Diagnostic utility of 16S rRNA meta genomic next-generation sequencing in clinical Infectious Diseases practice: a retrospective study from South India

Objectives 16S rRNA sequencing is an emerging diagnostic tool for bacterial syndromes caused by fastidious pathogens and in culture negative infections. However, it can pose significant challenges from pre-analytic to post-analytic phase due to sampling issues, lack of an approved platform and test characteristics. We aimed to evaluate the diagnostic performance of 16S rRNA sequencing in sterile site samples compared to conventional microbiological techniques [CMT] and a composite reference standard [CRS] Methods We conducted a retrospective study at a tertiary hospital from January 2022 to July 2024. We included clinical data of patients with sterile site samples processed for both CMT and 16S rRNA sequencing. Sequencing used the Credence Genomics pipeline. Results 166 samples met the inclusion criteria: 97 tissue, 35 pus, and 34 fluid samples. Pathogen detection rate was 42.8% [71/166] by CMT and 58.4% [97/166] by 16S rRNA. Concordance between methods was 50.6%. Sensitivity and specificity of 16S rRNA against CMT was 60.6% and 43.2%; and 69.4% and 66.7% against CRS respectively. CMT showed 54% sensitivity and 92.9% specificity against CRS. Combined testing improved sensitivity to 84.7% and accuracy was 78.9%. Conclusion 16S rRNA sequencing provides incremental diagnostic sensitivity over conventional microbiological techniques but at the cost of reduced specificity. Its routine frontline use as a standalone diagnostic tool is not supported by our findings. Instead, it may be best reserved for selected culture-negative cases with high clinical suspicion, where results can be interpreted in conjunction with clinical, radiological, and microbiological data by experienced clinicians.