Rising Menace of Scrub Typhus – Current Status and Challenges

Scrub typhus, an age old disease, is caused by the intracellular bacterium Orientia tsutsugamushi . It has reemerged in recent years due to factors like climatic changes and human encroachment because of rampant urbanization. The disease is endemic in the area known as the ‘tsutsugamushi triangle’ and has recently spread its fangs into various other continents like South America and Africa. Although the disease is endemic in India, there is a lack of appropriate sero-epidemiology in community settings. It is one of the essential causes of acute undifferentiated fever in tropical locations and, if untreated, can cause mortality ranging from 2-30% of cases. Early diagnosis is an important parameter in administering the non beta-lactam regimen to prevent complications and mortality. Yet, there is a lack of accurate and rapid methods for diagnosis in the early stage of the disease, more so in rural areas where the disease is supposed to be predominant. The gold standard diagnostic test has its problems. Recently, there have been reports of drug resistance to the standard scrub typhus regimen. There is a gap of a decade in the research into this entity. Thus, a new look into the disease, its epidemiology and the challenges in its diagnostic scenario is an apt topic for discussion.


INTRODUCTION
Scrub typhus was a disease of the preantibiotic era, especially during military operations, but even today causes one million cases annually worldwide. 1In the recent years it has reemerged as a significant public health issue due to multiple factors like climatic changes, human activities disturbing the ecological balance, beta-lactam antibiotics overuse and urbanization of rural areas. 2 This disease is an underdiagnosed entity due to a lack of typical pathognomonic clinical features coupled with inadequate availability of appropriate laboratory methods.Thus there is limited epidemiological data regarding the disease globally.

Epidemiology of Scrub typhus
Scrub typhus is caused an intracellular Gram negative small pleomorphic coccobacilli named Orientia tsutsugamushi, 3 which was previously under Rickettsia family.It is named as 'tsutsugamushi' (dangerous bug), after a jungle mite or chigger, which acts as a reservoir and transmits the disease to man by biting through pores or hair follicles on exposed skin.Human encroachment during deforestation, logging, road building, military operations, rice cultivation, etc., bring us close to infected chiggers, the parasitic stage in the vector Leptotrombidium mite.Climatic change due to global warming also has a role in the reemergence of this disease (Figure 1). 4 Tropical regions have appropriate temperatures and humidity for chigger activity and maintaining the pathogen in transovarian and transstadial transmission in the mites.Researchers have also noted horizontal transmission of Orientia among mites. 5Thus, the disease occurs around the year.However, in temperate zones like in the northern part of Japan, the mite activity is seasonal corresponding to the disease. 6crub typhus is endemic to a region denoted as the ''tsutsugamushi triangle''.South East Asian region is endemic for scrub typhus. 7ut in recent times, the disease has surfaced in other non endemic areas like Europe, Chile, Peru, Middle East, African peninsula and sometimes with a different species of the bacteria, like O.chuto seen in the Middle East (Figure 2). 8In India, several states have varying ecological profile, like Haryana, Jammu, and Kashmir, Himachal Pradesh, Uttaranchal in north; Kerala, Tamil Nadu in South; Bihar, West Bengal, Assam in East; Maharashtra in West report this disease. 9,10Sero-prevalence of Jan 2020 year; F>M this infection in India is between 9.3% and 27.9%, and the mortality rate is around 30% among untreated individuals, as noted in passive national surveillance systems. 7,11Among acute onset febrile illness, scrub typhus constitutes about 25.3% of cases in India (Table 1). 12rientia shows many genetic and antigenic variations resulting from variations in tsa gene, which codes for 56-kDa type specific antigen. 1,13There are around 30 serological types like kato, karp, kuroki, gilliam and kawasaki that as detected by immunoperoxidase reaction 14 Litchfield strain is a novel strain detected in Australia. 15The correlation between this antigenic diversity and virulence is still unclear.

Clinical presentation
Fever is the commonest presentation, seen in 95-100% of cases. 16,17Scrub typhus accounts for a significant chunk of "fever of unknown origin" in endemic regions.Even the term "typhus" is derived from Greek terminology 'Typos' meaning 'fever with stupor' The age group of 50-60 is commonly afflicted while, sex preponderance varies across different countries. 3A papular lesion is formed at the chigger bite site, which becomes larger with time, followed by necrosis and crusting in the centre and finally developing a black eschar, which is a pathognomonic feature of scrub typhus.The presence of eschar is specific (98.9%) for diagnosis of this disease but is limited by sensitivity, which varies between 7%-97%. 79][20] Scrub typhus can present in varied forms ranging in severity from asymptomatic to multi organ failure. 21Common symptoms are myalgia, headache, nausea, vomiting, abdominal pain, cough, generalized lymphadenopathy and skin rash in varying combinations. 3,22,23Owing to its mimicking signs and symptoms, it took almost 30 years to prove the original finding of Coyttarus (1578) that typhoid and typhus were different diseases.Untreated cases may develop several complications generally occurring after the first week of illness.Various complications like acute renal failure, jaundice with rising liver enzymes, pneumonitis and acute respiratory Tsutugamushi triangle, an area approximating about 8 million km 2 is endemic for scrub typhus.This extends in North from the Russian Far East, to Pakistan in the west, Australia in the south, and the Japan in the east.Seroprevalence of scrub typhus detected in recent studies, 63 have been tabulated which ranges from mere 1.1% in Vietnam to 68.4 % in Japan.But apart from this, other area in globe like -Chile, United Arab Emirates, African countries ( Camroon, Kenya, congo, Tanjania) also have reported cases of scrub typhus.distress syndrome, septic shock, myocarditis, meningoencephalitis and reversible deafness have been noted in prior studies. 18,20Renal involvement can be expected in about 9% of patients.Patients of meningoencephalitis often have CSF changes indistinguishable from viral or tuberculous meningitis.Unusual presentations include conjunctival hyperemia or erosion, gastrointestinal mucosal erosion without any predilecting site and acute abdomen. 24,25Septic shock ensues with further organ damage to liver, lungs, kidneys along with DIC. 26,16,27,28Elevated transaminases, thrombocytopenia and leukocytosis are the biochemical investigations pertinent to diagnosing the disease when used in combination (specificity and positive predictive value for diagnosis -80%). 29ortality from this disease varies from 7-30%, 30 but much less in children. 31The possible patient factors associated with complicated cases are -age (≥60 years), patients without eschar, WBC counts >10000/mm and serum albumin level ≤3.0 g/dL. 31,2Being a great mimicker, diagnosis in the early stages is challenging yet important for successfully treating scrub typhus.The median case fatality rate is reduced to 1.4% in treated patients from 6% seen in late or untreated ones. 11,29,32Diagnosis is based on clinical suspicion with appropriate lab investigation.

Lab diagnosis of scrub typhus Serological assays
Serological tests that detect antibodies to against scrub typhus, like Weil Felix test, ELISA, Immunofluorescence and immunoperoxidase tests are the commonly performed tests for lab diagnosis.IgM is preferred over IgG detection as it can help diagnose recent infections.But, all there tests have many issues that needs addressing.A ≥ 4-fold increase in antibody titer between two consecutive samples is diagnostic, 33 but often not practical.Secondly, a baseline titer (cut-off) is to be established in the geographical setting based on the endemicity of the disease for appropriate reporting, which is often lacking.There is wide variation across India in cut-off values of the various serological tests (Table 2).Then again, most serological tests use an antigen cocktail of Karp, Kato, and Gilliam serotypes.But there are many other antigenic variations apart from these three, differing in different geographical regions of the world. 1 For example, in mites collected from a single field in Malaysia, eight different serotypes were found. 34Boryong is the commonest serotype in South Korea in three-fourths of total isolates. 35imilarly, Kawasaki or Kuroki serotypes accounted for >90% of Kyushu island isolates of Japan. 36In India, data on serotype prevalence in different areas is still lacking.Thus, common serotypes must be explored and included as the antigen for serological testing purposes.

IFA
IFA, the gold standard test for detection suffers from many pitfalls.For example, in a Korean study, IFA had false negative results in six patients with a typical eschar which was positive for O. tsutsugamushi DNA. 37Further, it is labor intensive, needs resource settings and can have interoperator variations. 7

Rapid test
The dot blot immunoassay dipstick is rapid, semi-quantitative, accurate and easy to use inexpensive point of care test that can also be used in rural settings. 19,38Rapid immunochromatographic test is another POC test with higher sensitivity and specificity of 96.8% and 93.3%, respectively when used for detection of IgM. 391][42] ICT kits can be used with another method like LAMP/ PCR assays for improving accuracy (Table 3).

Molecular assays
PCR, either conventional, nested or real-time PCR can be used for diagnosis of scrub typhus. 43,44Q-PCR is faster, has higher sensitivity and specificity and produces quantitative results than other methods. 457][48] Q PCR with 16S r RNA as the desired target has the highest sensitivity and accuracy compared to other targets and also when compared with Immunofluorescence assay for diagnosis.Specimens from which PCR can be done are eschar, whole blood, clots or buffy coat.Immunohistochemical staining and PCR from eschar material are more sensitive and remains positive even after treatment. 37,49All the PCR assays remain positive only during the period of rickettsemia.Common genetic targets for OT detection are-tsa gene encoding the 56-kDa type-specific antigen; htrA gene coding for 47-kDa periplasmic serine protease 48 ; groEL gene -Hsp60; 16S rRNA. 50,51Although the56-kDa antigen is highly specific, 52,53 but variability in sequence can affect the annealing of the primer and reduce test sensitivity. 44Assay targeting the 16S rRNA gene showed a higher sensitivity than 56-kDa gene. 51As O. tsutsugamushi genome has a high degree of genetic variations, improving specificity of the detection by using multiple genes approach either by conventional or real-time PCR is the need of the hour.

LAMP assay
LAMP assay with the groEL gene of Orientia tsutsugamushi has been tried. 37LAMP assay has many advantages such as not needing a thermal cycler and visual result reading.But, clinical use warrants further validation.A study has also shown that limit of detection with LAMP assay is 14 copies/μL compared with three copies/ μL for real-time PCR.

STIC criteria for diagnosis of scrub typhus
Gold standard or reference test for scrub diagnosis includes IFA or IIP assays, which have many limitations for accurate diagnosis.Bayesian model showed that the IFA IgM assay has sensitivity and specificity of 70.0% and 83.8%, respectively. 41eeping this in mind, STIC criteria (Figure 3) using a battery of tests with high specificity has been proposed as an alternative reference comparator for accurate diagnosis. 54

Treatment of scrub typhus
The treatment options for scrub typhus are-doxycycline and tetracycline.Azithromycin, ciprofloxacin and rifampicin are effective alternatives where there is poor response to doxycycline.In pregnant women and children less than 8 years old, azithromycin is the preferred regimen.Severe disease needs to be treated with intravenous chloramphenicol with intravenous tetracycline.A recent multicentric study has concluded that combination therapy of intravenous doxycycline and azithromycin is a better treatment option for severe scrub typhus than any agent alone. 55There is no significant difference in outcome when azithromycin therapy is compared with other antibiotics singly or in combination in paediatric patients as noted in a recent meta-analysis. 56[59]

Prophylaxis of scrub typhus
Different localities have different antigenic variants of O. tsutsugamushi strains showing no to weak cross-protection.][62] WHO recommends single oral dose of tetracycline, doxycycline or chloramphenicol every 5 days for a total of 35 days for prophylaxis against Orientia infection 63 as opposed to CDC which opines that such a prophylactic treatment may only delay the disease and also hinder diagnosis.Other safety measures include avoiding exposure to vegetation by using full-sleeved clothing, mats to sit on the grass, using shoes, cleaning the garments with insect repellant after a possible exposure to get rid of mites, and rodent control. 63

CONCLUSION
Scrub typhus, a disease of wars, has raised its fangs with growing climate change and human activities encroaching on the habitat of the mite reservoir.Despite its long presence, there needs to be more data citing its actual prevalence, serotypes involved and determinants of clinical course, especially in India.One of the important reasons for this is the lack of a diagnostic test with desirable accuracy.Molecular methods are helpful early in the disease and are yet to be widely used for diagnosis.Adopting a rapid, accurate test protocol for clinical diagnosis of scrub typhus is necessary.Further clinical trials and research is needed for evaluating various regimens used for scrub typhus, keeping in mind the evolving drug resistance and its intracellular persistence causing relapses.

Figure 1 .Table 1 .
Figure 1.The epidemiological triad of scrub typhus The agent O. tsutsugamushi is maintained in its reservoir mites by transstadial and transovarian transmission and transmitted to man by bite of the larvae (chigger) in cases of human encroachment into mite habitat.Man to man transmission is not seen.

Figure 2 .
Figure 2. The Tsutsugamushi triangle and other area in the world where the disease has been reported.Tsutugamushi triangle, an area approximating about 8 million km 2 is endemic for scrub typhus.This extends in North from the Russian Far East, to Pakistan in the west, Australia in the south, and the Japan in the east.Seroprevalence of scrub typhus detected in recent studies,63 have been tabulated which ranges from mere 1.1% in Vietnam to 68.4 % in Japan.But apart from this, other area in globe like -Chile, United Arab Emirates, African countries ( Camroon, Kenya, congo, Tanjania) also have reported cases of scrub typhus.

Figure 3 .
Figure 3. STIC criteria for diagnosis of scrub typhus

Table 2 .
Cut off value calculated in different studies from various hospital based studies across India

Table 3 .
Various methods for laboratory diagnosis of scrub typhus Indirect diagnostic methods comprise of method which detect antibodies developed against Orientia tsutsugamushi while direct methods detect the organism from the samples either by culture, animal inoculation or the DNA of the bacteria by amplification methods

Table 4 .
The sensitivity, specificity, Positive predictive value and Negative predictive value of the common tests available for scrub typhus from various studies conducted from low and middle income country settings Test for Scrub