Development of a rapid diagnostic test based on loop-mediated isothermal amplification to identify the most frequent non-typhoidal Salmonella serovars from culture.

Identification of Salmonella serovars is performed by conventional seroagglutination or sequencing. These methods are labor-intensive and require technical experience. An easy-to-perform assay allowing the timely identification of the most common non-typhoidal serovars (NTS) is needed. In this study, a molecular assay based on loop-mediated isothermal amplification (LAMP) targeting specific gene sequences of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Derby, and S. Choleraesuis has been developed for rapid serovar identification from cultured colonies. A total of 318 Salmonella strains and 25 isolates of other Enterobacterales species that served as negative controls were analyzed. All S. Enteritidis (n = 40), S. Infantis (n = 27), and S. Choleraesuis (n = 11) strains were correctly identified. Seven out of 104 S. Typhimurium and 10 out of 38 S. Derby strains missed a positive signal. Cross-reactions of the gene targets were only rarely observed and restricted to the S. Typhimurium primer set (5 false-positives). Sensitivity and specificity of the assay compared to seroagglutination were as follows: 100% and 100% for S. Enteritidis, 93.3% and 97.7% for S. Typhimurium, 100% and 100% for S. Infantis, 73.7% and 100% for S. Derby, and 100% and 100% for S. Choleraesuis, respectively. With results available in just a few minutes of hands-on time and a test run time of 20 min, the LAMP assay developed here may be a useful tool for the rapid identification of common Salmonella NTS in daily routine diagnostics.

Screening of Klebsiella pneumoniae Isolates for Carbapenemase and Hypervirulence-Associated Genes by Combining the Eazyplex® Superbug CRE and hvKp Assays.

The acquisition of hypervirulence-associated genes by carbapenemase-producing Klebsiella pneumoniae is being increasingly observed, and easy-to-use diagnostic tests are needed for the surveillance of the hypervirulent K. pneumoniae (hvKp). In this pilot study, 87 K. pneumoniae isolates from invasive infections collected in 2022 and 2023 were analysed using the LAMP-based eazyplex® Superbug CRE and hvKp assays for the simultaneous identification of carbapenemases and virulence genes (rmpA/A2, iuC, iroC, ybt, clb). Nine isolates showed a Kleborate virulence score of 4 or 5 (10.3%). The time for the results of the eazyplex® assays ranged from 6.5 to 13 min, and the total turnaround time, including sample preparation, was less than 30 min. Five isolates, three of which produced New Delhi metallo-beta lactamase (NDM), were subjected to whole-genome sequencing (WGS) analysis for further characterisation. The eazyplex® test results for beta-lactamase and virulence genes were confirmed. The eazyplex® hvKp, currently only available as a Research Use Only assay, may be a useful tool for the rapid identification of hvKp without significant additional workload when combined with the eazyplex® Superbug CRE assay for the detection of carbapenemases.

Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures.

Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.