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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 4  |  Page : 351-359

Clinical and laboratory evaluation of cured and non-cured patients with cutaneous leishmaniasis treated by Glucantime


1 Leishmaniasis Research Center; Department of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
2 Research Center for Modeling in Health, Kerman University of Medical Sciences, Kerman, Iran
3 Leishmaniasis Research Center, Kerman, Iran
4 Kerman Oral and Dental Diseases Research Center, Kerman, Iran
5 Department of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
6 Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran
7 School of Medicine, Bam University of Medical Sciences, Bam; Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Submission19-Jul-2018
Date of Acceptance08-Apr-2019
Date of Web Publication30-Nov-2020

Correspondence Address:
Dr. I Sharifi
Ph.D, Professor, Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.302039

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  Abstract 

Background & objectives: Insufficient treatment of cutaneous leishmaniasis (CL) by conventional drugs is a major barrier in control strategies. This study was aimed to evaluate Glucantime efficacy and the susceptibility of Glucantime unresponsive and responsive CL isolates in the field and laboratory.
Methods: Chi-square test (x[2]) was used to determine the significance of difference between proportions in Glucantime-treated patients. The inhibitory activity of various concentrations of Glucantime against Leishmenia tropica stages was evaluated by a colorimetric cell viability MTT and macrophage assays. Mixed model, t-test and ANOVA were performed to determine the significance of difference between various concentrations of Glucantime unresponsive or responsive isolates and untreated control group and p <0.05 was defined as significant level. Altogether, 89.8% of the patients were cured by Glucantime, whilst 10.2% remained non-cured.
Results: The overall Glucantime efficacy in different age groups and genders was similar. The IC50 values of promastigotes and amastigotes for Glucanime unresponsive isolates were 2.1 and 2.6 times higher than the equivalent rates obtained for responsive cases, respectively. The overall mean number of amastigotes within macrophages in unresponsive isolates was significantly higher (32.68 ± 1.24) than that in responsive ones (18.68 ± 1.52, p <0.001). Glucantime unresponsive and responsive field isolates of anthroponotic CL (ACL) caused by L. tropica strongly correlated to in vitro assays.
Interpretation & conclusion: Monitoring of Glucantime unresponsiveness by the health surveillance system is extremely important, where anthroponotic transmission occurs in humans. Hence, physicians should be aware of such clinical unresponsive presentations with ACL for antimonial therapeutic failure to improve management of disease in endemic regions.

Keywords: Cutaneous leishmaniasis; Glucantime unresponsiveness; in vitro evaluation


How to cite this article:
Ezatkhah F, Sharifi I, Babaei Z, Baneshi M R, Zolala F, Kermanizadeh A, Keyhani A, Sharifi M, Dezaki E S, Aflatoonian M R, Aflatoonian B, Khatami M, Bamorovat M. Clinical and laboratory evaluation of cured and non-cured patients with cutaneous leishmaniasis treated by Glucantime. J Vector Borne Dis 2019;56:351-9

How to cite this URL:
Ezatkhah F, Sharifi I, Babaei Z, Baneshi M R, Zolala F, Kermanizadeh A, Keyhani A, Sharifi M, Dezaki E S, Aflatoonian M R, Aflatoonian B, Khatami M, Bamorovat M. Clinical and laboratory evaluation of cured and non-cured patients with cutaneous leishmaniasis treated by Glucantime. J Vector Borne Dis [serial online] 2019 [cited 2021 Nov 28];56:351-9. Available from: https://www.jvbd.org/text.asp?2019/56/4/351/302039


  Introduction Top


Leishmaniasis is a vector-borne disease and occurs in five World Health Organization (WHO) regions. Current data by Global Health Observatory Office indicated that an estimated 431 million people with cutaneous leishmaniasis (CL) in over 82 countries are at risk[1],[2],[3], with an overall prevalence of 12 million cases and incidence rate of approximately 1.2 million new patients annually[1]. Iran is among the seven most affected countries where most of the global CL cases (90%) occur[4]. Current reports indicate that the number of active and inactive cases have reached hyperendemic points, notably in the conflict zones of the Middle East countries. Therefore, the overall global burden of CL disease would remarkably increase by a factor of 6 to 10[1],[5],[6].

There are three distinct classical forms of leishmaniasis: visceral (VL), cutaneous (CL) and muco-cutaneous (MCL). Cutaneous leishmaniasis is the public form which comprises approximately 75% of the cases. This clinical form is a global challenge and causes a spectrum of disease syndromes from self-healing to chronic disfiguring manifestations and with a significant social stigma[1],[2].

Vector and reservoir control is difficult by the diverse ecology of over 100 species which are implicated in propagation and transmission of Leishmania species[7]. Currently, there is no safe vaccine available to control various forms of the disease in many clinical and epidemiological settings[8]. At present, leishmaniasis is essentially controlled through the use of chemotherapy. Although, pentavalent antimonial drugs (SbV) have been used as the first-line treatment for nearly 70 yrs[1],[9],[10]. resistance has frequently been reported, particularly against L. tropica and L. donovani, the two Old World anthroponotic CL (ACL) and anthroponotic VL (AVL), respectively[9][11],[12],[13],[14] Moreover, the use of second-line drugs is limited due to low efficacy and high cost[3],[15].

Since humans are the only known main reservoir host for the two anthroponotic Leishmania species, control strategies are basically focused on active case-detection, early diagnosis and appropriate treatment modality[3]. Therefore, these two species have developed resistance to antimonials in current practice; although, the spatial distribution and the extent of unresponsiveness to any single drug vary importantly[9],[16]. Moreover, the existing treatments are expensive and often associated with low efficacy, long duration of action and along with significant adverse effects[10],[17]

In Iran, ACL and ZCL due to L. tropica and L. major are the two causes of human CL, respectively. They affect 18 out of 31 provinces with considerable social and public health impact in terms of medical burden[18], that is why Iran has been among the seven most affected countries in the world[19]. Hyperendemic foci of ACL are merely restricted in southeastern Iran, notably in Bam and Kerman counties[20]. The typical presentations are manifested as localized forms, readily respond to conventional treatment (responsive cases) and comprise approximately 90-95% of the total forms[21]. In contrast, atypical cases of ACL, often persist as non-healing and chronic forms which include nearly 5-10%, last for several years and do not respond properly to standard therapies[22]. Such non-healing forms have been implicated in occasional cases of leishmaniasis recidivans[23].

Recent documents indicated that an increasing number of ACL cases have developed unresponsiveness to Glucantime, the drug of choice against all the clinical and epidemiological forms of leishmaniasis in Iran[10],[18],[24]. Overall, 12% of ACL isolates did not respond to treatment with Glucantime in Mashhad[25], 11% of the cases were clinically unresponsive to the same drug in Bam[21] and 11.3% in Kerman county in previous reports[24]. Other studies have also reported some levels of treatment failure in ZCL endemic areas in Iran[27],[28],[29],[30]. Therefore, the present study was aimed to evaluate the current trend of Glucantime efficacy, clinical presentations and also to compare the susceptibility of ACL Glucantime-unrespon- sive and responsive isolates in field and experimental models. This observation is highly essential for improvining the therapeutic measures in endemic areas.


  Material & Methods Top


Ethical consideration

Approval of the Ethical Committees of the Leishmaniasis Research Center and Kerman University of Medical Sciences (Project No. 91/351 and Ethic No. IR.KMU. REC. 1391. 357) was obtained for the present study. Written informed consent of the patients was obtained. Patients with CL were treated, accordingly. In addition, patients suspected of having secondary complications were referred to the county hospital levels for further diagnosis and proper medications.

Study site

A treatment health clinic has been assigned since 2005, after the massive earthquake and the sustained ACL epidemic in Bam, for the management of the CL patients. This clinic was used as a headquarters for controlling all the activities including physical examination, clinical evaluation ofthe lesions, and detection ofthe cases by active and passive approaches, diagnosis and treatment of the patients and along with prophylactic measures. The clinic was well-equipped and a team of expert physicians and well-trained health personnel was in charge of various activities in the clinic. The National Guidelines Protocol for control of CL was initially implemented in this clinic, and then assigned to endemic areas in various provinces of the country. Identification of the CL isolates was carried out at Leishmaniasis Research Center, School of Medicine, Kerman University of Medical Sciences in Kerman.

Non-healed patients

Non-healing or unresponsive cases (refractory) were defined as the patients who did not heal or respond to two full courses of treatment by systemic (intramuscular, IM) injection of 20 mg/kg/day for three weeks or local (intralesional) route of Glucantime administration every week for total of 12 times along with cryotherapy by liquid nitrogen according to the National Guidelines and in line with WHO protocol for the treatment of leishmaniasis[3],[18]. Patients with <5 lesions and >5 lesions were treated intralesionally (along with cryotherapy) or intramuscularly alone, respectively.

Healed patients

In contrast to the refractory patients, responsive cases (healing) were classified as those patients who showed complete re-epithelialization of the lesion and received either one or two full courses of Glucantime[18]. Treatment evaluation was achieved following six months of active and/or passive follow-up examination for both unresponsive and responsive cases.

Clinical isolates and sampling

This work was performed as a cross-sectional and also experimental study on clinical field isolates. Overall, among 973 CL cases, 30 clinical isolates; consisting of 10 unresponsive and 20 responsive patients were randomly selected. The cutaneous lesions were cleaned by ethyl alcohol for direct smear preparations. Tissue scrapings were taken from the periphery of active le sions by a scalpel and blade (No.15), smeared on to glass slides, fixed with methanol, stained by standard Giemsa and thoroughly examined by a light microscope for the presence of amastigotes (Leishman bodies). Simultaneously, tissue scrapings were inoculated into tubes containing Novy- MacNeal-Nicolle medium (NNN), pH 7.2 at 24 ± l °C for one week, then subcultured into RPMI 1640 medium (Gibco, UK), containing 10% heat-inactivated (56 °C for 30 min) fetal calf serum (FCS), streptomycin (200 μg/ml) and penicillin (200 units/ml) and incubated at 24 ± 1 °C for mass production of promastigotes.

Species identification

Parasite promastgote DNA, extracted using Qiagen kit, was subj ected to amplify a variable region of the kine- toplast mini circle DNA (kDNA) for species identification. Nested-PCR was performed in two consecutive rounds as described earlier[31]. The final PCR products were separated on 1.5% agarose gel electrophoresis and visualized under UV transilluminator (Uvitech, Cambridge, UK).

Drug preparation

Glucantime was provided by Sanofi-Aventis, France through the Ministry of Health and Medical Education in Iran. It was prepared in the RPMI 1640 medium just prior to assays. Various concentrations of SbV were diluted in the medium to prepare final concentrations of 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg/ml[32].

Promastigote susceptibility assay

The susceptibility was assessed according to the method described elsewhere. Briefly, serial dilutions of Glucantime in the medium were performed in a 96-well micro titer plate as previously mentioned. Then the promastigotes at log phase were added to each well and incubated at 24 ± 1 °C for standard time of 72 h. Organisms were cultured at the same conditions with no drug used as the untreated control and the medium with no promastigotes and Glucantime used as a blank. All experiments were repeated thrice. Colorimetric cell viability assay was performed as described earlier[32]. Finally, absorbance was read by an ELISA reader (BioTek- ELX800) at 492 nm. The IC50 value (50% inhibitory concentration) was determined by the probit analysis using SPSS software.

Amastigote susceptibility assay

Macrophage cell line J774-A1 (ECACC no. 91051511) was purchased from Pasture Institute, Tehran, Iran. Macrophages were added to complete RPMI 1640 medium as previously mentioned. Amastigote viability test was performed by adding 90 μl of trypan blue solution (0.2%) in physiological saline containing and 0.01% sodium azide to 10 μl of cell suspension (10[6]/ml). After two min, the cells were counted under a light microscope and viability was determined as follows:

% Viability = Live cells/All counted cells *100

Then, for each intra-macrophage amastigote assay, 200 μl of murine macrophages cell line (10[6] cells/ml) were attached to the 8-chamber slide (Lab-Tek, Nunc International NY, USA) and incubated at 37 °C and 5% CO2 for 24 h. Afterwards, the infected murine macrophages with amastigotes at different concentrations were incubated for additional 72 h. The experimental treatments were repeated in triplicates for unresponsive and responsive isolates. Treated slides were dried, fixed with methanol, stained by Giemsa and examined under a light microscope. Drug activity was evaluated by the mean number of amastigotes in 100 macrophages in triplicates and also by the calculated IC50 values.

Statistical analysis

Data analysis was performed by SPSS software ver. 18 (Chicago, Illinois, USA). Chi-square (x[2]) was used to determine the significance of difference between proportions and mixed-model, t-test and ANOVA (analysis of variances) were used to determine the efficacy of Glucantime between groups and any possible different effects among various concentrations of Glucantime-un- responsive or responsive isolates and the untreated control group. Pearson correlation coefficient was carried out among the mean number of amastigotes for the patients with unresponsive and responsive isolates. P <0.05 was considered as a significant difference. Probit procedure of the SPSS was used to calculate the IC50 values.


  Results Top


Demographic and clinical data

Over a 4-year period (2014-2017), 973 cases of CL were evaluated in southeastern Iran. The mean number of lesions was 1.5 (range: 1-10), equally distributed among the unresponsive and responsive patients with ACL. The mean diameter of lesions in non-cured and cured cases was 35 and 16 mm, respectively. Most of the lesions were single (64.5%) and the rest have two or more lesions (35.5%). Altogether, 89.8% of the patients were cured by Glucantime, whilst 10.2% remained uncured. The overall Glucantime efficacy in different age groups and genders was same [Table 1]. Majority of the patients were treated by intralesional route plus cryotherapy (n = 691; 71%), while the remaining were treated by the intramuscular route (n = 282; 29%) [Table 2]. The duration of the lesion in responsive patients was 4-8 months, but it was significantly longer in unresponsive cases (average 23 months). Totally, all the representative isolates (n = 127) including 30 specimens which were experimentally evaluated by the in vitro drug susceptibility assays and 97 of the other CL isolates were identified to be L. tropica.
Table 1: Demographic characteristics and clinical cure to Glucantime in patients with anthroponotic cutaneous leishmaniasis in southeastern Iran, 2014–2017

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Table 2: Clinical cure and non-cure to Glucantime in patients with anthroponotic cutaneous leishmaniasis by intralesional coupled with cryotherapy and systemic administration

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Clinical presentation

Of the patients 94.5% demonstrated localized and typical clinical presentation in both the healed and non- healed cases [Figure 1], whereas 5.5% displayed atypical clinical feature; of which lupoid leishmaniasis, lymphedema and lymphadenic forms were predominant [Figure 2].
Figure 1: Representative images of the non-healing skin lesions taken from Glucantime unresponsive patients with anthroponotic cutaneous leishmaniasis in endemic areas of Bam, southeastern Iran (a-i). [i]: A leishmaniasis recidivans (lupoid leishmaniasis) cutaneous lesion as charaterized by reactivation of satellitelike papules surrounding the scar of primary lesion[23].

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Figure 2: Representative images of the healing skin lesions taken from Glucantime responsive patients with anthroponotic cutaneous leishmaniasis from endemic areas of Bam, southeastern Iran (a-i). Image (g) is taken from reference [33] .

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Promastigote susceptibility assay

Initially, the effect of Glucatime on L. major promasti-gotes was investigated. Glucantime decreased promasti- gotes viability in a dose-response manner. The OD values of unresponsive cases as measured by enzyme-linked immunosorbent assay (ELISA) were significantly higher than the corresponding values for responsive isolates at various concentrations. These OD values were subjected to calculate the 50% inhibitory concentrations (IC50) for both the non-healed and healed isolates [Figure 3].
Figure 3: Comparison of optical density (OD) between various concentrations of Glucantime in unresponsive and responsive isolates of patients with anthroponotic cutaneous leishmaniasis on the multiplication rate of promastigotes by colorimetric assay.

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The overall OD values for unresponsive promastigote isolates were significantly higher compared to the corresponding responsive ones (p <0.001). The mean inhibitory effect of Glucantime among various concentrations of unresponsive and responsive isolates and also with the untreated control group was significantly higher (p <0.001, [Figure 4]).
Figure 4: Inhibitory effect of various concentrations of Glucantime in unresponsive and responsive isolates of patients with anthroponotic cutaneous leishmaniasis on the multiplication rate of promastigotes by colorimetric assay.

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Amastigote susceptibility assay

Leishmaniacidal activity of Glucantime was assessed by the mean multiplication rate of the clinical stage inside the macrophages. Glucantime unresponsive and responsive isolates behaved in similar manner at lower concentrations (6.25, 12.5 and 25 μg/ml) compared with the respective untreated control isolates [Table 3]. The mean multiplication rate of amastigotes in responsive isolates significantly decreased at 50 μl/ml and higher concentration (p <0.001), while similar effect was obtained at 100 μl/ml and higher concentrations in Glucantime unresponsive isolates (p <0.001). The overall mean numbers of amastigotes within macrophages in unresponsive and responsive isolates were 32.68 ± 1.24 and 18.68 ± 1.52, respectively (p <0.001).
Table 3: The effect of various concentrations of Glucantime on the mean multiplication rate of amastigotes in unresponsive and responsive isolates of patients with anthroponotic cutaneous leishmaniasis

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The IC50 values of promastigote and amastigote stages in unresponsive and responsive isolates were 489.6 μg/ ml and 235.9 μg/ml vs. 190.8 μl/ml and 74.6 μl/ml, respectively (p <0.001, [Table 4]). Overall, the IC50 value of promastigotes for the drug unresponsive isolates was approximately 2.1 times higher than the equivalent rates obtained for responsive cases. Similarly, the IC50 value of amastigotes obtained from unresponsive cases was 2.6 times higher than responsive cases. There was a positive correlation between the number of infected macrophages and the mean number of amastigotes in both unresponsive (r = 0.75) and responsive (r = 0.60) isolates [Figure 5], that represents multiplication of the L. tropica amastigotes within macrophages. Although, macrophages harbor the amastigote stages, they ultimately disrupt, despite the elaborated armature and offensive mechanisms involved in releasing oxidative metabolites and various ranges of proteolytic enzymes.
Table 4: Comparison of the IC50 values (μg/ml) for Glucantime among unresponsive and responsive isolates of patients with anthroponotic cutaneous leishmaniasis

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Figure 5: Multiplication of the Leishmania tropica amastigotes within macrophages. (a) A newly infected macrophage; (b) A fully infected macrophage; (c and d) Disruption of infected macrophages.

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On the basis of nested PCR analysis, all the 30 clinical isolates displayed 750 bp fragments which corresponded to L. tropica species [Figure 6]. The representative pictures of skin lesions taken from Glucantime unresponsive patients with ACL in endemic areas of Bam, southeastern Iran are presented in [Figure 1]. All of these non-healing presentations were primarly treated by two sessions of Glucantime either intramuscularly alone or intraleisionally along with cryotherapy, but no cure was obtained. The mean duration of lesion was 22 months at the time of clinical evaluation for unresponsive patients as compared to nine months for responsive cases. The latter patients were followed up to complete cure.
Figure 6: Agarose gel electrophoresis of variable mini circle regions of kinetoplast DNA (kDNA) by nested PCR method (products of the second round). L: DNA ladder (100bp); N: negative control (distilled water); 1: positive control (Leishmania major, 560 bp); 2: positive control (L. tropica, 750 bp); 2–9: L. tropica representative isolates from southeastern Iran.

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  Discussion Top


In the present study, Glucantime demonstrated a great level of treatment efficacy against ACL in the southeast of Iran; although various side effects have been associated with its parenteral application[34]. All the age and sex groups in unresponsive and responsive cases displayed similar pattern of response to Glucantime. The main reason for generation of non-healing forms of CL in the area under study is not well clear. Some authors have introduced intrinsic evidences supporting the idea that defects in the immune response and species and even strain specificity of the causative agent might play crucial roles in creation of such non-healing cases[35]. Underlying complications including diabetes mellitus and opium addiction are common phenomenon and relatively high in the CL endemic area in southeastern Iran. Numerous reports showed that various defensive cell types are affected by these complications and predispose individuals to severe forms of CL. These factors possibly contribute to the chronicity of the CL lesion in non-healing form[36].

Majority of CL lesions are self-healing and cure over time. In ACL foci, a proportion of cases relapse to lupoid leishmaniasis following recovery. This clinical form is a common presentation which lasts for several sessions of anthroponotic transmission cycle from human-to- human[23]. Varying ranges of Glucantime unresponsiveness from different foci of CL have been reported. The overall failure rate was 10.2% for two sessions of Glucantime therapy in patients with ACL in this study. This level of failure is much lower than the patients with CL in central, southern and northern (range 16-59%) Iran[37], but it is comparable to those efficacy rates in Kerman and Mashhad[13],[24],[26].

Drug unresponsiveness primarly remains a major barrier in the treatment and control of ACL and AVL caused by L. tropica and L. donovani, respectively[17]. In the present study, there was a significant difference in the IC50 values among unresponsive and responsive isolates, when comparing extracellular promastigotes with intra-macrophage amastigotes. Based on the previous findings, these two stages of Leishmania species are basically different in sensitivity to SbV[38]. In clinical isolates, the overall IC50 values in unresponsive isolates were significantly higher than those in responsive cases. Similarly, in healing and refractory patients, the IC50 values of promastigotes were significantly higher than the corresponding amastigote stage. The insensitivity of promastigotes to SbV at the same concentration is multifactorial[17]. Several reports have indicated that Leishmania species promastigotes are 2-10 fold less sensitive to SbV than the amastigote stages[9]. Finding of the present study confirmed that amastigotes as clinical stage; despite of being intracellular have greater intrinsic susceptibility to Glucantime than promastigotes. Previous investigations documented that amastigotes have a superior ability to concentrate drugs than promastigotes[9].

Experimental studies have performed to assess the sensitivity of various forms ofLeishmania to SbV including promastigotes cultured in the media, amastigote-mac- rophage model and axenic amastigotes cultured[39],[40],[41]. In the repeated experiments, amastigotes of L. major in murine macrophages were more than 3-fold less sensitive to Pentostam than L. donovani amastigotes. In the present study, there was a strong consistency between the in vitro results and clinical responsive or unresponsive isolates to Glucantime.

Several risk factors are probably responsible for the emergence of such drug failure; although, the exact mechanism and correlation between drug unresponsive isolates and treatment failure remain unclear[42]. Certainly, the determinants of the resistance to particular drug including SbV agents are essentially complex[17],[43],[44],[45]. One of the major factors which influences the activity of SbV is the interaction with the harboring macrophages by accumulation or metabolic mechanisms[9],[17]. Inability of the host immune response, inadequate treatment regimens, species and strain sensitivity to particular drugs, pharmacological deficiencies in the host, drug lot and manufacturer, could influence the outcome of treatment and eventually their therapeutic impacts[19],[43],[46],[47].

Leishmania tropica is considerably heterogeneous as confirmed by various intrinsic methods such as biochemical, immunological and molecular techniques[48]. Anthroponotic CL is associated with diverse and complex clinical and chronic forms which mimic variety of microbial infections in endemic areas as demonstrated in this study. These so-called non-healing presentations last several seasons of transmission cycles in which humans remain the only reservoir host for female phlebotomine sandflies. Therefore, precise clinical assessment of lesions along with appropriate laboratory identification would be critical for selecting proper therapeutic measures in preventing disfiguring consequences and consequent social stigmatization[35],[36],[49].


  Conclusion Top


The results represent that Glucantime demonstrated a successful treatment rate of 89.8%, whilst the nonhealing forms comprised 10.2%. Both isolates responded to Glucantime experimentally, but in different manner; that was, the effect initiated at 50 μl/ml for Glucantime responsive isolates; while it was 100 μg/ml for unresponsive ones, obtained from the patients with ACL. Glucantime unresponsive or responsive isolates were all identified to be ACL caused by L. tropica and strongly correlated to in vitro assays. Monitoring of SbV unre- sponsiveness by the health surveillance system personnel is extremely crucial, where anthroponotic transmission occurs in humans. Hence, physicians and health personnel should be aware of such clinical unresponsive presentations in patients with ACL for antimonial treatment failure to improve management of disease in endemic regions.

 
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    Figures

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    Tables

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