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Quantitative PCR for detection and discrimination of the bloodborne pathogen Staphylococcus epidermidis in platelet
Cherie Cameron Mastronardi1, Sandra Ramírez-Arcos
1Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON K1G 4J5, Canada.
Abstract:
Bacterial contamination of blood components is the major microbiological cause of transfusion-associated morbidity, with Staphylococcus epidermidis being the most frequently isolated organism from contaminated platelet preparations (PPs). We have recently shown that S. epidermidis forms biofilms during platelet storage, which might account for reported missed detection during routine screening. In this study, we developed a highly sensitive and specific multiplex quantitative PCR (QPCR) assay to detect S. epidermidis in PPs at levels of 10(2)-10(3) cfu/mL. A specific primer pair and hydrolysis probe were designed to amplify an internal region of the cell division divIVA gene that is unique to S. epidermidis. In addition, an internal sequence of the virulence gene icaA, which is involved in the synthesis of the S. epidermidis biofilm matrix, was selected to allow for differentiation of potentially biofilm-forming S. epidermidis isolates. A conserved region of the 8 alleles of the HLA-DQalpha1 locus present in residual white blood cells in PPs was selected as an internal control for the assay. The specificity of this assay was confirmed, as other staphylococcal species that were tested with the optimized parameters were not detected. This QPCR assay could be adaptable for the detection of other bloodborne bacterial pathogens.
Insights
A new quantitative PCR assay can detect Staphylococcus epidermidis in platelet preparations, even when it forms biofilms. This method improves detection of bacterial contamination in blood products, enhancing transfusion safety.
Area of Science:
- Microbiology
- Molecular Biology
- Blood Transfusion Safety
Background:
- Bacterial contamination of blood components, particularly platelet preparations (PPs), is a significant cause of transfusion-associated morbidity.
- Staphylococcus epidermidis is the most common organism isolated from contaminated PPs.
- S. epidermidis forms biofilms during storage, potentially evading standard detection methods.
Purpose of the Study:
- To develop a sensitive and specific multiplex quantitative PCR (QPCR) assay for detecting S. epidermidis in PPs.
- To differentiate biofilm-forming S. epidermidis isolates using the icaA virulence gene.
- To establish an internal control for assay validation using HLA-DQalpha1 locus.
Main Methods:
- Designed a QPCR assay targeting the divIVA gene unique to S. epidermidis.
- Incorporated amplification of the icaA gene for biofilm-forming strain differentiation.
- Utilized HLA-DQalpha1 locus as an internal control for residual white blood cells.
- Validated assay specificity against other staphylococcal species.
Main Results:
- The developed QPCR assay demonstrated high sensitivity, detecting S. epidermidis at levels of 10(2)-10(3) colony-forming units/mL.
- The assay successfully differentiated S. epidermidis isolates based on the presence of the icaA gene.
- Specificity was confirmed, with no cross-reactivity observed with other tested staphylococcal species.
Conclusions:
- The multiplex QPCR assay provides a sensitive and specific method for detecting S. epidermidis in platelet preparations.
- This assay can identify potentially biofilm-forming strains, addressing a limitation in current screening.
- The assay's adaptability suggests potential for detecting other bacterial pathogens in blood products, improving transfusion safety.
