MouldAntigenDetect – validation of fungi specific (ELISA) – correlation with cell count (microscopy, qPCR) and colony forming units (CFU) under controlled, reproducible conditions

Status:

completed 12/2021

Aims:

Bioaerosols in workplaces can cause serious illness in workers at high concentrations and repeated exposure. Sampling and exposure assessment is required to evaluate protective and reduction measures. Different detection methods have different strengths, weaknesses, detection limits and specificities. The aim of the study "SchimmelAntigenDetect" was to evaluate six ELISAs specific for mould antigens, developed by the Institute for Prevention and Occupational Medicine (IPA) in Bochum using airborne dust samples.

Activities/Methods:

Different concentrations of mould spores were necessary for the evaluation (detection limit). On the one hand, the specificity has already been shown in publications, on the other hand, it was also tested in this study using defined mixtures of fungal spores and samples from workplaces. In order to test the ELISA and obtain references to the concentrations of the airborne dust samples, all samples were analyzed using four analytical methods. The ELISA results were compared with microscopic spore counting, colony forming units and assessment of SSU-copy numbers using qPCR. Airborne dust was sampled by filtration comprising GSP 10L sampling heads, Gilian 10i pumps and Teflon filters. In a bioaerosol chamber, spore aerosols of A. amoenus, A. amstelodami, A. fumigatus, C. herbarum, P. chrysogenum and W. sebi were produced as pure cultures or as a mixture under controlled conditions. For aerosol generation, two aerosol generators (LSA, RBG) were used to produce five concentrations for each organism. For analysis by ELISA, two extraction procedures, with and without cell disruption, were tested. In the complementary project "AntigenSampling", samples from workplaces were taken in Austria. These were also evaluated with the ELISAs developed by the IPA and the other tree analytical methods. In the project, five concentrations could be produced in the bioaerosol test system for five of the six fungal organisms. For the high concentrations, an aerosol generator not previously tested for fungal spores was used (RBG1000). Since A. fumigatus is a risk level 2 organism and special equipment is needed to generate spore dust, which was not available in the level 2 laboratory, only three concentrations were tested for this organism. In addition, A. fumigatus was omitted from the spore mixtures. Therefore, the mixture samples were prepared from five organisms and used in four concentrations. For each organism and concentration at least five filters were produced.

Results:

The comparison of the simultaneously sampled filters shows stable conditions in the test system, but already high fluctuations in the results of the qPCR. The analysis with colony forming units (CFU), spore counting and ELISA provided stable replicates. This result is also evident in the direct comparison of the analytical methods to each other and the calculation of the correlation coefficients. The ELISA developed by the IPA showed the highest agreement with spore count, followed by CFU. In terms of sensitivity, CFU showed the lowest detection limit at high germination rates after liquid cultivation. The detection limit of ELISAs depended on the spore size; while large spores were within the measurable range from a spore count of 10² / mL, small spores required up to 106 spores / mL. Extraction by cell disruption showed no positive influence on the detection limit for most of the organisms. The evaluation of the mixed samples shows that the ELISA were able to distinguish the different fungi from each other. The results showed a high agreement with the CFU. The antigens or genes of the different fungal species could also be detected in the workplace samples using both ELISAs and specific qPCR. In summary, the authors conclude that the fungus-specific ELISAs developed by the IPA are a very good complement to classical methods and advocate their use in workplaces. The relatively high detection limit for certain small spores compared to CFU and thus lower sensitivity should be taken into consideration and a combination of analytical methods should be used. The joint use with routinely performed, sensitive methods such as CFU would cover a large analytical breadth and increase the specificity and thus the significance of the data.

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