Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice

Abstract

Background Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear. Methods We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019. Results A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2–31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens. Conclusion 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

A case of paradoxical reaction after treatment of generalized tuberculous lymphadenopathy in a peritoneal dialysis patient

“…One recently published retrospective analysis has reported highest yield (37%) in cardiovascular infections. 17 Similarly, another study from Indonesia reported positive nucleic acid amplification test (using 16S ribosomal RNA) with 83.1% sensitivity in AFB smear-negative patients with pulmonary TB. 18 With regard to PD patients, there are a number of case reports where pathogen was detected by 16S ribosomal RNA and cultures were negative.…”

Clinical Use of a 16S Ribosomal RNA Gene-Based Sanger and/or Next Generation Sequencing Assay to Test Preoperative Synovial Fluid for Periprosthetic Joint Infection Diagnosis

“…Prior to the introduction of NGS into the described assay, it was exclusively based on Sanger sequencing, and only samples with Ct values equal or below 32 cycles were sent to sequencing, as described in ( 21 ). Considering the higher sensitivity of NGS, the threshold for sequencing was increased to 34 cycles.…”

Determining the Clinical Utility of 16S rRNA Sequencing in the Management of Culture-Negative Pediatric Infections

“…16S rRNA sequencing is a broad-range PCR used in identifying bacterial pathogens to the species level by sequencing the highly-conserved bacterial 16S rRNA region [ 1 , 2 ]. Previous studies have demonstrated this molecular application has reasonable concordance when compared to conventional bacterial cultures and has clinical significance [ 4 , 5 , 6 ]. This technology is especially beneficial, but not limited, to more invasive, difficult-to-diagnose and complicated culture-negative infections where pathogen identification is of high importance.…”

“…Several studies have also shown its clinical impact in the management of culture-negative endocarditis [ 12 , 13 , 14 , 15 ]. To date, there is a paucity of data on the clinical utility of 16S rRNA sequencing in pediatric infections [ 2 , 6 ]. Few studies highlight the diagnostic utility of 16S rRNA sequencing in select pediatric infections [ 16 , 17 , 18 , 19 , 20 ].…”