Targeted NGS-based Analyses of Pneumocystis jiroveci reveals geographically clustered haplotypes

Pneumocystis jiroveci 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 in NP/throat swabs. Given that Pneumocystis consists of an environmental ascus form and a trophic from (the latter is the form that attaches to lung epithelium), it is possible that life-from specific assays may be useful in non-bronchoscopic diagnosis. However, to accomplish this goal, these assays require genotypic information as the current fungal genomic data is largely from the US and Europe. To genotype Pneumocystis across the globe we developed a NGS based genotyping assay focused on genes expressed in the ascus as well as trophs using PERCH throat swabs from Africa, Bangla-desh, and Thailand as well as North American samples. The NGS panel reliably detected 21 fungal targets in these samples and revealed unique haplotypes in genes expressed in trophs in-cluding Meu10, an ascospore assembly gene, as well as in two in the mitochondrial gene ATP8, as well as intergenic region between COX1 and ATP8. This assay can be used for enhanced Pneumo-cystis epidemiology to study outbreaks but also permit more accurate RT-CPR or CRISPR based assays to improve non-bronchoscopic diagnoses of this under-reported fungal pathogen.