Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

Accurate species identification is a prerequisite for successful management of tuberculosis and non-tuberculous mycobacterial (NTM) diseases. The novel FluoroType Mycobacteria assay combines three established GenoType DNA strip assays (CM, AS, and NTM-DR), allowing detection of Mycobacterium tuberculosis and 32 NTM species/subspecies in a single assay with automatic detection and result analysis. We evaluated the clinical performance of the FluoroType assay and its feasibility in replacing the GenoType Mycobacterium CM assay as the initial method for mycobacterial identification. A total of 191 clinical mycobacterial cultures were analyzed in this study: 180 identified for one mycobacterial species, 6 for multiple, and 5 for no mycobacterial species. Positive percent agreement (PPA) for the FluoroType assay was 87.8% (n = 158), with full agreement for 23/29 species. Weakest PPA was observed for Mycobacterium gordonae (50%, n = 9/18), Mycobacterium interjectum (40%, n = 2/5), and Mycobacterium intracellulare (42%, n = 5/12). Clinical and mixed cultures containing multiple mycobacterial species gave equally single species and genus level identifications (n = 30). No cross-reactivity with non-mycobacterial species was observed (n = 22). In a separate in silico analysis of 2016-2022 HUS area (Finland) register data (n = 2,573), the FluoroType assay was estimated to produce 18.8% (n = 471) inadequate identifications (genus/false species) if used as the primary identification method compared to 14.2% (n = 366) with the GenoType CM assay. The FluoroType assay was significantly more convenient in terms of assay workflow and result interpretation compared to the entirely manual and subjective GenoType CM assay. However, the feasibility of the assay should be critically assessed with respect to the local NTM species distribution. IMPORTANCE: This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.