High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran
Abstract
Background and Objectives: The three old world Leishmania species i.e., L. major, L. tropica, and L. infantum are considered as potential etiological agents of the various clinical forms of leishmaniasis in Iran. Different species co-exist in some areas. Accurate differentiation between the species is essential for choosing an appropriate therapy. Conventional and gold standard methods for the detection and characterization of parasites are time-consuming, laborious, and have low sensitivity. A polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis has been employed for detection and species identification. Most of the studies suffer from the use of multiple targets and/ or requiring more than one reaction to identify a single sample. The present study aimed to design a PCR method based on the amplification of kinetoplast DNA minicircles (kDNA) and HRM analysis of the amplicons for rapid discrimination of the three mentioned species.
Materials and Methods: DNA from reference strains including L. major, L. tropica, and L. infantum and fifty-eight strains subjected to PCR-HRM analysis targeting kDNA. All the samples were also analyzed by conventional kDNA- PCR.
Results: The PCR-HRM analysis allowed discrimination between the three Old World species. The normalized HRM curves for the amplicons of kDNA indicated a unique and repeatable melting plot for each species, even in combination with human and mouse genomic DNA. Conventional kDNA- PCR could not properly discriminate L. tropica from L. infantum.
Conclusion: PCR- HRM analysis of kDNA proved to be fast and accurate for discrimination of L. major, L. tropica, and L. infantum.
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12. Khademvatan S, Neisi N, Maraghi S, Saki J. Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran. Ann Trop Med Parasitol 2011; 105: 559-565.
13. Losada-Barragan M, Cavalcanti A, Umana-Perez A, Porrozzi R, Cuervo-Escobar S, Vallejo AF, et al. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples. Vet Parasitol 2016; 226: 57-64.
14. Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F, De Silva D, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 2009; 10: 5.
15. Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floeter-Winter LM. High resolution melting analysis targeting hsp70 as a fast and efficient method for the discrimination of Leishmania species. PLoS Negl Trop Dis 2016; 10(2):e0004485.
16. Hernández C, Alvarez C, González C, Ayala MS, León CM, Ramírez JD. Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay. Parasit Vectors 2014; 7: 501.
17. Aghaei AA, Rassi Y, Sharifi I, Vatandoost H, Mollaie H, Oshaghi M, et al. First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran. Asian Pac J Trop Med 2014; 7: 93-96.
18. Kuang Z, Zhang C, Pang H, Ma Y. A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene. Acta Trop 2018; 178: 103-106.
19. Ceccarelli M, Galluzzi L, Migliazzo A, Magnani M. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS One 2014; 9(2):e88845.
20. Schönian G, Schweynoch C, Zlateva K, Oskam L, Kroon N, Gräser Y, et al. Identification and determination of the relationships of species and strains within the genus Leishmania using single primers in the polymerase chain reaction. Mol Biochem Parasitol 1996; 77: 19-29.
21. Dabirzadeh M, Mirmohammad Sadeghi H, Baghaie M, Hejazi H. Genetic polymorphism of Leishmania major in two hyper endemic regions of Iran revealed by PPIP-PCR and ITS-RFLP. Arch Iran Med 2012; 15: 151-156.
22. Nicolas L, Milon G, Prina E. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 2002; 51: 295-299.
23. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vectors 2018; 11: 273.
24. Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ, Vardarli AT, et al. A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. PLoS Negl Trop Dis 2013; 7(5):e2205.
25. Talmi-Frank D, Nasereddin A, Schnur LF, Schonian G, Toz SO, Jaffe CL, et al. Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis 2010; 4(1): e581.
26. Brewster S, Aslett M, Barker D. Kinetoplast DNA minicircle database. Parasitol Today 1998; 14: 437-438.
27. Mohaghegh M, Fata A, Salehi G, Berenji F. Molecular identification of Leishmania species using samples obtained from negative stained smears. Iran J Parasitol 2013; 8: 337-341.
28. Abdolmajid F, Ghodratollah SS, Hushang R, Mojtaba MB, Ali MM, Abdolghayoum M. Identification of Leishmania species by kinetoplast DNA-polymerase chain reaction for the first time in Khaf district, Khorasan-e-Razavi province, Iran. Trop Parasitol 2015; 5: 50-54.
29. Mahmoodi MR, Mohajery M, Afshari JT, Shakeri MT, Panah MJY, Berenji F, et al. Molecular identification of Leishmania species causing cutaneous leishmaniasis in Mashhad, Iran. Jundishapur J Microbiol 2010; 3: 195-200.
30. Ceccarelli M, Galluzzi L, Diotallevi A, Andreoni F, Fowler H, Petersen C, et al. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis. Parasit Vectors 2017; 10: 239.
2. Shirian S, Oryan A, Hatam GR, Daneshbod Y. Three Leishmania/L. species--L. infantum, L. major, L. tropica--as causative agents of mucosal leishmaniasis in Iran. Pathog Glob Health 2013; 107: 267-272.
3. de Vries HJ, Reedijk SH, Schallig HD. Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol 2015; 16: 99-109.
4. Kumar R, Bumb RA, Ansari NA, Mehta RD, Salotra P. Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite identification and characterization using molecular and immunologic tools. Am J Trop Med Hyg 2007; 76: 896-901.
5. El-Beshbishy HA, Al-Ali KH, El-Badry AA. Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province, western Saudi Arabia. Int J Infect Dis 2013; 17(5):e334-338.
6. Azmi K, Nasereddin A, Ereqat S, Schnur L, Schonian G, Abdeen Z. Methods incorporating a polymerase chain reaction and restriction fragment length polymorphism and their use as a ‘gold standard’in diagnosing Old World cutaneous leishmaniasis. Diagn Microbiol Infect Dis 2011; 71: 151-155.
7. Mouttaki T, Morales-Yuste M, Merino-Espinosa G, Chiheb S, Fellah H, Martin-Sanchez J, et al. Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays. Parasit Vectors 2014; 7: 420.
8. Mohammadi AM, Khamesipour A, Khatami A, Javadi A, Nassiri-Kashani M, Firooz A, et al. Cutaneous leishmaniasis in suspected patients referred to the center for research and training in skin diseases and leprosy, tehran, iran from 2008 to 2011. Iran J Parasitol 2013; 8: 430-436.
9. Pita-Pereira D, Lins R, Oliveira MP, Lima RB, Pereira BA, Moreira OC, et al. SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases. Parasit Vectors 2012; 5: 15.
10. Ghasemian M, Gharavi MJ, Akhlaghi L, Mohebali M, Meamar AR, Aryan E, et al. SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples. J Parasit Dis 2016; 40: 81-87.
11. Weirather JL, Jeronimo SM, Gautam S, Sundar S, Kang M, Kurtz MA, et al. Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J Clin Microbiol 2011; 49: 3892-3904.
12. Khademvatan S, Neisi N, Maraghi S, Saki J. Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran. Ann Trop Med Parasitol 2011; 105: 559-565.
13. Losada-Barragan M, Cavalcanti A, Umana-Perez A, Porrozzi R, Cuervo-Escobar S, Vallejo AF, et al. Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples. Vet Parasitol 2016; 226: 57-64.
14. Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F, De Silva D, et al. Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 2009; 10: 5.
15. Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floeter-Winter LM. High resolution melting analysis targeting hsp70 as a fast and efficient method for the discrimination of Leishmania species. PLoS Negl Trop Dis 2016; 10(2):e0004485.
16. Hernández C, Alvarez C, González C, Ayala MS, León CM, Ramírez JD. Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay. Parasit Vectors 2014; 7: 501.
17. Aghaei AA, Rassi Y, Sharifi I, Vatandoost H, Mollaie H, Oshaghi M, et al. First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran. Asian Pac J Trop Med 2014; 7: 93-96.
18. Kuang Z, Zhang C, Pang H, Ma Y. A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene. Acta Trop 2018; 178: 103-106.
19. Ceccarelli M, Galluzzi L, Migliazzo A, Magnani M. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS One 2014; 9(2):e88845.
20. Schönian G, Schweynoch C, Zlateva K, Oskam L, Kroon N, Gräser Y, et al. Identification and determination of the relationships of species and strains within the genus Leishmania using single primers in the polymerase chain reaction. Mol Biochem Parasitol 1996; 77: 19-29.
21. Dabirzadeh M, Mirmohammad Sadeghi H, Baghaie M, Hejazi H. Genetic polymorphism of Leishmania major in two hyper endemic regions of Iran revealed by PPIP-PCR and ITS-RFLP. Arch Iran Med 2012; 15: 151-156.
22. Nicolas L, Milon G, Prina E. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 2002; 51: 295-299.
23. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vectors 2018; 11: 273.
24. Toz SO, Culha G, Zeyrek FY, Ertabaklar H, Alkan MZ, Vardarli AT, et al. A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. PLoS Negl Trop Dis 2013; 7(5):e2205.
25. Talmi-Frank D, Nasereddin A, Schnur LF, Schonian G, Toz SO, Jaffe CL, et al. Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis 2010; 4(1): e581.
26. Brewster S, Aslett M, Barker D. Kinetoplast DNA minicircle database. Parasitol Today 1998; 14: 437-438.
27. Mohaghegh M, Fata A, Salehi G, Berenji F. Molecular identification of Leishmania species using samples obtained from negative stained smears. Iran J Parasitol 2013; 8: 337-341.
28. Abdolmajid F, Ghodratollah SS, Hushang R, Mojtaba MB, Ali MM, Abdolghayoum M. Identification of Leishmania species by kinetoplast DNA-polymerase chain reaction for the first time in Khaf district, Khorasan-e-Razavi province, Iran. Trop Parasitol 2015; 5: 50-54.
29. Mahmoodi MR, Mohajery M, Afshari JT, Shakeri MT, Panah MJY, Berenji F, et al. Molecular identification of Leishmania species causing cutaneous leishmaniasis in Mashhad, Iran. Jundishapur J Microbiol 2010; 3: 195-200.
30. Ceccarelli M, Galluzzi L, Diotallevi A, Andreoni F, Fowler H, Petersen C, et al. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis. Parasit Vectors 2017; 10: 239.
| Files | ||
| Issue | Vol 13 No 1 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i1.5505 | |
| Keywords | ||
| Leishmania major; Leishmania tropica; Leishmania infantum; High resolution melting; Kinetoplast DNA; Minicircles | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Fayaz S, Fard-Esfahani P, Bahrami F, Parvizi P, Ajdary S. High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran. Iran J Microbiol. 2021;13(1):137-144.
