Natural Leishmania (Leishmania) mexicana infection and biting activity of anthropophilic sand fly Lutzomyia ayacuchensis in the Ecuadorian Andes.

To elucidate the transmission mode of Andean cutaneous leishmaniasis (Andean-CL), natural Leishmania infection and biting activity of sand flies were tested in a selected sylvatic focus of the endemic area of the Ecuadorian Andes. Monthly sand fly collections and dissections were conducted during 12 months from July 2018 to June 2019. The Leishmania positive specimens/slides with innumerable amounts of actively mobile flagellates made us easy to detect positive sand flies. The promastigotes observed located in the anterior and posterior midgut, without the hindgut localization. The parasite isolated was identified as L. (L.) mexicana by cytochrome b gene analysis. No other Leishmania or flagellate species parasitic in sand flies was observed in the area. Only Lu. ayacuchensis was caught throughout. Monthly microscopic examination of Lu. ayacuchensis revealed 0.75-8.33% of natural L. (L.) mexicana infection rates. Higher Leishmania infection months were present at the end of the wet season of the Andes, while higher sand fly numbers occurred during the dry season. Diurnal biting (blood meal seeking) activity of sand flies started around 17:30 before sunset, increased between 18:00 and 19:30, and thereafter decreased drastically probably because of low temperature (15-18 °C) in the area. The results provide information important for the planning of vector control strategy and management of the disease in the Andean-CL endemic area of Ecuador.

Anthropophilic phlebotomine sand fly Lutzomyia species and search for the natural Leishmania infections in an area endemic for cutaneous leishmaniasis in Ecuador.

By employing protected human bait landing and modified Shannon light trap, a total of 1924 phlebotomine sand fly Lutzomyia spp. were captured in an area from which L. (V.) guyanensis was reported as the causative parasite of cutaneous leishmaniasis (CL). The sand flies captured alive were dissected and identified at species level, based mainly on their spermathecae. At the same time, the sand flies dissected were searched for the Leishmania parasites by microscopic-test, and later on by PCR-test. No positive sand flies were detected by both tests, while considerable numbers of anthropophilic sand fly species of the genus Lutzomyia were observed as probable vectors of the Leishmania parasite in the areas. Those were eight species, Lu. robusta, Lu. trapidoi, Lu. maranonensis, Lu. gomezi, Lu. shannoni, Lu. migonei, Lu. punctigeniculata and Lu. spathotrichia. Among them, the first two species Lu. robusta and Lu. trapidoi were most dominant, suggesting probable vectors of the Leishmania parasite prevailing in the area. Lu. punctigeniculata and Lu. spathotrichia were for the first time recorded for the Manabí province, Ecuador. These findings provide basic information useful for future planning of the control and management of the disease in the areas, though further study to incriminate the vector sand fly remains.

PCR-RFLP analyses of Leishmania species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador.

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.

Leishmaniasis caused by Leishmania (Viannia) guyanensis in north-central Pacific region of Ecuador: A clinico-epidemiological feature.

The current four year study was undertaken to investigate the clinical and epidemiological features of Leishmania (Viannia) guyanensis infections in Valle Hermoso, Santo Domingo de Los Tsachilas province, north-central Pacific areas of Ecuador. A total of 155 parasitologically confirmed (Leishmania-amastigote-positive) clinical cases diagnosed at a rural health center during January 2014-December 2017 were analyzed thoroughly. Molecular characterization of the causative Leishmania parasites from different endemic sites within the study areas was performed by PCR amplification of cytochrome b (cyt b) sequencing. All the FTA-card and/or smear impregnated materials tested were characterized, and identified as L. (V.) guyanensis, without detecting any other Leishmania species. The following features were described: 1) the majority of patients were suffered from a single ulcer lesion (simple and mild to chronic), followed by multiple lesions, including recidiva cutis-"like" and Chiclero's ulcer-"like" clinical forms; 2) the majority (65.70%) of lesions were less than 10 mm in size, and distributed mainly on the upper body regions (arm, forearm, face, and neck including ear and head); 3) about 30% (29.68%) of the subjects tested were less than 10 years of age, strongly suggesting the intra- and/or peri-domestic transmission of the disease in the areas. The current clinico-epidemiological feature detected emphasizes the need for further such investigations of the L. (V.) guyanensis infections prevalent at different Pacific ecoregions of Ecuador, including Amazon regions.

Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador: the vector Lutzomyia sand flies and reservoir mammals.

The vector Lutzomyia sand flies and reservoir host mammals of the Leishmania parasites, causing the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador were thoroughly reviewed, performing a survey of literatures including our unpublished data. The Peruvian L. (V.) peruviana, a principal Leishmania species causing Andean-CL in Peru, possessed three Lutzomyia species, Lu. peruensis, Lu. verrucarum and Lu. ayacuchensis as vectors, while the Ecuadorian L. (L.) mexicana parasite possessed only one species Lu. ayacuchensis as the vector. Among these, the Ecuadorian showed a markedly higher rate of natural Leishmania infections. However, the monthly and diurnal biting activities were mostly similar among these vector species was in both countries, and the higher rates of infection (transmission) reported, corresponded to sand fly's higher monthly-activity season (rainy season). The Lu. tejadai sand fly participated as a vector of a hybrid parasite of L. (V.) braziliensis/L. (V.) peruviana in the Peruvian Andes. Dogs were considered to be principal reservoir hosts of the L. (V.) peruviana and L. (L.) mexicana parasites in both countries, followed by other sylvatic mammals such as Phyllotis andium, Didelphis albiventris and Akodon sp. in Peru, and Rattus rattus in Ecuador, but information on the reservoir hosts/mammals was extremely poor in both countries. Thus, the Peruvian disease form demonstrated more complicated transmission dynamics than the Ecuadorian. A brief review was also given to the control of vector and reservoirs in the Andes areas. Such information is crucial for future development of the control strategies of the disease.

Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador: the causative Leishmania parasites and clinico-epidemiological features.

This study provides comprehensive information on the past and current status of the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador, mainly focusing on the causative Leishmania parasites and clinico-epidemiological features. Available information and data including our unpublished works were analyzed thoroughly. Endemic regions of the Andean-CL (uta) in Peru run from the north Piura/Cajamarca to the south Ayacucho at a wide range of the Pacific watersheds of the Andes through several departments, while in Ecuador those exist at limited and spotted areas in the country's mid-southwestern two provinces, Azuay and Chimborazo. The principal species of the genus Leishmania are completely different at subgenus level, L. (Viannia) peruviana in Peru, and L. (Leishmania) mexicana and L. (L.) major-like (infrequent occurrence) in Ecuador. The Peruvian uta is now prevalent in different age and sex groups, being not clearly defined as found in the past. The precise reasons are not known and should be elucidated further, though probable factors, such as emergence of other Leishmania parasites, non-immune peoples' migration into the areas, etc., were discussed briefly in the text. The Andean-CL cases in Ecuador are more rural than before, probably because of a rapid development of the Leishmania-positive communities and towns, and the change of life-styles of the inhabitants, including newly constructed houses and roads in the endemic areas. Such information is helpful for future management of the disease, not only for Leishmania-endemic areas in the Andes but also for other endemic areas.

Leishmaniases in Ecuador: Comprehensive review and current status.

This article reviews current knowledge about leishmaniases in Ecuador, proceeding from 1920, when the first human case was described, to the present, mainly focusing on the recent research events published. Regarding basic situations, it appears that 23 of Ecuador's 24 provinces have leishmaniasis-case reports. The disease is one of the mandatory notification infectious diseases in the country since 2005. All the 21,305 cases notified to the Ministry of Public Health, during the period from 2001 through 2014, were said to involve different clinical features of cutaneous leishmaniasis (CL) but not visceral (VL). Eight Leishmania species, L. (Viannia) guyanensis, L. (V.) panamensis, L. (V.) braziliensis, L. (Leishmania) mexicana, L. (L.) amazonensis, L. (L.) major-like, L. (V.) naiffiand L. (V.) lainsoni were characterized. The last two species were most recently reported from the Ecuadorian Amazon regions. Of the 73 Ecuadorian Lutzomyia species (43 man-biting species) recorded, only four, Lu. trapidoi, Lu. gomezi, Lu. ayacuchensis, and Lu. tortura were incriminated as vectors of the Leishmania parasites. Current knowledge on the reservoir hosts of Leishmania in Ecuador is extremely poor. Recently, in Ecuador different kinds of molecular techniques were developed for diagnosis and mass screening of the disease, employing various materials derived from patients and sand fly vectors. These are PCR-RFLP, colorimetric FTA-LAMP etc. Brief comments and recommendations were also given, for future research and control of leishmaniases in Ecuador.

Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis.

A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.

Diffuse and disseminated cutaneous leishmaniasis: clinical cases experienced in Ecuador and a brief review.

BACKGROUND: In Ecuador, cutaneous leishmaniasis (CL) is prevalent countrywide, but only one case of diffuse-CL and two cases of disseminated-CL were experienced during our research activities more than 30 years from 1982 to date. These three patients suffered from multiple lesions distributed at a wide range of the body surface, revealing difficulty to clinically differentiate each other. METHODS: There is a considerable confusion of the use and/or differentiation of the terminologies (terms) between the two disease forms, diffuse-CL and disseminated-CL. One of the aims of the present study is to clarify the difference between the two disease forms, mainly based on the cases experienced in Ecuador. RESULTS: The disseminated-CL case newly reported here was clinically very similar to the diffuse-CL case, but the former showed the following marked differences from the latter: (1) the organisms isolated were identified as the parasites of Leishmania (Viannia) guyanensis/panamensis, which are also known as the causative agents of disseminated-CL in different endemic countries of the New World; (2) the patient was sensitive against antimonials; and (3) mucosal involvement was observed, which is never observed in diffuse-CL. CONCLUSIONS: In the text, three clinical cases, one diffuse-CL and two disseminated-CL, were presented. Furthermore, a bibliographic comparison of the features between the two disease forms was made, and a brief comment was also given.

First Human Cases of Leishmania (Viannia) lainsoni Infection and a Search for the Vector Sand Flies in Ecuador.

An epidemiological study of leishmaniasis was performed in Amazonian areas of Ecuador since little information on the prevalent Leishmania and sand fly species responsible for the transmission is available. Of 33 clinical specimens from patients with cutaneous leishmaniasis (CL), causative parasites were identified in 25 samples based on cytochrome b gene analysis. As reported previously, Leishmania (Viannia) guyanensis and L. (V.) braziliensis were among the causative agents identified. In addition, L. (V.) lainsoni, for which infection is reported in Brazil, Bolivia, Peru, Suriname, and French Guiana, was identified in patients with CL from geographically separate areas in the Ecuadorian Amazon, corroborating the notion that L. (V.) lainsoni is widely distributed in South America. Sand flies were surveyed around the area where a patient with L. (V.) lainsoni was suspected to have been infected. However, natural infection of sand flies by L. (V.) lainsoni was not detected. Further extensive vector searches are necessary to define the transmission cycle of L. (V.) lainsoni in Ecuador.

An analysis of reported cases of leishmaniasis in the southern Ecuadorian Amazon region, 1986-2012.

An analysis of reported cases of cutaneous leishmaniasis (CL) was performed using the data registered in the southern Ecuadorian Amazon region during 27 years from 1986 to 2012. The cases/subjects with both the suspected CL lesions and the amastigote-positive results were recruited for the analysis. The yearly occurrence of cases showed a markedly higher number during the six years, 1988 and 1993. After 1994 when the insecticide spraying campaign using helicopter in 1993-1994, the number dropped remarkably. Then, the yearly occurrence gradually fluctuated from 101 cases in 1996 to 11 in 2009, maintaining a low number of cases after the campaign. The monthly occurrence of cases showed a markedly high number during March and August, suggesting a correlation to the rainy season (months) in the areas. A statistical significance was found between the monthly average number of the CL case and the average precipitation (p=0.01474). It was suggested that the time of transmission of CL would depend on the rainy seasons at each endemic area of Ecuador, which has a diverse climatic feature depending on the geographic regions. Such information at given leishmaniasis-endemic areas of Ecuador would be important for the future planning of the disease control. Molecular analysis and characterization of clinical samples revealed the presence of Leishmania (Viannia) braziliensis.

Genotyping of sand fly species in Peruvian Andes where leishmaniasis is endemic.

Genotyping of sand fly species circulating in Peru was established on the basis of PCR-restriction fragment length polymorphisms (RFLPs) of the 18S ribosomal RNA (rRNA) gene. The sequences of 18S rRNA gene fragments from 12 Lutzomyia and 1 Warileya species were determined and their RFLP-patterns were analyzed. Consequently, RFLP analysis with the restriction enzyme AfaI and then HapII or KpnI, followed by XspI successfully differentiated them. Intraspecific genetic diversity affecting RFLP-patterns was not detected in the specimens collected from 24 areas of 8 departments. The genotyping was applied to the surveillance of sand flies collected from Andean areas where leishmaniasis is endemic, and its usability was verified. The present method promises to be a powerful tool for the classification and surveillance of sand flies circulating in Peru.

Natural infections of man-biting sand flies by Leishmania and Trypanosoma species in the northern Peruvian Andes.

The natural infection of sand flies by Leishmania species was studied in the Andean areas of Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured by human bait and Center for Disease Control (CDC) light trap catches at Nambuque and Padregual, Department of La Libertad, Peru, and morphologically identified. Among 377 female sand flies dissected, the two dominant man-biting species were Lutzomyia (Helcocyrtomyia) peruensis (211 flies) and Lutzomyia (Helcocyrtomyia) caballeroi (151 flies). Another sand fly species captured by light trap was Warileya phlebotomanica (15 flies). The natural infection of sand flies by flagellates was detected in 1.4% of Lu. (H.) peruensis and 2.6% of Lu. (H.) caballeroi, and the parasite species were identified as Le. (V.) peruviana and Trypanosoma avium, respectively, by molecular biological methods. The results indicated that the vector species responsible for the transmission of leishmaniasis in the study areas is Lu. (H.) peruensis. In addition, the presence of Trypanosoma in man-biting sand fly species means that more careful consideration is necessary for vector research in areas of Andean Peru where leishmaniasis is endemic.

Use of FTA cards for direct sampling of patients' lesions in the ecological study of cutaneous leishmaniasis.

The FTA card (Whatman) was assessed for its utility as a molecular epidemiological tool in collecting samples from patients with leishmaniasis in Peru because the card has a variety of merits; it is less invasive for patients and easy to handle for both physicians and other medical personnel for sample collection or diagnosis, in addition to its simplicity and easy countrywide and/or intercountry transportation for analysis. Samples were collected from 132 patients suspected of having leishmaniasis, and Leishmania species were successfully identified in samples from 81 patients in 15 departments of Peru by cytochrome b and mannose phosphate isomerase gene analyses. Of these, 61.7% were identified as Leishmania (Viannia) peruviana, 22.2% as L. (V.) braziliensis, 12.3% as L. (V.) guyanensis, 2.5% as L. (V.) shawi, and 1.2% as L. (V.) lainsoni. The three predominant species, L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis, were mainly found in the Andean highlands, in the tropical rainforest, and in northern and central rainforest regions, respectively. This is the first time L. (V.) shawi has been identified outside Brazil. The present study showed that the FTA card will be a useful tool for the ecological study of different forms of leishmaniasis. Furthermore, collecting samples directly from patients' lesions by using the FTA card eliminates (i) the possibility of contamination of Leishmania isolates during short- and/or long-term passages of culture in vitro in each laboratory and (ii) pain and suffering of patients from taking samples by skin biopsy.