In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy

Abstract

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment’s onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy.

1. Introduction

Leishmaniases are parasitic diseases transmitted by sandflies showing heterogeneous clinical manifestation, depending on the Leishmania species and host health status. About 20 species of Leishmania, mainly belonging to the subgenera Leishmania and Viannia, can parasitize humans. A comprehensive updated taxonomy of trypanosomatidae, including the genus Leishmania, has been recently published [1]. Clinical manifestations range from cutaneous lesions (cutaneous leishmaniasis, CL) to severe systemic multiorgan disease (visceral leishmaniasis, VL). In the Old World (i.e., southern Europe, the Middle East, Asia, and Africa) the etiological agents of CL are Leishmania (Leishmania) species, such as L. (L.) donovani, L. (L.) infantum, L. (L.) major, L. (L.) aethiopica, and L. (L.) tropica [2]. The lesions generally occur on skin portions easily accessible to sand flies, such as the face or limbs. The CL lesions can manifest as a single nodular or ulcerative lesion at the site of parasite inoculation, called localized cutaneous leishmaniasis (LCL) or as multiple lesions caused by infection propagation, named diffuse cutaneous leishmaniasis (DCL) [3]. In the Mediterranean basin, CL often manifests as a single, painless lesion caused by L. (L.) infantum [4], which represents the etiological agent of CL and VL in humans, as well as canine leishmaniasis (CanL).

In the Old World, intralesional pentavalent antimony compounds (i.e., sodium stibogluconate and Meglumine Antimoniate) are among the first choice for the treatment of uncomplicated CL, while liposomal amphotericin B or other systemic treatments can be used for complicated CL or for immunosuppressed patients [5]. Antimonials are used also in the standard therapy for CanL, either alone, or in combination with allopurinol [6]. Notably, treatment failure due to resistance to antimonials has been described in different studies, mostly in the treatment of L. (L.) donovani VL in the Indian subcontinent [7], and rarely in the Mediterranean region [8], where treatment efficiency exceeding 95% in HIV-negative individuals has been reported [9].

In recent years, new evidence of CL cases in north-eastern Italy has been documented [10]. However, to the best of our knowledge, human CL cases not responding to antimonials have never been notified in Italy so far. Here, we report an autochthonous CL case due to L. (L.) infantum in central Italy, with unsatisfactory response to treatment with intralesional pentavalent antimony compounds.

2. Description of the Case

A 61-year-old male was referred from the San Salvatore–Muraglia hospital (Pesaro, Italy), in May 2019 as a suspected case of CL, with a skin lesion on the dorsal left forearm with diameter of 5 × 3 cm. The patient reported not having traveled abroad in the previous years. Physical examination did not reveal any clinical sign of pathology, except for the abovementioned lesion. The lesion was biopsied by a dermatologist in aseptic conditions, with histological report of skin characterized by intense chronic limphoplasmocytoid inflammation and presence of histiocytes containing several Leishmania amastigotes. The ensuing pathological diagnosis was cutaneous leishmaniasis of the left forearm. Laboratory results showed 5.81 × 10⁶ erythrocytes, Hb 17.1 g/dL, MCV 88, normal white blood cells, and platelet count and biochemical parameters within normal range. Renal and liver function were also normal, and no autoantibodies were detected. The patient had just been tested for polyglobulia by a hematologist, and he was in clinical follow-up. No evidence of BCR-ABL or Jak2 mutations was reported.

The patient was treated with a local infiltration of Meglumine Antimoniate (Glucantime^® vials 1.5 g/5 mL—1 vial every week for a total of 5 weeks) in the skin lesion and along its margins. Twelve weeks after the beginning of therapy the clinical result was not deemed satisfactory by the managing clinicians (Figure 1).

After 12 weeks from the beginning of treatment, the patient underwent a second skin biopsy. Maintaining aseptic conditions and after the expression patient’s informed consent, two 5 mm diameter biopsies were collected from the nodular lesion. One biopsy was fixed in 4% formalin and sent for microscopic analysis which showed persistence of histiocytes containing Leishmania amastigotes. The second biopsy was collected in 5 mL sterile Tobie medium, disrupted by pipetting and divided into two aliquots: one was used for parasite isolation as described previously [11,12] and the other for DNA extraction. The DNA was extracted with the DNeasy Blood & Tissue kit (Qiagen) and amplified by a real-time PCR assay (qPCR-ML) as previously described [13,14,15]. The qPCR-ML assay gave positive amplification results indicating the presence of Leishmania spp DNA in the skin sample. Melting analysis [13] allowed amplicons to be assigned to Leishmania (Leishmania) subgenus (Figure 2A). The ITS1-PCR RFLP analysis, performed as described by Schönian et al. [16], enabled the identification of L. (L.) infantum species (Figure 2B). The species identification was confirmed by partial sequencing of glucose-6-phosphate isomerase gene and successive alignment against Leishmania sequences using the BLASTN algorithm, followed by construction of the phylogenetic tree (Figure 3).

Regarding parasite isolation, after 7 days of culture, the liquid phase of Evans’ modified Tobie’s medium (EMTM) presented numerous motile promastigotes, confirming the presence of viable parasites in the bioptic sample (Figure 4).

Furthermore, the isolate was genotyped by analyzing the nucleotide polymorphism 390 T > G in the malic enzyme gene by High Resolution Melt (HRM) analysis, as described previously [18]. The HRM analysis showed a 390 G genotype (Figure 5), which is not associated with the zymodemes MON-1 (the most common in the Mediterranean basin), MON-72, 201 [18].

To determine whether unresponsiveness to Glucantime^® was due to the presence of resistant parasites, the IC₅₀ of SbIII (the active trivalent antimonial form) was determined in the promastigotes isolated from the lesion. Briefly, late log/stationary phase promastigotes were resuspended in complete RPMI-PY medium [19] at a density of 2.5 × 10⁶ parasites/mL in 96-well plates (100 μL/well). The promastigotes were treated with potassium antimonyl tartrate trihydrate (SbIII) (Sigma-Aldrich) at concentrations of 1, 5, 25, 125, 625 μM for 72 h at 26 °C. The reference strain L. (L.) infantum MHOM/TN/80/IPT1 was included as control. Moreover, the promastigotes were also treated with Miltefosine (Sigma–Aldrich). Each condition was repeated sixfold. To evaluate the promastigote viability, the CellTiter 96 H Aqueous Non-Radioactive Cell Proliferation Assay (Promega) was carried out. The parasites derived from the unresponsive patient were significantly less susceptible to SbIII than the reference strain parasites (p < 0.01, unpaired t-test with Welch’s correction) (

Table 1. In vitro susceptibility tests.

Treatment	L. (L.) infantum Clinical Isolate IC50 (μM)	L. (L.) infantum MHOM/TN/80/IPT1 IC50 (μM)	Unpaired t-Test with Welch’s Correction p-Value
Potassium antimonyl tartrate trihydrate (SbIII)	27.06 ± 2.70	13.22 ± 2.11	<0.01
Miltefosine	1.31 ± 0.14	4.27 ± 0.40	<0.01

). On the contrary, promastigotes from the clinical isolate appeared more sensitive to Miltefosine compared to the reference strain.

Due to the unsatisfactory clinical response to pentavalent antimony compounds and the in vitro demonstration of low susceptibility to antimonials of the infecting parasitic strain, a new therapy with intravenous liposomal Amphotericin B was administered at the dose of 3 mg/Kg in daily doses for five days, with two further doses at an interval of two weeks. Liposomal Amphotericin B treatment was effective, with healing of the lesion after two months and no major side-effect was reported during the follow-up. The patient did not return with any relapse in the following 6 months.

3. Discussion

A high circulation of Leishmania strains causing cutaneous leishmaniasis has been recently observed in northeastern Italy [10]. In the index case, a lack of travel history, the molecular identification of L. (L.) infantum and the fact that the patient resided in the Marche region (central Italy) where L. (L.) infantum is endemic, strongly suggest an autochthonous origin of the infection.

The diagnosis of CL was initially performed by histology with the detection of leishmanial amastigotes in tissue sections. However, due to treatment unresponsiveness, a second bioptic sample was taken and histological diagnosis was repeated, followed by molecular diagnosis and parasite isolation and characterization.

Treatment failure in leishmaniasis can be caused by drug resistance of the infecting parasite, host factors such as immunity and nutritional status, individual variation in pharmacokinetics, or other drug-related responses, or whether the parasite resides in tissues accessible to drugs [7]. Antimonials are often the first choice in the treatment of CL and they can be administered intralesionally as well as systemically [20]. In Old World CL, intralesional antimonials have shown >90% cure rates but most of the data are related to L. (L.) major infections. [20]. In fact, treatment data are scarce for L. (L.) infantum CL lesions [21]. To confirm the first diagnosis and to investigate the lack of response to treatment in the index case, the parasitic strain isolated from the lesion was further characterized with molecular tools and its susceptibility to antimonials was evaluated in vitro. Pentavalent antimonials (SbV) such as Meglumine Antimoniate need to be reduced to a trivalent antimonial (SbIII) in order to be active. Since promastigotes cannot reduce SbV to SbIII, the susceptibility test was directly performed with SbIII, the active trivalent antimonial form [22].

The L. (L.) infantum clinical isolate showed significantly lower susceptibility to antimonials than the reference strain (MHOM/TN/80/IPT1), which is itself a VL strain less susceptible to antimonials with respect to CL causing species [23]. At the same time, the clinical isolate exhibited a higher sensitivity to Miltefosine when compared to the reference strain.

Treatment of CanL with Glucantime^® is a common practice in many Mediterranean countries, where repeated treatments of dogs have been shown to produce a reservoir of L. (L.) infantum parasites with a decreased sensitivity to antimonials [24,25]. Nevertheless, the clinical isolate of our index case was genotyped as a strain not related to zymodemes MON-1, 72, 201 (polymorphism 390 G in malic enzyme), which include the most common zymodeme circulating in dogs in the Mediterranean basin (MON-1). Therefore, it is unlikely that the strain has canine origin. Since the lower susceptibility to SbIII could not be explained with canine origin of the strain, further investigation on genes involved in inducing antimony-resistant parasites (e.g., AQP1, MRPA, γ-GCS, TR, TDR1) will be needed.

According to the European guidelines [5], CL lesions > 4 cm should be treated with systemic therapy. However, Since the patient presented a single lesion that was slightly above this index, local therapy with Glucantime^® was attempted to avoid the toxic effects of the systemic therapy. Nonetheless, after the intralesional therapy, according to the clinician’s judgment, a satisfactory clinical response was not achieved. Subsequent investigations revealed low susceptibility to SbIII. Therefore, the incomplete response to treatment could be due to both an insufficient therapeutic dose and the low susceptibility of the parasite.

In the Old World, Glucantime^® failure due to parasite resistance has been reported mostly in L. (L.) donovani strains circulating in the Indian subcontinent. The L. (L.) infantum CL case described here showed decreased susceptibility to Meglumine Antimoniate. Due to the scarcity of data regarding treatment of L. (L.) infantum CL lesions, it is possible that some similar cases may not have been reported. Additional studies are needed to confirm the epidemiological and clinical relevance of these findings.