deutonymph, tritonymph, and adult. the larvae, commonly referred to as chiggers (figure 4), are the only ectoparasitic stage feeding on the body fluids of rodents and other opportunistic mammals. thus, they are the only stage in the life of mites that transmit the infection. wild rats of the genus rattus are the principal natural hosts of the chiggers. chiggers feed only once on a mammalian host. the feeding usually takes 2 to 4 days. in contrast to most parasites, they do not feed on blood, but instead on the body fluid through the hair follicles or skin pores. they possess a special feeding apparatus called a stylostome, which is a tube formed by solidified saliva. their saliva can dissolve the host tissue around the feeding site, so that they ingest the liquefied tissue. o. tsutsugamushi is present in the salivary glands of mites and is released into the host tissue during this feeding. cellular invasion mechanism of cell invasion by o. tsutsugamushi. orientia tsutsugamushi initially attacks the myelocytes (young white blood cells) in the area of inoculation, and then the endothelial cells lining the vasculature. the process of cellular invasion is shown in figure 5. in the blood circulation, it targets professional phagocytes ("cell eaters", white blood cells) such as dendritic cells and macrophages in all organs as the secondary targets. the parasite first attaches itself to the target cells using surface proteoglycans present on the host cell and bacterial surface proteins such as type specific protein 56 (or type specific antigen, tsa56) and surface cell antigens (scaa and scac, which are membrane transporter proteins). these proteins interact with the host fibronectin to induce phagocytosis (the process of ingesting the bacterium). the ability to actually enter the host cell depends on integrin-mediated signaling and reorganisation of the actin cytoskeleton. orientia tsutsugamushi has a special adaptation for surviving in the host cell by evading the host immune reaction. once it interacts with the host cells, it causes the host cell membrane to form a transportation bubble called a clathrin-coated vesicle by which it gets transported into the cytoplasm. inside the cytoplasm, it makes an exit from the vesicle (now known as an endosome) before the endosome is destroyed (in the process of cell-eating called autophagy) by the lysosomes. it then moves towards the nucleus, specifically at the perinuclear region, where it starts to grow and multiply. unlike other closely related bacteria which use actin-mediated processes for movement in the cytoplasm (called intracellular trafficking or transport), o. tsutsugamushi is unusual in using microtubule-mediated processes similar to those employed by viruses such as adenoviruses and herpes simplex viruses. further, the escape (exocytosis) from an infected host cell is also unusual. it forms another vesicle using the host cell membrane, gives rise to a small bud, and releases itself from the host cell surface while still enclosed in the vesicle. the membrane-bound bacterium is formed by interaction between cholesterol-rich lipid rafts as well as htra, a 47-kda protein on the bacterial surface. however, the process of budding and importance of the membrane-bound bacterium are not yet understood. strains orientia tsutsugamushi is a diverse species of bacteria. ida a. bengtson of the united states public health service was the first to note the existence of different strains using antigen-antibody interaction (complement fixation test) in 1944. she observed that different strains had varying degree of virulence, and that the antibodies in the blood sera of patients cross-react to different strains. by 1946, she established that there were three principal strains (serotypes), namely karp (from new guinea), gilliam (from india) and seerangay (from british malaya). akira shishido described the kato strain, in addition to gilliam and karp, in japan in 1958. since then, six basic antigenic strains are recognised, namely gilliam, karp, kato, shimokoshi, kawasaki, and kuroki. karp is the most abundant strain, accounting for about 50% of all infections. in korea, the major strain is boryong. as of 2009, more than 20 different strains have been established in humans based on antigenic variation using serological tests such as complement fixation and immunofluorescence assay. the number is much higher if the strains in rodents and mites are taken into account. for example, a study in japan in 1994 reported 32 strains, 14 from human patients, 12 from wild rodents, and 6 from trombiculid mites. the different strains exert different levels of virulence, and the most virulent is kn-3, which is predominant among wild rodents. another study in 1996 reported 40 strains. genetic methods have revealed even greater complexity than had been previously described (for example, gilliam is further divided into gilliam and jg types). due to immunological differences of the serotypes, simultaneous and repeated infection with different strains is possible. antigenic variation orientia tsutsugamushi has four major surface-membrane proteins (antigens) having molecular weights 22 kda, 47 kda, 56 kda and 110 kda. a 56-kda type specific antigen (tsa56) is the most important because it is not produced by any other bacteria, and is responsible for making the genetic diversity in different strains. it accounts for about 10–15% of the total cell proteins. the 22-kda, 47-kda or 110-kda antigens are not strain specific so that tsa56 is the main target in sophisticated diagnostic tests such as immunoblotting, elisa and dna analysis. the protein assists the adhesion and entry of the bacterium into host cells, as well as evasion of the host's immune reaction. it varies in size from 516 to 540 amino acid residues between different strains, and its gene is approximately 1,550 base pairs long. its gene contains four hypervariable regions, indicating that it synthesises many antigenically different proteins. there are also 11-kda and 60-kda proteins inside the bacterium which are very similar to groes and groel of the bacterium escherichia coli, but not that of rickettsia species. groes and groel are heat shock proteins belonging to the family of molecular chaperones in bacteria. dna analyses have shown that the groes and groel genes are indeed present in o. tsutsugamushi with slight variation in different strains, and they produce the 11-kda and 60-kda proteins. disease further information: scrub typhus orientia tsutsugamushi causes a complex and potentially life-threatening disease known as scrub typhus. infection starts when chiggers bite on the skin during their feeding. the bacteria are deposited at the site of feeding (inoculation), where they multiply. they cause progressive tissue damage (necrosis), which leads to formation of an eschar on the skin. necrosis progresses to inflammation of the blood vessels, called vasculitis. this in turn causes inflammation of the lymph nodes, called lymphadenopathy. within a few days, vasculitis extends to various organs including the liver, brain, kidney, meninges and lungs. the disease is responsible for nearly a quarter of all the febrile (high fever) illness in endemic areas. mortality in severe cases or due to improper treatment or misdiagnosis may be as high as 30–70%. about 6% of infected people die untreated, and 1.4% of the patients die even with medical treatment. moreover, the death rate can be as high as 14% with neurological problems and 24% with multi-organ dysfunction among treated patients. in cases of misdiagnosis and failure of treatment, systemic complications rapidly develop including acute respiratory distress syndrome, acute kidney failure, encephalitis, gastrointestinal bleeding, hepatitis, meningitis, myocarditis, pancreatitis, pneumonia, septic shock, subacute thyroiditis, and multiple organ dysfunction syndrome. harmful effects involving multiple organ failure and neurological impairment are difficult to treat, and can cause lifelong debilitation or be directly fatal. the central nervous system is often affected and results in various complications including cerebellitis, cranial nerve palsies, meningoencephalitis, plexopathy, transverse myelitis, and guillan-barré syndrome. death rates due to complications can be up to 14% in brain infections, and 24% with multiple organ failure. in india, scrub typhus has become the major cause of acute encephalitis syndrome, which was earlier caused mainly by a viral infection, japanese encephalitis. epidemiology map showing the tsutsugamushi triangle. countries with human cases are labeled with a star. the world health organization in 1999 stated that: scrub typhus is probably one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization in the region. the absence of definitive signs and symptoms combined with a general dependence upon serological tests make the differentiation of scrub typhus from other common febrile diseases such as murine typhus, typhoid fever and leptospirosis quite difficult. scrub typhus is historically endemic to the asia-pacific region, covering the russian far east and korea in the north, to northern australia in the south, and afghanistan in the west, including islands of the western pacific oceans such as japan, taiwan, philippines, papua new guinea, indonesia, sri lanka, and the indian subcontinent. this geographic region is popularly called the tsutsugamushi triangle as shown in figure 6. however, it has spread to africa, europe and south america. one billion people are estimated to be at risk of infection at any moment and an average of one million cases occur every year in the tsutsugamushi triangle. the burden of scrub typhus in rural areas of asia is huge, accounting for up to 20% of febrile sickness in hospital, and seroprevalence (positive infection on blood test) over 50% of the population. more than one-fifth of the population carry the bacterial antibodies, i.e. they had been infected, in endemic areas. south korea has the highest level incidence (with its highest of 59.7 infections out of 100,000 people in 2013), followed by japan, thailand, and china at top of the list. the age group of 60–69 years is at highest risk of infection. higher infection (57.3%) is seen in females compared to males (42.7%). farmers are most vulnerable, accounting for 70% of the cases in china. the disease is more prevalent in rural areas, but there is a rapid increase in urban areas. for example, in korea, the annual incidence increased 21-fold between 2003 and 2013 in metropolitan areas. symptom eschar due to o. tsutsugamushi infection on the shoulder (a, b) of a female and on the penis (c, d) of a male. the main symptom of o. tsutsugamushi infection is high (febrile) fever; however, the symptom is similar to other vector-borne tropical diseases such as malaria, leptospirosis, typhoid, murine typhus, chikungunya, and dengue fever. this makes precise clinical diagnosis difficult, which often leads to misdiagnosis. the initial indications are fever with chills, associated with headache, muscle pain (myalgia), sweating and vomiting. the appearance of symptoms (the incubation period) takes between 6 and 21 days. a simple visual diagnosis is the presence of an inflamed scar-like scab called eschar, which is regarded as "the most useful diagnostic clue in patients with acute febrile illness". eschar is formed on the skin where an infected mite bit, usually seen in the armpit, groin or any abdominal area (figure 7). in rare cases, it can be seen on the cheek, ear lobe and dorsum of the feet. but, the problem is that eschar is not always present; at the highest record, only 55% of scrub typhus patients had eschar during an outbreak in south india. also, eschar is not specific to scrub typhus, occurring in other rickettsial diseases such as rocky mountain spotted fever, brazilian spotted fever, and indian tick typhus. using dna analysis by advanced polymerase chain reaction, different rickettsial infections can be identified from eschars. blood test suspected infections are confirmed with serological tests. o. tsutsugamushi is most often detected from blood serum using the weil–felix test. weil–felix is the simplest and most rapid test, but it is not sensitive or specific, as it detects any kind of rickettsial infection. more sensitive tests such as rapid immunochromatographic test (rict), immunofluorescence assays (ifa), elisa, and dna analysis using polymerase chain reaction (pcr) are used. ifa is regarded as the gold standard test, as it gives a reliable result; however, it is expensive and not specific for different rickettsial bacteria. elisa and pcr can detect o. tsutsugamushi-specific proteins such as the tsa56 and groel, so that they are highly specific and sensitive. on the other hand, they are highly sophisticated and expensive techniques. treatment orientia tsutsugamushi infection can be treated with antibiotics such as azithromycin, chloramphenicol, doxycycline, rifampicin, roxithromycin, and tetracyclin. doxycycline is the most commonly used and is considered as the drug of choice because of its high efficacy and quick action. but, in pregnant women and babies, it is contraindicated, and azithromycin is the drug