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.