Leishmania Research Articles

Investigating in vitro anti-leishmanial effects of silibinin and silymarin on Leishmania major

Cutaneous leishmaniosis is an important zoonotic disease caused by various Leishmania species. The aim of this study was to investigate the effects of silibinin and silymarin on the in vitro growth and proliferation of promastigotes and amastigotes of Leishmania major compared to glucantime-treated parasites. The promastigotes and amastigotes of this parasite were treated with the two drugs, silibinin and silymarin, in several concentrations (25–100 μM). The highest effect on promastigotes was for silymarin in concentration of 100 μM with 90% and 91% death rate at hours 48 and 72, respectively. Regarding amastigotes, the highest effect at 48 hours was for silibinin in concentration of 100 μM with 35% death rate. However, at 72 hours, silymarin showed the highest effect with 63% death rate in concentration of 100 μM. The highest observed maximal 50% lethal concentration (LC50) for promastigotes was for silymarin with 19.34 μM at 48 hours and 18.22 μM at 72 hours. Likewise, maximal LC50 for amastigotes was for silymarin with 191 μM at 48 hours and 24.27 μM at 72 hours. Our findings demonstrated that both medications have suitable effects like Glucantime® on the parasite in vitro. Therefore, clinical assessment of the anti-leishmanial activity of silibinin and silymarin for treating the dermal lesions caused by L. major is recommended.

Leishmania amazonensis isolated from human visceral leishmaniasis: histopathological analysis and parasitological burden in different inbred mice.

Leishmania amazonensis is a major etiological agent of human cutaneous leishmaniasis in the Americas; nevertheless there are some reports of this species causing visceral disease in dogs and men. In the present work we have studied a Leishmania strain isolated from a human case of visceral leishmaniasis. We have infected different mouse strains and analyzed the development of the disease, studying the parasite's ability to visceralize and whether this ability is influenced by host genetics. Female BALB/c, C57BL/6, C57BL/10, CBA, DBA/2, and C3H/He mice were subcutaneously infected with 10⁴ L. amazonensis amastigotes. BALB/c, C57BL/6 and C57BL/10 mice were found to be very susceptible to infection, showing lesions that developed to necrosis and ulceration. CBA mice developed a late but severe lesion. DBA/2 mice developed only discrete lesions, while C3H/He mice did not develop any lesions. All mouse strains except C3H/He showed some degree of visceralization, presenting parasites in the spleen, while BALB/c, C57BL/6 and CBA presented parasites also in the liver. Moreover, most of the strains presented high parasite load at the infection site, whereas DBA and C3H/He mice showed low or no parasite load 90 days after infection, respectively. Histopathology corroborates the results, showing that susceptible mice presented an inflammatory reaction with parasites in the skin, lymph nodes and spleen, while strains that are more resistant presented low parasitism and discrete inflammatory reaction. Results indicate that this isolate is extremely virulent, can easily visceralize and that the pathogenesis of leishmaniasis is, at least in part, related to the genetic background of the host.

Evaluation of Leishmania major infectivity preserved in RPMI1640 media containing human urine in vivo

فعالية طفيلي اللشمانيا تنخفض من خلال تمريره وحفظه في الاوساط الزرعية. يستعمل الوسط الزرعي الحاوي على 10% ادرار الانسان بدلا من مصل جنين البقر لصعوبة الحصول عليه وثمنه المرتفع, وان الوسط البديل يبقي الطفيلي محافظا على فعاليته بعد تمريره في هذا الوسط ولعشرة تمريرات من خلال احداث الاصابة التجريبية في الحيوانات المختبرية لثمانية مجاميع من الفئران وبلوغه الاحشاء الداخلية لها (الطحال) في اليوم 30بعد الاصابة, فقد لوحظ عدم وجود فرق احصائي معنوي على مستوى ( ( 0.05> p في وسادة القدم للمجاميع المصابة بعد التمرير الثالث الى التمرير العاشر الوسط الزرعية المقارنة مع حيوانات السيطرة المصابة بعد التمرير الاول من عزل الطفيلي على الوسط الزرعي الحاوي على مصل جنين البقر. لم يلاحظ وجود فرق معنوي على مستوى ( ( 0.05> p في اعداد الطفيلي(Leishmania donovani bodies ) في طبعات الطحال بين المجاميع الثمانية المصابة بالمقارنة مع حيوانات السيطرة وهذا يشير الى عدم تغيير فعالية الطفيلي بالرغم تمريره بالوسط الزرعي .الهدف من هذه الدراسة هو توفير وسط بديل عن مصل جنين البقر رخيص الثمن ومتوفر ويحافظ على فعالية وحيوية الطفيلي دون الحاجة الى تنشيطه بعد تكرار تمريره بالوسط الزرعي وهذا مهم في البحوث المختبرية الهادفة لإيجاد لقاح او علاج حالات الاصابة بالطفيلي

Epidemiology of Infantile Visceral Leishmaniasis in Western Algerian And The Convenience of Serum For The Disease Diagnosis by PCR and Immunochromatography.

Epidemiological situation of infantile visceral leishmaniasis (IVL), which is a public health problem in Algeria, is almost unknown in the cities of Western part of the country. The aim of this study was to analyze the epidemiological, clinical, biological, therapeutic, and evolutionary aspects of IVL in Western Algeria, to evaluate the performance of the immunochromatography as a rapid diagnostic test of the disease, and to propose a diagnosis approach by real-time polymerase chain reaction (RT-PCR) assay from the serum. This prospective study was performed on 63 suspicious cases of visceral leishmaniasis collected from the infectious diseases department at the Pediatric Hospital of Oran from January 2012 to July 2017. For each patient, the epidemiological parameters, and the clinical and biological data were collected. Bone marrow and blood samples were drawn from all cases. Bone marrow was performed to research amastigote forms of Leishmania and to identify the species by PCR-sequencing. Blood samples were used to detect anti-Leishmania antibodies as well as parasite DNA. Patients from the Western regions were mostly from rural areas. Sensitivity of RT-PCR from the bone marrow and from serum was 95.45% and 94.44%, respectively. The immunochromatography allowed the disease’s diagnosis for 11 cases whose myelogram did not confirm the presence of the amastigote forms of Leishmania. Immunochromatography was revealed to be a good technique for disease diagnosis regarding the strongly evocative clinical signs. The results also suggest the interest of the RT-PCR assay from patient serum as a non-invasive sample, in the detection of parasite DNA.

Rural area of the Brazilian Pantanal wetlands associated with the occurrence of anti-<i>Leishmania</i> spp. antibodies in dogs

Neste trabalho foi realizada uma avaliação sobre a presença de anticorpos anti-Leishmania infantum chagasi em cães domésticos das áreas urbanas e rurais da região do Pantanal brasileiro usando técnicas sorológicas. Um total de 429 cães foram amostrados em três áreas do bioma do Pantanal, incluindo os municípios de Poconé, Santo Antônio de Leverger e Barão de Melgaço, em Mato Grosso, e o município de Corumbá, em Mato Grosso do Sul. A reação de imunofluorescência indireta (RIFI) foi utilizada para detectar anticorpos (ponto de corte de 40) de Leishmania infantum chagasi como antígeno. Devido à possibilidade de reação cruzada entre as espécies do gênero Leishmania, as amostras positivas na RIFI para L. infantum chagasi foram também avaliadas na RIFI utilizando L. amazonensis e L. braziliensis como antígenos. As amostras positivas na RIFI para L. infantum chagasi foram avaliadas utilizando o ensaio de imunoadsorção ligado à enzima (ELISA). Os resultados mostraram a presença de anticorpos contra L. infantum chagasi em 23 (5,36%; IC 95%: 3,50% -8,05%) cães e pelo menos um cão soro-reativo foi encontrado em todos os municípios avaliados neste estudo. Os títulos de anticorpos variaram de 40 a 5.120 e todas as amostras positivas na RIFI foram positivas no ELISA. Entre os 23 cães positivos, nove também reagiram para L. amazonensis e L. braziliensis. A ocorrência de anticorpos anti-L. infantum chagasi em cães foi maior nas áreas rurais (7,06%) do que nas áreas urbanas (2,50%) (P < 0,05). Com base neste estudo, concluímos que cães de áreas rurais do Pantanal tiveram contato com espécies de Leishmania, o que é uma informação relevante, dada a sua importância para a saúde pública.

Curvas de fusión de regiones genómicas específicas: una herramienta prometedora para el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia.

Introducción. La leishmaniasis cutánea es una enfermedad causada por parásitos del género Leishmania que tiene gran incidencia en Colombia. El diagnóstico y la identificación de la especie infecciosa son factores críticos en el momento de escoger e iniciar el tratamiento. Actualmente, los métodos de diagnóstico y tipificación requieren procedimientos complejos, por lo que es necesario validar nuevos marcadores moleculares y métodos que simplifiquen el proceso.Objetivo. Desarrollar una herramienta basada en la reacción en cadena de la polimerasa (PCR) con curvas de fusión (High Resolution Melting; PCR-HRM) para el diagnóstico y tipificación de las tres especies de Leishmania de importancia epidemiológica en casos de leishmaniasis cutánea en Colombia.Materiales y métodos. Los genomas de Leishmania panamensis, L. braziliensis y L. guyanensis se compararon mediante métodos bioinformáticos. Las regiones específicas de especie identificadas se validaron mediante PCR. Para los marcadores seleccionados se diseñó una PCR-HRM y se estimaron algunos parámetros de validez y seguridad usando aislamientos de pacientes colombianos caracterizados previamente mediante PCR y análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism - RFLP; PCR-RFLP) del gen hsp70.Resultados. El análisis genómico comparativo mostró 24 regiones específicas de especie. Sin embargo, la validación mediante PCR solo identificó un marcador específico para cada especie de Leishmania. Los otros marcadores mostraron amplificación cruzada. El límite de detección para los tres marcadores seleccionados fue de un parásito, mientras que la sensibilidad, la especificidad, el valor predictivo positivo y el negativo fueron de 91,4, 100, 100 y 75 %, respectivamente.Conclusiones. Las tres regiones seleccionadas pueden emplearse como marcadores moleculares en el diagnóstico y tipificación de las especies causantes de la leishmaniasis cutánea en Colombia.

Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses.

The use of natural products as adjuvants has emerged as a promising approach for the development of effective vaccine formulations. Pentalinonsterol (PEN) is a recently isolated compound from the roots of Pentalinon andrieuxii and has been shown to possess antileishmanial activity against Leishmania spp. The objective of this study was to examine the immunomodulatory properties of PEN and evaluate its potential as an adjuvant. Macrophages and bone-marrow-derived dendritic cells (BMDCs) were stimulated with PEN and tested for gene expression, cytokine production, and their ability to activate T cells in vitro. PEN was also evaluated for its ability to generate antigen-specific Th1 and Th2 responses in vivo, following ovalbumin (OVA) immunization using PEN as an adjuvant. The results obtained demonstrate that PEN enhances the expression of NF-κB and AP1 transcription factors, promotes gene expression of Tnfα, Il6, Nos2, and Arg1, and upregulates MHCII, CD80, and CD86 in macrophages. PEN also enhanced IL-12 production in BMDCs and promoted BMDC-mediated production of IFN-γ by T cells. Further, mice immunized with OVA and PEN showed enhanced antigen-specific Th1 and Th2 cytokines in their splenocytes and lymph node cells, as well as increased levels of IgG1 and IgG2 in their sera. Taken together, this study demonstrates that PEN is a potent immunomodulatory compound and potentially can be used as an adjuvant for vaccine development against infectious diseases.

Evaluation of the Cytotoxicity Effect of Chaerophyllum Extract on Leishmania major and J774 Cell Line in Vitro

Evaluation of the Cytotoxicity Effect of Chaerophyllum Extract on Leishmania major and J774 Cell Line in Vitro

A Case of African Visceral Leishmaniasis (VL) Treated with a Combination of Liposomal Amphotericin-B and oral Miltefosine

A 36-year-old Nigerian man was assessed for chronic recurrent fever, generalized weakness, body aches, epistaxis and bleeding gums of 3-month duration. Despite receiving treatment for suspected malaria twice and undergoing multiple courses of antibiotics for suspected infections, his symptoms persisted. Examination revealed hepatosplenomegaly, lymphadenopathy, and severe pallor. Investigations revealed severe anemia, pancytopenia, raised globulin levels. Bone marrow biopsy revealed Leishmania Donovani (LD) bodies. He received treatment with a short duration of high-dose liposomal Amphotericin B followed by outpatient oral Miltefosine to which he responded successfully.

Estudios sobre la sensibilidad cutánea en la leishmaniosis tegumentaria en Costa Rica

This paper presents some aspects rdating the cutaneous activity of leishmaniasis in Costa Rica. The A. considers in the first place the bahavior of the antigens of Leishmania brasiliensis and L. enriettii with relation to cutaneom sensitivity. He made a total of 158 intradermal-reactions using both antigenes in order to compare the antigenic re!ationship between the two species of the genus Leishmania . He found that the cutaneous activity was manifested with equal sensitivity to both antigens. The allergic reaction was greater with the Leishmania brasiliensis antigen in 37 per cent of the cases. The cutaneous sensitivity was equal in 61 per cent of the cases and only 2 per cent the allergic response was higher for the Leishmania enriettii antigen. The A. also noted among the local reactions that all the patients treated with the Leishmania brasiliensis antigen cxperienced a greater itching than those treated with the Leishmania enriettii antigen. The positivity to the heterologous antigen showd that the Leishmania enriettii presents common antigens with the L. brasiliensis and that these are responsible for the crossed-reactions in the cases of the leishmaniasis of the skin. These are same facts that have been observe in the other species of leishmanias which are parasitic to man. Another point considered is the early beginning and the duration of the skin sensitivity. He found in 20 patientes with lesions of 8 days to one month duration, that 89 per cent showed a positive reaction. In two cases in which the innoculation was done experimentaly with Leishmania brasiliensis , he found that the beginning of the cutaneous sensitivity is independent of the incubation period and the time of evolution and is related only to the allergic background of each particular person. The A. observed also that in Costa Rica, as in other countries in America, the sensitivity which is acquired with leishmaniasis persists for the rest of the life and is manifested with a positive skin-reaction regardless of the last period of activity. He made intradermal-reactions in patients with cutaneous and muco-cutaneous lesions and found that the most positive reactions were given by patients with muco-cutaneous lesions. The A. made skin-reactions in 13 patients with skin diseases of other etiology without obtaining positive reactions. In 131 patients without history of leishmaniasis but living in endemic areas he found 3 who gave positive skin-reactions. The A. thinks, as Pessoa (23), that those persons had a sub-clinical infection which did not give a skin evidence but an allergic one. T he A. found a sensitivity of 96 per cent in a group of 71 patients whose clinical diagnosis was laishmaniasis. He observed false negative reactions in patients with viral diseases such as measles, and probably yellow fever, varicela and mumps. Finaly, he studied the cutaneous reactivity in the geographical regio n of Moravia de Chirripo and he found an allergic index of 37,74 per cent. Most of the reactors were from Pacuare; in this place the index was as high as 44 per cent. This is the most important endemic area in Moravia.

The Physiology of Leishmania.

The metabolism of all members of the genus Leishmania is characterized by aerobic fermentations, which lead to the excretion of various partly oxidized metabolic endproducts. The flagellates utilize several carbohydrates freely, but hardly anything is known about the intermediate carbohydrate metabolismo At suitably low pH they are able to consume intermediates of the Krebs cycle, but the presence of a functional tricarboxylic acid cycle has not yet been demonstrated conclusively. Recent data on lipid metabolism and biosynthesis have revealed interesting paralleles to phytoflagellates, as have newer studies on nucleic acids. The composition of the proteins of Leishmania is essentially unknown, but the fact that under certain experimental conditions numerous amino acids are leaked into the medium, indicates that probably all common amino acids are leaked into the medium, indicates that probably all common amino acids are present. The respiration of the flagellates is probably characterized by heavy metal catalysis, but it is uncertain whether or not they contain a fully functional cytochrome system.

Especies de flebotomíneos (Diptera: Psychodidae) recolectados en reservas naturales de las regiones del Darién y del Pacífico en Colombia

Introduction: The departments of Choco and Antioquia in Colombia show climatic and vegetation conditions favoring the establishment of vector species of the genus Lutzomyia and the transmission of Leishmania spp. to human populations entering conserved forest environments. Objective: To report the species of Phlebotomine sandflies present in three natural reserves in the Darien and Pacific regions of Colombia. Materials and methods: Sand flies were collected specifically in the natural reserves El Aguacate (Acandi, Choco), Nabuga (Bahia Solano, Choco) and Tulenapa (Carepa, Antioquia). Sand flies were collected with CDC light traps, active search in resting places and Shannon traps. The taxonomic determination of species was based on taxonomic keys. For some species of taxonomic interest, we evaluated the partial sequences of the 5’ region of COI gene. Results: A total of 611 adult sand flies were collected: 531 in Acandi, 45 in Carepa and 35 in Bahia Solano. Seventeen species of the genus Lutzomyia , three of the genus Brumptomyia and one of the genus Warileya were identified. The genetic distances (K2P) and grouping supported (>99%) in the neighbor joining dendrogram were consistent for most established molecular operational taxonomic units (MOTU) of the Aragaoi group and clearly confirmed the identity of Lu. coutinhoi . Conclusion: Species that have importance in the transmission of leishmaniasis in Acandi, Bahia Solano and Carepa were identified. The presence of Lu. coutinhoi was confirmed and consolidated in Colombia.

Laboratory-acquired blood-borne parasites from accidental exposure

Background Infection caused by blood protozoa is becoming very common, especially in developed countries because of immunosuppressed persons and travelers. Therefore, this review aimed to illustrate the dangers of blood protozoa exposure among persons working in medical laboratories, especially when dealing with samples containing blood protozoa such as Babesia, Leishmania, Plasmodium, and Trypanosoma spp. Conclusion As protozoa multiply rapidly in humans even with a small inoculum, they can cause illness, in contrast to helminths. Diagnoses of infection can be confirmed through dermal scraping, examination of the stained slides by light microscopy, obtaining biopsy specimens for thin, stained smear, and needle aspiration for cultures of Leishmania spp. In addition, protocols should be provided for handling specimens that could carry viable organisms, using protective laboratory equipment.

Cutaneous Leishmania in Wadi Hadramout, Yemen

Cutaneous Leishmania in Wadi Hadramout, Yemen

Visceral Leishmaniasis with renal involvement or Systemic lupus Erythematosus: Clinicians dilemma

Indian subcontinent is responsible for the largest proportion of global Visceral Leishmaniasis. 35 year old man watchman by occupation hailing from Nepal came with complaints of high grade fever on and off since two months. He also had pedal oedema, breathlessness, oliguria and distension of abdomen. On examination patient had pallor, pedal oedema, ascites and splenomegaly. His investigations showed proeteinuria (1.5 gm/day), raised Creatinine and pancytopenia. Bone marrow aspiration showed multiple Leishmania Donovani(LD) bodies, amastigote form scattered intracellular/extracellular form. Bone marrow Biopsy was suggestive of normocellular marrow with histiocytic infiltration with LD bodies. Patient was treated with Amphotericin B infusions. Patients condition deteriorated due to amphotericin induced nephrotoxicity. Report of ANA +2 (1:160) nucleolar was then received. Patient was given methyl prednisolone injection 1 gm /day for 5 days. Patient succumbed to hypo-proteinemia, sepsis and renal impairment. Visceral Leishmaniasis and Systemic Lupus Erythematosus (SLE) with flare both can mimic each other due to overlapping clinical features.SLE can have simultaneous infection with VL in endemic areas. A clinician will have to use is detailed history, thorough examination, battery of laboratory investigations and his clinical acumen to arrive at diagnosis and rule out the mimicker.

Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

Resequencing and assembly of seven complex loci to improve the leishmania major (Friedlin strain) reference genome

Resequencing and assembly of seven complex loci to improve the leishmania major (Friedlin strain) reference genome

Leishmania disease gap analysis study – Pakistan

Leishmania disease gap analysis study – Pakistan

Macrophage cell responses in cutaneous leishmaniasis caused by Leishmania donovani

Macrophage cell responses in cutaneous leishmaniasis caused by Leishmania donovani

Molecular characterization of Leishmania infantum strains by kinetoplast DNA RFLP-PCR.

Multilocus enzyme electrophoresis is the tool most frequently used to classify Leishmania spp., although it is time consuming and, sometimes, a not enough discriminative method. In the present study a kinetoplast DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to characterize 16 zymodeme MON-1 Leishmania infantum strains: 15 were from dogs housed in public kennels of 7 geographical areas in the Emilia-Romagna region, Northern Italy, 1 was the L. infantum reference strain MHOM/TN/1980/IPT1. Six enzymatic patterns were observed. Kinetoplast DNA RFLP-PCR confirmed to have a good discriminatory power within the same zymodeme and proved to be useful for comparing few strains or discriminating between relapse and reinfection in the same host. We therefore recommend it use for discriminating between relapse and reinfection in the same host rather than supporting large-scale epidemiological studies.