DNA Load Analysis Using Real Time PCR In Comparison With Immunohistochemical Findings of Dry Type Cutaneous Leishmaniasis; Before and After Treatment by Imiquimode,Glucantime and Combination of Both Drugs

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 <em>Leishmania tropica</em> ITS (internal transcribed spacer) gene. After doing PCR, the obtained results were analyzed using 2^-ΔΔC_T 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 <em>Leishmania</em> 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.