P-2166. Sensitivity and Specificity of Nanopore Sequencing for Detection of Methicillin-Resistant <i>Staphylococcus aureus and</i> Methicillin-Resistant <i>Staphylococcus epidermidis</i> in Positive Blood Culture Samples

Abstract Background Nanopore sequencing enables rapid DNA/RNA analysis without amplification. Implementing this for point-of-care diagnostics reduces time delays in traditional AST methods, potentially enhancing patient outcomes. We aim to evaluate the performance of Oxford Nanopore Technologies sequencing from positive blood culture for bacterial identification and antimicrobial susceptibility prediction in S. aureus and S. epidermidis by detecting the mecA gene. Methods We conducted a cross-sectional descriptive study on individuals with positive blood cultures identifying S. aureus and S. epidermidis over a 12-month period from April 2023 to March 2024. Bacterial DNA was extracted from the blood culture specimens, and then sequenced using the PromethION Flow Cell to identify the organisms and mecA genes, comparing the results to routine blood cultures and standard AST. Results 108 blood culture samples were initially collected. After applying the exclusion criteria, 80 samples qualified for DNA extraction and sequencing. 45 samples were identified as S. aureus (6 MRSA and 39 MSSA) and 35 as S. epidermidis (27 MRSE and 8 MSSE). Notably, 67.5% of samples contained less than 0.5 ng/μl of DNA. Sensitivity and specificity of mecA gene detection were 35.5% (95% CI: 18.6%–52.3%) and 83.7% (95% CI: 73.3%–94.0%), respectively, with an accuracy of 65.0% (95% CI: 54.5%–75.5%). Upon excluding samples with DNA less than 0.5 ng/μl accuracy improved to 73.1% (95% CI: 56.0%–90.1%). The specimen that showed a false-positive result will proceed to phenotypic confirmation using methicillin-infused mannitol-salt agar. After phenotypic confirmation, the accuracy improved to of 88.5% (95% CI: 76.2%–100.0%). In this study, we also found 5 samples in which the mecA gene was shown to be genotypically oxacillin-sensitive after phenotypic confirmation. The median time for detecting the mecA gene was 198 minutes (IQR: 37.5–458.7) after the onset of sequencing. Conclusion Our study on nanopore sequencing for MRSA and MRSE detection from blood cultures found promising but limited sensitivity and specificity in detecting mecA gene. Challenges in DNA extraction from Gram-positive bacteria affected overall accuracy. Further improvements are needed for enhanced diagnostic reliability. Disclosures All Authors: No reported disclosures