Sepsis is a leading cause of death and disability in Australia. Microbiological diagnosis aiding appropriate antibiotic selection is critical in preventing rapid progression and death. For standard organisms, current culture-based diagnostics take greater than 48 hours, while fastidious or atypical organisms may take even longer, if they can be cultured at all. Every hour without appropriate treatment increases the risk of mortality in sepsis, thus rapid diagnostic approaches need to be considered, such as MinION nanopore real-time sequencing. Here we present the application of both the MinION sequencer and digital droplet PCR (ddPCR) to provide a rapid diagnosis and enhanced characterisation in a case of severe, atypical bacterial sepsis.
A woman with a history of a dog bite was admitted to the intensive care unit with severe sepsis, including multi-organ failure, severe acute kidney injury and haemolysis. EDTA whole blood samples were sent for MinION sequencing. The sample was sequenced overnight on the MinION using the rapid 1D library preparation protocol. A ddPCR assay was designed from the generated sequence reads to provide quantification of bacterial loads within the samples.
The initial MinION run produced primarily human sequences, but also generated 19 reads of a combined length of approximately 20Kb which were identified as belonging to Capnocytophaga canimorsus, a fastidious, gram-negative rod which is typical commensal flora in dogs’ mouths. Absolute quantification showed a 100-fold higher bacterial load in whole blood collected at presentation, compared to the plasma derived from the same sample, followed by a dramatic decrease in an EDTA collected 5 days later. In total, the time to diagnosis from obtaining the EDTA sample using the MinION was 20 hours, compared to over 5 days using traditional culturing methods. The result provided confidence in the selection of beta-lactam antibiotics and de-escalation of other classes.
New technologies can provide a supporting role to traditional techniques in improving and enhancing sepsis diagnosis to rationalise antibiotic therapy.