RESUMO
Sequence analysis of a Triatoma dimidiata salivary gland cDNA library resulted in the identification of two transcripts (Td60 and Td101) homologous to triabin, an inhibitor of thrombin in Triatoma pallidipennis saliva. In the present study, a recombinant protein of Td60, designated dimiconin, was expressed in Escherichia coli and its activity was characterized. The resulting protein inhibited the intrinsic but not extrinsic blood coagulation pathway, suggesting that dimiconin is not a thrombin inhibitor. Measurement of the enzymatic activity of coagulation factors using chromogenic substrates revealed that dimiconin efficiently inhibited factor XIIa (FXIIa) activity in a dose-dependent manner. In addition, pre-incubation of dimiconin with FXII effectively inhibited FXIIa activity whereas dimiconin did not affect already activated FXIIa, indicating that dimiconin inhibits the activation of FXII but not the enzymatic activity of FXIIa. These results show that dimiconin is an inhibitor of the contact phase initiated by FXII activation in the blood coagulation cascade, which differs from the bioactivity of triabin.
Assuntos
Anticoagulantes , Coagulação Sanguínea/efeitos dos fármacos , Fator XIIa/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Triatoma/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Doença de Chagas/transmissão , Proteínas de Insetos/genética , Insetos Vetores/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Triatoma/genéticaRESUMO
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.
Assuntos
Leishmania , Leishmaniose Cutânea , Psychodidae , Animais , Humanos , Peru , PrevalênciaRESUMO
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.
Assuntos
Leishmania/classificação , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/patologia , Parasitologia/métodos , Manejo de Espécimes/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Citocromos b/genética , Feminino , Humanos , Lactente , Recém-Nascido , Leishmania/genética , Masculino , Manose-6-Fosfato Isomerase/genética , Pessoa de Meia-Idade , Peru , Proteínas de Protozoários/genética , Adulto JovemRESUMO
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.
RESUMO
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.
Assuntos
Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Phlebotomus/parasitologia , Animais , Equador/epidemiologia , Humanos , Leishmaniose Cutânea/transmissãoRESUMO
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.
Assuntos
Mordeduras e Picadas de Insetos , Insetos Vetores/parasitologia , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Equador/epidemiologia , Humanos , Leishmania mexicana/genética , Leishmania mexicana/isolamento & purificaçãoRESUMO
In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.
Assuntos
Citocromos b/genética , DNA de Protozoário/química , Leishmania/classificação , Filogenia , Animais , Sequência de Bases , Sequência Consenso , Humanos , Leishmania/enzimologia , Leishmania/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Trypanosomatina/classificação , Trypanosomatina/genéticaRESUMO
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.
Assuntos
Núcleo Celular/genética , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/parasitologia , Mitocôndrias/genética , Animais , Pareamento Incorreto de Bases , DNA de Cinetoplasto , Equador , Humanos , Leishmania/isolamento & purificação , Leishmania/fisiologia , Leishmaniose Cutânea/transmissão , Leishmaniose Mucocutânea/transmissão , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Psychodidae/fisiologiaRESUMO
Surveillance of the distribution of sand fly species is important for prediction of the risk and expansion of Leishmania infection in endemic and surrounding areas. In the present study, a simple and reliable method of typing New World Lutzomyia species circulating in endemic areas in Ecuador was established by using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. PCR-RFLP of 18S ribosomal RNA (rRNA) genes with the restriction enzyme AfaI and subsequently HinfI successfully identified seven sand fly species in nine endemic areas in Ecuador. Although intraspecific genetic-diversity affecting the RFLP-patterns was detected in a species, the patterns were species specific. The method promises to be a powerful tool for the classification of New World Lutzomyia species.
Assuntos
Variação Genética , Leishmaniose/epidemiologia , Filogenia , Psychodidae/genética , Animais , Análise por Conglomerados , Primers do DNA/genética , Equador/epidemiologia , Polimorfismo de Fragmento de Restrição/genética , Psychodidae/classificação , RNA Ribossômico 18S/genética , Especificidade da EspécieRESUMO
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.
Assuntos
Leishmania/classificação , Leishmania/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Psychodidae/parasitologia , Animais , Equador/epidemiologia , Feminino , Geografia , Humanos , Masculino , Peru/epidemiologiaRESUMO
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 10â¯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.
Assuntos
Leishmania guyanensis , Leishmaniose Mucocutânea/epidemiologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Citocromos b/genética , Equador/epidemiologia , Feminino , Humanos , Lactente , Leishmania guyanensis/genética , Leishmania guyanensis/isolamento & purificação , Leishmaniose Mucocutânea/transmissão , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.
Assuntos
Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Leishmaniose/transmissão , Reação em Cadeia da Polimerase/métodos , Psychodidae/parasitologia , Animais , Citocromos b/química , Citocromos b/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Equador/epidemiologia , Leishmania/genética , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Insetos Vetores/parasitologia , Leishmania , Leishmaniose Cutânea/epidemiologia , Psychodidae/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Equador/epidemiologia , Feminino , Humanos , Lactente , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/transmissão , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Adulto JovemRESUMO
The saliva of hematophagous arthropods injected during blood feeding contains potent pharmacologically active components to counteract the host hemostatic and inflammatory systems. In the present study, dominant salivary gland transcripts of Panstrongylus chinai, a vector of Chagas disease, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library. This analysis showed that 56.5% of the isolated transcripts coded for putative secreted proteins, of which 73.7% coded for proteins belonging to the lipocalin family. The most abundant transcript of lipocalin family proteins was a homologue of pallidipin 2, an inhibitor of collagen-induced platelet aggregation of Triatoma pallidipennis. In addition, homologues of triafestin, an inhibitor of the kallikrein-kinin system of T. infestans, were identified as the dominant transcript. Other salivary transcripts encoding lipocalin family proteins had homology to triplatin (an inhibitor of platelet aggregation) and others with unknown function. Other than lipocalin family proteins, homologues of a Kazal-type serine protease inhibitor (putative anticoagulant), a hemolysin-like protein (unknown function), inositol polyphosphate 5-related protein (a regulator of membrane phosphoinositide), antigen 5-related protein (unknown function) and apyrase (platelet aggregation inhibitor) were identified.
Assuntos
Vetores de Doenças , Inibidores da Agregação Plaquetária/análise , Agregação Plaquetária/genética , Glândulas Salivares/fisiologia , Proteínas e Peptídeos Salivares/genética , Fatores de Transcrição/genética , Triatoma/genética , Sequência de Aminoácidos , Animais , Doença de Chagas/transmissão , Biblioteca GênicaRESUMO
The dataset in this report is related to the research article with the title: "Salivary gland transcripts of the kissing bug, Panstrongylus chinai, a vector of Chagas disease" (Kato et al., 2017) [1]. Lipocalin family proteins were identified as the dominant component in P. chinai saliva, and phylogenetic analysis of the salivary lipocalins resulted in the formation of five major clades. For further characterization, each clade of P. chinai lipocalin was s alignment and phylogenetic analyses together with homologous triatomine lipocalins; pallidipin 2, an inhibitor of collagen-induced platelet aggregation identified from saliva of Triatoma pallidipennis (clade I), pallidipin-like salivary lipocalin from Triatoma dimidiata (clade II), salivary lipocalin from T. dimidiata (clade III), triatin-like salivary lipocalin identified in the saliva of T. dimidiata (clade IV), and lipocalin-like TiLipo37 from Triatoma infestans (clade V).
RESUMO
A 35-year-old man with a 19-year history of slowly evolving diffuse cutaneous leishmaniasis was treated with oral miltefosine, 50 mg three times a day. The patient responded after four months of miltefosine treatment with clearance of all nodular lesions and plaques from the entire body surface and had negative slit-skin smears and cultures for Leishmania. However, two months after stopping miltefosine, skin lesions reappeared and parasites were observed in samples. The relapsed lesions did not respond to an additional two-month course of miltefosine. No laboratory or clinical adverse events to miltefosine were observed. Parasites from skin lesions were cultured and identified as Leishmania (Leishmania) mexicana by isoenzyme electrophoresis.