{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T05:14:33Z","timestamp":1786943673013,"version":"build-2736575974"},"reference-count":65,"publisher":"American Society for Clinical Investigation","issue":"8","license":[{"start":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T00:00:00Z","timestamp":1755043200000},"content-version":"vor","delay-in-days":163,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["R01AI120033"],"award-info":[{"award-number":["R01AI120033"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000050","name":"NHLBI","doi-asserted-by":"crossref","award":["R35HL139930"],"award-info":[{"award-number":["R35HL139930"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000272","name":"NIHR","doi-asserted-by":"crossref","award":["project 134342"],"award-info":[{"award-number":["project 134342"]}],"id":[{"id":"10.13039\/501100000272","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,4,15]]},"abstract":"<jats:sec>\n                    <jats:title>BACKGROUND<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Pneumocystis jirovecii<\/jats:italic>\n                      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\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>jirovecii<\/jats:italic>\n                      diagnosis using swabs and serum. Herein, we describe the development of an ultrasensitive RT-PCR\u2013coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>METHODS<\/jats:title>\n                    <jats:p>\n                      Mouse analyses employed an RT-PCR CRISPR assay to analyze\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>murina<\/jats:italic>\n                      transcripts in WT and\n                      <jats:italic>\n                        Rag2\n                        <jats:sup>\u2013\/\u2013<\/jats:sup>\n                      <\/jats:italic>\n                      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\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>jirovecii<\/jats:italic>\n                      transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from patients who were infected with\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>jirovecii<\/jats:italic>\n                      and those who were not.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>RESULTS<\/jats:title>\n                    <jats:p>\n                      The\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>murina<\/jats:italic>\n                      assays sensitively detected\n                      <jats:italic>Pneumocystis<\/jats:italic>\n                      RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>jirovecii<\/jats:italic>\n                      with greater sensitivity (96.3% versus 66.7%) and specificity (100% versus 90.6%) than RT-qPCR compared with mitochondrial large subunit rRNA gene (\n                      <jats:italic>mtLSU<\/jats:italic>\n                      ) standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% versus 26.7%) in adult serum specimens.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>CONCLUSION<\/jats:title>\n                    <jats:p>\n                      Since 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\n                      <jats:italic>Pneumocystis<\/jats:italic>\n                      diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>FUNDING<\/jats:title>\n                    <jats:p>The 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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1172\/jci177241","type":"journal-article","created":{"date-parts":[[2025,3,3]],"date-time":"2025-03-03T12:00:32Z","timestamp":1741003232000},"source":"Crossref","is-referenced-by-count":3,"title":["CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis"],"prefix":"10.1172","volume":"135","author":[{"given":"Brady M.","family":"Youngquist","sequence":"first","affiliation":[{"name":"Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayanda Trevor","family":"Mnguni","sequence":"additional","affiliation":[{"name":"Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,"},{"name":"Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa."},{"name":"Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5631-2543","authenticated-orcid":true,"given":"Dora","family":"Pungan","sequence":"additional","affiliation":[{"name":"Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel PJ","family":"Lai","sequence":"additional","affiliation":[{"name":"Department of Infectious Disease, Imperial College London, London, United 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