Line Immunoblot Assay for Tick-Borne Relapsing Fever and Findings in Patient Sera from Australia, Ukraine and the USA

Abstract

Tick-borne relapsing fever (TBRF) is caused by spirochete bacteria of the genus Borrelia termed relapsing fever Borreliae (RFB). TBRF shares symptoms with Lyme disease (LD) caused by related Lyme disease Borreliae (LDB). TBRF and LD are transmitted by ticks and occur in overlapping localities worldwide. Serological detection of antibodies used for laboratory confirmation of LD is not established for TBRF. A line immunoblot assay using recombinant proteins from different RFB species, termed TBRF IB, was developed and its diagnostic utility investigated. The TBRF IBs were able to differentiate between antibodies to RFB and LDB and had estimated sensitivity, specificity, and positive and negative predictive values of 70.5%, 99.5%, 97.3%, and 93.4%, respectively, based on results with reference sera from patients known to be positive and negative for TBRF. The use of TBRF IBs and analogous immunoblots for LD to test sera of patients from Australia, Ukraine, and the USA with LD symptoms revealed infection with TBRF alone, LD alone, and both TBRF and LD. Diagnosis by clinical criteria alone can, therefore, underestimate the incidence of TBRF. TBRF IBs will be useful for laboratory confirmation of TBRF and understanding its epidemiology worldwide.

Lyme Disease: Diversity of Borrelia Species in California and Mexico Detected Using a Novel Immunoblot Assay

“…Recombinant target proteins were obtained by cloning hybrid gene constructs or portions of genes into pET vectors, expressing the gene products in Escherichia coli (GenScript, Piscataway, NJ, USA), then isolating the proteins to >90% purity, as previously described [1,2]. Bbsl recombinant proteins were derived from several US and European species of Bbsl including Bbss strains B-31 and 297 for the detection of the following targets: P18, P23 (OspC), P28, P30, P31 (OspA), P34 (OspB) P39 (BmpA), P41, P45, P58, P66, P93, the variable surface antigen of Bbss (VlsE), and C6 (a hybrid protein containing the immunodominant region of VlsE from different Bbsl species), as previously described [1].…”

“…garinii, B. mayonii, and B. valaisiana. Recombinant proteins from different RFB species were derived for the detection of four target antigens, BipA, GlpQ, fHbp, and FlaB, as previously described [2]. The targeted RFB species were B. hermsii, B. miyamotoi, B. turicatae, and B. turcica.…”

“…Antigen strips for Bbsl and RFB immunoblots were prepared as previously described [1,2]. In brief, purified proteins and control proteins were diluted (7-19 ng protein/line) and sprayed in straight lines on nitrocellulose sheets (Amersham Protran, GE Healthcare Life Science) using a BioDot liquid dispenser (BioDot, Irvine, CA, USA).…”

“…Borrelia spirochetes are a significant cause of disease worldwide. Borrelia species fall within the family Spirochaetaceae and are characterized as Gram-negative, helical bacteria moving via periplasmic axial filaments [1][2][3]. Currently, there are at least 53 known Borrelia species categorized into three groups: approximately 22 species fall within the Lyme Disease group (B. burgdorferi sensu lato, Bbsl), and approximately 29 fall within the Relapsing Fever Borrelia (RFB) group.…”

“…To assess the impact of testing limitations and to determine levels of exposure to Bbsl and RFB species in California and Mexico, we employed a recently developed modified Western blot procedure, termed the line immunoblot, which uses recombinant antigens from common strains and species of the Bbsl and RFB groups for the serological diagnosis of LD and RF [1,2]. Testing was conducted over an eight-month period in 2019 on patients with suspected tickborne disease.…”