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Rapid characterisation of microbial pathogens in healthcare-associated infection samples

The GridION device

Healthcare-associated infections (HAIs) are responsible for mortality and morbidity in hospitals and healthcare facilities worldwide, with over 4 million cases and approximately 37,000 deaths reported annually in Europe alone1. Of particular concern is the consistent and continued increase in cases exhibiting antimicrobial resistance (AMR). The World Health Organization (WHO) states that ‘antimicrobial resistance threatens the very core of modern medicine and the sustainability of an effective, global public health response to the enduring threat from infectious diseases2.

 
Individuals recovering from surgery are particularly vulnerable to infections, and surgical site infections are a major source of morbidity. Up to 25% of those undergoing pancreatic head resection experience infections at the surgical site post-operatively2. After pancreatic surgery, bile duct samples are taken for screening for the presence of microbes, while prophylactic antibiotics are administered to reduce risk of infection until the microbial profile of the samples can be determined. Unfortunately, the time to result may be days or weeks using standard culture techniques, and AMR testing is not routine in most clinical microbiology laboratories. This means that antibiotics may be administered inappropriately or unnecessarily during the post-operative period. In addition, not all pathogen species are culturable, which limits the scope of detection using culture-based methods.


Yonkus et al.3 investigated the potential of using nanopore sequencing for improving post-operative outcomes after pancreatic surgery. They used nanopore technology to characterise the bacterial and fungal pathogens present in 42 bile duct aspirate samples, comparing the results to traditional culture methods. Sequencing libraries were prepared using the Rapid PCR Barcoding Kit, which enables sample preparation in 15 minutes followed by PCR, and sequenced on a GridION. Using both standard culture methods and sequencing, 22 samples were found to feature bacterial DNA, whilst AMR was identified in 21. A wide range of AMR phenotypes were predicted, including resistance to aminoglycosides, beta-lactams, lincosamides, macrolides, streptogramins, and tetracyclines. However, the number of predicted AMR phenotypes was significantly higher using sequencing than standard culture techniques — 10.3 versus 2.7. 


The bacterial species present in the samples were dominated by Bifidobacterium spp., Enterococcus spp., Klebsiella spp., Lactobacillus spp., and Streptococcus spp., with nanopore sequencing detecting more species than standard culture methods  — 10.5 versus 4.4 species. In addition to bacterial pathogens, both methods could identify fungal pathogens present in samples, with Candida spp. being the dominant species found. The authors noted that nanopore sequencing ‘had perfect correlation with SCs [standard cultures] regarding microbial detection’.

'SC [standard culture] is not necessarily the ideal “gold standard” to use for microbial profiling; NS [nanopore sequencing] techniques have been shown to be less biased and more complete3


The median time to result using standard culture techniques was 98 hours, but with nanopore sequencing it was only eight hours. Nanopore sequencing could detect more bacterial species and AMR than standard culture and was significantly faster. In addition, by identifying negative samples considerably earlier, this study demonstrates the potential to prevent the use of unnecessary prophylactic antibiotics. The authors remarked that ‘Although NS [nanopore sequencing] has a higher up-front cost, it has the potential to reduce healthcare costs through a reduction in SSI [surgical site infections] and through a reduction of complications from prolonged antibiotics’. HAIs cost the US healthcare system approximately 3.5–10 billion USD annually, with a single case of surgical site infection costing up to 90,000 USD. For vulnerable patients, such as those recovering from surgery, a serious infection can lead to death, especially where AMR is present, necessitating the need for fast characterisation of strains present.

The authors concluded that nanopore sequencing had a ‘perfect  negative predictive value’ for standard culture, which means that in future ‘it can potentially have immediate clinical impact as it can reduce the number of patients on prophylactic antibiotics unnecessarily’.

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References

1.    Report on the burden of endemic health care-associated infection worldwide. World Health Organization. 2011. Available at: https://apps.who.int/iris/bitstream/handle/10665/ 80135/?sequence=1 [Accessed: 02 May 2023].
2.    Global action plan on antimicrobial resistance. World Health Organization. 2016. Available at: https://www.who.int/publications/i/item/9789241509763 [Accessed: 06 April 2023]
3.    Yonkus, J. A. et al. “Answers in hours”: A prospective clinical study using nanopore sequencing for bile duct cultures. Surgery. 171(3): 693-702 (2022). DOI: https://doi.org/10.1016/j.surg.2021.09.037