Inhibition of Legionella pneumophila PCR in respiratory samples: a quantitative approach
Monika Kern1, Stephan Böhm, Ludwig Deml
1Institute for Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg, Germany. monika.kern@klinik.uni-regensburg.de
Abstract:
Impurities in complex biological samples that persist through nucleic acid preparation can inhibit PCR or reduce the sensitivity and efficiency of PCR amplification and thus affect reliable results in PCR diagnostics. To obtain information on the incidence of inhibition events and on the magnitude of the loss of sensitivity, respectively, we devised a relative inhibition assay and examined 100 samples from patients with respiratory tract infections. As a reference, samples were spiked with Legionella (L.) pneumophila. Detection was by standard nucleic acid purification and subsequent real-time PCR. By comparing the crossing points of the fluorescent curves to those of an L. pneumophila standard dilution series, we were able to quantify the respective degrees of inhibition into several categories. We found complete inhibition in 2% of the samples. 12% were not reliably detected. 65% of the tested samples showed moderate to strong inhibition, but were still reliably detected, whereas in 21% of the samples no inhibition was observed. Except for a significantly higher inhibition in tracheal aspirates than in BAL samples, the degree of inhibition did not correlate with the physical properties of the respective sample. The relative inhibition assay established an unexpectedly broad distribution of the inhibition-degrees in inflammatory respiratory materials.
Insights
Many respiratory samples contain impurities that inhibit PCR testing, potentially affecting diagnostic accuracy. A new assay revealed significant PCR inhibition in 65% of patient samples, highlighting the need for robust diagnostic methods.
Area of Science:
- Molecular Biology
- Diagnostic Microbiology
- Clinical Chemistry
Background:
- Nucleic acid preparation can leave impurities that inhibit Polymerase Chain Reaction (PCR).
- PCR inhibition reduces diagnostic sensitivity and efficiency, impacting reliable results in PCR diagnostics.
- Understanding the prevalence and extent of PCR inhibition in clinical samples is crucial.
Purpose of the Study:
- To quantify the incidence and magnitude of PCR inhibition in respiratory tract infection samples.
- To evaluate the reliability of PCR diagnostics in the presence of sample-derived inhibitors.
- To assess the correlation between inhibition levels and sample properties.
Main Methods:
- Development of a relative inhibition assay using spiked Legionella (L.) pneumophila.
- Analysis of 100 respiratory patient samples using standard nucleic acid purification and real-time PCR.
- Quantification of inhibition by comparing PCR crossing points to a standard dilution series.
Main Results:
- Complete PCR inhibition was observed in 2% of samples.
- 12% of samples were not reliably detected due to inhibition.
- 65% of samples exhibited moderate to strong inhibition but were still reliably detected.
- 21% of samples showed no inhibition.
- Inhibition levels did not correlate with sample physical properties, except for higher inhibition in tracheal aspirates compared to BAL samples.
Conclusions:
- A significant proportion of respiratory samples (65%) demonstrate moderate to strong PCR inhibition.
- The developed relative inhibition assay reveals a broad distribution of inhibition degrees in inflammatory respiratory materials.
- These findings underscore the challenge of PCR inhibition in respiratory diagnostics and the need for methods to mitigate its effects.
