CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis

Abstract

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.

Keywords: Diagnostics; Infectious disease; Mitochondria; Mouse models; Pulmonology.

Figure 1. Study Participants from Cape Town, South Africa.

After enrollment and screening, RT-PCR CRISPR was evaluated by blind analysis of BAL and serum from a cohort of adult patients from South Africa who were 27 HIV positive with and without PCP confirmed by Pneumocystis jirovecii immunofluorescence. Study participants were enrolled with dyspnea and hypoxemia (sO₂ ≤ 94% or PaO₂≤ 10kPa) and an abnormal chest X-ray. BAL and serum were obtained from patients at baseline before treatment initiation, and diagnosis was achieved from collected BAL specimens using the P. jirovecii immunofluorescence assay.

Figure 2. Overview of the RT-PCR CRISPR assay workflow for P. jirovecii diagnosis.

(A) RNA isolates from oropharyngeal swab or serum specimens were subjected to RT-PCR to amplify a target mRNA differentially expressed in the fungal trophic form required for active infection. These amplicons were recognized by a Cas12a/gRNA complex that cleaved and derepressed a quenched fluorescent probe in proportion to amplicon abundance. (B) DNA and mRNA phenotypes expected in children with P. jirovecii colonization and infection events and (C) characteristics of conventional qPCR and proposed RT-PCR CRISPR assays for P. jirovecii infection.

Figure 3. Sp and Gsc1 assay performance in serial BAL and serum from P. murina–infected mice.

(A) Scheme showing mouse infection and sampling time course with analysis of P. murina ascus- and trophic-life form transcripts Sp and Gsc1. Sp and Gsc1 assay signal in mouse (B and C) lung RNA, (D and E) BAL and (F and G) serum at 2-, 4-, and 6-weeks after inoculation with P. murina. Graphs indicate mean ± SD values of triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-sample Welch’s t test corrected for multiple comparisons by the Holm-Šidák method (WT versus Rag2^–/–) or performed without correction (4 versus 6 weeks after infection).

Figure 4. Characterization of Nad4 and Gsc1 assay performance in spiked samples.

(A) Ranked list of the most abundant and differentially detected P. jirovecii RNAs identified by sequencing of BAL samples of two patients who were positive for P. jirovecii after subtractive hybridization to remove host-derived RNA transcripts. (B) Genomic organization of enriched P. jirovecii mitochondrial genes and alignment of the P. jirovecii Nad4 primer and gRNA sequences with corresponding sequence regions of other Pneumocystis species (red text denotes sequence mismatches). LoD analyses for the (C) Nad4 and (D) Gsc1 CRISPR assays and (E) a matching Nad4 RT-qPCR assay, and the linear detection range data for the (F) Nad4, (G) Gsc1 CRISPR assays, and for (H) RT-qPCR Nad4. Species specificity of the P. jirovecii (I) Nad4 and (J) Gsc1 assays when analyzing samples spiked with corresponding sequences from other respiratory pathogens. NTC, no template control. Graphs indicate mean ± SD values of triplicate analyses. Standard curve graphs indicate the linear regression line of the data, its 95% CI, and Pearson coefficient.

Figure 5. Characterization of Nad4 assay performance with infant oropharyngeal swab and adult BAL samples.

(A) Heatmap of CRISPR and RT-qPCR assay positive (red) and negative (blue) results for Nad4 and Gsc1 in infant oropharyngeal swab and adult BAL samples from patients with and without P. jirovecii infection. Nad4 levels detected in (B) infant oropharyngeal swab and (C) adult BAL samples from North America, where positive signal was defined as signal that exceeded a threshold of the mean plus 3 times the SD of triplicate NTC samples (vertical dashed lines). (D and E) Heatmap of CRISPR and RT-qPCR assay positive (red) and negative (blue) results for Nad4 and Gsc1 in adult BAL and serum samples from PCP-positive and -negative cases determined by immunofluorescence assay (IFA). Nad4 levels detected in (F) adult BAL and (G) adult serum samples from patients in South Africa, where positive signal was defined as signal that exceeded a threshold of the mean plus 3 times the SD of triplicate NTC samples (vertical dashed lines).