Evaluation of an amplicon-based nanopore sequencing test for fungal diagnostics.

This study describes an amplicon-based fungal nanopore sequencing test based on a fragment of the ribosomal operon covering the ITS1, ITS2, D1 and D2 region within one single PCR fragment. First, it was shown that the test can discriminate between at least 28 different fungal genera, and an accuracy at genus level of 94.4% was observed for a collection of 89 reference samples. Species level agreement was evaluated based on a subset (n = 73) of these samples and turned out to be 80.8%. Next, the suitability of the test for use in diagnostics was evaluated by sequencing a set of clinical samples known to contain one or more fungi (n = 31). The overall positive agreement between sequencing and standard diagnostic testing (culture, PCR and antigen testing) turned out to be 75.6%, and false negative sequencing results could be explained by either low abundance of the fungus in the sample or potential primer mismatches. This study shows that amplicon-based fungal nanopore sequencing can contribute to clinical diagnostics but should be used complementary with conventional methods.