The Accuracy of Diagnosis and Genotyping of Leishmania Species Based on Spliced Leader Mini-Exon Gene by Nuclear Magnetic Resonance and Sequencing Assays
-
Mahyar Khorram
,
Heidar Masjedi
,
Fatemeh Tabrizi
,
Mitra Sadat Rezaee
,
Payam Tabarsi
,
Majid Marjani
,
Mihan Pourabdoullah Toutkaboni
,
Fatemeh Maryam Sheikholeslami
Abstract
Background: We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene.
Methods: Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCR-RFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (1H-NMR).
Results: Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, L-Tyrosine in L. tropica (P<0.01).
Conclusion: The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene. SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata.
1. Shirzadi MR, Esfahania SB, Mohebali M, et al. Epidemiological status of leishmaniasis in the Islamic Republic of Iran, 1983-2012. East Mediterr Health J. 2015;21(10):736-42.
2. Razavi MR, Shirzadi MR, Mohebali M, et al. Human Cutaneous Leishmaniosis in Iran, Up to Date-2019. J Arthropod Borne Dis. 2021;15(2):143-51.
3. Sharifi I, Khosravi A, Aflatoonian MR, et al. Cutaneous leishmaniasis situation analysis in the Islamic Republic of Iran in preparation for an elimination plan. Front Public Health. 2023;11:1091709.
4. Lin C, Wei Z, Cheng KK, et al. 1H NMR-based Investigation of Metabolic Response to Electro-Acupuncture Stimulation. Sci Rep. 2017;7(1):6820.
5. Mahdy MA, Al-Mekhlafi AM, Abdul-Ghani R, et al. First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen. PLoS One . 2016;11(3):e0151265.
6. Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. 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. 2003;41(7):3147-53.
7. Al-Nahhas SA, Kaldas RM. Characterization of Leishmania Species Isolated from Cutaneous Human Samples from Central Region of Syria by RFLP Analysis. ISRN Parasitol. 2013;2013:308726.
8. Tielens AG, van Hellemond JJ. Surprising variety in energy metabolism within Trypanosomatidae. Trends Parasitol. 2009;25(10):482-90.
9. Verner Z, Cermakova P, Skodova I, Kovacova B, Lukes J, Horvath A. Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei. Mol Biochem Parasitol. 2014;193(1):55-65.
10. Schonian G, Nasereddin A, Dinse N, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003;47(1):349-58.
11. El Tai NO, El Fari M, Mauricio I, et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001;97(1):35-44.
12. Albuquerque A, Campino L, Cardoso L, Cortes S. Evaluation of four molecular methods to detect Leishmania infection in dogs. Parasit Vectors. 2017;10(1):57.
13. Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71(3):267-75.
14. Alcolea PJ, Alonso A, Degayon MA, et al. In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus. BMC Genom. 2016;17:375.
15. Gupta N, Goyal N, Singha UK, Bhakuni V, Roy R, Rastogi AK. Characterization of intracellular metabolites of axenic amastigotes of Leishmania donovani by 1H NMR spectroscopy. Acta Trop. 1999;73(2):121-33.
16. Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. J Parasit Dis.2020; 44(2):253–272.
17. Matsumoto T, Hashiguchi Y, Gomez EA, et al. Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador. Trans R Soc Trop Med Hyg. 1999;93(6):606-7.
18. Bhattacharyya R, Das K, Sen S, Roy S, Majumder HK. Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction. FEMS Microbiol Lett. 1996;135(2-3):195-200.
19. Inga R, De Doncker S, Gomez J, et al. Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia. Mol Biochem Parasitol. 1998;92(2):219-28.
20. Hitakarun A, Tan-ariya P, Siripattanapipong S, et al. Comparison of PCR methods for detection of Leishmania siamensis infection. Parasit Vectors. 2014;7:458.
21. Roelfsema JH, Nozari N, Herremans T, Kortbeek LM, Pinelli E. Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients. Exp Parasitol. 2011;127(1):36-41.
22. Harris E, Kropp G, Belli A, Rodriguez B, Agabian N. Single-step multiplex PCR assay for characterization of New World Leishmania complexes. J Clin Microbiol. 1998;36(7):1989-95.
23. Arjmand M, Madrakian A, Khalili G, Najafi Dastnaee A, Zamani Z, Akbari Z. Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy. Iran Biomed J. 2016;20(2):77-83.
24. Tabrizi F, Seyyed Tabaei SJ, Ali Ahmadi N, Arefi Oskouie A. A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica. Iran J Med Sci. 2021;46(1):43-51.
25. Westrop GD, Williams RA, Wang L, et al. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism. PLoS One. 2015;10(9):e0136891.
26. Alcolea PJ, Alonso A, Garcia-Tabares F, et al. An insight into differential protein abundance throughout Leishmania donovani promastigote growth and differentiation. Int Microbiol. 2023;26(1):25-42.
27. Alcolea PJ, Alonso A, Garcia-Tabares F, Mena MDC, Ciordia S, Larraga V. An Insight into the Constitutive Proteome Throughout Leishmania donovani Promastigote Growth and Differentiation. Int Microbiol. 2019;22(1):143-54.
28. Alcolea PJ, Alonso A, Garcia-Tabares F, Torano A, Larraga V. An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes. PLoS One. 2014;9(12):e113837.
29. Mirzapour A, Badirzadeh A, Ashrafmansouri M, et al. Super Infection of Cutaneous Leishmaniasis Caused by Leishmania major and L. tropica to Crithidia fasciculata in Shiraz, Iran. Iran J Public Health. 2019;48(12):2285-92.
2. Razavi MR, Shirzadi MR, Mohebali M, et al. Human Cutaneous Leishmaniosis in Iran, Up to Date-2019. J Arthropod Borne Dis. 2021;15(2):143-51.
3. Sharifi I, Khosravi A, Aflatoonian MR, et al. Cutaneous leishmaniasis situation analysis in the Islamic Republic of Iran in preparation for an elimination plan. Front Public Health. 2023;11:1091709.
4. Lin C, Wei Z, Cheng KK, et al. 1H NMR-based Investigation of Metabolic Response to Electro-Acupuncture Stimulation. Sci Rep. 2017;7(1):6820.
5. Mahdy MA, Al-Mekhlafi AM, Abdul-Ghani R, et al. First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen. PLoS One . 2016;11(3):e0151265.
6. Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. 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. 2003;41(7):3147-53.
7. Al-Nahhas SA, Kaldas RM. Characterization of Leishmania Species Isolated from Cutaneous Human Samples from Central Region of Syria by RFLP Analysis. ISRN Parasitol. 2013;2013:308726.
8. Tielens AG, van Hellemond JJ. Surprising variety in energy metabolism within Trypanosomatidae. Trends Parasitol. 2009;25(10):482-90.
9. Verner Z, Cermakova P, Skodova I, Kovacova B, Lukes J, Horvath A. Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei. Mol Biochem Parasitol. 2014;193(1):55-65.
10. Schonian G, Nasereddin A, Dinse N, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003;47(1):349-58.
11. El Tai NO, El Fari M, Mauricio I, et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001;97(1):35-44.
12. Albuquerque A, Campino L, Cardoso L, Cortes S. Evaluation of four molecular methods to detect Leishmania infection in dogs. Parasit Vectors. 2017;10(1):57.
13. Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71(3):267-75.
14. Alcolea PJ, Alonso A, Degayon MA, et al. In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus. BMC Genom. 2016;17:375.
15. Gupta N, Goyal N, Singha UK, Bhakuni V, Roy R, Rastogi AK. Characterization of intracellular metabolites of axenic amastigotes of Leishmania donovani by 1H NMR spectroscopy. Acta Trop. 1999;73(2):121-33.
16. Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. J Parasit Dis.2020; 44(2):253–272.
17. Matsumoto T, Hashiguchi Y, Gomez EA, et al. Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador. Trans R Soc Trop Med Hyg. 1999;93(6):606-7.
18. Bhattacharyya R, Das K, Sen S, Roy S, Majumder HK. Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction. FEMS Microbiol Lett. 1996;135(2-3):195-200.
19. Inga R, De Doncker S, Gomez J, et al. Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia. Mol Biochem Parasitol. 1998;92(2):219-28.
20. Hitakarun A, Tan-ariya P, Siripattanapipong S, et al. Comparison of PCR methods for detection of Leishmania siamensis infection. Parasit Vectors. 2014;7:458.
21. Roelfsema JH, Nozari N, Herremans T, Kortbeek LM, Pinelli E. Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients. Exp Parasitol. 2011;127(1):36-41.
22. Harris E, Kropp G, Belli A, Rodriguez B, Agabian N. Single-step multiplex PCR assay for characterization of New World Leishmania complexes. J Clin Microbiol. 1998;36(7):1989-95.
23. Arjmand M, Madrakian A, Khalili G, Najafi Dastnaee A, Zamani Z, Akbari Z. Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy. Iran Biomed J. 2016;20(2):77-83.
24. Tabrizi F, Seyyed Tabaei SJ, Ali Ahmadi N, Arefi Oskouie A. A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica. Iran J Med Sci. 2021;46(1):43-51.
25. Westrop GD, Williams RA, Wang L, et al. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism. PLoS One. 2015;10(9):e0136891.
26. Alcolea PJ, Alonso A, Garcia-Tabares F, et al. An insight into differential protein abundance throughout Leishmania donovani promastigote growth and differentiation. Int Microbiol. 2023;26(1):25-42.
27. Alcolea PJ, Alonso A, Garcia-Tabares F, Mena MDC, Ciordia S, Larraga V. An Insight into the Constitutive Proteome Throughout Leishmania donovani Promastigote Growth and Differentiation. Int Microbiol. 2019;22(1):143-54.
28. Alcolea PJ, Alonso A, Garcia-Tabares F, Torano A, Larraga V. An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes. PLoS One. 2014;9(12):e113837.
29. Mirzapour A, Badirzadeh A, Ashrafmansouri M, et al. Super Infection of Cutaneous Leishmaniasis Caused by Leishmania major and L. tropica to Crithidia fasciculata in Shiraz, Iran. Iran J Public Health. 2019;48(12):2285-92.
| Files | ||
| Issue | Vol 18 No 3 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v18i3.13756 | |
| Keywords | ||
| Proton nuclear magnetic resonance Leishmaniasis Spliced leader miniexon gene Genotyping C. fasciculata | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Khorram M, Masjedi H, Tabrizi F, Rezaee MS, Tabarsi P, Marjani M, Pourabdoullah Toutkaboni M, Sheikholeslami F. The Accuracy of Diagnosis and Genotyping of Leishmania Species Based on Spliced Leader Mini-Exon Gene by Nuclear Magnetic Resonance and Sequencing Assays. Iran J Parasitol. 2023;18(3):331-341.
