Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics

Abstract

<jats:title>Abstract</jats:title> <jats:p>The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.</jats:p>

<p>Metagenomic Next-Generation Sequencing in Diagnosis of a Case of <em>Pneumocystis jirovecii</em> Pneumonia in a Kidney Transplant Recipient and Literature Review</p>

“…To date, there have been 5 reports on successful use of mNGS to diagnose pulmonary infections involving P. jirovecii (Table 2). [14][15][16][17][18] While the sample size is small (1 to 13 specimens each), these studies involve the use of different types of specimens (BALF, sputum, lung biopsy and blood) and different NGS platforms. The numbers of sequence reads for P. jirovecii are highly variable (2 to 303,572) among studies though all are less than the P. jirovecii reads identified from our case (1,665,693).…”

“…This method has been successfully used as a diagnostic tool for various infectious diseases including PCP. [14][15][16][17] Its application to diagnose PCP in renal transplant recipients has been attempted in one study, 18 and its value awaits further evaluation. Here we report a case of PCP diagnosed by mNGS following negative results in multiple conventional diagnostic tests.…”

Pneumocystis jirovecii in Patients With Cystic Fibrosis: A Review

“…Finally, all these genotypic characteristics were obtained using a unilocus approach and Sanger's method of sequencing, while more informative assays such as multilocus sequence typing (MLST) combined with NGS are available today (Alanio et al, 2016 ; Charpentier et al, 2017 ). Moreover, shotgun metagenomics may represent an alternative method to identify pulmonary mycobiota including P. jirovecii organisms (Irinyi et al, 2020 ).…”

Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection

“…[7]. In particular, Nanopore technology provides the speed that can shorten sample-to-answer time, which is important for the severely ill. From literature, several research groups explored the use of Nanopore sequencing for pneumonia diagnosis, utilizing both metagenomic and targeted approaches [8][9][10][11]. In this study, we adopted the targeted approach because it enables higher depth of microbial reads and thus allowing more room for multiplexing which significantly lowers the cost.…”