Broad-spectrum analysis for pathogens in patients with respiratory tract infections is becoming more relevant as the number of potential infectious agents is still increasing. Here we describe the new multiparameter RespiFinder assay, which is based on the multiplex ligation-dependent probe amplification (MLPA) technology. This assay detects 15 respiratory viruses in one reaction. The MLPA reaction is preceded by a preamplification step which ensures the detection of both RNA and DNA viruses with the same specificity and sensitivity as individual monoplex real-time reverse transcription-PCRs. The RespiFinder assay was validated with 144 clinical samples, and the results of the assay were compared to those of cell culture and a respiratory syncytial virus (RSV)-specific immunochromatography assay (ICA). Compared to the cell culture results, the RespiFinder assay showed specificities and sensitivities of 98.2% and 100%, respectively, for adenovirus; 96.4% and 100%, respectively, for human metapneumovirus; 98.2% and 100%, respectively, for influenza A virus (InfA); 99.1% and 100%, respectively, for parainfluenza virus type 1 (PIV-1); 99.1% and 80%, respectively, for PIV-3; 90.1% and 100%, respectively, for rhinovirus; and 94.6% and 100%, respectively, for RSV. Compared to the results of the RSV-specific ICA, the RespiFinder assay gave a specificity and a sensitivity of 82.4% and 80%, respectively. PIV-2, PIV-4, influenza B virus, InfA H5N1, and coronavirus 229E were not detected in the clinical specimens tested. The use of the RespiFinder assay resulted in an increase in the diagnostic yield compared to that obtained by cell culture (diagnostic yields, 60% and 35.5%, respectively). In conclusion, the RespiFinder assay provides a user-friendly and high-throughput tool for the simultaneous detection of 15 respiratory viruses with excellent overall performance statistics.

Eumycetoma is a chronic infectious disease characterized by a large subcutaneous mass, often caused by the fungus Madurella mycetomatis. A combination of surgery and prolonged medication is needed to treat this infection with a success rate of only 30%. There is, therefore, an urgent need to find more effective drugs for the treatment of this disease. In this study, we screened 800 diverse drug-like molecules and identified 215 molecules that were active in vitro. Minimal inhibitory concentrations were determined for the 13 most active compounds. One of the most potent compounds, a fenarimol analogue for which a large analogue library is available, led to the screening of an additional 35 compounds for their in vitro activity against M. mycetomatis hyphae, rendering four further hit compounds. To assess the in vivo potency of these hit compounds, a Galleria mellonella larvae model infected with M. mycetomatis was used. Several of the compounds identified in vitro demonstrated promising efficacy in vivo in terms of prolonged larval survival and/or reduced fungal burden. The results presented in this paper are the starting point of an Open Source Mycetoma (MycetOS) approach in which members of the global scientific community are invited to participate and contribute as equal partners. We hope that this initiative, coupled with the promising new hits we have reported, will lead to progress in drug discovery for this most neglected of neglected tropical diseases.

A novel olfactory method for bacterial species identification using an electronic nose device called the MonoNose was developed. Differential speciation of microorganisms present in primary cultures of clinical samples could be performed by real-time identification of volatile organic compounds (VOCs) produced during microbial replication. Kinetic measurements show that the dynamic changes in headspace gas composition are orders of magnitude larger than the static differences at the end of fermentation. Eleven different, clinically relevant bacterial species were included in this study. For each of the species, two to eight different strains were used to take intra-species biodiversity into account. A total of 52 different strains were measured in an incubator at 37°C. The results show that the diagnostic specificities varied from 100% for Clostridium difficile to 67% for Enterobacter cloacae with an overall average of 87%. Pathogen identification with a MonoNose can be achieved within 6-8 h of inoculation of the culture broths. The diagnostic specificity can be improved by broth modification to improve the VOC production of the pathogens involved.

Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.

Objectives Eumycetoma is currently treated with a combination of itraconazole therapy and surgery, with limited success. Recently, olorofim, the lead candidate of the orotomides, a novel class of antifungal agents, entered a Phase II trial for the treatment of invasive fungal infections. Here we determined the activity of olorofim against Madurella mycetomatis, the main causative agent of eumycetoma. Methods Activity of olorofim against M. mycetomatis was determined by in silico comparison of the target gene, dihydroorotate dehydrogenase (DHODH), and in vitro susceptibility testing. We also investigated the in vitro interaction between olorofim and itraconazole against M. mycetomatis. Results M. mycetomatis and Aspergillus fumigatus share six out of seven predicted binding residues in their DHODH DNA sequence, predicting susceptibility to olorofim. Olorofim demonstrated excellent potency against M. mycetomatis in vivo with MICs ranging from 0.004 to 0.125 mg/L and an MIC90 of 0.063 mg/L. Olorofim MICs were mostly one dilution step lower than the itraconazole MICs. In vitro interaction studies demonstrated that olorofim and itraconazole work indifferently when combined. Conclusions We demonstrated olorofim has potent in vitro activity against M. mycetomatis and should be further evaluated in vivo as a treatment option for this disease.