In Vitro Sensitivity Of Ethiopia Cutaneous Leishmania Strains Tomiltefosine Paromomycinamphotericin Band Sodium Stibogluconate Using Amastigotemacrophage Model.

BackgroundrnEthiopian cutanneous leishmaniasis (ECL) is a public health and social problem with a sequel of severernand mutilating skin lesions. It is manifested in three forms: LCL, MCL and DCL. Treatmentrnunresponsiveness to the first line drug sodium stibogluconate has been a frequent complain.rnObjectivesrnTo describe the in vitro susceptibilities of Leishmania strains of Ethiopian CL patients to the fourrnantileishmanial drugs: miltefosine, paromomycin, amphotericin B and sodium stibogluconatern(SbV) and identify the most efficacious and/or potent drug against them.rnMaterials and methodrnStationery phase promastigotes (SPPs) of seven ECL Leishmania strains were allowed to infect andrnreplicate as amastigotes in peritoneal macrophages of CD1 mice in RPMI 1640 complete media withrnHiFCS and antibiotics in ratios ranging from 5-10 to 1 macrophage in a 16 well culture chamber slide.rnInfection rate and optimal ratio were determined microscopically. 24 ECL strains and one L.aethiopicarn(MHOM/ET/82) reference strain were included for the in vitro sensitivity test to four drugs: miltefosinernparomomycin, amphotericin B, and sodium stibogluconate (SbV). The 24 strains were isolates of LCLrn(n=8), DCL (n=7) and MCL (n=9) patients. The SPPs of the strains and L.aethiopica were allowed torninfect the macrophages at 7:1 promastigote to macrophage optimal ratio. Each strain was divided intorntreated experimental and untreated control replicates. The experimental replicates were treated with thernfour drugs each in threefold diluted six concentrations in duplicates while the controls in duplicates leftrnuntreated. Then, both treated and untreated replicates were incubated for 5days with drug and media alonernreplacement on the 3rnrdrnday, respectively. At the end of 5rnthrnday, slides were methanol fixed and Giemsarnstained. Amastigote density in both treated and untreated controls counted microscopically. ICrnvaluesrnwere determined using Prism. Strains variation in sensitivity was plotted using non-parametric box andrnwhisker plot. Comparison of sensitivity variation among the three groups was determined using KurskalWallisrnrnnon-parameteric test at the critical α vaule=0.05. Efficacy and potency were compared betweenrnthe drugs using four strains : the reference strain, LCL strain (LDS119), MCL strain (LDS229/08), andrnDCL strain (LDS118) using Prism. AI was measured as a function of both the reference strain and mostrnsensitive local strain of the three CL patients.rnResultrnDuring optimization, infection rate (>80%)was attained at 7:1 promastigote to macrophage ratio. Thernoverall ICrnmean ±SD in µg/ml for paromomycin, SbV, Miltefosine and amphotericin B, respectivelyrnwere 13.63±18.74, 10.23±8.12, 5.88±4.79 and 0.16±0.18. Overall strains dispersion in sensitivity rankedrnfrom the highest to lowest in the same order. The overall dispersion in ICrn50rnvalues was statisticallyrnsignificantly (Pparomomycin>miltefosine>amphotericin B. Strains of each form exhibited statisticallyrnsignificant (P0.05) difference in sensitivity dispersion to all the 4rndrugs. Miltefosine (ICrn7.61, 0.83, 6.29, and 7.1µg/ml), the most effecacious drug across all the fourrnstrains (the reference, LCL, MCL and DCL, respectively)the followed by amphotericin B againstrnreference and LCL strain at ICrn50rn0.5121 and 0.0081µg/ml whereas paromomycin against the MCL and thernDCL strain at ICrn50rn50rn16.18 and 24.13µg/ml, respectively. The SbV was the least effecacious against thernreference and LCL at ICrn11.65 µg/ml and 1.01 µg/ml while amphotericin B against MCL and DCLrnstrains at ICrn50rn50rn0.32µg/ml and 0.07 µg/ml, respectively. Amphotericin B was the most potent followed byrnmiltefosine across all the strains while paromomycin was the 3rnrdrnagainst the reference, MCL and DCL atrnICrn50rnof 13.26, 6.29 and 7.1 µg/ml respectively and SbV 3rnrdrnagainst LCL strain at ICrnof 0.01µg/ml. SbVrnwas the least potent drug. AI values of the drugs were not sufficient enough to show whether the testedrnstrains were resistant to any of the four drugs.rnConclusions and suggestions for future workrn50rnThe promising efficacy of miltefosine needs further in vitro/in vivo study. The lack of significantrnsensitivity difference between strains of the three CL patients might imply the role of individual immunernresponse and needs further investigation. AI computation might need standardization.

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