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.

Macrolide Resistance in Mycoplasma pneumoniae, Midwestern United States, 2014 to 2021.

The epidemiology of macrolide resistance in Mycoplasma (Mycoplasmoides) pneumoniae in the United States is incompletely described. Using a PCR assay targeting common mutations associated with macrolide resistance in M. pneumoniae (23S rRNA gene, A2063G/A2064G), the frequency of macrolide resistance was estimated to be 10% based on analysis of 114 samples tested from January 2014 to September 2021 at Mayo Clinic Laboratories. Seasonality data showed the highest rates of M. pneumoniae infection in the fall/early winter.