Performance of molecular and serologic tests for the diagnosis of scrub typhus.

Kavitha Kannan,Rebecca John,Kavitha Saravu,Sowmya Sathyendra,Ramya Iyadurai,John Antony Jude Prakash,Balaji Veeraraghavan,Karthik Gunasekaran,Binesh Lal Yesudhason,Divya Dayanand,Anand Zachariah,Debasree Kundu,George M Varghese,Alice Joan Mathuram,M N Sumana ,Linda Rose Jose ,M Kavitha ,Kundavaram Pp Abhilash ,Asha Mary Abraham ,Ooriapadickal Cherian Abraham ,Samuel George Hansdak

doi:10.1371/journal.pntd.0008747

Abstract

Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its non-specific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluates the diagnostic performance of various tests. The present study aims at assessing the accuracy of various rapid diagnostic tests, serologic tests, and nucleic acid amplification methods on well-characterized patient samples. Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood, eschar or tissue were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative real time PCR using 47kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil-Felix test(WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT).Among the 316 participants, 158 had confirmed scrub typhus (cases) and 158 were controls. ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies had excellent discriminative potential with sensitivities and specificities of 92%, 94% and 92%, 92% respectively. The sensitivity and specificity of IFA were found to be 95% and 74% respectively. IgM serology had a false positivity rate of 8% with other acute febrile illnesses such as dengue, leptospirosis and spotted fever due to the nonspecific binding of the pentavalent IgM. LAMP assay had 91.7% sensitivity and 77.2% specificity while qPCR provided excellent sensitivity (97%) and perfect specificity. In conclusion, ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies have excellent sensitivity and specificity while the accuracy of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories particularly for diagnosis of early disease with less than 7 days duration. This study provides a comprehensive evaluation of all currently available diagnostic tests for scrub typhus.

Highlights

IntroductionScrub typhus, caused by the bacterium Orientia tsutsugamushi and transmitted by the bite of the larval stage of trombiculid mites (chiggers), is the most common and clinically important rickettsial infection worldwide, especially in several Asian countries including India
Scrub typhus, caused by the bacterium Orientia tsutsugamushi and transmitted by the bite of the larval stage of trombiculid mites, is the most common and clinically important rickettsial infection worldwide, especially in several Asian countries including India
Non-specific binding of the IgM antibody, attributed to its pentavalent structure is probably the cause of this false positivity rate

Summary

Introduction

Scrub typhus, caused by the bacterium Orientia tsutsugamushi and transmitted by the bite of the larval stage of trombiculid mites (chiggers), is the most common and clinically important rickettsial infection worldwide, especially in several Asian countries including India. An estimated one billion people are at risk in endemic regions, with nearly a million cases occurring each year [1]. As most cases occur in rural areas with poor diagnostics, this is almost certainly a gross under estimation. Sero-epidemiologic studies estimate an increasing prevalence ranging from 9–31% across Asia [2, 3]. The symptoms and signs of scrub typhus are non-specific and often resemble other tropical infections such as dengue, malaria, typhoid and leptospirosis which are endemic to these regions. The case fatality rate can be as high as 30–50% [7]

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