Commercial bacterial and fungal broad-range PCR (Micro-Dx™) used on culture-negative specimens from normally sterile sites: diagnostic value and implications for antimicrobial treatment.

The aim of this study was to evaluate the clinical value of partial 16S/18S rRNA gene sequencing with the commercial kit Micro-Dx™ used with the SelectNA™plus instrument on culture-negative samples. A retrospective study of microbiological and clinical data from a 2.5-year period was performed. Assessment of the clinical relevance of the 16S/18S rRNA gene sequencing results was based on evaluation of the results in the clinical context and changes in antimicrobial therapy. Included were 529 samples from 223 patients, representing 251 episodes. In 191 samples (36.1%), bacterial/fungal DNA was detected. Positive results were judged clinically relevant in 79 (31.5%) episodes. Antimicrobial treatment was adjusted according to the 16S/18S rRNA gene sequence analysis result in 42 (16.7%) episodes. The results from 16S/18S rRNA gene sequence analysis were highly clinically relevant. These findings support the use of this analysis in a routine setting.

Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting.

OBJECTIVES: To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm). METHODS: Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme. RESULTS: Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain. CONCLUSIONS: Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Whole genome sequencing as a tool for phylogenetic analysis of clinical strains of Mitis group streptococci.

Identification of Mitis group streptococci (MGS) to the species level is challenging for routine microbiology laboratories. Correct identification is crucial for the diagnosis of infective endocarditis, identification of treatment failure, and/or infection relapse. Eighty MGS from Danish patients with infective endocarditis were whole genome sequenced. We compared the phylogenetic analyses based on single genes (recA, sodA, gdh), multigene (MLSA), SNPs, and core-genome sequences. The six phylogenetic analyses generally showed a similar pattern of six monophyletic clusters, though a few differences were observed in single gene analyses. Species identification based on single gene analysis showed their limitations when more strains were included. In contrast, analyses incorporating more sequence data, like MLSA, SNPs and core-genome analyses, provided more distinct clustering. The core-genome tree showed the most distinct clustering.