Document Type : Original Research

Authors

1 Dept. of Pathology, Leishmania Research Center, Afzalipour Medical School, Kerman University of Medical Science, Kerman, Iran

2 Dept. of Pathology, Afzalipour Medical School, Kerman University of Medical Science, Kerman, Iran

3 Dept. of Dermatology, Afzalipour Medical School, Kerman University of Medical Science, Kerman, Iran

4 Dept. of Biotechnology, Pasteur Institute of Iran, Tehran, Iran & Shahid-Sadoughi University of Medical Sciences, Yazd, Iran

5 Pharmaceutics Research Center, Kerman University of Medical Science, Kerman, Iran

6 Dept. of Pharmacology, Afzalipour Medical School, Kerman University of Medical Science, Kerman, Iran

7 Dept. of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Background and Objectives: Kerman Province, especially city of Bam in the southeast part of Iran, is epidemics for dry type cutaneous leishmaniasis (DTCL). This study was conducted to compare the effect of different treatments on parasite DNA load following therapies using Real-Time PCR method.
Materials and Methods: Fifteen patients were divided into three groups under therapy with intralesional meglumine antimoniate, topical imiquimod and combination of both drugs. After obtaining consent from patients, punch biopsies were taken before and after treatment .To compare the amount of DNA load a relative quantitative Real-Time PCR method was designed and set up using Leishmania tropica ITS (internal transcribed spacer) gene. After doing PCR, the obtained results were analyzed using 2^-ΔΔCT method and relativity of DNA load before and after treatment were calculated.
Results: The highest falling of DNA load was for imiquimod (mean 4/7 cases), glucantime (mean 2/2 cases) and combination therapy (mean 2/4). From clinical point of view combination therapy had the best response. On the other hand, the overall IHC findings showed good response based on decreased CD1a epidermal, increased CD1a dermal, decreased CD68 macrophages and increased CD3 and CD20 of dermis.
Conclusion: We set up a new method to compare Leishmania DNA load using the stable human gene of beta actin for normalization. We concluded that imiquimod was immune modulator and had synergistic effects on the best parasitocidal drug of glucantime for better response.
 

Keywords

  1. McPherson RA, Pincus MR. Henry's clinical diagnosis and management by laboratory methods. 22th ed. New York: Saunders; 2011.
  2. Nadim A, Aflatoonian M. Anthroponotic cutaneous leishmaniasis in the city of Bam, southeast Iran. Iranian J Public Health. 1995;24(1-2):15-24.
  3. Meymandi SS, Bahmanyar M, Dabiri S, Aflatonian MR, Bahmanyar S, Meymandi MS. Comparison of cytologic giemsa and real-time polymerase chain reaction technique for the diagnosis of cutaneous leishmaniasis on scraping smears. Acta Cytologica 2010;54(4):539-45.
  4. Khosravi S, Hejazi SH, Hashemzadeh M, Eslami G, Darani HY. Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp. J Vector Borne Dis 2012;49(1):15-8.
  5. Medeiros AC, Rodrigues SS, Roselino AM. Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis. Braz J Med Biol Res 2002;35(4):421-4.
  6. Santos DO, Coutinho CER, Madeira MF, Bottino CG, Vieira RT, Nascimento SB, et al. Leishmaniasis treatment—a challenge that remains: a review. Parasitol Res 2008;103(1):1-10.
  7. Shamsi Meymandi S, Zandi S, Aghaie H, Heshmatkhah A. Efficacy of CO2 laser for treatment of anthroponotic cutaneous leishmaniasis, compared with combination of cryotherapy and intralesional meglumine antimoniate. J Eur Acad Dermatol Venereol 2011;25(5):587-91.
  8. Ashutosh, Sundar S, Goyal N. Molecular mechanisms of antimony resistance in Leishmania. J Med Microbiol 2007;56(Pt 2):143-53.
  9. Gupta AK, Cherman AM, Tyring SK. Viral and nonviral uses of imiquimod: a review. J Cutan Med Surg 2004;8(5):338-52.
  10. Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, et al. Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PLoS One. [Research Support, Non-U.S. Gov't] 2010;5(4):e10107.
  11. van der Meide W, Guerra J, Schoone G, Farenhorst M, Coelho L, Faber W, et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol. [Comparative Study Evaluation Studies Research Support, Non-U.S. Gov't] 2008;46(1):73-8.
  12. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-8.
  13. Shamsi Meymandi S, Javadi A, Dabiri S, Shamsi Meymandi M, Nadji M. Comparative histological and immunohistochemical changes of dry type cutaneous leishmaniasis after administration of meglumine antimoniate, imiquimod or combination therapy. Arch Iran Med 2011 ;14(4):238-43.
  14. Mahboudi F, Abolhassan M, Yaran M, Mobtaker H, Azizi M. Identification and differentiation of Iranian Leishmania species by PCR amplification of kDNA. Scand J Infect Dis 2001;33(8):596-8.
  15. Manna L, Reale S, Viola E, Vitale F, Foglia Manzillo V, Pavone LM, et al. Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol 2006;142(3-4):271-80.
  16. Schulz A, Mellenthin K, Schonian G, Fleischer B, Drosten C. Detection, differentiation, and quantitation of pathogenic Leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J Clin Microbiol 2003;41(4):1529-35.
  17. Gerlag DM, Boyle DL, Rosengren S, Nash T, Tak PP, Firestein GS. Real-time quantitative PCR to detect changes in synovial gene expression in rheumatoid arthritis after corticosteroid treatment. Ann Rheum Dis 2007;66(4):545-7.
  18. Firooz A, Khamesipour A, Ghoorchi MH, Nassiri-Kashani M, Eskandari SE, Khatami A, et al. Imiquimod in combination with meglumine antimoniate for cutaneous leishmaniasis: a randomized assessor-blind controlled trial. Arch Dermatol 2006;142(12):1575-9.
  19. Arevalo I, Ward B, Miller R, Meng TC, Najar E, Alvarez E, et al. Successful treatment of drug-resistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator. Clin Infect Dis 2001;33(11):1847-51.
  20. Croft SL, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev 2006;19(1):111-26.
  21. Sereno D, Holzmuller P, Mangot I, Cuny G, Ouaissi A, Lemesre JL. Antimonial-Mediated DNA Fragmentation in Leishmania infantum Amastigotes. Antimicrob Agents Chemother 2001;45(7):2064-9.
  22. Lacarrubba F, Nasca MR, Micali G. Advances in the use of topical imiquimod to treat dermatologic disorders. Ther Clin Risk Manag 2008;4(1):87-97.
  23. Buates S, Matlashewski G. Treatment of experimental leishmaniasis with the immunomodulators imiquimod and S-28463: efficacy and mode of action. J Infect Dis 1999;179(6):1485-94.
  24. Hadighi R, Mohebali M, Boucher P, Hajjaran H, Khamesipour A, Ouellette M. Unresponsiveness to glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites. PLoS Med 2006;3(5):e162.
  25. Von Stebut E. Immunology of cutaneous leishmaniasis: the role of mast cells, phagocytes and dendritic cells for protective immunity. Eur J Dermatol 2007;17(2):115-22.
  26. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 2001;25(4):386-401.