BACKGROUNDPneumocystis jirovecii pneumonia (PCP) is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are difficult to obtain. Oropharyngeal swabs and serum could improve the PCP diagnostic workflow, and we hypothesized that CRISPR could enhance assay sensitivity to allow robust P. jirovecii diagnosis using swabs and serum. Herein, we describe the development of an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum.METHODSMouse analyses employed an RT-PCR CRISPR assay to analyze P. murina transcripts in WT and Rag2-/- mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks after infection. Human studies used an optimized RT-PCR CRISPR assay to detect P. jirovecii transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from patients who were infected with P. jirovecii and those who were not.RESULTSThe P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with P. jirovecii with greater sensitivity (96.3% versus 66.7%) and specificity (100% versus 90.6%) than RT-qPCR compared with mitochondrial large subunit rRNA gene (mtLSU) standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% versus 26.7%) in adult serum specimens.CONCLUSIONSince swabs are routinely collected in pediatric patients with pneumonia and serum is easier to obtain than BAL, this assay approach could improve the accuracy and timing of pediatric and adult Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.FUNDINGThe work was supported by the NIH (R01AI120033), NHLBI (R35HL139930), the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program, and by research funding provided by National Institute of Allergy and Infectious Diseases (NIAID) (R01AI144168, R01AI175618, R01AI173021). This research was also funded by the NIHR (project 134342) using UK aid from the UK government to support global health research.

Pneumocystis jirovecii is the most common cause of fungal pneumonia in children under the age of 2 years. However, the inability to culture and propagate this organism has hampered the acquisition of a fungal genome as well as the development of recombinant antigens to conduct seroprevalence studies. In this study, we performed proteomics on Pneumocystis-infected mice and used the recent P. murina and P. jirovecii genomes to prioritize antigens for recombinant protein expression. We focused on a fungal glucanase due to its conservation among fungal species. We found evidence of maternal IgG to this antigen, followed by a nadir in pediatric samples between 1 and 3 months of age, followed by an increase in prevalence over time consistent with the known epidemiology of Pneumocystis exposure. Moreover, there was a strong concordance of anti-glucanase responses and IgG against another Pneumocystis antigen, PNEG_01454. Taken together, these antigens may be useful tools for Pneumocystis seroprevalence and seroconversion studies.

Pneumocystis pneumonia (PCP) is a serious fungal lung infection caused by Pneumocystis jirovecii in immunosuppressed individuals. The lack of viable in vitro/ex vivo PCP models has greatly hindered the progress in studying the biology of these fungi, the host/pathogen interactions, as well as antifungal susceptibility testing. In this study, we show the utility of precision-cut lung slices (PCLS) to support the survival of Pneumocystis murina in vitro. We cultured PCLS tissue derived from wild type and immunocompromised mice with a P. murina inoculum in submerged or air-liquid interface models for up to 14 days. We isolated total RNA from the cultured lung tissues at days 0, 3, 7, 10, and 14 and analyzed for the expression of host lung genes and P. murina genes (Gsc1 for asci and Sp for trophs) by real-time quantitative PCR. When cultured in media alone, P. murina died gradually within a few days. However, when cultured on PCLS, both the troph and ascus forms survived throughout the incubation period of 2 weeks. Moreover, immunohistochemistry staining of P. murina inoculated PCLS sections using polyclonal anti-Pneumocystis sera and showed evidence of fungal aggregation and possible biofilm formation. Additionally, in vitro (PCLS) antibiotic susceptibility testing using commonly used antifungal drugs confirmed successful targeting of the troph and ascus forms by trimethoprim sulfamethoxazole and the ascus form by echinocandins.IMPORTANCEOur study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

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.

Pneumocystis is the most common fungal pulmonary infection in children under the age of 5 years. In children with primary immunodeficiency, Pneumocystis often presents at 3–6 months of age, a time period that coincides with the nadir of maternal IgG and when IgM is the dominant Ig isotype. Because B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal-specific IgMs in humans and mice and that these IgM specificities would predict T cell antigens. We detected fungal-specific IgMs in human and mouse sera and utilized immunoprecipitation to determine whether any antigens were similar across donors. We then assessed T cell responses to these antigens and found anti-Pneumocystis IgM in WT mice, Aicda–/– mice, and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide-specific lung T cell responses after P. murina infection. After mice were immunized with 2 of the antigens, adoptive transfer of vaccine-elicited CD4+ T cells showed effector activity, suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline-encoded IgM B cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK+CD8+ T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK+ Taa cells as a potential target to address age-associated dysfunctions of the immune system.Copyright © 2020 Elsevier Inc. All rights reserved. PMID: 33271118