Background
Leishmaniasis, a parasitic disease caused by the protozoan
Leishmania, affects millions of the world’s poorest, and is ranked among the top three most common travel acquired dermatoses [
1,
2]. Parenteral drugs available in North America include formulations of Amphotericin B (AB), pentavalent antimonials and pentamidine, while oral drugs, including miltefosine and azole antifungal compounds, are options for oral treatment of CL [
3]. Pentavalent antimonials, including sodium stibogluconate and meglumine antimoniate, are regarded as first-line treatment against New World CL [
2‐
4]. This class of drugs is highly toxic, often difficult to access, and requires enhanced clinical monitoring or hospitalization to prevent irreversible toxicities to the heart, liver, kidney, and pancreas [
2‐
4]. Clinically significant adverse events, including but not limited to, severe thrombocytopenia and pancreatitis, are common reasons for treatment interruption with antimonials. Miltefosine is a highly effective oral alternative to antimonials for the treatment of CL [
3], though its use is limited by prohibitively high cost. Amphotericin B (AB) is a polyene antifungal that targets the sterol rich membranes of
Leishmania spp. by producing ion-channel pores spanning the lipid bilayer, and increasing cell membrane permeability to small ions and solute molecules, resulting in cell death [
5,
6]. Four formulations of AB, including amphotericin B deoxycholate, liposomal amphotericin, cholesterol dispersion amphotericin, and lipid complex amphotericin are used as second-line treatment of CL, and vary in treatment efficacy [
3,
5‐
8]. Severe side effects of AB, especially renal toxicity are mostly associated with amphotericin B deoxycolate compared to other amphotericin formulations [
3,
5‐
8].
Antifungal azoles such as itraconozole, ketonazole, and fluconazole (FZ), have been evaluated in clinical trials of CL treatment, and species-specific results suggest that these comparatively well-tolerated oral regimens also demonstrate efficacy, particularly for Old World CL [
3,
4,
9‐
11]. FZ inhibits C14α-demethylase of the ergosterol biosynthetic pathway [
7]. The oral formulation, long half-life, and high skin-to-plasma concentrations make it a popular alternative in the treatment of CL [
9]. Widespread use of FZ for CL treatment is limited by a lack of large randomized controlled trials demonstrating efficacy, and the need for high-dose administration in order to achieve cure [
3,
9‐
11]. Current clinical management guidelines indicate that “no ideal or universally applicable therapy for CL has been identified”, and that selection of therapy should be individualized [
3]. Given the absence of a first-line agent and considering the many factors such as patient preference, lesion localization, cost, ease of administration, probable efficacy, likelihood of subsequent mucosal disease, age, existing co-morbidities, and drug accessibility, treatment should be individualized, and this process would be enhanced by drug susceptibility platforms to inform clinical decision-making [
3]. Geographic and anticipated species-specific response to therapy should also be considered when selecting a therapeutic agent, particularly given high rates of failure of antimonials in pockets of endemicity [
3,
12,
13]. However, few objective parasitologic data exist to guide the clinical decision-making process when selecting a therapeutic agent a priori. Estimating the likelihood of drug failure is largely informed by physician experience and clinical and epidemiological data, rather than objective parasitologic metrics, as one would achieve through standardized in-vitro drug susceptibility testing (as is done for countless other microbial infections).
At present, in-vitro systems for assessing predominantly Old World strains of
Leishmania spp
. susceptibility include: agar dilution, broth microdilution, flow cytometry, reporter gene assays, enzymatic determination, H
3-thymidine incorporation, and colorimetric assays including the use of resazurin based Alamar Blue [
14‐
18]. These techniques are primarily used in research laboratories, and have yet to be validated for routine clinical use due to their time-consuming nature, and requirement of substantial technical expertise and laboratory infrastructure. Another challenge to routine drug susceptibility testing is the biphasic life cycle of
Leishmania, where the larger, motile (and therefore more visible by light microscopy) promastigote stage inhabits the sandfly midgut, while the 2-μm amotile amastigote resides inside the mammalian host macrophages, where it evades immune detection. Intracellular amastigotes harvested from macrophages remain the gold standard for testing drug susceptibility in-vitro [
14]. However, given that log-phase promastigotes are generally more resistant to anti-
Leishmania drugs than amastigotes [
14‐
17], and detectable in a cell-free culture system incubated at room temperature, promastigotes are surrogates of an isolate’s susceptibility pattern, independent of cell-mediated parasiticidal mechanisms [
14].
The Sensititre™ YeastONE™ YO9 Susceptibility Plate (Thermo Scientific), used for routine quantitative antifungal susceptibilities (MIC) in non-fastidious yeast, such
Candida spp. and
Cryptococcus spp., contains the following antifungals: anidulafungin, amphotericin B, micafungin, caspofungin, 5-flucytosine, posaconazole, voriconazole, itraconazole and fluconazole [
19]. The alamarBlue® technology is a colorimetric growth indicator based on detectable metabolic activity, and remains constant with extended incubation times (as are required for culture of
Leishmania), and across inoculation media [
19,
20]. Thus, it could be adapted for use in
Leishmania susceptibility testing within an antifungal-based panel, such as the Sensititre™ YeastOne™ YO9 Susceptibility Plate.
Given the scarcity of well tolerated, easily accessible, and inexpensive therapies coupled with the necessity to treat active lesions of Latin American Viannia strains to potentially minimize the risk of downstream mucosal disease, as well as the propensity of strains to fail all available therapies with sufficient frequency, a user-friendly drug susceptibility testing platform with potential for clinical implementation should be developed. In order to address several aspects of this existing knowledge and care gap, we adapted the Sensititre™ YeastONE™ YO9 Susceptibility Plate to examine AB and FZ susceptibility profiles in log-phase Leishmania spp. promastigotes. We herein report the use of this commercialized antifungal drug susceptibility platform as proof-of-concept to assess drug susceptibility profiles of Leishmania strains imported to Canada and available via the American Type Culture Collection (ATCC®).
Discussion
Treatment of CL is hindered by many factors including, but not limited to: variability in clinical response to treatment with partial correlation to infecting species and region of acquisition; toxicity, expense, and inaccessibility of therapeutics with little pharmacologic innovation over decades; the absence of large-scale therapeutic clinical trials; and the lack of objective laboratory criteria by which to inform likelihood of clinical response and decision-making at the bedside. Clinicians treating patients with CL are provided with little objective parasitologic data to compel selection of one drug over another, and must present patients with clinical guidelines that incorporate a number of contingencies into the decision-making process. Azole antifungals are easily accessible, inexpensive, well tolerated, and supported by several reported trials of efficacy [
3,
9], but clinical response to this class of medication can be highly variable compared to other systemic options such as amphotericin and miltefosine, which are more toxic. Development and validation of an objective drug susceptibility system to approximate probable clinical response to therapy should be encouraged, and we have herein demonstrated, as proof-of-concept, that the Sensititre™ YeastOne™ YO9 system is potentially adaptable for routine clinical laboratory testing of AB susceptibility in clinical strains of
Leishmania. Adaptation of an existing commercialized system guarantees a standard of quality assurance associated with GCP/GLP manufacturing processes, while reducing errors of reproducibility and accuracy compared to systems developed on a smaller, ad hoc, non-commercial scale, as in many research laboratories [
14‐
19]. As per TREK Diagnostic Systems, the Sensititre™ YeastOne™ YO9 system provides results within 96 h from inoculation to final reading of MICs [
19]. The clinical utility of this plate is highlighted by its cost-effectiveness, time efficiency, and low burden of technical expertise required. The cost per plate including 9 dehydrated drugs with varying concentrations and media is $30 USD with a maximum of 2 h of technical support to set-up and read the plate. In comparison, other in-vitro platforms require individual drug procurement, which translates to well over $300 USD excluding reagents [
14‐
19]. Moreover, such ad hoc investigational systems lack a standard of quality assurance inherent to commercialization and licensure, and are subject to cross contamination [
14‐
19]. Additionally, time for technical support including experimental set-up, monitoring and reading of plates exceeds 2 h, and requires additional training for outcomes measured by flow cytometry, fluorescence-activated cell sorting (FACS), electron microscopy, zone of inhibition (ZI) analysis, and motile cell counts for disk diffusion and broth dilution methods, respectively [
14‐
18]. Lastly, final MIC readings in such ad hoc systems often exceed 96 h, and, in some cases, require up to 20 days [
14‐
18]. Overall, the Sensititre™ YeastOne™ YO9 system provides a more efficient and objective measure of analysis, which can be compared between laboratories. Other advantages include the less labor intensive technologies such as the plate-impregnated alamarBlue® technology, which eliminates the need for microscopy.
Clinical case reports and studies have demonstrated the efficacy of high-dose FZ in the treatment of CL in both New and Old World strains of
Leishmania [
3,
9‐
11]. A study conducted in Saudi Arabia demonstrated a 79% cure rate of CL due to
L. major at 12-weeks following initiation of 6-weeks of 200-mg daily FZ [
10]. In another trial, treatment of localized CL due to
L. major with FZ at 400 mg/day for 42 days led to an 81% cure rate at 6-weeks [
11]. In a study of CL due to
L. V. braziliensis, clinical cure was observed more rapidly and to a greater extent (mean duration of treatment 4-weeks; 100% cure) when FZ was prescribed at 8-mg/kg per day compared to a lower dose 5-mg/kg/day regimen (mean duration of treatment 7.5-weeks; 75% cure) [
9]. Recent data surrounding the treatment of
L. V. braziliensis and
L. V. guyanensis from the Brazilian Amazon demonstrate sub-optimal cure rates with FZ 6.5–8 mg/kg/day for 28 days and 450 mg/day for 30 days, respectively [
24‐
26], thus reinforcing the need for a standardized objective marker of expected clinical response, one component of which could be a drug susceptibility testing system that would be functional across species and geographic origins. The isolates tested in our study exhibited FZ MICs ≥256 μg/mL in every case, corroborating the observed clinical requirement of high concentrations of FZ for leishmanicidal effect. Further testing of clinical and ATCC® strains of
Leishmania against higher concentrations of FZ will be important to determine the adaptability of an in vitro system for FZ susceptibility testing.
Liposomal amphotericin B is a less toxic alternative to antimonial treatment of CL and mucosal leishmaniasis (ML), however, further studies on the optimal dosage are required given species-specific cure rates and variation in reports of total treatment dosages [
26‐
28]. Currently, treatment of CL or ML caused by
L. V. brazilienisis with AB requires a minimum 3 mg/kg dosage over many days [
26,
29,
30]. MIC values obtained in this study support average parasiticidal concentrations of 0.5 μg/mL for AB, which equates to a total dose below the recommended dosing schedule, suggesting that a lower, less toxic dose of AB might be effective clinically. Further testing to determine serum blood concentrations to correlate in-vitro drug susceptibility to clinical dosing of AB in cases of CL is necessary and warranted [
26,
30].
AB has been proven effective in low dosages against the promastigote form of the parasite in visceralizing species, such as
L. donovani [
26,
29‐
31]
, and this clinical phenomenon is reflected in our data as well, given that a greater proportion of non-visceralizing strains demonstrated growth at higher concentrations of AB compared to visceralizing strains. Although we did not observe any differences in actual MIC values to AB, the proportionate growth of visceralizing compared to non-visceralizing isolates highlights a potential trend that is supported clinically, and will be further validated using amastigotes in the next phase of this work.
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