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Development and evaluation of a molecular viability assay for Pneumocystis carinii
N Maher1, S Vermund, M Lasbury
1Division of Geographic Medicine, Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Abstract:
Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 10(6) trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.
Insights
A new molecular viability assay detects viable Pneumocystis carinii (PCP) organisms using mRNA detection. This method accurately identifies live Pneumocystis carinii in air and respiratory samples, crucial for understanding PCP transmission.
Area of Science:
- Medical Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Pneumocystis carinii pneumonia (PCP) is a significant opportunistic illness in individuals with AIDS.
- Detecting viable Pneumocystis carinii in environmental and clinical samples is challenging.
- Current methods cannot reliably determine the viability of detected Pneumocystis carinii.
Purpose of the Study:
- To develop and validate a molecular viability assay for Pneumocystis carinii.
- To differentiate between viable and non-viable Pneumocystis carinii organisms.
- To assess the utility of the assay for environmental and clinical sample analysis.
Main Methods:
- Development of a reverse transcription-PCR (RT-PCR) assay targeting Pneumocystis carinii mRNA.
- Utilizing primers specific to a heat shock protein 70 family member.
- Validation using ethidium bromide staining to confirm viability and testing against non-viable organisms (heat, desiccation, UV).
- Assessing specificity against common airborne fungi.
Main Results:
- The assay detected as few as 100 viable Pneumocystis carinii trophozoites.
- No signal was detected from 10^6 non-viable trophozoites.
- The assay successfully distinguished Pneumocystis carinii from other fungi.
- The assay demonstrated potential for detecting viable organisms in bioaerosols and respiratory specimens.
Conclusions:
- A novel molecular viability assay for Pneumocystis carinii has been successfully developed and evaluated.
- This assay can accurately detect viable Pneumocystis carinii in environmental and clinical samples.
- The assay holds promise for improving the detection and understanding of PCP transmission and infection.