First case report on molecular detection of Trypanosoma lewisi in an urban rat in Kelantan, Malaysia: An accidental finding

Objective: This case report highlights the first detection of Trypanosoma lewisi, a blood protozoan parasite found in an urban rat in Kota Bharu, Kelantan. Materials and Methods: Rat trapping was carried out within the Kota Bharu vicinity near a local wet market. A total of 38 rats were captured and subjected to peripheral blood smearing using Giemsa stain. Positive rats were sent for histopathological analysis for the evaluation of the organ samples. Results: The presence of trypanosomes was found in one sample from a blood smear. This was connected to a histological lesion on kidney tissues, which revealed a high concentration of trypanosomes. Additionally, the positive sample was confirmed as T. lewisi based on molecular diagnosis via polymerase chain reaction and subsequent sequencing and phylogenetic analysis. Conclusions: This finding serves as a baseline for further surveillance on T. lewisi population among urban rats in Kelantan and possible zoonotic transmission to humans.


Introduction
Trypanosomes are flagellated blood protozoa that can infect both warm-blooded and cold-blooded mammals [1]. It is a monophyletic genus of obligate intercellular parasites that induce trypanosomiasis in insects and vertebrates [2]. The organism is divided into two groups which are salivarian and stercorarian parasites. Both parasites are usually found in the digestive tract of the vector [3]. The parasite can cause severe diseases, such as human African trypanosomiasis (HAT) and Chagas disease in South and Central America. Not just in humans, this infection also can cause a decline in animal health, such as Surra and Nagana disease in horses [4]. Rodent species are primarily infected with Trypanosoma lewisi, which is commonly transmitted by several species of rat flea such as Ceratophyllus fasciatus, Nosopsyllus fasciatus, and Xenopsylla cheopis, respectively [5]. T. lewisi infection can be acquired by contact with feces of infected fleas or by ingestion of infected fleas [6]. This organism also can infect other mammals sporadically [7]. Trypanosoma lewisi can be found in urban and wild rats worldwide, especially in Asia [8,9]. Previously, it was thought that T. lewisi is a host-restricted parasite and does not possess a zoonotic risk to humans. However, several cases reported that the organism is an opportunistic pathogen that can cause infection to humans, including one fatal case [10][11][12].
Human infection with T. lewisi may show non-specific symptoms such as prolonged fever, appetite loss, and severe convulsion [11][12][13]. However, the effects of T. lewisi infestation on rodents are not well known. Therefore, knowing the zoonotic potential to humans with a close approximation to rodent population, here we report the first case of T. lewisi infection found in a wild rat in Kelantan, Malaysia. The finding is supported with blood smear results, histopathology, and confirmation by

Ethical approval
This study was conducted after approval by the Animal Ethics Committee of Universiti Malaysia Kelantan with the reference no: UMK/FPV/ACUE/FYP/10/2021.

Sample collection and identification of trypanosome
A total of 38 rats, 26 male and 12 female, were caught at random in the wet markets of Kota Bahru, Kelantan, Malaysia, to determine the incidence of leptospirosis and ectoparasites in the population of urban rats (manuscripts in preparation). All rats were euthanized using 2% (v/v) of carbon dioxide in a chamber (Dira Resources, Malaysia), and the blood was collected shortly via intracardiac puncture. A peripheral blood smear was conducted using Giemsa stain for blood routine examination. Several organs (liver, spleen, lungs, and kidneys) were harvested for subsequent diagnostic work-ups, including histopathology examination.

Detection of trypanosomes using universal trypanosome species (KIN) and T. lewisi-specific primer (TRYP1)
Suspected rats with trypanosomes were subjected to PCR to confirm the species using two sets of primers: KIN and TRYP1, respectively. The sequence of the primers is shown in Table 1. The thermocycler conditions for KIN primers are initial denaturation at 94°C for 3 min, denaturation at 94°C for 45 sec, annealing at 58°C for 3 min, and extension at 72°C for 3 min. The steps were repeated in 35 cycles and followed by further extension at 72°C for 3 min. For TRYP1 primer, the thermocycling parameters are initial denaturation at 94°C for 2 min, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, and the steps were repeated in 35 cycles followed by further extension step at 72°C for 10 min. The PCR products were visualized on a 2% (w/v) agarose gel stained with 1.0 μl Midori green and photographed under UV light. Sample with a positive result at PCR level was sequenced, and phylogenetic analysis using Molecular Evolutionary Genetics Analysis version 10.0 (PSU, USA) was constructed.

Results and Discussion
Out of 38 captured rats, 1 female brown rat (Case 1: Rattus norvegicus) had multiple polymorphisms of trypanosomes observed on a thin blood smear (Fig. 1). The organism was described as an individual, slender, tadpole-like form with a thin posterior end, oval kinetoplast, elongated nucleus, and a free flagellum known as adult trypomastigote. The appearance of adult trypomastigotes is suggestive for T. lewisi [15]. An unusual shape of trypanosomes with divided chromatins was found in the blood smear dividing stage epimastigotes. Both forms are usually present in acute infection in rats [16].
Interestingly, in histopathology lesions, we found abundant epimastigotes in kidney tubules (Fig. 2). However, no evidence of inflammatory and degenerative tissue changes was observed in the kidney, and other organs showed normal tissue appearance (figures not shown). Furthermore, the sequencing and phylogenetic analysis of the PCR positive result was confirmative to T. lewisi (Fig. 3).
Although T. lewisi is a common parasitic protozoan found in rats worldwide, to our knowledge, this is the first evidence on T. lewisi found in a rodent reported in Kelantan, Malaysia. This rat was captured in a wet market  within the vicinity of Kota Bharu. Previously, several studies described Trypanosoma spp. in urban rats in Malaysia at a low prevalence rate [17,18]. These studies reflected the presence of Trypanosome in rodents that live closer to the human setting, which may potentially expose the organism to the susceptible human host. The presence of T. lewisi polymorphism in the blood of rats in our study shows the establishment of an infective stage with the presence of both epimastigotes (development stage) and trypomastigotes (infectious stage; adult stage). This finding is interesting because epimastigotes are usually seen in the midgut of invertebrate vectors such as flies [19] and rarely in maintenance hosts [20]. It may be possible that stages of T. lewisi development occur in both maintenance host and invertebrate vectors compared to its closest relatives, such as Trypanosoma cruzi [21]. Apart from blood smear data, PCR and phylogenetic analyses (Fig. 3) further confirmed the evidence of T. lewisi presence in the rat. Although TRYP1 primers are specific for T. lewisi, the outcome from the PCR must be sequenced for confirmation as TRYP1 primers showed high cross-reactivity against other Trypanosoma species and other organisms related to rodents [3]. Nevertheless, both PCR are proven to be useful for screening of T. lewisi in rodents. The presence of epimastigotes in kidney cells revealed an exciting characteristic of the organism (Fig. 2). This showed that although T. lewisi is an obligate hemoparasitism, it is possible to migrate to multiple organs and exhibit a visceral form, as previously reported in Trypanosoma evansi [22]. The histological examination revealed T. lewisi in the interstitium of the kidney in our study. However, the parasites did not induce significant lesions to the organ. A study by Biswas et al. [23] showed that bandicoot rats affected with trypanosomiasis typically exhibited inflammatory, degenerative, and necrotic changes in multiple organs suggesting acute infection. These lesions were not seen in our case. However, different rat species may have varying levels of immunogenicity, and minor tissue alterations could imply that the brown rat is more tolerant to T. lewisi infection and hence acts as a principal reservoir or carrier for the parasite.
The occurrence of T. lewisi found in this study raises a public health concern among Kota Bharu residents, with its zoonotic potential to humans, possibly underreported. The organism is rapidly divided into mature parasites less than 24 h post-infection [24]. It possibly transmits vertically from mother to fetuses [25], further disseminating the organism within the rats' population within a short time and to humans, subsequently. For this reason, a rodent surveillance program is warranted to estimate the prevalence of T. lewisi in the wild rat's population in Kota Bharu and alert the health authorities on its effect on humans. Additionally, molecular surveys on the type of vectors carrying T. lewisi are indicated to evaluate the tendency of transmission between rodent-borne trypanosomes and humans. Such studies can help provide essential information on the diversity of these vectors toward designing an effective preventive strategy to eliminate the possible infection.

Conclusion
Trypanosoma lewisi is a common parasitic infection in wild rodents which potentially transmit to humans. This is the first case report of trypanosomiasis in rats in the Malaysian state of Kelantan. Further surveillance is needed to ascertain the prevalence of T. lewisi in rodent populations to assess the risk of human infection.