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Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?
C Oliver Morton1, P Lewis White2, Rosemary A Barnes3
1Western Sydney University, Sydney, Australia.
Abstract:
A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.
Insights
Polymerase chain reaction (PCR) assays for invasive aspergillosis (IA) show variable accuracy. Genus-specific PCR targeting rRNA genes is preferred for improved detection of Aspergillus species, though false positives can occur.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Polymerase chain reaction (PCR) tests for invasive aspergillosis (IA) offer diverse technical approaches but lack standardization.
- Methodological recommendations for PCR testing vary based on sample type, prioritizing sensitivity for blood and specificity for invasive samples.
Purpose of the Study:
- To evaluate the analytical specificity of various PCR methods for detecting IA.
- To determine the range of Aspergillus species detectable and assess cross-reactivity with other fungi.
Main Methods:
- A multicenter study blind-tested a panel of DNA from various fungal species using different PCR methods.
- Positivity rates were calculated, and regression analysis explored associations between technical specifications and assay performance.
Main Results:
- Aspergillus genus-specific PCR assays demonstrated higher accuracy (71.8%) compared to species-specific assays (47.2%).
- Genus-specific assays targeting rRNA genes showed a positive association with accuracy but also with false positivity.
- Commonly missed species by genus-specific assays included A. lentulus, A. versicolor, and A. terreus.
Conclusions:
- Current PCR assays are most effective for detecting Aspergillus fumigatus, with less reliable detection of other Aspergillus species.
- Utilizing genus-specific PCR assays targeting rRNA genes is recommended for improved diagnostic performance in IA, despite potential for false positives.
