CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis
Bibliographic record
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".