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Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes
Dora Pungan1, Taylor Eddens2, Kejing Song1
1Center for Translational Research in Infection and Inflammation, John W. Deming Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA.
Abstract:
Pneumocystis jirovecii is an important etiological agent of pneumonia that is underdiagnosed due to the inability to culture the organism. The 2019 PERCH study identified Pneumocystis as the top fungal cause of pneumonia in HIV-negative children using a PCR cutoff of 104 copies of Pneumocystis per mL of sample in nasopharyngeal/oropharyngeal (NP/OP) specimens. Given that Pneumocystis consists of an environmental ascus form and a trophic from (the latter is the form that attaches to the lung epithelium), it is possible that life-form-specific molecular assays may be useful for diagnosis. However, to accomplish this goal, these assays require genotypic information, as the current fungal genomic data are largely from the US and Europe. To genotype Pneumocystis across the globe, we developed an NGS-based genotyping assay focused on genes expressed in asci as well as trophs using PERCH throat swabs from Africa, Bangladesh, and Thailand, as well as North American samples. The NGS panel reliably detected 21 fungal targets in these samples and revealed unique genotypes in genes expressed in trophs, including Meu10, an ascospore assembly gene; two in mitochondrial gene ATP8, and the intergenic region between COX1 and ATP8. This assay can be used for enhanced Pneumocystis epidemiology to study outbreaks but also permits more accurate RT-CPR- or CRISPR-based assays to be performed to improve the non-bronchoscopic diagnosis of this under-reported fungal pathogen.
Insights
Pneumocystis jirovecii pneumonia is underdiagnosed. A new NGS assay identified unique genotypes in fungal pathogens from global samples, enabling better diagnostics and epidemiology.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Disease Epidemiology
Background:
- Pneumocystis jirovecii causes pneumonia but is difficult to culture, leading to underdiagnosis.
- The 2019 PERCH study highlighted P. jirovecii as a major fungal pneumonia cause in children.
- Genomic data for P. jirovecii are limited globally, hindering diagnostic assay development.
Purpose of the Study:
- To develop a next-generation sequencing (NGS) based genotyping assay for P. jirovecii.
- To analyze P. jirovecii genotypes from diverse global samples, including different life forms.
- To enable improved molecular diagnostic and epidemiological studies of P. jirovecii.
Main Methods:
- Developed an NGS panel targeting genes expressed in P. jirovecii asci and trophs.
- Utilized PERCH throat swabs from Africa, Bangladesh, Thailand, and North America.
- Analyzed 21 fungal targets, including specific genes and intergenic regions.
Main Results:
- The NGS assay successfully detected P. jirovecii in diverse global samples.
- Identified unique genotypes in troph-expressed genes, including Meu10, ATP8, and the COX1-ATP8 intergenic region.
- Demonstrated the assay's capability to differentiate genotypes across geographic locations.
Conclusions:
- The developed NGS assay provides crucial genotypic information for P. jirovecii.
- This tool can enhance global P. jirovecii epidemiology and outbreak investigations.
- Facilitates the development of more accurate non-bronchoscopic diagnostic assays, such as RT-CPR and CRISPR-based methods.
