Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae), the Cayenne tick: phylogeography and evidence for allopatric speciation.

BACKGROUND: Amblyomma cajennense F. is one of the best known and studied ticks in the New World because of its very wide distribution, its economical importance as pest of domestic ungulates, and its association with a variety of animal and human pathogens. Recent observations, however, have challenged the taxonomic status of this tick and indicated that intraspecific cryptic speciation might be occurring. In the present study, we investigate the evolutionary and demographic history of this tick and examine its genetic structure based on the analyses of three mitochondrial (12SrDNA, d-loop, and COII) and one nuclear (ITS2) genes. Because A. cajennense is characterized by a typical trans-Amazonian distribution, lineage divergence dating is also performed to establish whether genetic diversity can be linked to dated vicariant events which shaped the topology of the Neotropics. RESULTS: Total evidence analyses of the concatenated mtDNA and nuclear + mtDNA datasets resulted in well-resolved and fully congruent reconstructions of the relationships within A. cajennense. The phylogenetic analyses consistently found A. cajennense to be monophyletic and to be separated into six genetic units defined by mutually exclusive haplotype compositions and habitat associations. Also, genetic divergence values showed that these lineages are as distinct from each other as recognized separate species of the same genus. The six clades are deeply split and node dating indicates that they started diverging in the middle-late Miocene. CONCLUSIONS: Behavioral differences and the results of laboratory cross-breeding experiments had already indicated that A. cajennense might be a complex of distinct taxonomic units. The combined and congruent mitochondrial and nuclear genetic evidence from this study reveals that A. cajennense is an assembly of six distinct species which have evolved separately from each other since at least 13.2 million years ago (Mya) in the earliest and 3.3 Mya in the latest lineages. The temporal and spatial diversification modes of the six lineages overlap the phylogeographical history of other organisms with similar extant trans-Amazonian distributions and are consistent with the present prevailing hypothesis that Neotropical diversity often finds its origins in the Miocene, after the Andean uplift changed the topology and consequently the climate and ecology of the Neotropics.

The population structure of Lutzomyia verrucarum (Diptera: Psycodidae), a Bartonella bacilliformis and Leishmania peruviana vector in Peru.

The population genetic structure of Lutzomyia verrucarum (Townsend), a sand fly disease vector of Carrion's disease and cutaneous leishmaniasis in the Peruvian Andes, was characterized by sequencing 653 bp of cytochrome b and 1,125 bp of the NADH dehydrogenase subunit 4 genes of its mitochondrial genome. DNA sequence variation within and between valleys was compared in a sample of 220 sand flies from three valleys (Purisima, Huaylas, and Conchucos) and five departments (Amazonas, Cajamarca, Piura, Lima, and Huancavelica). Gene network and phylogenetic analyses indicated a high similarity of haplotypes collected within a single valley (0-0.52% nucleotide divergence). Flies from each valley had unique genotypes not shared with specimens from other valleys or from more distant regions (0.8-3.1% nucleotide divergence). Mountain ranges and geographic distance appear to have impeded migration (N(m) = < 0.18) between valleys and separated populations into discrete genetic units.

Description of the female of the Peruvian sand fly Lutzomyia reclusa (Diptera: Psychodidae: Phlebotominae).

The female of the phlebotomine sand fly Lutzomyia reclusa Fernández & Rogers 1991 [= Pintomyia (Pifanomyia) reclusa (Fernández & Rogers) sensu Galati], is described for the first time, based on specimens collected in the Department of Cajamarca, in northern Peru. The female can be recognized from other species of the series pia, species group Verrucarum, by wing venation with beta shorter than half of alpha, labrum just shorter than head width but longer than flagellomere 1, palpomere 5 much longer than palpomere 3, arrangement of cibarial armature, and form of spermathecae and relative size of spermathecal ducts. Diagnostic characters and measurements of the male of Lu. reclusa are provided as well.

Molecular detection of Bartonella species in ticks from Peru.

A total of 103 ticks, collected from canines, horses, donkeys, and snakes from Peru, were screened for the presence of Bartonella DNA by polymerase chain reaction analysis. Bartonella DNA was detected in two ticks using Bartonella 16S-23S intergenic spacer region primers and in an additional two ticks using Bartonella NADH dehydrogenase gamma subunit gene (nuoG) primers. Bartonella rochalimae Eremeeva et al., B. quintana Schmincke, and B. elizabethae Daly et al. DNA was detected in a Rhipicephalus sanguineus Latreille (Acari: Ixodidae) female tick removed from a dog and B. quintana DNA was present in a Dermacentor nitens Neumann (Acari: Ixodidae) pool of five larvae, one nymph, and one adult male tick collected from donkeys. This is the first study to report the detection of B. rochalimae, B. quintana, and B. elizabethae DNA in ticks from Peru. Further investigations must be performed to decipher the role ticks may play in the transmission of Bartonella species.

Natural infections of Pintomyia verrucarum and Pintomyia maranonensis by Leishmania (Viannia) peruviana in the Eastern Andes of northern Peru.

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.

Prevalence of Genetically Complex Leishmania Strains With Hybrid and Mito-Nuclear Discordance.

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.

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.

Comparative Analysis of Bacterial Communities in Lutzomyia ayacuchensis Populations with Different Vector Competence to Leishmania Parasites in Ecuador and Peru.

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.

Nuclear and kinetoplast DNA analyses reveal genetically complex Leishmania strains with hybrid and mito-nuclear discordance in Peru.

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.

First Report of Bartonella SP. In Sand Flies (Diptera: Psychodidae: Phlebotominae) From Southern Mexico.

The genus Bartonella encompasses several zoonotic species that cause emerging infectious diseases in humans. These species are transmitted to humans by hematophagous arthropods. In South America, sand flies are the most important vectors of Bartonella bacilliformis, the etiological agent of Verruga peruana. Due to the importance of bartonellosis in public health, the aim of this study was to detect Bartonella in wild populations of phlebotomine sand flies in southern Mexico. In total, 29 sand flies belonging to 6 species were collected. Only 2 specimens were positive, representing a prevalence of 8.69% (2/23). This appears to be the first report of Bartonella sp. in phlebotomine sand flies outside the endemic area of Verruga peruana in South America. Further studies are needed to clarify the relation between this bacterium and sand flies in Mexico.

Identification of Bartonella rochalimae in Guinea Pigs (Cavia porcellus) and Fleas Collected from Rural Peruvian Households.

In the present study, we tested 391 fleas collected from guinea pigs (Cavia porcellus) (241 Pulex species, 110 Ctenocephalides felis, and 40 Tiamastus cavicola) and 194 fleas collected from human bedding and clothing (142 Pulex species, 43 C. felis, five T. cavicola, and four Ctenocephalides canis) for the presence of Bartonella DNA. We also tested 83 blood spots collected on Flinders Technology Associates (FTA) cards from guinea pigs inhabiting 338 Peruvian households. Bartonella DNA was detected in 81 (20.7%) of 391 guinea pig fleas, in five (2.6%) of 194 human fleas, and in 16 (19.3%) of 83 guinea pig blood spots. Among identified Bartonella species, B. rochalimae was the most prevalent in fleas (89.5%) and the only species found in the blood spots from guinea pigs. Other Bartonella species detected in fleas included B. henselae (3.5%), B. clarridgeiae (2.3%), and an undescribed Bartonella species (4.7%). Our results demonstrated a high prevalence of zoonotic B. rochalimae in households in rural areas where the research was conducted and suggested a potential role of guinea pigs as a reservoir of this bacterium.

Further insight into the geographic distribution of Leishmania species in Peru by cytochrome b and mannose phosphate isomerase gene analyses.

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.

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.

The Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group of ticks: phenotypic plasticity or incipient speciation?

BACKGROUND: The goal of this study was to reassess the taxonomic status of A. maculatum, A. triste and A. tigrinum by phylogenetic analysis of five molecular markers [four mitochondrial: 12S rDNA, 16S rDNA, the control region (DL) and cytochrome c oxidase 1 (cox1), and one nuclear: ribosomal intergenic transcribed spacer 2 (ITS2)]. In addition, the phenotypic diversity of adult ticks identified as A. maculatum and A. triste from geographically distinct populations was thoroughly re-examined. RESULTS: Microscopic examination identified four putative morphotypes distinguishable by disjunct geographical ranges, but very scant fixed characters. Analysis of the separated mitochondrial datasets mostly resulted in conflicting tree topologies. Nuclear gene sequences were almost identical throughout the geographical ranges of the two species, suggesting a very recent, almost explosive radiation of the terminal operational taxonomic units. Analysis of concatenated molecular datasets was more informative and indicated that, although genetically very close to the A. maculatum - A. triste lineage, A. tigrinum was a monophyletic separate entity. Within the A. maculatum - A. triste cluster, three main clades were supported. The two morphotypes, corresponding to the western North American and eastern North American populations, consistently grouped in a single monophyletic clade with many shared mitochondrial sequences among ticks of the two areas. Ticks from the two remaining morphotypes, south-eastern South America and Peruvian, corresponded to two distinct clades. CONCLUSIONS: Given the paucity of morphological characters, the minimal genetic distance separating morphotypes, and more importantly the fact that two morphotypes are genetically indistinguishable, our data suggest that A. maculatum and A. triste should be synonymized and that morphological differences merely reflect very recent local adaptation to distinct environments in taxa that might be undergoing the first steps of speciation but have yet to complete lineage sorting. Nonetheless, future investigations using more sensitive nuclear markers and/or crossbreeding experiments might reveal the occurrence of very rapid speciation events in this group of taxa. Tentative node dating revealed that the A. tigrinum and A. maculatum - A. triste clades split about 2 Mya, while the A. maculatum - A.triste cluster radiated no earlier than 700,000 years ago.

Identifying Trypanosoma cruzi discreet typing units in triatomines collected in different natural regions of Perú.

INTRODUCTION: Trypanosoma cruzi has been divided by international consensus into six discrete typing units (DTU): TcI, TcII, TcIII, TcIV, TcV y TcVI. The factors determining the dynamics of T. cruzi genotypes vector transmission of Chagas' disease in the different geographical regions of Perú are still unknown. OBJECTIVE: To detect and type T. cruzi DTUs from the faeces of seven species of triatomines (Panstrongylus chinai, P. geniculatus, P. herreri, Rhodnius robustus, R. pictipes, Triatoma carrioni and T. infestans) captured in eight departments from different natural regions of Perú. MATERIALS AND METHODS: We examined 197 insects for detecting trypanosomes. DNA was extracted from each insect intestinal contents and PCR amplification of kDNA, SL-IR, 24Sα rRNA and 18Sα RNA was performed for detecting T. cruzi DTUs. RESULTS: Five T. rangeli and 113 T. cruzi infections were detected; 95 of the latter were identified as TcI (two in P. chinai, one in P. geniculatus, 68 in P. herreri, four in R. pictipes, seven in R. robustus, one in T. carrioni, 12 in T. infestans), five as TcII (four in P. herreri, one in T. infestans), four as TcIII (three in P. herreri, one in R. robustus) and four TcIV infections in P. herreri. CONCLUSIONS: This is the first study which has attempted a large-scale characterization of T. cruzi found in the intestine of epidemiologically important vectors in Perú, thus providing basic information that will facilitate a better understanding of the dynamics of T. cruzi vector transmission in Perú.

First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.

A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool.

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per µl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.

Identification of causative Leishmania species in Giemsa-stained smears prepared from patients with cutaneous leishmaniasis in Peru using PCR-RFLP.

A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis.

Genetic divergence in populations of Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis, in Ecuador and Peru.

Haplotype and gene network analyses were performed on mitochondrial cytochrome oxidase I and cytochrome b gene sequences of Lutzomyia (Lu.) ayacuchensis populations from Andean areas of Ecuador and southern Peru where the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and populations from the northern Peruvian Andes, for which transmission of Leishmania by Lu. ayacuchensis has not been reported. The haplotype analyses showed higher intrapopulation genetic divergence in northern Peruvian Andes populations and less divergence in the southern Peru and Ecuador populations, suggesting that a population bottleneck occurred in the latter populations, but not in former ones. Importantly, both haplotype and phylogenetic analyses showed that populations from Ecuador consisted of clearly distinct clusters from southern Peru, and the two populations were separated from those of northern Peru.