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Leishmania (Viannia) spp. can harbor a double-stranded RNA virus known as Leishmania RNA virus 1 (LRV-1), whose presence has been reported in nine countries across the Americas and seven Leishmania species. Here, we studied 100 Leishmania (Viannia) isolates from patients with cutaneous leishmaniasis collected from different endemic areas in Panama from 2016 to 2022. We identified L. (V.) panamensis, L. (V.) guyanensis, L. (V.) braziliensis/guyanensis hybrid, and L. (V.) panamensis sp.1. (genetic variant). LRV-1 was detected by RT-PCR in 9% of L. (Viannia) isolates (eight cases in L. (V.) panamensis, and one in L. (V.) guyanensis). Phylogenetic analysis based on sequencing data classified all LRV-1 isolates within genotype A, suggesting that LRV phylogenetic proximity is closely aligned with geographical distribution or to the phylogenetic proximity of the Leishmania host in the case of the L. (V.) panamensis and L. (V.) guyanensis in Panama.

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We detected Leishmania RNA virus 1 (LRV1) in 11 isolates of Leishmania (Viannia) panamensis collected during 2014-2019 from patients from different geographic areas in Panama. The distribution suggested a spread of LRV1 in L. (V.) panamensis parasites. We found no association between LRV1 and an increase in clinical pathology.

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According to the last leishmaniasis report from the Pan American Health Organization (2021) so far Panama is considered free of visceral leishmaniasis (VL). Although the presence of potential vectors and reservoirs involved in the VL transmission cycle have been described in some rural regions of the country, no cases have been reported in humans and domestic or wild animals. Dogs play an important role in the urban transmission of VL; therefore, it is important to detect possible cases of canine visceral leishmaniasis (CVL) in the country. In this sense,this study reports for the first time the Leishmania (Leishmania) infantum infection in imported dogs in Panama. Eleven dogs with clinical suspicion of CVL were evaluated by parasitological (bone marrow aspirate smear), serological (indirect immunofluorescence and/or reference immunochromatographic rapid test) and molecular tests (conventional PCR). The dogs included in this study were analyzed during the period from 2013 to 2020. All dogs presented clinical manifestations compatible with CVL. The samples were initially evaluated by smears and/or rapid serological tests by private practice veterinarians, and later confirmed by serological and/or molecular tests at the national reference laboratory for Leishmania diagnosis. The diagnosis was confirmed in 5/11 dogs by serological, parasitological and/or conventionals PCR targeting kDNA minicircle and Hsp70 gene. Leishmania (L.) infantum species was identified in 3/5 dogs by PCR-RFLP and by sequencing Hsp70-PCR products. This study evidenced the need to increase awareness of private practitioners as well as public health veterinarians on visceral leishmaniasis (VL), and to consider this parasitosis in the differential diagnosis of dogs with clinical and epidemiological characteristics compatible with the disease.

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Didelphis marsupialis has been reported as a competent reservoir for trypanosomatid parasites infections. The aim of this study was to measure Trypanosoma cruzi, T. rangeli, and Leishmania spp. infection rates and to characterize discrete typing units (DTUs) of T. cruzi in D. marsupialis from two Chagas disease endemic sites in Panama. Blood from 57 wild-caught D. marsupialis were examined from two rural communities, Las Pavas (N = 18) and Trinidad de las Minas (N = 39). Twenty-two (38.60%) opossums were positive for flagellates by general hemoculture. T. cruzi infection was confirmed by positive hemoculture and/or kDNA based PCR performed in 31/57 (54.39%) blood samples from opossums. T. rangeli infection was confirmed by hemoculture and/or TrF/R2-Primer PCR assay applied on 12/57 (21.05%) blood samples. Nine (15.79%) D. marsupialis harbored T. cruzi/T. rangeli coinfections. All opossums tested negative for Leishmania spp. by PCR assays based on kDNA and HSP70 gene amplification. There was a significant association between T. cruzi infection and site (Fisher exact test, p = 0.02), with a higher proportion of T. cruzi infected opossums in Las Pavas (77.78%, n = 14/18) compared to Trinidad de las Minas (43.59%, n = 17/39). A significant association was found between habitat type and T. cruzi infection in opossums across both communities, (X2 = 6.91, p = 0.01, df = 1), with a higher proportion of T. cruzi infection in opossums captured in forest remnants (76%, 19/25) compared to peridomestic areas (37.5%, 12/32). T. rangeli detection, but not T. cruzi detection, may be improved by culture followed by PCR. TcI was the only DTU detected in 22 T. cruzi samples using conventional and real-time PCR. Eight T. rangeli positive samples were characterized as KP1(-)/lineage C. Trypanosome infection data from this common synanthropic mammal provides important information for improved surveillance and management of Chagas disease in endemic regions of Panama.

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Introducción: La leishmaniasis cutánea (LC) es una enfermedad zoonótica endémica en Panamá. Su agente causal son protozoarios del género Leishmania y la transmiten insectos flebotominos. Objetivo: Evaluar los factores de riesgos asociados con la LC y la diversidad de flebotominos en dos comunidades rurales de Panamá Oeste. Metodología: Se seleccionaron dos comunidades endémicas para LC: Trinidad de las Minas (TM), de alta incidencia y Las Pavas (LP), de baja incidencia. Los factores de riesgo asociados con la LC fueron evaluados mediante una encuesta aplicada a100 personas (TM: n=50; LP: n=50). Se colectaron flebotominos con trampas CDC durante tres noches consecutivas en temporada lluviosa y seca. Resultados: La mayoría de las personas confirmó conocer sobre la LC (TM: 96% y LP: 68%). No se encontraron diferencias significativas entre las características sociodemográficas, estructura de las viviendas, composición del peridomicilio y abundancia/diversidad de animales domésticos en ambas comunidades. El reporte de perezosos cercanos al peridomicilio fue mayor en TM (70%) vs LP (32%). La especie de flebotomino antropofílica más abundante durante la temporada seca fue Lutzomyia gomezi (TM: 40.1% y LP: 10.4%). Durante la temporada lluviosa fue Nyssomyia trapidoi (43.4%) en TM y Psychodopygus panamensis (75.7%) en LP. Las especies zoofílicas más comunes en ambas comunidades fueron Trichopygomyia triramula y Pressatia dysponeta. Conclusión: La mayor incidencia de LC en TM podría estar condicionada a su ecología montañosa, con una cobertura boscosa cercana más extensa y una mayor frecuencia de mamíferos reservorios silvestres. Se confirmó la presencia de vectores de LC en el peridomicilio de ambas comunidades. (provisto por Infomedic International)

Introduction: Cutaneous leishmaniasis (CL) is a zoonotic disease endemic in Panama. Its causal agent are protozoa of the genus Leishmania and is transmitted by phlebotomine sandflies. Objective: To evaluate the risk factors associated with CL and the diversity of phlebotomine sandflies in two rural communities in western Panama. Methodology: Two CL endemic communities were selected: Trinidad de las Minas (TM), with high incidence and Las Pavas (LP), with low incidence. The risk factors associated with CL were assessed by means of a survey applied to 100 people (TM: n=50; LP: n=50). Phlebotomine sandflies were collected with CDC traps during three consecutive nights in rainy and dry season. Results: The majority of people confirmed knowledge about CL (TM: 96% and LP: 68%). No significant differences were found between sociodemographic characteristics, housing structure, peridomicile composition and abundance/diversity of domestic animals in both communities. The report of sloths near the peridomicile was higher in TM (70%) vs LP (32%). The most abundant anthropophilic phlebotomine species during the dry season was Lutzomyia gomezi (TM: 40.1% and LP: 10.4%). During the rainy season it was Nyssomyia trapidoi (43.4%) in TM and Psychodopygus panamensis (75.7%) in LP. The most common zoophilic species in both communities were Trichopygomyia triramula and Pressatia dysponeta. Conclusion: The higher incidence of CL in TM could be conditioned to its mountainous ecology, with a more extensive nearby forest cover and a higher frequency of wild mammal reservoirs. The presence of CL vectors in the peridomicile of both communities was confirmed. (provided by Infomedic International)

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Macrophages play important roles in the innate and acquired immune responses against Leishmania parasites. Depending on the subset and activation status, macrophages may eliminate intracellular parasites; however, these host cells also can offer a safe environment for Leishmania replication. In this sense, the fate of the parasite may be influenced by the phenotype of the infected macrophage, linked to the subtype of classically activated (M1) or alternatively activated (M2) macrophages. In the present study, M1 and M2 macrophage subsets were analyzed by double-staining immunohistochemistry in skin biopsies from patients with American cutaneous leishmaniasis (ACL) caused by L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis ,and L. (L.) infantum chagasi. High number of M1 macrophages was detected in nonulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi (M1 = 112 ± 12, M2 = 43 ± 12 cells/mm2). On the other side, high density of M2 macrophages was observed in the skin lesions of patients with anergic diffuse cutaneous leishmaniasis (ADCL) (M1 = 195 ± 25, M2 = 616 ± 114), followed by cases of localized cutaneous leishmaniasis (LCL) caused by L. (L.) amazonensis (M1 = 97 ± 24, M2 = 219 ± 29), L. (V.) panamensis (M1 = 71 ± 14, M2 = 164 ± 14), and L. (V.) braziliensis (M1 = 50 ± 13, M2 = 53 ± 10); however, low density of M2 macrophages was observed in NUCL. The data presented herein show the polarization of macrophages in skin lesions caused by different Leishmania species that may be related with the outcome of the disease.

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Isolates from 475 cutaneous leishmaniasis (CL) patients from three endemic regions were studied by three typing techniques. The molecular analysis from lesion scrapings based on hsp70 PCR-restriction fragment length polymorphism (RFLP) showed that 78.1% (371/475) restriction patterns corresponded to Leishmania (Viannia) panamensis, 19% (90/475) to Leishmania (Viannia) guyanensis, and 3.0% (14/475) to Leishmania (Viannia) braziliensis. Promastigotes isolated by culture from lesions of 228 patients (48.0%, 228/475) were identified by multi-locus enzyme electrophoresis. Of them, 95.2% (217/228) were typified as L. (V.) panamensis, 1.3% (3/228) as L. (V.) guyanensis, 2.2% (5/228) as L. (V.) braziliensis, and 1.3% (3/228) as hybrids (L. [V.] braziliensis/L. [V.] panamensis). However, a partial sequencing analysis of the hsp70 gene from 77 selected samples showed 16.9% (13/77) typified as L. (V.) panamensis, 68.8% (53/77) as Leishmania (V.) sp., 1, 3.9% (3/77) as L. (V.) guyanensis, 1.3% (1/77) as L. (V.) braziliensis outlier, 2.6% (2/77) as Leishmania (Viannia) naiffi, 2.6% as (2/77) Leishmania (V.) sp., and 2 and 3.9% (3/77) hybrid isolates of L. (V.) braziliensis/L. (V.) guyanensis. These results confirm L. (V.) panamensis as the predominant species and cause of CL lesions in Panama and that L. (V.) guyanensis, L. (V.) braziliensis, and L. (V.) naiffi are circulating to a lower degree. Furthermore, the determination of parasite isolates belonging to atypical clusters and hybrid isolates suggests the circulation of genetic variants with important implications for the epidemiology and clinical follow-up of CL in Panama. No evidence of the existence of parasites of the Leishmania (Leishmania) mexicana complex in Panamanian territory was found in this study.

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AIMS: Leishmaniasis is considered a disease with multiple clinical/immunopathological characteristics, depending on the immunity of the host and the species of the parasite. In Panama, the most prevalent species that causes localized cutaneous leishmaniasis (LCL) is Leishmania (Viannia) panamensis, and its immune response is poorly studied. Therefore, we evaluated by immunohistochemistry, the in situ immune response during this infection. METHODS AND RESULTS: Biopsies from Panamanian patients with LCL were collected and processed by histological techniques. Infection by L. (V.) panamensis was demonstrated by isolation in culture and molecular characterization by Hsp70-RFLP. The in situ immune response was assessed by immunohistochemistry. The immune response was characterized by predominance of T cells, mainly CD8 cells that showed positive correlation with IFN-γ and Granzyme B. CD4 cells presented positive correlation with both IFN-γ and IL-13, pointed by mixed cellular immune response. Regulatory response was characterized by FoxP3 cells, which showed positive correlation to IL-10 but not with TGF-ß. CONCLUSIONS: L. (V.) panamensis infection triggers a mixed cellular immune response, characterized by the presence of pro-inflammatory, anti-inflammatory and regulatory elements in the skin lesion of Panamanian patients. These data contribute to a better understanding of the immunopathogenesis of Leishmania Viannia infection in Panama.

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[This corrects the article DOI: 10.1371/journal.pone.0169047.].

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Cutaneous leishmaniasis (CL) is an endemic disease in the Republic of Panama, caused by Leishmania (Viannia) parasites, whose most common clinical manifestation is the presence of ulcerated lesions on the skin. These lesions usually present a chronic inflammatory reaction, sometimes granulomatous, with the presence of lymphocytes, plasma cells and macrophages. This study describes the histopathological characteristics found in the skin lesions of patients with CL caused by Leishmania (V.) panamensis in Panama. We analyzed 49 skin biopsy samples from patients with clinical suspicion of CL, by molecular tests (PCR for subgenus Viannia and HSP-70) and by Hematoxylin-Eosin staining. Samples were characterized at the species level by PCR-HSP-70/RFLP. From the 49 samples studied, 46 (94%) were positive by PCR and were characterized as Leishmania (V.) panamensis. Of these, 48% were positive by Hematoxylin-Eosin staining with alterations being observed both, in the epidermis (85%) and in the dermis (100%) of skin biopsies. The inflammatory infiltrate was characterized according to histopathological patterns: lymphohistiocytic (50%), lymphoplasmacytic (61%) and granulomatous (46%) infiltration, being the combination of these patterns frequently found. The predominant histopathological characteristics observed in CL lesions caused by L. (V.) panamensis in Panama were: an intense inflammatory reaction in the dermis with a combination of lymphohistiocytic, lymphoplasmacytic and granulomatous presentation patterns and the presence of ulcers, acanthosis, exocytosis and spongiosis in the epidermis.

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