A real-time polymerase chain reaction assay for detection of Pneumocystis from bronchoalveolar lavage fluid
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Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment
Published on: June 23, 2019
Rodney C Arcenas1, James R Uhl, Seanne P Buckwalter
1Division of Clinical Microbiology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Abstract:
Pneumocystis jiroveci is an important cause of pneumonia in immunocompromised individuals. This organism cannot be cultured, and therefore, diagnosis relies on microscopic identification of the organism using stains or antibodies. Although simple, these tests are insensitive and require expertise for accurate interpretation. We developed a real-time polymerase chain reaction (PCR) assay that provides sensitive and objective detection of Pneumocystis from bronchoalveolar lavage fluid. Primers and fluorescence resonance energy transfer probes were developed that target the cdc2 gene of P. jiroveci. Assay sensitivity is 6 copies of target per microliter of sample. No cross-reactivity occurs with other pathogens, and the PCR assay has a 21% increase in clinical sensitivity as compared with Calcofluor white staining. The real-time PCR assay provides a sensitive, rapid, and objective method for the detection of Pneumocystis from bronchoalveolar lavage fluid.
Insights
A new real-time polymerase chain reaction (PCR) assay offers sensitive and objective detection of Pneumocystis pneumonia in immunocompromised patients. This molecular method improves upon traditional staining techniques for diagnosing this opportunistic infection.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Pneumocystis jiroveci causes pneumonia in immunocompromised individuals.
- Current diagnostic methods like staining are insensitive and require expertise.
- P. jiroveci cannot be cultured in vitro.
Purpose of the Study:
- To develop a sensitive and objective diagnostic assay for P. jiroveci.
- To improve the detection of Pneumocystis pneumonia.
Main Methods:
- Development of a real-time polymerase chain reaction (PCR) assay.
- Design of primers and fluorescence resonance energy transfer (FRET) probes targeting the cdc2 gene of P. jiroveci.
- Testing the assay on bronchoalveolar lavage (BAL) fluid samples.
Main Results:
- The real-time PCR assay demonstrated high sensitivity (6 copies/µL).
- The assay showed no cross-reactivity with other common respiratory pathogens.
- Clinical sensitivity increased by 21% compared to Calcofluor white staining.
Conclusions:
- Real-time PCR is a sensitive, rapid, and objective method for detecting P. jiroveci in BAL fluid.
- This molecular assay offers a significant improvement over conventional diagnostic methods.
- The developed PCR assay aids in the timely diagnosis of Pneumocystis pneumonia.
