Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory

Abstract

We evaluated the performance of 16S and internal transcribed spacer (ITS) region amplification and sequencing of rDNA from clinical specimens, for the respective detection and identification of bacterial and fungal pathogens. Direct rDNA amplification of 16S and ITS targets from clinical samples was performed over a 4-year period and reviewed. All specimens were from sterile sites and submitted to a reference laboratory for evaluation. Results of 16S and ITS were compared to histopathology, Gram and/or calcofluor stain microscopy results. A total of 277 16S tests were performed, with 64 (23%) positive for the presence of bacterial DNA. Identification of an organism was more likely in microscopy positive 16S samples 14/21 (67%), compared to 35/175 (20%) of microscopy negative samples. A total of 110 ITS tests were performed, with 14 (13%) positive. The yield of microscopy positive ITS samples, 9/44 (21%), was higher than microscopy negative samples 3/50 (6%). Given these findings, 16S and ITS are valuable options for culture negative specimens from sterile sites, particularly in the setting of positive microscopy findings. Where microscopy results are negative, the limited sensitivity of 16S and ITS in detecting and identifying an infectious agent needs to be considered.