Research Article Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory Michael Payne, 1 Robert Azana, 2 and Linda M. N. Hoang 1,2 1 Department of Pathology and Laboratory Medicine, UBC, Vancouver, BC, Canada V6T 2B5 2 BC Centre for Disease Control Public Health Laboratory, Vancouver, BC, Canada V5Z 4R4 Correspondence should be addressed to Linda M. N. Hoang; linda.hoang@bccdc.ca Received 9 August 2015; Accepted 14 November 2015 Copyright © 2016 Michael Payne et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. e 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. 1. Introduction Rapid identification of pathogens from clinical specimens is important for the selection of correct treatment, as well as for patient prognosis. However, in some specimens cultures remain negative and are ideal candidates for use of molecular methods for amplification and identification of the potential pathogens [1]. e 16S rRNA gene in bacteria and the 18S rRNA gene, with associated internal transcribed spacer (ITS) regions, in fungi are common gene targets used in the microbiology laboratory for gene amplification followed by sequencing for organism identification. ese assays were ini- tially developed as a method for the identification and classifi- cation of organisms from culture specimens [2–4]. However, these assays have been shown to successfully amplify and identify pathogens from clinical specimens where cultures are negative, and organisms may be fastidious or nonviable from antibiotic exposure [1]. Amplification of 16S and ITS DNA directly from clinical specimens has both challenges and limitations. Polymicrobial infections or nonsterile sites typically cause difficulties inter- preting sequencing results [5]. Specificity of results may also be difficult to determine as contamination of the sample can occur during collection or in the laboratory. Also, amplification of microorganism DNA from the sample may not necessarily ensure the organism identified is the causal pathogen. e negative predictive value of 16S and ITS is difficult to determine and may not be ideal, particularly in smear negative samples [1]. Also, 16S and ITS are labour- intensive procedures requiring advanced infrastructure and technical expertise and can add considerably to the workload of a laboratory [5]. Finally, standardization for 16S and ITS is poor, with many different primer/probe targets, specimen processing methods, and reference databases in use [6, 7]. Given the lack of information on test performance from direct clinical specimens, the results of 16S and ITS tests performed over the past 4 years were reviewed. rough this review we aimed to better define the correlation with microscopy results and overall performance of 16S and ITS testing from direct clinical specimens. 2. Materials and Methods Since 2008, the British Columbia Centre for Disease Control Public Health Laboratory (PHL) has offered 16S and ITS Hindawi Publishing Corporation Canadian Journal of Infectious Diseases and Medical Microbiology Volume 2016, Article ID 4210129, 6 pages http://dx.doi.org/10.1155/2016/4210129