Development of a rapid antigen-based lateral flow assay for tick-borne spotted fever rickettsioses

Introduction: Rickettsiae are Gram-negative and obligately intracellular bacteria primarily targeting microvascular endothelial cells. The incidence of tick-borne spotted fever rickettsioses (SFRs) has risen dramatically in the last two decades, possibly due to changes in climate and expansion of the tick population. Despite the availability of (non-standard) antibiotics to which rickettsiae are sensitive, SFRs continue to cause severe illness and death in otherwise-healthy individuals due to lack of a timely and reliable diagnostic test. At present, detection of antibodies against rickettsiae in the serum by indirect immunofluorescence assay is the gold standard diagnostic test. Pathogenic rickettsiae primarily infect the microvascular endothelial cells and may not circulate in large numbers in the blood until the disease has progressed to a severe phase of infection. Since no convenient, specific, acute-phase laboratory diagnostic tests are available for SFRs, these diseases are not only notoriously difficult to diagnose but also severely underreported. We recently identified a potential multi-pathogen diagnostic biomarker for SFRs, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Originally identified in R. conorii-infected endothelial cell supernatant, we have demonstrated that RC0497 also is present in the circulating blood of an infected mouse model of Mediterranean spotted fever, one of the most severe form of SFRs.

Materials and

Methods: We employed guinea pig and mouse experimental models of Rocky Mountain Spotted Fever (RMSF) and Mediterranean Spotted Fever (MSF), respectively. To determine how early RC0497 can be detected in the serum over the course of SFR, we employed a mouse model of MSF based on intravenously inoculating C3H/HeN mice with R. conorii. Stable isotope dilution (SID)-parallel reaction monitoring was used to estimate the concentration of RC0497 in sera of R. rickettsii-infected guinea pigs and R. conorii-infected mice at 0.1 to 1.1 ng/ml. Commercially-produced rabbit polyclonal antibodies against recombinant RC0497 were immobilized on the LFA test line and covalently conjugated onto carboxylated polystyrene europium chelate nanoparticles. A POC-compatible, low-cost, time-resolved europium fluorescence LFA reader was used to analyze the LFA strips.

Results, Conclusions, and Discussions: We developed a lateral flow assay (LFA) based on europium chelate nanoparticle reporters, and evaluated its performance with serum samples of experimentally-infected animals and with human serum spiked with recombinant RC0497. Interestingly, 21 of 22 (95.5%) serum samples from R. rickettsii-infected guinea pigs and R. conorii-infected mice yielded positive results. All uninfected samples were negative, suggesting high specificity. Mice infected with a lethal dose of R. conorii and treated with doxycycline on day 3 post-infection (p.i.) displayed significantly decreased rickettsial loads, comparable to the sublethal infection group on day 5 p.i.. A panel of human serum samples spiked with recombinant RC0497 was analyzed by LFA, demonstrating a limit of detection at 0.64 ng/mL. This is the first demonstration of a rapid antigen-detection test prototype based on a lateral flow assay platform for diagnosing life-threatening spotted fever rickettsioses in a timely manner. Furthermore, these findings suggest that the timely detection of RC0497 offers guidance for treatment, leading to a significant improvement in infection outcomes.

Acknowledgements (Optional): This research was funded by grants from the DoD (W81XWH2010834) and CDC (1U01CK000512).