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2. Ecological divergence and hybridization of Neotropical Leishmania parasites

Author

Van den Broeck, Frederik, Savill, Nicholas J., Imamura, Hideo, Sanders, Mandy, Maes, Ilse, Cooper, Sinclair, Mateus, David, Jara, Marlene, Adaui, Vanessa, Arevalo, Jorge, Llanos-Cuentas, Alejandro, Garcia, Lineth, Cupolillo, Elisa, Miles, Michael, Berriman, Matthew, Schnaufer, Achim, Cotton, James A., and Dujardin, Jean-Claude

Published
2020

19. Ecological divergence and hybridization of Neotropical Leishmania parasites

Author

Van den Broeck, Frederik, primary, Savill, Nicholas J., additional, Imamura, Hideo, additional, Sanders, Mandy, additional, Maes, Ilse, additional, Cooper, Sinclair, additional, Mateus, David, additional, Jara, Marlene, additional, Adaui, Vanessa, additional, Arevalo, Jorge, additional, Llanos-Cuentas, Alejandro, additional, Garcia, Lineth, additional, Cupolillo, Elisa, additional, Miles, Michael, additional, Berriman, Matthew, additional, Schnaufer, Achim, additional, Cotton, James A., additional, and Dujardin, Jean-Claude, additional

Published
2019
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20. Epítopes clase II de Células T predichos a partir del secretoma de Leishmania braziliensis evocan respuesta de Interferon-gamma en células mononucleares periféricas de sangre, en pacientes con Leishmaniasis cutánea activa y pacientes curados con historia de Leishmaniasis cutánea

Author

Enciso, Javier, H. Gutierrez, Andres, Cruz, María, Liu, Rui, Tassone, Ryan, Falconi-Agapito, Francesca, Terry, Frances, Arévalo, Jorge, S. De Groot, Anne, and Adaui, Vanessa

Subjects
Interferon-gamma, células mononucleares periféricas, Epítopes, Leishmaniasis cutánea, Leishmaniasis cutánea activa, Células T, sangre, Leishmania braziliensis
Abstract

En nuestro país, en la última década, se han detectado 8000 casos por año de leishmaniasis cutánea (LC), siendo Leishmania braziliensis considerada una de las especies más importantes, debido a su prevalencia, asociación a falla terapéutica, y riesgo de generar leishmaniasis mucosa.Actualmente no existe una vacuna efectiva contra LC para uso en humanos, a pesar de los esfuerzos a nivel mundial. Estudios en vacunas basadas en epítopes facilitaron el desarrollo de herramientas inmuno-informáticas para predecir epítopes potencialmente inmunogénicos. Pocos estudios han utilizado métodos inmunoinformáticos con el fin de identificar epítopes de células T, a partir del proteoma de Leishmania, además de su validación experimental. La mayoría de estudios se enfocaron en especies de Leishmania del Viejo Mundo, mientras que pocos en especies del Nuevo Mundo, particularmente L. braziliensis. 2017-09-08

Published
2017

21. Desarrollo de una técnica de diagnóstico molecular de Escherichia coli enterotoxigénica (ETEC) en heces basada en la amplificación isotérmica con recombinasa y polimerasa (RPA)

Author

Cabrera, Luis, Adaui, Vanessa, and Ochoa, Theresa J.

Subjects
recombinasa y polimerasa (RPA), diagnóstico molecular, amplificación isotérmica, Escherichia coli enterotoxigénica|heces
Abstract

Introducción: La Escherichia coli enterotoxigénica (ETEC) causa diarrea en niños menores de 5 años. Su diagnóstico se basa en la detección de las toxinas LT o ST. Actualmente, las técnicas moleculares son usadas, pero su aplicación en campo es difícil. La amplificación isotérmica con recombinasa y polimerasa (RPA) es una técnica reciente que se usa en la detección de varios patógenos. La RPA tiene potencial para su uso en campo porque puede funcionar entre 37° y 42°C, la amplificación es rápida y los reactivos pueden ser almacenados sin refrigeración por varias semanas. Objetivo: Desarrollar un método para el diagnóstico de Escherichia coli enterotoxigénica (ETEC) en muestras de heces mediante la detección simultánea de los genes para las toxinas LT y ST usando la amplificación con recombinasa y polimerasa (RPA). Métodos: Se diseñaron y seleccionaron cebadores o “primers” para los genes que codifican para las toxinas LT y ST (STIa y STIb) adecuados para su detección simultánea por RPA. Los productos de amplificación fueron relevados por electroforesis en gel de agarosa. Los ADN extraídos de la cepa ETEC H10407 y de muestras de heces infectadas con esa cepa fueron usados como control positivo. Las condiciones de reacción óptimas (agitación, temperatura y tiempo) fueron determinadas. La especificidad (ausencia de reacción cruzada con otros patógenos) y la sensibilidad (límite de detección) analíticas fueron evaluadas. Finalmente, se determinó la sensibilidad y la especificidad diagnóstica, y sus intervalos de confianza al 95% (IC 95%), de la técnica de RPA en muestras de heces previamente recolectadas usando la PCR en tiempo real como estándar de oro. Resultados: En total, se diseñaron 32 “primers” para las tres toxinas de ETEC. Para la amplificación con RPA, dos de estos “primers” fueron seleccionados para la toxina LT, mientras que se usaron “primers” previamente reportados para las toxinas STIa y STIb (Guion et al., 2008). Los tres genes pudieron ser amplificados con RPA, pero solo se logró la detección simultánea para LT y STIa. Por otro lado, el paso de agitación no afectó la eficacia de amplificación, la RPA funcionó entre 30 y 50°C y el tiempo mínimo de reacción fue de 5 min. Además, no hubo reacción cruzada con otros enteropatógenos. El límite de detección para los genes de las toxinas LT y STIa fue 10 y 100 UFC/rxn, respectivamente. Finalmente, al evaluar 298 muestras de heces con RPA y PCR en tiempo real, la sensibilidad fue 91.57% (IC 95%: 87.52 – 94.64%) y la especificidad fue 94.59% (IC 95%: 81.81 – 99.34%) para la detección de ETEC. Conclusiones: Se desarrolló una técnica para la detección de ETEC usando RPA. Las condiciones óptimas y los límites de detección están dentro de lo esperado. Las altas sensibilidad y especificidad hacen que la técnica sea adecuada para su uso. Su implementación en campo requiere mejorar la detección del producto amplificado con oligocromatografía. 2017-09-08

Published
2017

23. Influence of Leishmania (Viannia) Species on the Response to Antimonial Treatment in Patients with American Tegumentary Leishmaniasis

Author

Arevalo, Jorge, Ramirez, Luis, Adaui, Vanessa, Zimic, Mirko, Tulliano, Gianfranco, Miranda-Verástegui, César, Lazo, Marcela, Loayza-Muro, Raúl, De Doncker, Simonne, Maurer, Anne, Chappuis, Francois, Dujardin, Jean-Claude, Llanos-Cuentas, Alejandro, Arevalo, Jorge, Ramirez, Luis, Adaui, Vanessa, Zimic, Mirko, Tulliano, Gianfranco, Miranda-Verástegui, César, Lazo, Marcela, Loayza-Muro, Raúl, De Doncker, Simonne, Maurer, Anne, Chappuis, Francois, Dujardin, Jean-Claude, and Llanos-Cuentas, Alejandro

Abstract

Background. Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species. Methods. Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerasechain-reaction restriction fragment—length polymorphism analysis. Results. The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P = .03). Conclusions. Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatment

Published
2017

26. Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused byLeishmania braziliensisin Peru and Bolivia

Author

Adaui, Vanessa, primary, Lye, Lon-Fye, additional, Akopyants, Natalia S., additional, Zimic, Mirko, additional, Llanos-Cuentas, Alejandro, additional, Garcia, Lineth, additional, Maes, Ilse, additional, De Doncker, Simonne, additional, Dobson, Deborah E., additional, Arevalo, Jorge, additional, Dujardin, Jean-Claude, additional, and Beverley, Stephen M., additional

Published
2015
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27. An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis

Author

Cruz, Israel, primary, Millet, Aurélie, additional, Carrillo, Eugenia, additional, Chenik, Mehdi, additional, Salotra, Poonam, additional, Verma, Sandeep, additional, Veland, Nicolás, additional, Jara, Marlene, additional, Adaui, Vanessa, additional, Castrillón, Carlos, additional, Arévalo, Jorge, additional, Moreno, Javier, additional, and Cañavate, Carmen, additional

Published
2013
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28. Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

Author

Jara, Marlene, primary, Adaui, Vanessa, additional, Valencia, Braulio M., additional, Martinez, Dalila, additional, Alba, Milena, additional, Castrillon, Carlos, additional, Cruz, Maria, additional, Cruz, Israel, additional, Van der Auwera, Gert, additional, Llanos-Cuentas, Alejandro, additional, Dujardin, Jean-Claude, additional, and Arevalo, Jorge, additional

Published
2013
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31. Multilocus genotyping reveals a polyphyletic pattern among naturally antimony-resistant Leishmania braziliensis isolates from Peru

Author

Adaui, Vanessa, primary, Maes, Ilse, additional, Huyse, Tine, additional, Van den Broeck, Frederik, additional, Talledo, Michael, additional, Kuhls, Katrin, additional, De Doncker, Simonne, additional, Maes, Louis, additional, Llanos-Cuentas, Alejandro, additional, Schönian, Gabriele, additional, Arevalo, Jorge, additional, and Dujardin, Jean-Claude, additional

Published
2011
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32. Comparative Gene Expression Analysis throughout the Life Cycle of Leishmania braziliensis: Diversity of Expression Profiles among Clinical Isolates

Author

Adaui, Vanessa, primary, Castillo, Denis, additional, Zimic, Mirko, additional, Gutierrez, Andres, additional, Decuypere, Saskia, additional, Vanaerschot, Manu, additional, De Doncker, Simonne, additional, Schnorbusch, Kathy, additional, Maes, Ilse, additional, Van der Auwera, Gert, additional, Maes, Louis, additional, Llanos-Cuentas, Alejandro, additional, Arevalo, Jorge, additional, and Dujardin, Jean-Claude, additional

Published
2011
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35. Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

Author

Moens, Britta, primary, López, Giovanni, additional, Adaui, Vanessa, additional, González, Elsa, additional, Kerremans, Lien, additional, Clark, Daniel, additional, Verdonck, Kristien, additional, Gotuzzo, Eduardo, additional, Vanham, Guido, additional, Cassar, Olivier, additional, Gessain, Antoine, additional, Vandamme, Anne-Mieke, additional, and Van Dooren, Sonia, additional

Published
2009
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37. Influence ofLeishmania (Viannia)Species on the Response to Antimonial Treatment in Patients with American Tegumentary Leishmaniasis

Author

Arevalo, Jorge, primary, Ramirez, Luis, additional, Adaui, Vanessa, additional, Zimic, Mirko, additional, Tulliano, Gianfranco, additional, Miranda‐Verástegui, César, additional, Lazo, Marcela, additional, Loayza‐Muro, Raúl, additional, Doncker, Simonne De, additional, Maurer, Anne, additional, Chappuis, Francois, additional, Dujardin, Jean‐Claude, additional, and Llanos‐Cuentas, and Alejandro, additional

Published
2007
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40. Real-Time PCR Assay for Detection and Quantification of Leishmania(Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters

Author

Jara, Marlene, Adaui, Vanessa, Valencia, Braulio M., Martinez, Dalila, Alba, Milena, Castrillon, Carlos, Cruz, Maria, Cruz, Israel, Van der Auwera, Gert, Llanos-Cuentas, Alejandro, Dujardin, Jean-Claude, and Arevalo, Jorge

Abstract

ABSTRACTEarlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmaniain human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania(Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmaniadetection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PDqPCR assays were highly correlated (r= 0.87; P< 0.0001), but the former showed higher sensitivity (P= 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P= 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P= 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania(Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.

Published
2013
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41. Evaluation of a Microculture Method for Isolation of LeishmaniaParasites from Cutaneous Lesions of Patients in Peru

Author

Boggild, Andrea K., Miranda-Verastegui, Cesar, Espinosa, Diego, Arevalo, Jorge, Adaui, Vanessa, Tulliano, Gianfranco, Llanos-Cuentas, Alejandro, and Low, Donald E.

Abstract

ABSTRACTTraditional culture of Leishmaniaspp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-µl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, <0.001). The mean times to culture positivity were 4.2 days by microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmaniaparasites from cutaneous lesions than traditional culture.

Published
2007
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43. Influence of Leishmania (Viannia) Species on the Response to Antimonial Treatment in Patients with American Tegumentary Leishmaniasis

Author

Arevalo, Jorge, Ramirez, Luis, Adaui, Vanessa, Zimic, Mirko, Tulliano, Gianfranco, Miranda-Verástegui, César, Lazo, Marcela, Loayza-Muro, Raúl, De Doncker, Simonne, Maurer, Anne, Chappuis, Francois, Dujardin, Jean-Claude, Llanos-Cuentas, Alejandro, Arevalo, Jorge, Ramirez, Luis, Adaui, Vanessa, Zimic, Mirko, Tulliano, Gianfranco, Miranda-Verástegui, César, Lazo, Marcela, Loayza-Muro, Raúl, De Doncker, Simonne, Maurer, Anne, Chappuis, Francois, Dujardin, Jean-Claude, and Llanos-Cuentas, Alejandro

Abstract

Background. Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species. Methods. Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerasechain-reaction restriction fragment—length polymorphism analysis. Results. The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P = .03). Conclusions. Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatment

44. Toxicity assessment of synthetic chalcones with antileishmanial potential in BALB/c mice.

Author

Cancino K, Castro I, Yauri C, Jullian V, Arévalo J, Sauvain M, Adaui V, and Castillo D

Subjects
Animals, Mice, Mice, Inbred BALB C, Antiprotozoal Agents therapeutic use, Chalcone, Chalcones toxicity, Leishmaniasis
Abstract

Objective: To evaluate the toxicity of three synthetic chalcones administered intraperitoneally to BALB/c mice., Materials and Methods: The median lethal dose (LD50) was estimated by Dixon's Up-and-Down method. Subchronic toxicity of chalcones was evaluated at 20 and 40 mg/kg for 21 days. Behavioral, physiological, biochemical, and histological toxic effects were evaluated., Results: Chalcone 43 produced mucus in feces, visceral damage (liver) and alterations in organ coefficient (kidney, p = 0.037 and brain, p = 0.008) when compared to the control group. In addition, histological analysis showed that this chalcone produced edema, inflammation and necrosis in the evaluated organs, although there was no significant difference with the control. None of the biochemical parameters differed significantly between the treatment groups at 40 mg/kg dose and the control., Conclusions: The LD50 for all three chalcones was greater than 550 mg/kg of body weight. Chalcones 40 and 42 were found to be relatively non-toxic. Both can be considered safe for intraperitoneal application in BALB/c mice and, consequently, are potential candidates for use in the treatment of leishmaniasis.

Published
2021
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45. A novel marker, ARM58, confers antimony resistance to Leishmania spp.

Author

Nühs A, Schäfer C, Zander D, Trübe L, Tejera Nevado P, Schmidt S, Arevalo J, Adaui V, Maes L, Dujardin JC, and Clos J

Abstract

Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.

Published
2013
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