J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 132 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Evaluation of Correlation between the In Vitro Susceptibility of Field Isolates of Leishmania major and Clinical Outcomes of Meglumine Antimoniate Therapy in Fars Province, Iran Nafiseh Ghobakhloo 1, 2, *Mohammad Hossein Motazedian 1, 3, Behrad Pourmohammadi 4, Zahra Yousefi 1 1Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran 4Department of Public Health, School of Health, Semnan University of Medical Sciences, Semnan, Iran (Received 1 Mar 2015; accepted 5 Apr 2016) Abstract Background: This study was designed to detect whether there is a correlation between in vitro susceptibility of field isolates of Leishmania major and the clinical outcomes of meglumine antimoniate (Glucantime®) therapy, the main- stay of cutaneous leishmaniasis treatment in Iran. Methods: Forty-three patients infected with L. major were enrolled in this study from October 2009 to March 2010 and categorized as responsive or unresponsive to Glucantime® treatment after receiving the appropriate therapy. Then, intracellular amastigote approach was conducted on these field strains to investigate in vitro drug susceptibility as well. Results: At clinical level, out of 43 patients, 15 were clinically non-responsive and 28 were responsive to antimony therapy. All those 28 clinically sensitive strains were susceptible to antimony in the in vitro assay, whereas merely 11 isolates from 15 non-healing isolates were resistant in vitro. Finally, a good correlation (78.9%) with high sensi- tivity, specificity (100/73) between clinical outcomes and the in vitro susceptibility test was achieved. Conclusion: The intracellular amastigote model could be an appropriate assay for evaluation of the in vivo drug sen- sitivity of field isolates. However, more comprehensive studies with larger sets of isolates are needed to confirm the- se preliminary data. Keywords: In vitro susceptibility, Antimonial resistance, Leishmania major, Iran Introduction The most common form of leishmaniasis, Zoonotic Cutaneous leishmaniasis (ZCL), caused by L. major, is found in many re- gions of the middle east including Iran (Razmjou et al. 2009, Jacobson 2011, Alvar et al. 2012, Akhoundi et al. 2013). Since their discovery over 6 decades ago, the antimonial compounds have played vital roles in treat- ment of all forms of leishmaniasis (Croft et al. 2006). The emergence of antimony re- sistance as the mainstay of treatment, how- ever, has represented critical health problems in most endemic areas including Iran (Hadighi et al. 2006, Sundar and Goyal 2007, Pour- mohammadi et al. 2011, Mohammadzadeh et al. 2013). Since there is no effective vaccine for prevention, disease control is essentially based on chemotherapy. As resistance phenomenon can cause se- rious effects on disease treatment and control, this issue is an urgent concern (Sundar et al. 2014). Hence, estimating the efficacy of stand- ard drugs and determining the prevalence of *Corresponding author: Dr Mohammad Hossein Motazedian, E-mail: motazedm@sums.ac.ir, motaze -di- an33@yahoo.com 3Basic Sciences in Infectious Diseases, Research Center, Shiraz University of Medical Sciences, Shiraz, Iran J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 133 http://jad.tums.ac.ir Published Online: March 14, 2017 resistance in endemic regions seem to be ex- tremely essential (Croft 2001). Unfortunate- ly, there is no applicable in vitro test for evaluating the clinical resistance. In recent years, some promastigote and intracellular amastigote assays have been established to investigate the effectiveness of anti-Leishma- nia drugs. Fumarola et al. (2004) for screen- ing new compounds against L. infantum in- vestigated intracellular amastigotes than pro- mastigotes. Sereno et al. (2007) used green fluorescent protein or luciferase for screen- ing the drug resistance. An in vitro intra- cellular model for screening the Leishmania field isolates were applied by da Luz et al. (2009) and they mentioned that this model needed to be evaluated and standardized for the field isolates. An intracellular model for drug screening also was recommended by Vermeersch et al. (2009) using reference strain of L. donovani in different in vitro models. Regardless of the advantages and disad- vantages mentioned for these in vitro meth- ods, the intracellular amastigote model is a more reliable in vitro assay for measuring treatment failure in the field isolates (Maia et al. 2013). In this context, there are some previous reports on clinical evaluation of drug sensi- tivity, but these studies did not use the standard drug dosage as recommended by WHO and a few studies have been con- ducted to evaluate the in vitro susceptibility of Leishmania spp. to antimony (Hadighi et al. 2006, Pourmohammadi et al. 2011). We aimed in this study, to establish a fea- sible and suitable approach for detecting re- sistant strains at the clinical level for imple- mentation of a rational therapy and mapping the prevalence of resistance in Fars Prov- ince, south part of Iran, evaluating these re- sistance isolates in the in vitro assay, and determining if this test could be extendable to the in vivo assay, which has not been ap- plied to L. major yet. Materials and Methods Clinical isolates Forty-three field strains enrolled in this study were collected previously from pa- tients infected with L. major in Fars Prov- ince between 2009 and 2010. The study was conducted under full respect to Ethics State- ments and after appropriate informed con- sent from the patients (Pourmohammadi et al. 2011). Briefly, individuals suspected of ZCL were referred to Valfajr Health Center in Shiraz and infection with L. major was confirmed by PCR. Then, patients were treated with intramuscular Glucantime® at 20mg/kg/ day dose for 20 days as a standard protocol of WHO. After finishing the treatment course, they were followed up for six weeks and three month. Patients were considered unre- sponsive to treatment if amastigotes were found on light microscope examination of Geimsa stained slides prepared from the lesion edge. Reference sensitive strain The reference strain (MRHO/IR/75/ER), sensitive to Glucantime® treatment, used in this experiment was originally bought from the Pasture Institute of Iran was. In addition, a clinical isolate, which showed high rate of resistance to Glucantime® in both clinical level and in vitro assay, was used as a refer- ence resistant strain in this work. Anti-Leishmania drug Meglumine antimonite (Glucantime®) was prepared from Sigma-Aldrich Chemical Com- pany (011M0125V). The stock solution was produced by dissolving of drug powder in DMEM medium, to reach four different drug dilutions l0. With respect to finding a more suitable drug dose for establishing in vitro assay, which could be more extendable to clinical consequences, we used four different concentrations of Glucantime® obtained J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 134 http://jad.tums.ac.ir Published Online: March 14, 2017 through further dilution in complete medium (15 μg/ml, 30 μg/ml, 45 μg/ml and 60 μg/m). The mouse macrophage cell line (J774) This cell line was bought from national cell bank of Pasture Institute of Iran and grew in DMEM medium supplemented with 12% FBS, 100IU/ml of penicillin, and 100 g/ ml of streptomycin and incubated at 37 °C under 5% CO2 atmosphere until it reached to logarithmic phase of growth. The in vitro intracellular susceptibility assay The efficacy of meglumine antimonite of filed isolates was conducted on the mouse macrophage cell line (J774) as previously de- scribed (Faraut-Gambarelli et al. 1997). Brief- ly, the numbers of 5× 10 5 of J774 cells in log phase of growth were seeded in each 8- well chamber slides with cover slips (Nunc, 177445). Then they were left for cells adhe- sion for 1 h at 37 °C and 5% CO2. Adherent macrophages were infected with late station- ary phase of promastigotes at a ratio of 10:1 (promastigote/cell) for 4 h. After that, the excess promastigotes, which were not able to infect macrophages, were removed and each well was replaced with 400-μl medium con- taining Glucantime®. Meanwhile, all exper- iments were done in triplicate for each drug concentration. There was a control well against three treated wells contained only infected macrophage and 400μl of complete medium without any drug. Lastly, after 5 days incu- bation, all wells were fixed with absolute methanol and stained with 10% Geimsa for microscopic examination. Finally, parasite bur- dens as the percentage of infected macrophag- es× (mean number of amastigotes/ macrophage) were calculated and compared to the burdens for the untreated infected control wells. Statistical analysis Based on the reduction of total parasite burdens, 50% inhibitory concentrations (IC50) were calculated for each well. At least, re- garding to categorizing strains to resistant and sensitive isolates, IC50 of each isolate was compared with IC50 of reference (sensi- tive and resistant) isolates by the chi-square test and McNemar. If there was a significant difference between IC50 of reference strains and each field isolate, this clinical strain was characterized as resistant to drug in vitro as- say and vice versa. For the determining of correlation between clinical outcomes and in vitro sensitivity test, we used Kappa coeffi- cient that a P< 0.05 was considered statisti- cally significant. Statistical analysis was per- formed by the use of SPSS version 16.0 (Chicago, IL, USA). Results Out of 43 ZCL patients, 15 cases (34.9%) were non-responsive (non-healing) and 28 (65.1%) were responsive (healing) after com- plete treatment with Glucantime® at clinical level. In this experiment, IC50 values of field isolates represented ranging from (15 μg/ml to 60 μg/ml), while the IC50 was (˂15 μg/ ml) for reference sensitive strain and was (˃60 μg/ml) for reference resistant strain. The comparative results between clinical outcomes and in vitro sensitivity test are sum- marized in Table 1. In the highest drug dose (60 μg/ml), all of responsive field strains rep- resented sensitivity profiles in vitro as well, although four of fifteen strains which were determined as resistant at clinical level were sensitive in the in vitro assay at this drug dose. Furthermore, the result of McNemar and Kappa coefficient test (P< 0.05) conduct- ed for finding the sensitivity and specificity and agreement between clinical outcomes and in vitro assay is shown in Table 1. Over- all, a best sensitivity, specificity (100/73) and strong correlation (78.9%) was observed es- pecially in terms of using 60μg/ml of Glu- cantime® between the results of clinical se- quence and the in vitro test. J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 135 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 1. The comparison between the susceptibility of in vitro and clinical outcomes of Leishmania major field isolates to Glucantime® by McNemar and Kappa Discussion Regarding the increasing rate of antimony failures in ZCL patients in Iran, this study was done to set out a reliable in vitro method to determine the antimonial susceptibility of clinical isolates of L. major in Fars Province, as there was no report of a comprehensive work on this issue in Iran. In fact, for the first time we aimed to assess this test on L. major clinical strains. Since the intracellular amastigote, method had the best agreement with clinical responses rather than using pro- mastigote form, we decided to evaluate the susceptibility of antimony in this stage of par- asite as well (Maia et al. 2013, Coelho et al. 2014). In fact, the current results revealed a strong correlation (coefficient: r= 0.7860) and the best sensitivity, specificity between the clin- ical outcomes of antimony therapy and the in vitro antimonial susceptibility test. Indeed, this study was conducted on L. major clini- cal strains and has great corroboration with previous researches on various species of Leish- mania around the world (Fumarola et al. 2004, Vermeersch et al. 2009, Aït-Oudhia et al. 2012, Maia et al. 2013, Coelho et al. 2014). Like our findings, in another research conducted on 26 clinical isolates of L. tropica in Iran by Hadighi et al. there was an excellent correlation between clinical outcomes and the in vitro susceptibility test in amastigote-marophage model (Hadighi et al. 2006). Based on these findings, this in vitro assay can be considered as a valid meth- od for evaluation of drug sensitivity in both species of Lesihmania causing cutaneous leish- maniasis Iran. Despite these supportive investigations, however, no potential agreement was noticed between the clinical response of L. braziliensis, and L. donovani isolates, and the intracellu- lar amasigote in vitro system in studies per- formed in various endemic regions around the globe (Rojas et al. 2006, Yardley et al. 2006, Rijal et al. 2007). Overall, these agree- ments and contradictions between different reports can be explained by some factors that may affect the results of the in vitro assays. Firstly, partially distinctive genetic make-ups of Leishmania species would be a reasonable proof to clarify these disagreements among several surveys carried out on different species of Leishmania (Croft et al. 2006). However, our findings obtained from field strains of L.major were relatively in agreement with the results from other species of Leishmania (L. tropica) in Iran (Hadighi et al. 2006). Moreover, clinical definition of resistance seems to be an important clue for justifying Drug dose In vitro assay Clinical out- comes Sensitive n (%) Clinical out- comes Resistant (%) P-value for McNemar Sensitivi- ty/ Specificity Kappa Total 15 μg/ml Sensitive 20 (71.4) 6 (40) 0.79 71/60 0.35 (.04) 26(60.5) Resistant 8 (28.6) 9 (60) 17(39.5) 30 μg/ml Sensitive 26 (92.9) 4 (26.7) 0.69 93/73 0.68 (<0.001) 30(69.8) Resistant 2 (7.1) 11 (73.3) 13(30.2) 45 μg/ml Sensitive 28 (100) 6 (40) .03 100/60 .66 (< 0.001) 34(79.1) Resistant 0 (0) 9 (60) 9(29.9) 60 μg/ml Sensitive 28 (100) 4 (26.7) .125 100/73 .78 (< 0.001) 32(74.4) Resistant 0 (0) 11 (73.3) 11(25.6) J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 136 http://jad.tums.ac.ir Published Online: March 14, 2017 these discrepancies among different studies (Lira et al. 1999, Croft 2001, Rijal et al. 2007). In the present work, the patients were cate- gorized as responsive and unresponsive after six weeks and following three months of a standard therapy protocol; while, in another study by Luz et al. (2009) this period was extended to 6 and 12 months following the therapy. There were also some differences among various studies in the operational protocols such as time of drugging, the pro- mastigote-macrophage ratio, and timing of the cessation of the experiment (da Luz et al. 2009). Obviously, if we applied another ap- proach, perhaps the correlation value would have been changed. Therefore, in this con- text, we made a big effort to standardize the in vitro procedures through investigating var- ious factors, which could consequently make the results extendable to clinical outcomes. In the present experiment, out of 15 unre- sponsive clinical isolates to Glucantime®, 11 cases were also resistant in the in vitro assay. This evidence can be associated clearly with the intrinsic natures of various Leishmania strains, which make them show original sen- sitivity or resistance to antimonial compo- nents (Polonio and Efferth 2008, Jeddi et al. 2011). Since L. major causes the ZCL dis- ease, it is assumed that these parasites have never been exposed to drugs, and so these 11 clinical isolates which are resistant at both clinical and in vitro levels, are inherently re- sistance to antimony. Beyond the genetic fac- tors, some researches have focused on re- vealing the antimony resistant aspects and have demonstrated that host factors such as genetics and particularly the immune status may have crucial roles on the efficacy of an- timony on Leishmania parasites in vivo (Mur- ray and Delph-Etienne 2000, Campino et al. 2006, Sundar and Goyal 2007). According to this fact, we observed some clinical strains, cured after complete therapy with Glucan- time®, while they showed a resistant pheno- type in the in vitro susceptibility test. Similar finding was reported by Vanaerschot et al. (2013) on L. donovani, and they infered that perhaps the immune components will act in a great synergy with the drug in order to defend more strongly against Leishmania cells. Therefore, recognition of some isolates as responsive to therapy at clinical level cat- egorized as resistant in the in vitro assay is more likely due to lack of immune system effects in the in vitro tests. Furthermore, even different strains of one species of Leish- mania may use multiple resistant mechanisms against antimony components in diverse cir- cumstances, in vitro and in vivo (Ouellette et al. 2004, Chakravarty and Sundar 2010). There- fore, it seems to be a good explanation to de- scribe why some of our isolates showed re- versed phenotypes in vitro and at clinical levels. For example, there were some isolates, which were not responsive to therapy at clinical level, while these were responsive in vitro and vice versa. Perhaps they behaved differently and used various anti-drug mechanisms in differ- ent conditions. Conclusion In the absence of a gold standard method for monitoring drug sensitivity, based on find- ings of this paper, it is highly recommended that this intracellular-amastigote in vitro as- say can be utilized as a reliable method for both monitoring the antimony efficacy of field strains of L. major and epidemiological studies in different endemic areas of Iran. However, with respect to multi factorial phe- nomenon of antimony resistance in different field strains, further researches should be un- dertaken with more samples and in other en- demic areas of Iran to confirm our prelimi- nary data. Acknowledgements This investigation received financial support J Arthropod-Borne Dis, March 2017, 11(1): 132–138 N Ghobakhloo et al.: Evaluation of Correlation … 137 http://jad.tums.ac.ir Published Online: March 14, 2017 from the Vice Chancellery of Research Af- fairs of Shiraz University of Medical Sci- ences for financial support (Grant No. 5804). The authors wish to express their gratitude to S Habibi physician and D Pour Kamal staff of Valfajr Health Center for their useful co- operation in this study. We would also like to thank Motahareh Motazedian for editing the English manuscript. The authors declare that there is no conflict of interest related to this study. References Aït-Oudhia K, Gazanion E, Sereno D, Oury B, Dedet J, Pratlong F, Lachaud L (2012) In vitro susceptibility to anti- monials and amphotericin B of Leish- mania infantum strains isolated from dogs in a region lacking drug selection pressure. Vet Parasitol. 187(3): 386–393. 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