Further insights into the eco-epidemiology of American cutaneous leishmaniasis in the Belem metropolitan region, Pará State, Brazil

Abstract INTRODUCTION: In the Belém Metropolitan Region (BMR), Pará State, Brazil, American cutaneous leishmaniasis (ACL) is endemic; however, very little is known regarding its causative agents. Therefore, we used our standard diagnostic approach combined with an RNA polymerase II largest subunit (RNAPOIILS)-polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphism (PCR-RFLP) to identify Leishmania spp. ACL agents in this region. METHODS: Thirty-two Leishmania spp. isolates from patients with ACL in the BMR during 1995-2018 were analyzed. Leishmania spp. DNA samples were amplified using the primers RPOR2/RPOF2, and the 615-bp PCR products were subjected to enzymatic digestion using TspRI and HgaI endonucleases. RESULTS: ACL etiological agents in the BMR comprised Leishmania (Viannia) lindenbergi (43.7%) followed by Leishmania (Viannia) lainsoni (34.4%), Leishmania (Leishmania) amazonensis (12.5%), and Leishmania (Viannia) braziliensis (9.4%). CONCLUSIONS: To our knowledge, the results of the study revealed for the first time that L. (V.) lindenbergi and L. (V.) lainsoni are the main ACL agents in BMR.

ACL behaves as a primary zoonosis of wild mammals in Brazil, and the transmission of Leishmania species occurs through the bites of infected females of different phlebotomine vectors (Diptera: Psychodidae) [5][6][7][8] . ACL has an occasional but endemic character in the Belém Metropolitan Region (BMR), Pará State, in the Brazilian Amazon that is mainly associated with three Leishmania species, including L. (L.) amazonensis 9 , L. (V.) lainsoni 10 , and L. (V.) lindenbergi 11 . Over the years, the BMR has experienced an increase in growth rate with intense urban construction and displacement of populations to areas neighboring secondary native forest areas, favoring human contact with the enzootic cycles of these Leishmania species.
The identification of potential ACL agents is a key step in surveillance strategies, and the existing knowledge and molecular tools available for the identification and characterization of Leishmania species must be improved and harmonized 12 . In this sense, species typing has evolved into a molecular approach. An overview of the different methods and targets currently available can be found elsewhere 13 .
Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) has been widely applied to characterize New World Leishmania species, and has been focused on different targets of kinetoplast or genomic DNA [14][15][16][17][18][19] . Due to the high inter/intraspecific diversity/polymorphism in the parasites, these techniques do not usually show continental reproducibility, and regional-scale assays must be improved to validate established protocols. To this end, a set of targets that encode the genes of the RNA polymerase II largest subunit (RNAPOIILS; considered phylogenetically informative as defined by parsimony criteria) has been used to explore the relationships between Leishmania species 20 . We used our standard diagnostic approach combined with the RNAPOIILS-PCR-RFLP assay (previously designed to identify Amazonian/Guianan Leishmania species) 19 to identify Leishmania spp. that act as potential ACL agents in the BMR. These results provide crucial new insights into the eco-epidemiology of ACL in this region.

Study area
The BMR comprises a cluster of socioeconomic integrated municipalities located in the northeastern Pará State, Brazil (Belém [the State capital], Ananindeua, Marituba, Benevides, Santa Izabel do Pará, Santa Bárbara do Pará, and Castanhal), with a resident population of approximately 2,505,242 inhabitants 21 and a territorial area of 6.890 km² (Figure 1). The landscape has an extensive alluvial plain, with a typically equatorial climate and average temperatures ranging from 24°C to 26°C and humidity above 80%. The annual precipitation is approximately 2500 mm, with a rainy season from January to June. The vegetation is mainly secondary forest, although some original remnants still cover ~31% of the region, which is composed of upland (terra firme), floodplain (várzea), and wetland (igapó) forests 22 .

Patients with ACL examined at the Ralph Lainson Leishmaniases
Lab (with the BMR as the geographical local of presumed infection from 1995 to 2018) were also screened following our standard diagnostic approach comprising clinical-epidemiological investigation and laboratory diagnosis. The patients were diagnosed by parasitological demonstrations (Giemsa-stained smears of exudates from cutaneous lesions) and by the interpretation of the Montenegro skin test (inactivated promastigotes of L. (V.) braziliensis -MHOM/BR/M17323 -1×10 7 parasites/mL), as previously described 4,23,24 . In vitro/in vivo parasite isolation (inoculating exudates from cutaneous lesions in Difco B 45 media and/or in the hind foot of Mesocricetus auratus) was also performed routinely 25 . The ACL-confirmed cases received systemic therapy (meglumine antimoniate) at a dose of 12 mg Sb5/kg/day in two series of 22 days, with an interval of 7-10 days between each series 23 .
All Leishmania spp. isolates obtained from ACL cases of localized cutaneous leishmaniasis (LCL) clinical form originating from the BMR from 1995 to 2018 were included in the analysis. Of the 32 cultured samples, 16 were from the municipality of Belém, seven from Ananindeua, four from Benevides, three from Santa Izabel, one from Santa Bárbara do Pará, and one from Castanhal. No isolate was registered in the municipality of Marituba (Figure 1).

DNA extraction/quantification
DNA was extracted from positive culture samples using the commercial Reliaprep gDNA Tissue Miniprep System Kit (Promega, USA). After performing cell lysis using proteinase K, the samples were placed in a column surrounded by a silica membrane, and during centrifugation, the DNA adhered to the membrane. After several washes, the extracted DNA was eluted in 150 μL of elution buffer. DNA sample quantification was performed using a Qubit 2.0 fluorometer (Invitrogen, USA).

RNAPOIILS-PCR-RFLP
The methodology was adapted from Simon et al. 19 with minor modifications. In brief, Leishmania DNA amplification was performed using the primers RPOF2 (5′-AGAACATGGGCGGCC-3′) and RPOR2 (5′-CGAGGGTCACGTTCTTG-3′), which amplified a 615-bp fragment of the RNAPOIILS gene. The reaction was performed in a final volume of 50 μL containing 0.2 μM of each  DNTP (dATP, dCTP, dGTP, and dTTP) (Quatro G), 0.01 μM of each primer (Invitrogen), 2.5 U of Taq DNA polymerase (Invitrogen), and 10 μL of extracted DNA (1 ng/μL). The reactions were performed in an Eppendorf (Mastercycler® personal) thermal cycler programmed for an initial denaturation temperature of 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The final extension step was maintained for 5 min at 72°C. The PCR products were applied to a 1% agarose gel and stained with Safe Dye (Kasvi) to confirm proper amplification.
A total of 15 μL of the PCR product was digested with 10 U of TspRI (New England Biolabs) (2 h at 65°C) or with 2 U of HgaI (New England Biolabs) endonucleases (2 h at 37°C), following the manufacturer's recommendations. Both digestions (HgaI and TspRI) were performed separately for each 15 μL of PCR product in a final volume of 20 μL. The digestion mixtures were individually applied to 3% agarose gels and stained with Safe dye.

Molecular characterization of Leishmania spp. isolates
The molecular characterization of Leishmania spp. isolates from patients with ACL in the BMR was based on the RNAPOIILS-PCR-RFLP analysis in accordance with previously published studies 19,30

Clinical and epidemiological features of ACL in the BMR
Thirty-two Leishmania spp. isolates were obtained within the historical series analyzed from cutaneous lesions of the LCL clinical form of patients with ACL. Most lesions (75%; 24/32) were single (ranging from to 1-2) and localized to the arm and/or leg (81%; 26/32), with reactive Montenegro skin tests ranging at 5-32 mm. The ACL sample comprised patients with a mean age of 32.5 ±18.9 years, predominantly male (81%; 26/32), with histories of activities in forested areas ( Table 2). All ACL-confirmed cases showed satisfactory treatment responses with no history of relapse for one year post-treatment.

DISCUSSION
DNA-based methods have been extensively used since the 1980s for the characterization of Leishmania spp. However, these methods are currently restricted to referral hospitals and research centers with well-equipped laboratories. Currently available techniques include direct sequencing of a PCR product, use of species-specific restriction sites via PCR-RFLP, PCR fingerprinting, random amplified polymorphic DNA, or high-resolution melting. Of these, only PCR-RFLP and sequence analysis coupled with the appropriate target in the genome are suitable for the discrimination of all Leishmania species. In many cases, a combination of different markers must be applied to achieve a definitive taxonomic resolution 13,14 .
The characterization of Leishmania spp. has traditionally been performed in the Ralph Lainson Leishmaniases Lab (since the 1970s) using a combined methodology that takes into consideration the parasite's behavior within experimentally infected invertebrate (phebotomines) and vertebrate (hamsters) hosts in the Dfico B 45 culture medium, but has been improved with the "gold standard" MLEE and IFAT-Mabs analyses 8,[26][27][28][29] . We extended the applicability of a simple and direct molecular tool that was originally proposed (and recently revised) for French Guiana 19,30 , and used it to identify (to date) the coexisting human-pathogenic dermotropic Leishmania species in the BMR. This methodology has already been used to identify Leishmania isolates from patients with ACL in our immunopathology research laboratory 31 as well as from phlebotomines 24,29 . The sensitivity of RNAPOIILS-PCR-RFLP was 100%, as expected for isolates. High specificity was also presumed, since the RNAPOIILS-PCR-RFLP for Leishmania profiles is distinct from that for other microorganisms. For non-isolated samples, future steps will include the analysis of Giemsa-stained lesion imprint slides, which have a presumed sensitivity of approximately 90% 19 , to improve sensitivity for Leishmania DNA detection. These samples can be preliminarily screened with markers targeting kDNA.  The present study represents the first systematic study to examine, mainly through molecular methods, the repertoire of Leishmania species occurring in this region, and to our knowledge, revealing for the first time that L. (V.) lindenbergi (43.7%) and L. (V.) lainsoni (34.4%) are the main ACL agents in the BMR, followed by L. (L.) amazonensis (12.5%) and L. (V.) braziliensis (9.4%). From an eco-epidemiological point of view, it is interesting to note that these Leishmania spp. enzootics in the BMR remain established in residual forest fragments with ecological conditions, such as the presence of phlebotomine potential vectors 37,38 , which favor Leishmania life cycles. Clinical and socioeconomic data show ACL in the BMR as a predominantly accidental disease with an occupational/eco-touristic character, since it has mainly been associated with middle-aged men exposed to peri-urban forest environments. Occasional ACL cases of patients with no history of forest exposure (such as elderly housewives) have drawn attention to its potential for intra/peridomiciliary transmission.  36 . Underreporting of ACL due to this parasite is possible, since some methodologies currently employed for Leishmania identification may not distinguish this species from others. Therefore, increasing efforts to develop novel techniques for species identification in other Amazonian regions may expand our knowledge on the geographical range of L. (V.) lindenbergi.
In this study, we record the first three cases of ACL due to L. (V.) braziliensis in the BMR, with strains from the municipalities of Belém, Benevides, and Castanhal being compatible with the MHOM/BR/1975/M2903 RNAPOIILS-PCR-RFLP profile, thus, distinct from that of L. (V.) lindenbergi. The ecological scenario of L. (V.) braziliensis is not yet well understood in this region, as the well-known vectors Psychodopygus wellcomei/Psychodopygus complexus have not yet been recorded in surveyed forest fragments in the BMR 11,37,38 . Alternative transmission (most likely by other 'psychodopygians'), thus, cannot be ruled out. Psychodopygus davisi, for instance, is a very active human-biting phlebotomine species present in the BMR that was found to be infected with L. (V.) braziliensis in the southern Pará State 47 .
In summary, the results of this study provide a better understanding of the ACL epidemiological scenario in the BMR. Strong ecological transformations have occurred in this region over the years, although these changes do not appear to limit the enzootic cycles of the Leishmania species already identified here.