GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus.

Nitaya Indrawattana,Manas Chongsa-Nguan,Wanpen Chaicumpa,Watee Seesuay,Nawannaporn Sae-Lim,Nitat Sookrung,Pisinee Aiumurai,Onrapak Reamtong

doi:10.3390/diagnostics12010136

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.

Highlights

Scrub typhus is a febrile illness caused by an obligate intracellular and highly pleomorphic Gram-negative bacterium of the Rickettsiales order, Rickettsiaceae family, named Orientia tsutsugamushi
Appropriate clone of the transformed BL21 (DE3) E. coli was grown under 0.4 mM isopropyl-β-D thiogalactopyranoside (IPTG) induction condition and the 6× His tagged-recombinant protein was purified from the bacterial inclusion body (IB) by using a metal affinity resin
The overall results in this study indicate that the so-developed ICT antigen detection test kit (AgTK) can be used for point-of-care scrub typhus diagnosis in the resource-limited areas where there are high incidences of undifferentiated febrile illnesses that require specific and differential diagnoses

Summary

Introduction

Scrub typhus is a febrile illness caused by an obligate intracellular and highly pleomorphic Gram-negative bacterium of the Rickettsiales order, Rickettsiaceae family, named Orientia tsutsugamushi. Orientia tsutsugamushi consists of six prototypic strains, including Gillian, Karp, Kato, Shimokoshi, Kawasaki and Kuroki [4,5]. The saliva of the chiggers contains an enzyme that can damage human skin, causing a typical necrotic lesion at the wound called an eschar [7]. Delayed diagnosis and treatment may lead to bacterium mediated-vascular injury in various vital organs, including the liver, kidneys, meninges and brain [9]; and disseminated intravascular coagulopathy (DIC) with platelet consumption, vascular leakage, pulmonary edema, shock and organ failure, which may eventually result in death [10,11]

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