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General primer-mediated PCR for detection of Aspergillus species
W J Melchers1, P E Verweij, P van den Hurk
1Department of Medical Microbiology, University Hospital Nijmegen, The Netherlands.
Abstract:
A PCR assay was developed for the diagnosis of invasive aspergillosis in immunocompromised patients. For this purpose, the complete nucleotide sequences of the genes encoding the 18S rRNA of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, and Aspergillus flavus were elucidated and aligned to the sequences of Aspergillus fumigatus and other clinically relevant prokaryotic and eukaryotic microorganisms. Genus-specific sequences could be identified in the V7 to V9 region of 18S rRNA. By using hot-start PCR, Southern blot hybridization, and restriction enzyme analysis, Aspergillus-specific and -sensitive determination was achieved. Five of six immunosuppressed mice experimentally infected with A. fumigatus developed infection, and rRNA could be detected in each case, even in livers with the absence of positive cultures. Aspergillus species were detected by PCR in four neutropenic patients with proven aspergillosis, although Aspergillus species had been isolated from only one bronchoalveolar lavage (BAL) fluid sample. Aspergillus species were detected by PCR in two more patients suspected of having infection. Positive PCR signals were obtained from the BAL samples of 3 of 8 neutropenic patients who had developed pulmonary infiltrates, but none were obtained from the samples of 14 nonimmunosuppressed patients. These results indicate the potential value of PCR to detect Aspergillus species in BAL samples and, therefore, to identify neutropenic patients at risk for invasive aspergillosis.
Insights
A new PCR assay accurately detects Aspergillus species in immunocompromised patients, aiding early diagnosis of invasive aspergillosis. This molecular test shows promise for identifying at-risk individuals, even when traditional cultures are negative.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive aspergillosis is a serious fungal infection, particularly in immunocompromised patients.
- Accurate and early diagnosis is crucial for effective treatment and improved patient outcomes.
- Current diagnostic methods, including culture, have limitations in sensitivity and speed.
Purpose of the Study:
- To develop and validate a Polymerase Chain Reaction (PCR) assay for the specific and sensitive detection of Aspergillus species.
- To evaluate the utility of the PCR assay in diagnosing invasive aspergillosis in immunocompromised patients and experimentally infected mice.
Main Methods:
- Sequencing and alignment of 18S rRNA genes from various Aspergillus species and other microorganisms.
- Design and application of genus-specific PCR primers targeting the V7-V9 region of 18S rRNA.
- Validation using Southern blot hybridization, restriction enzyme analysis, and testing of clinical samples (bronchoalveolar lavage - BAL).
Main Results:
- The PCR assay demonstrated specificity for Aspergillus species.
- Detection of Aspergillus rRNA in experimentally infected mice, including cases with negative cultures.
- Successful detection of Aspergillus species by PCR in neutropenic patients with proven or suspected invasive aspergillosis, often where cultures were negative.
Conclusions:
- The developed PCR assay is a sensitive and specific tool for detecting Aspergillus species in clinical samples.
- This molecular method holds significant potential for the early diagnosis of invasive aspergillosis in immunocompromised and neutropenic patients.
- PCR can identify patients at risk for invasive aspergillosis, complementing traditional diagnostic approaches.