CRISPR-Mediated Detection of Pneumocystis in Non-Invasive Specimens for Pneumocystis Pneumonia Diagnosis

Brady M. Youngquist, PhD Student, Department of Molecular Biology and Biochemistry, Tulane University

Tony Y. Hu, Professor, the Weatherhead Presidential Chair in Biotechnology Innovation and director of the Tulane Center for Cellular & Molecular Diagnostics, Tulane University

Editor: Jing Cao, PhD, DABCC, FADLM, Medical Director of mass spectrometry at William P. Clements Jr. University Hospital and Director of Point-of-Care Testing at Children’s Health; Associate Professor of Pathology at the University of Texas Southwestern Medical Center.

The March issue of the Journal of Clinical Investigation published a study led by NACCCA member, Dr. Tony Ye Hu at Tulane University on a CRISPR-based test that diagnoses Pneumocystis jirovecii pneumonia (PCP), a life-threatening fungal infection primarily affecting children and immunocompromised patients, more quickly and less invasively.

PCP is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are obtained through bronchoscopy, an expensive and invasive procedure. Herein the authors developed an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant oropharyngeal swabs and adult BAL and serum. CRISPR enhances assay sensitivity to allow robust P. jirovecii diagnosis.

This study involves mouse analyses that employed an RT-PCR CRISPR assay to analyze P. murina transcripts in wild-type and Rag2–/– mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks post-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.

Authors reported that the P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay identified infants infected with P. jirovecii with greater sensitivity (96.3% vs. 66.7%) and specificity (100% vs. 90.6%) than RT-qPCR, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% vs. 26.7%) in adult serum specimens.

Since swabs are routinely collected in pediatric pneumonia patients 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, eliminating the need for invasive bronchoscopy procedures currently used for diagnosis.

In addition to diagnosing patients, this test can be used on throat swabs already gathered in clinics to improve epidemiology and mapping of pneumocystis in the United States. Looking ahead, the research team sees potential for the CRISPR diagnostic platform to detect other respiratory infections.

Reference

Brady M Youngquist, Tony Y Hu et al. CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis. J Clin Invest; 2025 Mar 3: e177241.

https://www.jci.org/articles/view/177241/pdf

CRISPR介导的非侵入性标本中肺孢子菌的检测用于肺孢子菌肺炎诊断

三月刊的《临床调查杂志》发表了一项由杜兰大学的 NACCCA 成员 Tony Ye 胡 博士领导的一项基于 CRISPR 的测试研究，该测试可以更快和侵入性更小地诊断耶氏肺孢子菌肺炎 （PCP），这是一种危及生命的真菌感染，主要影响儿童和免疫功能低下的患者。PCP 是导致真菌性肺炎的主要原因，但其诊断主要依赖于支气管镜检查获得的支气管肺泡灌洗液 （BAL） 标本, 这是一种昂贵且侵入性强的手术。本文作者开发了一种超灵敏的 RT-PCR 偶联 CRISPR 检测方法，在婴儿口咽拭子、成人 BAL 和血清中具有很高的活性感染特异性。

CRISPR 提高了检测的灵敏度，以实现高性能的 P. jirovecii 诊断。本研究涉及小鼠分析，采用 RT-PCR CRISPR 测定法分析感染后 2 周、 4 周和 6 周野生型和 Rag2–/– 小鼠肺 RNA、BAL 和血清中的鼠假单胞菌转录物。人体研究使用优化的 RT-PCR CRISPR 检测法检测婴儿口咽拭子样本、成人血清和成人 BAL 样本中的 P. jirovecii 转录本。

作者报道，鼠源检测在整个感染过程中灵敏地检测到感染小鼠血清中的肺孢子菌 RNA。口咽拭子检测结果显示, CRISPR检测方法在感染耶氏假单胞菌的婴儿中比 RT-qPCR 具有更高的敏感性 （96.3% vs. 66.7%） 和特异性 （100% vs. 90.6%），并且 在成人血清标本中, CRISPR检测 结果的敏感性（93.3% vs. 26.7%）也高于RT-qPCR 检测结果。由于在小儿肺炎患者中常规采集拭子样本，并且血清比 BAL 更容易获得，因此这种检测方法可以通过实现对活性感染的特异性来提高儿童和成人肺孢子菌诊断的准确性和时效性，并可能避免对 BAL 标本的要求，从而消除目前用于诊断的侵入性支气管镜检查程序的需要。

除了诊断患者外，该检测方法还可用于诊所已经收集的咽拭子，以改善美国肺孢子病的流行病学和分布图。展望未来，该研究团队认为 CRISPR 诊断平台有潜力推广到其他呼吸道感染的检测。