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Aspergillosis causes significant health risks to both birds and mammals. The outcome of these infections is often poor due to delayed diagnosis and treatment failure. We investigated 152 cases of aspergillosis from birds and mammals in Belgium. Most samples originated from the taxonomic orders Artiodactyla (40.1 %) and Columbiformes (19.7 %). Five isolates (3.3 %) showed phenotypical resistance against at least one medical azole. Three of these isolates were pan-azole resistant bearing the TR34/L98H mutation. The predominance of this resistance mutation supports an environmental route for exposure and resistance selection, highlighting the importance of the One Health concept.

IntroductionDuring 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.MethodsIn 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.ResultsA 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

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.

Background: There is evidence that global anthropogenic climate change may be impacting floral phenology and the temporal and spatial characteristics of aero‐allergenic pollen. Given the extent of current and future climate uncertainty, there is a need to strengthen predictive pollen forecasts. Methods: The study aims to use CatBoost (CB) and deep learning (DL) models for predicting the daily total pollen concentration up to 14 days in advance for 23 cities, covering all five continents. The model includes the projected environmental parameters, recent concentrations (1, 2 and 4 weeks), and the past environmental explanatory variables, and their future values. Results: The best pollen forecasts include Mexico City (R2(DL_7) ≈.7), and Santiago (R2(DL_7) ≈.8) for the 7th forecast day, respectively; while the weakest pollen forecasts are made for Brisbane (R2(DL_7) ≈.4) and Seoul (R2(DL_7) ≈.1) for the 7th forecast day. The global order of the five most important environmental variables in determining the daily total pollen concentrations is, in decreasing order: the past daily total pollen concentration, future 2 m temperature, past 2 m temperature, past soil temperature in 28–100 cm depth, and past soil temperature in 0–7 cm depth. City‐related clusters of the most similar distribution of feature importance values of the environmental variables only slightly change on consecutive forecast days for Caxias do Sul, Cape Town, Brisbane, and Mexico City, while they often change for Sydney, Santiago, and Busan. Conclusions: This new knowledge of the ecological relationships of the most remarkable variables importance for pollen forecast models according to clusters, cities and forecast days is important for developing and improving the accuracy of airborne pollen forecasts. [ABSTRACT FROM AUTHOR]

Azole resistance in Aspergillus fumigatus (ARAf) is becoming a worldwide health threat due to increasing occurrence in the environment. However, environmental surveillance programs are not commonly in place and are lacking in Belgium. Since no data on the occurrence of ARAf and the presence of hotspots for the selection of azole resistance is available in Belgium, a first study on the prevalence of ARAf in the environment was conducted. A total of 232 air and compost or soil samples were taken from two composting facilities, and from horticultural and agricultural crops. The azole susceptibility pattern was determined using the EUCAST method (E. Def. 9.4), and the c yp 51A gene and its promotor region were sequenced in A. fumigatus isolates with phenotypic azole resistance. Six pan-azole-resistant A. fumigatus isolates were identified, originating from compost and horticultural crops. Four isolates carried the TR34/L98H mutation, and one isolate carried the TR46/Y121F/T289A mutation. However, we did not observe any ARAf isolates from agricultural crops. In conclusion, this study reported the first TR34/L98H and TR46/Y121F/T289A mutation isolated from a composting facility and horticulture in Belgium. The implementation of standardization in environmental surveillance of A. fumigatus on a European level would be beneficial in order to identify hotspots. [ABSTRACT FROM AUTHOR]

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.

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.

Abstract 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.

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.

Abstract 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.

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.

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.

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.