Review
Diagnosis of scrub typhus
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Expert Rev. Anti Infect. Ther. 12(12), 1533–1540 (2014)
Jeshina Janardhanan1, Paul Trowbridge2, George M Varghese*1 1 Medicine Unit I and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India 2 Department of Infectious Diseases, Tufts University, Boston, MA, USA *Author for correspondence: Tel.: +91 9487393015 Fax: +0416 2232035 [email protected].
Scrub typhus is an acute febrile illness that, if untreated, can result in considerable morbidity and mortality. One of the primary reasons for delays in the treatment of this potentially fatal infection is the difficulty in diagnosing the condition. Diagnosis is often complicated because of the combination of non-specific symptoms that overlap with other infections commonly found in endemic areas and the poor available diagnostics. In the majority of the endemic settings, diagnosis still relies on the Weil–Felix test, which is neither sensitive nor specific. Other methods of testing have become available, but at this time, these remain insufficient to provide the rapid point-of-care diagnostics that would be necessary to significantly change the management of this infection by providers in endemic areas. This article reviews the currently available diagnostic tools for scrub typhus and their utility in the clinical setting. KEYWORDS: 56-KDa type-specific antigen • ELISA • immunofluorescence assay • Orientia tsutsugamushi • rapid diagnosis • scrub typhus
Scrub typhus is an acute febrile illness, affecting multiple organ systems, and is caused by the obligate intracellular bacteria Orientia tsutsugamushi. This organism is transmitted through the bite of chiggers, the larval stage of the mite Leptotrombidium, commonly encountered in rural areas. Orientia belongs to the Rickettsiaceae family and has a larger chromosome than other members of the family. It represents the most highly repeated bacterial genome sequenced so far with approximately 47% of the genome being repetitive sequences formed from integrative and conjugative elements, transposable elements and pseudogenes. The massive intragenomic deletions, duplications and rearrangements, along with acquisition of foreign genes, have contributed to a high degree of plasticity within the Orientia genome [1,2]. This disease is endemic to a region known as the ‘tsutsugamushi triangle’, which extends from Japan and Russia in the North to Northern Australia in the South and the Arabian peninsula in the West. Within this region of endemicity, which contains more than half of the world’s population, it has been previously estimated that around 1 million cases of this disease occur annually and 1 billion people are at risk of infection [3]. Notably, this estimate was made before the recognition of the re-emergence of this disease in several areas, and, in some regions, scrub typhus can account for up to 50% of undifferentiated febrile illnesses admitted to the hospital during the rainy season [4].
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In addition to the massive scope of this infection, scrub typhus is often co-endemic with other diseases found in these regions such as dengue, malaria, leptospirosis and typhoid with which it shares similar presentations [5]. By itself scrub typhus was a major concern in the preantibiotic era, as the case fatality rate approached 60% [5], and it caused thousands of fatalities during the Second World War. Fortunately, since the advent of antibiotics, scrub typhus outcomes have significantly improved when appropriate treatment is started in a timely manner. However, given the overlap in the clinical presentation of this infection with other diseases common to the area of endemicity, the administration of appropriate treatment may be delayed by difficulty in reaching a diagnosis. This difficulty with diagnosing this potentially fatal illness is added by the lack of good point-of-care testing. Clinical features
The diagnosis of scrub typhus requires a high degree of clinical suspicion, prompting the clinician to request appropriate laboratory investigations and confirmatory tests. Patients usually present with an acute undifferentiated febrile illness, headache, myalgia, breathlessness, lymphadenopathy and varying involvement of organs such as the liver, lung and kidney. Organ involvement may be subtle and not evident initially unless carefully looked for. Patients may also have an eschar. The eschar
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in scrub typhus is a small, necrotic lesion, which may resemble a cigarette burn mark [6].These are often found on the groin, axillae, genitalia or neck. Although pathognomonic for scrub typhus, this may easily be overlooked. Previous reports have placed the incidence of eschar in acute scrub typhus at anywhere from 10 to 90% [7,8]. Most patients with scrub typhus develop thrombocytopenia and blood counts reveal leukocytosis in about half of the patients. Differential counts often reveal early lymphopenia with late lymphocytosis. Abnormal liver function tests with transaminase (AST/ALT) elevation of more than twice the normal limit is seen in majority of the patients [9]. Renal function impairment or cerebrospinal fluid abnormalities may be seen in up to quarter of the patients [10]. Chest x-ray may reveal localized infiltrates. Severe complications of the disease include acute respiratory distress syndrome, hepatitis, renal impairment, hypotension, meningitis, myocarditis and even death [6,11]. A high degree of clinical suspicion and familiarity with the various manifestations of scrub typhus is necessary to allow for early diagnosis and the timely initiation of appropriate therapy. However, given the impressive spectrum of presentation with this infection, even with a high index of clinical suspicion and the best clinical knowledge, diagnosis remains difficult. Serological diagnosis
The mainstay of diagnosis of scrub typhus has been and remains serology. In primary scrub typhus infection, a significant IgM antibody titer is mounted by the end of the first week, whereas IgG antibodies typically appear by the end of the second week. With reinfection, however, IgM antibody titers may be variable and IgG detectable by the sixth day of infection [12]. Weil–Felix testing has been in use for many years, though it is neither sensitive nor specific. This test uses a cross-reacting proteus OXK strain for the serodiagnosis of the disease, but this test has a sensitivity of only around 50% in the second week of infection [13]. Typically, a minimum titer of 1:80 or a fourfold increase over previous levels is taken as significant [14]. The current serological gold standard and reference test is the indirect immunofluorescent antibody (IFA) test, which uses fluorescence-labeled anti-human immunoglobulin to detect antibodies in the serum of the patient that have bound to immobilized bacterial antigen on a slide. Although considered the gold standard for the diagnosis of scrub typhus, several major limitations exist for this testing method. Although demonstration of a fourfold increase in antibody titers between acute and convalescent serum samples is diagnostic, such a diagnosis remains retrospective and is of little practical relevance for guiding initial management. Another major limitation of this test is the requirement of fluorescent microscopes, which are often not available in the resource-limited areas where scrub typhus is endemic [15]. Furthermore, the technique is expensive, requiring considerable technical expertise. Several routes have been taken to attempt to address the shortcomings of this testing method. IFA has been further 1534
adapted into a microimmunofluorescence format where lesser volumes of serum and antigen could be used [16]. This was found to be more convenient and allowed for simultaneous testing for multiple strains. Indirect immunoperoxidase (IIP) was also developed as a modification of the IFA, using peroxidase-labeled antibodies instead of fluorescein, thus negating the need for a fluorescent microscope [17]. Evaluation of IIP has shown that it is equivalent to IFA. Unfortunately, the sensitivity in both cases is affected by the strain variation and though the need for a fluorescent microscope is negated by this method, the need for technical expertise remains unchanged [18]. In a comparison of IFA using venous blood and finger prick filter paper blood spots, the sensitivity and specificity for classifying patients as having scrub typhus was found to be 95 and 88%, respectively. The use of blood spots facilitates transport of specimens to laboratories with IFA facilities; however, a decrease in accuracy was noted, regardless of the temperature at which specimens were stored, as storage time increased [19]. Finally, attempts have also been made to allow IFA to be used in a more clinically relevant manner, using a single acute serum sample for the diagnosis of scrub typhus. However, an extensive review of IFA methodologies and cut-offs on single acute serum samples for diagnosis of scrub typhus failed to form a consensus, questioning the suitability of using the test in this manner and even the use of this test as a reference standard. The most common cut-off was 1:400, but values ranged significantly from 1:10 to 1:400 [20]. Owing to the non-availability of IFA in common laboratories and the low sensitivity of Weil–Felix testing, ELISA is currently the preferred method of serological diagnosis. The 56-kDa type-specific antigen (TSA) is best suited for diagnostic testing, as it is the major immunodominant surface protein, containing both group- and strain-specific epitopes, and it is abundant in patient sera. The 56-kDa TSA is also highly variable, accounting for strain differentiation and genetic diversity in O. tsutsugamushi. The US Naval Medical Research Institute developed the r56, or recombinant 56-kDa antigen, which is now being used in several rapid diagnostic assays and ELISAs [21]. This antigen was found to be much more convenient to use than native antigens because large quantities of the antigen could be prepared safely using Escherichia coli without the need for bio-safety level 3 facilities, and the antigen product could be stored for a longer period. Using r56, IgM capture ELISA has been reported to have a sensitivity and specificity of 96.3 and 99%, respectively. As well, it has been reported that this assay could serologically confirm infection with a single serum specimen taken during any phase of acute infection or early convalescence, except for the first 2–3 days after the onset of illness [22]. Evaluation of this method in various scrub typhus endemic locations has shown sensitivities ranging from 85 to 100% with specificities of 97–99% [22,23]. Despite many advantages with this testing method, ELISA testing remains very expensive to perform and a time-intensive process. Other serological testing methods, such as complement fixation, hemagglutination and western blot have not fared better Expert Rev. Anti Infect. Ther. 12(12), (2014)
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Diagnosis of scrub typhus
than IFA. Complement fixation was one of the earliest tests used for serodiagnosis of scrub typhus, and it has been reported to be highly specific but with poor sensitivity. Being strain specific, complement fixation also requires antigens from all of the strains endemic to a particular region to be used for efficient detection [24]. SDS-PAGE immunoblot testing using the 56-kDa antigen has been used and found to be highly sensitive and specific, but, similar to use of ELISA, it is time consuming and hence rarely used in serodiagnosis [25,26]. A passive hemagglutination using recombinant 56-kDA antigen fused to a maltose-binding protein and adsorbed to sheep erythrocytes was compared to IFA, and an overall sensitivity and specificity of 98.9% was observed [27]. However, even the best functioning serological testing method, with a high sensitivity and specificity, remains dependent on detecting antibodies. After the onset of symptoms, it may take several days to weeks for the antibody titers to reach detectable levels, which in turn delays appropriate treatment. Convalescent phase serum samples for confirmation testing may not always be available, which further affects the reliability of these tests. Rapid diagnostic tests
Further developments in the diagnostics of scrub typhus are the various rapid assays that have been made available, such as the lateral flow immunochromatographic test (ICT) or immunoblot test and the latex agglutination test. These tests build off of the same basic concepts as other serologic tests, but they use the recombinant 56-kDa TSA of only the most common subgroups – Karp, Kato and Gilliam and have shown a wide range of sensitivities and specificities in various studies. ICTs typically have shown sensitivities ranging from 74 to 90% and specificities from 86 to 99% for the detection of IgM [28,29]. Evaluation of one IgM ICT (Panbio, Australia) has demonstrated higher specificity but continues to have sensitivities too low for clinical purposes [30]. Another ICT, constructed using a recombinant chimeric protein containing epitopes from Karp and TA763 strains, showed a higher sensitivity of 90% but only a moderate specificity of 85% [31]. Dot blot immunoassays have been investigated to be pointof-care tests as they can be rapidly and easily performed, are relatively inexpensive and can be accurate. However, false positives have been reported in special populations, such as those with high-titer IgM antibody to unrelated antigens, and interpretation of the results of not only persons with a history of scrub typhus but also those who were presently sick with an unrelated illness was found be difficult [32,33]. A dipstick test using a dot blot immunoassay format was also developed in Thailand for the serodiagnosis of scrub typhus in specific, and it was also found to have a similar sensitivity of 87% and specificity of 94%, compared to IIP test [34]. Latex agglutination testing, which was developed into a simple and rapid diagnostic tool for scrub typhus, when compared to IFA had an overall sensitivity, specificity and accuracy of 89.1, 98.2 and 93.6%, respectively. An evaluation of this test on scrub typhus confirmed samples from Thailand identified a informahealthcare.com
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minimum cut-off titer of 1:16. This test was demonstrated to be rapid, simple and easy to use and made an excellent replacement for the Weil–Felix screening test. However, so far, reports of a successful evaluation of this test on a larger scale for the application of routine laboratory testing are not available [35]. Overall, though showing some promise, the major issue affecting these rapid diagnostic kits is the low sensitivities, which is in part due to the antigenic diversity of strains in different geographical locations. A recent study found that the inclusion of chimeric recombinant antigen cr56 and other recombinant antigens, r21 and kr56 from Korean endemic strains in ELISA, dot blot assay and rapid diagnostic test, significantly increased the sensitivity and specificity of detection for Korean isolates [36]. Generally, inclusion of recombinant antigens from region-specific strains has been found to increase detection sensitivities in those specific regions [36,37]. However, most of the currently available diagnostic kits rely on antigens from Karp, Kato and Gilliam strains, allowing other strains to be potentially undetected. In addition, the circulating strains in many areas remain uninvestigated. Furthermore, none of these assays are used routinely for diagnosis, and they have largely only been evaluated in a few East Asian countries. Although the performance of some of these assays has been as good as or better than the IFA or IIP and the technology shows promise, a reproducible, quantifiable assay using these methods for diagnosis of scrub typhus is still lacking. Molecular diagnostic tests
Although the first molecular biology-based method used for the detection of scrub typhus was PCR RFLP of the outer membrane protein gene, the most commonly used technique is a nested polymerase chain reaction targeting a 483 bp region located on the 56-kDa TSA gene. This technique was first described by Furuya et al. [38] and has since then become a widely used technique for diagnosing scrub typhus from blood, buffy coat and eschar samples with varying degrees of sensitivities ranging from 62 to 90 and 100% specificity [39–41]. Several studies have found PCR to be more sensitive than IFA, and particularly eschar PCR has been found to be more sensitive than using blood or buffy coat given the high number of organisms in an eschar and because of false negatives observed due to inhibition of PCR by hemoglobin and other components in blood [42]. However, though eschar PCR is certainly the most sensitive of the techniques, the absence of an eschar on many patients limits the utility of this test. The majority of available literature evaluating conventional PCR has shown lower sensitivities than nested PCR. However, many of these studies relied on the internal primers used for nested PCR, which may explain these outcomes. A few studies have reported that standard PCR targeting the 56-kDa gene shows sensitivities higher than nested PCR, while retaining the 100% specificity [7,43,44]. Furthermore, compared to nested PCR, conventional PCR takes half the time and reagents and carries less risk of contamination during the transfer steps. In a recent study, we optimized and evaluated an earlier described 1535
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conventional PCR targeting the 56-kDa TSA, and this was found to be more sensitive and economical than N-PCR with a similar specificity [41]. A major outstanding advantage of using PCR techniques is that detection of the diseases is possible before antibodies have become detectable by serology, allowing the infection to be diagnosed as it is happening to the patient rather than days or weeks later. Furthermore, despite concerns that direct detection of the organism could be diminished by antibiotic use before testing as may be the case for cultures, eschar tissues have been even reported to give positive results up to 7 days after the administration of antibiotics [45]. Looking at other targets aside from the 56-kDa TSA, highly sensitive quantitative PCRs that detect partial sequences of the Orientia 47-kDa outer membrane protein antigen/high temperature requirement A gene and the 60-kDa heat shock protein gene, groEL, were developed and evaluated [39,46–49]. However, though the Q-PCR is highly sensitive and specific, even detecting very low numbers of copies, it remains too expensive for routine diagnosis in an endemic setting. However, these tests are currently prohibitively expensive in many endemic areas, although this may change in the future. Another widely evaluated molecular detection technique is the loop-mediated isothermal PCR assay (LAMP). This is an isothermal amplification technique using a robust DNA polymerase and a set of three primer pairs that produce a specific double hairpin DNA template with high specificity and efficiency. However, LAMP assays targeting the groEL gene have only shown a sensitivity of 70% and specificity of 85% when compared to IgM ELISA, and a sensitivity and specificity of 83 and 88%, respectively, when compared to nested PCR for diagnosing acute scrub typhus infection [50]. A recent study from Thailand comparing the diagnostic capabilities of four rapid ICTs to LAMP assay again found low sensitivities of individual ICTs, ranging from 46 to 68%, with specificities ranging from 68 to 95%. The sensitivity of LAMP assay was also found to be low at 52% with a specificity of 94%. However, a combination of LAMP with the ICT was found to slightly increase the sensitivity to 67% with a specificity of 94%. Unfortunately, this study highlights the limitations of the currently available rapid tests and LAMP assays, which otherwise have the advantage of being able to be performed in endemic settings using minimal equipments [51]. Culture
Being an obligate intracellular pathogen, culturing O. tsutsugamushi is a long and tedious process requiring biosafety level 3 containment and experienced personnel. Culture has been attempted from blood, buffy coat and skin biopsies and has been performed in mice, mammalian cells, embryonated chicken eggs, Vero (African green monkey kidney) or L (mouse fibroblast) cells. Buffy coat is typically separated from ethylenediaminetetraacetic acid blood tubes and inoculated into a Vero or L929 monolayers in minimum essential medium supplemented with fetal bovine serum and glutamine. This is then incubated for at least 2 weeks 1536
at 37˚C in a 5% CO2 incubator. The requirement for a biocontainment facility and the potential contamination with other organisms is a major issue in culturing this organism [52]. Owing to the tedious nature, long incubation times and low sensitivity, culture can hardly be considered a diagnostic tool in a clinical setting. By and large, culture is used only for research purposes in reference laboratories. Immunohistochemistry
Immunohistochemical staining has been successfully used in other rickettsial infections, such as rickettsial pox and Rocky Mountain spotted fever, but has been only rarely used in the diagnosis of scrub typhus. Evaluation of IHC on eschars and maculopapular skin lesions has found that although the specificity on both eschar and skin lesion was 100%, the sensitivity for skin lesions was only 65%. The sensitivity was 100% when IHC was performed on eschar samples. However, as previously mentioned, eschars can be an unreliable physical finding, present in a highly variable percentage of patients, limiting the utility of this testing [53]. Notably, however, IHC has also been used to show the presence of intracellular bacteria in the endothelial cells of organs like the heart, lung, brain and kidneys of soldiers suspected to have died of scrub typhus during World War II and the Vietnam War. However, these samples are not taken from typical patients who are still alive. Choosing the appropriate diagnostic test
Scrub typhus is grossly under-diagnosed because of its nonspecific clinical presentation and poor available diagnostics. All of the currently available diagnostic tests have traits that make them either undesirable or unobtainable for the majority of facilities within endemic area. Serological testing is still the most common method for diagnosis. All serological methods, however, share the same flaw, in that they rely on the availability of the human body’s response to infection, antibodies, to detect infection. The process of producing antibodies within the body requires time, and often rechecking of titers, making all of these methods ineffective, to a greater or lesser extent, at diagnosing illness in a timely manner, particularly in very acute infections. Aside from this class-wide flaw, individual tests have their own strengths and weaknesses. While the Weil–Felix test is certainly widely available, cheap and easily performed even in resource-limited settings, it is of limited utility because of its extremely poor sensitivity. Some of the rapid serologic diagnostic assays, most notably the latex agglutination test, are certainly an improvement over the Weil– Felix test, but, by virtue of their very methodology, which includes use of only the most common Orientia strain antigens, also have sub-optimal sensitivities, and they remain untested on a larger scale. IFA remains the gold standard for diagnosis. However, because this method requires fluorescent microscopes and expertise for testing, which are usually not available in most areas where the disease is prevalent, this method is of limited utility. ELISA show promising results, with both a high sensitivity and specificity. Although easily available rapid diagnostic tests can be Expert Rev. Anti Infect. Ther. 12(12), (2014)
Diagnosis of scrub typhus
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Table 1. Currently available diagnostics for scrub typhus. Test
Sensitivity
Specificity
Advantage
Disadvantage
Ref.
Weil–Felix test
50–60%
95%
Cheap Widely available Easily performed
Very low sensitivity Serology not positive until days to weeks into infection
[13,14]
ELISA
97%
99%
Highly sensitive and specific
Expensive Time-consuming Serology may not be positive until days into infection
[22,23]
IFA
80–91%
80–100%
Highly sensitive and specific
Requires specialized equipment Requires experienced personnel
[16,20]
Nested PCR
60–90%
100%
Expensive
Conventional PCR
80–95%
100%
Quantitative PCR
83%
100%
Highly specific Positive earlier than serologic testing Can remain positive even after antibiotic treatment is started
PCR [39–41] [41,43,44]
Requires specialized equipment
[46–49]
Requires experienced personnel IFA: Immunoflourescent antibody.
used as a screening test in the community setting, IgM ELISA test is the most practically useful test for confirmation. Non-serological methods of testing share the advantage of testing directly for the presence of the organism rather than the body’s response to it. This allows these methods to potentially be of greater utility early in the infection when the body has not yet had a chance to produce antibodies. Culture, however, takes sufficiently long as to negate these advantages. Also, the low sensitivity of this method makes it a poor diagnostic tool. PCR testing does show promise, but issues remain around the obtainable sensitivities for affordable tests and the costs of more effective assays. Certainly, Q-PCR could hold potential for becoming a good confirmatory test in the future, but its high cost and limited availability remain prohibitive for most areas of endemicity right now. There are several challenges in developing a field usable rapid assay for diagnosis of scrub typhus. Most assays have focused on the 56-kDa TSA protein, as this protein is specific to O. tsutsugamushi bacteria. Analysis of this major outer membrane protein has revealed that the fragment contains four variable domains and four conserved domains. Sera from most patients with scrub typhus can recognize this protein, as a result of which it is widely used as a diagnostic antigen in many countries. However, there are several antigenic variants in O. tsutsugamushi based on the 56-kDa TSA, and so all of the TSAs derived from the endemic strains in a geographical location must be included for the assay to be sensitive to picking up these local variations. In addition to being sensitive to local strain variations, an ideal rapid diagnostic kit must also be cost–effective with a longer shelf life, distinguish between active and past infection and allow the use of direct sample without processing. It should also be suitable for use in rural clinical informahealthcare.com
sites, as scrub typhus is largely a rural disease, without requiring the use of any specialized equipment, facilities or a trained technician. Several rapid diagnostic kits are commercially available at this time and have shown some promise. However, further evaluation of these currently available kits based on the geographic diversity of the strains is essential. The overall picture of the currently available diagnostics is provided in TABLE 1. Conclusion
Overall, the diagnosis of scrub typhus remains solely dependent on maintaining a higher degree of clinical suspicion and working with limited diagnostic tests. At this point, we remain largely reliant on acute and convalescent antibody titer for serological confirmation, which limits the utility of testing. Further complicating this picture is the geographic strain heterogeneity, which may lower the sensitivity of these already substandard tests. Although several serological tests are currently commercially available, most of the tests have a low sensitivity or are too expensive and impractical for routine application, and the further evaluation of these tests is hampered by the imperfect gold standard and the geographic strain variation. Among all the available serological tests, IgM ELISA remains the most useful confirmatory test. Non-serologic testing methods, though having some advantages, similarly remain infeasible at this time due to expense and limited evaluation. With no single diagnostic test capable of rapidly detecting this deadly infection, the development of a highly sensitive and specific point-of-care test is of utmost urgency. Expert commentary
Scrub typhus is a serious multi-system infection causing significant morbidity and mortality. The understanding of the 1537
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immunopathogenesis and disease mechanisms are evolving. Diagnosis of this infection is often delayed because of the combination of non-specific symptoms, limited awareness among clinicians and the poor available diagnostics. However, promoting awareness among clinicians and reliable methods for rapid diagnosis will be the key to improve outcome in these patients. Current effort in developing rapid and point-of-care diagnostics and evidence-based management strategies in endemic regions will address the gap in identification and management of this significant public health problem.
not far from scientific reach. Progress in nanotechnology and sensitive platforms can make the point-of-care a reality. Furthermore, improved culture techniques and molecular diagnostics will strengthen the confirmatory diagnosis of this condition. Advances in diagnostic testing may permit clinicians to diagnose the condition promptly, which will result in improved outcomes. These essential changes may transform the clinical landscape and provide optimal care for patients with scrub typhus. Financial & competing interests disclosure
Five-year view
Looking forward into the immediate future, major advances in rapid diagnosis and management are expected. Aided by the new technologies to sequence the organism and understand the immunodominant proteins, newly identified diagnostic targets for developing rapid and point-of-care tests are
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Scrub typhus is a grossly under-diagnosed disease because of its non-specific clinical presentation and poor available diagnostics. • Serological testing is still the most common method for diagnosis. • Immunofluorescent antibody testing remains the gold standard for serologic diagnosis. However, it requires specialized equipments and expertise, which is usually not available in scrub typhus endemic settings. • Evaluation of currently available rapid diagnostic kits are hampered by the imperfect gold standard and the geographic strain variation. • Among all the available serological tests, IgM ELISA remains the most useful confirmatory test, which is still an expensive and timeconsuming test. • With no single diagnostic test capable of rapidly detecting this deadly infection, the development of a highly sensitive and specific pointof-care test is of utmost urgency.
Beran GW, Ed.) CRC Press; Boca Raton, FL: 1994. p. 463-74
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Ching WM, Rowland D, Zhang Z, et al. Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi. Clin Diagn Lab Immunol 2001;8:409-14 Blacksell SD, Jenjaroen K, Phetsouvanh R, et al. Accuracy of AccessBio immunoglobulin M and total antibody rapid immunochromatographic assays for the diagnosis of acute scrub typhus infection. Clin Vaccine Immunol 2010;17: 263-6
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Weddle JR, Chan TC, Thompson K, et al. Effectiveness of a dot-blot immunoassay of anti–Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus. Am J Trop Med Hyg 1995;53:43-6 Watt G, Strickman D, Kantipong P, et al. Performance of a dot blot immunoassay for the rapid diagnosis of scrub typhus in a longitudinal case series. J Infect Dis 1998;177:800-2 Chinprasatsak S, Wilairatana P, Looareesuwan S, 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 Health 2001;32:132-6 Wongchotigul V, Waicharoen S, Riengrod S, et al. Development and evaluation of a latex agglutination test for the rapid diagnosis of scrub typhus. Southeast Asian J Trop Med Public Health 2005;36:108-12 Kim YJ, Yeo SJ, Park SJ, et al. Improvement of the diagnostic sensitivity of scrub typhus using a mixture of recombinant antigens derived from Orientia tsutsugamushi serotypes. J Korean Med Sci 2013;28:672-9 Cao M, Guo H, Tang T, et al. Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus. Am J Trop Med Hyg 2007;76:553-8 Furuya Y, Yoshida Y, Katayama T, et al. Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase
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Prakash JA, Kavitha ML, Mathai E. Nested polymerase reaction on blood clots for gene encoding 56Kda antigen and serology for the diagnosis of scrub typhus. Indian J Med Microbiol 2011;29:47-50
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Fournier PE, Siritantikorn S, Rolain JM, et al. Detection of new genotypes of Orientia tsutsugamushi infecting humans in Thailand. Clin Microbiol Infect 2008;14: 168-73
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illness. Am J Trop Med Hyg 2013;89: 308-10 50.
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Diagnosis of scrub typhus
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Expert Rev. Anti Infect. Ther. 12(12), 1533–1540 (2014)
Jeshina Janardhanan1, Paul Trowbridge2, George M Varghese*1 1 Medicine Unit I and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India 2 Department of Infectious Diseases, Tufts University, Boston, MA, USA *Author for correspondence: Tel.: +91 9487393015 Fax: +0416 2232035 [email protected].
Scrub typhus is an acute febrile illness that, if untreated, can result in considerable morbidity and mortality. One of the primary reasons for delays in the treatment of this potentially fatal infection is the difficulty in diagnosing the condition. Diagnosis is often complicated because of the combination of non-specific symptoms that overlap with other infections commonly found in endemic areas and the poor available diagnostics. In the majority of the endemic settings, diagnosis still relies on the Weil–Felix test, which is neither sensitive nor specific. Other methods of testing have become available, but at this time, these remain insufficient to provide the rapid point-of-care diagnostics that would be necessary to significantly change the management of this infection by providers in endemic areas. This article reviews the currently available diagnostic tools for scrub typhus and their utility in the clinical setting. KEYWORDS: 56-KDa type-specific antigen • ELISA • immunofluorescence assay • Orientia tsutsugamushi • rapid diagnosis • scrub typhus
Scrub typhus is an acute febrile illness, affecting multiple organ systems, and is caused by the obligate intracellular bacteria Orientia tsutsugamushi. This organism is transmitted through the bite of chiggers, the larval stage of the mite Leptotrombidium, commonly encountered in rural areas. Orientia belongs to the Rickettsiaceae family and has a larger chromosome than other members of the family. It represents the most highly repeated bacterial genome sequenced so far with approximately 47% of the genome being repetitive sequences formed from integrative and conjugative elements, transposable elements and pseudogenes. The massive intragenomic deletions, duplications and rearrangements, along with acquisition of foreign genes, have contributed to a high degree of plasticity within the Orientia genome [1,2]. This disease is endemic to a region known as the ‘tsutsugamushi triangle’, which extends from Japan and Russia in the North to Northern Australia in the South and the Arabian peninsula in the West. Within this region of endemicity, which contains more than half of the world’s population, it has been previously estimated that around 1 million cases of this disease occur annually and 1 billion people are at risk of infection [3]. Notably, this estimate was made before the recognition of the re-emergence of this disease in several areas, and, in some regions, scrub typhus can account for up to 50% of undifferentiated febrile illnesses admitted to the hospital during the rainy season [4].
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10.1586/14787210.2014.974559
In addition to the massive scope of this infection, scrub typhus is often co-endemic with other diseases found in these regions such as dengue, malaria, leptospirosis and typhoid with which it shares similar presentations [5]. By itself scrub typhus was a major concern in the preantibiotic era, as the case fatality rate approached 60% [5], and it caused thousands of fatalities during the Second World War. Fortunately, since the advent of antibiotics, scrub typhus outcomes have significantly improved when appropriate treatment is started in a timely manner. However, given the overlap in the clinical presentation of this infection with other diseases common to the area of endemicity, the administration of appropriate treatment may be delayed by difficulty in reaching a diagnosis. This difficulty with diagnosing this potentially fatal illness is added by the lack of good point-of-care testing. Clinical features
The diagnosis of scrub typhus requires a high degree of clinical suspicion, prompting the clinician to request appropriate laboratory investigations and confirmatory tests. Patients usually present with an acute undifferentiated febrile illness, headache, myalgia, breathlessness, lymphadenopathy and varying involvement of organs such as the liver, lung and kidney. Organ involvement may be subtle and not evident initially unless carefully looked for. Patients may also have an eschar. The eschar
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in scrub typhus is a small, necrotic lesion, which may resemble a cigarette burn mark [6].These are often found on the groin, axillae, genitalia or neck. Although pathognomonic for scrub typhus, this may easily be overlooked. Previous reports have placed the incidence of eschar in acute scrub typhus at anywhere from 10 to 90% [7,8]. Most patients with scrub typhus develop thrombocytopenia and blood counts reveal leukocytosis in about half of the patients. Differential counts often reveal early lymphopenia with late lymphocytosis. Abnormal liver function tests with transaminase (AST/ALT) elevation of more than twice the normal limit is seen in majority of the patients [9]. Renal function impairment or cerebrospinal fluid abnormalities may be seen in up to quarter of the patients [10]. Chest x-ray may reveal localized infiltrates. Severe complications of the disease include acute respiratory distress syndrome, hepatitis, renal impairment, hypotension, meningitis, myocarditis and even death [6,11]. A high degree of clinical suspicion and familiarity with the various manifestations of scrub typhus is necessary to allow for early diagnosis and the timely initiation of appropriate therapy. However, given the impressive spectrum of presentation with this infection, even with a high index of clinical suspicion and the best clinical knowledge, diagnosis remains difficult. Serological diagnosis
The mainstay of diagnosis of scrub typhus has been and remains serology. In primary scrub typhus infection, a significant IgM antibody titer is mounted by the end of the first week, whereas IgG antibodies typically appear by the end of the second week. With reinfection, however, IgM antibody titers may be variable and IgG detectable by the sixth day of infection [12]. Weil–Felix testing has been in use for many years, though it is neither sensitive nor specific. This test uses a cross-reacting proteus OXK strain for the serodiagnosis of the disease, but this test has a sensitivity of only around 50% in the second week of infection [13]. Typically, a minimum titer of 1:80 or a fourfold increase over previous levels is taken as significant [14]. The current serological gold standard and reference test is the indirect immunofluorescent antibody (IFA) test, which uses fluorescence-labeled anti-human immunoglobulin to detect antibodies in the serum of the patient that have bound to immobilized bacterial antigen on a slide. Although considered the gold standard for the diagnosis of scrub typhus, several major limitations exist for this testing method. Although demonstration of a fourfold increase in antibody titers between acute and convalescent serum samples is diagnostic, such a diagnosis remains retrospective and is of little practical relevance for guiding initial management. Another major limitation of this test is the requirement of fluorescent microscopes, which are often not available in the resource-limited areas where scrub typhus is endemic [15]. Furthermore, the technique is expensive, requiring considerable technical expertise. Several routes have been taken to attempt to address the shortcomings of this testing method. IFA has been further 1534
adapted into a microimmunofluorescence format where lesser volumes of serum and antigen could be used [16]. This was found to be more convenient and allowed for simultaneous testing for multiple strains. Indirect immunoperoxidase (IIP) was also developed as a modification of the IFA, using peroxidase-labeled antibodies instead of fluorescein, thus negating the need for a fluorescent microscope [17]. Evaluation of IIP has shown that it is equivalent to IFA. Unfortunately, the sensitivity in both cases is affected by the strain variation and though the need for a fluorescent microscope is negated by this method, the need for technical expertise remains unchanged [18]. In a comparison of IFA using venous blood and finger prick filter paper blood spots, the sensitivity and specificity for classifying patients as having scrub typhus was found to be 95 and 88%, respectively. The use of blood spots facilitates transport of specimens to laboratories with IFA facilities; however, a decrease in accuracy was noted, regardless of the temperature at which specimens were stored, as storage time increased [19]. Finally, attempts have also been made to allow IFA to be used in a more clinically relevant manner, using a single acute serum sample for the diagnosis of scrub typhus. However, an extensive review of IFA methodologies and cut-offs on single acute serum samples for diagnosis of scrub typhus failed to form a consensus, questioning the suitability of using the test in this manner and even the use of this test as a reference standard. The most common cut-off was 1:400, but values ranged significantly from 1:10 to 1:400 [20]. Owing to the non-availability of IFA in common laboratories and the low sensitivity of Weil–Felix testing, ELISA is currently the preferred method of serological diagnosis. The 56-kDa type-specific antigen (TSA) is best suited for diagnostic testing, as it is the major immunodominant surface protein, containing both group- and strain-specific epitopes, and it is abundant in patient sera. The 56-kDa TSA is also highly variable, accounting for strain differentiation and genetic diversity in O. tsutsugamushi. The US Naval Medical Research Institute developed the r56, or recombinant 56-kDa antigen, which is now being used in several rapid diagnostic assays and ELISAs [21]. This antigen was found to be much more convenient to use than native antigens because large quantities of the antigen could be prepared safely using Escherichia coli without the need for bio-safety level 3 facilities, and the antigen product could be stored for a longer period. Using r56, IgM capture ELISA has been reported to have a sensitivity and specificity of 96.3 and 99%, respectively. As well, it has been reported that this assay could serologically confirm infection with a single serum specimen taken during any phase of acute infection or early convalescence, except for the first 2–3 days after the onset of illness [22]. Evaluation of this method in various scrub typhus endemic locations has shown sensitivities ranging from 85 to 100% with specificities of 97–99% [22,23]. Despite many advantages with this testing method, ELISA testing remains very expensive to perform and a time-intensive process. Other serological testing methods, such as complement fixation, hemagglutination and western blot have not fared better Expert Rev. Anti Infect. Ther. 12(12), (2014)
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Diagnosis of scrub typhus
than IFA. Complement fixation was one of the earliest tests used for serodiagnosis of scrub typhus, and it has been reported to be highly specific but with poor sensitivity. Being strain specific, complement fixation also requires antigens from all of the strains endemic to a particular region to be used for efficient detection [24]. SDS-PAGE immunoblot testing using the 56-kDa antigen has been used and found to be highly sensitive and specific, but, similar to use of ELISA, it is time consuming and hence rarely used in serodiagnosis [25,26]. A passive hemagglutination using recombinant 56-kDA antigen fused to a maltose-binding protein and adsorbed to sheep erythrocytes was compared to IFA, and an overall sensitivity and specificity of 98.9% was observed [27]. However, even the best functioning serological testing method, with a high sensitivity and specificity, remains dependent on detecting antibodies. After the onset of symptoms, it may take several days to weeks for the antibody titers to reach detectable levels, which in turn delays appropriate treatment. Convalescent phase serum samples for confirmation testing may not always be available, which further affects the reliability of these tests. Rapid diagnostic tests
Further developments in the diagnostics of scrub typhus are the various rapid assays that have been made available, such as the lateral flow immunochromatographic test (ICT) or immunoblot test and the latex agglutination test. These tests build off of the same basic concepts as other serologic tests, but they use the recombinant 56-kDa TSA of only the most common subgroups – Karp, Kato and Gilliam and have shown a wide range of sensitivities and specificities in various studies. ICTs typically have shown sensitivities ranging from 74 to 90% and specificities from 86 to 99% for the detection of IgM [28,29]. Evaluation of one IgM ICT (Panbio, Australia) has demonstrated higher specificity but continues to have sensitivities too low for clinical purposes [30]. Another ICT, constructed using a recombinant chimeric protein containing epitopes from Karp and TA763 strains, showed a higher sensitivity of 90% but only a moderate specificity of 85% [31]. Dot blot immunoassays have been investigated to be pointof-care tests as they can be rapidly and easily performed, are relatively inexpensive and can be accurate. However, false positives have been reported in special populations, such as those with high-titer IgM antibody to unrelated antigens, and interpretation of the results of not only persons with a history of scrub typhus but also those who were presently sick with an unrelated illness was found be difficult [32,33]. A dipstick test using a dot blot immunoassay format was also developed in Thailand for the serodiagnosis of scrub typhus in specific, and it was also found to have a similar sensitivity of 87% and specificity of 94%, compared to IIP test [34]. Latex agglutination testing, which was developed into a simple and rapid diagnostic tool for scrub typhus, when compared to IFA had an overall sensitivity, specificity and accuracy of 89.1, 98.2 and 93.6%, respectively. An evaluation of this test on scrub typhus confirmed samples from Thailand identified a informahealthcare.com
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minimum cut-off titer of 1:16. This test was demonstrated to be rapid, simple and easy to use and made an excellent replacement for the Weil–Felix screening test. However, so far, reports of a successful evaluation of this test on a larger scale for the application of routine laboratory testing are not available [35]. Overall, though showing some promise, the major issue affecting these rapid diagnostic kits is the low sensitivities, which is in part due to the antigenic diversity of strains in different geographical locations. A recent study found that the inclusion of chimeric recombinant antigen cr56 and other recombinant antigens, r21 and kr56 from Korean endemic strains in ELISA, dot blot assay and rapid diagnostic test, significantly increased the sensitivity and specificity of detection for Korean isolates [36]. Generally, inclusion of recombinant antigens from region-specific strains has been found to increase detection sensitivities in those specific regions [36,37]. However, most of the currently available diagnostic kits rely on antigens from Karp, Kato and Gilliam strains, allowing other strains to be potentially undetected. In addition, the circulating strains in many areas remain uninvestigated. Furthermore, none of these assays are used routinely for diagnosis, and they have largely only been evaluated in a few East Asian countries. Although the performance of some of these assays has been as good as or better than the IFA or IIP and the technology shows promise, a reproducible, quantifiable assay using these methods for diagnosis of scrub typhus is still lacking. Molecular diagnostic tests
Although the first molecular biology-based method used for the detection of scrub typhus was PCR RFLP of the outer membrane protein gene, the most commonly used technique is a nested polymerase chain reaction targeting a 483 bp region located on the 56-kDa TSA gene. This technique was first described by Furuya et al. [38] and has since then become a widely used technique for diagnosing scrub typhus from blood, buffy coat and eschar samples with varying degrees of sensitivities ranging from 62 to 90 and 100% specificity [39–41]. Several studies have found PCR to be more sensitive than IFA, and particularly eschar PCR has been found to be more sensitive than using blood or buffy coat given the high number of organisms in an eschar and because of false negatives observed due to inhibition of PCR by hemoglobin and other components in blood [42]. However, though eschar PCR is certainly the most sensitive of the techniques, the absence of an eschar on many patients limits the utility of this test. The majority of available literature evaluating conventional PCR has shown lower sensitivities than nested PCR. However, many of these studies relied on the internal primers used for nested PCR, which may explain these outcomes. A few studies have reported that standard PCR targeting the 56-kDa gene shows sensitivities higher than nested PCR, while retaining the 100% specificity [7,43,44]. Furthermore, compared to nested PCR, conventional PCR takes half the time and reagents and carries less risk of contamination during the transfer steps. In a recent study, we optimized and evaluated an earlier described 1535
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conventional PCR targeting the 56-kDa TSA, and this was found to be more sensitive and economical than N-PCR with a similar specificity [41]. A major outstanding advantage of using PCR techniques is that detection of the diseases is possible before antibodies have become detectable by serology, allowing the infection to be diagnosed as it is happening to the patient rather than days or weeks later. Furthermore, despite concerns that direct detection of the organism could be diminished by antibiotic use before testing as may be the case for cultures, eschar tissues have been even reported to give positive results up to 7 days after the administration of antibiotics [45]. Looking at other targets aside from the 56-kDa TSA, highly sensitive quantitative PCRs that detect partial sequences of the Orientia 47-kDa outer membrane protein antigen/high temperature requirement A gene and the 60-kDa heat shock protein gene, groEL, were developed and evaluated [39,46–49]. However, though the Q-PCR is highly sensitive and specific, even detecting very low numbers of copies, it remains too expensive for routine diagnosis in an endemic setting. However, these tests are currently prohibitively expensive in many endemic areas, although this may change in the future. Another widely evaluated molecular detection technique is the loop-mediated isothermal PCR assay (LAMP). This is an isothermal amplification technique using a robust DNA polymerase and a set of three primer pairs that produce a specific double hairpin DNA template with high specificity and efficiency. However, LAMP assays targeting the groEL gene have only shown a sensitivity of 70% and specificity of 85% when compared to IgM ELISA, and a sensitivity and specificity of 83 and 88%, respectively, when compared to nested PCR for diagnosing acute scrub typhus infection [50]. A recent study from Thailand comparing the diagnostic capabilities of four rapid ICTs to LAMP assay again found low sensitivities of individual ICTs, ranging from 46 to 68%, with specificities ranging from 68 to 95%. The sensitivity of LAMP assay was also found to be low at 52% with a specificity of 94%. However, a combination of LAMP with the ICT was found to slightly increase the sensitivity to 67% with a specificity of 94%. Unfortunately, this study highlights the limitations of the currently available rapid tests and LAMP assays, which otherwise have the advantage of being able to be performed in endemic settings using minimal equipments [51]. Culture
Being an obligate intracellular pathogen, culturing O. tsutsugamushi is a long and tedious process requiring biosafety level 3 containment and experienced personnel. Culture has been attempted from blood, buffy coat and skin biopsies and has been performed in mice, mammalian cells, embryonated chicken eggs, Vero (African green monkey kidney) or L (mouse fibroblast) cells. Buffy coat is typically separated from ethylenediaminetetraacetic acid blood tubes and inoculated into a Vero or L929 monolayers in minimum essential medium supplemented with fetal bovine serum and glutamine. This is then incubated for at least 2 weeks 1536
at 37˚C in a 5% CO2 incubator. The requirement for a biocontainment facility and the potential contamination with other organisms is a major issue in culturing this organism [52]. Owing to the tedious nature, long incubation times and low sensitivity, culture can hardly be considered a diagnostic tool in a clinical setting. By and large, culture is used only for research purposes in reference laboratories. Immunohistochemistry
Immunohistochemical staining has been successfully used in other rickettsial infections, such as rickettsial pox and Rocky Mountain spotted fever, but has been only rarely used in the diagnosis of scrub typhus. Evaluation of IHC on eschars and maculopapular skin lesions has found that although the specificity on both eschar and skin lesion was 100%, the sensitivity for skin lesions was only 65%. The sensitivity was 100% when IHC was performed on eschar samples. However, as previously mentioned, eschars can be an unreliable physical finding, present in a highly variable percentage of patients, limiting the utility of this testing [53]. Notably, however, IHC has also been used to show the presence of intracellular bacteria in the endothelial cells of organs like the heart, lung, brain and kidneys of soldiers suspected to have died of scrub typhus during World War II and the Vietnam War. However, these samples are not taken from typical patients who are still alive. Choosing the appropriate diagnostic test
Scrub typhus is grossly under-diagnosed because of its nonspecific clinical presentation and poor available diagnostics. All of the currently available diagnostic tests have traits that make them either undesirable or unobtainable for the majority of facilities within endemic area. Serological testing is still the most common method for diagnosis. All serological methods, however, share the same flaw, in that they rely on the availability of the human body’s response to infection, antibodies, to detect infection. The process of producing antibodies within the body requires time, and often rechecking of titers, making all of these methods ineffective, to a greater or lesser extent, at diagnosing illness in a timely manner, particularly in very acute infections. Aside from this class-wide flaw, individual tests have their own strengths and weaknesses. While the Weil–Felix test is certainly widely available, cheap and easily performed even in resource-limited settings, it is of limited utility because of its extremely poor sensitivity. Some of the rapid serologic diagnostic assays, most notably the latex agglutination test, are certainly an improvement over the Weil– Felix test, but, by virtue of their very methodology, which includes use of only the most common Orientia strain antigens, also have sub-optimal sensitivities, and they remain untested on a larger scale. IFA remains the gold standard for diagnosis. However, because this method requires fluorescent microscopes and expertise for testing, which are usually not available in most areas where the disease is prevalent, this method is of limited utility. ELISA show promising results, with both a high sensitivity and specificity. Although easily available rapid diagnostic tests can be Expert Rev. Anti Infect. Ther. 12(12), (2014)
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Table 1. Currently available diagnostics for scrub typhus. Test
Sensitivity
Specificity
Advantage
Disadvantage
Ref.
Weil–Felix test
50–60%
95%
Cheap Widely available Easily performed
Very low sensitivity Serology not positive until days to weeks into infection
[13,14]
ELISA
97%
99%
Highly sensitive and specific
Expensive Time-consuming Serology may not be positive until days into infection
[22,23]
IFA
80–91%
80–100%
Highly sensitive and specific
Requires specialized equipment Requires experienced personnel
[16,20]
Nested PCR
60–90%
100%
Expensive
Conventional PCR
80–95%
100%
Quantitative PCR
83%
100%
Highly specific Positive earlier than serologic testing Can remain positive even after antibiotic treatment is started
PCR [39–41] [41,43,44]
Requires specialized equipment
[46–49]
Requires experienced personnel IFA: Immunoflourescent antibody.
used as a screening test in the community setting, IgM ELISA test is the most practically useful test for confirmation. Non-serological methods of testing share the advantage of testing directly for the presence of the organism rather than the body’s response to it. This allows these methods to potentially be of greater utility early in the infection when the body has not yet had a chance to produce antibodies. Culture, however, takes sufficiently long as to negate these advantages. Also, the low sensitivity of this method makes it a poor diagnostic tool. PCR testing does show promise, but issues remain around the obtainable sensitivities for affordable tests and the costs of more effective assays. Certainly, Q-PCR could hold potential for becoming a good confirmatory test in the future, but its high cost and limited availability remain prohibitive for most areas of endemicity right now. There are several challenges in developing a field usable rapid assay for diagnosis of scrub typhus. Most assays have focused on the 56-kDa TSA protein, as this protein is specific to O. tsutsugamushi bacteria. Analysis of this major outer membrane protein has revealed that the fragment contains four variable domains and four conserved domains. Sera from most patients with scrub typhus can recognize this protein, as a result of which it is widely used as a diagnostic antigen in many countries. However, there are several antigenic variants in O. tsutsugamushi based on the 56-kDa TSA, and so all of the TSAs derived from the endemic strains in a geographical location must be included for the assay to be sensitive to picking up these local variations. In addition to being sensitive to local strain variations, an ideal rapid diagnostic kit must also be cost–effective with a longer shelf life, distinguish between active and past infection and allow the use of direct sample without processing. It should also be suitable for use in rural clinical informahealthcare.com
sites, as scrub typhus is largely a rural disease, without requiring the use of any specialized equipment, facilities or a trained technician. Several rapid diagnostic kits are commercially available at this time and have shown some promise. However, further evaluation of these currently available kits based on the geographic diversity of the strains is essential. The overall picture of the currently available diagnostics is provided in TABLE 1. Conclusion
Overall, the diagnosis of scrub typhus remains solely dependent on maintaining a higher degree of clinical suspicion and working with limited diagnostic tests. At this point, we remain largely reliant on acute and convalescent antibody titer for serological confirmation, which limits the utility of testing. Further complicating this picture is the geographic strain heterogeneity, which may lower the sensitivity of these already substandard tests. Although several serological tests are currently commercially available, most of the tests have a low sensitivity or are too expensive and impractical for routine application, and the further evaluation of these tests is hampered by the imperfect gold standard and the geographic strain variation. Among all the available serological tests, IgM ELISA remains the most useful confirmatory test. Non-serologic testing methods, though having some advantages, similarly remain infeasible at this time due to expense and limited evaluation. With no single diagnostic test capable of rapidly detecting this deadly infection, the development of a highly sensitive and specific point-of-care test is of utmost urgency. Expert commentary
Scrub typhus is a serious multi-system infection causing significant morbidity and mortality. The understanding of the 1537
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immunopathogenesis and disease mechanisms are evolving. Diagnosis of this infection is often delayed because of the combination of non-specific symptoms, limited awareness among clinicians and the poor available diagnostics. However, promoting awareness among clinicians and reliable methods for rapid diagnosis will be the key to improve outcome in these patients. Current effort in developing rapid and point-of-care diagnostics and evidence-based management strategies in endemic regions will address the gap in identification and management of this significant public health problem.
not far from scientific reach. Progress in nanotechnology and sensitive platforms can make the point-of-care a reality. Furthermore, improved culture techniques and molecular diagnostics will strengthen the confirmatory diagnosis of this condition. Advances in diagnostic testing may permit clinicians to diagnose the condition promptly, which will result in improved outcomes. These essential changes may transform the clinical landscape and provide optimal care for patients with scrub typhus. Financial & competing interests disclosure
Five-year view
Looking forward into the immediate future, major advances in rapid diagnosis and management are expected. Aided by the new technologies to sequence the organism and understand the immunodominant proteins, newly identified diagnostic targets for developing rapid and point-of-care tests are
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Scrub typhus is a grossly under-diagnosed disease because of its non-specific clinical presentation and poor available diagnostics. • Serological testing is still the most common method for diagnosis. • Immunofluorescent antibody testing remains the gold standard for serologic diagnosis. However, it requires specialized equipments and expertise, which is usually not available in scrub typhus endemic settings. • Evaluation of currently available rapid diagnostic kits are hampered by the imperfect gold standard and the geographic strain variation. • Among all the available serological tests, IgM ELISA remains the most useful confirmatory test, which is still an expensive and timeconsuming test. • With no single diagnostic test capable of rapidly detecting this deadly infection, the development of a highly sensitive and specific pointof-care test is of utmost urgency.
Beran GW, Ed.) CRC Press; Boca Raton, FL: 1994. p. 463-74
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