Detection of Francisella tularensis in Biological Specimens Using a Capture Enzyme-Linked Immunosorbent Assay, an Immunochromatographic Handheld Assay, and a PCR

Abstract

The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis 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 Burkholderia spp., was observed. The detection limit of the assay was 10 3 to 10 4 bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-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 of 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 cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.Within the genus Francisella there are two known species, Francisella tularensis and Francisella philomiragia, which have a 16S rRNA gene sequence similarity of more than 98% (6). F. tularensis, a gram-negative, small (0.2 to 0.7 by 0.2 m) facultative intracellular bacterium,

Serosurveillance forFrancisella tularensisAmong Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

“…In the present study, the cutoff value in the cELISA for animal specimens was set as 40%, thus cross-reaction of antibodies to F. novicida was negligible. This might be supported by the previous report that showed no cross-reaction between F. tularensis and F. novicida (Grunow et al 2000). Antibodies detected in the wild animals by the cELISA are therefore considered to be specific to F. tularensis LPS.…”

An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

“…Although this method is not as sensitive for the detection of F. tularensis bacteria in blood as it is for the detection of bacteria in PBS solution, the detection limit is still comparable to most ELISA methods [24,53]. However, our technology has the advantage of greater safety because we are detecting bacterial peptides after the bacteria have been inactivated by lysis and proteolysis.…”

“…The singly-charged, epitope-containing tryptic peptide (aa 49-61 NIVAIEGGEDVTK) from the IglC protein was observed at m/z = 1344.7 ( Figure 4). Therefore the detection limit for bacteria in PBS solution is determined to be 8 CFU bacteria on target by using 50% of the antibody beads incubated with 50μL of a bacterial solution in PBS, at a concentration of 320 CFU mL -1 , which is as low as the most sensitive detection systems yet developed [51,52]. It should be noted that 8 CFU does not represent the lowest possible detection limit in real applications, but is an estimate of the lowest possible detection limit of our method, as the tryptic digestion of the protein equivalent of 8 CFU might be difficult.…”

“…[10], which reduces the specificity and could lead to false positives. More recently, another type of serological test, an enzyme-linked immunosorbent assay (ELISA), achieved a detection limit of 10 3 bacteria mL -1 in PBS and 10 4 bacteria mL -1 in human serum [24]. However, when samples are contaminated with other microorganisms [25], accurate diagnosis with ELISA or culturing methods is very difficult due to their low specificities.…”

Tularemia: current diagnosis and treatment options

“…One product is a handheld immunochromatographic assay that has a limit of detection of 10 6 -10 7 bacteria/ml in human serum [60]. Further research will hopefully yield diagnostic products with both speed and accuracy.…”