mass Spectrometry Application.

Online MALDI-TOF mass spectrometry applications, such as MSI-2, have been shown to help identify dermatophytes, but recurrent errors are still observed between phylogenetically close species. The objective of this study was to assess different approaches to reduce the occurrence of such errors by adding new reference spectra to the MSI-2 application. Nine libraries were set up, comprising an increasing number of spectra obtained from reference strains that were submitted to various culture durations on two distinct culture media: Sabouraud gentamicin chloramphenicol medium and IDFP Conidia medium. The final library included spectra from 111 strains of 20 species obtained from cultures on both media collected every three days after the appearance of the colony. The performance of each library was then analyzed using a cross-validation approach. The spectra acquisitions were carried out using a Microflex Bruker spectrometer. Diversifying the references and adding spectra from various culture media and culture durations improved identification performance. The percentage of correct identification at the species level rose from 63.4 to 91.7% when combining all approaches. Nevertheless, residual confusion between close species, such as Trichophyton rubrum, Trichophyton violaceum and Trichophyton soudanense, remained. To distinguish between these species, mass spectrometry identification should take into account basic morphological and/or clinico-epidemiological features.

The taxonomy of the Trichophyton rubrum complex: a phylogenomic approach.

The medically relevant Trichophyton rubrum species complex has a variety of phenotypic presentations but shows relatively little genetic differences. Conventional barcodes, such as the internal transcribed spacer (ITS) region or the beta-tubulin gene, are not able to completely resolve the relationships between these closely related taxa. T. rubrum, T. soudanense and T. violaceum are currently accepted as separate species. However, the status of certain variants, including the T. rubrum morphotypes megninii and kuryangei and the T. violaceum morphotype yaoundei, remains to be deciphered. We conducted the first phylogenomic analysis of the T. rubrum species complex by studying 3105 core genes of 18 new strains from the BCCM/IHEM culture collection and nine publicly available genomes. Our analyses revealed a highly resolved phylogenomic tree with six separate clades. Trichophyton rubrum, T. violaceum and T. soudanense were confirmed in their status of species. The morphotypes T. megninii, T. kuryangei and T. yaoundei all grouped in their own respective clade with high support, suggesting that these morphotypes should be reinstituted to the species-level. Robinson-Foulds distance analyses showed that a combination of two markers (a ubiquitin-protein transferase and a MYB DNA-binding domain-containing protein) can mirror the phylogeny obtained using genomic data, and thus represent potential new markers to accurately distinguish the species belonging to the T. rubrum complex.

A Polyphasic Approach to Classification and Identification of Species within the Trichophyton benhamiae Complex.

In recent years, considerable advances have been made in clearing up the phylogenetic relationships within the Arthrodermataceae family. However, certain closely related taxa still contain poorly resolved species boundaries. Here, we tried to elucidate the species composition of the Trichophyton benhamiae species complex using a combined approach consisting of multi-gene phylogenetic analysis based on internal transcribed spacer (ITS) and beta-tubulin (BT) gene regions, morphological analysis, and spectral comparison using MALDI-ToF. We confirmed the existence of 11 different monophyletic clades within the complex representing either species or genetically distinct groups within species. MALDI-ToF spectrometry analysis revealed that most of these clades were readily distinguishable from one another; however, some closely related sister clades, such as T. europaeum and T. japonicum, were often misidentified as their counterpart. The distinct "yellow" and "white" phenotypes of T. benhamiae do not have a clear genetic basis and should thus be considered as different morphotypes of the same species. Strains traditionally considered T. benhamiae can be divided into three main clades: (i) T. benhamiae, (ii) T. europaeum/T. japonicum, and (iii) the phylogenetically distant T. africanum. While T. europaeum and T. japonicum are distinguishable based on their genotype, spectral and morphological analysis did not provide clear delimiting characteristics.

Identification of Molds with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Performance of the Newly Developed MSI-2 Application in Comparison with the Bruker Filamentous Fungi Database and MSI-1.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a promising tool for the rapid and efficient identification of molds, but improvements are still necessary to achieve satisfactory results when identifying cryptic species. Here, we aimed to validate a new web application, MSI-2, which replaces MSI-1, an application that was built and deployed online in 2017. For the evaluation, we gathered 633 challenging isolates obtained from daily hospital practice that were first identified with DNA-based methods, and we submitted their corresponding mass spectra to three identification programs (Bruker, MSI-1, and MSI-2). The MSI-2 application had a better identification performance at the species level than MSI-1 and Bruker, reaching 83.25% correct identifications, compared with 63.19% (MSI-1), 38.07% (Bruker with a 1.7 threshold), and 21.8% (Bruker with a 2.0 threshold). The MSI-2 application performed especially well for Aspergillus and Fusarium species, including for many cryptic species, reaching 90% correct identifications for Aspergillus species and 78% for Fusarium species compared to 69% and 43% with MSI-1. Such an improvement may have a positive impact on patient management by facilitating the identification of cryptic species potentially associated with a specific antifungal resistance profile.

Clinical Origin and Species Distribution of Fusarium spp. Isolates Identified by Molecular Sequencing and Mass Spectrometry: A European Multicenter Hospital Prospective Study.

Fusarium spp. are widespread environmental fungi as well as pathogens that can affect plants, animals and humans. Yet the epidemiology of human fusariosis is still cloudy due to the rapidly evolving taxonomy. The Mass Spectrometry Identification database (MSI) has been developed since 2017 in order to allow a fast, accurate and free-access identification of fungi by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the MSI database user network, we aim to study the species distribution of Fusarium spp. isolates in an international multicenter prospective study. This study also allowed the assessment of the abilities of miscellaneous techniques to identify Fusarium isolates at the species level. The identification was performed by PCR-sequencing and phylogenic-tree approach. Both methods are used as gold standard for the evaluation of mass spectrometry. Identification at the species complex was satisfactory for all the tested methods. However, identification at the species level was more challenging and only 32% of the isolates were correctly identified with the National Center for Biotechnology Information (NCBI) DNA database, 20% with the Bruker MS database and 43% with the two MSI databases. Improvement of the mass spectrometry database is still needed to enable precise identification at the species level of any Fusarium isolates encountered either in human pathology or in the environment.

Intra- and inter-laboratory comparison of mDixon and FastFung broths for Malassezia antifungal susceptibility testing.

BACKGROUND: Malassezia spp. antifungal susceptibility testing (AFST) capacities are limited by the lack of efficient and standardised AFST procedure, mainly because of the fastidious cultivation of these yeast. OBJECTIVES: This study aimed to compare the FastFung broth (FFB) to modified Dixon broth (mDIXB) for the in vitro AFST of Malassezia spp. Fluconazole, ketoconazole, voriconazole and terbinafine MICs against a 19 Malassezia strains, including 6 M furfur, 4 M pachydermatis, 5 M sympodialis and 4 M slooffiae. METHODS: The essential agreement (EA) between the two assays, and the intra- and inter-laboratory agreement of each assay were assessed. RESULTS: The MIC data obtained in our study were comparable to those reported in the literature. FFB showed to enhance Malassezia growth and displayed 100% (±2-fold dilution) EAs demonstrating similar performances to mDIXB. In addition, the MIC data obtained by using the FFB were reproducible between laboratories with EAs ranging from 94.7% to 100%. CONCLUSIONS: Therefore, FFB is a suitable alternative to mDXB for Malassezia spp. AFST.

Superficial mycoses in Belgium: Burden, costs and antifungal drugs consumption.

BACKGROUND: Monitoring of superficial mycoses requires more attention due to their important incidence, health costs and antifungal drugs consumption. OBJECTIVES: The objectives were to estimate the burden of superficial mycoses in Belgium and to assess trends in associated antifungal consumption. METHODS: The burden of dermatophytoses (including onychomycosis), as well as skin and genital candidiasis, was estimated using disability-adjusted life years (DALY). Moreover, trends in systemic and topical antifungal consumption in ambulatory care were examined for the period 2010-2017, together with their associated costs. RESULTS: Due to their high incidence and long treatment duration, dermatophytoses represented the bulk of the burden, accounting for 92.2% of the total DALYs of superficial mycoses. Terbinafine was the most prescribed antifungal in terms of doses (35.4% of the total doses) while fluconazole was the most delivered drug in terms of packages (29.1% of the total packages). More than 70% of the prescriptions were made by general practitioners while consumption varied according to age and gender of the patients. A global 12% decrease in antifungal prescriptions was observed between 2011 and 2017. However, this reduction would result mainly from packaging changes and increased self-medication. A significant decrease in itraconazole treatments was notably compensated by an increased prescription of fluconazole packages. CONCLUSION: This study emphasises that dermatological presentations of superficial mycoses are the most important in terms of both burden and antifungal consumption in Belgium. Further reduction in antifungals use can be achieved by applying the adequate treatment after identification of the causative agent.

Lineages Within the Trichophyton rubrum Complex.

The most important species of the Trichophyton rubrum group are T. rubrum, causing mainly skin and nail infections, and T. violaceum which is mostly scalp-associated. The status of a third species, T. soudanense, has been under debate. With a polyphasic approach, using molecular phylogenetic techniques, MALDI-TOF mass spectrometry and physiological and morphological analysis, we re-evaluated the T. rubrum complex. Our results support four genetic lineages within the complex each with a distinct morphology and identifiable via MALDI-TOF MS: T. rubrum, T. violaceum, T. soudanense and the T. yaoundei clade. However, ITS and Bt2 sequencing data could not confirm these taxa as four monophyletic species. Our results also suggest that strains formerly identified as T. kuryangei and T. megninii should be considered in future taxonomic studies.

Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of Exophiala jeanselmei in Indoor Air of Air-Conditioned Offices.

Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing fungi, even though this fraction could impact health. Among the contaminants suspected to be part of this fraction, Exophiala jeanselmei is an interesting case study. Known to be pathogenic, this black yeast grows in humid environments such as air-conditioning systems, where it has been previously detected using classical culture-based methods. However, until now, this fungus was never detected in indoor air in contact with these air-conditioning systems. This study shows the first detection of E. jeanselmei in indoor air collected from offices in contact with contaminated air-conditioning reservoirs. While its presence in indoor air could not be demonstrated with culture-based methods, it was found by real-time PCR and massive parallel sequencing. The latter also allowed obtaining a broader view on the fungal diversity in the tested samples. Similar approaches were applied on water samples collected from the conditioning reservoirs to trace the source of contamination. The comparison of results obtained with both methods confirmed that the molecular tools could improve indoor air monitoring, especially of dead and/or uncultivable contaminants or when competition between species could occur.

Species Distinction in the Trichophyton rubrum Complex.

The Trichophyton rubrum species complex comprises commonly encountered dermatophytic fungi with a worldwide distribution. The members of the complex usually have distinct phenotypes in culture and cause different clinical symptoms, despite high genome similarity. In order to better delimit the species within the complex, molecular, phenotypic, and physiological characteristics were combined to reestablish a natural species concept. Three groups, T. rubrum, T. soudanense, and T. violaceum, could be distinguished based on the sequence of the internal transcribed spacer (ITS) ribosomal DNA barcode gene. On average, strains within each group were similar by colony appearance, microscopy, and physiology, but strains between groups showed significant differences. Trichophyton rubrum strains had higher keratinase activity, whereas T. violaceum strains tended to be more lipophilic; however, none of the phenotypic features were diagnostic. The results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and amplified fragment length polymorphism (AFLP) were partially consistent with the ITS data but failed to distinguish the species unambiguously. Despite their close similarity, T. violaceum, T. soudanense, and T. rubrum can be regarded as independent species with distinct geographical distributions and clinical predilections. Trichophyton soudanense is pheno- and genotypically intermediate between T. rubrum and T. violaceum For routine diagnostics, ITS sequencing is recommended.

Relationships between aeroallergen levels and hospital admissions for asthma in the Brussels-Capital Region: a daily time series analysis.

BACKGROUND: Outdoor pollen grain and fungal spore concentrations have been associated with severe asthma exacerbations at the population level. The specific impact of each taxon and the concomitant effect of air pollution on these symptoms have, however, still to be better characterized. This study aimed to investigate the short-term associations between ambient concentrations of various aeroallergens and hospitalizations related to asthma in the Brussels-Capital Region (Belgium), an area recording especially high rates of admissions. METHODS: Based on administrative records of asthma hospitalizations and regular monitoring of 11 tree/herbaceous pollen taxa and 2 fungal spore taxa, daily time series analyses covering the 2008-2013 period were performed. Effects up to 6 days after exposure were captured by combining quasi-Poisson regression with distributed lag models, adjusting for seasonal and long-term trends, day of the week, public holidays, mean temperature and relative humidity. Effect modification by age and air pollution (PM, NO2, O3) was tested. RESULTS: A significant increase in asthma hospitalizations was observed for an interquartile range increase in grass (5.9%, 95% CI: 0.0, 12.0), birch (3.2%, 95% CI: 1.1, 5.3) and hornbeam (0.7%, 95% CI: 0.2, 1.3) pollen concentrations. For several taxa including grasses, an age modification effect was notable, the hospitalization risk tending to be higher in individuals younger than 60 years. Air pollutants impacted the relationships too: the risk appeared to be stronger for grass and birch pollen concentrations in case of high PM10 and O3 concentrations respectively. CONCLUSIONS: These findings suggest that airborne grass, birch and hornbeam pollen are associated with severe asthma exacerbations in the Brussels region. These compounds appear to act in synergy with air pollution and to more specifically affect young and intermediate age groups. Most of these life-threatening events could theoretically be prevented with improved disease diagnosis/management and targeted communication actions.

New insights in dermatophyte research.

Dermatophyte research has renewed interest because of changing human floras with changing socioeconomic conditions, and because of severe chronic infections in patients with congenital immune disorders. Main taxonomic traits at the generic level have changed considerably, and now fine-tuning at the species level with state-of-the-art technology has become urgent. Research on virulence factors focuses on secreted proteases now has support in genome data. It is speculated that most protease families are used for degrading hard keratin during nitrogen recycling in the environment, while others, such as Sub6 may have emerged as a result of ancestral gene duplication, and are likely to have specific roles during infection. Virulence may differ between mating partners of the same species and concepts of zoo- and anthropophily may require revision in some recently redefined species. Many of these questions benefit from international cooperation and exchange of materials. The aim of the ISHAM Working Group Dermatophytes aims to stimulate and coordinate international networking on these fungi.

Comparative long-term trend analysis of daily weather conditions with daily pollen concentrations in Brussels, Belgium.

A clear rise in seasonal and annual temperatures, a gradual increase of total radiation, and a relative trend of change in seasonal precipitation have been observed for the last four decades in Brussels (Belgium). These local modifications may have a direct and indirect public health impact by altering the timing and intensity of allergenic pollen seasons. In this study, we assessed the statistical correlations (Spearman's test) between pollen concentration and meteorological conditions by using long-term daily datasets of 11 pollen types (8 trees and 3 herbaceous plants) and 10 meteorological parameters observed in Brussels between 1982 and 2015. Furthermore, we analyzed the rate of change in the annual cycle of the same selected pollen types by the Mann-Kendall test. We revealed an overall trend of increase in daily airborne tree pollen (except for the European beech tree) and an overall trend of decrease in daily airborne pollen from herbaceous plants (except for Urticaceae). These results revealed an earlier onset of the flowering period for birch, oak, ash, plane, grasses, and Urticaceae. Finally, the rates of change in pollen annual cycles were shown to be associated with the rates of change in the annual cycles of several meteorological parameters such as temperature, radiation, humidity, and rainfall.

Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study.

BACKGROUND: Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR®Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii. METHODS: In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii. RESULTS: Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples. CONCLUSION: We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems.

Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination.

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.