(STILL BEING EDITED – May 2018)
Historically, most scrub typhus case diagnoses were serological in nature since isolation or visualization of the causative organism was and is very difficult. Thus, serological methods provided the bulk of procedures that have been used in the diagnosis of scrub typhus. These serological approaches have been successively improved from the earliest Weil-Felix test to the most recent approaches. Serological methods still represent the most widely used procedures. Efforts continue to be made to improve the accuracy and especially the speed with which these methods are applied.
1) THE WEIL-FELIX TEST
The Weil-Felix test was first developed in 1916 for use in the diagnosis of typhus. The introduction of the Weil-Felix assays and, in particular, the introduction of use of the OX-K (Kingsbury) strain of Proteus in 1934 facilitated serodiagnosis of human scrub typhus (Mackie, 1946, p. 196). The Weil-Felix test (WF) is based on the agglutination of the OXK strain of Proteus mirabilis. Among diagnostic tests for scrub typhus, it remains the simplest detection method. As such, the WF continues the be widely used for clinical identification, especially within resource-limited settings of less well equipped hospitals in the Tsutsugamushi triangle. As such, it remains an important tool in the diagnosis of scrub typhus.
Diagnosis is based upon the ability of a sample of patient serum to promote seroagglutination when challenged by a set of OX antigens in a tube or on a laboratory slide.
The figure on the left represents a classic Weil-Felix test of a scrub typhus patient’s sera using Proteus mirabilis OX-K antigens and a 35 well U-bottomed plate. Horizontal rows reflect doubling (2-fold) dilutions of patient’s serum, 1:10 through 1:640. Horizontal rows 1 and 5 are negative serum controls. Rows 2, 3 and 4 read 1: 10 (acute sample), 1:40 and > or = 1:640 respectively and result would be reported as a diagnostic (4-fold or greater) rise in titer. Note: negative wells of non-agglutinated antigen show as sharply defined round buttons, whereas positive agglutination appears as clumped antigen and is more evident upon inspection using a visual plate reader. Weil-Felix was performed using the method of Gaultney, Wende and Williams, 1971. Many tests in 2018 would be done using a commercially prepared card containing the OX-K antibodies that would be agitated or rocked to facilitate agglutination.
Although very inexpensive (a reason for the still common use of this test to this day), the Weil-Felix test had come to be considered inaccurate, lacking sensitivity and specificity. In 1987 the WHO task force on serological diagnosis of scrub typhus formally discouraged its use (World Health Organization, 1987). Nevertheless, it has been used for decades to diagnose scrub typhus.
2) IMMUNOFLUORESCENCE ASSAY (IFA)
Introduction of the fluorescence microscope and consequently the immunofluorescence assay (IFA) in the 1960’s (203) and its micromodification the microimmunofluorescence assay or MIF (Robinson et al) permitted the accurate even though delayed serodiagnosis, especially when paired acute and convalescent sera were used to show diagnostic specific rises in antibody titer. In that era the MIF was primarily a research tool generally not available to clinicians. This issue of test availability and accuracy became more important soon after World War II when effective antibiotics (chloramphenicol and subsequently the tetracyclines) came available to effectively treat the disease once recognized (Smadel, refs). Thus misdiagnosis or failure to treat the disease could prove fatal. Identification of group-specific IgM antibodies to the various strains of O. tsutsugamushi, possible using the MIF, provided strong evidence of recent active infection. The sensitivity and specificity of MIF with paired sera has been reported to be 91% and 96% for IgG and 85% and 98% for IgM, respectively, using a 1:400 cutoff [Coleman, 2002, ref 202]. This assay has recently become commercially available (Fuller Laboratory, Fullerton, CA). Still, questions of standardization of the IFA/MIF assay remain, however (Blacksell, 2007, ref 201). [Suggest photo insert of slide and fluorescence test]
3) ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
A scrub typhus group-specific enzyme-linked immunosorbent assay or ELISA was developed in the late 1970s. It has served primarily for screening large numbers of sera reactive with single strains of O. tsutsugamushi or mixtures and can measure scrub typhus-specific IgM and IgG [Dasch, 1979, 208 ]. Either purified whole-cell preparations or lysed pressure cell extracts of the purified organisms have been used as ELISA antigens [31 ,141 ]. The sensitivity and specificity from a study on Thai patients were 97% and 89% for IgG (1:1600 cutoff) and 94% and 91% for IgM (1:400 cutoff) [202 ]. More recently the ELISA has incorporated recombinant proteins such as 56-, 47- and 22-kDa recombinents, as well as surface cell antigens [209 –212 ].
Some useful references: Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Laongnualpanich A, Basnyat B, Day NPJ, et al. Diagnostic accuracy of the InBios scrub typhus detect enzyme-linked immunoassay for the detection of IgM antibodies in Northern Thailand. Clin Vaccine Immunol. 2015;23(2): 148–154.
4) INDIRECT IMMUNOPEROXIDASE TEST (IP)
In the early 1980’s a practical adaptation of the MIF, the indirect immunoperoxidase test (IP) was initially introduced in Japan and shown to correlate closely with the MIF in sensitivity and specificity (Yamamoto, 1982; Kelly, et al, 1988). The IP test required only a common light microscope to quantify specific antibody. Although quite promising, the limited availability of reagents proved problematic and distribution of kits and supplementary reagents was primarily limited to Malaysia and Thailand. Still, the avid interest of the user state-run laboratories demonstrated the need for commercial availability for a simple diagnostic test (Kelly, 1990).
5) IMMUNOCHROMATOGRAPHIC DIPSTICK TEST KIT
In the 1990’s a first generation commercially available immunochromatographic dipstick test kit using native scrub typhus antigens was introduced and proven to be reliable, sensitive, and specific (Integrated Diagnostics, Baltimore, MD, USA). It could measure specific IgM associated with new infections (Koay, 1993). [Suggest photo of developed stick(s) ;needs a ref for st test]. More recently a second-generation immunochromatographic rapid diagnostic test using a mixture of O. tsutsugamushi 56 kDa-derived recombinant protein antigens has been introduced (Blacksell, 2010). Commercial development and production of recombinant protein antigens has made it possible to develop specific and sensitive RDTs. A mixture of three recombinant 56-kDa antigens from Karp, Kato, and Gilliam strains of O. tsutsugamushi was recently used to produce the Scrub Typhus CareStart tests manufactured by AccessBio (Somerset, NJ, USA). The lateral-flow-format showed good sensitivity and specificity for detection of IgM (96.8% and 93.3%), but lower specificity for detecting total antibodies (71.4%), though the sensitivity was high (97.6%) [215 ]. More recently, a second-generation lateral-flow-format product, ScrubTyphus Detect (ST Detect), manufactured by InBios (Seattle, WA, USA) using a mixture of 4 recombinant 56-kDa antigens from Karp, Kato, Gilliam and TA763 strains of O. tsutsugamushi, showed improved sensitivity for IgM detection of 99.25 and 100% among Indian and Thai patients, respectively [216 ,217 ]. RDTs produce rapid results and follow a simple protocol with no need for sophisticated electrical equipment; they meet the requirements of a field-deployable, point-of-car diagnostic assay for the early diagnosis of scrub typhus with military relevance, and are also attractive for use in rural areas where the use of diagnostics like ELISA and IFA may not be available.
reference: Lee K, Moon C, Oh WS, Sohn KM, Kim B. Diagnosis of scrub typhus: Introduction of the immunochromatographic test in Korea. Korean J Intern Med. 2014;29: 253–255.
Watthanaworawit W, Turner P, Turner C, Tanganuchitcharnchai A, Jintaworn S, Hanboonkunupakarn B, et al. Diagnostic accuracy assessment of immunochromatographic tests for the rapid detection of antibodies against Orientia tsutsugamushi using paired acute and convalescent specimens. Am J Trop Med. Hyg. 2015;93(6): 1168–1171.
Chinprasatsak S, Wilairatana P, Looareesuwan S, Chappuis F, Akkhavong K, Laferl H, et al. Evaluation of a newly developed dipstick test for the rapid diagnosis of scrub typhus in febrile patients. Southeast Asian J Trop Med Public Heal. 2001;32(1): 132–136.
Kim Y, Park S, Premaratna R, Selvaraj S, Park S, Kim S, et al. Clinical evaluation of rapid diagnostic test kit for scrub typhus with improved performance. 2016;(12): 1190–1196.
Stephen S, Kim S, Pradeep J, Kim YJ, Kim EY, Kim MW, et al. Evaluation of ImmuneMed scrub typhus rapid test kit, for diagnosis of scrub typhus. J Vector Borne Dis. 2016(53): 283–287.