Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR

The early detection of Francisella tularensis, the causative agent of tular emia, is important for adequate treatment by antibiotics and the outcome of the disease, Here we describe a new capture enzyme-linked immunosorbent as say (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp, holarctica and Francisella tularens is subsp, tularensis, No cross-reactivity with Francisella tularensis subsp , novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp,, Yersinia spp,, Escherichia coli, and Bu rkholderia spp,. was observed. The detection limit of the assay was 10(3) t o 10(4) bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA, In addition, a novel immunochromatographic membra ne-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that o f the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus), Whereas all infected samples were recognized by the cELI SA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conc lusion, the HHA can be used as a very fast and simple approach to perform f ield diagnosis to obtain a first hint of an infection with F. tularensis, e specially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.