Targeted Metagenomic Sequencing-based Approach Applied to 2146 Tissue and Body Fluid Samples in Routine Clinical Practice.

BACKGROUND: The yield of next-generation sequencing (NGS) added to a Sanger sequencing-based 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) assay was evaluated in clinical practice for diagnosis of bacterial infection. METHODS: PCR targeting the V1 to V3 regions of the 16S rRNA gene was performed, with amplified DNA submitted to Sanger sequencing and/or NGS (Illumina MiSeq) or reported as negative, depending on the cycle threshold value. A total of 2146 normally sterile tissues or body fluids were tested between August 2020 and March 2021. Clinical sensitivity was assessed in 579 patients from whom clinical data were available. RESULTS: Compared with Sanger sequencing alone (400 positive tests), positivity increased by 87% by adding NGS (347 added positive tests). Clinical sensitivity of the assay that incorporated NGS was 53%, which was higher than culture (42%, P < .001), with an impact on clinical decision-making in 14% of infected cases. Clinical sensitivity in the subgroup that received antibiotics at sampling was 41% for culture and 63% for the sequencing assay (P < .001). CONCLUSIONS: Adding NGS to Sanger sequencing of the PCR-amplified 16S rRNA gene substantially improved test positivity. In the patient population studied, the assay was more sensitive than culture, especially in patients who had received antibiotic therapy.

Comparison of the BioFire Joint Infection Panel to 16S Ribosomal RNA Gene-Based Targeted Metagenomic Sequencing for Testing Synovial Fluid from Patients with Knee Arthroplasty Failure.

The diagnosis of periprosthetic joint infection (PJI) is challenging, often requiring multiple clinical specimens and diagnostic techniques, some with prolonged result turnaround times. Here, the diagnostic performance of the Investigational Use Only (IUO) BioFire Joint Infection (JI) Panel was compared to 16S rRNA gene-based targeted metagenomic sequencing (tMGS) applied to synovial fluid for PJI diagnosis. Sixty synovial fluid samples from knee arthroplasty failure archived at -80°C were tested. Infectious Diseases Society of America (IDSA) diagnostic criteria were used to classify PJI. For culture-positive PJI with pathogens targeted by the JI panel, JI panel sensitivity was 91% (21/23; 95% confidence interval [CI], 73 to 98%), and tMGS sensitivity was 96% (23/24; 95% CI, 80 to 99%) (P = 0.56). Overall sensitivities of the JI panel and tMGS for PJI diagnosis were 56% (24/43; 95% CI, 41 to 70%) and 93% (41/44; 95% CI, 82 to 98%), respectively (P < 0.001). JI panel and tMGS overall specificities were 100% (16/16; 95% CI, 81 to 100%) and 94% (15/16; 95% CI, 72 to 99%), respectively. While the clinical sensitivity of the JI panel was excellent for on-panel microorganisms, overall sensitivity for PJI diagnosis was low due to the absence of Staphylococcus epidermidis, a common causative pathogen of PJI, on the panel. A PJI diagnostic algorithm for the use of both molecular tests is proposed.