Miltefosine Resistant Field Isolate From Indian Kala-Azar Patient Shows Similar Phenotype in Experimental Infection

Emergence of resistance to drugs used to treat the Indian Kala-azar patients makes control strategy shattered. In this bleak situation, Miltefosine (MIL) was introduced to treat mainly antimonial unresponsive cases. Within years, resistance to MIL has been reported. While checking the MIL sensitivity of the recent KA clinical isolates (n = 26), we came across one isolate which showed four times more EC50 for MIL than that of MIL-Sensitive (MIL-S) isolates and considered as putative MIL-Resistant (MIL-R). The expressions of LdMT and LdRos3 genes of this isolate were found down regulated. Th1/Th2 cytokines, ROS and NO, FACS dot plots and mitochondrial trans membrane potential measurement were performed. In vivo hamster model with this MIL-R isolate showed much lesser reduction in liver weight (17.5%) compared to average reduction in liver weight (40.2%) of the animals infected with MIL-S isolates. The splenic and hepatic stamps smears of MIL-R infected hamsters revealed the retention of parasite load of about 51.45%. The splenocytes of these animals failed to proliferate anti leishmanial T-cells and lack of cell mediated immunity hampered recovery. Thus, these phenotypic expressions of experimental model may be considered similar to that of the MIL unresponsive patients. This is first such kind of report.

this deadly disease. For this, each and every field isolate of the patients should be characterized extensively and it is not done in many places including India.
For MIL transport in Leishmania parasites, a two-subunit amino phospholipid translocase, Leishmania donovani miltefosine transporter (LdMT) and its specific beta subunit LdRos3, internalizes the drug 23 . Phospholipid vesicles (liposomes) employed as carrier systems for MIL, reduces its toxic side effects 23 and there have connection between the expression levels of both proteins and the parasite sensitivity towards the drug 24,25 .
Understanding the mechanism of resistance, factors related to it and control strategy to develop thereafter against the resistant parasites, it is prerequisite to study MIL-R isolates in vitro and/or in vivo. In recent report, two Leishmania isolates were identified as MIL-Resistant (MIL-R) by the in vitro and genome study and these isolates were collected from confirmed Indian KA patients 26 . Few years back, researchers in the field generated a MIL-R Leishmania parasite by step-wise increment of drug pressure in vitro and reported that in case of MIL-Sensitive (MIL-S) Leishmania parasite, the effect of MIL was mediated through Apoptosis-like death but not in MIL-R Leishmania parasite 27 . Till date, there is no report of animal models for in vivo characterization of the MIL-R isolates. The phenotypic expression observed in the animal model may be similar to that of the unresponsive MIL patients and would be instrumental in developing the control strategy.
In the present study, we have characterized all the clinical isolates of Indian KA at species level because of the fact that though Leishmania donovani historically known as the causative agent for Indian KA or VL 3,4 , other species (L. tropica) is found to be associated with the disease 5,28 . Thus, before going for any typological work with any clinical isolate, it became mandate to ascertain its identification at species level with the help of species specific markers [e.g., rRNA gene-internal transcribed spacers (ITS), heat shock protein of 70 kDa (hsp70), Major surface protease msp (gp63) gene and genes encoding cysteine proteinase B etc] [29][30][31][32] . As a part of our epidemiological search for Indian KA, we rigorously characterized all the recently collected clinical isolates through Randomly amplified polymorphic DNA (RAPD) analysis and performed Restriction Fragment Length Polymorphism (RFLP) analysis with the help of several species specific markers (ITS1 and hsp70) to ascertain their species identity 4,5 . All the isolates used here (n = 26) were collected from confirmed Indian KA and typed as Leishmania donovani 4,5 . Then we have checked the drug sensitivity of all isolates (n = 26) for MIL and found one as putative MIL-R field isolate of KA (L. donovani) with four times more EC 50 for MIL than that of MIL-Sensitive (MIL-S) isolates. We have opted for in vivo evaluation of this field isolate (study code T9) in hamster model. Infection with this MIL-R isolate in hamsters showed 17.5% reduction in liver weight compared to average reduction in liver weight by 40.2% of the animals infected with MIL-S isolates. MIL-R infected animals revealed the retention of parasite load in spleen and liver by about 51.45% respectively. The splenocytes of these animals failed to proliferate anti leishmanial T-cells and this T-cell anergy hampered recovery mimicking the scenario with MIL unresponsive patients. Thus, we are reporting for the first time, the phenotypic expressions of confirmed MIL-R L.donovani isolate in hamster model.

Identification of the clinical isolates at species level by Restriction Fragment Length
Polymorphism (RFLP) method. The Internal Transcribed Spacer 1 (ITS1) RFLP 33

and the Heat Shock
Protein 70 (hsp70) RFLP 32 are well-known molecular markers for the characterization of Leishmania parasites at species level. The ITS1 region and hsp70 region of all the samples were amplified separately and subjected to ITS1 RFLP and hsp70 RFLP analysis individually. Species-specific RFLP patterns were obtained for L.donovani WHO strain (DD8) and L. tropica WHO strain (K27) respectively. A portion of the isolates of the present study, had been characterized earlier by RAPD (n = 9) 4 and RFLP (n = 25) 5, 34 methods. We noticed that the additional clinical isolate (n = 1) of the present study have shown similar ITS1 RFLP ( Supplementary Fig. S1) and hsp70 RFLP profile ( Supplementary Fig. S2) as that of L. donovani standard strain DD8. Thus, the clinical isolates along with the putative MIL-R (study code T9) included in the present study were identified as L. donovani.
In vitro MIL susceptibility assay. MIL susceptibility was determined at intracellular amastigote stage for the field isolates of KA. In the present study, we have used RAW 264.7 cells as host cell and percentage of infected MØs ranged from 80 to 89 and the number of amastigotes/100 MØs ranged from 90 to 98. When the intracellular amastigotes of the KA isolates were subjected to test the susceptibility towards MIL, the EC 50 values ranged from 1.74 ± 0.10 to 5.35 ± 0.83 μM with a mean EC 50 of 3.32 ± 0.07 μM for MIL-sensitive (MIL-S) isolates while only one field isolate, T9 showed EC 50 of 13.47 ± 0.87 μM (Table 1) which was approximately 4 times more EC 50 value compared to the mean EC 50 value of the MIL-S isolates. On the other hand, the AG83 (MHOM/IN/1983/AG83) isolate was used as a reference strain as it has been thoroughly worked out by workers as sensitive towards both of the drugs: MIL 35 and Sodium Stibo Gluconate (SSG) 36 . Our single MIL-resistant (MIL-R) isolate from the field, showed approximately 3 times higher EC 50 value compared to the EC 50 value of the L. donovani MIL-S isolate (AG83).
This observation corroborated the previous report of two MIL-R isolates 26 , where the researchers suggested that the IC 50 value of MIL-R field isolates approximately 2 times more higher than the IC 50 value of the L. donovani MIL-S standard strain (DD8) 26 although it is also reported that, the in vitro activity regarding the effectiveness of any anti-leishmanial drugs against Leishmanial parasites may be dependent on the host cell 37 . Our putative MIL-R isolate (T9) is SSG-Sensitive (SSG-S) with EC 50 value 4.69 ± 0.30 μg/ml and the result was also expressed in terms of Activity Index (AI) using AG83 as reference isolate of KA. Isolates with an AI ≥ 3.0 are considered as SSG-Resistant (SSG-R) and an AI < 3 are considered as SSG-Sensitive (SSG-S) 34,38 . The AI of the MIL-R isolate is 2.51. Therefore it was identified as SSG-S.
Expression of cytokines level. In case of MØs infected with MIL-S clinical isolates ( Fig. 1a panels AG83,   T8), the expression level of IL-10 were gradually decreased with increasing concentrations of MIL. On the other hand, there was no significant decrease in the expression level of IL-10 release from MØs infected with putative MIL-R isolate (T9) and then treated with MIL ( Fig. 1a).
On the other hand, the expression levels of IL-12 ( Fig. 1b panels AG83, T8) and TNF-α ( Fig. 1c panels AG83, T8) releases from MØs infected with MIL-S KA isolates following drug treatment (MIL), were gradually increased with increasing concentrations of MIL. On contrary, there is no significant change in the expression levels of IL-12 ( Fig. 1b, panel T9) and TNF-α (Fig. 1c, panel T9) releases from MØs infected with putative MIL-R isolate and then treated with MIL with respect of its infected control group.

Measurement of nitric oxide (NO) and reactive oxygen species (ROS).
The in vitro MIL sensitivity assay and cytokines data showed the response patterns of the studied clinical isolates towards MIL. We carried out experiments to understand the status of NO and ROS in infected and drug treated macrophages because they are the essential leishmanicidal molecules as stated earlier 39 .
In case of MIL-S clinical isolates (AG83 & T8) infected MØs, the generation levels of NO ( Fig. 2a panels AG83, T8) and ROS ( Fig. 2b panels AG83, T8) were gradually enhanced with increasing concentrations of MIL. There was no significant increase in the NO (Fig. 2a, panel T9) and ROS (Fig. 2b, panel T9) releases from MØs infected with putative MIL-R isolate (T9) and treated with MIL with respect to infected control group.
Detection of the mitochondrial trans membrane potential (ΔΨm) and viable Leishmania isolates following MIL treatment. JC-1 was used as a probe for the measurement of ΔΨm by flow cytometry.
We observed that the MIL-S clinical isolates (AG83, T8) showed sensitivity to MIL as evident from the noteworthy decrease in ΔΨm after 72 h of MIL treatment (Fig. 3a,b panels AG83, T8). When the putative MIL-R isolate (T9) was exposed to similar MIL treatment, the red/green fluorescence ratio was not decreased significantly due to the less sensitivity towards MIL (Fig. 3a,b panel T9).
The flow cytometric analysis with FITC Annexin V/PI double staining result revealed that when MIL-S clinical isolates were exposed to MIL, around 7% viable population of cells were observed (Fig. 3c,d panels AG83, T8). On contrary, after MIL exposure to T9 (putative MIL-R) about 67% viable population of cells were observed (Fig. 3c,  Parasite load of liver and spleen were expressed as Leishman-Donovan Unit (LDU). In liver, the parasite load after MIL treatment in experimental animal groups (AG83-TRE & T8-TRE) showed about 3-7% retention compared to that of the infected groups (Fig. 5c, panels AG83, T8) while group T9-TRE animals (infected with putative MIL-R isolate and then treated with MIL) showed retention of 32-62% (average 51.54%) parasites in the liver (Fig. 5c, panel T9). The retention of parasite load after MIL treatment in experimental animal groups (AG83-TRE & T8-TRE) of animals was about 3-8% in spleen compared to that of the infected groups ( Fig. 5d panels AG83, T8). T9-TRE animals (infected with putative MIL-R isolate and then treated with MIL) showed retention of 36-69% (average 51.35%) parasites in spleen (Fig. 5d, panel T9).
T-cell proliferation assay. The splenocytes of treated MIL-S groups (AG83-TRE & T8-TRE) stimulated with SLA showed a significantly higher level of T-cell proliferation than that of the infected groups and the splenocytes of T9-TRE group animals failed to proliferate anti leishmanial T-cell against leishmanial antigen at significantly higher level than T9-INF group ( Supplementary Fig. S5).

Measurement of antileishmanial antibody responses.
To understand the disease dynamics, anti leishmanial antibodies were measured using the sera from all the groups of animals. The presence of anti leishmanial IgG2 antibodies in the sera of these animals were detected together with IgG1 ( Fig. 6a-c). Following MIL treatment, in respect to the infected groups of hamsters (AG83-INF and T8-INF groups), the level of IgG1 isotype of treated groups [AG83-TRE (P = 0.001 at 10 −1 dilution), T8-TRE (P = 0.0039 at 10 −1 dilution)] were decreased significantly (Fig. 6a,b) except for T9-TRE group (hamsters infected with putative MIL-R isolate T9 and then treated with MIL) (Fig. 6c). On the other hand, MIL treated hamsters groups: AG83-TRE (P < 0.0001 at 10 −3 dilution) and T8-TRE (P < 0.0001 at 10 −3 dilution) showed significantly increased level of IgG2 in respect of AG83-INF and T8-INF groups (Fig. 6a,b). In contrast, there was no significant change in the level of IgG2 between T9-TRE and T9-INF group (Fig. 6c).

Analysis of Th1/Th2 mRNA cytokines levels by Real-time PCR.
Real-time PCR data revealed that there was noteworthy increased expression levels in the mRNA transcripts of IFN-γ (Fig. 7a, panel AG83), iNOS (Fig. 7b, panel AG83) and significant decreased expression levels in the mRNA transcripts of TGF-β (Fig. 7c, panel AG83) in the MIL treated MIL-S group of animals (AG83-TRE). T9-TRE group of animals showed no significant change in the expression levels of mRNA transcripts of Th1, Th2 cytokines and iNOS with respect to T9-INF group (Fig. 7a-c, panel T9).

Discussion
The common pentavalent antimonials like sodium stibo gluconate (SSG) used for the treatment of VL, necessitate prolonged course of treatment and is losing its efficacy due to increasing parasite resistance. This has emerged as a major difficulty in the treatment and control of VL 9 . The progression of SSG resistance in the endemic region specially in Bihar, directed that resistance could also come out to the other established anti leishmanial drugs and this may be due to poverty, illiteracy, poor health and HIV/VL-coinfection 6    have been executed previously to demonstrate the role of probable factors in the treatment failure and mechanism of resistance towards the drug MIL 22,27 . Function of fatty acid and steroid metabolism in addition to the expression levels of two MIL transporter proteins, LdMT and its specific beta subunit LdRos3 24, 25 seem responsible for the resistance. It was further suggested that LdMT gene mutation could be employed as a molecular marker for MIL-R L. donovani isolates 26 .
Earlier studies confirmed that the biochemical actions for SSG uptake are catalyzed either by thiol metabolising genes or antimony transporter genes and several types of ATP Binding Cassette (ABC) transporters are related to multi-drug resistance (MDR) 36 . We have noticed the increment of 3.4 times of MRPA gene in the putative MIL-R corroborating the observations that in in vitro conditions, the MDR-related proteins [Multidrug Resistant Protein A (MRPA) or P-glycoprotein (PGPA)] have been amplified in different Leishmania spp. in response to different drugs 42 . The parasites are not able to metabolize MIL itself and can extrude via either exocytosis or probably by ABC transporter protein, such as P-glycoprotein (mdr1) 43 . These studies considerably increased our knowledge about Miltefosine resistance in clinical isolates of KA. Further studies need to be performed in the natural populations of L. donovani to examine the epidemiology of resistance in order to diminish the harshness of KA. In order to divulge the mechanism of resistance, establishment of in vivo animal model is essential. The phenotypic expression in experimental infection model may be extrapolated to that of the KA patients unresponsive to Miltefosine.
By testing the MIL susceptibility of all the clinical isolates of KA and PKDL in vitro amastigote-macrophage model, we identified one putative MIL resistant (MIL-R) field isolate (study code T9), which is typified as L. donovani by ITS1 RFLP and hsp70 RFLP methods. This finding was supported through the measurement of Th1, Th2 cytokines and level of ROS and NO release from MIL induced macrophages infected with this isolate and then treated with the drug. The measurement of ΔΨm and FITC-conjugated Annexin-V and PI double staining results further corroborated our claim. Experimental MIL-resistant L. donovani isolates showed down regulated expression of LdMT and LdRos3 transporters 25 . Our study with single field isolate, resistant to MIL also showed the down regulation in the expression of these transporters. The MIL-R fields isolate also showed approximately 3.4 times higher MRPA expression level than MIL-S isolate (Supplementary Fig. S4).   Hamster is a superior model for VL and expands a progressive, fatal disease which is very closely related to the human symptoms of the disease 44,45 . The splenic and hepatic stamps smears revealed the retention of parasite load after MIL treatment in the AG83-TRE and T8-TRE groups of animals of about 3-8%. On contrary, animals infected with MIL-R isolate and then treated with the drug showed retention of 36-69% (average 51.35%) parasites in spleen and 32-62% (average 51.54%) parasites in the liver.
Cell mediated immunity impaired in Leishmania infection is characterized by marked T-cell anergy specific for Leishmanial antigen 46 . After checking the effects of MIL on treated animals, we became interested to see whether the T-cell anergy occurred during progressive infection, could be reversed by the treatment of MIL. The splenocytes of T9-TRE group of animals failed to proliferate anti leishmanial T-cell in response to leishmanial antigen.
Active VL is also associated with the production of an altered level of antibody 44 . Significant increase in IgG2 levels in cured animals is surrogate marker of enhanced cell mediated immunity 45 . Our study revealed that MIL treated hamsters groups: AG83-TRE and T8-TRE showed significantly increased level of IgG2, indirectly indicating development of an effective Th1 type immune response 44,47 . In contrast, there was no enhancement of cell mediated immunity in the treated MIL-R infected (T9-TRE) group. This observation further supported through the measurement of mRNA transcripts of Th1 and Th2 cytokines.
Our observations strongly suggested that out of three groups of experimental animals, AG83-TRE and T8-TRE groups of animal were almost cured with MIL treatment but T9-TRE group of animals did not realize recovery establishing the animal model for MIL-R faithfully.
The whole genome analysis of this clinical isolate is in progress.

Ethics Statements. Bone marrow aspirates were collected from KA patients and approved by the Ethical
Committee of the Calcutta National Medical College, Kolkata. The written consent was obtained from legal guardian of the patient (as it was the case of a minor). In the present study, all methods were carried out in accordance with the relevant guidelines.  Quantification of nitric oxide (NO). NO production was evaluated by the Griess Reagent as described previously 39 . Measurement of mitochondrial trans membrane potential. Transmembrane potential (ΔΨm) was evaluated using JC-1, a lipophilic cationic dye 39 39 . Briefly, untreated, MIL-treated (72 hrs treatment) promastigotes were washed with PBS. The pellets were resuspended in 1X binding buffer at a concentration of 1 × 10 6 /ml followed by incubation with 5 μl of FITC Annexin V and 1 µg/ml PI for 25 min in dark. Then 400 μl of 1X binding buffer was added and samples were analyzed by Flow Cytometry within 1 hr.

Measurement of reactive oxygen species (ROS
RNA isolation and semi quantitative RT-PCR analysis of the genes responsible for MIL transport. RNA was isolated from samples by disrupting in Trizol solution 51 and newly prepared cDNA were then amplified by taking 0.5 μl of cDNA with1 μl 10 mM dNTP, 1.5 μl 50 mM MgCl 2 , 0.5 μl Taq polymerase as well as gene specific primers (Supplementary Table S2). Amplification reactions were performed with cycling conditions for genes of interest were 5 min at 95 °C, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at (55°-60 °C) for 30 s and extension at 72 °C for 30 s. PCR-amplified respective gene products were checked by Agarose gel electrophoresis. For Densitometry analyses, the ImageJ software (National Institute of Health) was used and the same band area in Agarose gel was used to find out band intensity and normalized for GAPDH.
Statistical analyses for the in vitro study. Results of all in vitro studies are expressed as mean ± SD.
Student's t test for significance was carried out using GraphPad prism software and P value of <0.05 was considered to be significant. Correlation between the EC 50 and other parameters were determined by Spearman rank correlation coefficient and expressed as r 36 .
Animals for in vivo study. Golden

Selection of MIL doses for in vivo experiments. Hamsters were challenged with freshly transformed
Leishmania T9, T8 and AG83 parasites (10 7 parasites/animal) via intra cardiac route 52 . After 8 weeks of infection, infected animals were treated with MIL at dose of 40 mg/kg (body weight) for 10 consecutive days and were sacrificed on days 45 post treatment 44 . Hamsters have been grouped in the following ways: Normal: 5 healthy control without challenge infection and treatment with MIL; Infected: each 10 animals were infected with T9, T8 and AG83 respectively and would be represented as T9-INF, T8-INF and AG83-INF respectively; Treated: among 10 infected animals of each group, 5 were treated with MIL. These groups henceforth would be written as T9 -TRE, T8-TRE and AG83-TRE respectively.
Collection of blood and preparation of serum. Blood was collected from hamsters as illustrated earlier 39 and kept overnight at 4 °C. Then serum was prepared from collected blood sample.

Determination of Organomegaly and Parasite Burden in spleen and liver. Weight and parasitic
burden of spleen and liver from experimental groups of animals were assessed after sacrifice. Splenic and hepatic parasite burden of hamsters of different groups were determined by microscopic evaluation of Giemsa stained tissue imprints method 39 . Total parasite load in each organ is expressed in LDU unit (Leishman-Donovan Unit). 1 LDU = amastigote per nucleated cell x organ weight in milligram.
T-cell proliferation assay. Splenocytes from studied groups of hamsters were prepared after Ficoll density gradient centrifugation and dissolved in complete RPMI medium and then plated in 96-well plates (at a concentration of 10 5 cells/well) therefore allowed to proliferate for 72hrs at 37 °C with 5% CO 2 either in the presence or absence of SLA (5 μg/ml) or ConA (5 μg/ml). Cells were treated with MTT (0.5 mg/ml) as described earlier 39 . Then Isopropanol-HCl mixture (0.04%) was used to solubilise the MTT crystals and the absorbance at 570 nm was interpreted at an ELISA plate reader (DTX 800 multimode detector, Beckman Coulter, California).
Measurement of anti leishmanial antibody responses. Serum samples were collected from different groups of hamsters and examined to find out the parasite SLA-specific antibody titer. IgG1 and IgG2 present in the collected sera were measured as stated earlier 39 .
Real-time PCR to estimate expressions mRNAs of Th1/Th2 cytokines levels. RNA was isolated from the splenocyte of hamsters and 50-100 ng of total RNA was used for synthesis of cDNA. RT-qPCR was done as described elsewhere 44 . Briefly, it was carried out with 7 μl of SYBR green PCR master mix, 1 μl of cDNA from RT reaction mix and gene specific primers in a final volume of 15 μl. PCR was conducted under the following conditions: initial denaturation at 95 °C for 10 min followed by 40 cycles, each consisting of denaturation at 95 °C for 15 s, annealing at 58 °C for 1 min and extension at 72 °C for 40 s per cycle using the ABI 7500 Real time PCR system and data were analyzed by the comparative CT method 45,54 . cDNAs from infected hamsters were used as comparator samples. All quantifications were normalized to the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT).
Scientific RepoRts | 7: 10330 | DOI:10.1038/s41598-017-09720-1 Statistical analysis for in vivo study. Statistical level of significance between different groups was calculated by unpaired two-tailed Student's t-test with GraphPad Prism software (San Diego, California, USA). P < 0.05 were considered to be significant for all analyses.