TY - JOUR
T1 - Development of a rapid antigen-based lateral flow assay for tick-borne spotted fever rickettsioses
AU - Willson, Richard
AU - Zhao, Yingxin
AU - Brosamer, Kristen
AU - Pal, Yogita
AU - Blanton, Lucas S.
AU - Arroyave, Esteban
AU - Roach, Carson
AU - Walker, David H.
AU - Kourentzi, Katerina
AU - Fang, Rong
N1 - Publisher Copyright:
© 2025 Willson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2025/1
Y1 - 2025/1
N2 - Tick-borne spotted fever rickettsioses (SFRs) continue to cause severe illness and death in otherwise-healthy individuals due to lack of a timely and reliable diagnostic laboratory test. We recently identified a diagnostic biomarker for SFRs, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Here, we developed a prototype laboratory test that targets RC0497 for diagnosis of SFRs. The concentrations of RC0497 in sera of Rickettsia rickettsii-infected guinea pigs and R. conorii-infected mice were determined by stable isotope dilution–parallel reaction monitoring mass spectrometry (SID-PRM-MS), ranging from 0.1 to 1.1 ng/ml. Using europium chelate nanoparticle reporters, we developed a lateral flow assay (LFA) and evaluated the test with a panel of serum samples of mock and experimentally infected animals. Interestingly, 21 of 22 (95.5%) serum samples from R. rickettsii-infected guinea pigs and R. conorii-infected mice yielded positive results with a ratio of test line / control line greater than the cutoff value determined for non-infected animals. All uninfected samples were in agreement with the intended results, suggesting that the initially assessed specificity of the test is 100%, among these samples. Mice infected with a lethal dose of R. conorii and treated with doxycycline on day 3 post-infection (p.i.), upon RC0497 detection by LFA, displayed significantly decreased rickettsial loads, comparable to the sublethal infection group on day 5 p.i.. A panel of human serum samples spiked with various concentrations of recombinant RC0497 were analyzed by LFA, suggesting that the limit of detection of the LFA was 0.64 ng/mL. These findings suggest that the timely detection of RC0497 by a europium LFA offers guidance for treatment, leading to a significant improvement in infection outcomes. This work, for the first time, shows significant promise for a rapid and easy-to-use platform offering a timely diagnostic assay for severe SFRs.
AB - Tick-borne spotted fever rickettsioses (SFRs) continue to cause severe illness and death in otherwise-healthy individuals due to lack of a timely and reliable diagnostic laboratory test. We recently identified a diagnostic biomarker for SFRs, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Here, we developed a prototype laboratory test that targets RC0497 for diagnosis of SFRs. The concentrations of RC0497 in sera of Rickettsia rickettsii-infected guinea pigs and R. conorii-infected mice were determined by stable isotope dilution–parallel reaction monitoring mass spectrometry (SID-PRM-MS), ranging from 0.1 to 1.1 ng/ml. Using europium chelate nanoparticle reporters, we developed a lateral flow assay (LFA) and evaluated the test with a panel of serum samples of mock and experimentally infected animals. Interestingly, 21 of 22 (95.5%) serum samples from R. rickettsii-infected guinea pigs and R. conorii-infected mice yielded positive results with a ratio of test line / control line greater than the cutoff value determined for non-infected animals. All uninfected samples were in agreement with the intended results, suggesting that the initially assessed specificity of the test is 100%, among these samples. Mice infected with a lethal dose of R. conorii and treated with doxycycline on day 3 post-infection (p.i.), upon RC0497 detection by LFA, displayed significantly decreased rickettsial loads, comparable to the sublethal infection group on day 5 p.i.. A panel of human serum samples spiked with various concentrations of recombinant RC0497 were analyzed by LFA, suggesting that the limit of detection of the LFA was 0.64 ng/mL. These findings suggest that the timely detection of RC0497 by a europium LFA offers guidance for treatment, leading to a significant improvement in infection outcomes. This work, for the first time, shows significant promise for a rapid and easy-to-use platform offering a timely diagnostic assay for severe SFRs.
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U2 - 10.1371/journal.pone.0312819
DO - 10.1371/journal.pone.0312819
M3 - Article
C2 - 39823491
AN - SCOPUS:85215402865
SN - 1932-6203
VL - 20
JO - PLoS ONE
JF - PLoS ONE
IS - 1
M1 - e0312819
ER -