Development of a novel mycobiome diagnostic for fungal infection.

BACKGROUND: Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. METHODS: Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. RESULTS: The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. CONCLUSIONS: This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Aspergillus sensitisation detection using point-of-care lateral flow assay in moderate to severe asthma.

Allergic fungal airway diseases are associated with asthma exacerbations and poor control. However, the early identification of allergic Aspergillus airway diseases can be challenging, especially in resource-poor countries. We aimed to evaluate the clinical utility of the point-of-care Aspergillus IgG-IgM lateral flow assay in diagnosing Aspergillus airway diseases in patients with moderate-severe asthma. Patients with moderate-severe asthma, severe asthma with fungal sensitisation (SAFS) and allergic bronchopulmonary aspergillosis (ABPA) were recruited. Clinical information was extracted from clinical records. Blood samples were collected for serological tests. Serum samples were evaluated using Aspergillus immunochromatographic test (ICT). A total of 65 patients were recruited into the study, of whom 23.1% had clinical diagnosis of ABPA, 18.5% had SAFS and 58.5% had moderate-to-severe asthma who did not fit ABPA or SAFS criteria. The ICT test gave a sensitivity of 69 [95% confidence interval: 51-88]% and a specificity of 77 [60-88]% in predicting a positive Aspergillus IgG test. The sensitivity and specificity for a positive Aspergillus IgE were 77 [59-88]% and 86 [71-94]%, respectively. The majority (sensitivity: 87 [62-96]%) of patients with ABPA had positive ICT results, with a specificity of 70%. The negative predictive value was high (95 [82-99]%) with a low negative likelihood ratio (< 0.2), making it potentially useful in ruling out ABPA. The ICT assay may be valuable in ruling out ABPA in resource-limited countries where serological investigations are less feasible. The ICT assay may be particularly useful in ruling out ABPA and warrants further validation.

Allergic Aspergillus diseases can make patients with asthma more unwell, but this is difficult to diagnose, especially in developing countries where tests are not widely available. We have found that a cheap, easy-to-use and fast test may be useful in diagnosing allergic Aspergillus diseases.