CRISPR-Mediated Detection of <i>Pneumocystis</i> Transcripts in Bronchoalveolar, Oropharyngeal, and Serum Specimens for <i>Pneumocystis</i> Pneumonia Diagnosis

Pneumocystis jirovecii pneumonia (PCP) is the culprit of many cases of fungal pneumonia, especially among immunocompromised individuals. Detection of this pathogen is routinely achieved via bronchoalveolar lavage (BAL), which is difficult to obtain and delays prompt diagnosis. Researchers developed a novel CRISPR-Cas12a assay that detects Pneumocystis RNA in easily accessible specimens like oropharyngeal swabs (OPS) and serum, offering a clinically practical alternative to invasive BAL procedures.The study included a pediatric cohort of hospitalized infants at Children’s Hospital New Orleans, where oropharyngeal swabs were collected from both PCP-positive and PCP-negative patients to evaluate CRISPR assay performance on noninvasive samples. The adult cohort consisted of patients at University Medical Center and affiliates, with bronchoalveolar lavage and serum samples collected from confirmed PCP cases and controls. Adult subgroups included HIV-positive individuals, solid organ transplant recipients, and other immunocompromised patients.Preclinical analysis included wild-type and Rag2−/− knockout mice infected with Pneumocystis murina. In mice, targets were assessed in lung tissue, BAL, and serum at weeks 2, 4, and 6 after infection. Clinical application used infant oropharyngeal samples and adult BAL/serum specimens from patients with confirmed PCP vs control noninfected participants. Reverse transcription polymerase chain reaction (RT-PCR) amplification of mRNA (eg, Sp, Gsc1) was followed by Cas12a/RNA detection of cleaved fluorescent reporters. Human samples underwent comparison of novel CRISPR assay vs traditional reverse transcription quantitative polymerase chain reaction PCR (RT-qPCR) (mtLSU marker) on OPS, serum, and BAL.In the mouse model, the CRISPR assay demonstrated sensitive detection of Pneumocystis transcripts in serum at all infection time points. In pediatric oropharyngeal swabs, the assay achieved 96.3% sensitivity and 100% specificity, outperforming RT-qPCR (66.7% sensitivity, 90.6% specificity). In adult serum, CRISPR sensitivity was 93.3% compared with just 26.7% with RT-qPCR.The findings support CRISPR-based molecular diagnostics as a powerful tool to detect active PCP infection in both pediatric and adult patients, without the need for bronchoscopy. Transcript-level targeting of trophic-form mRNA improves discrimination of active infection vs colonization, facilitating early intervention in both pediatric and adult patients.This study presents a significant leap forward in PCP diagnostics. This enhanced approach shows exceptional sensitivity on easily obtainable specimens, while detecting trophic-form mRNA adds specificity for active disease.