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Detection of Aspergillus fumigatus by polymerase chain reaction
C Spreadbury1, D Holden, A Aufauvre-Brown
1Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Hammersmith Hospital, United Kingdom.
Abstract:
Aspergillus fumigatus is an opportunistic nosocomial pathogen causing an often fatal pneumonia, invasive aspergillosis (IA), in immunosuppressed patients. Oligonucleotide primers were used to amplify a 401-bp fragment spanning the 26S/intergenic spacer region of the rDNA complex of A. fumigatus by the polymerase chain reaction (PCR). The primers were highly sensitive and specific: as little as 1 pg of A. fumigatus genomic DNA could be detected, and the primers only amplified DNA from A. fumigatus and not any other fungal, bacterial, viral, or human DNA tested. Using the PCR, we were able to detect A. fumigatus DNA in lung homogenates from immunosuppressed mice experimentally infected with A. fumigatus but not from immunosuppressed uninfected controls. There was 93% correlation between the culture results and the PCR results. In a retrospective clinical study, the sensitivity of the PCR for the detection of A. fumigatus in clinical samples was confirmed by positive amplification in three of three culture-positive respiratory samples from confirmed cases of IA. Because isolation of Aspergillus spp. may reflect contamination and colonization without infection, the feasibility of using the PCR was evaluated by analyzing culture-negative samples from both immunosuppressed patients at high risk for IA and immunocompetent patients with other lung infections. Only 2 of 10 patients were culture negative and PCR positive in the high-risk group, and 2 of 7 patients were culture negative and PCR positive in the immunocompetent group. The results indicate that PCR detection might be a valuable adjunct to current laboratory methods to diagnose IA.
Insights
A novel polymerase chain reaction (PCR) assay accurately detects Aspergillus fumigatus DNA, offering a sensitive tool for diagnosing invasive aspergillosis (IA) in high-risk patients.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Aspergillus fumigatus is a significant opportunistic pathogen causing invasive aspergillosis (IA), a life-threatening pneumonia in immunocompromised individuals.
- Current diagnostic methods, like culture, can be limited by sensitivity and may not distinguish colonization from infection.
Purpose of the Study:
- To develop and validate a highly sensitive and specific polymerase chain reaction (PCR) assay for the detection of Aspergillus fumigatus.
- To evaluate the utility of this PCR assay as an adjunct diagnostic tool for invasive aspergillosis.
Main Methods:
- Oligonucleotide primers were designed to amplify a specific 401-bp fragment of the 26S/intergenic spacer region of the A. fumigatus rDNA complex.
- The PCR assay's sensitivity and specificity were tested using known amounts of A. fumigatus genomic DNA and various other DNA sources.
- The assay was validated in experimentally infected mice and retrospectively in human clinical samples, including culture-negative specimens.
Main Results:
- The PCR assay demonstrated high sensitivity, detecting as little as 1 pg of A. fumigigatus DNA, and was specific for A. fumigatus.
- A. fumigatus DNA was detected in lung homogenates of infected mice but not in controls, correlating with culture results (93% agreement).
- In clinical samples, the PCR assay showed promise in detecting A. fumigatus in culture-negative cases, suggesting its potential beyond traditional methods.
Conclusions:
- The developed PCR assay is a sensitive and specific molecular tool for detecting Aspergillus fumigatus.
- This PCR method may serve as a valuable adjunct to conventional laboratory diagnostics for invasive aspergillosis, particularly in differentiating infection from colonization.