First Report of an Asymptomatic Leishmania (Viannia) shawi Infection Using a Nasal Swab in Amazon, Brazil

The state of Pará has recorded seven Leishmania species that cause tegumentary leishmaniasis (TL). Leishmania species induce distinct immunological responses from the host and exhibit resistance to Glucantime, the first-line drug treatment for TL in Brazil. Objective: Identify the etiology of TL in an Amazonian city in the state of Pará. Material and methods: Eleven patients with TL were recruited and nasal swabs, lesion swabs, and skin fragments samples were collected. In the control group (n = 6), only the nasal swabs were collected. Polymerase Chain Reaction (PCR) amplification of the gene region hsp70-234 was performed using the extracted DNA from the samples, from which nine patients with TL and five in the control group were positive. Products were sequenced, mounted in CAP3 software, aligned using MAFFT v.7.221, edited in Geneious software v.8.1.7, and compared and aligned with sequences available in GenBank using the BLAST tool. Results: For patients with TL, six molecular diagnosis at the species level (L. (Viannia) braziliensis (n = 5/9), L. (Viannia) shawi (n = 1/9)) and three at the genus level (Leishmania sp. (n = 3/9)) were obtained. In the control group, four individuals were infected with Leishmania sp. (n = 4/5) and L. (V.) shawi (n = 1/5). Conclusion: This is the first report of L. (V.) shawi infection in the mucosal secretion of a healthy person in Brazil. Moreover, genetic variants were identified in the haplotypes of L. (V.) braziliensis in the gene sequence hsp70-234.


Introduction
In the Brazilian Amazon, several species of Leishmania cause TL. The state of Pará, the second-largest extension in this region, is the only state in Brazil to record seven dermotropic species of Leishmania pathogenic to humans, presenting variable levels of pathogenicity and drug resistance [1,2].
Lesional pleomorphism in patients with TL results from a balance in the parasitehost relationship. Leishmania species induce different immunological responses in the host. Three important species are associated with the severe evolution of the disease: Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, and Leishmania (Leishmania) amazonensis [3,4].
Some Leishmania species are more prone to hematogenous dissemination. L. (V.) braziliensis causes more frequently disseminated conditions, being the main species causing mucosal leishmaniasis [4]. L. (V.) braziliensis can be detected in the nasal mucosa of both sick and healthy people living in areas with endemic TL [5]. L. (V.) guyanensis, which is predominant in certain endemic areas of northern Brazil, notably in the states of Amazonas and Amapá [6], can also cause mucosal lesions [7] and is highly resistant to Glucantime, the first-line drug treatment for TL in Brazil [2]. Meanwhile, L. (L.) amazonensis can induce severely disseminated disease and anergic diffuse leishmaniasis [8]. Other pathogenic species, including Leishmania (Viannia) lainsoni, Leishmannia (Viannia) shawi, L. (Viannia) naiffi, and L. (Viannia) lindenbergi, do not usually cause complications. Leishmania (V.) lainsoni and L. (V.) naiffi usually present as a single lesion. L. (V.) lindenbergi has localized cutaneous lesions. No case of the mucocutaneous disease has been reported in any of the species [9].
L. (V.) shawi was isolated for the first time from sloths and monkeys from the Serra dos Carajás, Pará, Brazil, which is one of the etiological agents of TL in Brazil, and is associated with the cutaneous form of TL, usually observed as a single lesion without mucous or other complications [9,10]. L. (V.) shawi has been identified in sandflies, Lutzomyia (Nyssomyia) whitmani [11], and Lutzomyia gomezi [12]. There are reports of L. (V.) shawi in the Amazon region identified in TL carriers [2,13].
The molecular characterization of Leishmania sp. that causes TL in the Amazon is important not only to define a prognosis for the patient but also to guide the therapeutic approach. In addition, it is essential to evaluate the distribution of Leishmania sp. and the occurrence of variants, and alert surveillance services to support transmission prevention [6,14]. The great challenge in the Amazon is to provide access and resources of scientific advances in communities to improve molecular diagnostic techniques [15]. Molecular methods have received increasing attention due to the possibility of distinguishing species in material obtained directly from the lesion. The enzymatic techniques that are effective for species distinction depend on the isolation of Leishmania, which makes their use in routine diagnostics impossible. The PCR in its various versions and with different markers are able to discriminate Leishmania species, but the sequencing of products with certain markers is what confirms the species distinction. Among the most used markers in PCR are G6PhD, ITS1, and Hsp70-234 [16]. In the Brazilian Amazon region, it obtained excellent results using the marker Hsp70-234 [6,17]. The study aimed to identify the etiology of TL in an Amazonian city in the state of Pará. Despite the difficulties in performing the study during the COVID-19 pandemic, molecular analysis of several clinical samples revealed human infection by L. (V.) shawi in nasal swabs for the first time. Moreover, we discuss the importance of this finding on the epidemiological surveillance of TL.

Study Site
The research was conducted at the Municipal Hospital of Tomé-Açu. The city of Tomé-Açu is located in the northeastern region of the state of Pará, Brazilian Amazon, and is an important agricultural and mining center in the state ( Figure 1) [18,19]. In the municipality of Tomé-Açu there are two rainy seasons a year, one from December to May, representing 80% of the rainfall, and another from June to November. The locality features the following general characteristics: average annual temperature of 26 • C and rainfall average of 2300 mm/year [19].

Participant Recruitment
Participants were recruited in two stages: the first stage from October 2019 to February 2020, and the second from August 2020 to October 2020. The stages chosen were related to the increase in cases during the rainy season, and also to changes in health service due to the established pandemic.
Men and women over 18 years of age with a confirmed diagnosis of TL and controls were included in the study. The controls were individuals without injury, companions of the patients with TL who agreed to participate in the research, or those who live with the patients.

Participant Recruitment
Participants were recruited in two stages: the first stage from October 2019 to February 2020, and the second from August 2020 to October 2020. The stages chosen were related to the increase in cases during the rainy season, and also to changes in health service due to the established pandemic.
Men and women over 18 years of age with a confirmed diagnosis of TL and controls were included in the study. The controls were individuals without injury, companions of the patients with TL who agreed to participate in the research, or those who live with the patients.

Participants and Sample Types
For patients with TL (n = 11), nasal swab, lesion swab, and skin fragment samples were collected. In the control group (n = 6), only nasal swabs were collected. All participants gave their consent for inclusion in the study. The study was conducted according to the Declaration of Helsinki, and the protocol was approved by the Research Ethics Committee of the Instituto Evandro Chagas (Opinion number: 3.601.679).

Sample Collection
Sample collection was performed after site cleaning and antisepsis, in the following order: (1) nasal swab, (2) lesion swab, and (3) skin biopsy. For the nasal swab, the absorbent segment of the swab was inserted into the anterior third of the nose and rotated clockwise (360°) over the nasal mucosa. The same procedure was performed in both teins with the same swab. For the lesion swab, the absorbent segment was pressed parallel to the edge of the lesion and rotated on its surface clockwise [20,21]. Meanwhile, the skin sample was obtained using a disposable punch (5 mm) after local anesthesia (2% lidocaine). This was performed on the infiltrated and eysthematous edge of the lesion [14].

Sample Storage
The end rod of each swab containing the sample was cut with the aid of sterile scissors and placed in a 1.5 mL microtube containing 500 μL of NET (NaCl 0.15 mM; EDTA

Participants and Sample Types
For patients with TL (n = 11), nasal swab, lesion swab, and skin fragment samples were collected. In the control group (n = 6), only nasal swabs were collected. All participants gave their consent for inclusion in the study. The study was conducted according to the Declaration of Helsinki, and the protocol was approved by the Research Ethics Committee of the Instituto Evandro Chagas (Opinion number: 3.601.679).

Sample Collection
Sample collection was performed after site cleaning and antisepsis, in the following order: (1) nasal swab, (2) lesion swab, and (3) skin biopsy. For the nasal swab, the absorbent segment of the swab was inserted into the anterior third of the nose and rotated clockwise (360 • ) over the nasal mucosa. The same procedure was performed in both teins with the same swab. For the lesion swab, the absorbent segment was pressed parallel to the edge of the lesion and rotated on its surface clockwise [20,21]. Meanwhile, the skin sample was obtained using a disposable punch (5 mm) after local anesthesia (2% lidocaine). This was performed on the infiltrated and eysthematous edge of the lesion [14].

Sample Storage
The end rod of each swab containing the sample was cut with the aid of sterile scissors and placed in a 1.5 mL microtube containing 500 µL of NET (NaCl 0.15 mM; EDTA 50 mM; Tris-HCl 0.1 M, pH 7.5). Skin samples were also preserved in NET. All tubes containing NET samples were stored at 4-8 • C.

DNA Extraction
DNA extraction from the samples was performed using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's recommendations, with a final volume of 25 µL.

Phylogenetic Sequencing and Analysis
PCR-hsp70-234 products were purified with the EXOSAP-ITTM Express PCR Product Cleanup (AppliedBioystems ® , University Park, IL, USA) and prepared for sequencing using the BigDye Terminator Kit v3.1 (Thermo Fisher Scientific ® , Waltham, MA, USA) according to the manufacturer's instructions. The forward and reverse sequences were obtained using the ABI3500XL automatic DNA analyzer. The sequences obtained were mounted in CAP3 software (Iowa State University, Ames, IA, USA), aligned using MAFFT v.7.221 (Universitu of Osaka, Osaka, Japan [22], edited in Geneious v.8.1.7 software (Biomatters Ltd., Auckland, New Zealand) [23], and compared with sequences available in GenBank using the BLAST tool. Phylogenetic analyses were performed in three stages: (i) determination of the evolutionary model and likelihood analysis using the IQ-TREE v.

Results
Among the 11 patients with TL included in the study, six received molecular diagnosis at the species level (L. (V.) braziliensis (n = 5/9), L. (V.) shawi (n = 1/9)), and three at the genus level (Leishmania sp. (n = 3/9)). For two individuals, it was not possible to amplify the deoxyribonucleic acid (DNA) of the respective samples. Furthermore, the sensitivity of PCR to amplify hsp70-234 varied according to the sample collection. The highest number of positive results was obtained from the biopsy samples (n = 7/11), followed by injury swabs (n = 6/11) and nasal swabs (n = 4/11). Species identification also occurred in a greater proportion of the biopsy samples (n = 6/7). In the group of healthy individuals (n = 6), five had hsp70-234-positive PCR results in nasal secretion swabs, where L. (V.) shawi (n = 1/5) and Leishmania sp. (n = 4/5) were identified (Table 1) The nine patients with a molecular diagnosis of TL had ulcerated lesions and mostly localized lesions in the lower limbs (n = 7/9). The size of the lesions varied between 2 cm and 32 cm, and smaller lesions (2-6 cm in diameter) were more frequent (n = 4/9). The sociodemographic and clinical data of the nine individuals, for whom molecular diagnosis was possible, are shown in Table 2.
Eight nucleotide sequences were obtained: six were identified as L. (V.) braziliensis, with four haplotypes; and two were L. (V.) shawi, corresponding to only one haplotype (Table 3).

Discussion
A higher amplification rate of the gene region hsp70-234 was observed in DNA samples from skin biopsy, which also favored the discrimination of Leishmania species using sequence analysis. Swab is a collection method considered useful for public health because it is painless and easy to perform and can be performed by trained professionals of

Discussion
A higher amplification rate of the gene region hsp70-234 was observed in DNA samples from skin biopsy, which also favored the discrimination of Leishmania species using sequence analysis. Swab is a collection method considered useful for public health because it is painless and easy to perform and can be performed by trained professionals of the health surveillance team and not necessarily physicians. Since it is not invasive, swab sampling offers more comfort and less risks, and is important for health surveillance in places with structural difficulties [21,[25][26][27][28]. Moreover, the efficacy of swab compared to biopsy can be used for the discrimination of Leishmania species, and swab could be indicated as an aid in health services, in support of the diagnosis of the disease, especially when the parasitological examination of the exudate obtained through scarification of the lesion (blade-colored smear) is negative [29].
In this study, Leishmania DNA was found in nasal secretions, including the first report of L. (V.) shawi in the nasal mucosa of a healthy woman, accompanied by a male patient infected with L. (V.) shawi. The species was identified in patients with TL at the study site [2], and in other locations in the Brazilian Amazon, in the city of Roraima [13]. In the municipality of Tomé-Açu, the sandfly species are present in extradomiciliary environments [18].
Leishmania RNA virus 1 (LRV1) was found in patients diagnosed with TL due to L. (V.) shawi infection in the Brazilian Amazon [30]. Leishmania infection by LRV1, a double-taped RNA virus of the Totiviridae family, is associated with increased mucosal injury [29]. There are strong indications that viruses infecting Leishmania spp. can increase the survival of the parasite in humans and influence the pathogenicity of the disease, as well as the drug resistance of the parasite [31,32]. The frequency of this virus is twice as high in mucosal lesions than in cutaneous lesions [32,33]. LRV1 mainly infects L. (V.) braziliensis and L. (V.) guyanensis, both of which are capable of producing mucous lesions and are resistant to the first-line of treatment for TL-Glucantime ® [2]. These two species were reported in the same area as in the present study. We previously identified cases of human CL caused by L. (V.) shawi [1].
The findings of this study provide a warning for the possible association between L. (V.) shawi and mucous lesions, since L. (V.) braziliensis can be found both in normal mucosa and in injured mucosa [5]. Experimental studies showed that infection by L. (V.) shawi without symptoms in mice can occur through the activation of antigens capable of inducing a protective immune profile, which is important for the development of vaccines against TL [34,35]. These observations allow for further studies in the fields of genomics and bioinformatics in this species of Leishmania.
The nucleotide sequences of L. (V.) shawi identified in this study (MZ399593 and MZ399589) corresponded to haplotype V and were grouped in the same clade as other sequences obtained from GenBank, suggesting that there was no genetic variation in the sample group.
The presence of the parasite in individuals without active disease can be observed in endemic areas, especially in the blood samples, nasal mucosa smears, conjunctiva, or tonsils. The presence of Leishmania in mucous tissues without signs and symptoms may also be considered a common feature of the natural history of infection by Leishmania species of the subgenus Viannia [36]. Studies on the role of asymptomatic Leishmania carriers in the transmission of leishmaniasis are limited, and whether community and population control interfere with the transmission requires further understanding [37].
L. (V.) braziliensis was the most frequent species in the individuals and was also found in the mucosal secretion of patients, but only L. (V.) shawi was detected both in a male patient (skin lesion) and in a healthy individual (nasal swab). We have previously identified a high frequency of L. (V.) braziliensis in skin biopsies of TL carriers in the city of Tomé-Açu [2]. In this same locality, we detected canine infection by L. (V.) braziliensis and reported the first case of L. (V.) infection. guyanensis in dogs [17].
In this small series of cases, the clinical profile (single ulcerated lesion) and demographics of the patients resembled that in the commonly observed areas with endemic TL [38]. Multiple injuries were identified in three patients, which may have occurred due to infection with L. (V.) braziliensis.
Normally, species from the same geographical regions tend to cluster in the same clade [39]. Therefore, the topology of the phylogenetic tree estimated for the L. (V.) braziliensis sequences grouped into distinct clades may be the result of possible genetic mutations in the locality. In the Nordeste region of Brazil, there are reports of genetic variability and polymorphisms of the species, resulting in phylogenetic differences, although they present close genetic content [40,41].

Conclusions
Here, we report the first L. (V.) shawi infection in mucosal secretion in Brazil; particularly, from a healthy woman living in the same household with one of the patients with TL participating in the study also infected with L. (V.) shawi. This finding represents a warning for the possible association between L. (V.) shawi and mucous lesions in the Amazon. We also highlighted the importance of metagenomic studies in investigating Leishmania sp. infection by LRV1 in cases of mucosal leishmaniasis, which could elucidate the pathogenic action of L. (V.) shawi and other Leishmania species.
Four haplotypes or genetic variants of L. (V.) braziliensis based on the gene sequence hsp70-234 were identified, and the same genetic variability was not detected in L. (V.) shawi. The relationship between genetic variants and the severity of TL still needs to be studied, since mutations could influence the evolution and outcome of TL treatment. Clinical studies to establish this relationship are necessary and cannot be dispensed with the molecular characterization of Leishmania.