Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

The responses of aquatic organisms to chronic exposure to environmental concentrations of toxicants, often found in mixtures, are poorly documented. Here passive sampler extracts were used in experimental contamination of laboratory channels, to investigate their effects on natural biofilm communities. A realistic mixture of pesticides extracted from Polar Organic Chemical Integrative Samplers was used to expose biofilms in laboratory channels to total pesticide concentrations averaging 0.5 ± 0.1 μg l⁻¹. The level of exposure was representative of field conditions in terms of relative proportions of the substances but the exposure concentration was not maintained (decreasing concentrations between contamination occasions). The impact on the structural as well as the functional characteristics of the autotrophic and heterotrophic components was determined, using biofilm grown in uncontaminated conditions (reference site) and in sites exposed to pesticides (contaminated site). The exposure imposed did not significantly modify the structure or functions of reference biofilms, nor did it modify tolerance as measured by mixture EC₅₀ (EC₅₀ mix). In contrast, the communities from the more contaminated downstream section lost tolerance following decreased dose exposure, but community composition remained fairly stable. Overall, these results indicate that low levels of contamination did not lead to strong changes in community structure, and 14-day changes in tolerance seemed to depend mainly on physiological adaptation, suggesting that other environmental factors or longer-lasting processes prevailed. This study reports the first attempt to use passive sampler extracts as a realistic composite contaminant for experimental exposure of biofilms, with promising perspectives in further ecotoxicology studies.

The Morcille River located in the Beaujolais vineyard area (Eastern France) is subjected to strong vine-growing pressure leading to the contamination by a range of herbicides and fungicides of the surrounding freshwater environment. Particularly high concentrations of norflurazon, desmethyl norflurazon and tebuconazole were recorded in spring 2010 at the downstream site of the river. Despite their occurrence in rivers, scarce toxicity data are available for these products, in particular in the case of desmethyl norflurazon (main norflurazon degradation product). Furthermore, the toxicity data are generally available only for single compounds and are issued from single species toxicity tests, leading to a lack of ecological relevance. Consequently, this study was undertaken to evaluate the toxic effects of norflurazon, desmethyl norflurazon and tebuconazole singly and in a ternary mixture on fluvial biofilm. Toxicity tests were performed in microplates for 48 h. Photosynthetic endpoints were measured using pulse amplitude-modulated fluorometry; diatom densities and taxonomic composition were determined. After 48 h of exposure, significant effects on optimal quantum yield (F v/F m) for desmethyl norflurazon and mixture were observed.

BackgroundIndoor 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.MethodsIn this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii.ResultsUsing 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-0996-4) contains supplementary material, which is available to authorized users.

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.

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.

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