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.

Keywords:
American cutaneous leishmaniasis; Leishmania spp; Molecular characterization; PCR-RFLP; Amazonian Brazil

INTRODUCTION

American cutaneous leishmaniasis (ACL) is a parasitic protozoan disease caused by different Leishmaniinae parasites (Kinetoplastida: Trypanosomatidae) and is widely distributed in most Latin American countries. There are at least 15 recognized species within the subgenera Leishmania (Leishmania), L. (Viannia), and L. (Mundinia) that may give rise to human diseases^1,2. Seven well-known Leishmania species have been identified as ACL agents in Brazil; Leishmania (V.) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, L. (V.) naiffi, L. (V.) lindenbergi, and L. (L.) amazonensis³. More recently, a new subspecies, L. (V.) shawi santarensis, as well as the first hybrid parasite, L. (V.) guyanensis / L. (V.) shawi shawi, have been found in the Brazilian Amazon⁴.

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)^5-8. 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.) amazonensis⁹, L. (V.) lainsoni¹⁰, and L. (V.) lindenbergi¹¹. 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 harmonized¹². In this sense, species typing has evolved into a molecular approach. An overview of the different methods and targets currently available can be found elsewhere¹³.

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^14-19. 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 species²⁰. 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.

METHODS

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 inhabitants²¹ 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) forests²².

FIGURE 1:
Study area; the Belém Metropolitan Region, located in the northeastern Pará State, Brazil. ( ) number of Leishmania isolates (1995-2018) in each municipality.

Surveying the ACL epidemiology in the BMR

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 - 1×10⁷ parasites/mL), as previously described^4,23,24. In vitro/in vivo parasite isolation (inoculating exudates from cutaneous lesions in Difco B⁴⁵ media and/or in the hind foot of Mesocricetus auratus) was also performed routinely²⁵. 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 series²³.

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. The following World Health Organization (WHO) Leishmania reference strains preserved in the Ralph Lainson Leishmaniases Lab (Instituto Evandro Chagas) cryobank that had previously been characterized were selected for this analysis: L. (L.) amazonensis (IFLA/BR/1967/PH8), L. (V.) braziliensis (MHOM/BR/1975/M2904), L. (V.) guyanensis (MHOM/BR/1975/M4147), L. (V.) naiffi (MDAS/BR/1979/M5533), L. (V.) lainsoni (MHOM/BR/1981/M6426), L. (V.) shawi (MCEB/BR/1984/M8408), and L. (V.) lindenbergi (MHOM/BR/1998/M15732)⁴. However, considering the known genetic diversity of some Leishmania species in the Brazilian Amazon, especially L. (V.) braziliensis and L. (V.) guyanensis, another strain of L. (V.) braziliensis (MHOM/BR/1975/M2903), which differs from the above reference strains, was included in this analysis as well as another strain of L. (V.) guyanensis (MHOM/BR/1997/M16342)(Table 1).

TABLE 1:
Leishmania spp. WHO reference strains (and closely related others) used for the RNA polymerase II largest subunit (RNAPOIILS) - polymerase chain reaction followed by analysis of restriction fragment length polymorphism (PCR-RFLP) and their respective digestion profiles with TspRI and HgaI endonucleases

Species	Host	WHO code	Locality	RNAPOIILS-PCR-RFLP profile
L. (V.) braziliensis	Homo sapiens	MHOM/BR/1975/M2903	Parauapebas - PA	TspRI SG3 - HgaI SG6
L. (V.) braziliensis	Homo sapiens	MHOM/BR/1975/M2904	Parauapebas - PA	TspRI SG3 - HgaI SG2*
L. (V.) guyanensis	Homo sapiens	MHOM/BR/1975/M4147	Almeirim - PA	TspRI SG2 - HgaI SG2
L. (V.) guyanensis	Homo sapiens	MHOM/BR/1997/M16342	Rio Preto da Eva - AM	TspRI SG2 - HgaI SG2
L. (V.) lainsoni	Homo sapiens	MHOM/BR/1981/M6426	Benevides - PA	TspRI SG3 - HgaI SG4
L. (V.) naiffi	Dasypus novemcintus	MDAS/BR/1979/M5533	Almeirim - PA	TspRI SG3 - HgaI SG3
L. (V.) shawi	Sapajus apella	MCEB/BR/1984/M8408	Parauapebas - PA	TspRI SG3 - HgaI SG5
L. (V.) lindenbergi	Homo sapiens	MHOM/BR/1996/M15729	Belém - PA	TspRI SG3 - HgaI SG2*
L. (L.) amazonensis	Bichromomyia flaviscutellata	IFLA/BR/1967/PH8	Belém - PA	TspRI SG1- HgaI SG1

• L.: Leishmania; V.: Viannia; WHO: World Health Organization; PA: Pará State, Brazil; AM: Amazonas State, Brazil. *These different Leishmania species present the same RNAPOIILS-PCR-RFLP profile, requiring additional methods for unambiguous characterization.

Molecular characterization through RNAPOIILS-PCR-RFLP analysis was based on the reactivity profile of the above WHO Leishmania reference strains for both TspRI and HgaI endonucleases. Therefore, TspRI digestion identified three subgroups (SGs): SG1 (no restriction site), corresponding to L. (L.) amazonensis; SG2, consisting of L. (V.) guyanensis; and SG3, consisting of L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, L. (V.) lindenbergi, and L. (V.) shawi, while HgaI digestion identified six SGs: SG1 (no restriction site), corresponding to L. (L.) amazonensis; SG2, consisting of L. (V.) guyanensis, L. (V.) braziliensis (MHOM/BR/1975/M2904), and L. (V.) lindenbergi; SG3, consisting of L. (V.) naiffi; SG4, consisting of L. (V.) lainsoni; SG5, consisting of L. (V.) shawi; and SG6, consisting of L. (V.) braziliensis (MHOM/BR/1975/M2903). All seven reference strains could be distinguished from one another using the combined analysis of TspRI and HgaI digestion profiles, with the exception of L. (V.) braziliensis (MHOM/BR/1975/M2904) and L. (V.) lindenbergi (Table 1 and Figure 2).

FIGURE 2:
RNA polymerase II largest subunit (RNAPOIILS)-polymerase chain reaction followed by analysis of restriction fragment length polymorphism (PCR-RFLP) of Leishmania spp. digestion profiles (TspRI and HgaI endonucleases). (A): TspRI digestion subgroups (SGs): WM, molecular weight marker (in base pairs) (1kb-Ludwig Biotec); SG1 (no restriction site), corresponding to L. (L.) amazonensis; SG2, consisting of L. (V.) guyanensis; and SG3, consisting of L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, L. (V.) lindenbergi, and L. (V.) shawi. (B): HgaI digestion SGs: SG1 (no restriction site), corresponding to L. (L.) amazonensis; SG2, consisting of L. (V.) guyanensis, L. (V.) braziliensis (MHOM/BR/1975/M2904), and L. (V.) lindenbergi; SG3, consisting of L. (V.) naiffi; SG4, consisting of L. (V.) lainsoni; SG5, consisting of L. (V.) braziliensis (MHOM/BR/1975/M2903); and SG6, consisting of L. (V.) shawi.

RESULTS

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.

TABLE 2:
Leishmania spp. isolates from patients with American cutaneous leishmaniasis from the Belém Metropolitan Region (1995-2018), characterized by RNA polymerase II largest subunit (RNAPOIILS)-polymerase chain reaction followed by analysis of restriction fragment length polymorphism (PCR-RFLP).

Mnemonic	Infection site	Sex	Age	Lesions	MST (mm)	WHO code	RNAPOIILS-PCR-RFLP profile	Leishmaniaspecies
SNFS	Ananindeua	M	22	2 (leg)	n.a.	MHOM/BR/1995/M15265	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
EBR	Ananindeua	F	17	1 (leg)	n.a.	MHOM/BR/1995/M15418	TspRI SG1- HgaI SG1	L. (V.) amazonensis
LSM	Ananindeua	M	21	1 (leg)	n.a.	MHOM/BR/1996/M15720	TspRI SG1- HgaI SG1	L. (V.) amazonensis
JMS	Belém	M	20	1 (arm)	17 x 17	MHOM/BR/1996/M15279	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
OPR	Benevides	M	19	2 (hand)	32 x 32	MHOM/BR/1996/M15732	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
WNSS	Belém	M	19	1 (arm)	n.a.	MHOM/BR/1996/M15740	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
AOM	Belém	M	20	1 (leg)	10 x 10	MHOM/BR/1996/M15819	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
PCB	Belém	M	43	1 (back)	n.a.	MHOM/BR/1999/M18048	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
LFB	Belém	F	38	1 (arm)	7 x 9	MHOM/BR/1999/M18054	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
PGP	Belém	M	50	2 (leg)	12 x 12	MHOM/BR/2000/M18820	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
MDSA	Belém	M	20	1 (arm)	n.a.	MHOM/BR/2000/M19418	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
WGH	Benevides	M	41	2 (leg)	8 x 8	MHOM/BR/2000/M19477	TspRI SG3 - HgaI SG6	L. (V.) braziliensis
VLS	Castanhal	M	21	1 (arm)	n.a.	MHOM/BR/2000/M19480	TspRI SG3 - HgaI SG6	L. (V.) braziliensis
PAN	Santa Barbara	M	45	1 (arm)	9 x 9	MHOM/BR/2001/M20321	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
JBS	Santa Izabel	M	32	1 (nose)	n.a.	MHOM/BR/2002/M21484	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
LSB	Ananindeua	M	16	1 (leg)	14 x 14	MHOM/BR/2003/M22090	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
FDS	Belém	M	30	2 (leg)	10 x 10	MHOM/BR/2009/M26488	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
EAO	Belém	M	44	1 (head)	10 x 10	MHOM/BR/2011/M28690	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
EMO	Belém	M	44	1 (leg)	10 x 10	MHOM/BR/2012/M29080	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
DGA	Benevides	M	20	2 (arm)	10 x 10	MHOM/BR/2012/M29084	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
ELG	Belém	M	25	1 (face)	14 x 14	MHOM/BR/2013/M29809	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
SGA	Benevides	F	62	1 (leg)	8 x 8	MHOM/BR/2013/M29986	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
OMP	Ananindeua	M	33	1 (leg)	13 x 14	MHOM/BR/2013/M30178	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
CPL	Belém	M	7	2 (face/arm)	5 x 5	MHOM/BR/2014/M30464	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
HMM	Belém	F	19	1(leg)	n.a.	MHOM/BR/2014/M30800	TspRI SG1- HgaI SG1	L. (V.) amazonensis
THS	Belém	F	79	1 (arm)	15 x 15	MHOM/BR/2014/M30921	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
WNSC	Belém	M	7	2 (face)	30 x 30	MHOM/BR/2015/M31232	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
RFF	Santa Izabel	M	89	1 (face)	n.a.	MHOM/BR/2015/M31234	TspRI SG3 - HgaI SG4	L. (V.) lainsoni
OSP	Santa Izabel	M	44	1 (arm)	5 x 5	MHOM/BR/2015/M31462	TspRI SG1- HgaI SG1	L. (V.) amazonensis
PLBS	Ananindeua	F	16	1 (leg)	9 x 9	MHOM/BR/2015/M31702	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi
FEGA	Belém	M	41	1 (arm)	12 x 12	MHOM/BR/2016/M31799	TspRI SG3 - HgaI SG6	L. (V.) braziliensis
WFS	Ananindeua	M	36	1 (leg)	10 x 10	MHOM/BR/2018/M32884	TspRI SG3 - HgaI SG2	L. (V.) lindenbergi

• WHO: World Health Organization; MST: Montenegro skin test; n.a.: not available.

Identification of Leishmania spp. isolates through RNAPOIILS-PCR-RFLP analysis

The RNAPOIILS-PCR-RFLP analysis of Leishmania spp. DNA products allowed the identification of 14 (43.7%) isolates of L. (V.) lindenbergi, 11 (34.4%) of L. (V.) lainsoni, four (12.5%) of L. (L.) amazonensis, and three (9.4%) of L. (V.) braziliensis (Table 2). The geographic distributions of the known presumptive contamination sites showed Leishmaniaspecies distributions (at the municipality level) as follows: Belém 16 (50%) [L.(V.)lindenbergi seven, L. (V.) lainsoni seven,L. (L.) amazonensisone, and L. (V.) braziliensis one], Ananindeua seven (21.8%) [L.(V.)lindenbergi three, L. (V.) lainsoni two, and L. (L.) amazonensis two], Benevides four (12.5%) [L.(V.)lindenbergi three and L. (V.) braziliensis one], Santa Izabel do Pará three (9.5%) [L.(V.)lainsoni two and L. (L.) amazonensis one], Santa Bárbara do Pará one (3.1%) [L.(V.)lindenbergi], and Castanhal one (3.1%) [L.(V.)braziliensis] (Figure 3).

FIGURE 3:
Distribution of Leishmania species in the municipalities of the Belém Metropolitan Region.

The combined analysis of the TspRI and HgaI digestion profiles of the isolates of L. (V.) lindenbergi, L. (V.) lainsoni, and L. (L.) amazonensis presented the same RNAPOIILS-PCR-RFLP profiles as their respective WHO Leishmania reference strains. However, considering that the two L. (V.) braziliensis WHO Leishmania reference strains (MHOM/BR/1975/M2904 and MHOM/BR/1975/M2903) showed two distinct patterns upon HgaI digestion, all L. (V.) lindenbergi isolates could be distinguished from L. (V.) braziliensis MHOM/BR/1975/M2903, but not from the MHOM/BR/1975/M2904 strain. Likewise, all three isolates of L. (V.) braziliensis were identified based on the MHOM/BR/1975/M2903 RNAPOIILS-PCR-RFLP profile (Table 2). To clarify this ambiguous reactivity (TspRI and HgaI) between L. (V.) lindenbergi and L. (V.) braziliensis (MHOM/BR/1975/M2904), the biological behavior of experimentally infected hamsters was checked for all 14 isolates of L. (V.) lindenbergi after the parasite was isolated, which revealed that none of these isolates could produce apparent ulcerated cutaneous lesion at the “hamster” inoculation site (hindfoot), suggesting a typical behavior of L. (V.) lindenbergi infection and not of L. (V.) braziliensis infection.

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 B⁴⁵culture medium, but has been improved with the “gold standard” MLEE and IFAT-Mabs analyses^8,26-29. 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 laboratory³¹ 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 RNAPOIILS-PCR-RFLP profiles of L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, and L. (V.) naiffi have been published previously¹⁹, although, to our knowledge, this is the first analysis of L. (V.) shawi and L. (V.) lindenbergi, thus, providing an extension of the species-specific distinction power of this methodology. The analysis of some Leishmania species with high polymorphic genetic potential can be challenging. In this sense, L. (V.) guyanensis from eastern and western Amazonia are antigenically distinct when analyzed by IFAT-Mabs³², and a degree of intra-specific heterogeneity has been observed in distinct populations from French Guiana using a small subunit and internal transcribed spacer 1 in the rRNA genes-PCR-RFLP analysis³³. Despite these observations, no intra-specific variation was observed in the RNAPOIILS-PCR-RFLP analysis.

Additionally, in the present study, L. (V.) braziliensis, a widely distributed and potentially polymorphic species^34,35, revealed different RNAPOIILS-PCR-RFLP profiles of two samples from the same geographical area in the “Serra dos Carajás” southeastern region of the Pará State (MHOM/BR/1975/2904 [Serra Norte N1] and MHOM/BR/1975/M2903 [Serra Norte H7]), with the profile of the former being indistinguishable from that of L. (V.) lindenbergi. This indicates a limitation of PCR-RFLP analysis when referring to an ecological region with a considerable genetic diversity of Leishmania parasites. This was also observed in another study in the Brazilian Amazon that failed to distinguish L. (V.) lindenbergi from L. (V.) guyanensis through hsp70 PCR-RFLP (with HaeIII digestion). In this scenario, the authors utilized 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase MLEE, the analysis of partial sequences of hsp70, isocitrate dehydrogenase, and mannose phosphate isomerase genes for final identification³⁶. However, in the present study, the biological behavior (experimental infection in “hamster”) of all 14 isolates of suspected L. (V.) lindenbergi when the parasite was isolated revealed that none of these isolates could produce conspicuous ulcerated cutaneous lesions at the inoculation site (footpad) of laboratory animals, thus, confirming the typical biological behavior of L. (V.) lindenbergi infection. Another point that contradicts this high number (14) of isolates being L. (V.) braziliensis is that in over 40 years of research at the BMR, we have never seen a case of mucocutaneous leishmaniasis originating from this region (Silveira, personal communication).

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.

The adaptation of L. (V.) lindenbergi and L. (V.) lainsoni enzootic cycles to the current ACL ecological scenario in the BMR is interesting. These species are together responsible for the majority (81.3%) of all ACL cases examined in this study. While L. (V.) lainsoni has already been found in the Brazilian Amazon States of Pará, Amapá³⁹, Rondônia⁴⁰, and Acre⁴¹ as well as in other South American countries/territories, such as Peru⁴², Bolivia⁴³, Colombia⁴⁴, Suriname⁴⁵, French Guiana¹⁹, and Ecuador⁴⁶, L. (V.) lindenbergi has, until recently, not been recorded outside its type-locality, the BMR. The first report of L. (V.) lindenbergi causing ACL occurred in Rondônia State, Brazilian western Amazon region³⁶. 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 State⁴⁷.

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.

ETHICAL STANDARDS

Procedures involving humans were submitted and approved by the Comitê de Ética em Pesquisa - CEP (Ethics in Research Committee), under protocol CAAE 95080418.0000.0019. Procedures involving access to stored material of non-human vertebrates were submitted and approved by the Comissão de Ética no Uso de Animais - CEUA (Ethics in Animal Use Commission), under protocol CEUA/IEC/SVS/MS n.42/2018.

ACKNOWLEDGMENTS

We wish to thank to Ghislaine Prévot and Marine Ginouvès (Département de Médecine, Université de Guyane, Cayenne, French Guiana) for their advice on the RNAPOIILS-PCR-RFLP training given to one of us (TVS). We are also grateful to the Ralph Lainson's Leishmaniasis Lab team, in particular to Ana Camila Oliveira Alves and Ádria de Oliveira Castro for their technical support given to the molecular analysis; to Domingas Ribeiro Everdosa and João Batista Palheta da Luz for their technical support with the laboratory diagnosis of the patients, and to Raimundo Nonato Barbosa Pires for his technical support and curatorial care with the Leishmania cryobank.

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Publication Dates

• Publication in this collection
  11 Dec 2020
• Date of issue
  2020

History

• Received
  22 May 2020
• Accepted
  19 Oct 2020