GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus

Abstract

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

Keywords

• antigen detection test kit
• scrub typhus
• GroEL chaperonin
• <i>Orientia tsutsugamushi</i>
• hybridoma
• immunochromatography (ICT)