Study level
- Master of Philosophy
- Honours
Faculty/School
Faculty of Health
Topic status
We're looking for students to study this topic.
Supervisors
- Position
- Professor
- Division / Faculty
- Faculty of Health
External supervisors
- Prof Robert Flower (Australian Red Cross Lifeblood)
- Dr Eileen Roulis (Australian Red Cross Lifeblood)
Overview
Bacterial sepsis is second only to ABO incompatibility as a cause of death from transfusion. Bacterial contamination of platelets is recognised as the most significant residual infectious risk of transfusion in developed countries. Bacterial Contamination Screening (BCS) has been required for testing of pooled and apheresis platelets manufactured by the Blood Service since April 2008. International microbiological culture studies suggest that the incidence of bacterial contamination ranges from 1:3000 to 1:1000 units of apheresis platelets and 1 in 600 to 1 in 200 units of pooled platelets (a pool consists of platelets from 4 donors). The majority of organisms identified by BCS are slow growing anaerobic skin flora and are not usually associated with post transfusion septic reactions. However, a minority of platelets have grown clinically significant organisms. These infections were likely due to transient or occult bacteraemia in the donor and could have led to potentially serious septic transfusion reactions. All platelet donations are subjected to BCS within 24 hours of donation and are cultured for up to 7 days – which is past the platelet expiry date of five days. Standard microbiological culture is sufficient for detection of the majority of bacterial organisms detected in donations. However, up to 97% of bacterial organisms remain intractable to traditional culture methods due to fastidious nutrient requirements and the transfusion transmission risk attributed to these organisms is unknown. Previous studies have demonstrated the utility of next-generation sequencing (NGS) and metagenomic analysis to investigate the bacterial diversity in various blood components and products.
This pilot study aims to characterise the most common genera of organisms found in pooled platelet samples flagged for BCS and determine the relative frequency of these organisms in the donor population. Additionally, this study will assess the potential transfusion risk of detected organisms and determine whether concurrent metagenomic analysis of BCS samples in addition to traditional culture methods would be beneficial for transfusion risk reduction.
Project methodology
Samples: Ethical approval has been sought for this study and both recently obtained and archival BCS samples will be used. Approximately 100 pooled, BCS flagged samples will be analysed, with 5 BCS unflagged pooled platelet samples serving as diversity controls.
Sample testing: The pooled samples will be subjected to whole genomic DNA (gDNA) extraction using the QIASymphony robotic extractor (QIAGEN – Hilden, Germany) and extracted samples stored at -30oC. PCR will be performed on the gDNA samples using previously published primers targeting variable domains 3 and 4 of the 16S ribosomal RNA gene (16S rRNA) which is specific to the bacterial domain. NGS libraries will be prepared from PCR products using the Nextera XT library preparation kit (Illumina, USA) and sequenced on the MiSeq platform using a 600 cycle kit (Illumina, USA). Generation of FASTA files will be performed on board the MiSeq platform.
Data Analysis: Metagenomic analyses will be performed on de-multiplexed FASTA files using QIIME (Quantitative Insights Into Microbial Ecology) software. QIIME will be used to perform quality filtering, OTU picking, taxonomic assignment, diversity analyses and phylogenetic reconstruction on the dataset, as well as generating OTU cluster diagrams and phylogenetic trees.
Resources: All resources (PCR reagents, sequencing reagents) and pooled platelet samples for BCS will be supplied by the Australian Red Cross Lifeblood.
Keywords
Contact
Phone: 07 3838 9020
Email: rflower@redcrossblood.org.au