Description of human infestations by ticks in Panama and Costa Rica

Tick bites in humans are associated with the transmission of pathogens, anaphylactic shock, paralysis, and secondary infections. In this work we described six cases of tick bites in patients from Panama and Costa Rica. These bites were provoked for adults of Ornithodoros puertoricensis, Amblyomma cf. oblongoguttatum, Amblyomma ovale, and Ixodes cf. boliviensis, and a nymph of Amblyomma mixtum. The relationships of these species of ticks with the environment are explained. Among the reactions observed in the patients there are blisters, maculo-papular rash, granuloma, lymphadenopathy, and erythema migrans-like rash, but none referred symptoms compatible with an infectious disease. In the collected ticks a PCR battery was developed to rule out Borrelia, Rickettsia, Anaplasmataceae, and Coxiella diseases to the ticks from the cases 1, 2, 3 and 4, with negative results. Although no pathogen infections were evidenced, these finding indicate that the tick bites constitute a public health problem what goes unnoticed in many countries.


Background
In clinical practices, the tick bites are related to the risks to transmission of viral, bacteria or protozoa diseases, according to the country or geographic distribution of the ticks (1,2). In fact, ticks are associated with a greater variety of pathogens than other groups of arthropods, including mosquitoes (3,4). However, in addition to their known vector capacity, the bites can provoke other manifestations that vary to mild or severe, e.g. allergies, toxicosis, paralysis, and a tickinduced red-meat allergy (alpha-gal syndrome) (5)(6)(7)(8). These manifestations are produced by different components of tick saliva, that in general, have properties anticoagulant, antiplatelet, vasodilator and vasoconstrictors, antiinflammatory, lytic, and immunomodulatory (9,10). In fact, the sialome of ticks is composed by a variety of multifunctional p r o t e i n s , p e p t i d e s a n d n o n -p e p t i d i c m o l e c u l e s , prostaglandins, among other components (11)(12)(13). As a whole, the saliva of the ticks allows "adapting" and handling the immune response the host for a prolonged feeding, including inhibits pain, itch, haemostasis, modulates angiogenesis and bite-site healing; which causes some of the damages mentioned above (14,15).
In some cases, tick bites produce reactions that can be confused with infections caused by pathogens (16)(17)(18). For example, rash caused by some pathogenic Rickettsia and Borrelia species are among the symptoms of infection after tick bites; these could be similar to reactions caused by the effect of the saliva of the ticks (6,19,20). In this sense, reactions to tick bites must be differentiated from signs of pathogenic microorganism infection itself , such as exanthematous eruptions, inoculation eschar, erythema migrans and lymphadenopathy, which are common to various tick-borne diseases, such as Lyme Disease (LD), Tick-Borne Lymphadenopathy (TIBOLA), and other rickettsial diseases (17,18,21,22). Moreover, the Southern Tick-Associated Rash Illness (STARI), a condition caused by Amblyomma americanum bites and whose etiological agent is unknown, has been confused with LD due to the circular rash similar to that caused by this disease. This condition may be accompanied by symptoms as fatigue, headache, fever, and muscle aches (18). Therefore, in medical practice, it is

Description of human infestations by ticks in Panama and
Costa Rica  6 Venzal necessary to know the differences between the manifestations caused by the saliva of ticks, and those related to pathogens.
In Central America, tick bites to humans have been linked to the transmission of tick-borne relapsing fever (TBRF), ehrlichiosis, mild rickettsiosis, severe and fatal cases of Rickettsia rickettsii spotted fever, as well as allergies, dermal lesions, and paralysis (23)(24)(25)(26)(27)(28)(29)(30). Despite the harmful effects caused by ticks, many aspects related to tick bites remain unknown to physicians and patients in this region, which can lead to confusion or misdiagnosis (31). It is possible that in Central America, Panama and Costa Rica are the countries with the most data related to ticks that parasitize humans and tick-borne diseases (TBD) (30); even so, few details are known about the species that parasitize people and even less about the effects they can cause. Therefore, the objective of this paper is to describe tick bites in humans and the related reactions in Panama and Costa Rica.

Case reports
Case 1: On August 2018, a 42 year-old man suffered three tick bites on his thigh while sleeping in a cabin in a rural town, in the province of Colón, Panamá. The three bites turned into blisters within hours (Fig. 1). Even after two days, the blisters emanated blood, and the affected person reported intense itching and slight pain around the blisters. In addition to the blisters and the irritation, he did not refer symptoms, and did not seek medical attention. The blisters diminished over the weeks, and the effect of the bites disappeared after 3-4 months. During a search into the cabin, several engorged (five adults) and non-engorged ticks (three nymphs and two adults) were collected in crevices in the walls and the ground. No other blood-sucking arthropods were found (e.g. bed bugs or kissing bugs) in the cabin. The ticks were preserved in 85% ethanol and sent to the laboratory of Ectoparasites in the Department of Medical Entomology of the Gorgas Memorial Institute for Health Studies, for their identification and analyses. Ticks were identified as Ornithodoros puertoricensis using molecular methods previously described (32).  (Fig. 2a). The tick was extracted immediately and preserved in 80% ethanol. She did not refer symptoms in the following weeks, and did not need to apply any treatment on the bite site; however, a month later, a granuloma was evidenced in the area of the bite (Fig.  2b). In the same laboratory to the case 1, the tick was identified as Amblyomma ovale male, according to Bermúdez et al. (33). During laboratory observation, the mouthparts were found to be complete. year-old woman went hiking in the afternoon in a forest area of Ancón community, a suburban area in City of Panamá, Panamá. The following morning, she noticed a tick attached to right site of her nape, extracted it, and preserved in 80% ethanol. Afterwards, the woman noticed that a maculo-papular erythema developed at the site of the bite. During the follow 8 days, the lesion gradually expanded to the surrounding area, from 3 cm (2 days after tick removal) to 10 cm (8 days after tick removal) (Fig. 3). Additionally , the woman had pruritus and lymphadenopathy at the anterior cervical single chain lymph node, which was tender on palpation and warm to touch. She did not refer other symptoms in the following days. However, according to the recommendations to prevent tick-borne rickettsiosis in Panama (34), she was treated with doxycycline (100 mg, orally, every 12 hours for 7 days), and topic clotrimazol, neomicina and betametasona for the itch. After 5 days of treatment, the patient reported improvement of the pruritus, a decrease in the maculopapular lesion, and disappearance of the cervical adenopathy. In laboratory, the tick was identified as Amblyomma cf. oblongoguttatum female (33), and during the observation, the mouthparts were complete. rd Case 4: On May 3 2021, a 71 year-old man visited his farm in El Valle de Antón, province of Coclé, Panamá. Five days later, he noted an engorged nymph attached to the inner side of his left ankle. An erythematous and purple plaque was observed at the site of the bite, which diminished within a few weeks (Fig. 4). After removing the tick, the patient only felt a little itching and a slight pain to the touch, so he did not apply any topical treatment, and did not reported additional symptoms or discomfort in the following weeks. In laboratory , the tick was kept alive in a controlled environmental incubator (78% humidity, 26°C), and after 15 days emerged an Amblyomma mixtum male (33). Case 5: On August 16th 2020, a 6 year-old girl was walking in a forest area of San Isidro, province of Heredia, a rural town close to the Braulio Carrillo National Park (=1400 meters of high), Costa Rica. This same day, she got itchy and her parents noticed a tick attached to the chest (Fig. 5). The tick was extracted immediately and preserved in 80% ethanol. She did not refer fever or symptoms in the following weeks, therefore, did not need to treatment. The tick was sent to the laboratory of Parasitology Laboratory of the National University of Costa Rica (LP-UN), for their identification, corresponding to an Ixodes cf. boliviensis female (33,35). During the identification it was observed that the chelicerae were broken. Case 6: On August 2021 a 16 year old boy, was at the morning in his yard in Rio Segundo, province of Alajuela, near to the International Airport of Costa Rica. At night he felt itch and found the tick on his arm. Afterwards, he noticed a rash similar to erythema migrans-like rash in his arm, around the site of the bite (Fig. 6). The diameter of the rash was 4-5 cm and it took one week to disappear. There was not any fever or rash after the extraction of the tick. No medical treatment was necessary. The tick was sent to the laboratory of Parasitology Laboratory of National University of Costa Rica (LP-UNA), and identified as A. ovale male (33).

Figure 6: Erythema migrans-like caused by Ambyomma ovale male
Molecular assays in the ticks: To exclude potential TBD, ticks from the cases 1, 2, 3 and 4, were bisected longitudinally using sterile scalpels and washed with distilled water to remove ethanol. DNA was extracted using DNeasy™ tissue extraction kit (Qiagen, Hilden, Germany), following the manufacturer's criteria. Extracted DNA was tested by a battery of PCR protocols targeting bacterial 16S rRNA, Rickettsia, Coxiella, and Borrelia using specific primers and published protocols for each agent (36)(37)(38)(39). The primers used for detecting these pathogens, and the corresponding product sizes are listed in Table 1. DNA of the target pathogens was not detected in any of the ticks analyzed.

Discussion
We described six cases of tick bites in people from different urban, rural and wild regions in Panamá and Costa Rica. In both countries, the environmental and anthropic differences of each area influence in the diversity of ticks (40,41). In this sense, in the lowlands of these countries the most of the anthropophilic Ixodidae and Argasidae ticks, correspond to the genera Amblyomma and Ornithodoros, respectively, and there is less information of Rhipicephalus biting human (33). In the highlands (+1400 meters over sea level) there are few reports of ticks biting humans and correspond to I. cf. boliviensis and Ornithodoros kelleyi (33,42).
Among the manifestations found in the patients were blisters, maculo-papular localized rash, granuloma, lymphadenopathy or erythema migrans-like rash, which are among the most commonly reported reactions to the tick bites (15,43,44). In our group of affected, these manifestations lasted to heal from weeks to several months after the removal of the ticks, but were not accompanied by symptoms consistent with infections; in fact, after months of having contact with the ticks, the patients did not manifest discomfort.
Although dermatoses caused by tick saliva is related with the inflammatory response of the host or with the sensitivity of each person, there are other factors that can exacerbate the reaction, as the number, stage, and the tick species, as well the time of exposure to the bites, and/or constant and repetitive ticks bites (45,46). Furthermore, the biological differences between Argasidae and Ixodidae lead to different reactions in parasitized people.
In general, Argasidae do not penetrate deeply into the skin, and feed during the night when the hosts are sleeping. In this sense, although cases of Ornithodoros bites have been registered in people who visit caves or guano areas, most of the reports are related to intradomiciliary conditions (5,24). The ingestion of blood is rapidly (for less than an hour) in nymphs and adults, but the feeding of the larvae can last 2-4 days (47,48). Consequently, Ornithodoros may go unnoticed when biting, in particular in human dwellings, and are therefore reported less frequently than hard ticks (46,49). These characteristics are consistent with the anamnesis of case 1, since the affected person did not observe the ticks while they were feeding. The bites showed the characteristic formation of blisters keeps the blood of the hosts flowing. Although these lesions can be considered "benign", some cases associated with certain species of Ornithodoros can be extremely painful and produce severe effects, causing strong allergic reactions, and even anaphylactic shocks (24,43,46,(50)(51)(52). Moreover, some Ornithodoros species (e.g. O. talaje, O. rudis, among others) are also related to the transmission of TBRF, which is among the most important neglected diseases in the region (26).
The rest of the affected were parasitized by three species of Ixodidae and, as expected, the degree of alteration was different according to the ticks and the person. With some exceptions related to pets or work activities in farms or veterinary clinics, Ixodidae bites are most related to outdoors activities (53,54). Ixodidae feeding for several days and can penetrate deeply into the dermis or hypodermis, depending on the species of tick and the location on the skin (48). Beside to the damages to the skin, during this prolonged feeding, the amount of saliva inoculate on their host is variable, as well is the variety of its components (55). These "cocktail" of components affect the inflammatory response and are responsible of cutaneous and subcutaneous manifestations, and possibly causes swollen glands (31). Moreover, in the case of vasodilators or other components of the saliva, their persistence produce bruising around the bite, which cause redness and subsequent clearing for would lyse of the blood cells.
Regarding to our observation in the case 2, the patient affected had little exposure to the tick and did not present other symptoms; even so, the lesion at the bite site remained as a granuloma for several weeks. The granulomas associated with tick bites may be caused by the breakdown of the mouthparts (hypostoma and chelicera) within the skin, or for the long-term reaction to the salivary , causing inflammation around them (16,56). In this patient, it is possible that the inflammation was due to a local reaction to the bite, as has been suggested by other authors (31,56). This is similar to case 5, as the time between possible exposure and a removal of the tick was short; but, despite the chelicerae was broken, not granuloma was evidenced in the following days. Regarding to the case 3, since the affected person noticed the tick at least 12 hours after hiking, so it is possible that the parasitism lasted longer, which produced greater effects and symptoms as the lymphadenopathy. In this patient, the absence of fever or malaise could rule out a disease transmitted by the tick; though the norms in Panama recommend the use of doxycycline when suspected of a potential rickettsial infection (34). All these cases, the bites were caused by single adult ticks.
In the case 4, the bite was caused by a nymph that remained attached for almost a week, and there were still no significant symptoms. Because of their small size, immature ticks are more difficult to locate and extract, and several bites could cause an allergic reaction. Granulomatous lesions caused by bites of nymphs of A. mixtum (referred to as A. cajennense) have been described that took more than 2 years to heal (53). Furthermore, with the case 4, it was possible to confirm that humans are suitable hosts and allow the complete development of A. mixtum nymphs, a fact reported also for tis species and A. coelebs nymphs in Brazil (53,54).
On the other hand, in the case 6, the manifestations in the skin are consistent with erythema migrans-like rash, which could be defined as a skin lesion that expands around the tick bite, and that can remain for a period of days to weeks. Although this type of manifestation was considering as one of the characteristic of LD, the absence of systemic symptoms (e.g. fever, sickness) in following months to the extraction of the tick, indicate a reaction produced by the tick saliva. Until now, LD has been no confirming in Costa Rica, despite the report of a human suspected case (57), and for the presence of the Borrelia burgdorferi complex in I. cf. boliviensis from Panama (35). LD is related to the transmission of Ixodes ricinus group in North Hemisphere. This is the first report of a erythema migrans-like rash caused by A. ovale in Costa Rica. This tick species has been related to a case of paralysis in a U.S. soldier in Panama, and has been implicated to the transmission of Rickettsia parkeri (23,58).
Finally, despite the harmful effects of the tick bites, these are generally underestimated by the people affected and by physicians. Even when the bites are not associated with the transmission of pathogens, represent discomfort that can last longer than it should if those affected are not treated in time, or cause severe complications as such anaphylactic shock, paralysis or secondary infections and myiases, which can further complicate the healing of the affected tissue (23,43,53). In other words, tick bites can affect the quality of life of those who suffer from them, if they are not treated properly (31). Moreover, the presence of cutaneous or subcutaneous manifestations could be confused with TBD, which can cause erroneous reports due to the lack of conclusive diagnoses. All these facts indicate that the tick bites constitute a public health problem what goes unnoticed in many countries.

Acknowledgments
Authors thanks to Alejandro Cabezas-Cruz and Álvaro Faccini-Martínez for their comments and suggestions of the manuscript.

Institutional Review Board Statement
This work was authorized by the Gorgas Memorial Institute's Bioethics Committee of Investigation (GMIBCI) under reference No. 111/CBI/ICGES21.

Informed Consent Statement
All of the people affected for tick-bites were notified and permission to use photos and general information was granted for their inclusion in this paper, according to the regulations of GMIBCI.

Conicts of Interest
The authors declare no conflict of interest.