**2. Chiggers as vectors of infectious diseases**

Although different microorganisms have been detected in different species of chiggers, their role as vectors of infectious diseases has been only demonstrated for scrub typhus.

#### **2.1. Scrub typhus**

fluids of the dermal layer (but not blood). Ears, head, armpits, abdomen, genitalia, and the area around the tail are preferred in animals [4,27]. In humans, bites occur mainly in body exposed areas and at sites where the clothing constricts [17,28]. Once engorged (development to subsequent stage cannot take place unless larvae have fed on the host), larvae fall to the ground and develop to the nymphal stages and subsequently to adults (900–1,200 μm).

Trombiculid mites live in moist soil covered with vegetation such as grassy and weedy areas. In general, optimal living conditions require a relative air humidity of 80% (what explains that chiggers are not typically found on vegetation higher than 30 cm off the ground) and neutral to slightly alkaline soil. The optimum activity of chiggers occurs at

Trombiculid mites often form localized "mite islands" (or "mite focus", "larvae focus") in suitable areas inhabited by potential hosts [30]. Therefore, chiggers have a patchy distribution on the vegetation. Mite islands are quite clearly defined, and larvae could not be detected in their immediate vicinity [26,31]. A possible explanation for this focalization may be that chiggers apparently do not move more than a few meters from where they hatched. Chiggers would temporarily disperse if a host approached. On the contrary, if physical contact were not managed or if the host were not close enough for them to drop on it, chiggers would invariably

The life cycle of trombiculid mites has been mainly studied in the laboratory. The most outstanding feature of the life cycle is the constant duration of quiescent periods and the variable duration of active stages. Trombiculid mites usually have one generation per year, but with overlapping generations, each well synchronized with the seasons because they can overwinter in most stages (egg, larva, deutonynph, and adult) and because the adult mites have a long life span [33]. In boreal species, an egg-to-egg cycle ranges from 150 to 400 days, but it is shorter in tropical species [23]. In nature, the life cycle is supposed to be completed in 2–12 months or longer, depending on the species and environmental conditions. In temperate areas, there may be 1 to 3 generations per year, whereas in tropical regions the life cycle is shorter and continuous throughout the year [1]. In Europe, the duration has been estimated

As mentioned above, trombiculid mites only act as parasites during their larval stage. Thus, the greatest attention has been paid to chiggers. In addition, adults and deutonymphs of the majority of trombiculid species have never been observed on the soil surface (in fact, their habitats are mostly unknown). Therefore, the taxonomy of trombiculid mites is based solely on their larvae [34]. It is estimated that only the postlarval stage of less than 10% of the total of Trombiculidae species are known [7]. This is the case of some tropical species in contrast to

It is well known that when feeding on hosts, chiggers develop a characteristic feeding tube (stylostome) in the host's skin. The stylostome is mostly formed of the larval salivary secretions solidifying in the host's epidermis [7]. Larva cuts the stratum corneum with its rather short

the difficulty of finding active postlarval instars in northern countries [23].

and promptly return to the cluster and would continue waiting [32].

in five to seven months under favorable conditions [26].

**1.3. Feeding process**

temperatures of 25–30°C [26,29].

178 An Overview of Tropical Diseases

Scrub typhus or tsutsugamushi disease [from Japanese words meaning disease (*tsutsuga*) mite (*mushi*) is a life-threatening arthropod-borne bacterial infection that presents as an acute undifferentiated febrile illness widely endemic in Asian Pacific regions. The disease is transmitted to humans by chiggers of *Leptotrombidium* spp. and is caused by the bacterium *Orientia tsutsugamushi*. Noteworthy are the reported cases that suggest other *Orientia* species as etiological agents of scrub typhus-like disease.

The disease was widely reported in soldiers during World War II [40] and now is an important illness for travelers to the endemic regions [41]. More than half (55%) of the world population lives in areas where scrub typhus is endemic, so over one billion people are at risk of acquiring the infection [42]. Approximately one million new cases have been estimated to occur annually [43]. However, this is surely an underestimation because recognition of the disease is difficult due to its overlapping clinical spectrum with other common causes of fever in this population, the lack of awareness among affected people, and the limitations of current diagnostic methods [44].

#### *2.1.1. Etiology and epidemiology*

The etiological agent of scrub typhus, *O. tsutsugamushi* (previously known as *Rickettsia orientalis* or *Rickettsia tsutsugamushi*), is an *α*-proteobacteria that was reclassified as a new genus separate from *Rickettsia* based on phenotypic and genotypic differences [45]. *Orientia* differs from *Rickettsia* in the structure of the cell wall, antigenic profile, and genome size, which is almost twice the size of the *Rickettsia* genome.

There are three prototype strains: Gilliam, Karp, and Kato; however, more than 20 antigenically distinct serotypes are present in endemic areas [46], and currently over 70 strains of *O. tsutsugamushi* are known [47]. As chigger mites are habitat specific, *O. tsutsugamushi* strains could have evolved mostly in separate biotopes, resulting in different serotypes depending on their location [48]. The general course and the prognosis of the disease is determined by the strain of *O. tsutsugamushi* implicated [49], although multiple factors such as the patient's age, genetic factors, and previous immunity are also involved [50]. It is very likely that chiggers, as it is assumed for ticks [51], have potential immunomodulatory effects in their saliva that could affect the pathogenesis, immunity, and outcome of the disease [47].

Chiggers act as reservoir and vector of *O. tsutsugamushi*, being wild rodents the main hosts. Infected mites maintain the infection through the trombiculids' life cycle by transstadial and transovarial transmission [52]. Reverse transfer from infected animals to chiggers occurs infrequently, and the bacteria transmitted in this manner are not usually passed on to the next generation [53]. Chiggers cofeeding on rodents seems to be more relevant for effective mouseto-mite transmission of *Orientia* than feeding on rickettsemic hosts [54]. Nevertheless, the disease can only be transmitted to humans by chiggers already transovarially infected by *O. tsutsugamushi* [49].

Endemic regions are characterized by rice fields, scrubland, and the presence of primary deforestation [55,56]. Chiggers harboring the bacterium bite exposed individuals in vulnerable niches such as forests and infested undergrowth during occupational or recreational activities. *Leptotrombidium* chiggers feed on lymph and tissue fluids of the dermal layer for a period of 2–4 days [57]. Following the bite, the pathogen multiplies at the site of inoculation and subsequently induces local (eschar) and systemic manifestations of infection [58].

Several studies suggest the evidence of human infection with more than one strain of *O. tsutsugamushi* [59]. It could be explained by bites from different chiggers, each one infected with one strain or, alternatively, by the bite of individual chiggers infected with multiple strains [60].

Seasonal occurrence of scrub typhus is determined by the time of appearance of chiggers because humans are infected through bites of the larva. In temperate zones, scrub typhus season is observed mainly in the autumn but also in the spring [61]. More than 45 species of trombiculid mites are known to be infected with *O. tsutsugamushi* in nature, but only *Lepto‐ trombidium pallidum*, *Leptotrombidium akamushi*, *Leptotrombidium scutellare*, *Leptotrombidium deliense*, *Leptotrombidium arenicola*, *Leptotrombidium imphalum*, *Leptotrombidium chiangraiensis*, *Leptotrombidium fletcheri*, *Leptotrombidium gaohuensis*, and *Leptotrombidium pavlovsky* are proven to transmit scrub typhus [1,13,14]. Principal vector species differ according to endemic areas (Table 2): *L. akamushi*, *L. pallidum*, and *L. scutellare* mediate scrub typhus in temperate zones, such as Japan and Korea, whereas *L. deliense* and *L. arenicola* are the principal vectors in tropical and subtropical regions or Southeast Asia and the Southwest Pacific [49] (Figure 2).

**Figure 2.** *Leptotrombidium intermedium* (left) and *Leptotrombidium pallidum* (right). Provided by Dr. Shatrov.

Scrub typhus is confined to a 13,000,000-km2 definite geographic region, the "tsutsugamushi triangle," where it is widely distributed (Figure 3). It extends from northern Japan, Korea, and far-eastern Russia in the North, to northern Australia in the South and to Pakistan and Afghanistan in the West, as well as the islands of the western Pacific and Indian Oceans, including Taiwan, Philippines, New Guinea, Indonesia, and Sri Lanka [62].

**Figure 3.** Tsutsugamushi triangle.

*2.1.1. Etiology and epidemiology*

180 An Overview of Tropical Diseases

*tsutsugamushi* [49].

strains [60].

almost twice the size of the *Rickettsia* genome.

The etiological agent of scrub typhus, *O. tsutsugamushi* (previously known as *Rickettsia orientalis* or *Rickettsia tsutsugamushi*), is an *α*-proteobacteria that was reclassified as a new genus separate from *Rickettsia* based on phenotypic and genotypic differences [45]. *Orientia* differs from *Rickettsia* in the structure of the cell wall, antigenic profile, and genome size, which is

There are three prototype strains: Gilliam, Karp, and Kato; however, more than 20 antigenically distinct serotypes are present in endemic areas [46], and currently over 70 strains of *O. tsutsugamushi* are known [47]. As chigger mites are habitat specific, *O. tsutsugamushi* strains could have evolved mostly in separate biotopes, resulting in different serotypes depending on their location [48]. The general course and the prognosis of the disease is determined by the strain of *O. tsutsugamushi* implicated [49], although multiple factors such as the patient's age, genetic factors, and previous immunity are also involved [50]. It is very likely that chiggers, as it is assumed for ticks [51], have potential immunomodulatory effects in their saliva that

Chiggers act as reservoir and vector of *O. tsutsugamushi*, being wild rodents the main hosts. Infected mites maintain the infection through the trombiculids' life cycle by transstadial and transovarial transmission [52]. Reverse transfer from infected animals to chiggers occurs infrequently, and the bacteria transmitted in this manner are not usually passed on to the next generation [53]. Chiggers cofeeding on rodents seems to be more relevant for effective mouseto-mite transmission of *Orientia* than feeding on rickettsemic hosts [54]. Nevertheless, the disease can only be transmitted to humans by chiggers already transovarially infected by *O.*

Endemic regions are characterized by rice fields, scrubland, and the presence of primary deforestation [55,56]. Chiggers harboring the bacterium bite exposed individuals in vulnerable niches such as forests and infested undergrowth during occupational or recreational activities. *Leptotrombidium* chiggers feed on lymph and tissue fluids of the dermal layer for a period of 2–4 days [57]. Following the bite, the pathogen multiplies at the site of inoculation and

Several studies suggest the evidence of human infection with more than one strain of *O. tsutsugamushi* [59]. It could be explained by bites from different chiggers, each one infected with one strain or, alternatively, by the bite of individual chiggers infected with multiple

Seasonal occurrence of scrub typhus is determined by the time of appearance of chiggers because humans are infected through bites of the larva. In temperate zones, scrub typhus season is observed mainly in the autumn but also in the spring [61]. More than 45 species of trombiculid mites are known to be infected with *O. tsutsugamushi* in nature, but only *Lepto‐ trombidium pallidum*, *Leptotrombidium akamushi*, *Leptotrombidium scutellare*, *Leptotrombidium deliense*, *Leptotrombidium arenicola*, *Leptotrombidium imphalum*, *Leptotrombidium chiangraiensis*, *Leptotrombidium fletcheri*, *Leptotrombidium gaohuensis*, and *Leptotrombidium pavlovsky* are proven to transmit scrub typhus [1,13,14]. Principal vector species differ according to endemic areas

subsequently induces local (eschar) and systemic manifestations of infection [58].

could affect the pathogenesis, immunity, and outcome of the disease [47].

Several reports of scrub cases typhus-like infections have been described in unusual areas, indicating that a wider geographic distribution should be taken into account [47]. Thus, the recent isolation of *Orientia chuto* in a febrile patient who acquired the infection in the United Arab Emirates, the detection of another divergent *Orientia* sp. in a patient in Chile, and the serologic diagnoses of scrub typhus acquired in Africa reveal that the geographical range accepted until now may be an underrepresentation [63,64]. To date, the genetic diversity of the genus *Orientia* is being reviewed because until recently *O. tsutsugamushi* has been consid‐ ered the sole species of the genus. Apart from *Leptotrombidium* spp., other trombiculid mites such as *Neotrombicula japonica* and *Eushoengastia koreaensis* have also been implicated as possible vectors of this disease [15,65].

The disease is considered rural, and the risk of infection is closely related to occupation. In areas where scrub typhus is prevalent, most cases are acquired through agricultural exposure. Most travel acquired cases of scrub typhus are associated with outdoor activities such as camping, rafting, or trekking in endemic areas [50]. Outbreaks related to military operations have been reported [66]. The impact of scrub typhus in pregnancy is less explored. Acute scrub typhus can be transmitted vertically but congenital malformation due to infection *per se* has not been demonstrated [67].

#### *2.1.2. Clinical features and pathogenesis*

Scrub typhus ranges in severity from mild and self-limiting to fatal depending on the duration of the illness, the strain of *O. tsutsugamushi*, the immune status, and other factors of the patients [14]. After an incubation period of 10–12 days (can vary between 5 and 20 days), the onset of the disease is characterized by an eschar and regional lymphadenopathy followed subse‐ quently by fever, general malaise, headache, and myalgia. The disease is characterized by focal or disseminated vasculitis and perivasculitis, which may involve the lungs, heart, liver, spleen, and central nervous system [68]. Progression of scrub typhus is accompanied by generalized lymphadenopathy, rash, cough, and interstitial pneumonia, acute respiratory distress syn‐ drome, gastrointestinal symptoms, meningoencephalomyelitis, myocarditis, acute renal failure, hypotensive shock, and disseminated intravascular coagulation may occur in severe cases [14,47,69].

The fever appears abruptly frequently accompanied by headache, myalgia, and malaise, with peaks on the 3rd–4th day of the disease and persists for more than 3 weeks in untreated cases. About a week after the onset of the symptoms, the eschar, which is not always present, is developed. It represents localized cutaneous necrosis at the site of mite feeding and is a typical scrub typhus marker, which is considered almost diagnostic [67]. It starts as a small papule that enlarges and subsequently undergoes central necrosis, and it eventually acquires a blackened crust with an erythematous halo that resembles a cigarette burn (Figure 4).

The common sites for finding an eschar are trunk, arms, and legs, but it also appears on the scalp, axilla, genitalia, waist, and other exposed parts of the body [14,49]. The prevalence of eschars in patients diagnosed by scrub typhus ranges from 7% to 97% [67,70]. These differences may be may be due to the difficulty in detecting small eschars in dark-skinned individuals and atypical appearance of eschars in areas of damp and moist skin. Multiple eschars have been

Several reports of scrub cases typhus-like infections have been described in unusual areas, indicating that a wider geographic distribution should be taken into account [47]. Thus, the recent isolation of *Orientia chuto* in a febrile patient who acquired the infection in the United Arab Emirates, the detection of another divergent *Orientia* sp. in a patient in Chile, and the serologic diagnoses of scrub typhus acquired in Africa reveal that the geographical range accepted until now may be an underrepresentation [63,64]. To date, the genetic diversity of the genus *Orientia* is being reviewed because until recently *O. tsutsugamushi* has been consid‐ ered the sole species of the genus. Apart from *Leptotrombidium* spp., other trombiculid mites such as *Neotrombicula japonica* and *Eushoengastia koreaensis* have also been implicated as

The disease is considered rural, and the risk of infection is closely related to occupation. In areas where scrub typhus is prevalent, most cases are acquired through agricultural exposure. Most travel acquired cases of scrub typhus are associated with outdoor activities such as camping, rafting, or trekking in endemic areas [50]. Outbreaks related to military operations have been reported [66]. The impact of scrub typhus in pregnancy is less explored. Acute scrub typhus can be transmitted vertically but congenital malformation due to infection *per se* has

Scrub typhus ranges in severity from mild and self-limiting to fatal depending on the duration of the illness, the strain of *O. tsutsugamushi*, the immune status, and other factors of the patients [14]. After an incubation period of 10–12 days (can vary between 5 and 20 days), the onset of the disease is characterized by an eschar and regional lymphadenopathy followed subse‐ quently by fever, general malaise, headache, and myalgia. The disease is characterized by focal or disseminated vasculitis and perivasculitis, which may involve the lungs, heart, liver, spleen, and central nervous system [68]. Progression of scrub typhus is accompanied by generalized lymphadenopathy, rash, cough, and interstitial pneumonia, acute respiratory distress syn‐ drome, gastrointestinal symptoms, meningoencephalomyelitis, myocarditis, acute renal failure, hypotensive shock, and disseminated intravascular coagulation may occur in severe

The fever appears abruptly frequently accompanied by headache, myalgia, and malaise, with peaks on the 3rd–4th day of the disease and persists for more than 3 weeks in untreated cases. About a week after the onset of the symptoms, the eschar, which is not always present, is developed. It represents localized cutaneous necrosis at the site of mite feeding and is a typical scrub typhus marker, which is considered almost diagnostic [67]. It starts as a small papule that enlarges and subsequently undergoes central necrosis, and it eventually acquires a

blackened crust with an erythematous halo that resembles a cigarette burn (Figure 4).

The common sites for finding an eschar are trunk, arms, and legs, but it also appears on the scalp, axilla, genitalia, waist, and other exposed parts of the body [14,49]. The prevalence of eschars in patients diagnosed by scrub typhus ranges from 7% to 97% [67,70]. These differences may be may be due to the difficulty in detecting small eschars in dark-skinned individuals and atypical appearance of eschars in areas of damp and moist skin. Multiple eschars have been

possible vectors of this disease [15,65].

not been demonstrated [67].

182 An Overview of Tropical Diseases

cases [14,47,69].

*2.1.2. Clinical features and pathogenesis*

**Figure 4.** Eschar and erythema on the fifth day of illness in the left arm of a patient of a 36-year-old patient (photo provided by Dr. Takahashi).

reported in 0.6% to 2.2% of patients with confirmed scrub typhus [70]. Uncommonly, a maculopapular rash with centrifugal distribution may appear a week after the onset of these symptoms, starting on the chest, abdomen, or whole trunk and spreading to the limbs. Rash lasts a few days to a week [13,71]. Regional lymphadenopathy, characterized by tenderness and enlargement of the draining lymph node around the primary eschar, arises at the end of the first week after the disease onset [13]. Generalized lymphadenopathy appears 2–3 days later in some cases [72].

From the second week onwards, a proportion of patients (especially those untreated) will evidence of severe systemic infection. The extended vasculitis helps to explain the great diversity of clinical manifestations that have been described [49]. Respiratory symptoms, including interstitial pneumonia, acute respiratory distress, and pulmonary edema, are frequent. In fact, about 40% of scrub typhus patients complain of cough at the time of admis‐ sion. Gastrointestinal symptoms comprise nausea, vomiting, abdominal pain, diarrhea, or gastrointestinal bleeding. Alterations in liver function and pancreatitis are also common. The central nervous system (CNS) is frequently affected. Indeed, *O. tsutsugamushi* is detected in the cerebrospinal fluid of 24% of the patients with no clinical signs of CNS involvement. Transient hearing loss, eye manifestations, confusion, neck stiffness, delirium, and mental changes occur frequently. Patients usually suffered from acute diffuse encephalomyelitis, encephalopathy, meningitis, or meningoencephalitis. Regarding the cardiovascular system, myocarditis, vasculitis, pericarditis, and rhythm abnormalities are often seen, but congestive heart failure is rare. Acute renal failure develops frequently in severe cases but may also occur in mild cases [13,14,61,62,67,69]. The case fatality rate in untreated patients is estimated in appropriately 10%, ranging from 0% to 30% [67].

At the beginning of the infection, *O. tsutsugamushi* mainly infects dendritic cells in the eschar [58]. The systemic dissemination of *O. tsutsugamushi* is suggested to be lymphogenous to the regional lymph nodes, followed by spread to target organs via the blood. This pathway was suggested based on the early development of lymphadenopathy in the regional drainage of the eschar as well as on animal experiments and clinical observations [14,47]. Once *O. tsutsu‐* *gamushi* infection progresses, the main target cells are vascular endothelial cells and macro‐ phages of the reticuloendothelial system, although cardiac myocytes can also been infected [14]. The endothelial cells seem to have a central role in the systemic inflammation because *in vitro*-infected human dermal microvascular endothelial cells are activated to express interleu‐ kin (IL)-8 and monocyte chemoattractant protein just after the infection. Moreover, soluble endothelial cell-specific adhesion molecules (sE-selectin) are highly concentrated in serum at the early stage of the disease.

The basic histopathologic findings reveal multiplication of *O. tsutsugamushi* in the endothelial cells lining the small blood vessels, perivasculitis and focal interstitial mononuclear cell infiltrations, and edema. Perivasculitis may involve the lung, heart, brain, kidneys, gastroin‐ testinal tract, liver, spleen, and lymph node [73].

### *2.1.3. Diagnosis and treatment*

Due to the severity of *Orientia* infection, treatment has to be started as soon as possible, even before having a conclusive microbiological diagnosis.

As in other infectious diseases, the gold standard of the diagnosis of scrub typhus is the isolation of the etiological agent by culture. Isolation of *O. tsutsugamushi* can be done in cell culture or in inoculated mice. Yolk sacs of 5- to 7-day-olds have been widely used in the past, but it was replaced by cell culture systems [74]. Currently, culture in HeLa, Vero, BHK, L929, ECV304, and HMEC-1 cell lines is the reference method for isolating *O. tsutsugamushi* from clinical samples [45,49,75,76]. These techniques are restricted to biosafety level 3 facilities and personnel with extensive experience. A positive result is given in an average time of 28 days, being inappropriate for the routine diagnosis of the disease. The shell-vial culture technique makes the detection of the microorganism possible in 48–72 h, allowing an early diagnosis before seroconversion [74,77]. *O. tsutsugamushi* can also be isolated by inoculating patient blood into mice, but results are not available in time to guide clinical management [78]. Mouse inoculation remains helpful when isolation of the organism from postmortem tissues is required [74].

The mainstay in scrub typhus diagnostics remains serology [79,80]. Nevertheless, despite their widespread use, all currently available serologic tests have limitations. The Weil–Felix OX-K agglutination reaction was the earliest serological tests used for clinical diagnosis of scrub typhus. It is inexpensive, easy to perform, and results are available overnight; however, it lacks specificity and sensitivity [46]. To date, the gold standard assay for the serologic detection of scrub typhus antibodies is the indirect immunofluorescence assay (IFA) [79]. Most frequently, IFA uses antigen from serotypes Karp, Kato, and Gilliam [46]. IFA is sensitive, and results are available in a couple of hours. Although it is accepted that a ≥4-fold increase in antibody titer between two consecutive samples (acute and convalescent-phase) is diagnostic, this is a retrospective diagnosis and cannot guide initial treatment [79]. Anyway, IFA is expensive and requires a level of technical expertise and equipment that may not be available in rural areas. Indirect immunoperoxidase is an alternative that eliminates the expense of a fluorescent microscope by substituting peroxidase for fluorescein [80].

The development of PCR amplification-based approaches have been incorporated to the diagnoses of infectious diseases even in nonreference laboratories. PCR has potential benefits in detecting *Orientia*-DNA before antibody response occurs. However, the high resource costs and training required for this technique make them impractical in many areas where scrub typhus is endemic. Moreover, the most appropriate specimen to use remains unclear. The PCR of eschar material yields more sensitive results than blood and remains positive even after the initiation of treatment. However, eschar-based PCR would diagnose a small amount of the cases in a scenario with a prevalence of eschars as few as 7%. Buffy coat could improve sensitivity compared with whole blood, but the use of blood-based assays is limited to the time window of rickettsemia [81,82]. Moreover, low copy numbers is an important handicap of DNA-based approaches. The optimal PCR target for diagnosing scrub typhus stays also uncertain. A target gene enabling specific but sensitive detection as well as sufficiently broad coverage of genotypes of *O. tsutsugamushi* is needed. A nested-PCR assay targeting the 56-kDa gene is highly specific, but sequence variability of this gene may affect primer annealing and, therefore, test sensitivity [83]. 16S rDNA-based *Orientia*-specific PCR may show a broader detection spectrum than an assay based on a more variable species-specific target, such as the 56-kDa gene [47]. Real-time PCR assays targeting the 47-kDa outer membrane protein and the *groEL* genes of *O. tsutsugamushi* are also very sensitive tools for the diagnosis of scrub typhus [78,84]. Recently, loop-mediated isothermal PCR assay (LAMP) targeting the *groEL* gene has shown diagnostic accuracy similar to real-time and nested conventional PCR assays [84]. This assay is simple and less expensive and can be considered a valid molecular method for the early diagnosis of scrub typhus.

*gamushi* infection progresses, the main target cells are vascular endothelial cells and macro‐ phages of the reticuloendothelial system, although cardiac myocytes can also been infected [14]. The endothelial cells seem to have a central role in the systemic inflammation because *in vitro*-infected human dermal microvascular endothelial cells are activated to express interleu‐ kin (IL)-8 and monocyte chemoattractant protein just after the infection. Moreover, soluble endothelial cell-specific adhesion molecules (sE-selectin) are highly concentrated in serum at

The basic histopathologic findings reveal multiplication of *O. tsutsugamushi* in the endothelial cells lining the small blood vessels, perivasculitis and focal interstitial mononuclear cell infiltrations, and edema. Perivasculitis may involve the lung, heart, brain, kidneys, gastroin‐

Due to the severity of *Orientia* infection, treatment has to be started as soon as possible, even

As in other infectious diseases, the gold standard of the diagnosis of scrub typhus is the isolation of the etiological agent by culture. Isolation of *O. tsutsugamushi* can be done in cell culture or in inoculated mice. Yolk sacs of 5- to 7-day-olds have been widely used in the past, but it was replaced by cell culture systems [74]. Currently, culture in HeLa, Vero, BHK, L929, ECV304, and HMEC-1 cell lines is the reference method for isolating *O. tsutsugamushi* from clinical samples [45,49,75,76]. These techniques are restricted to biosafety level 3 facilities and personnel with extensive experience. A positive result is given in an average time of 28 days, being inappropriate for the routine diagnosis of the disease. The shell-vial culture technique makes the detection of the microorganism possible in 48–72 h, allowing an early diagnosis before seroconversion [74,77]. *O. tsutsugamushi* can also be isolated by inoculating patient blood into mice, but results are not available in time to guide clinical management [78]. Mouse inoculation remains helpful when isolation of the organism from postmortem tissues is

The mainstay in scrub typhus diagnostics remains serology [79,80]. Nevertheless, despite their widespread use, all currently available serologic tests have limitations. The Weil–Felix OX-K agglutination reaction was the earliest serological tests used for clinical diagnosis of scrub typhus. It is inexpensive, easy to perform, and results are available overnight; however, it lacks specificity and sensitivity [46]. To date, the gold standard assay for the serologic detection of scrub typhus antibodies is the indirect immunofluorescence assay (IFA) [79]. Most frequently, IFA uses antigen from serotypes Karp, Kato, and Gilliam [46]. IFA is sensitive, and results are available in a couple of hours. Although it is accepted that a ≥4-fold increase in antibody titer between two consecutive samples (acute and convalescent-phase) is diagnostic, this is a retrospective diagnosis and cannot guide initial treatment [79]. Anyway, IFA is expensive and requires a level of technical expertise and equipment that may not be available in rural areas. Indirect immunoperoxidase is an alternative that eliminates the expense of a fluorescent

the early stage of the disease.

184 An Overview of Tropical Diseases

*2.1.3. Diagnosis and treatment*

required [74].

testinal tract, liver, spleen, and lymph node [73].

before having a conclusive microbiological diagnosis.

microscope by substituting peroxidase for fluorescein [80].

The diagnosis and subsequently the antibiotic treatment are often missed or made late due to the lack of effective commercially available diagnostic tests and the lack of specificity of the early clinical presentation. It is important to remark that treatment must begin whenever scrub typhus is clinically suspected, without waiting for microbiological confirmation. It is well known that delayed treatment leads to complications such as adult respiratory distress syndrome, disseminated intravascular coagulation, acute renal failure, meningitis, menin‐ goencephalitis, and gastrointestinal tract bleeding [57]. Bacterial proliferation and the time of antibiotic treatment are very important predictors of lethality.

The clinical discrimination of scrub typhus from other undifferentiated fevers is often very difficult because the clinical symptoms are similar. In patients presenting an eschar and/or rash, and generalized or regional lymphadenopathy in a endemic area, scrub typhus should be considered in the differential diagnosis along with rickettsialpox, Mediterranean spotted fever, dengue, leptospirosis, and murine typhus [55,71].

Mortality in the pre-antibiotic era was variable and in some series approached 60%, but specific and effective antimicrobial chemotherapy is now available [80]. Doxycycline and chloram‐ phenicol are both effective oral or intravenous agents against scrub typhus, dissipating fever in 24 h in most patients [71]. Although the disease can be treated effectively with these antibiotics, reinfection and relapse frequently occur due to the wide variety of antigenically distinct serotypes [85]. Azithromycin and rifampicin are alternative drugs [61].

Currently, effective chemoprophylaxis or vaccination approaches for dealing with *O. tsutsu‐ gamushi* infection are still not available [42]. A prophylactic vaccine to scrub typhus is a public health priority because of its high incidence, high mortality, nonspecific clinical presentation, lack of sensitive diagnostic tests, and emergence of antibiotic resistance. The development of an effective and safe vaccine has to be strongly focused on T cell-mediated immunity, empirical testing of the immunogenicity of proteins encoded by conserved genes, and assessment of protection in relevant animal models that truly mimic human scrub typhus resistance [57]. Therefore, prevention of scrub typhus is based mainly on avoiding the chigger bites and the use of repellents during travel in rural areas of endemic countries [61]. Wearing protective clothing and self-examination after visiting arthropod-vector infested areas are also recom‐ mended [86].

#### **2.2. Other chigger-borne infectious diseases**

Nowadays, *O. tsutsugamushi* remains as the unique agent whose transmission by chigger bites has been confirmed. Nevertheless, trombiculid mites inhabit areas where the presence of several arthropod-borne microorganisms, their vectors, and reservoirs has been demonstrated. Thus, the vector competence of chiggers has long been investigated worldwide.

There are a lot of references in the old scientific literature that associate chiggers with the transmission of several pathogens, being *N. autumnalis* the most reported species. However, the majority of them correspond to secondary anecdotal information and present poor or no details [87]. In the 2000s, *Anaplasma phagocytophilum*-DNA was detected in unfed *N. autumna‐ lis* chiggers collected on vegetation in a mountainous area from the North of Spain [88]. This finding remains doubtful taking into consideration that the infection occurred in unfed larvae, so chiggers are speculated to be true carriers of the bacteria and inherited it through transo‐ varial transmission. The presence of rickettsiae was also investigated in chiggers of the same mountainous area of Spain. Amplicons compatible with infection by *Rickettsia* spp. were detected by molecular techniques in *Neotrombicula inopinata* collected over vegetation [89]. Up to date, these results remain unconfirmed. The vector competence of *N. autumnalis* chiggers for the transmission of *Borrelia burgdorferi* sensu lato (s.l.) has been also investigated. This bacterium was screened by PCR and further DNA hybridization in questing larvae collected on vegetation and feeding larvae removed from trapped micromammals in Germany [87]. Borrelial DNA was amplified in chiggers from 1 larva feeding on a white-toothed shrew (*Crocidura russula*), from a pool of 4 larvae feeding on a *Borrelia garinii*-infected laboratory mouse, and from 1 nymph that had previously fed as a larva on a *Borrelia afzelii*-positive laboratory gerbil. Therefore, the vector competence of *N. autumnalis* remains unclear. The presence of *B. burgdorferi* s.l. and *A. phagocytophilum* DNA was also been investigated by PCR and reverse line blotting in chiggers found on wild birds captured in the western Carpathian Mountains (Czech Republic) [24]. *B. garinii* and *B. valaisiana* were found in a pool of 5 chiggers from the genus *Neotrombicula* collected from a Eurasian Blackcap (*Sylvia atricapilla*). Regarding *A. phagocytophilum*, DNA was detected in none of the samples [87]. Trombiculid mites have also been associated to *Bartonella* spp. A new strain of *Bartonella* sp. was isolated from the gray squirrels *Sciurus carolinensis* in Georgia [90]. Then this bacterium was studied in ectoparasites removed from gray squirrels by PCR. None of the mites tested (*Eutrombicula splendens*, *Myiatrombicula cynos*, and *Neotrombicula whartoni*y) were positive, whereas 6 *Bartonella* spp. strains were detected, 2 in fleas and 4 in lice [91]. Furthermore, *Leptotrombidium* mites have been reported as carriers of *Bartonella tamiae* [92], species isolated from patients from Thailand [93].

Several rickettsiae previously found in humans as *Rickettsia akari*, *Rickettsia japonica*, *Rickettsia conorii*, *Rickettsia felis*, *Rickettsia typhi*, and *Rickettsia* sp. closely related to TwKM02, *Rickettsia australis*, and Cf15 were detected using molecular methods in trombiculid mites removed from wild rodents collected in Korea [94]. Although the rickettsial DNA was detected in mites, it has yet to be determined whether the DNA was amplified from the meal of an infected animal or from the mite tissue itself. Tsui *et al*., (2007), identified TwKM02 and TwKM03 closely related to *R. australis* and *R. felis* URRWXCal2, respectively, in *Leptotrombidium* chiggers collected in Taiwan [95].

Chiggers are also suspected to be vectors of viral diseases [96]. The role of *L. scutellare* as possible vector of a Hantavirus causing epidemic hemorrhagic fever with renal syndrome (HFRS) in China was hypothesized [97]. The authors suggested that this mite could be naturally infected by HFRS virus and transmitted to vertebrates by biting and to its offspring via transovarian transmission. On the other hand, although the spread of Hantavirus had been thought to be exclusively by rodent excrement and urine, Hantavirus-RNA was detected in *Leptotrombidium* mites from Texas (2 larvae and 1 free-living predatory stage), suggesting a possible role in the transmission of Hantavirus pulmonary syndrome [98].
