Determination of Optimal Diagnostic Cut-Offs for the Naval Medical Research Center Scrub Typhus IgM ELISA in Chiang Rai, Thailand.

Meghna Phanichkrivalkosil,Suthatip Jintaworn,Allen L Richards,Nicholas P J Day,Tri Wangrangsimakul,Ampai Tanganuchitcharnchai,Daniel H Paris,Pacharee Kantipong,Wirongrong Chierakul,Achara Laongnualpanich,Stuart D Blacksell

doi:10.4269/ajtmh.18-0675

Abstract

.In this diagnostic accuracy study, we evaluated data from 135 febrile patients from Chiang Rai, to determine the optimal optical density (OD) cutoffs for an in-house scrub typhus IgM ELISA. Receiver operating characteristic curves were generated using a panel of reference assays, including an IgM immunofluorescence assay (IFA), PCR, in vitro isolation, presence of an eschar, or a combination of these. Altogether, 33 patients (24.4%) were diagnosed as having scrub typhus. Correlation between positivity by IFA and increasing OD values peaked at a cutoff of 2.0, whereas there was little association between positivity by culture or eschar with increasing ELISA cutoffs—cutoffs of 3.0 and 4.0 were demonstrated to be optimal for the total absorbance of the OD at dilutions 1:100, 1:400, 1:1,600, and 1:6,400, for admission and convalescent samples, respectively. The optimal cutoff at a 1:100 dilution was found to be between 1.85 and 2.22 for admission samples and convalescent-phase samples, respectively. Sensitivities for the cutoffs varied from 57.1% to 90.0% depending on the reference test and sample timing, whereas specificities ranged from 85.2% to 99.0%. We therefore recommend a cutoff of around 2.0, depending on the sensitivity and specificity desired in clinical or epidemiological settings. The results demonstrate the ELISA to be a valuable diagnostic tool, suitable for use in resource-limited endemic regions, especially when used in combination with other diagnostic modalities such as the presence of an eschar.

Highlights

Scrub typhus is a major cause of acute febrile illness in the Asia-Pacific region, accounting for up to 20–50% of febrile hospital admissions in endemic areas.[1,2,3] Orientia tsutsugamushi, the causative agent in the region, is transmitted to humans through the bite of the larval stage of infected chigger mites.[4,5] Clinical manifestations commonly observed include fever, eschar, rash, headache, myalgia, malaise, and regional lymphadenopathy
The following reference diagnostic assays were performed to determine the final scrub typhus infection status of the patients included in the study: 1) in vitro isolation of O. tsutsugamushi from buffy coat samples performed using a previously described method[16]; PCR assays including the 56 kDa nested PCR assay,22 2) 47 kDa– based quantitative real-time PCR assay[23] and 3) GroEL-based qPCR assay[24]; and 4) the immunofluorescence assay (IFA) based on O. tsutsugamushi pooled Karp, Kato, Gilliam whole cell antigens for scrub typhus.[19]
The conventional IFA IgM cutoff titer of 3 1:400 in the admission sample or a 4-fold rise to 3 1:200 in the convalescentphase sample may lead to a high rate of false positivity.[8,20]

Summary

Introduction

Scrub typhus is a major cause of acute febrile illness in the Asia-Pacific region, accounting for up to 20–50% of febrile hospital admissions in endemic areas.[1,2,3] Orientia tsutsugamushi, the causative agent in the region, is transmitted to humans through the bite of the larval stage of infected chigger mites.[4,5] Clinical manifestations commonly observed include fever, eschar, rash, headache, myalgia, malaise, and regional lymphadenopathy. The disease may progress to multi-organ failure and death.[6] With the exception of the eschar, scrub typhus is difficult to differentiate clinically from other febrile illnesses such as dengue, murine typhus, or leptospirosis. If diagnostic facilities are available, serological or molecular tests can be performed to enhance the clinical diagnosis, but both diagnostic methods have inherent weaknesses.[7]

Methods

Results

Conclusion