Fungi of the healthy and immunocompromised human airway: enhanced stringency analysis for taxon identification reveals covariation with bacterial populations

Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing data because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources. Results: Here we analyzed fungal lineages present in the human airway using an improved method for contamination filtering. We compared fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We used deep sequencing to characterize ribosomal rRNA gene segments from fungi (ITS amplicon) and bacteria (16S amplicon) in DNA extracted from bronchiolar lavage samples (n=89) and oropharyngeal wash (n=36). We introduce the use of DNA quantification data, which is routinely acquired during DNA library preparation, to annotate sequencing data and improve the identification and filtering of the contamination background. Conclusions: Using the abundance-weighted method, we quantified fungi authentically present in samples, showing increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in the subjects with increasingly severe pulmonary and immunologic deficits. We analyzed covariation of fungal and bacterial taxa, and found that oropharyngeal communities rich in Candida were also enriched in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to identify and characterize fungal communities in the human respiratory tract more accurately and explore their linkage to bacterial communities and to disease.