Rapid Antigen-Based Assay Detects Tick-Borne Diseases Before Symptoms Appear

Tick-borne spotted fever rickettsioses (SFRs) continue to pose a significant threat to otherwise healthy individuals, causing severe illness and death due to the lack of a reliable and timely diagnostic laboratory test. The absence of specific, convenient, acute-phase diagnostic tests for SFRs contributes to their difficulty in diagnosis and underreporting. Currently, the gold standard for diagnosing tick-borne rickettsioses involves detecting antibodies against rickettsiae in the serum through indirect immunofluorescence assay (IFA). However, this antibody response typically appears 7 to 10 days after illness onset, when fatalities often occur. While molecular detection of bacterial DNA and immunohistochemical analysis of rickettsial antigens in skin biopsies may provide early diagnosis, the occurrence of skin rashes and their timing are unreliable. Furthermore, many hospital laboratories do not perform rapid molecular diagnostic testing for SFRs. Researchers have now identified a biomarker for SFRs, leading to the development of a prototype laboratory test aimed at detecting this biomarker for more accurate diagnosis.

Researchers at University of Houston (Houston, TX, USA) have identified a rickettsial protein, RC0497, in R. conorii-infected endothelial cell supernatant. RC0497, a putative N-acetylmuramoyl-L-alanine amidase, is highly conserved across various SFG species. The researchers showed that RC0497 is present in the blood of both infected mouse models of Mediterranean spotted fever (MSF) and MSF patients. In this study, the researchers tested the hypothesis that detecting RC0497 through a rapid lateral flow assay (LFA) could provide a timely diagnostic tool, potentially improving treatment outcomes for severe SFRs. The team used two experimental models of SFRs—R. conorii-infected C3H/HeN mice and R. rickettsii-infected guinea pigs—which replicate the pathophysiological changes seen in patients with Rocky Mountain spotted fever (RMSF). They measured RC0497 concentrations at various disease stages by mass spectrometry (MS) using blood samples from infected animals.

Using specific antibodies against RC0497 and europium chelate nanoparticle reporters, the researchers developed a europium nanoparticle LFA (EuNP LFA) to detect this biomarker for SFRs. The assay demonstrated high analytical sensitivity and specificity in serum samples from experimentally infected animals at the early stages of infection, when antibiotics could still control rickettsiae. The results suggest that detecting RC0497 in a timely manner using the europium LFA could guide treatment, significantly improving infection outcomes. This represents the first rapid antigen-detection test prototype based on a lateral flow assay platform for diagnosing life-threatening SFRs in a timely manner.

The research confirmed that a rickettsial antigen, detectable by a simple test, is present in the circulation of infected animals. Unlike serological tests used to detect past exposure to potentially nonpathogenic rickettsial species, the RC0497 LFA can distinguish active infections. This will enhance clinical diagnosis and epidemiological surveillance. Although further validation with patient specimens is needed, the team has established proof-of-concept for an LFA that could potentially be developed into a point-of-care diagnostic tool for use in real-world settings.