Related Experiment Video
Updated: Oct 7, 2025
Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip
Published on: March 29, 2024
Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in
Jie Yi1,2,3, Nan Wang4,5,6, Jie Wu1,2,3
1State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China.
Abstract:
Background: Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR. Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR. Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR. Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.
Insights
Droplet digital PCR (ddPCR) offers superior sensitivity for detecting Pneumocystis jirovecii DNA, especially in low-load infections, compared to quantitative PCR (qPCR). This makes ddPCR a more reliable tool for diagnosing Pneumocystis pneumonia (PCP) in immunocompromised patients.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Pneumocystis jirovecii causes life-threatening Pneumocystis pneumonia (PCP) in immunocompromised individuals.
- Accurate detection of P. jirovecii is crucial for managing opportunistic infections.
- Quantitative PCR (qPCR) is limited by low fungal load and PCR inhibitors.
Purpose of the Study:
- To develop and evaluate a droplet digital PCR (ddPCR) method for P. jirovecii DNA detection.
- To compare the sensitivity of ddPCR against qPCR in respiratory specimens.
- To assess ddPCR's utility for P. jirovecii quantification in clinical settings.
Main Methods:
- Developed a ddPCR assay for P. jirovecii DNA detection in respiratory samples.
- Tested bronchoalveolar fluid (BALF) and sputum samples from patients with suspected or diagnosed PCP using both ddPCR and qPCR.
- Utilized metagenomic next-generation sequencing (mNGS) for discrepant results and analyzed treatment impact on fungal load.
Main Results:
- ddPCR demonstrated higher sensitivity than qPCR, detecting P. jirovecii in four BALF samples missed by qPCR.
- ddPCR identified P. jirovecii in eight sputum samples negative by qPCR.
- ddPCR detected P. jirovecii post-treatment when qPCR showed undetectable levels, indicating superior sensitivity for low fungal loads.
Conclusions:
- ddPCR is more sensitive than qPCR for detecting P. jirovecii DNA, particularly in samples with low pathogen burden.
- ddPCR offers a viable and reliable alternative to qPCR for P. jirovecii testing in immunocompromised patients.
- The enhanced sensitivity of ddPCR aids in more accurate diagnosis and monitoring of Pneumocystis pneumonia.
