Molecular detection of Aspergillus in respiratory samples collected from patients at higher risk of chronic pulmonary aspergillosis

Oliveira, M.; Pinto, M.; Simões, H.; Gomes, J.P.; Veríssimo, C.; Sabino, R.

http://hdl.handle.net/10400.18/8508

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Abstract(s)

Objective: Aspergillosis diagnosis depends on the detection of Aspergillus in biological samples ─ usually using cultural and immunoenzyme techniques ─ but their sensitivity and specificity varies. We aimed to study the prevalence of Aspergillus in patients at higher risk of chronic pulmonary aspergillosis (i.e., HIV-infected patients and individuals with active or previous tuberculosis), and to determine the potential role of molecular approaches to increase detection of Aspergillus in respiratory samples. Methods: The DNA extracted from 43 respiratory samples that had been previously analyzed by immunoenzyme and/or cultural techniques was amplified by real-time multiplex PCR, and the results of these methods were compared. We also sequenced the ITS1 region and the calmodulin gene in 10 respiratory samples to perform a pilot metagenomic study to understand the ability of this methodology to detect potential pathogenic fungi in the lung mycobiome. Results: Real-time Aspergillus PCR test exhibited a higher positivity rate than the conventional techniques used for aspergillosis diagnosis, particularly in individuals at risk for chronic pulmonary aspergillosis. The metagenomic analysis allowed for the detection of various potentially pathogenic fungi. Conclusions: Molecular techniques, including metagenomics, have great ability to detect potentially pathogenic fungi rapidly and efficiently in human biological samples.

Highlights: Aspergillus real-time PCR detect showed higher sensitivity than conventionalmethodologies; Aspergillusreal-time might be a particularly useful tool to help in the diagnose of CPA; Metagenomic allows the quick and easy detection of potentially pathogenic fungi; Metagenomic permits identification of a high fungal variety in respiratory samples.