Species Identification and Genotyping of Cutaneous Leishmaniasis in Clinical Samples Based on ITS1-PCR- Sequencing in Southeast Iran
Abstract
Background: Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases in many regions of Iran. It has a major role in deprived societies. We aimed to identify Leishmania species based on molecular as ITS1-rDNA-PCR internal transcribed spacer 1 (ITS1) region, microscopy, and culture techniques in diagnosing cutaneous leishmaniasis.
Methods: From April 2018 to May 2020, we conducted a cross-sectional study involving 32 patients with suspected CL lesions in Sistan and Baluchistan Province, located in southeast Iran. Samples were subjected to microscopic examination, culture, and PCR amplification targeting the internal transcribed spacer 1 (ITS1) region. DNA sequencing was performed on PCR-positive samples for species identification and phylogenetic analysis.
Results: PCR demonstrated superior sensitivity (93.75%, 30/32) compared to culture (56.25%, 18/32) and microscopic examination (53.1%, 17/32). Molecular analysis revealed that L. major was the predominant causative agent of CL in the study area, with L. tropica occurring less frequently. Sequencing and phylogenetic analysis of the ITS1 region showed high intraspecies similarity among L. tropica isolates, while L. major isolates exhibited greater genetic diversity.
Conclusion: This study shows the co-existence of L. major and L. tropica in Mirjaveh, southeast Iran, with L. major as the primary cause. While L. tropica isolates displayed high genetic similarity, L. major samples were more diverse, indicating different epidemiological patterns. These findings highlight the importance of molecular methods for accurately identifying Leishmania species and understanding CL epidemiology in the region.
2. WHO Expert Committee on the Control of the Leishmaniases & World Health Or-ganization. (2010). Control of the leish-maniases: report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases, Geneva, 22-26 March 2010. World Health Organization.
3. Cecílio P, Cordeiro-da-Silva A, Oliveira F (2022). Sand flies; Basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites. Commun Biol, 5(1): 305.
4. Alemayehu B, Alemayehu M (2017). Leish-maniasis: a review on parasite, vector, and reservoir host. Health Sci J, 11(4): 519.
5. Moreira OC, Yadon ZE, Cupolillo E (2018). The applicability of real-time PCR in the diagnostic of cutaneous leishmania-sis and parasite quantification for clini-cal management: current status and per-spectives. Acta Trop, 184: 29-37.
6. Sharifi-Rad M, Dabirzadeh M, Sharifi I, Babaei Z (2016). Leishmania major: Genet-ic profiles of the parasites isolated from Chabahar, Southeastern Iran by PPIP-PCR. Iran J Parasitol, 11(3): 290-295.
7. Ghatee MA, Taylor WR, Karamian M (2020). The geographical distribution of cutaneous leishmaniasis causative agents in Iran and its neighboring countries, a review. Front Public Health, 8: 11.
8. Mahmoudzadeh-Niknam H, Abrishami F, Doroudian M et al. (2011). The problem of mixing up Leishmania isolates in the laboratory: suggestion of ITS1 gene se-quencing for verification of species. Iran J Parasitol, 6(1): 41-48.
9. Galluzzi L, Ceccarelli M, Diotallevi A, Me-notta M, Magnani M (2018). Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vectors, 11(1): 273.
10. Schönian G, Lukeš J, Stark O, Cotton JA (2018). Molecular evolution and phylog-eny of Leishmania. Drug Resistance in Leishmania Parasites: Springer; pp. 19-57.
11. Schönian G, Kuhls K, Mauricio I (2011). Molecular approaches for a better un-derstanding of the epidemiology and population genetics of Leishmania. Para-sitology, 138(4): 405-425.
12. Yang BB, Guo XG, Hu XS et al. (2010). Species discrimination and phylogenetic inference of 17 Chinese Leishmania iso-lates based on internal transcribed spacer 1 (ITS1) sequence. Parasitol Res, 107(5): 1049-1065.
13. Parvizi P, Alaeenovin E, Kazerooni P, Ready P (2013). Low diversity of Leish-mania parasites in sandflies and the ab-sence of the great gerbil in foci of zo-onotic cutaneous leishmaniasis in Fars province, southern Iran. Trans R Soc Trop Med Hyg, 107(6): 356-362.
14. El Tai NO, El Fari M, Mauricio I et al. (2001). Leishmania donovani: intraspecific polymorphisms of Sudanese isolates re-vealed by PCR-based analyses and DNA sequencing. Exp Parasitol, 97(1): 35-44.
15. Talmi-Frank D, Nasereddin A, Schnur LF et al. (2010). Detection and identifica-tion of high-resolution mania by high-resolution melt analysis. PLoS Negl Trop Dis, 4(1): e581.
16. Dabirzadeh M, Baghaie M, Hejazi H (2012). Genetic polymorphism of Leishmania major in two hyperendemic regions of Iran revealed by PPIP-PCR and ITS-RFLP. Arch Iran Med, 15(3): 151-156.
17. Dabirzadeh M, Hashemi M, Maroufi Y (2016). Study of genetic variation of Leishmania major based on internal tran-scribed spacer 1 (ITS1) in Chabahar, Iran. Jundishapur J Microbiol, 9(6): e33498.
18. Fazaeli A, Fouladi B, Sharifi I (2009). Emergence of cutaneous leishmaniasis in a border area at the south-east of Iran: an epidemiological survey. J Vector Borne Dis, 46(1): 36-42.
19. Mohammadpour I, Hatam GR, Handjani F, et al (2019). Leishmania cytochrome b gene sequence polymorphisms in south-ern Iran: relationships with different cu-taneous clinical manifestations. BMC In-fect Dis, 19(1): 98.
20. Sharifi-Rad M, Dabirzadeh M, Sharifi I, Babaei Z (2016). Leishmania major: Genet-ic profiles of the parasites isolated from Chabahar, Southeastern Iran by PPIP-PCR. Iran J Parasitol, 11(3): 290–295.
21. Hajjaran H, Vasigheh F, Mohebali M, Re-zaei S, Mamishi S, Charedar S (2011). Di-rect diagnosis of Leishmania species on serosity materials punctured from cuta-neous leishmaniasis patients using PCR-RFLP. J Clin Lab Anal, 25(1): 20-24.
22. Karamian M, Kuhls K, Hemmati M, Ghatee MA (2016). Phylogenetic structure of Leishmania tropica in the new endemic focus Birjand in East Iran in compari-son to other Iranian endemic regions. Acta Trop, 158: 68-76.
23. Seyedi-Rashti M, Keighobadi K, Nadim A (1984). Urban cutaneous leishmaniasis in Kerman, Southeast Iran. Bull Soc Pathol Exot Filiales, 77(3):312-9.
24. Ghatee MA, Sharifi I, Mirhendi H, Kanan-nejad Z, Hatam G (2013). Investigation of a double-band electrophoretic pat-tern of ITS-rDNA region in Iranian iso-lates of Leishmania tropica. Iran J Parasitol, 8(2): 264–272.
25. Hamid K, Ezat-Aldin J, Mona S (2011). Bi-onomics of phlebotomine sand flies (Dip-tera: Psychodidae) as vectors of leishmani-asis in the County of Iranshahr, Sistan-Baluchistan Province, Southeast of Iran. Iran J Clin Infect Dis, 6(3): 112-116.
26. Motazedian H, Noamanpoor B, Ardehali S (2002). Characterization of Leishmania parasites isolated from provinces of the Islamic Republic of Iran. East Mediterr Health J, 8(2-3): 338-344.
27. Ghatee MA, Sharifi I, Kuhls K et al (2014). Heterogeneity of the internal tran-scribed spacer region in Leishmania tropi-ca isolates from southern Iran. Exp Para-sitol, 144: 44-51.
Files | ||
Issue | Vol 53 No 11 (2024) | |
Section | Original Article(s) | |
Keywords | ||
Cutaneous leishmaniasis Iran Polymerase chain reaction Sequence |
Rights and permissions | |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |