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We report the novel use of cryosurgery to treat cutaneous feline leishmaniosis (FeL) in a domestic cat from mid-western Venezuela. Amastigotes, evident by microscopy in aspirates from the nodular, erythematous nose lesions, were identified as Leishmania mexicana by cytochrome b gene sequence analysis. Lesions resolved completely without relapse after 14 months.

Nous décrivons une nouvelle utilisation de la cryochirurgie pour traiter la leishmaniose féline cutanée (FeL) chez un chat domestique du centre-ouest du Venezuela. Les amastigotes, observés par microscopie dans les cytoponctions des lésions nodulaires et érythémateuses du nez, ont été identifiés comme Leishmania mexicana par analyse de la séquence du gène du cytochrome b. Les lésions ont complètement disparu sans rechute après 14 mois.

Describimos el uso novedoso de la criocirugía para tratar la leishmaniosis cutánea felina (FeL) en un gato doméstico del medio oeste de Venezuela. Los amastigotes, evidentes por microscopía en los aspirados de las lesiones nasales nodulares eritematosas, se identificaron como Leishmania mexicana mediante el análisis de la secuencia del gen del citocromo b. Las lesiones se resolvieron completamente sin recidiva tras 14 meses.

Neste estudo, relatamos a utilização inédita de criocirurgia para tratar leishmaniose felina cutânea (FeL) em um gato doméstico no centro-oeste da Venezuela. Amastigotas, evidentes à microscopia de aspirados da lesão nodular e eritematosa na região nasal, foram identificadas como Leishmania Mexicana por sequenciamento do gene do citocromo b. As lesões se resolveram completamente sem recidiva após 14 meses.

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The natural infection of sand flies by Leishmania was investigated in Andean areas located between the Central and Eastern Cordilleras of northern Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured at five locations along the Utcubamba River in the Department of Amazonas, and morphologically identified under a microscope. Among 422 female sand flies dissected, the most dominant species was Pintomyia verrucarum (320 flies), followed by Pi. maranonensis (83 flies), Pi. robusta (13 flies), and Lutzomyia castanea (6 flies). Genetic analysis of sand flies from these areas together with those from other areas revealed that individuals of Pi. verrucarum were closely related regardless of morphological variation of their spermathecae. On the other hand, individuals of Pi. maranonensis collected in the study area were distant from those of other areas with genetic distances over the intraspecific level but mostly below the interspecific level, suggesting the unique characteristics of sand flies in this area. The natural infection of sand flies by flagellate parasites was detected mainly in the hindgut of each one of Pi. verrucarum and Pi. maranonensis. Both parasite species were identified as L. (V.) peruviana based on cytochrome b and mannose phosphate isomerase gene analyses. In addition, parasite species obtained from the lesion of a patient with cutaneous leishmaniasis in the study area in this period was identified as L. (V.) peruviana. These results strongly suggest that Pi. verrucarum and Pi. maranonensis are responsible for the transmission of L. (V.) peruviana in these areas. This is the first report of the natural infection of Pi. maranonensis by L. (V.) peruviana.

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Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis. Although Leishmania species have been conventionally identified by multilocus enzyme electrophoresis, genetic analysis targeting kinetoplast and nuclear DNA (kDNA and nDNA, respectively) is now widely used for this purpose. Recently, we conducted countrywide epidemiological studies of leishmaniasis in Ecuador and Peru to reveal prevalent species using PCR-RFLP targeting nDNA, and identified unknown hybrid parasites in these countries together with species reported previously. Furthermore, comparative analyses of kDNA and nDNA revealed the distribution of parasites with mismatches between these genes, representing the first report of mito-nuclear discordance in protozoa. The prevalence of an unexpectedly high rate (~10%) of genetically complex strains including hybrid strains, in conjunction with the observation of mito-nuclear discordance, suggests that genetic exchange may occur more frequently than previously thought in natural Leishmania populations. Hybrid Leishmania strains resulting from genetic exchanges are suggested to cause more severe clinical symptoms when compared with parental strains, and to have increased transmissibility by vectors of the parental parasite species. Therefore, it is important to clarify how such genetic exchange influences disease progression and transmissibility by sand flies in nature. In addition, our aim was to identify where and how the genetic exchange resulting in the formation of hybrid and mito-nuclear discordance occurs.

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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered.

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An 88-year-old man with mutilating mucosal leishmaniasis (ML) involving septal perforation, with granulomas in the pharynx and larynx, was treated with oral miltefosine, 50 mg three times/day for 28 days. Miltefosine, an antineoplastic agent, is considered an alternative option for the treatment of ML, showing efficacies of 75-92% in Bolivia, Brazil, and Argentina. The patient denied having previous cutaneous (CL) leishmaniasis, and no CL lesions were recognized by physical examination. Parasites obtained from mucosal lesions were identified by cytochrome b gene sequencing as Leishmania guyanensis. Clinical cure was observed 2 months posttreatment, and no evidence of reactivation was observed in the 3-year follow-up. Adverse effects such as nausea, loss of appetite, and epigastric pain were experienced during treatment with miltefosine. There is a need for improved access to miltefosine in leishmaniasis-endemic areas of Latin America and a greater awareness of ML and its treatment among physicians working in endemic countries.

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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.

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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.

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Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.

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To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.

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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.

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