RESUMO
BACKGROUND: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. METHODS: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. RESULTS: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. CONCLUSIONS: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.
Assuntos
Leishmania tropica , Leishmaniose Cutânea , Humanos , Citocromos b/genética , DNA Ribossômico , Leishmaniose Cutânea/epidemiologia , Leishmania tropica/genética , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
La leishmaniasis cutánea (LC) es una patología cutánea inmunomediada causada principalmente por Leishmania (L.) major, Leishmania tropica, Leishmania braziliensis, L. mexicana y L. amazonensis. La carga de la LC en términos de morbilidad y estigmas sociales se concentra en ciertos países en desarrollo de Asia, África y Sudamérica. Las personas con LC asintomática representan una gran proporción de los individuos infectados en las zonas endémicas que no presentan ninguna lesión y pueden controlar la infección mediante mecanismos aún no del todo conocidos. En la actualidad, no existe ninguna medida profiláctica de control aprobada para la LC. Este reporte epidemiológico reveló que la LC prevalecía en las áreas geográficas de Selva y Sierra de Perú, mientras que departamentos como Ancash y La Libertad, áreas de la Costa, también estuvieron involucradas en el reporte de casos en los últimos años. El patrón de casos de LC reportados en los distintos departamentos del país tuvo un comportamiento muy parecido en el periodo comprendido entre 2016 y 2021, predominando en las primeras 4 o 5 semanas epidemiológicas la mayor frecuencia de pacientes enfermos con lesiones dérmicas compatibles y corroboradas con LC. La enfermedad es endémica en muchas partes del país, por eso se necesitan más esfuerzos y enfoques integrados para combatir esta patología(AU)
Cutaneous leishmaniasis (CL) is an immune-mediated skin pathology caused mainly by Leishmania (L.) major, Leishmania tropica, Leishmania braziliensis, L. mexicana and L. amazonensis. The burden of CL in terms of disease and social stigma is concentrated in certain developing countries in Asia, Africa, and South America. People with asymptomatic CL represent a large proportion of infected individuals in endemic areas who do not have any lesions and can control the infection through mechanisms that are not fully understood. Currently, there is no approved prophylactic control measure for CL. This epidemiological report revealed that CL was prevalent in the Selva and Sierra geographic areas of Peru, while departments such as Ancash and La Libertad, coastal areas, were also involved in the reporting of cases in recent years. The pattern of CL cases reported in the different departments of the country had a very similar behavior in the period between 2016 and 2021, with the highest frequency of sick patients with skin lesions compatible and corroborated with CL predominating in the first 4 or 5 epidemiological weeks. The disease is endemic in many parts of the country, so more efforts and integrated approaches are needed to combat this pathology(AU)
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Leishmania tropica , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Cutânea/epidemiologia , Peru/epidemiologia , Estudos RetrospectivosRESUMO
Leishmania parasites cause leishmaniasis, one of the most epidemiologically important neglected tropical diseases. Leishmania exhibits a high ability of developing drug resistance, and drug resistance is one of the main threats to public health, as it is associated with increased incidence, mortality, and healthcare costs. The antimonial drug is the main historically implemented drug for leishmaniasis. Nevertheless, even though antimony resistance has been widely documented, the mechanisms involved are not completely understood. In this study, we aimed to identify potential metabolite biomarkers of antimony resistance that could improve leishmaniasis treatment. Here, using L. tropica promastigotes as the biological model, we showed that the level of response to antimony can be potentially predicted using 1H-NMR-based metabolomic profiling. Antimony-resistant parasites exhibited differences in metabolite composition at the intracellular and extracellular levels, suggesting that a metabolic remodeling is required to combat the drug. Simple and time-saving exometabolomic analysis can be efficiently used for the differentiation of sensitive and resistant parasites. Our findings suggest that changes in metabolite composition are associated with an optimized response to the osmotic/oxidative stress and a rearrangement of carbon-energy metabolism. The activation of energy metabolism can be linked to the high energy requirement during the antioxidant stress response. We also found that metabolites such as proline and lactate change linearly with the level of resistance to antimony, showing a close relationship with the parasite's efficiency of drug resistance. A list of potential metabolite biomarkers is described and discussed.
Assuntos
Antimônio/toxicidade , Antiprotozoários/toxicidade , Resistência a Medicamentos , Leishmania tropica/metabolismo , Metaboloma , Metabolismo Energético , Leishmania tropica/efeitos dos fármacos , Pressão Osmótica , Estresse OxidativoRESUMO
We report the case of a patient with cutaneous leishmaniasis who showed a rapidly progressing ulcerative lesion after traveling to multiple countries where different Leishmania species are endemic. Diagnosis of Leishmania tropica, an exotic species in Mexico was established by using serological and molecular tools.
Assuntos
Doenças Transmissíveis Importadas/diagnóstico , Leishmania tropica , Leishmaniose Cutânea/diagnóstico , Doença Relacionada a Viagens , Adulto , Doenças Transmissíveis Importadas/parasitologia , Humanos , Leishmania tropica/genética , Leishmania tropica/imunologia , Leishmaniose Cutânea/parasitologia , MasculinoRESUMO
BACKGROUND: Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. METHODS: All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. RESULTS: Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n = 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n = 48, 53.93%) than L. tropica (n = 4, 4.49%) (Mann-Whitney U test: p < 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. CONCLUSION: L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.
Assuntos
Leishmania major/genética , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Animais , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Doenças Endêmicas , Feminino , Haplótipos , Humanos , Irã (Geográfico) , Leishmania major/isolamento & purificação , Leishmania tropica/isolamento & purificação , Leishmania tropica/ultraestrutura , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/patologia , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , População RuralRESUMO
ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.
Assuntos
Humanos , Animais , Masculino , Feminino , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Leishmania major/genética , População Rural , Haplótipos , Polimorfismo de Fragmento de Restrição , Leishmania tropica/isolamento & purificação , Leishmania tropica/ultraestrutura , Reação em Cadeia da Polimerase , DNA de Protozoário/isolamento & purificação , DNA de Protozoário/genética , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/epidemiologia , Leishmania major/isolamento & purificação , Doenças Endêmicas , Irã (Geográfico)RESUMO
In Sweden, leishmaniasis is an imported disease and its epidemiology and incidence were not known until now. We conducted a retrospective, nationwide, epidemiological study from 1993 to 2016. Probable cases were patients with leishmaniasis diagnoses reported to the Swedish Patient registry, collecting data on admitted patients in Swedish healthcare since 1993 and out-patient visits since 2001. Confirmed cases were those with a laboratory test positive for leishmaniasis during 1993-2016. 299 probable cases and 182 confirmed cases were identified. Annual incidence ranged from 0.023 to 0.35 per 100 000 with a rapid increase in the last 4 years. Of 182 laboratory-verified cases, 96 were diagnosed from 2013 to 2016, and in this group, almost half of the patients were children under 18 years. Patients presented in different healthcare settings in all regions of Sweden. Cutaneous leishmaniasis was the most common clinical manifestation and the majority of infections were acquired in Asia including the Middle East, specifically Syria and Afghanistan. Leishmania tropica was responsible for the majority of cases (42%). A combination of laboratory methods increased the sensitivity of diagnosis among confirmed cases. In 2016, one-tenth of the Swedish population were born in Leishmania-endemic countries and many Swedes travel to these countries for work or vacation. Swedish residents who have spent time in Leishmania-endemic areas, could be at risk of developing disease some time during their lives. Increased awareness and knowledge are needed for correct diagnosis and management of leishmaniasis in Sweden.
Assuntos
Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/parasitologia , Leishmania tropica , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ásia/etnologia , Criança , Pré-Escolar , Doenças Transmissíveis Importadas/diagnóstico , Feminino , Grécia/etnologia , Humanos , Incidência , Lactente , Leishmaniose Cutânea/diagnóstico , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Estudos Retrospectivos , América do Sul/etnologia , Espanha/etnologia , Suécia/epidemiologia , Viagem/estatística & dados numéricos , Trabalho/estatística & dados numéricos , Adulto JovemRESUMO
Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.
Assuntos
Doenças Transmissíveis Importadas/diagnóstico , Leishmania braziliensis/isolamento & purificação , Leishmania tropica/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/veterinária , Análise de Sequência de DNA , Pré-Escolar , Doenças Transmissíveis Importadas/parasitologia , Humanos , Japão , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Nanoporos , Síria , VenezuelaRESUMO
Phlebotominae representa um dos grupos de insetos de grande importância médica, especialmente para o Brasil, estando associado à transmissão de agentes das leishmanioses tegumentar e visceral. A geração de conhecimentos sobre a morfologia e morfometria de ambos, machos e fêmeas de flebotomíneos vetores de leishmaniose tegumentar americana (LTA), poderão indicar estruturas diagnósticas importantes como subsídios para estudos filogenéticos que permitam avaliar a relação vetor -patógeno no processo de coevolução. Com isso o trabalho teve como objetivo analisar comparativamente as seguintes espécies de flebotomíneos: Nyssomyia intermedia, Ny. neivai, Ny. whitmani, Migonemyia migonei e Bichromomyia flaviscutellata, importantes vetores de Leishmania spp., causadores da LTA no Brasil. As análises morfométricas foram capazes de separar os gêneros. A análise comparativa dos flebotomíneos por morfometria geométrica e clássica, permitiu aprofundar a discussão sobre a posição taxonômica de Ny. intermedia e Ny. neivai, consideradas por alguns autores como espécies crípticas, onde houve diferença significativa em várias das estruturas analisadas, como no tamanho do centroide das asas e a razão entre os filamentos genitais e a ponta dos filamentos genitais. Através das análises de morfometria clássica e do tamanho dos centróides e as formas médias das asas, as espécies de Nyssomyia se apresentam mais próximas feneticamente de Mg. migonei, vetores de Leishmania (Viannia) braziliensis, do que de Bi. flaviscutellata, vetora de Leishmania (Leishmania) amazonensis, o que também corrobora com a separação dos gêneros Nyssomyia e Bichromomyia.
Assuntos
Leishmania tropica , Leishmaniose , Psychodidae , BrasilRESUMO
Cutaneous leishmaniasis (CL) is a widespread tropical infection which has a high incidence rate in Iran. Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in various parts of Iran with a high incidence rate. The aim of this study was to evaluate the reappraisal of the diagnosis and epidemiology of CL in Iran, by different clinical, parasitological and molecular assays among patients suspected of CL referred to the Department of Parasitology, at the Pasteur Institute of Iran during 2006-2009. Two hundred samples from patients with ulcerative skin lesions were collected, clinical analyses were applied, data questionnaire was completed and samples were examined for CL by using both direct microscopic and culture methods. Moreover, PCR assay was applied for detection of Leishmania species in CL isolates resulting from parasitological assay. Clinical observation revealed that the majority (58%) of lesions was single; double lesions were observed in 22% of patients, and only 20% of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5%) by direct smear and 40% by cultivation assay. Most patients (21.3%) had a travel history to the Isfahan province, one of the most important endemic areas of CL located in center of Iran. PCR assay by kDNA indicated 32 and 18 out of 50 isolates respectively had similar patterns with standard L. major and L. tropica. In conclusion, clinical manifestations and an appropriate diagnostic assay with a parallel molecular characterization of CL may lead to a screening evaluation of disease, prognosis, treatment and control strategies.