Occurrence of Leishmania infection in the immediate geographic region of Ji-Paraná, Rondônia State, Brazil

Abstract INTRODUCTION: This study evaluated the epidemiology of American cutaneous leishmaniasis in the immediate region of Ji-Paraná, Rondônia State. METHODS: Samples and epidemiological data were collected from 105 patients. RESULTS: Leishmania infection was observed in 58 (55.2%) patients, and Leishmania braziliensis was present in 82.9% of the 41 sequenced samples. Infected patients were predominantly male (93.1%). Leishmania infection was twice as prevalent among rural inhabitants versus urban inhabitants. Lesions were more frequent in the upper limbs (arms/hands, 41.82%). CONCLUSIONS: The present data corroborate the zoonotic profile of cutaneous leishmaniasis; this information could help to improve surveillance and control strategies.

Leishmaniasis, a disease caused by Leishmania, is widely distributed worldwide. Brazil is one of the six countries with the highest rates of both American cutaneous leishmaniasis (ACL) and visceral leishmaniasis (VL), with approximately 20,000 and 3,400 reported cases per year, respectively 1 .
The North Region of Brazil has experienced a particularly high number of leishmaniasis cases; a total of 6,681 ACL cases and 496 VL cases were reported in 2019 alone 1 . ACL is highly endemic in Rondônia State, and the primary source of human infection is the bite from infected female sand flies in forest environments. Rondônia records an average of 1,000 cases per year, with an incidence rate of 45 cases per 100,000 inhabitants. Seven Leishmania species have been recorded as agents of infection, with Leishmania braziliensis (Vianna) being the most prevalent 2 .
Rondônia is geographically divided into six immediate regions: Porto Velho, Ariquemes, Jarú, Ji-Paraná, Cacoal, and Vilhena ( Figure 1B). In the immediate region of Ji-Paraná, where this work was conducted, 139 cases of ACL were registered in 2019 alone 1 . Despite the high incidence of ACL in Rondônia, studies on leishmaniasis in patients have been limited to the municipalities of Porto Velho and Monte Negro 2,3 . Thus, the present study aimed to identify the Leishmania species responsible for ACL cases in the immediate region of Ji-Paraná and to use the data collected from diagnosed patients to determine the epidemiological profile of ACL in this region.
The immediate region of Ji-Paraná is composed of 13 municipalities located in the central region of Rondônia ( Figure 1C). Human samples (skin lesions) were collected between December 2016 and November 2018 in Ji-Paraná, at Padre Adolfo Lutz Tropical Disease Research Institute. This institute is the reference center responsible for assisting and diagnosing suspected ACL cases. The institute performs direct examination using a sterile scalpel to collect samples from the edge of ulcerated lesions (scarification). The collected sample material was smeared on slides, stained with Giemsa, and observed at 100X magnification using optical microscopy.
Patients with skin lesions were invited to participate in the study, and those who agreed to receive and signed the Free and Informed Consent Form (ICF). Subsequently, a trained technician from the institute collected biological samples from the participating patients. Samples were stored in 50 µL of phosphate-buffered saline (PBS), identified, and frozen at -20 °C for subsequent DNA extraction. Patients also answered an epidemiological questionnaire that identified their sex; age; living area (rural or urban); their involvement in hunting, fishing, or eco-tourism; and the proximity of their home to forests. Moreover, a clinical examination was performed on all patients to collect data on the presence, number, and location of lesions.
The obtained sequences were visualized and edited using Phred, Phrap, and Consed software and then submitted to BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with sequences from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Epidemiological data were analyzed using descriptive statistics. For categorical variables, we computed the data as proportion and percentage of patients in each group. For numerical variables, we computed the data as median and interquartile range (IQR). The odds ratio (OR) of infection for Leishmania was obtained for its association with sex; age; living area (rural or urban); their involvement in hunting, fishing, or eco-tourism; and the proximity of their home to forests. We computed the OR using conditional maximum likelihood estimation. All analyses were performed using the free program language Python 3.8. Data and codes have been provided as supplementary files (Supplementary TABLE). 2/41). Leishmania braziliensis was the most prevalent species and was found in patients from every municipality. Leishmania guyanensis was detected in patients from the municipalities of Ji-Paraná and Ouro Preto do Oeste, and L. naiffi was detected solely in patients from the municipality of Ouro Preto do Oeste ( Figure 1D).
According to our data, Leishmania prevalence was higher in men: 93.10% (54/58) of the positive samples belonged to men; thus, the OR of being positive for Leishmania was highest in this group. Individuals from rural areas were more likely (OR: 2.27) to be positive for Leishmania than those who lived in urban areas (Table 1). Furthermore, our data indicate that individuals whose primary occupation is ecotourism were approximately twice as likely to be positive for Leishmania relative to individuals who were in other occupations ( Table 1).
This study delineates the epidemiological framework of CL in the immediate region of Ji-Paraná. The study highlighted a pattern of L. braziliensis infection in seven municipalities in this region. Indeed, this is the most prevalent species in Brazilian cases; human cases have been reported in every state of the Brazilian Amazon, and the prevalence of this Leishmania species is highest in states such as Acre, Pará, and Roraima 6,7 . In Rondônia, L. braziliensis has already been registered in the immediate regions of Porto Velho and Ariquemes 2,3 , as well as in the Ji-Paraná region. Leishmania braziliensis is a common species widely distributed in Brazil, and this success could be related to a variety of sand fly species that have vector competence for this protozoan 8 . In Rondônia, there is an abundance of Psychodopygus davisi (Root) and Psychodopygus hirsutus hirsutus (Mangabeira), which have been detected with promastigote forms and Leishmania DNA 9,10 , and may contribute to the transmission cycle of the parasite in the region.
We observed two cases of L. guyanensis in two municipalities; its prevalence in Rondônia is low relative to that in L. braziliensis 2,3 . Leishmania guyanensis is widely distributed in the Amazon Basin, and its distribution is certainly related to the presence and abundance of Nyssomyia umbratilis (Ward & Fraiha) populations 11,12,13 . In Amazonas, L. guyanensis is the primary agent of ACL, and N. umbratilis, its main vector, is present in high abundance 13 . Studies indicate that N. umbratilis is present in low abundance in Rondônia 9,10 .
The presence of L. naiffi DNA in two samples from Ouro Preto do Oeste demonstrates that this species is in circulation in the region and that human hosts play a role in its transmission cycle. Information about the presence of this species in Rondônia is sparse; this species has been reported in sand fly species 4 and in only one human. This may be due to the fact that L. naiffi occurs at a low frequency and has low pathogenicity in Rondônia 14 . Consequently, people may not seek health assistance, which would cause the distribution of Leishmania species such as L. naiffi, L. guyanensis, and L. braziliensis to be underestimated, which could have affected the results. In the immediate region of Ji-Paraná, data indicated that the probability of Leishmania infection was double among rural inhabitants versus urban inhabitants, that infections were most prevalent among people aged 30-50 years old, and that infections were more common in men than in women. This profile could be explained by the fact that people aged 30-50 years are the most economically active, and that rural residents generally live and work near forests or forest fragments, which greatly increases their exposure to sand fly bites. This pattern has been observed in Rondônia and other states in the Brazilian Amazon 9,10,11,12,13,14 .
The highest proportion of infections occurred as single lesions, and the parts of the body most affected were the arms, legs, head, and feet, which corroborate the findings of other studies 12,13,14 . These parts of the body are the most affected because they remain exposed to vectors for many hours of the day when individuals work in forest environments (primarily in the occupations of logging and hunting) 7,11,12 .
A pattern of infection was also observed in people who practiced ecotourism but did not live in rural areas. In Rondônia, the presence of cases in urban areas could be attributable to cultural practices such as farm visits, camping, and fishing during holidays. People who practice these leisure activities generally remain in close proximity to forest environments for many hours; therefore, their risk may have increased exposure to sand fly bites and possible infection by Leishmania species 13 .
In summary, the obtained data indicate a zoonotic profile of the ACL in the immediate region of Ji-Paraná, in which people present lesions primarily on the exposed parts of the body. Our data also demonstrate that the likelihood of ACL infection may be related to sex, place of residence, and occupation because, for example, living in a rural area increases the OR of being positive for Leishmania. The prevalence of Leishmania species may be related to sand fly vectors and reservoir distributions, but studies conducted in the future will need to evaluate these relationships. All information presented herein could help to improve surveillance and control strategies aimed at reducing the number of CL cases in the municipalities of the immediate region of Ji-Paraná and in other regions of Rondônia State.