Next-generation 16S-rRNA sequencing for early identification of external ventricular drain-related infections in a critical care setting: a retrospective cohort study with external validation

Abstract Introduction: External ventricular drain (EVD)-associated infections (EVDIs) are difficult to distinguish from aseptic inflammation, leading to excessive broad-spectrum antibiotic use while slow culture results hinder timely decisions. Next-generation 16S-rRNA sequencing offers rapid bacterial detection with the potential to improve surveillance and antibiotic stewardship. We aimed to evaluate the diagnostic performance of 16S-rRNA sequencing compared to conventional CSF cultures and its ability to guide EVDI-targeted antibiotic therapy. Methods: Adult (≥ 18 years) patients treated with EVDs in the intensive care unit (ICU) at Karolinska University Hospital, Stockholm, Sweden, between November 2021 and October 2024, were included. Infection classification was based on independent, manual reviews of medical records by three physicians, with inter-reviewer agreement evaluated using Cohen’s κ statistic. The performance of 16S-rRNA sequencing (index test) was compared to bacterial cultures for EVDI diagnosis. Additionally, a 16S-guided treatment protocol was developed and its potential impact on antibiotic stewardship was assessed, then validated in an external cohort of 135 patients from Norrlands University Hospital. Results: Of the 265 patients treated with EVDs during the study period, 191 had paired results from 16S-rRNA sequencing and bacterial cultures. Among these, 8 cases of verified infection (VI) and 61 cases of suspected infection (SI) were identified. Both methods identified 7 of 8 VI cases, yielding a per-patient sensitivity of 87.5%. On a per-sample basis, 16S-rRNA sequencing and culture demonstrated sensitivities of 92.9% vs. 91.7%, specificities of 92.7% vs. 97.8%, a negative predictive values (NPV) of of 99.7%, and a positive predictive values (PPV) of 35.1% vs. 61.1%, respectively. Implementing a 16S-rRNA sequencing-guided treatment protocol could reduce antibiotic exposure by 43–71%, and a 40-55% reduction in the number of treated patients was estimated in an external cohort. Conclusion: Our findings suggest that 16S-rRNA sequencing is a valuable adjunct to traditional EVDI surveillance, primarily functioning as a rapid rule-out test to support withholding or de-escalation of broad-spectrum antibiotic treatment if results are readily available. Furthermore, exploratory findings suggest that semi-quantitative reads ratios may help differentiate clinically relevant findings from contamination.