Poor Agreement Between Next-Generation DNA Sequencing and Bacterial Cultures in Orthopaedic Trauma Procedures

Abstract

Background: Next-generation DNA sequencing (NGS) detects bacteria-specific DNA corresponding to the 16S ribosomal RNA gene and can identify bacterial presence with greater accuracy than traditional culture methods. The clinical relevance of these findings is unknown. The purpose of the present study was to compare the results from bacterial culture and NGS in order to characterize the potential use of NGS in orthopaedic trauma patients.Methods: A prospective cohort study was performed at a single academic, level-I trauma center. Three patient groups were enrolled: (1) patients undergoing surgical treatment of acute closed fractures (presumed to have no bacteria), (2) patients undergoing implant removal at the site of a healed fracture without infection, and (3) patients undergoing a first procedure for the treatment of a fracture nonunion who might or might not have subclinical infection. Surgical site tissue was sent for culture and NGS. The proportions of culture and NGS positivity were compared among the groups. The agreement between culture and NGS results was assessed with use of the Cohen kappa statistic.Results: Bacterial cultures were positive in 9 of 111 surgical sites (110 patients), whereas NGS was positive in 27 of 111 surgical sites (110 patients). Significantly more cases were positive on NGS as compared with culture (24% vs. 8.1%; p = 0.001), primarily in the acute closed fracture group. No difference was found in terms of the percent positivity of NGS when comparing the acute closed fracture, implant removal, and nonunion groups. With respect to bacterial identification, culture and NGS agreed in 73% of cases (k = 0.051; 95% confidence interval, 20.12 to 0.22) indicating only slight agreement compared with expected chance agreement of 50%.Conclusions: NGS identified bacterial presence more frequently than culture, but with only slight agreement between culture and NGS. It is possible that the increased frequency of bacterial detection with molecular methods is reflective of biofilm presence on metal or colonization with nonpathogenic bacteria, as culture methods have selection pressure posed by restrictive, artificial growth conditions and there are low metabolic activity and replication rates of bacteria in biofilms. Our data suggest that NGS should not currently substitute for or complement conventional culture in orthopaedic trauma cases with low suspicion of infection.Level of Evidence: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence. F racture-related infection negatively affects patient quality of life and is costly to the health-care system 1 . Current clinical tests to evaluate and diagnose fracture-related infection are imperfect because of the limitations associated with culture-based techniques 2 . In this regard, molecular methods for detecting bacterial presence have the potential to improve our ability to manage fracture-related infections.Bacteria exist in planktonic and biofilm phenotypes 2 . The biofilm form, occurring in the pres...

Clinical Application and Influencing Factor Analysis of Metagenomic Next-Generation Sequencing (mNGS) in ICU Patients With Sepsis

“…This indicates that mNGS may miss pathogens, be inconsistent with blood culture, have false negative results, and need for higher sequencing depth detection ( Duan et al., 2021a ; Gu W., et al., 2021 ). Natoli et al. (2022) also agreed that any contamination with the blood culture between sample processing and data analysis could bias the final results seriously.…”

“…This indicates that mNGS may miss pathogens, be inconsistent with blood culture, have false negative results, and need for higher sequencing depth detection (Duan et al, 2021a;Gu W., et al, 2021). Natoli et al (2022) also agreed that any contamination with the blood culture between sample processing and data analysis could bias the final results seriously. Boers et al (Boers et al, 2019) also showed that the above reasons lead to the discovery of non-existent bacterial genera, false correlations between microbes and their hosts, and the inability to detect true correlations.…”

Diagnosis of Occult Infection Using Fracture-Related Infection Criteria at the Time of Nonunion Repair

“…Implant sonification and histological analysis have been demonstrated to improve diagnosis of FRI. 34,35 By contrast, next-generation sequencing has shown inconsistent agreement with cultures taken during surgery, and it does not currently have evidence to support its routine use. 36…”

Microbe-metabolite interaction networks, antibiotic resistance, and in vitro reconstitution of the penile prosthesis biofilm support a paradigm shift from infection to colonization

“…We note that this limitation is not unique to the present study, but rather is currently the case for the microbiome field in general. Evidence in related subjects such as orthopedic trauma and urinary tract infections, suggests that there is currently insufficient evidence for NGS as a substitute or complement to conventional culture, and additional studies are necessary to determine its clinical utility 20 , 21 . Although there was considerable overlap between the NGS and cuture results in device biofilms in the present study, the clinical utility of NGS for the diagnosis of device-associated infection or to inform treatments for this condition requires further investigation.…”