Clinical Research and Public HealthInfectious diseasePulmonology Open Access | 10.1172/JCI177241

Brady M. Youngquist,1 Ayanda Trevor Mnguni,2,3,4 Dora Pungan,5 Rachel PJ Lai,6 Guixiang Dai,5 Chun Fai Ng,7 Amy Samson,2 Yasmean Abdelgaliel,1 Christopher J. Lyon,1 Bo Ning,1 Shahid Husain,7 Sean Wasserman,8 Jay K. Kolls,5 and Tony Y. Hu1

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

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1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Mnguni, A. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

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1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

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1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Dai, G. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Ng, C. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Samson, A. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Abdelgaliel, Y. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Lyon, C. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Ning, B. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Husain, S. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Wasserman, S. in: JCI | PubMed | Google Scholar

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Kolls, J. in: JCI | PubMed | Google Scholar |

1Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

2Centre for Infectious Diseases Research in Africa, University of Cape Town, Cape Town, South Africa,

3Africa Mycology Unit, University of Cape Town, Cape Town, South Africa and Department of Internal Medicine, University of Cape Town, Cape Town, South Africa.

4Department of Internal Medicine, University of Stellenbosch, Stellenbosch, South Africa.

5Center for Translational Research in Infection and Inflammation, School of Medicine, Tulane University, New Orleans, Louisiana, USA.

6Department of Infectious Disease, Imperial College London, London, United Kingdom.

7Multi-Organ Transplant Program, Division of Infectious Diseases, Department of Medicine, University Health Network/ University of Toronto, Toronto, Ontario, Canada.

8St. George’s University, London, United Kingdom.

Address correspondence to: Jay K. Kolls 333 S. Liberty St., JBJ Rm 375, New Orleans, Louisiana, 70112, USA. Phone: 504.988.0455; Email: jkolls1@tulane.edu. Or to: Tony Hu 333 S. Liberty St., JBJ Rm 474, New Orleans, Louisiana, 70112, USA. Phone: 504.605.8004; Email: tonyhu@tulane.edu.

Authorship note: BMY, ATM, and DP contributed equally to this work.

Find articles by Hu, T. in: JCI | PubMed | Google Scholar

Authorship note: BMY, ATM, and DP contributed equally to this work.

Published March 3, 2025 - More info

Published in Volume 135, Issue 8 on April 15, 2025
J Clin Invest. 2025;135(8):e177241. https://doi.org/10.1172/JCI177241.
© 2025 Youngquist et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 3, 2025 - Version history
Received: November 20, 2023; Accepted: February 17, 2025 View PDF Abstract

BACKGROUND. Pneumocystis 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.

METHODS. Mouse 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.

RESULTS. The 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.

CONCLUSION. 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 Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.

FUNDING. 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.

Introduction

Molecular epidemiology evidence indicates that Pneumocystis jirovecii pneumonia (PCP) is the leading cause of fungal pneumonia in HIV-negative infants under 2 years old (1, 2), but PCP is also clinically relevant in adults and children with immunodeficiencies or who are receiving immunosuppressive regimens (2). P. jirovecii infections that cause severe disease and require mechanical ventilation can have mortality rates of 20%–25% (3). Rapid PCP diagnosis is required for effective therapeutic intervention, but current diagnostic tests require an invasive bronchoalveolar lavage (BAL) procedure to obtain diagnostic specimens, which can delay diagnosis (4, 5), and use Grocott methenamine silver (GMS) or immunofluorescent staining methods or PCR of P. jirovecii-specific genomic DNA to detect P. jirovecii infection (6). However, there is evidence that an organism-specific diagnostic that uses minimally invasive or noninvasive samples is needed to improve diagnosis (7–9). Direct fluorescent antibody staining of induced and expectorated sputum has variable sensitivity for P. jirovecii and is primarily useful in patients who are HIV-positive, who have higher P. jirovecii burdens than other patients with PCP (10), while a blood-based 1,3 β-D-glucan test used to diagnose PCP lacks specificity for P. jirovecii (11, 12). PCR-based assays for PCP can be more rapid, sensitive, and specific than staining procedures, but also primarily rely on BAL specimens and can detect P. jirovecii colonization events (P. jirovecii detected without pneumonia or with pneumonia caused by another pathogen). This can reduce their diagnostic value (13), since PCR values can vary widely in infected individuals, preventing the use of a universal threshold for PCP diagnosis (1, 14, 15). PCR tests have also been used to detect P. jirovecii DNA in oral wash, induced sputum, and serum specimens, but these tests have variable sensitivity and may also detect colonization events (7, 16–19). There is, therefore, still an urgent need for PCP diagnostics that use less invasive specimen types to provide rapid and accurate results that can guide treatment decisions.

Current PCR tests that target P. jirovecii genomic DNA can also detect P. jirovecii colonization events; however, there is no accepted threshold to distinguish colonization from active infection (1, 14, 20). We hypothesized that assays that detect and quantify mRNA transcripts that distinguish the troph and ascus life stages of P. jirovecii, rather than overall pathogen abundance, could improve specific detection of active infection, since each stage exhibits distinct metabolic activity and behavior during colonization and active infection (21, 22). More sensitive assays may be required to detect such transcripts, however, particularly in less invasive samples where Pneumocystis mRNA may be less abundant or rapidly degraded by environmental hydrolases.

CRISPR reactions employed to enhance the sensitivity and specificity of nucleic acid amplification assays (23–25) have been applied to diagnose viral, bacterial, and fungal infections in minimally invasive sample types including blood, saliva, nasal swabs, and urine (25). Such approaches can substantially improve assay sensitivity and specificity, since target amplification and detection relies on specific binding of a reverse transcription (RT) primer (for an RNA target), PCR amplification primers, and a guide RNA sequence that mediates the binding and trans-cleavage activity of a target-specific CRISPR Cas complex that can be employed to cleave a quenched reporter nucleotide and amplify the assay readout signal (23, 26). We therefore developed RT-PCR CRISPR Cas12a assays to sensitively and specifically detect Pneumocystis mRNAs that differentially overexpressed in the troph and ascus stages of P. jirovecii and P. murina, a closely related species that causes fungal pneumonia in mice (21).

Here, we describe the development and characterization of these assays and their performance to detect these stage-selective mRNA targets in serum and BAL samples of P. murina–infected mice and oropharyngeal swab in infants with P. jirovecii infection, and serum and BAL specimens of cohorts of adult patients with PCP. Our results detected a differential increased expression of the troph versus ascus marker in immunocompromised Rag2–/– mice at increased risk for active infection versus WT mice. Similarly, we observed that the P. jirovecii troph marker exhibited greater specificity for adults and infants diagnosed with PCP, although both markers were overexpressed in these cases, and there was a clear signal separation in individuals diagnosed with active PCP and those without active disease. These results suggest that similar assays could be employed with oropharyngeal swabs and serum to improve the diagnosis and monitoring of PCP cases required to improve patient outcomes.

Results

Study design for RT-PCR CRISPR clinical validation. RT-PCR CRISPR assays were developed and used to blindly analyze 107 retrospectively collected oropharyngeal swab samples obtained from the PERCH cohort (1), an international case-control study designed to analyze the incidence of pathogens that cause pneumonia in infants (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI177241DS1). This study examined samples collected from children aged 1–59 months who were admitted to the hospital with severe pneumonia and age-matched healthy controls from the same general communities, and used quantitative PCR to detect the P. jirovecii gene mtLSU in extracted nucleic acid samples at a threshold of greater than 1 × 104 copies/mL as a classifier for active disease. CRISPR and RT-qPCR assay sensitivity and specificity results were calculated against the corresponding PERCH study mtLSU qPCR swab results. RT-PCR CRISPR assays were also employed to blindly evaluate 32 BAL samples from 12 individuals with PCP infection and 20 individuals without P. jirovecii infection, using residual BAL specimens from patients who had PCP-positive pneumonia (qPCR-positive for P. jirovecii mtLSU DNA) or from patients who were PCP negative undergoing clinical surveillance after lung transplant or for other conditions (Supplemental Table 2).

To assess the potential for blood-based PCP diagnosis, CRISPR and RT-qPCR assays were used to blindly analyze matched BAL and serum samples from a prospective cohort of 27 adult patients with HIV with suspected PCP who were enrolled in an observational cohort study at Khayelitsha District Hospital in Cape Town, South Africa (Figure 1). Study participants, who had dyspnea and hypoxemia (sO2 ≤ 94% or PaO2 ≤ 10kPa) with an abnormal chest X-ray, were provided with PCP treatment and underwent BAL collection to confirm PCP using a P. jirovecii immunofluorescence assay (IFA) and had serum collected at the same time.

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.

Development and optimization of CRISPR-enhanced RT-PCR assays for two P. murina mRNA targets. Pneumocystis-derived biomarkers that distinguish replicating troph and nonreplicating ascus spores could permit development of assays that distinguish Pneumocystis infection from colonization to guide treatment decisions (Figure 2). Since we previously reported that P. murina serine protease (Sp) and 1,3-β glucan synthase subunit (Gsc1) mRNA transcripts are differentially upregulated in its troph and ascus stages, we hypothesized that RT-PCR CRISPR-Cas12a assays might have the sensitivity necessary to detect them in serum to permit minimally invasive diagnosis. We used an in silico approach to identify primer pairs and gRNAs to amplify and detect target sequences within these mRNAs (Supplemental Table 3).

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.

RT-PCR conditions for these mRNA targets were optimized by analyzing the CRISPR signal produced when their amplicons were generated over a range of annealing temperatures with cDNA generated from lung tissue homogenates of P. murina–infected mice, as previously described (21). CRISPR signal-to-noise ratios defined by the signal generated with and without input template (Supplemental Figure 1, A and B) identified optimum annealing temperatures for Sp and Gsc1 amplification (57.5°C and 59.9°C) that were used in all further analyses. Subsequent analyses identified the determined reporter concentration (667 pM) that produced the highest signal-to-noise ratio for the least amount of input probe (Supplemental Figure 1, C and D), and the Cas12a/gRNA concentration (67 pM) that yielded optimum signal kinetics for the amount of input Cas12a and gRNA (Supplemental Figure 1, E and F). No substantial signal increases were observed in the absence of input template, consistent with minimal reporter degradation.

Linearity and limit of detection (LoD) values for these optimized Sp and Gsc1 RT-PCR CRISPR assays were then determined using serial dilutions of synthetic Sp or Gsc1 DNA fragments spiked into healthy serum (1 × 10–1 to 1 × 106 copies/μL) (Supplemental Figure 2, A and B). These Sp and Gsc1 assays detected positive signals in serum concentration standards spiked with 0.3 and 1 copies/μL, respectively and had strong linear correlations with the spiked-in target amount (R2 values of 0.990 and 0.983) from their LoDs to the highest analyzed target concentration (1 × 104 copies/ μL) (Supplemental Figure 2, C and D). Sp and Gsc1 assay signal also demonstrated strong species specificity since positive signal was not detected when these assays were used to analyze genomic RNA or DNA of an array of common viral and microbial respiratory pathogens, including the related human pathogen P. jirovecii (Supplemental Figure 2, E and F).

Sp and Gsc1 detection in BAL and serum of P. murina–infected WT and Rag2–/– mice. Sp and Gsc1 RT-PCR CRISPR assays were used to analyze lung tissue, BAL, and serum specimens collected from C57BL6/J WT and immunocompromised (Rag2–/–) mice sacrificed 2-, 4-, and 6-weeks after inoculation with P. murina (Figure 3A), as this model reflects critical aspects of human disease (27, 28). Lung tissue Sp mRNA expression was higher in Rag2–/– than WT mice, and Gsc1 mRNA expression was higher in the lungs of WT than Rag2–/– mice (Figure 3, B and C). Lung tissue Sp and Gsc1 signal did not vary over time in Rag2–/– mice, but both significantly decreased at 6 weeks after inoculation in the WT mice, potentially indicating infection clearance. Sp and Gsc1 signal was less reliably detected in the BAL and serum samples of these mice (Figure 3, D–G), particularly the WT mice. Sp and Gsc1 signal was consistently detected in Rag2–/– mouse BAL and serum specimens at 4 weeks after inoculation, but signal for both targets was more variable in the matching WT mouse samples and in samples collected at 2 weeks after inoculation in both groups. Sp and Gsc1 signals tended to be greater in Rag2–/– mouse BAL versus serum specimens, and Sp signal tended to be consistently greater than Gsc1 signal throughout infection, consistent with a reduced ability of the Rag2–/– mice to suppress their P. murina infections, as neither difference was detected in the WT mouse samples. Sp-positive Rag2–/– mouse BAL and serum samples also tended to be Gsc1-positive by week 2 after inoculation, with double-positive results detected in all Rag2–/– mouse BAL and serum samples by week 4 after inoculation. By contrast, BAL and serum samples of the WT mice tended to be Sp-negative and Gsc1-negative at week 2 after inoculation, sporadically positive for both markers at week 4 after inoculation, and mostly negative for both markers at week 6 after inoculation, consistent with greater containment of their P. murina infections.

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).

Development and optimization of CRISPR-enhanced RT-PCR assays for P. jirovecii RNA targets. We next translated this approach to detect troph and ascus targets of P. jirovecii, as this human pathogen is closely related to P. murina. However, while a P. jirovecii–specific Gsc1 primer and gRNA set produced strong signal, those generated for the Sp homolog of P. jirovecii did not produce detectable signal (data not shown), likely due to low confidence in P. jirovecii Sp sequence data or polymorphisms. We therefore, instead, identified P. jirovecii RNAs that were differentially expressed and abundantly detected in a RNA-seq dataset of BAL specimens from 2 patients who were immunocompromised and diagnosed with P. jirovecii infections (29). Similar to previous work indicating that mitochondrial transcripts are enriched in troph-derived P. murina RNA, P. jirovecii mitochondrial RNAs were the most abundant differentially enriched transcripts detected in these samples (Figure 4, A and B), consistent with a previous study indicating that the trophic form of P. jirovecii plays a dominant role in pulmonary infections and that troph-derived P. murina RNA is enriched for mitochondrial RNA transcripts (21). NADH-ubiquinone oxidoreductase chain 4 (Nad4) was selected for further analysis since primers to this RNA amplified a region containing a candidate gRNA sequence with a conserved protospacer adjacent motif (PAM) site required for efficient Cas12a target recognition and cleavage activity. These primers and gRNA sequences were designed to avoid known Nad4 SNPs that might affect their binding and detection and lack substantial homology with corresponding Nad4 sequences of other Pneumocystis species.

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 sequenc