Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.

BACKGROUND: Plasmodium vivax is the main causative agent of malaria in Panama. However, the prevalence of asymptomatic infections in the different endemic regions remains unknown. Understanding the epidemiological behavior of asymptomatic infections is essential for the elimination of malaria. This study aimed to determine the prevalence of asymptomatic malarial infections in one of the main endemic regions of Panama using multiplex real-time reverse transcription RT-MqPCR. METHODS: A cross-sectional study was conducted in three communities in the Guna Yala Comarca. A total of 551 thick blood smears and their respective samples on filter paper were collected from volunteers of different ages and sexes from June 20 to 25, 2016. Infections by the Plasmodium spp. were diagnosed using microscopy and RT-MqPCR. All statistical analyses were performed using the R software. RESULTS: The average prevalence of asymptomatic infections by P. vivax in the three communities detected by RT-MqPCR was 9.3%, with Ukupa having the highest prevalence (13.4%), followed by Aidirgandi (11.1%) and Irgandi (3.3%). A total of 74 samples were diagnosed as asymptomatic infections using RT-MqPCR. Light microscopy (LM) detected that 17.6% (13/74) of the asymptomatic samples and 82.4% (61/74) were diagnosed as false negatives. A 100% correlation was observed between samples diagnosed using LM and RT-MqPCR. A total of 52.7% (39/74) of the asymptomatic patients were female and 85.1% (63/74) were registered between the ages of 1 and 21 years. Factors associated with asymptomatic infection were community (aOR = 0.38 (95% CI 0.17-0.83), p < 0.001) and age aOR = 0.98 (95% CI 0.97-1.00), p < 0.05); F = 5.38; p < 0.05). CONCLUSIONS: This study provides novel evidence of the considerable prevalence of asymptomatic P. vivax infections in the endemic region of Kuna Yala, representing a new challenge that requires immediate attention from the National Malaria Program. The results of this study provide essential information for the health authorities responsible for developing new policies. Furthermore, it will allow program administrators to reorient and design effective malaria control strategies that consider asymptomatic infections as a fundamental part of malaria control and move towards fulfilling their commitment to eliminate it.

Epidemiology and Genetic Characterization of Leishmania RNA Virus in Leishmania (Viannia) spp. Isolates from Cutaneous Leishmaniasis Endemic Areas in Panama.

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.

Spatiotemporal Analysis of Malaria Transmission in the Autonomous Indigenous Regions of Panama, Central America, 2015-2022.

Despite ongoing efforts for elimination, malaria continues to be a major public health problem in the Republic of Panama. For effective elimination, it is key that malaria foci and areas of high transmission are identified in a timely manner. Here, we study malaria transmission records for the 2015-2022 period, a time when cases have increased by a factor of ten. Using several methods to study spatial and spatiotemporal malaria confirmed case clusters at the level of localities, including LISA and scan, we found that cases are clustered across indigenous villages located within the autonomous indigenous regions of Ngäbe-Buglé, Guna Yala, and Embera, with the latter on the eastern border of Panama (with Colombia). We discuss the different factors that might be shaping the marked increase in malaria transmission associated with these clusters, which include an inflow of malaria-exposed migrating populations hoping to reach the USA, insufficient health services, and the lack of culturally sensitive actionable tools to reduce malaria exposure among the ethnically diverse and impoverished indigenous populations of Panama.

Comparación de pruebas moleculares para el diagnóstico de Leishmania spp. en lesiones cutáneas con baja carga parasitaria

Introducción: La leishmaniasis cutánea (LC) es un problema grave de salud pública en Panamá. El diagnóstico de esta parasitosis ha sido siempre desafiante, no sólo debido a su similitud con otras infecciones dérmicas, sino también a características particulares de las lesiones, como cargas parasitarias bajas. ​Materiales y Métodos: En este estudio, se evaluaron mediante cuatro métodos moleculares, 235 muestras de ADN procedentes de lesiones con frotis negativos por LC obtenidas durante el período 2015-2019. Resultados: Los resultados señalan que las sensibilidades encontradas fueron de 75.6% (IC 0.6234-0.8709) para la PCR kDNA-Género específico, de 66.7% (IC 0.5359-0.776) para la PCR Hsp70-Género específico y de 77.6% (IC 0.645-0.8949) para la qPCR 18S ribosomal. Todas las pruebas obtuvieron un valor predictivo positivo de 100%, mientras que el valor predictivo negativo más alto fue con la qPCR (80.58%) y el más bajo con el PCR Hsp70-Género específico (73.2%). En cuanto a la precisión de diagnóstico se obtuvo un rango mayor del 82% en todas las pruebas evaluadas. Conclusión: Este estudio confirma la buena sensibilidad de la PCR kDNA-Viannia para el análisis de lesiones de LC con baja carga parasitaria. Esta metodología es relativamente fácil de estandarizar, por lo que se recomienda su uso en laboratorios clínicos regionales de Panamá. Aun cuando la qPCR 18S ribosomal presentó una sensibilidad relativamente menor, el uso de esta metodología debe ser también considerada por su facilidad de uso, menor tiempo de ejecución y capacidad de cuantificación. (provisto por Infomedic International)

Introduction: Cutaneous leishmaniasis (CL) is a serious public health problem in Panama. The diagnosis of this parasitosis has always been challenging, not only because of its similarity to other dermal infections, but also because of characteristics of the lesions, such as low parasite loads. Materials and Methods: In this study, 235 DNA samples from smear-negative lesions by LC obtained during the period 2015-2019 were evaluated by four molecular methods. Results: The results indicate that the sensitivities found were 75.6% (CI 0.6234-0.8709) for kDNA-Gene-specific PCR, 66.7% (CI 0.5359-0.776) for Hsp70-Gene-specific PCR and 77.6% (CI 0.645-0.8949) for 18S ribosomal qPCR. All tests obtained a positive predictive value of 100%, while the highest negative predictive value was with qPCR (80.58%) and the lowest with Hsp70-Gene-specific PCR (73.2%). In terms of diagnostic accuracy, a range greater than 82% was obtained in all the tests evaluated. Conclusion: This study confirms the good sensitivity of kDNA-Viannia PCR for the analysis of LC lesions with low parasite load. This methodology is relatively easy to standardize, so it is recommended for use in regional clinical laboratories in Panama. Although 18S ribosomal qPCR showed a relatively lower sensitivity, the use of this methodology should also be considered because of its ease of use, shorter execution time and quantification capacity. (provided by Infomedic International)

Insights into the Genetic Diversity of Leishmania (Viannia) panamensis in Panama, Inferred via Multilocus Sequence Typing (MLST).

Leishmaniasis is a disease caused by parasites of the genus Leishmania and transmitted by sand fly vectors. Tegumentary leishmaniasis is the most prevalent clinical outcome in Latin America, afflicting people from 18 countries. In Panama, the annual incidence rate of leishmaniasis is as high as 3000 cases, representing a major public health problem. In endemic regions, L. panamensis is responsible for almost eighty percent of human cases that present different clinical outcomes. These differences in disease outcomes could be the result of the local interplay between L. panamensis variants and human hosts with different genetic backgrounds. The genetic diversity of L. panamensis in Panama has only been partially explored, and the variability reported for this species is based on few studies restricted to small populations and/or with poor resolutive markers at low taxonomic levels. Accordingly, in this study, we explored the genetic diversity of sixty-nine L. panamensis isolates from different endemic regions of Panama, using an MLST approach based on four housekeeping genes (Aconitase, ALAT, GPI and HSP70). Two to seven haplotypes per locus were identified, and regional differences in the genetic diversity of L. panamensis were observed. A genotype analysis evidenced the circulation of thirteen L. panamensis genotypes, a fact that might have important implications for the local control of the disease.

Characterization of Leishmania spp. Causing Cutaneous Lesions with a Negative Parasitological Diagnosis in Panama.

A total of 123 DNA samples from Panamanian patients with cutaneous leishmaniasis (CL) lesions were evaluated. These samples were previously confirmed with CL by a specific KDNA-Viannia PCR but had a negative parasitological diagnosis (Group A). Epidemiological variables, such as age, sex, geographic origin, evolution time, and the number and location of the lesions, were analyzed. No significant differences (p < 0.05) were found when these variables were evaluated against a control panel of 123 CL lesion samples from CL patients with positive parasitological diagnoses (Group B). Of the 123 samples (Group A), 67% (82/123) gave positive results when re-analyzed by PCR-hsp70. An analysis of 69 of these samples via PCR-hsp70-RFLP showed that 59.4% (41/69) of the found restriction patterns corresponded to Leishmania (Viannia) panamensis and 40.6% (28/69) to Leishmania (Viannia) guyanensis. Finally, the sequence and phylogenetic analysis of 32 of the samples confirmed the species in 21 (65.6%, 21/32) samples, originally characterized as L. (V.) panamensis. However, 11 samples (34.4%, 11/32), initially identified via RFLP-Hsp70 as L. (V.) guyanensis, matched the sequence of a genetic variant known as Leishmania sp.1. These results point out the species/genetic variants of Leishmania in the case of CL lesions with an apparently low parasite load.

First report of imported canine visceral leishmaniasis cases in Panama, Central America: Public health implications.

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.

Genetic Diversity of Trypanosoma cruzi in Panama Inferred by Multi-locus Sequence Typing of Mitochondrial Genes.

The objective of this study was to provide information on Trypanosoma cruzi genetic diversity among isolates obtained from different biological sources circulating in endemic areas of Panama. Initial discrete typing units (DTUs) assignment was performed evaluating three single locus molecular markers (mini-exon, heat shock protein 60 and glucose-6-phosphate isomerase genes). Further diversity within TcI lineages was explored using a multi-locus sequence typing approach with six maxicircle genes. Haplotype network analysis and evolutionary divergency estimations were conducted to investigate the genetic relatedness between Panamanian TcI isolates and isolates from different endemic regions in the Americas. Our molecular approach validated that TcI is the predominant DTU circulating in Panama across different hosts and vector species, but also confirmed the presence of TcIII and TcVI circulating in the country. The phylogenetic tree topography for most Panamanian TcI isolates displayed a high level of genetic homogeneity between them. The haplotype network analysis inferred a higher genetic diversity within Panamanian TcI isolates, displaying eight different haplotypes circulating in endemic regions of the country, and revealed geographical structuring among TcI from different endemic regions in the Americas. This study adds novelty on the genetic diversity of T. cruzi circulating in Panama and complements regional phylogeographic studies regarding intra-TcI variations.

Surveillance and genotype characterization of zoonotic trypanosomatidae in Didelphis marsupialis in two endemic sites of rural Panama.

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.

Factores de riesgo asociados con la leishmaniasis cutánea en dos comunidades rurales de Panamá Oeste

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)

Plasmodium vivax Genetic Diversity in Panama: Challenges for Malaria Elimination in Mesoamerica.

Panama and all nations within the Mesoamerican region have committed to eliminate malaria within this decade. With more than 90% of the malaria cases in this region caused by Plasmodium vivax, an efficient national/regional elimination plan must include a comprehensive study of this parasite's genetic diversity. Here, we retrospectively analyzed P. vivax genetic diversity in autochthonous and imported field isolates collected in different endemic regions in Panama from 2007 to 2020, using highly polymorphic markers (csp, msp-1, and msp-3α). We did the analysis using molecular techniques that are cost-effective for malaria molecular surveillance within Mesoamerica. Thus, we used molecular analyses that are feasible for malaria molecular surveillance within the region, and that can provide useful information for policy and decision making about malaria elimination. We also evaluated if haplotypes established by combining the genotypes found in these genes were associated with relevant epidemiological variables and showed structure across the transmission foci that have been observed in Panama. Ten different haplotypes were identified, some of them strongly associated with geographical origin, age, and collection year. Phylogenetic analysis of csp (central repeat domain) revealed that both major variant types (vk210 and vk247) were circulating in Panama. Variant vk247 was restricted to the eastern endemic regions, while vk210 was predominant (77.3%) and widespread, displaying higher diversity (14 alleles) and geographically biased alleles. The regional implications of these molecular findings for the control of P. vivax malaria to achieve elimination across Mesoamerica are discussed.

Macrophage Polarization in the Skin Lesion Caused by Neotropical Species of Leishmania sp.

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.

Natural malaria infection in anophelines vectors and their incrimination in local malaria transmission in Darién, Panama.

BACKGROUND: More than 85% of the malaria cases in Panama occur in poor, rural and indigenous regions like Darien Province. Vector diversity, infection rate and spatial distribution are important entomological parameters of malaria transmission dynamics. Their understanding is crucial for the development of effective disease control strategies. The objective of this study was to determine the composition of Anopheles species, their natural infection rate and their geographic distribution to better understand the malaria transmission dynamics in Darién, Panama. METHODS: Anophelines mosquitoes were captured during the rainy and dry season of 2016. We selected five communities where adult anophelines were collected using CDC light-traps, and through protective human-baited traps. Detection of natural infection and Plasmodium genotype were detected via nested PCR through the amplification of ssrRNA and the circumsporozoite protein gene (csp), respectively. RESULTS: A total of 1,063 mosquitoes were collected mosquitoes were collected for the detection of natural infection with Plasmodium spp. Nine Anophelines species were identified, with the predominant species being: An. (Nys.) darlingi (45.0%) and An. (Nys.) albimanus (42.6%). Natural infection in An. (Nys.) albimanus with P. vivax was detected in one mosquito pool from the community Pueblo Tortuga (0.6%), three from Marraganti (1.7%), two from Bajo Chiquito (1.1%) and three pools from Alto Playona 3 (1.7%). For An. (Nys.) darlingi mosquitoes, we detected seven positive pools from the community Bajo Chiquito (4.0%), two pools from Marraganti (1.1%) and two pools from Alto Playona (1.1%). The P. vivax allelic variant VK210 was detected in infected mosquitoes. CONCLUSION: The results from this study provide new information on the transmission dynamics associated with anophelines vectors in the Darién region. This is the first report of natural P. vivax infection in An. (Nys.) darlingi and its incrimination as a potential malaria vector in this region of Panama. Additional studies are necessary to expand our knowledge and determine crucial parameters in malaria transmission in Darién, which in turn will aid the National Malaria Program in attaining an adequate malaria control strategy towards malaria elimination.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama.

BACKGROUND: The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES: To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS: We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS: We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS: Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

Molecular Identification of Parasites Causing Cutaneous Leishmaniasis in Panama.

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.

In situ study of cellular immune response in human cutaneous lesions caused by Leishmania (Viannia) panamensis in Panama.

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.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

Landscape disturbance impacts on Attalea butyracea palm distribution in central Panama.

BACKGROUND: Increased Attalea butyracea palm propagation, notable for its role as key habitat for the primary Chagas disease vector in Panama, has been linked to landscape disturbance in single-palm observations in this region. Close proximity of these palms to human dwellings is proposed to increase risk of Chagas disease transmission from sylvatic transmission cycles to domestic transmission involving human populations. This study examines the relationship between landscape disturbance and mature A. butyracea spatial distribution, density, and proximity to human populations and vector and reservoir species' movement corridors at a regional scale in a 300 km2 heterogeneous tropical landscape in central Panama. METHODS: We remotely identified the locations of over 50,000 mature A. butyracea palms using high-resolution WorldView2 satellite imagery. A local Getis-Ord Gi* spatial analysis identified significant clusters of aggregated palms. Associations between palm and cluster abundance and a landscape disturbance gradient, derived from official Panama land cover data, were tested using Chi-square tests for Homogeneity and Z-test for proportions. Kruskall-Wallis non-parametric analysis of variance tests were run to assess whether palm cluster area varied by disturbance level, or whether disturbance was associated with proximity of palms and palm clusters to susceptible populations or vector movement corridors. RESULTS: Our findings indicate a regional relationship between landscape disturbance and A. butyracea occurrence. We observe a significant increase in both individual and clustered A. butyracea in secondary forest, but a reduction of palms in agricultural settings. We do not detect evidence of any reduction in abundance of palms in residential settings. The majority of residential and commercial buildings in our study area are within vector flight distance of potential vector habitat in palm crowns. CONCLUSIONS: We observe probable anthropogenic elimination of A. butyracea palms in agricultural, but not residential, settings. Even in heavily deforested regions, significant concentrations of mature palms remain in close proximity to human establishments.

Plasmodium falciparum Genetic Diversity in Panamá Based on glurp, msp-1 and msp-2 Genes: Implications for Malaria Elimination in Mesoamerica.

Panamá, together with all the nations in Mesoamerica, has committed to eliminate malaria from the region by 2020. As these countries approach malaria elimination and local transmission decreases, an active molecular surveillance to identify genotypes circulating along the border areas is particularly needed to accurately infer infection origin, drug resistance and disease propagation patterns in the region. This study evaluated the genetic diversity and allele frequencies of msp-1, msp-2 and glurp genes using different molecular analyses (nested PCR, PCR-restriction fragment length polymorphism (RFLP) and sequencing) from 106 autochthonous and imported P. falciparum isolates collected from different endemic areas in Panamá between 2003 and 2019. We also explored if P. falciparum genotypes assessed with these molecular markers were associated with relevant malaria epidemiological parameters using a multiple correspondence analysis. A strong association of certain local haplotypes with their geographic distribution in endemic areas, but also with parasite load and presence of gametocytes, was evidenced. Few multiclonal infections and low genetic diversity among locally transmitted P. falciparum samples were detected, consequent with the low transmission intensity of this parasite in Panamá, a pattern likely to be extended across Mesoamerica. In addition, several imported cases were genetically dissimilar to local infections and representative of more diverse extra-continental lineages.

Long-term transmission patterns and public health policies leading to malaria elimination in Panamá.

BACKGROUND: The present study provides a countrywide perspective of the malaria situation in Panamá over a long-term framework, with the purpose of identifying historical malaria resurgence events and their potential causes. METHODS: A descriptive-ecological study was conducted by analysing demographic and epidemiological annual malaria time series data in Panamá (1884-2019) using several data sources. Malaria intensity indicators were calculated during the study period. The effects of El Niño Southern Oscillation on malaria transmission were also analysed using a retrospective analysis of malaria cases between 1957 and 2019. RESULTS: Several factors were identified responsible for malaria resurgence in Panamá, mostly related with Malaria Control Programme weakening. During the past 20 years (2000-2019) malaria has progressively increased in prevalence within indigenous settlements, with a predominance of male cases and a high proportion (15% of total cases) in children less than 5 years old. During this period, a significant and increasing proportion of the Plasmodium falciparum cases were imported. Retrospective analysis (1957-2019) evidenced that ENSO had a significant impact on malaria transmission dynamics in Panamá. CONCLUSIONS: Data analysis confirmed that although authorities have been successful in focalizing malaria transmission in the country, there are still neglected issues to be solved and important intercultural barriers that need to be addressed in order to achieve elimination of the disease by 2022. This information will be useful for targeting strategies by the National Malaria Elimination Programme.