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Oropharyngeal samples for detection of Pneumocystis carinii by DNA amplification
A E Wakefield1, R F Miller, L A Guiver
1Department of Paediatrics, John Radcliffe Hospital, Oxford.
Abstract:
Pneumocystis carinii pneumonia is a major complication of T-lymphocyte immune deficiency. Restriction of the disease to the alveolar spaces and failure to culture R. carinii has hindered simple diagnostic methods. We have developed a specific DNA amplification method for P. carinii and shown diagnostic sensitivity and specificity exceeding 95% for pneumocystis pneumonia when applied to bronchoscopic lavage and hypertonic saline induced sputum. We here report application of DNA amplification to simple oropharyngeal samples in 31 HIV-positive patients with respiratory illness. P. carinii-specific DNA was detected in 10 of 18 (56%) patients with pneumocystis pneumonia by ethidium bromide stained gels and 14 of 18 (78%) patients by the more sensitive technique of oligoblotting. P. carinii DNA was not detected in samples from 13 patients with other respiratory diagnoses. An oropharyngeal sample offers a simple specimen for detecting P. carinii by DNA amplification; refinements of technique and calibration may allow its development for accurate diagnostic and epidemiological work.
Insights
Diagnosing Pneumocystis pneumonia (PCP) in immunocompromised patients is challenging. A new DNA amplification method using oropharyngeal samples shows promise for accurate, non-invasive PCP detection.
Area of Science:
- Medical Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Pneumocystis carinii pneumonia (PCP) is a significant opportunistic infection in individuals with T-lymphocyte deficiencies.
- Current diagnostic methods for PCP are often invasive or lack sensitivity, hindering early detection.
- P. carinii resides in alveolar spaces, and traditional culture methods are unreliable.
Purpose of the Study:
- To evaluate the efficacy of a DNA amplification technique for detecting P. carinii in oropharyngeal samples.
- To assess the diagnostic potential of oropharyngeal sampling for Pneumocystis pneumonia in HIV-positive patients.
Main Methods:
- Development of a specific DNA amplification assay for P. carinii.
- Application of the assay to oropharyngeal samples from 31 HIV-positive patients with respiratory symptoms.
- Detection of P. carinii DNA using ethidium bromide stained gels and oligoblotting.
Main Results:
- P. carinii DNA was detected in 56% of PCP patients via standard gels and 78% via oligoblotting.
- No P. carinii DNA was found in samples from 13 patients with alternative respiratory diagnoses.
- Oligoblotting demonstrated higher sensitivity for P. carinii DNA detection in oropharyngeal samples.
Conclusions:
- Oropharyngeal samples are suitable for P. carinii detection using DNA amplification.
- This non-invasive method offers a simpler approach to diagnosing Pneumocystis pneumonia.
- Further refinement may enable accurate diagnostic and epidemiological applications.