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.

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.

Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

The calmodulin intergenic spacer as molecular target for characterization of Leishmania species.

BACKGROUND: Human leishmaniasis is a neglected disease caused by parasites of the genus Leishmania. Clinical aspects of this disease can vary significantly, reflecting the wide range of parasites in the genus Leishmania. Knowing accurately the Leishmania species infecting humans is important for clinical case management and evaluation of epidemiological risk. Calmodulin is an essential gene in trypanosomatids that modulates the calcium metabolism in various cellular activities. Despite its strong conservation in trypanosomatids, it has been recently observed that its untranslated regions (UTR) diverge among species. METHODS: In this study we analyzed the sequences and the absolute dinucleotide frequency of the intergenic spacer of the calmodulin gene (containing both, 3' and 5'UTR) in nine reference Leishmania species and ten clinical isolates obtained from patients with cutaneous leishmaniasis. RESULTS: We show that the short calmodulin intergenic spacers exhibit features that make them interesting for applications in molecular characterization and phylogenetic studies of Leishmania. Dendrograms based on sequence alignments and on the dinucleotide frequency indicate that this particular region of calmodulin gene might be useful for species typing between the Leishmania and Viannia subgenera. CONCLUSIONS: Mutations and composition of the calmodulin intergenic spacer from Leishmania species might have taxonomic value as parameters to define if an isolate is identical to a certain species or belongs to one of the two current subgenera.

A new endemic focus of Chagas disease in the northern region of Veraguas Province, Western Half Panama, Central America.

BACKGROUND: Chagas disease was originally reported in Panama in 1931. Currently, the best knowledge of this zoonosis is restricted to studies done in historically endemic regions. However, little is known about the distribution and epidemiology of Chagas disease in other rural areas of the country. METHODS AND FINDINGS: A cross-sectional descriptive study was carried out between May 2005 - July 2008 in four rural communities of the Santa Fe District, Veraguas Province. The study included an entomologic search to collect triatomines, bloodmeal type identification and infection rate with trypanosomes in collected vectors using a dot- blot and PCR analysis, genotyping of circulating Trypanosoma cruzi (mini-exon gene PCR analysis) and the detection of chagasic antibodies among inhabitants. The vector Rhodnius pallescens was more frequently found in La Culaca and El Pantano communities (788 specimens), where it was a sporadic household visitor. These triatomines presented darker coloration and larger sizescompared with typical specimens collected in Central Panama. Triatoma dimidiata was more common in Sabaneta de El Macho (162 specimens). In one small sub-region (El Macho), 60% of the houses were colonized by this vector. Of the examined R. pallescens, 54.7.0% (88/161) had fed on Didelphis marsupialis, and 24.6% (34/138) of T. dimidiata specimens collected inside houses were positive for human blood. R. pallescens presented an infection index with T. cruzi of 17.7% (24/136), with T. rangeli of 12.5% (17/136) and 50.7% (69/136) were mixed infections. In 117 T. dimidiata domestic specimens the infection index with T. cruzi was 21.4%. Lineage I of T. cruzi was confirmed circulating in these vectors. A T. cruzi infection seroprevalence of 2.3% (24/1,056) was found in this population. CONCLUSIONS: This is the first report of Chagas disease endemicity in Santa Fe District, and it should be considered a neglected public health problem in this area of Panama.

Molecular epidemiology of American tegumentary leishmaniasis in Panama.

American tegumentary leishmaniasis is an increasing public health problem in Panama. This study describes the clinical characteristics and the molecular epidemiology of leishmaniasis in Panama over a 5-year period (2004-2008). Additionally, we applied a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay to identify Leishmania species in clinical isolates, skin scrapings, and sandflies specimens. Whereas 60.3% of cases were detected with conventional parasitologic techniques (smear or in vitro culture), the PCR detected 72% positive patients. Our clinical-epidemiologic data corroborate the high incidence of L. (Viannia) panamensis and provide evidence of peridomestic and/or domestic transmission. Mucosal involvement was observed in 4.2% of the patients. The overall natural infection rate with Leishmania in 103 pools of sandflies was 0.46%. Lutzomyia gomezi and Lutzomya panamensis were the prevalent species incriminated as vectors at the capture sites in central Panama. This study contributes to a better knowledge of the current epidemiology of tegumentary leishmaniasis in Panama.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7 percent for trypanosome infections, a seroprevalence of 2.9 percent against T. cruzi and a predominance of T. rangeli infection (6.8 percent). The immunological and clinical implications of these findings are discussed.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal.

A total of 206 serum samples from children (3-14 years old) living in the Amador County (La Chorrera District, Province of Panama) were screened by indirect immunofluorescence antibody test (IFAT) for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR) followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%). The immunological and clinical implications of these findings are discussed.