Background Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. Objectives To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. Methods Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. Results The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. Conclusions The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

The following nutraceuticals: Moringa oleifera Lam., Garlic (Allium sativum) and Ginger (Zinger officinale) were screened for their possible use as Aflatoxin (Aspergillus flavus) reduction agents by extracting their photochemical constituent’s using aqueous and methanol extraction which were used as inhibitors of A. flavus . The results showed the presence of tannins, steroides, cardiac glycosides, anthraquinone, flavonoids, alkaloids terpenes and saponins in all the tested nutraceuticals. Methanol extraction had highest concentration of the phytochemical constituents in each and the growth inhibition zone of A. Flavus showed that the extracts of Moringa oleifera leaves, Garlic (Allium sativum L) and Ginger (Zingiber officinale) inhibit the growth of A. flavus in methanol extract, while aqueous extraction showed no zone of inhibition. The sensitivity results obtained revealed significant (p>0.05) difference on zones of inhibition at different concentrations of 12.5, 25, 50, 100 and 200mg/ml of the extracts of Moringa oleifera leaves (A), Garlic (B) and Ginger (C) and for methanol extracts (A), (B) and (C) at concentration of 12.5mg/ml. The highest zones of inhibition is extract (C) at 200mg/ml. The constituents in these nutraceuticals and the sensitivity results obtained indicates that these nutraceuticaals could be used as phytogenic feed additive for the inhibition of growth of mycotoxins in feeds