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    Presence of (A) 18S rRNA gene and (B) cyt b gene in skin and whole blood samples of patients with suspected cutaneous leishmaniasis. In (A), lane 1 = 100–base pair (bp) molecular marker; lane 2 = positive control (Leishmania equatorensis DNA); lines 3–5 = skin (s) and blood (b) samples; lane 6 = negative control. In (B), lane 1 = 100-bp molecular marker; lines 2–8 = skin (s) and blood (b) samples; lane 9 = negative control; lane 10 = positive control (L. equatorensis DNA). Number indicates the number of the patient in the study.

  • 1.

    Llanes A, Restrepo CM, del Vecchio G, Anguizola FJ, Lleonart R, 2015. The genome of Leishmania panamensis: insights into genomics of the L. (Viannia) subgenus. Sci Rep 5: 8550.

    • Search Google Scholar
    • Export Citation
  • 2.

    Reveiz L, Maia-Elkhoury AN, Nicholls RS, Romero GA, Yadon ZE, 2013. Interventions for American cutaneous and mucocutaneous leishmaniasis: a systematic review update. PLoS One 8: e61843.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ministerio de Salud Pública del Ecuador (MSP), 2013. Manual de procedimientos del Subsistema alerta acción SIVE-ALERTA. Available at: https://aplicaciones.msp.gob.ec/salud/archivosdigitales/documentosDirecciones/dnn/archivos/manual_de_procedimientos_sive-alerta.pdf. Accessed January 11, 2016.

    • Search Google Scholar
    • Export Citation
  • 4.

    Von Stebut E, 2015. Leishmaniasis. JDDG 13: 191201.

  • 5.

    Hartley MA, Drexler S, Ronet C, Beverley SM, Fasel N, 2014. The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis. Trends Parasitol 30: 412422.

    • Search Google Scholar
    • Export Citation
  • 6.

    Luna AM, Michelena MA, Fleming C, Arena G, Chiavassa AM, Rafti P, 2012. Mucocutaneous leishmaniasis with laryngeal involvement. Med Cutan Ibero Lat Am 40: 154157.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kato H, Calvopiña M, Criollo H, Hashiguchi Y, 2013. First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species. Acta Trop 128: 710713.

    • Search Google Scholar
    • Export Citation
  • 8.

    Calvopina M, Armijos R, Marco J, Uezato H, Kato H, Gómez E, Korenaga M, Barroso P, Mimori T, Cooper P, Nonaka S, Hashiguchi Y, 2006. Leishmania isoenzyme polymorphisms in Ecuador: relationships with geographic distribution and clinical presentation. BMC Infect Dis 6: 136139.

    • Search Google Scholar
    • Export Citation
  • 9.

    Mouttaki T, Morales-Yuste M, Merino-Espinosa G, Chiheb S, Fellah H, Martin-Sanchez J, Riyad M, 2014. Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays. Parasit Vectors 7: 420.

    • Search Google Scholar
    • Export Citation
  • 10.

    Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL, 2006. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44: 14351439.

    • Search Google Scholar
    • Export Citation
  • 11.

    Vergel C, Palacios R, Cadena H, Posso CJ, Valderrama L, Perez M, Walker J, Travi BL, Saravia NG, 2006. Evidence for Leishmania (Viannia) parasites in the skin and blood of patients before and after treatment. J Infect Dis 194: 503511.

    • Search Google Scholar
    • Export Citation
  • 12.

    Venazzi EA, Roberto AC, Barbosa-Tessmann IP, Zanzarini PD, Lonardoni MV, Silveira TG, 2007. Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis. Exp Parasitol 115: 399402.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ontario Public Health, 2014. Specimen Collection Guide for Cutaneous Leishmaniasis. Available at https://www.publichealthontario.ca/en/eRepository/Specimen_collection_guide_cutaneous_leishmaniasis.pdf. Accessed January 18, 2016.

    • Search Google Scholar
    • Export Citation
  • 14.

    Nzelu CO, Gomez EA, Cáceres AG, Sakurai T, Martini-Robles L, Uezato H, Mimori T, Katakura K, Hashiguchi Y, Kato H, 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop 132: 16.

    • Search Google Scholar
    • Export Citation
  • 15.

    Foulet F, Botterel F, Buffet P, Morizot D, Rivollet D, Deniau M, Pratlong F, Costa JM, Bretagne S, 2007. Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome b gene. J Clin Microbiol 45: 21102115.

    • Search Google Scholar
    • Export Citation
  • 16.

    Yang B-B, Chen D-L, Chen J-P, Liao L, Hu X-S, Xu J-N, 2013. Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference. Parasit Vectors 6: 32.

    • Search Google Scholar
    • Export Citation
  • 17.

    Romero GA, Guerra MV, Paes MG, Macedo VO, 2001. Comparison of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis and L. (V.) guyanensis in Brazil: therapeutic response to meglumine antimoniate. Am J Trop Med Hyg 65: 456465.

    • Search Google Scholar
    • Export Citation
  • 18.

    Bañuls AL, Hide M, Prugnolle F, 2007. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol 64: 1109.

    • Search Google Scholar
    • Export Citation
  • 19.

    Couppié P, Clyti E, Sobesky M, Bissuel F, Del Giudice P, Sainte-Marie D, Dedet JP, Carme B, Pradinaud R, 2004. Comparative study of cutaneous leishmaniasis in human immunodeficiency virus (HIV)-infected patients and non-HIV-infected patients in French Guiana. Br J Dermatol 151: 11651171.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mandal G, Mandal S, Sharma M, Charret KS, Papadopoulou B, Bhattacharjee H, Mukhopadhyay R, 2015. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl Trop Dis 9: e0003500.

    • Search Google Scholar
    • Export Citation
  • 21.

    Neitzke-Abreu HC, Venazzi MS, Bernal MV, Reinhold-Castro KR, Vagetti F, Mota CA, Silva NR, Aristides SM, Silveira TG, Lonardoni MV, 2013. Detection of DNA from Leishmania (Viannia): accuracy of polymerase chain reaction for the diagnosis of cutaneous leishmaniasis. PLoS One 8: e62473.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Diagnostic Efficacy of Molecular Techniques for Detection and Identification of Leishmania Species in Human Whole Blood and Skin Samples from Ecuador

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  • 1 Centro de Investigación Traslacional, Universidad de Las Américas (CIT-UDLA), Quito, Ecuador.
  • 2 Instituto Ecuatoriano de Seguridad Social, Quito, Ecuador.

Microscopic examination is the standard method for diagnosis of cutaneous and mucocutaneous leishmaniasis despite its low sensitivity. This study compared the diagnosis efficacy of microscopic examination versus polymerase chain reaction (PCR)–based methods and DNA sequencing using whole blood and skin lesion samples from patients with suspected leishmaniasis. The presence of Leishmania was determined by microscopy and amplification of 18S ribosomal RNA gene from blood and skin samples of 22 patients. Twenty individuals were positive for leishmaniasis. Microscopic analysis identified 85%, whereas PCR identified 100% of positive cases from skin and 90% from blood. Cytochrome b gene (cyt-b) amplification and sequencing identified Leishmania guyanensis, Leishmania shawi, and Leishmania naiffi from skin and blood samples. This study demonstrated the usefulness of whole blood and molecular techniques for the diagnosis and species identification of leishmaniasis.

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus.1 Leishmaniasis has become an important public health problem in tropical and subtropical regions of the world.2 In Ecuador, leishmaniasis infection has been reported in 23 of the 24 provinces (excluding only the Galápagos islands).3 The clinical presentation and disease progression of leishmaniasis depend on the causative species and the immune status of the patient.4 In Ecuador, human cutaneous and mucocutaneous leishmaniasis (CL and MCL, respectively) and their variations have been confirmed.3 CL is a common form of the disease characterized by the appearance of a localized skin lesion at the site of the vector-bitten area.5 In 5–20% of CL, the infection progresses to MCL, due to the dissemination of the parasite in the bloodstream, the lymphatic system, or by direct extension to mucosal membranes.6 A total of 15 species of Leishmania have been identified as responsible for the infection of humans.2 In Ecuador, only Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania major-like, Leishmania mexicana, Leishmania panamensis, and Leishmania naiffi have been identified.7,8

Traditionally, microscopic examination has been considered the standard method for diagnosis of CL and MCL. However, despite its high specificity (100%),9 its sensitivity is low (49–74.4%).10 More importantly, microscopy cannot identify specific Leishmania species causing the infection.9 More sensitive polymerase chain reaction (PCR)–based methods have been developed as an alternative for diagnosis and identification of Leishmania species in clinical samples.10 PCR-based diagnosis has been used with cutaneous lesions and isolated white blood cells,11,12 but not with whole blood.

Given the limitations of standard methods for the diagnosis and identification of Leishmania species in clinical samples, we compared the diagnostic efficacy of traditional microscopy to PCR-based methods and DNA sequencing using whole blood and skin samples from patients with suspected leishmaniasis. Twenty-two patients with clinical suspicion of CL were included in this study. Patients were recruited at a public clinic within a rural area endemic for leishmaniasis in “Pedro Vicente Maldonado” (0°08′31″, −79°05′33″) and a military reference hospital “Hospital de Especialidades de las Fuerzas Armadas No. 1” in Quito. All participants were tested by microscopy and molecular techniques. Samples were collected from cutaneous lesions according to the specimen collection guide for cutaneous leishmaniasis.13 In addition, 3 mL of venous blood was collected in tubes with ethylenediaminetetraacetic acid (Becton Dickinson, Franklin Lakes, NJ). Cutaneous and blood samples were stored at 4°C and processed within 24 hours of collection. DNA from cutaneous lesions and blood was isolated with the High Pure PCR Template Preparation Kit (Roche®, Quito, Ecuador). To diagnose leishmaniasis infection, PCR amplification of the 18S ribosomal RNA (rRNA) gene was performed as reported previously.14

Of the 22 recruited patients, only 10 had not received treatment of leishmaniasis at the time of sample collection. Table 1 compares the results of diagnosis for leishmaniasis by microscopy and by PCR amplification of the 18S rRNA gene from skin lesions. Data showed that seventeen skin lesions were positive for leishmaniasis by microscopy and PCR analysis. On the other hand, three skin lesions were positive by PCR and negative by microscopy. Finally, two skin samples were negative for both PCR and microscopy; these two patients were not under antileishmaniasis treatment at the time of recruitment. According to this table and considering microscopy as the gold standard, the sensitivity of PCR of skin lesions was 100% (17 of 17) and the specificity was 40% (two of five).

Table 1

Diagnostic efficacy of skin and whole blood PCR compared with microscopy for the diagnosis of cutaneous leishmaniasis

  MicroscopyTotalSensitivitySpecificity
+
Skin lesion PCR+173   
022217/17 = 100%2/5 = 40%
Whole blood PCR+162   
132216/17 = 94.1%3/5 = 60%

PCR = polymerase chain reaction.

Subsequently, we wanted to compare the efficacy of diagnosis between PCR analysis from two different biological samples, skin and whole blood, since blood is not routinely used for CL diagnosis. Data showed that PCR analysis of skin samples identified 20 positive cases, whereas blood PCR identified 18, Figure 1A. Skin and blood samples from two patients were negative for leishmaniasis by PCR. We also evaluated the agreement in the diagnosis of leishmaniasis between microscopy and blood PCR analysis. Data showed that 16 of the 17 patients that had a diagnosis of leishmaniasis by microscopy were positive by blood PCR (94.1%); on the other hand, of the 18 blood samples that were positive for leishmaniasis by blood PCR, 16 were positive by microscopy (88.9%); three patients were negative for leishmaniasis by blood PCR and microscopy. These data indicated good concordance between blood PCR and microscopic analysis for the diagnosis of leishmaniasis, 19/22 (86.4%). According to these results and considering microscopy as the gold standard, the sensitivity of PCR for whole blood was 94.1% (16 of 17) and the specificity was 60% (three of five), Table 1.

Figure 1.
Figure 1.

Presence of (A) 18S rRNA gene and (B) cyt b gene in skin and whole blood samples of patients with suspected cutaneous leishmaniasis. In (A), lane 1 = 100–base pair (bp) molecular marker; lane 2 = positive control (Leishmania equatorensis DNA); lines 3–5 = skin (s) and blood (b) samples; lane 6 = negative control. In (B), lane 1 = 100-bp molecular marker; lines 2–8 = skin (s) and blood (b) samples; lane 9 = negative control; lane 10 = positive control (L. equatorensis DNA). Number indicates the number of the patient in the study.

Citation: The American Society of Tropical Medicine and Hygiene 95, 4; 10.4269/ajtmh.16-0385

Previous studies indicate that variations on cyt-b gene distinguish Leishmania species.15,16 We amplified the cyt-b gene in skin and whole blood samples that were positive for 18S rRNA Leishmania gene (N = 20) as reported previously.15 Eight samples that were positive for 18S rRNA also amplified for the cyt-b gene; four from skin and four from blood, Figure 1B. To identify Leishmania species, quantitative PCR products were purified (Beckman Coulter, Pasadena, CA) and subsequently sequenced in a 3500 Genetic Analyzer sequencer (Applied Biosystems, Foster City, CA). Data were analyzed (SeqScape® v2.5 software, Foster City, CA) and the nucleotide sequences of the cyt-b gene for the various Leishmania species, available in the GenBank, were compared through sequence alignments and phylogenetic trees.15,16 In three different patients, we were able to identify species-specific sequences from the eight sequenced samples; L. guyanensis and Leishmania shawi were identified from two cutaneous samples, whereas L. naiffi was detected in blood.

The identification of Leishmania species in patients with CL has important implications related with the natural course of infection, its treatment, and the epidemiology of the disease.17 For instance, Leishmania tropica, L. major, Leishmania aethiopica, and L. mexicana complexes cause CL18; disseminated cutaneous leishmaniasis is caused by L. braziliensis, L. amazonensis, and L. guyanensis19; and MCL is usually caused by L. braziliensis19 and in rare cases by L. panamensis and L. guyanensis.17 Leishmania species also respond differently to treatment,17,18,20 for example, species-specific antimonial sensitivity has been determined in an in vitro study, in which L. tropica, L. braziliensis, and L. panamensis were 3, 2.3, and 6 times more resistant than L. major to this drug.20 Identification of Leishmania species is critical for the diagnosis and treatment of this infection. Use of present PCR diagnosis techniques will contribute to improve the clinical management of this parasitic disease as well as to better understand the anthroponotic transmission of the infection.11

Previous studies have used blood samples to identify Leishmania parasites by PCR.11,12,21 Venazzi and others,12 studying 68 patients with CL, detected parasite DNA in 3.4% of prepared blood samples. In that study, blood samples were treated with a solution containing 25 mM citric acid, 50 mM sodium citrate, and 81 mM glucose before DNA isolation. Vergel and others11 detected the presence of Leishmania DNA in 7.5% of skin lesions and in 19.2% in peripheral-blood monocytes isolated from 59 patients with confirmed diagnosis of CL. In a recent study, Neitzke-Abreu and others21 reported the identification of Leishmania DNA in 83.87% of skin lesions and 51.67% of peripheral-blood monocytes isolated from 106 patients with CL. The percentages of Leishmania DNA detected in the studies described above are lower than the percentage of Leishmania DNA detected in the skin and whole blood of the present study. It is possible that the observed differences for the detection of parasite DNA could be due to the methods used in previous studies to prepare the blood or to isolate the peripheral-blood monocytes. Herein, we did not manipulate the blood samples, and DNA was directly isolated from samples within 24 hours of collection. These data indicate that it is not necessary to either treat blood samples or isolate white blood cells from the samples of Leishmania-infected patients to be able to detect the parasite DNA. Rather, direct DNA isolation from whole blood could be a better source of DNA for leishmaniasis diagnosis. It is important to note that we were able to detect parasite DNA even in patients who had already started the conventional treatment with meglumine antimoniate.

Highest diagnostic sensitivity was obtained by 18s rRNA gene PCR analysis. Consequently, a potential gold standard for CL should be based on molecular analysis rather than microscopic examination. PCR analysis can also be successfully performed in whole blood samples from patients suspected of CL and MCL. Blood samples are more easily obtained and require less-invasive methods than skin scraping from suspected patients.

We acknowledge that there were limitations in the identification of specific Leishmania species for all the positive samples since cyt-b gene was amplified in eight of the 20 positive samples and in three of the samples in which the species of Leishmania were identified. It is possible that the number of copies of the gene in the clinical samples were not at the level of detection for PCR amplification. Since most patients were under treatment of leishmaniasis at the time of recruitment, it is likely that the parasitic load was decreased at the time of specimen collection. It is also possible that difficulties of the processing and purification of cyt-b-amplified products could have affected sample sequencing.

We conclude that it is critical to establish a simple and well-defined protocol for molecular analysis, not only for Leishmania detection, but also for species identification. The current study showed the feasibility and high efficacy of leishmaniasis diagnosis by the identification of the species in whole blood and skin samples using PCR amplification and sequencing. This study also reports for the first time the presence of L. shawi in Ecuador.

  • 1.

    Llanes A, Restrepo CM, del Vecchio G, Anguizola FJ, Lleonart R, 2015. The genome of Leishmania panamensis: insights into genomics of the L. (Viannia) subgenus. Sci Rep 5: 8550.

    • Search Google Scholar
    • Export Citation
  • 2.

    Reveiz L, Maia-Elkhoury AN, Nicholls RS, Romero GA, Yadon ZE, 2013. Interventions for American cutaneous and mucocutaneous leishmaniasis: a systematic review update. PLoS One 8: e61843.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ministerio de Salud Pública del Ecuador (MSP), 2013. Manual de procedimientos del Subsistema alerta acción SIVE-ALERTA. Available at: https://aplicaciones.msp.gob.ec/salud/archivosdigitales/documentosDirecciones/dnn/archivos/manual_de_procedimientos_sive-alerta.pdf. Accessed January 11, 2016.

    • Search Google Scholar
    • Export Citation
  • 4.

    Von Stebut E, 2015. Leishmaniasis. JDDG 13: 191201.

  • 5.

    Hartley MA, Drexler S, Ronet C, Beverley SM, Fasel N, 2014. The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis. Trends Parasitol 30: 412422.

    • Search Google Scholar
    • Export Citation
  • 6.

    Luna AM, Michelena MA, Fleming C, Arena G, Chiavassa AM, Rafti P, 2012. Mucocutaneous leishmaniasis with laryngeal involvement. Med Cutan Ibero Lat Am 40: 154157.

    • Search Google Scholar
    • Export Citation
  • 7.

    Kato H, Calvopiña M, Criollo H, Hashiguchi Y, 2013. First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species. Acta Trop 128: 710713.

    • Search Google Scholar
    • Export Citation
  • 8.

    Calvopina M, Armijos R, Marco J, Uezato H, Kato H, Gómez E, Korenaga M, Barroso P, Mimori T, Cooper P, Nonaka S, Hashiguchi Y, 2006. Leishmania isoenzyme polymorphisms in Ecuador: relationships with geographic distribution and clinical presentation. BMC Infect Dis 6: 136139.

    • Search Google Scholar
    • Export Citation
  • 9.

    Mouttaki T, Morales-Yuste M, Merino-Espinosa G, Chiheb S, Fellah H, Martin-Sanchez J, Riyad M, 2014. Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays. Parasit Vectors 7: 420.

    • Search Google Scholar
    • Export Citation
  • 10.

    Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL, 2006. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44: 14351439.

    • Search Google Scholar
    • Export Citation
  • 11.

    Vergel C, Palacios R, Cadena H, Posso CJ, Valderrama L, Perez M, Walker J, Travi BL, Saravia NG, 2006. Evidence for Leishmania (Viannia) parasites in the skin and blood of patients before and after treatment. J Infect Dis 194: 503511.

    • Search Google Scholar
    • Export Citation
  • 12.

    Venazzi EA, Roberto AC, Barbosa-Tessmann IP, Zanzarini PD, Lonardoni MV, Silveira TG, 2007. Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis. Exp Parasitol 115: 399402.

    • Search Google Scholar
    • Export Citation
  • 13.

    Ontario Public Health, 2014. Specimen Collection Guide for Cutaneous Leishmaniasis. Available at https://www.publichealthontario.ca/en/eRepository/Specimen_collection_guide_cutaneous_leishmaniasis.pdf. Accessed January 18, 2016.

    • Search Google Scholar
    • Export Citation
  • 14.

    Nzelu CO, Gomez EA, Cáceres AG, Sakurai T, Martini-Robles L, Uezato H, Mimori T, Katakura K, Hashiguchi Y, Kato H, 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop 132: 16.

    • Search Google Scholar
    • Export Citation
  • 15.

    Foulet F, Botterel F, Buffet P, Morizot D, Rivollet D, Deniau M, Pratlong F, Costa JM, Bretagne S, 2007. Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome b gene. J Clin Microbiol 45: 21102115.

    • Search Google Scholar
    • Export Citation
  • 16.

    Yang B-B, Chen D-L, Chen J-P, Liao L, Hu X-S, Xu J-N, 2013. Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference. Parasit Vectors 6: 32.

    • Search Google Scholar
    • Export Citation
  • 17.

    Romero GA, Guerra MV, Paes MG, Macedo VO, 2001. Comparison of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis and L. (V.) guyanensis in Brazil: therapeutic response to meglumine antimoniate. Am J Trop Med Hyg 65: 456465.

    • Search Google Scholar
    • Export Citation
  • 18.

    Bañuls AL, Hide M, Prugnolle F, 2007. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol 64: 1109.

    • Search Google Scholar
    • Export Citation
  • 19.

    Couppié P, Clyti E, Sobesky M, Bissuel F, Del Giudice P, Sainte-Marie D, Dedet JP, Carme B, Pradinaud R, 2004. Comparative study of cutaneous leishmaniasis in human immunodeficiency virus (HIV)-infected patients and non-HIV-infected patients in French Guiana. Br J Dermatol 151: 11651171.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mandal G, Mandal S, Sharma M, Charret KS, Papadopoulou B, Bhattacharjee H, Mukhopadhyay R, 2015. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl Trop Dis 9: e0003500.

    • Search Google Scholar
    • Export Citation
  • 21.

    Neitzke-Abreu HC, Venazzi MS, Bernal MV, Reinhold-Castro KR, Vagetti F, Mota CA, Silva NR, Aristides SM, Silveira TG, Lonardoni MV, 2013. Detection of DNA from Leishmania (Viannia): accuracy of polymerase chain reaction for the diagnosis of cutaneous leishmaniasis. PLoS One 8: e62473.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Manuel E. Baldeón, Centro de Investigación Traslacional, Universidad de Las Américas (CIT-UDLA), Calle Jose Queri, Entre Avenida Granados y Eloy Alfaro, S/N, Quito 170503, Ecuador. E-mail: manuel.baldeon@udla.edu.ec

Authors' addresses: Erika B. Muñoz, Stephanie Santander, Patricio Rojas-Silva, Paul A. Cardenas, Marco Fornasini, Sara C. Cifuentes, and Manuel E. Baldeón, Centro de Investigación Traslacional, Universidad de Las Américas, Quito, Ecuador, E-mails: erika.munoz@udla.edu.ec, chefasan@hotmail.com, patricio.rojas@udla.edu.ec, paul.cardenas@udla.edu.ec, marco.fornasini@udla.edu.ec, s.cifuentes@udlanet.ec, and manuel.baldeon@udla.edu.ec. Daniela Salvador, Emergencia, Instituto Ecuatoriano de Seguridad Social, Quito, Ecuador, E-mail: dani_salvador88@hotmail.com.

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