Investigating Transfusion-related Sepsis Using Culture-Independent Metagenomic Sequencing

Abstract

Background: Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration.

Methods: In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events.

Results: mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations.

Conclusions: Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

Keywords: healthcare infections; mNGS; metagenomic sequencing; platelet transfusion; septic transfusion.

Figure 1.

Acinetobacter septic transfusion investigation. A, Maximum likelihood phylogenetic tree based on single-nucleotide polymorphism alignments demonstrates relatedness of the novel Acinetobacter species isolated from both patient A and the residual transfused platelet product relative to closely related species within the Acinetobacter calcoaceticus/baumannii (ACB) complex. B, Abundance of ACB complex in the transfused platelet product and cocomponents (left panel) and in patient A’s plasma (right panel), determined by culture-independent metagenomic sequencing and measured in rpM. Abbreviation: rpM, reads per million.

Figure 2.

Pseudomonas septic transfusion investigation. Abundance of Pseudomonas aeruginosa in patient B’s plasma throughout the course of the fatal septic transfusion event, determined by culture-independent metagenomic sequencing and measured in rpM (lower panel). Procalcitonin level (ng/mL) over the course of the septic transfusion event is plotted in the upper panel. Abbreviation: rpM, reads per million.

Figure 3.

Klebsiella septic transfusion investigation. Abundance of Klebsiella pneumoniae in plasma from patients C (upper panel) and D (lower panel) during the course of related septic transfusion events, as determined by culture-independent metagenomic sequencing and measured in rpM. Patient C, who did not survive the event, had posttransfusion blood cultures return positive for K. pneumoniae that was highly related (1 single-nucleotide polymorphism across the 4.2 Mb core genome) to the K. pneumoniae isolated from the residual transfused platelet product. Patient D, who was receiving antibiotics with activity against Klebsiella prior to transfusion, survived but had negative posttransfusion blood cultures, precluding definitive confirmation of a related second septic transfusion event in the absence of culture-independent metagenomic sequencing. Abbreviation: rpM, reads per million.