Evaluation of the conserved subunit of the pathogenic <i>Leptospira</i> FlaB protein-based immunochromatographic test for the diagnosis of acute leptospirosis

We have computationally identified a conserved region of flagellin B (FlaB) protein that is present only in pathogenic <i>Leptospira</i> but absent in non-pathogenic <i>Leptospira</i> or other flagellated bacteria. The predicted FlaB subunit protein (sFlaB) comprised a sequence of 50-amino acids: ¹⁴⁰FARGSRVASMWFHMGPNQNQRERFYIGTMTSKALKLVKADGRPIAISSPG¹⁸⁹. The nucleotide sequence encoding sFlaB was amplified by polymerase chain reaction from <i>Leptospira</i> genomic DNA and cloned into a pET100 expression vector. The recombinant sFlaB was expressed in <i>Escherichia coli</i> BL21 and affinity purified to serve as an antigen to the immunochromatographic test (ICT) designed for specific IgM (ICT-IgM) and IgG (ICT-IgG) antibody detections. We evaluated 109 serum samples, including 46 from leptospirosis patients (29 acute sera and 17 convalescent sera) and 63 from patients with other acute febrile illnesses (46 acute sera and 17 convalescent sera). Compared to the paired serum results of the indirect immunofluorescent assay, the sensitivity of ICT-IgM and ICT-IgG was 80.40% and 71.73%, with specificities of 84.13% and 73.01%, respectively. For acute leptospirosis sera, ICT-IgM showed a sensitivity of 75.86% and a specificity of 89.13%, while ICT-IgG had a sensitivity of 68.97% and a specificity of 71.94%.IMPORTANCELeptospirosis is a zoonotic disease caused by pathogenic leptospires. The infected patient presents with a mild to severe febrile illness and may die while receiving inappropriate treatment. The microscopic agglutination test, the current gold standard method, is laborious and requires the use of live panel leptospires, which should only be done in a reference laboratory. In addition, the results of the paired serum samples are required for an accurate interpretation. Polymerase chain reaction (PCR) was used instead for diagnosis in the acute phase of infection. However, PCR requires an expensive machine and a specialist to analyze the results. Therefore, a simple and rapid test is needed for the early diagnosis of leptospirosis.