• View in gallery

    Calmodulin intergenic spacer restriction pattern from Leishmania reference strains with HaeIII enzyme. 1 = Leishmania braziliensis (MHOM/PA/02/WR2355); 2 = L. braziliensis (MHOM/BR/1975/M2903); 3 = Leishmania guyanensis (MHOM/BR/1975/M4147); 4 = Leishmania panamensis (MHOM/PA/86/WR746C); 5 = L. panamensis (MHOM/PA/98/WR2306); 6 = Leishmania peruviana (MHOM/PE/05/WR2771); 7 = Leishmania amazonensis (IFLA/BR/1967/PH8); 8 = Leishmania mexicana (MHOM/BZ/1982/BEL21); 9 = Leishmania infantum (MHOM/BR/1974/PP75); 10 = Leishmania lainsoni (MHOM/BR/1981/M6426).

  • View in gallery

    Calmodulin intergenic spacer restriction pattern from Leishmania (Viannia) reference strains with (A) BccI enzyme. M = Molecular size marker (100-bp ladder); 1 = Leishmania panamensis (MHOM/PA/98/WR2306); 2 = Leishmania guyanensis (MHOM/BR/1975/M4147); 3 = Leishmania braziliensis (MHOM/BR/1975/M2903). (B) Ava1 enzyme. M = Molecular size marker (100-bp ladder); 1 = Leishmania peruviana (MHOM/PE/05/WR2771); 2 = L. guyanensis (MHOM/BR/1975/M4147); 3 = L. braziliensis (MHOM/BR/1975/M2903); 4 = L. panamensis (MHOM/PA/98/WR2306). (C) Bsu36I enzyme M = Molecular size marker (100-bp ladder); 1 = L. guyanensis (MHOM/BR/1975/M4147); 2 = L. panamensis (MHOM/PA/98/WR2306); 3 = L. braziliensis (MHOM/BR/1975/M2903).

  • 1.

    Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M; WHO Leishmaniasis Control Team, 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization (WHO), 2010. Technical Report Series on the Control of the Leishmaniasis No. 949. Geneva, Switzerland: WHO.

  • 3.

    Arevalo J, Ramirez L, Adaui V, Zumic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A, Chappuis F, Dujardin JC, Llanos-Cuentas A, 2007. Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Infect Dis 195: 18461851.

    • Search Google Scholar
    • Export Citation
  • 4.

    Desjeux P, 2004. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27: 305318.

  • 5.

    González U, Pinart M, Rengifo-Pardo M, Macaya A, Alvar J, Tweed JA, 2009. Interventions for American cutaneous and mucocutaneous leishmaniasis. Cochrane Database Syst Rev 2: CD004834.

    • Search Google Scholar
    • Export Citation
  • 6.

    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
  • 7.

    Miranda A, Samudio F, Saldaña A, Castillo J, Brandão A, Calzada JE, 2014. The calmodulin intergenic spacer as molecular target for characterization of Leishmania species. Parasit Vectors 19: 735.

    • Search Google Scholar
    • Export Citation
  • 8.

    San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J, 2013. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Res Notes 6: 513.

    • Search Google Scholar
    • Export Citation
  • 9.

    Montalvo AM, Fraga J, Maes I, Dujardin JC, Van der Auwera G, 2012. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol 31: 14531461.

    • Search Google Scholar
    • Export Citation
  • 10.

    Van der Auwera G, Maes I, De Doncker S, Ravel C, Cnops L, Van Esbroeck M, Van Gompel A, Clerinx J, Dujardin JC, 2013. Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics. Euro Surveill 18: 20543.

    • Search Google Scholar
    • Export Citation
  • 11.

    Oddone R, Schweynoch C, Schönian G, de Sousa CS, Cupolillo E, Espinosa D, Arevalo J, Noyes H, Mauricio I, Kuhls K, 2009. Development of a multilocus microsatellite typing approach for discriminating strains of Leishmania (Viannia) species. J Clin Microbiol 47: 28182825.

    • Search Google Scholar
    • Export Citation
  • 12.

    Odiwuor S, Veland N, Maes I, Arévalo J, Dujardin JC, Van der Auwera G, 2012. Evolution of the Leishmania braziliensis species complex from amplified fragment length polymorphisms, and clinical implications. Infect Genet Evol 12: 19942002.

    • Search Google Scholar
    • Export Citation
  • 13.

    Miranda A, Saldaña A, González K, Paz H, Santamaría G, Samudio F, Calzada JE, 2012. Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America. Trans R Soc Trop Med Hyg 106: 544548.

    • Search Google Scholar
    • Export Citation
  • 14.

    Van Eys GJ, Schoone GJ, Kroon NC, Ebeling SB, 1992. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Mol Biochem Parasitol 51: 133142.

    • Search Google Scholar
    • Export Citation
  • 15.

    Piarroux R, Fontes M, Perasso R, Gambarelli F, Joblet C, Dumon H, Quilici M, 1995. Phylogenetic relationships between Old World Leishmania strains revealed by analysis of a repetitive DNA sequence. Mol Biochem Parasitol 73: 249252.

    • Search Google Scholar
    • Export Citation
  • 16.

    Dávila AM, Momen H, 2000. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol 94: 651654.

    • Search Google Scholar
    • Export Citation
  • 17.

    Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I, 2003. Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. J Clin Microbiol 41: 31473153.

    • Search Google Scholar
    • Export Citation
  • 18.

    Boité MC, Mauricio IL, Miles MA, Cupolillo E, 2012. New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis 6: e1888.

    • Search Google Scholar
    • Export Citation
  • 19.

    Tsukayama P, Núñez JH, De Los Santos M, Soberón V, Lucas CM, Matlashewski G, Llanos-Cuentas A, Ore M, Baldeviano GC, Edgel KA, Lescano AG, Graf PC, Bacon DJ, 2013. A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis. PLoS Negl Trop Dis 7: e1956.

    • Search Google Scholar
    • Export Citation
  • 20.

    Van der Auwera G, Ravel C, Verweij JJ, Bart A, Schönian G, Felger I, 2014. Evaluation of four single-locus markers for Leishmania species discrimination by sequencing. J Clin Microbiol 52: 10981104.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Calmodulin Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Leishmania Identification and Typing

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  • 1 Instituto Conmemorativo Gorgas de Estudios de la Salud, Panama City, Panama.
  • 2 Laboratorio Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil.

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

Leishmaniasis is a major neglected tropical disease affecting around 2 million people worldwide every year.1 The infections tend to cluster among the poorest and most isolated increasing the socioeconomic burden upon these vulnerable populations. There are more than 20 species of Leishmania capable of infecting humans and each having distinct epidemiological/demographic patterns, and nearly all have a zoonotic transmission with one main sylvatic reservoir.2 In the absence of vaccines and other effective preventive measures, the control of leishmaniasis is currently based on accurate diagnosis and prompt treatment.

A precise identification of the species has epidemiological, and particularly clinical importance, since the Leishmania species involved in human infections can influence the treatment decision and outcome.3 Furthermore, species identification is particularly relevant now that the disease appears to be currently underestimated and on the rise in several countries.1,4 There is also a renewed interest to evaluate the effectiveness of new drugs and/or treatment regimens, which requires accurate species identification.5,6 In this regard, several molecular markers and protocols have been described for Leishmania species identification and taxonomy. However, no single genetic marker has been shown to have the sufficient discriminatory power when dealing with closely related Leishmania species. We have recently reported that the calmodulin locus, located on chromosome 9 of Leishmania species, varies in copy number (2–3) and size of the intergenic spacers.7 The minor spacer between calmodulin copies of Leishmania presents sufficient variation in its sequence to allow clear discrimination between species, at least to the species/complex taxonomic level.7

We recognize, however, that our sequencing approach is not a feasible technology for many laboratories, particularly to those from developing countries/regions where Leishmania is endemic. In this sense, the aim of this study was to develop and evaluate a more practical polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) approach, based on part of the calmodulin gene for Leishmania identification and subsequent typing.

We initially tested the overall performance of our PCR assay using serial dilutions of DNA from different Leishmania species, and with the set of primers and methodology previously described.7 The PCR conditions were designed to amplify the shortest intergenic spacer (∼1,220 base pairs [bp]) between calmodulin-coding sequences. Leishmania DNA was extracted from cultures or skin scrapings using a commercial Kit (QIAGEN Hilden, Germany). We then performed in silico analysis of Leishmania calmodulin intergenic sequences deposited in GenBank–National Center for Biotechnology Information (accession numbers: JN966910 to JN966919, JQ302012, and JQ302013) using the software: “In silico simulation of molecular biology experiments” (available at the website http://insilico.ehu.es/) to search for restriction enzyme sites that allowed a clear distinction between Leishmania species based on the methodology previously described.8 Selected enzymes were then evaluated in vitro according to the manufacturers' recommendations, using as the target Leishmania reference strains (Table 1). Finally, we validated our methodology by analyzing 20 Leishmania human field isolates and by performing an in silico analysis of the calmodulin intergenic spacer sequences from Leishmania (Viannia) panamensis isolates available in GenBank (JN966920–JN966936).

Table 1

Leishmania reference strains evaluated in this study

SpeciesInternational code
Leishmania (Viannia) panamensisMHOM/PA/98/WR2306
L. (Viannia) panamensisMHOM/PA/86/WR746C
Leishmania (Viannia) peruvianaMHOM/PE/05/WR2771
Leishmania (Viannia) braziliensisMHOM/PA/02/WR2355
Leishmania (Viannia) braziliensisMHOM/BR/1975/M2903
Leishmania (Viannia) guyanensisMHOM/BR/1975/M4147
Leishmania (Viannia) lainsoniMHOM/BR/1981/M6426
Leishmania (Leishmania) infantumMHOM/BR/1974/PP75
Leishmania (Leishmania) amazonensisIFLA/BR/1967/PH8
Leishmania (Leishmania) mexicanaMHOM/BZ/1982/BEL21

To compare our typing results, reference strains and field isolates were also analyzed by a standard molecular methodology (hsp70-RFLP), performed as previously described.9 Sequencing of the shortest calmodulin intergenic region was performed on field isolates to validate the typing results.7 Use of clinical samples was approved by the National Review Board, (Comité Nacional de Bioética de la Investigación, Instituto Conmemorativo Gorgas de Estudios de la Salud, Panamá).

All Leishmania reference strains that were tested amplified a calmodulin PCR product around 1,220 bp (Supplemental Figure 1). Analytical sensitivity of our initial PCR ranged between 1.0 pg and 1.0 ng, depending on the Leishmania species tested (Supplemental Figure 2). This difference is likely related to the genomic organization of the calmodulin gene in different Leishmania species.7 Although the set of primers used can also amplify the calmodulin gene from other trypanosomatids (data not shown), the specificity of our PCR is based on the size of the expected amplified product, which in Leishmania is around 1,220 bp. Moreover, in the case of cutaneous leishmaniasis, the most common clinical form of the disease, the clinical sample sources are usually skin scrapings or biopsies that are unlikely to contain trypanosomatids other than Leishmania. However, the presence of other trypanosomatids circulating in Leishmania–endemic regions and the reports of the opportunistic presence of monogenetic trypanosomatids in immunosuppressed patients indicate that other trypanosomatids must be taken into account in any approach aiming to identify and characterize Leishmania. To address this situation, we performed an in silico analysis to observe the predicted restriction profile after cutting with different restriction enzymes the shortest intergenic region of calmodulin in different trypanosomatids. After amplification, several enzymes were capable of producing restriction patterns with different levels of discrimination between Leishmania species and/or complex. The best broad resolution was obtained with the enzyme HaeIII which yielded a clear distinction between reference strains from Leishmania (Leishmania) mexicana, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, Leishmania (Viannia) lainsoni, and the rest of the Viannia reference species analyzed (Figure 1). Interestingly, Leishmania peruviana (MHOM/PE/05/WR2771) presented a digestion profile different from the rest of the reference strains, but identical to the one presented by Leishmania (V.) braziliensis (MHOM/PA/02/WR2355) (Figure 1). To further investigate this result, we analyzed this L. (V.) braziliensis strain by hsp70-RFLP and by hsp70 sequencing.9,10 Leishmania (V.) braziliensis (MHOM/PA/02/WR2355) was classified as L. (V.) braziliensis by hsp70-RFLP, but was grouped by hsp70 sequencing as a “L. (V.) braziliensis outlier,” closer to the L. (V.) peruviana cluster (results not shown). In this regard, the classification of L. (V.) braziliensis and L. (V.) peruviana has generally been problematic. Previous isoenzyme and genetic analyses have grouped L. (V.) peruviana as a subcluster in the L. (V.) braziliensis complex; however, discrimination between these two strains has been hindered because many parasites of this complex seem to have a composite genotype from both species.11,12 Our finding reinforces the need to evaluate a larger sample of reference strains from both species.

Figure 1.
Figure 1.

Calmodulin intergenic spacer restriction pattern from Leishmania reference strains with HaeIII enzyme. 1 = Leishmania braziliensis (MHOM/PA/02/WR2355); 2 = L. braziliensis (MHOM/BR/1975/M2903); 3 = Leishmania guyanensis (MHOM/BR/1975/M4147); 4 = Leishmania panamensis (MHOM/PA/86/WR746C); 5 = L. panamensis (MHOM/PA/98/WR2306); 6 = Leishmania peruviana (MHOM/PE/05/WR2771); 7 = Leishmania amazonensis (IFLA/BR/1967/PH8); 8 = Leishmania mexicana (MHOM/BZ/1982/BEL21); 9 = Leishmania infantum (MHOM/BR/1974/PP75); 10 = Leishmania lainsoni (MHOM/BR/1981/M6426).

Citation: The American Society of Tropical Medicine and Hygiene 95, 2; 10.4269/ajtmh.15-0709

The closely related L. (V.) braziliensis, L. (V.) panamensis, and Leishmania (V.) guyanensis, presented the same digestion pattern after HaeIII digestion (Figure 1). However, if for medical or epidemiological reasons a distinction between these three Viannia species is needed, this can be readily accomplished in a subsequent digestion with the enzyme BccI (Figure 2A). Alternatively, due to the absence of a restriction site in L. (V.) panamensis, the enzyme Ava1 can differentiate this Viannia species from L. (V.) guyanensis and L. (V.) braziliensis, which have the same digestion pattern (Figure 2B). On the other hand, the enzyme Bsu36I makes a single cut in the L. (V.) panamensis calmodulin sequence, but does not recognize L. braziliensis/guyanensis (Figure 2C). All field isolates were identified as L. (V.) panamensis by the calmodulin-RFLP and hsp70-RFLP approaches. These typing results were further confirmed by sequencing the calmodulin minor spacer as described.7 In silico analysis of calmodulin intergenic spacer sequences available in GenBank (JN966920–JN966936) confirmed that these isolates were Leishmania (V.) panamensis. The enzyme HaeIII also clearly separates Leishmania from trypanosomatids of the genus Crithidia, Endotrypanum, Leptomonas, and Trypanosoma (Supplemental Figure 3). Therefore, trypanosomatids other than Leishmania that might be found in clinical samples can be easily distinguished by the calmodulin-RFLP approach itself.

Figure 2.
Figure 2.

Calmodulin intergenic spacer restriction pattern from Leishmania (Viannia) reference strains with (A) BccI enzyme. M = Molecular size marker (100-bp ladder); 1 = Leishmania panamensis (MHOM/PA/98/WR2306); 2 = Leishmania guyanensis (MHOM/BR/1975/M4147); 3 = Leishmania braziliensis (MHOM/BR/1975/M2903). (B) Ava1 enzyme. M = Molecular size marker (100-bp ladder); 1 = Leishmania peruviana (MHOM/PE/05/WR2771); 2 = L. guyanensis (MHOM/BR/1975/M4147); 3 = L. braziliensis (MHOM/BR/1975/M2903); 4 = L. panamensis (MHOM/PA/98/WR2306). (C) Bsu36I enzyme M = Molecular size marker (100-bp ladder); 1 = L. guyanensis (MHOM/BR/1975/M4147); 2 = L. panamensis (MHOM/PA/98/WR2306); 3 = L. braziliensis (MHOM/BR/1975/M2903).

Citation: The American Society of Tropical Medicine and Hygiene 95, 2; 10.4269/ajtmh.15-0709

We have developed and performed a preliminary validation of a molecular methodology (PCR-RFLP) based on the calmodulin gene for detecting and typing Leishmania species. Although we observed a relatively high experimental sensitivity for detecting Leishmania with our PCR approach, we recognize that the experimental method we used to evaluate the sensitivity does not mirror what happens biologically. Many factors such as the molecular target characteristics, the presence of PCR inhibitors in the clinical sample, and/or DNA extractions/PCR protocols can all significantly influence the overall PCR performance. Only 2–3 copies of calmodulin are present on chromosome 9 of the Leishmania genome, depending of the species. Since we designed the calmodulin-RFLP based on the amplification of the minor spacer between two copies of the calmodulin arrangement, only one copy from the Leishmania genome is targeted in this PCR approach. Other molecular markers such as the kinetoplastid or ribosomal genes are present in multicopies, whereas the heat shock protein gene is present in two tandem copies in chromosome 28 and a single copy in chromosomes 34 or 35. This fact, related to differences between the number of PCR targets in the Leishmania genome clearly influence the potential sensitivity of the test. In this sense, our methodology for species determination performed well with Leishmania DNA extracted from cultures, but the results were inconsistent when trying to detect and type Leishmania from DNA extracted directly from the cutaneous lesion samples (skin scraping or biopsies), probably due to the low number of parasites and calmodulin spacer targets in these biological samples.

The calmodulin-RFLP methodology was capable of distinguishing between most of the Leishmania reference strains and trypanosomatids so far tested. After amplification, an initial digestion with only one enzyme (HaeIII) allowed identification of most Leishmania species. Using different enzymes in a second digestion step, it is also possible to separate the other closely related Viannia species. The hsp70 gene is probably the molecular marker more widely used for Leishmania species identification, and currently the hsp70-RFLP approach has become a reference molecular method in many laboratories for these purposes.10 In this regard, an almost perfect agreement was observed between our calmodulin-RFLP methodology and the hsp70-RFLP protocol previously described.9 All field samples were typed as L. (V.) panamensis by both molecular methods. However, a bias due to the geographical origin of the samples, where L. (V.) panamensis predominates is likely.7,13 We also recognize that so far we have only tested a small number of reference strains and field isolates from a particular region. Some Viannia species such as Leishmania (Viannia) naiffi and Leishmania (Viannia) shawi were not evaluated in this study due to the lack of reference strains in our laboratory or the absence of calmodulin sequences from these two species in GenBank to perform an in silico analysis. This is an important limitation of our study considering that in some areas, such as the Amazon, sympatric circulation of Viannia species is very common. Thus, the potential of this marker as a complementary tool for Leishmania species identification and taxonomical analysis requires further field studies using a larger number of strains from different geographical areas.

Several molecular markers and PCR protocols have been developed for Leishmania species identification, strain typing, and consequent phylogenetic analysis.1417 Despite the molecular progress, the taxonomy and phylogenetic status of closely related Leishmania species within complexes and the classification of interspecies hybrids is still problematic. The evaluation of other variable genes has been recommended to address this issue. In this sense, the calmodulin-RFLP technique might provide additional information to understand the genetic complexity of Leishmania parasites.

Recent studies have demonstrated that multilocus sequence typing (MLST) based on sequence analysis of several internal control genes presents one of the highest discriminatory typing capacities for Leishmania species.1820 However, the establishment of MLST or any combination of molecular markers as a global and acceptable typing system requires a detailed evaluation of the number and type of Leishmania genes needed to achieve consistent typing results, as well as the validation of the methodology analyzing a wider range of Leishmania species from different geographical regions. In light of our results, the calmodulin intergenic spacer could be an interesting molecular marker to be considered in the standardization of a global system to type Leishmania. In conclusion, we have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for characterizing Leishmania field isolates.

ACKNOWLEDGMENTS

We wish to thank the Gorgas Memorial Institute of Health Studies in Panama for logistic support. We are grateful to all the members of the Parasitology Department, Gorgas Institute, Panama.

  • 1.

    Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M; WHO Leishmaniasis Control Team, 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization (WHO), 2010. Technical Report Series on the Control of the Leishmaniasis No. 949. Geneva, Switzerland: WHO.

  • 3.

    Arevalo J, Ramirez L, Adaui V, Zumic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A, Chappuis F, Dujardin JC, Llanos-Cuentas A, 2007. Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Infect Dis 195: 18461851.

    • Search Google Scholar
    • Export Citation
  • 4.

    Desjeux P, 2004. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27: 305318.

  • 5.

    González U, Pinart M, Rengifo-Pardo M, Macaya A, Alvar J, Tweed JA, 2009. Interventions for American cutaneous and mucocutaneous leishmaniasis. Cochrane Database Syst Rev 2: CD004834.

    • Search Google Scholar
    • Export Citation
  • 6.

    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
  • 7.

    Miranda A, Samudio F, Saldaña A, Castillo J, Brandão A, Calzada JE, 2014. The calmodulin intergenic spacer as molecular target for characterization of Leishmania species. Parasit Vectors 19: 735.

    • Search Google Scholar
    • Export Citation
  • 8.

    San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J, Bikandi J, 2013. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Res Notes 6: 513.

    • Search Google Scholar
    • Export Citation
  • 9.

    Montalvo AM, Fraga J, Maes I, Dujardin JC, Van der Auwera G, 2012. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol 31: 14531461.

    • Search Google Scholar
    • Export Citation
  • 10.

    Van der Auwera G, Maes I, De Doncker S, Ravel C, Cnops L, Van Esbroeck M, Van Gompel A, Clerinx J, Dujardin JC, 2013. Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics. Euro Surveill 18: 20543.

    • Search Google Scholar
    • Export Citation
  • 11.

    Oddone R, Schweynoch C, Schönian G, de Sousa CS, Cupolillo E, Espinosa D, Arevalo J, Noyes H, Mauricio I, Kuhls K, 2009. Development of a multilocus microsatellite typing approach for discriminating strains of Leishmania (Viannia) species. J Clin Microbiol 47: 28182825.

    • Search Google Scholar
    • Export Citation
  • 12.

    Odiwuor S, Veland N, Maes I, Arévalo J, Dujardin JC, Van der Auwera G, 2012. Evolution of the Leishmania braziliensis species complex from amplified fragment length polymorphisms, and clinical implications. Infect Genet Evol 12: 19942002.

    • Search Google Scholar
    • Export Citation
  • 13.

    Miranda A, Saldaña A, González K, Paz H, Santamaría G, Samudio F, Calzada JE, 2012. Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America. Trans R Soc Trop Med Hyg 106: 544548.

    • Search Google Scholar
    • Export Citation
  • 14.

    Van Eys GJ, Schoone GJ, Kroon NC, Ebeling SB, 1992. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Mol Biochem Parasitol 51: 133142.

    • Search Google Scholar
    • Export Citation
  • 15.

    Piarroux R, Fontes M, Perasso R, Gambarelli F, Joblet C, Dumon H, Quilici M, 1995. Phylogenetic relationships between Old World Leishmania strains revealed by analysis of a repetitive DNA sequence. Mol Biochem Parasitol 73: 249252.

    • Search Google Scholar
    • Export Citation
  • 16.

    Dávila AM, Momen H, 2000. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol 94: 651654.

    • Search Google Scholar
    • Export Citation
  • 17.

    Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I, 2003. Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. J Clin Microbiol 41: 31473153.

    • Search Google Scholar
    • Export Citation
  • 18.

    Boité MC, Mauricio IL, Miles MA, Cupolillo E, 2012. New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis 6: e1888.

    • Search Google Scholar
    • Export Citation
  • 19.

    Tsukayama P, Núñez JH, De Los Santos M, Soberón V, Lucas CM, Matlashewski G, Llanos-Cuentas A, Ore M, Baldeviano GC, Edgel KA, Lescano AG, Graf PC, Bacon DJ, 2013. A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis. PLoS Negl Trop Dis 7: e1956.

    • Search Google Scholar
    • Export Citation
  • 20.

    Van der Auwera G, Ravel C, Verweij JJ, Bart A, Schönian G, Felger I, 2014. Evaluation of four single-locus markers for Leishmania species discrimination by sequencing. J Clin Microbiol 52: 10981104.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Jose E. Calzada, Departamento de Parasitología, Instituto Conmemorativo Gorgas de Estudios de la Salud, Apartado Postal No. 0816-02593, Panama City, Republic of Panama. E-mail: jcalzada@gorgas.gob.pa† These authors contributed equally to this work.

Financial support: This investigation received administrative and financial support from the Gorgas Institute and from SENACYT, Panama (grant COL08-080).

Disclosure: Azael Saldaña and Jose E. Calzada are members of the Sistema Nacional de Investigación (SNI), SENACYT, Panama.

Authors' addresses: Aracelis Miranda, Kadir González, and Jose E. Calzada, Departamento de Parasitología, Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panama City, Panama, E-mails: amiranda@gorgas.gob.pa, kgonzalez@gorgas.gob.pa, and jcalzada@gorgas.gob.pa. Franklyn Samudio, Departamento de Parasitología, Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panama City, Panama, and Laboratorio Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil, E-mail: fsamudio@gorgas.gob.pa. Azael Saldaña, Departamento de Parasitología, Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), Panama City, Panama, and Centro de Investigación y Diagnóstico de Enfermedades Parasitarias (CIDEP), Facultad de Medicina, Universidad de Panamá, Panama City, Panama, E-mail: asaldana@gorgas.gob.pa. Adeilton Brandão, Laboratorio Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil, E-mail: abran@ioc.fiocruz.br.

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