https://orcid.org/0000-0001-6325-4508
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Abstract
Miltefosine, the only oral antileishmanial drug with regulatory approval, has expanded treatment options in several endemic settings but shows variable efficacy across Leishmania species, clinical forms, and host immune contexts. In Southeast Asia, where Leishmania (Mundinia) martiniquensis and L. (M.) orientalis are increasingly reported, amphotericin B formulations remain the main treatment despite toxicity, relapse, and implementation constraints. This review evaluates miltefosine’s clinical relevance, mechanisms of action, and resistance pathways, with emphasis on Thailand and neighboring Southeast Asian settings. A Thai compassionate-use case using miltefosine with liposomal amphotericin B for refractory L. martiniquensis infection achieved repeated clinical improvement and culture negativity after combination induction, although monotherapy was insufficient to maintain remission in advanced immunosuppression. Evidence from other endemic regions indicates that poor adherence, unregulated access, prolonged subtherapeutic exposure, and inadequate monitoring can reduce treatment durability and favor reduced susceptibility. Mechanistic studies in non-Mundinia species identify transport disruption, lipid and sterol remodeling, mitochondrial stress adaptation, redox buffering, and host-parasite effects as resistance-relevant axes, while regional data raise concern for amphotericin B-associated reduced miltefosine susceptibility in L. martiniquensis. Wider implementation should therefore be linked to species-resolved diagnostics, baseline susceptibility testing, longitudinal phenotype-genotype surveillance, drug stewardship, and One Health monitoring. Miltefosine should be considered a rational but carefully monitored therapeutic addition for emerging Southeast Asian leishmaniasis.
Author summary
Leishmaniasis is a neglected infectious disease affecting vulnerable populations worldwide. In Southeast Asia, autochthonous infections caused by Leishmania (Mundinia) martiniquensis and L. (M.) orientalis are increasingly reported, while available treatments remain toxic, costly, or difficult to administer. Miltefosine, the only oral drug approved for leishmaniasis, has been widely used in other endemic regions but remains underused in Southeast Asia. This review examines its mechanisms of action, resistance pathways, and potential role in Thailand. We discuss a compassionate-use case in which miltefosine combined with liposomal amphotericin B achieved repeated clinical improvement and parasite culture negativity in an immunosuppressed patient with limited treatment options, although miltefosine alone did not sustain remission. Incomplete or poorly monitored miltefosine use can reduce treatment durability and favor reduced susceptibility. Strategic implementation, including improved parasite identification, baseline susceptibility testing, genomic surveillance, and One Health vector and reservoir monitoring, could expand treatment options while protecting long-term efficacy in Southeast Asia.
Citation: Lerona PGE, Jitmuang A, Sarasombath PT, Chayakulkeeree M, Kumlert R, Siriyasatien P, et al. (2026) Miltefosine and emerging Leishmania (Mundinia) in Southeast Asia: Molecular insights, therapeutic challenges, and future strategic implementation. PLoS Negl Trop Dis 20(7): e0014555. https://doi.org/10.1371/journal.pntd.0014555
Editor: Sarman Singh, Advanced Centre for Chronic and Rare Diseases, INDIA
Published: July 31, 2026
Copyright: © 2026 Lerona et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Thailand Science Research and Innovation Fund and Chulalongkorn University (HEA_FF_69_197_3000_026 to KP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Leishmaniasis encompasses a spectrum of diseases caused by protozoan parasites of the genus Leishmania, transmitted primarily through the bite of infected phlebotomine sand flies [1]. Clinical manifestations range from self-limiting cutaneous ulcers to potentially fatal visceral disease involving the liver, spleen, and bone marrow [2]. Globally, over one billion people are estimated to be at risk, with 600,000–1 million new cases of cutaneous leishmaniasis (CL) and 50,000–90,000 new cases of visceral leishmaniasis (VL) reported annually [3]. CL is highly prevalent in the Americas, the Mediterranean basin, the Middle East, and Central Asia, whereas VL occurs predominantly in Brazil, East Africa, and the Indian subcontinent [4].
The geographic distribution and prevalence of Leishmania parasites are driven by interacting ecological, environmental, and socioeconomic factors [5,6]. For example, L. infantum is endemic in the Mediterranean basin and Latin America, where domestic dogs serve as major reservoir hosts and adapted sand fly vectors sustain transmission [6]. Socioeconomic conditions remain fundamental determinants of disease burden: poverty, malnutrition, inadequate housing, and limited access to healthcare increase vulnerability to infection, worsen clinical outcomes, and compound the stigma and economic exclusion associated with the disease [7]. Immunocompromised populations, particularly people living with HIV (PLWH), are at increased risk of disseminated or diffuse disease, chronic relapse, and reduced treatment efficacy [8]. Poverty-associated barriers to diagnosis, regulated drug access, and treatment completion can create conditions that favor antimicrobial resistance, although this evidence is broader than leishmaniasis alone. In leishmaniasis, irregular treatment, incomplete courses, and unsupervised access contributed to antimonial resistance in the Indian subcontinent, while analogous concerns have been raised for miltefosine because of its high cost, long elimination half-life, and 28-day regimen [9,10]. The documented distribution of a poor-quality generic miltefosine product without detectable active ingredient in Bangladesh further emphasizes the importance of drug-quality assurance in VL elimination programmes [11].
Miltefosine (hexadecylphosphocholine), originally developed as an anticancer agent [12], was repurposed as the first and only oral antileishmanial drug following successful clinical trials against L. donovani in India [13]. It received regulatory approval in India in 2002, with subsequent approvals in Germany (2004), Colombia (2005), and the United States (2014), and was added to the WHO Model List of Essential Medicines in 2011 [14,15]. Its oral bioavailability and suitability for outpatient administration offer substantial logistical advantages over parenteral regimens in settings with limited inpatient capacity [15]. Miltefosine is used in selected regional treatment regimens for VL and post-kala-azar dermal leishmaniasis (PKDL), and the WHO specifically recommends its use in combination with liposomal amphotericin B for HIV co-infected VL patients in East Africa and South-East Asia. Clinical efficacy varies across Leishmania species, geographic regions, and host immune status, with declining effectiveness, relapse, and gastrointestinal toxicity documented under routine programmatic conditions for L. donovani in the Indian subcontinent, although comparable clinical decline has not been directly demonstrated in Mundinia species [16–18]. Restricted global distribution, limited production capacity, teratogenic risk, and the emergence of resistance in endemic areas further constrain its utility, although clinical miltefosine resistance has not yet been directly demonstrated in Mundinia species [19,20].
In Southeast Asia, leishmaniasis caused by species within the subgenus Mundinia presents a distinct epidemiological and therapeutic landscape. Autochthonous infections caused by L. martiniquensis and L. orientalis have been increasingly reported in Thailand over the past two decades, with clinical presentations spanning VL, CL, diffuse CL, mucocutaneous leishmaniasis, and asymptomatic infections [21–24]. These Mundinia species diverge from the L. donovani complex in phylogeny, genome structure, and probable vectors [25]. Molecular surveys and experimental infections have implicated Culicoides biting midges (Diptera: Ceratopogonidae) as plausible vectors, distinguishing Mundinia transmission from classical sand fly-centered models [26–30]. A disproportionate burden falls on PLWH, who account for approximately half of reported Thai VL cases and experience high rates of relapse, disseminated disease, atypical manifestations, and treatment failure despite amphotericin B-based regimens [23,24,31–34]. Miltefosine is not currently included in Thailand’s National List of Essential Medicines [32,35,36], and circulating Leishmania populations in the region remain largely naïve to alkylphospholipid exposure [36,37]. The convergence of atypical vector biology, a vulnerable patient population, limited treatment options, and a miltefosine-naïve parasite population defines a therapeutic setting that differs fundamentally from the Indian subcontinent and Latin American contexts in which miltefosine was developed and deployed.
This review evaluates miltefosine as a rational addition to the limited therapeutic options for Leishmania (Mundinia) infections in Southeast Asia by integrating molecular, clinical, and ecological evidence. Mechanistic data derived primarily from L. donovani, L. infantum, L. major, and other well-studied species are assessed for their therapeutic and resistance implications, with explicit acknowledgment that direct validation in Mundinia species remains largely absent. This review includes a detailed clinical account of the first successful compassionate use of miltefosine in Thailand, administered with liposomal amphotericin B for refractory L. martiniquensis infection, based on institutional case records and clinical data provided by co-authors involved in the patient’s management. Clinical images from this case were previously published by Jitrukthai and Charatcharoenwitthaya [38]. This review further proposes a framework for evidence-based introduction of miltefosine in Southeast Asia, integrating species-resolved diagnostics, genomic surveillance, and One Health stewardship to preserve therapeutic efficacy while addressing the distinct epidemiological characteristics of this emerging disease setting.
2. Methods
2.1 Ethics statement
This review article synthesizes publicly available literature. The compassionate-use case discussed in this review was conducted with written informed consent obtained from the patient for the use and publication of clinical and medical information, which was approved by the Faculty of Medicine Siriraj Hospital, Mahidol University. The broader research initiative related to this work was conducted under ethical approval from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok (IRB No. 0286/67, COA Nos. 0757/2024, 0700/2025). No additional ethical approval was required.
2.2 Literature search and evidence synthesis
This narrative review was prepared through a targeted literature search of PubMed, Web of Science, Scopus, Google Scholar, and Crossref for articles relevant to miltefosine use, mechanisms of action, resistance, diagnostics, and implementation in leishmaniasis, with emphasis on Southeast Asia and Leishmania (Mundinia) species. Searches were conducted for articles published up to April 2026 using combinations of the terms “miltefosine,” “leishmaniasis,” “Leishmania,” “Mundinia,” “Leishmania martiniquensis,” “Leishmania orientalis,” “Leishmania siamensis,” “Thailand,” “Southeast Asia,” “drug resistance,” “miltefosine resistance,” “amphotericin B resistance,” “cross-resistance,” “miltefosine transporter,” “Ros3,” “ABC transporter,” “lipid metabolism,” “sterol biosynthesis,” “mitochondria,” “calcium homeostasis,” “redox stress,” “apoptosis-like cell death,” “immunomodulation,” “diagnostics,” “genomic surveillance,” “parasitomics,” and “One Health.” Additional sources were identified from reference lists of selected articles and WHO guidance documents.
Articles were prioritized when they addressed one or more of the following: clinical use of miltefosine in visceral, cutaneous, mucocutaneous, disseminated, or HIV-associated leishmaniasis; experimental or clinical evidence for miltefosine susceptibility or resistance; mechanisms of drug uptake, efflux, lipid remodeling, mitochondrial toxicity, redox buffering, host immunomodulation, or parasite survival adaptation; autochthonous leishmaniasis in Thailand or Southeast Asia; diagnostic or surveillance approaches relevant to Mundinia species; and implementation or stewardship considerations. Experimental evidence was appraised according to evidence type, distinguishing functional validation studies, laboratory-selected resistant lines, clinical isolate observations, omics associations, pharmacological associations, and hypotheses. Mechanistic findings derived from non-Mundinia species were interpreted as extrapolative unless direct evidence was available for L. martiniquensis, L. orientalis, or other Mundinia species.
3. Leishmania (Mundinia) and the emerging leishmaniasis landscape in Southeast Asia
3.1 Taxonomy and phylogenetic position of Mundinia
The genus Leishmania has undergone several taxonomic revisions as molecular approaches have refined our understanding of relationships previously inferred from morphology, clinical presentation, and geographic distribution [39]. Multilocus enzyme electrophoresis, ribosomal DNA sequencing, and whole-genome analyses have supported the current classification of the genus into four subgenera: Leishmania, Viannia, Sauroleishmania, and Mundinia (formerly known as the “Leishmania enriettii complex”) [25,39]. Comparative genomics and phylogenomic analyses generally place Mundinia as the earliest-diverging lineage within the genus Leishmania. Members of this subgenus possess relatively smaller genomes than many species belonging to the subgenera Leishmania and Viannia and may exhibit alternative vector associations, including suspected transmission by non-phlebotomine insects in some species [25]. Genomic reduction in Mundinia has been associated with gene losses exceeding gains and gene family contractions exceeding expansions at the Mundinia node. Reported changes include losses affecting parasite surface architecture, particularly β-amastins and lipophosphoglycan-modifying enzymes, as well as contractions in oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins [25]. Butenko and colleagues proposed that these genomic features may reflect adaptation to alternative hosts or vectors, resulting in altered host-parasite interactions and reduced reliance on certain previously utilized proteins [25].
Within the subgenus Mundinia, L. martiniquensis and L. orientalis constitute the primary human pathogens in Southeast Asia [31,40]. The first autochthonous case of VL in Thailand was reported in 1996 [41]. Several subsequent autochthonous cases were reported under the invalid designation L. siamensis, a nomen nudum, before this taxonomic confusion was resolved by Leelayoova and colleagues [42], who reclassified these cases into two distinct lineages: the “PG” lineage and the “TR” lineage. The “PG” lineage was later confirmed to be identical to L. martiniquensis, originally described in 1995 from cutaneous cases in Martinique [43,44], whereas the clinical isolate corresponding to the “TR” lineage was formally described as L. orientalis in 2018 [40]. The identification of these species in both the Caribbean and Southeast Asia has expanded the known geographic and evolutionary range of Leishmania (Mundinia) parasites [40,44]. Although VL caused by L. martiniquensis may resemble VL caused by the L. donovani complex, Mundinia species differ in phylogenetic position, genome architecture, and probable vector associations [25], indicating that therapeutic evidence derived from classical Leishmania complexes should be extrapolated cautiously when evaluating treatment options for Southeast Asian Mundinia infections [31].
3.2 Epidemiology in Thailand and the region
3.2.1 Clinical spectrum: VL, LCL, DCL, MCL, and HIV co-infection.
Leishmaniasis in Thailand has progressed from sporadic autochthonous reports to an emerging clinical and public health concern. Documented manifestations include visceral leishmaniasis (VL), localized cutaneous leishmaniasis (LCL), diffuse CL (DCL), mucocutaneous leishmaniasis (MCL), and asymptomatic infection [22,24]. Cases have been reported across northern provinces, including Chiang Rai and Lamphun; southern provinces, including Songkhla, Phang Nga, Trang, and Satun; and central provinces, including Bangkok, Lopburi, and Kanchanaburi [23,24,31,45–48]. Autochthonous leishmaniasis currently attributed to L. martiniquensis has been documented in Thailand and Myanmar, with Thai cases concentrated mainly in the southern region and Myanmar cases reported in Yangon, encompassing CL, VL, and asymptomatic presentations [31,49,50]. L. martiniquensis is the most common species, though L. orientalis, L. infantum, L. lainsoni, L. major, and L. donovani have also been sporadically detected [23,40,49]. Surveillance remains inconsistent as over 40 autochthonous cases have been identified in Thailand, but only 32 cases (26 VL, 6 CL) are listed in WHO databases [51], indicating probable underreporting and limited case detection [49]. In addition, a cross-sectional study among blood donors in Trang Province detected asymptomatic Leishmania infection in 19.0% of participants using direct agglutination testing (DAT) and/or nested PCR (nPCR), with L. martiniquensis predominating among nPCR-positive cases, suggesting that clinically recognized cases may underestimate local transmission in endemic areas [22].
Co-infection with HIV is a defining epidemiological feature of leishmaniasis in Thailand, where approximately half of all reported cases occur in PLWH [23,31]. In this population, Mundinia infection is associated with relapse, mortality, and concurrent VL and DCL manifestations that may be confused with systemic fungal infections [8,24,47,52]. L. martiniquensis can cause VL in immunocompetent hosts, but profound immunosuppression is associated with more severe disease, including VL with concomitant DCL [23,31,46]. MCL caused by L. martiniquensis has also been reported in a patient with HIV, demonstrating the parasite’s capacity for mucosal dissemination in the context of immunodeficiency [24,47]. This extensive clinical variability, combined with limited clinical awareness, directly contributes to underdiagnosis and misclassification in the region [24,47,53].
Clinical outcome in leishmaniasis is determined not only by systemic immunosuppression but also by the quality and localization of the host immune response [4,54]. Hyperergic inflammatory responses can drive mucosal or mucocutaneous tissue injury despite relatively low parasite burdens, reflecting pathology in which host-mediated inflammation contributes substantially to tissue damage [4,54,55]. Conversely, anergic or parasite-specific unresponsive states can permit diffuse cutaneous involvement, dissemination, and high parasite burdens with limited local inflammation; this pattern is well recognized in advanced HIV infection but may also occur without generalized immunodeficiency [54,56,57]. These immunopathological poles create distinct therapeutic problems: hyperergic disease requires clinical interpretation of tissue damage beyond parasite burden alone [54], whereas anergic disseminated disease increases the risk of poor drug response, persistent infection, and relapse [46,56,57]. For Southeast Asian Mundinia infections, where VL, DCL, and MCL have all been reported, treatment outcomes should therefore be interpreted in the context of both parasite species and host immune phenotype [24,31,46].
3.2.2 Transmission ecology: vectors, reservoirs, and One Health context.
The eco-epidemiology of leishmaniasis in Thailand remains poorly understood, primarily due to limited entomological surveillance that has impeded definitive vector incrimination and full characterization of transmission cycles [31]. Molecular surveys using ribosomal markers (ITS1, SSU rRNA, 3’UTR-HSP70-I) and, more recently, nanopore metabarcoding have detected L. martiniquensis and L. orientalis DNA in multiple Culicoides species, most frequently C. guttifer, C. peregrinus, and C. mahasarakhamense [26,28–30,33,58,59]. Parallel surveillance has detected parasite DNA in several phlebotomine sand flies, including Sergentomyia and Phlebotomus species [60–63]. Across available Thai entomological surveys, Leishmania DNA has been detected more consistently and at higher prevalences in Culicoides biting midges than in phlebotomine sand flies; however, this pattern may reflect differences in sampling intensity, trap placement, and microclimate ecology rather than definitive vector status [30,62]. Furthermore, detection rates are highly heterogeneous, with extensive surveys in known endemic provinces yielding entirely negative results, demonstrating the focal and patchy nature of transmission [64,65].
Whether these molecular detections represent true vector competence remains unresolved. C. peregrinus has been found naturally infected with promastigote-like flagellates in the foregut of wild-caught specimens, but infective metacyclic forms have not been demonstrated [58]. Recent microscopic dissections of field-caught midges similarly identified Leishmania DNA without viable promastigotes, while live Trypanosoma bennetti-related trypomastigotes were observed in the same specimens [30]. Laboratory infections of colonized C. sonorensis have demonstrated complete Mundinia development, metacyclogenesis, and transmission to mammals, whereas non-Mundinia species fail to establish mature infections in this midge [27,66]. These findings establish biological plausibility for a midge-mediated transmission cycle but have not yet met Killick-Kendrick’s vector incrimination criteria [67], which require confirmation of infective metacyclic stages in wild vectors and evidence of natural transmission [30].
Putative reservoir hosts further complicate transmission dynamics, with antileishmanial antibodies detected in buffaloes, cattle, cats, and dogs, and L. martiniquensis DNA recovered from black rats (Rattus rattus) [34,53,68]. These findings indicate exposure of domestic and synanthropic animals to Mundinia parasites and identify possible vertebrate hosts that may contribute to local parasite circulation, although reservoir competence remains unproven [28,31]. Reports of L. martiniquensis infection in cattle and equines outside Asia indicate a broad mammalian host range, but the relevance of these hosts to transmission in Southeast Asia remains unresolved [69–72]. This pattern is consistent with the broader ecology of zoonotic leishmaniasis, in which animal reservoirs can sustain parasite populations and enable human infection when competent vectors are present [6]. In Thailand, peridomestic agricultural activities, animal enclosures, proximity to termite mounds, and architectural factors such as soil flooring may intensify human-animal-vector contact, creating ecological conditions that favor localized endemicity [22,34].
3.3 Current therapeutic limitations
Amphotericin B and azole antifungals remain the primary treatments for leishmaniasis in Thailand; however, disease recurrence following treatment has been documented in multiple patient case reports [24,32,46]. Comparable outcomes have been reported globally, where liposomal amphotericin B monotherapy often fails to achieve a durable cure for VL in immunocompromised cohorts [73,74]. Consequently, current WHO guidelines conditionally recommend combination regimens (S1 Table), particularly liposomal amphotericin B combined with miltefosine, to enhance therapeutic response and reduce relapse rates in HIV-coinfected VL patients in the WHO South-East Asia Region [74]. In Thailand, miltefosine is currently unavailable and is not included in the National List of Essential Medicines, a status that limits routine public sector procurement [32,35,46]. Additional barriers to access include reliance on a single global supplier and historical minimum order batch requirements, while an uncertain market size may further reduce incentives for registration and inclusion in national formularies [19]. The convergence of limited therapeutic options, relapses in immunocompromised patients, incomplete eco-epidemiological characterization, and the absence of miltefosine from national guidelines defines a therapeutic gap that motivates the evaluation of miltefosine as an additional treatment option for Southeast Asia.
4. Miltefosine: Clinical evidence and regional therapeutic relevance
4.1 Clinical performance across species and regions
Miltefosine’s clinical efficacy varies substantially across Leishmania species, geographic regions, clinical forms, and treatment regimens (Table 1). Cure rates approach 90%–95% for VL caused by L. donovani in India under controlled conditions [13,75], but decline to 73.3% in Nepal [17] and 59.5% for L. infantum VL in Brazil [76]. In CL, outcomes are more variable and less directly comparable across studies because clinical forms, follow-up endpoints, and denominators differ. Cure rates range from 81.6% for L. (Viannia) panamensis in Colombia to 31.3% for L. (V.) braziliensis in Guatemala [77], with intermediate efficacy for L. tropica in Pakistan and Iran, at 52.6% and 63.0%, respectively [78,79], and 45.7% for L. aethiopica in Ethiopia [80]. DCL caused by L. mexicana and L. amazonensis responds poorly to miltefosine monotherapy, with cure rates of 0% and 7.7%, respectively [57].
In HIV-coinfected VL patients, miltefosine offers a more favorable safety profile than antimonials [81], but therapeutic responses remain heterogeneous, with reduced efficacy reported in Ethiopia [81] and more favorable outcomes in other endemic areas [82]. Combination therapy with liposomal amphotericin B substantially improves outcomes: a recent Indian trial reported cure rates rising from 85.3% with miltefosine monotherapy to 96% with combination treatment in HIV-VL patients [73], and similar improvements (80.8%–95.5%) have been observed in non-HIV-VL cohorts [83]. These findings establish combination therapy as the preferred regimen for immunocompromised VL patients in endemic regions where miltefosine is available.
The species- and form-specific heterogeneity summarized in Table 1 provides a cautionary comparator rather than direct evidence for L. (Mundinia) treatment in Southeast Asia. No clinical trial data on miltefosine efficacy against L. martiniquensis or L. orientalis are currently available, and outcomes from non-Mundinia species should be interpreted as indirect evidence given the phylogenetic, genomic, and phenotypic divergence of this subgenus [25]. The single documented Thai compassionate-use case, presented in Section 4.2, currently constitutes the only regional clinical evidence of miltefosine activity against a Mundinia infection.
4.2 First use of miltefosine for L. martiniquensis in Thailand: A compassionate-use case
This review includes the first detailed clinical account from Thailand of relapsing diffuse CL and VL caused by L. martiniquensis successfully managed using repeated induction courses of combined liposomal amphotericin B and miltefosine. Clinical images from this case were previously published by Jitrukthai and Charatcharoenwitthaya [38], whereas the treatment timeline, parasitological follow-up, and therapeutic interpretation presented here are based on institutional case records and clinical data provided by co-authors involved in the patient’s management.
A 54-year-old patient with HIV/AIDS from Southern Thailand presented with relapsing VL and diffuse CL. The isolate was designated MHOM/TH/2023/CULE10. Despite good adherence to antiretroviral therapy and virological suppression, he experienced multiple relapses following treatment with amphotericin B deoxycholate (1 mg/kg/day for one month, followed by monthly prophylaxis at 50 mg) and subsequently liposomal amphotericin B (5 mg/kg every other day, cumulative dose 30 mg/kg). These relapses manifested as diffuse cutaneous nodules, hepatosplenomegaly, ascites, and pancytopenia, indicating disease recurrence despite prior amphotericin induction and prophylaxis, with further treatment constrained by nephrotoxicity.
In accordance with World Health Organization recommendations and through coordination with the Thai Ministry of Public Health, oral miltefosine (100 mg/day) was provided via compassionate access and administered in combination with liposomal amphotericin B. Each induction with combined therapy resulted in rapid clinical improvement and culture negativity within two weeks, although Leishmania DNA remained detectable by qPCR during subsequent relapse episodes. Maintenance therapy with oral miltefosine alone did not sustain remission, with relapse occurring within one month in the context of advanced immunosuppression and limited treatment tolerance, indicating that sustained disease control required continued combination therapy or secondary prophylaxis.
Repeated cycles of combined liposomal amphotericin B and miltefosine achieved remission that was sustained for six months under monthly amphotericin prophylaxis. This case provides clinical evidence that combined miltefosine and liposomal amphotericin B can contribute to sustained remission of refractory L. martiniquensis infection in an HIV co-infected patient after amphotericin B monotherapy had failed, although miltefosine monotherapy alone was insufficient to maintain remission. The findings support combination-based induction, molecular confirmation of relapse, and supervised secondary prophylaxis as the operating framework for miltefosine use in Mundinia infections in Southeast Asia.
4.3 Therapeutic implications of miltefosine for Southeast Asian Mundinia leishmaniasis
The clinical evidence summarized above positions miltefosine as a plausible but still insufficiently validated therapeutic addition for Southeast Asian Mundinia leishmaniasis. Its oral administration, established use in other endemic settings, and WHO-recommended role in combination regimens for VL-HIV co-infection in East Africa and the WHO South-East Asia Region [74] provide a rationale for consideration in Thailand, where amphotericin B-based therapy remains constrained by toxicity, relapse, and access limitations [24,32,46,47]. However, the absence of clinical trial data for L. martiniquensis and L. orientalis means that efficacy estimates from L. donovani, L. infantum, Viannia, and other non-Mundinia species should be treated as indirect comparators rather than predictive evidence.
The Thai compassionate-use case supports the clinical plausibility of combined miltefosine and liposomal amphotericin B for refractory L. martiniquensis infection, particularly in the setting of HIV-associated relapse, but it does not establish generalizable efficacy or define optimal dosing, duration, or secondary prophylaxis. The therapeutic implication is therefore cautious and operational: miltefosine may expand treatment options in carefully selected cases, especially as part of supervised combination therapy, but its introduction should be linked to species-level diagnosis, relapse monitoring, and resistance preparedness. This requirement leads directly to the need to interpret miltefosine’s mechanisms of action and resistance as connected determinants of therapeutic durability.
5. Miltefosine’s mechanisms of action and resistance
5.1 Clinical emergence of reduced miltefosine susceptibility
Miltefosine efficacy declined after sustained programmatic deployment in the Indian subcontinent, where high initial cure rates against visceral leishmaniasis caused by L. donovani were followed by increasing relapse and treatment failure [17,18]. In India, long-term use was associated with a gradual increase in treatment failure compared with earlier trial outcomes, yielding a 90.3% cure rate even under strictly supervised, directly observed therapy in a clinical research setting [18]. In Nepal, relapse approached 20% within 12 months despite completion of therapy (Table 1) [17]. Relapse-associated L. donovani isolates from this period generally showed modest in vitro susceptibility shifts, often near twofold rather than uniform high-level resistance, indicating that clinical failure cannot be equated automatically with classical resistance phenotypes [85]. This distinction is central for Southeast Asia; while miltefosine remains a rational therapeutic addition for the region, its introduction must be accompanied by comprehensive clinical and parasitological surveillance. As established in other endemic zones, monitoring systems are required to definitively separate clinical relapse, intrinsic drug tolerance, pharmacokinetic failure, host immunological failure, and stable parasite resistance [86].
A terminal elimination half-life of 150–200 hours generates prolonged subtherapeutic exposure when treatment courses are incomplete, producing selection pressure favorable to the emergence of reduced susceptibility [87,88]. Programmatic risk factors compounding this pharmacokinetic vulnerability include high treatment cost, gastrointestinal toxicity, teratogenicity-related contraceptive requirements, unregulated retail availability in some endemic settings, and incomplete adherence to the 28-day oral regimen [9,11,18,19]. These factors are not parasite-intrinsic, but they shape the conditions under which parasite-intrinsic adaptations are selected [19].
Baseline miltefosine susceptibility of L. martiniquensis and L. orientalis clinical isolates from Southeast Asia has not been comprehensively characterized, and species-resolved susceptibility surveys remain to be performed [36,37,89]. Available experimental data suggest variable baseline susceptibility [36,89], and significantly reduced miltefosine sensitivity has been observed in specific amphotericin B-resistant L. martiniquensis lines [37], raising the possibility that resistance trajectories in Southeast Asia may follow molecular paths distinct from those documented in L. donovani and L. infantum [90]. The Indian subcontinent experience identifies the pharmacokinetic, programmatic, and parasite-intrinsic pressures that any new regional deployment must anticipate [19,88], but it does not predict which mechanisms will dominate in Mundinia infections in immunocompromised Southeast Asian patients. Wider miltefosine use in Southeast Asia should therefore be accompanied by species-level diagnosis, baseline susceptibility testing, treatment-response monitoring, and resistance-aware stewardship.
5.2 Integrated mechanisms of action and resistance
Miltefosine action and reduced susceptibility converge on the same biological systems: drug transport, membrane lipid organization, mitochondrial stress, redox buffering, cell death execution, and host-parasite interaction. Evidence across these axes comes from functional perturbation studies, laboratory-selected resistant lines, clinical isolates, and omics associations, which differ in causal strength. Table 2 summarizes these mechanisms according to molecular basis, supporting evidence type, species context, and evidentiary strength. Because most mechanistic data derive from non-Mundinia species, extrapolation to L. martiniquensis and L. orientalis requires explicit species-level caution.
5.2.1 Translocation dynamics: active uptake and efflux.
In L. donovani and L. infantum, plasma membrane inward translocation via the miltefosine transporter (MT) complex constitutes the primary determinant of intracellular drug accumulation and the most frequently disrupted target in highly resistant parasites [10,130]. MT, a P4-ATPase [95], functions cooperatively with its accessory β-subunit Ros3 [130] to mediate the ATP-dependent internalization of specific phospholipids and alkylphosphocholines (Fig 1A). In L. donovani, fluorescent alkylphosphocholine analogues accumulate to intracellular concentrations approximately 100-fold higher than the extracellular environment, consistent with active inward translocation rather than passive partitioning [131]. Functional complementation studies establish MT/Ros3 as the principal route of drug entry, since episomal restoration of wild-type MT or Ros3 re-establishes susceptibility in transporter-deficient L. infantum parasites [94,132]. Furthermore, metabolomic profiling of resistant L. infantum shows absent intracellular drug accumulation and the absence of the metabolic collapse otherwise observed in susceptible parasites, consistent with impaired uptake as the proximal resistance event [133].
(A) Miltefosine accumulation depends on active inward translocation across the plasma membrane through the miltefosine transporter (MT), a P4-ATPase that functions with the Ros3 β-subunit. ABC transporters can reduce intracellular drug retention by promoting outward transport or efflux, although their contribution varies by species, strain, and resistant background. (B) After membrane association, miltefosine partitions into sterol-rich microdomains, where physicochemical intercalation, kinetic retention, and altered sterol packing can perturb membrane organization [144,145]. These membrane effects are associated with disrupted lipid packing, altered phospholipid asymmetry, and downstream lipid remodeling, including reduced phosphatidylcholine (PC) and increased phosphatidylethanolamine (PE). The figure presents a generalized model synthesized from studies in non-Mundinia species and model membrane systems; direct validation of these processes in L. martiniquensis and L. orientalis remains limited. Created in BioRender. Lerona, P.G. (2026) https://BioRender.com/u6l8rje.
Loss-of-function changes in MT or Ros3 reduce miltefosine accumulation and constitute the best-validated resistance mechanism described to date [10,94,134]. Laboratory-selected resistant lines frequently acquire genetic lesions that disrupt this transport axis, employing inactivating missense point mutations [10,104,135] as well as nonsense mutations [10,135]. Whole-genome sequencing of laboratory-selected L. major miltefosine-resistant populations identifies multiple independent point mutations and small deletions in MT, demonstrating polyclonal mutational heterogeneity wherein individual clones within a single selection differ in both genotype and susceptibility phenotype [136]. Functionally validated alterations include a T420N substitution within the ATPase phosphorylation motif in in vitro-selected L. donovani [95,104], CRISPR-Cas9 disruption producing large structural deletions [137], an L832F substitution in a clinical L. infantum isolate [92], and frameshift mutations affecting LiMT and LiROS3 confirmed as causal by complementation [94,132]. Intrinsic tolerance in L. braziliensis arises from limited LbRos3 dosage that restricts plasma membrane localization of LbMT [100]. Clinical isolate observations qualify the laboratory picture: relapse-associated L. donovani isolates from India frequently retain wild-type MT and Ros3 sequences alongside baseline expression levels [97]. Additionally, aneuploidy-mediated dosage modulation involving the reduced copy number of chromosome 13 (encoding LdMT) provides an adaptive route under experimental drug pressure, as documented during the in vitro laboratory selection of Nepalese clinical strains [115,138]. Gain-of-function screening through Cos-Seq intrinsically excludes MT/Ros3 loss-of-function but identifies alternative resistance determinants linked to sterol biosynthesis and membrane lipid homeostasis [106], indicating that transporter-independent routes can also confer resistance.
ATP-binding cassette (ABC) transporters contribute to reduced intracellular drug retention through enhanced efflux [10,139]. Increased expression of ABCG4 and ABCG6 at the plasma membrane and flagellar pocket of laboratory-selected L. infantum and L. tropica lines correlates with reduced intracellular miltefosine retention and cross-tolerance to other alkylphospholipids [96,101,102], while independent L. donovani models demonstrate upregulation of different ABC classes, such as ABCG2, ABCG5, and ABCA7 [104]. Functional studies show that ATP hydrolysis is required for the efflux phenotype, since catalytic inactivation of ABCG6 abolishes the tolerance conferred by overexpression [96]. Pharmacological inhibition studies support an efflux contribution: beauvericin partially restored miltefosine susceptibility in L. tropica [96], and sitamaquine reversed miltefosine resistance through indirect modulation of ABC-dependent efflux rather than competitive transport [103]. The efflux contribution is not conserved across resistant backgrounds, however, as independent miltefosine-resistant L. donovani lines instead show downregulation of MDR1 and ABCG4 [140]. Furthermore, while increased ABCB2 (TAP1) abundance occurs in some miltefosine-resistant L. infantum clinical isolates [141], it is often observed alongside extensive remodeling of stress-response pathways rather than as an isolated dominant transport signature [117], and overexpression of P-glycoprotein-like transporters in arsenite-selected L. donovani does not confer cross-resistance to miltefosine [142]. Proteomic and transcriptomic associations with ABC transporter expression in clinical isolates therefore indicate variable and conditional involvement rather than a universal mechanism. Extracellular vesicle-mediated changes in cargo composition and morphology have been characterized in drug-resistant L. infantum. Miltefosine-resistant parasites produce larger extracellular vesicle subpopulations and exhibit a proteomic signature distinct from wild-type parasites and from antimony- or amphotericin B-resistant lines [143]. These changes suggest an auxiliary stress-response or remodeling phenotype, but their functional contribution to miltefosine resistance remains unresolved.
MT/Ros3 localization, transporter expression, and ABC efflux activity under miltefosine pressure remain unresolved in L. martiniquensis and L. orientalis. Candidate transporter variation has been described in amphotericin B-resistant L. martiniquensis, but its contribution to reduced miltefosine susceptibility requires functional testing [90].
5.2.2 Membrane intercalation and lipid remodeling.
Acute miltefosine exposure depletes phosphatidylcholine (PC) and increases phosphatidylethanolamine (PE) in L. donovani, producing a marked reduction of the PC/PE ratio and impaired membrane homeostasis, with elevated lysophospholipid levels consistent with enhanced phospholipid remodeling and membrane stress [107] (Fig 1B). These changes are attributed to disruption of Kennedy pathway activity (S1 Fig), particularly methylation-dependent steps and possibly cytidyltransferase activity, rather than simple substrate deprivation [107]. Although miltefosine inhibits choline transport in L. major, promastigotes of this species are not strict choline auxotrophs, indicating that reduced choline uptake alone is unlikely to explain parasite killing [146]. Short-term exposure also induces sterol accumulation, including increased ergosterol abundance, and disrupts sphingolipid metabolism, with sphingosine and ceramide elevation reported in L. donovani and L. major [105]. In L. mexicana, inhibition of alkyl-specific acyl-CoA acyltransferase has been described at concentrations exceeding antiproliferative thresholds, suggesting a secondary or context-dependent contribution [147]. Comparative preclinical and clinical studies show broad but variable miltefosine activity across CL-causing species, supporting a membrane-active mechanism with species-specific differences in potency and response [148,149].
In L. donovani, stably resistant promastigotes adapt primarily through reduced unsaturation of fatty-acid alkyl chains, decreased membrane fluidity, depleted C24-alkylated sterols, and increased incorporation of exogenous cholesterol [150]. Genetic and functional studies corroborate lipid metabolism as a determinant of reduced susceptibility: mutations altering L. infantum fatty-acid elongase (LinJ.14.0790) and long-chain acyl-CoA ligase (LinJ.13.0300) increase tolerance [135], while overexpression of lipase precursor-like protein (LinJ.31.0870) in L. donovani enhances resistance by promoting fatty-acid utilization and mitigating oxidative stress rather than altering drug transport [118]. In L. major, serine palmitoyltransferase-deficient promastigotes accumulate cholesterol, reduce ergosterol levels, and show diminished miltefosine susceptibility despite preserved drug uptake [105]. Loss of the miltefosine susceptibility locus containing NUC1 and NUC2 in L. infantum also increases basal sterol and glycerophospholipid content, consistent with membrane sequestration limiting effective drug action [98]. These findings indicate that membrane remodeling can reduce susceptibility, although the extent to which such changes act as primary resistance drivers rather than compensatory adaptations remains unresolved.
L. martiniquensis provides the clearest regional indication that sterol remodeling may intersect with miltefosine susceptibility. An in vitro-selected amphotericin B-resistant line and a clinical relapse isolate both exhibited elevated amphotericin B IC50 values together with reduced miltefosine susceptibility [37]. Genome analysis identified a stop-gained mutation in sterol C-24 reductase alongside missense mutations affecting an ABC transporter-like protein [90]. These findings make sterol and membrane remodeling plausible contributors to cross-resistance, but functional validation and clinical cohort evidence remain lacking. This cross-resistance phenotype is considered further in Section 5.3.
5.2.3 Intracellular ion homeostasis, mitochondrial toxicity, and redox buffering.
In L. donovani, miltefosine activates a sphingosine-dependent plasma membrane Ca2+ channel, leading to rapid acidocalcisome alkalinization and cytosolic Ca2+ overload [151] (Fig 2). Subsequent mitochondrial Ca2+ uptake accelerates electrochemical depolarization [152], while Ca2+ overload and mitochondrial dysfunction are expected to enhance reactive oxygen species production and contribute to bioenergetic collapse in kinetoplastids [153]. Miltefosine also directly inhibits mitochondrial cytochrome c oxidase, or complex IV, producing severe mitochondrial depolarization, ATP depletion, and metabolic collapse in L. donovani promastigotes [154]. Comparative kinetoplastid data support a mitochondrial contribution to drug action: bloodstream Trypanosoma brucei lacks a functional cytochrome-dependent electron transport chain [155], and this respiratory configuration is associated with lower susceptibility to miltefosine-mediated mitochondrial toxicity [154]. The relative order of membrane perturbation, complex IV inhibition, and redox collapse as initiating events remains unresolved [156]. Calcium imbalance should now be considered an additional candidate upstream stressor based on later work, but its position within this sequence remains to be established [151,152].
(A) Under basal conditions, acidocalcisomes maintain ionic balance through the coordinated activity of the vacuolar H⁺-ATPase, vacuolar H⁺-pyrophosphatase, and PMC1, a Ca2+-transporting ATPase, sustaining a proton motive force and an acidic lumen that supports Ca2+ sequestration. (B) Miltefosine-associated membrane perturbation can compromise proton retention, leading to proton leakage, loss of proton motive force, and passive Ca2+ release from acidocalcisomes into the cytosol, without implying direct inhibition of PMC1. (C) At the plasma membrane, miltefosine enhances Ca2+ influx through sphingosine-activated Ca2+ channels, while ATP depletion may limit PMC1-mediated Ca2+ extrusion, promoting cytosolic Ca2+ accumulation. (D) Increased cytosolic Ca2+ and ATP limitations may impair sarco/endoplasmic reticulum Ca2+-ATPase-mediated reuptake, contributing to endoplasmic reticulum Ca2+ dysregulation and organellar stress. This schematic represents a generalized model of miltefosine-associated Ca2+ disruption based mainly on studies in non-Mundinia species and related kinetoplastid calcium biology. Created in BioRender. Lerona, P.G. (2026) https://BioRender.com/t9n5kgx.
Quantitative proteomics of in vitro-selected miltefosine-resistant L. infantum revealed increased abundance of respiratory chain components across complexes III, IV, and V, including F0F1-ATP synthase, together with enrichment of fatty-acid β-oxidation enzymes, a pattern interpreted as maintenance of electron transport and ATP generation under drug pressure [113]. A multidrug-refractory mucosal L. braziliensis isolate showed elevated ATPase α subunit and mtHSP70 expression, indicating that mitochondrial stress-response proteins may also be altered in refractory clinical infection [157]. These changes may reflect mitochondrial adaptation in reduced-susceptibility contexts, but their causal contribution remains unresolved because most evidence is associative rather than functionally validated.
Iron superoxide dismutase A (FeSODA) is upregulated in miltefosine-resistant L. donovani and contributes to detoxification of drug-induced superoxide radicals [158,159]. Elevated cytosolic and mitochondrial tryparedoxin peroxidases also contribute to peroxide detoxification and redox buffering [110]. Proteomic profiling identified peroxiredoxin as a shared stress-response protein across L. infantum parasites resistant to miltefosine, antimonials, or paromomycin, consistent with generalized adaptation to oxidative stress rather than drug-specific selection [141]. The functional contribution of individual redox enzymes is context-dependent: partial downregulation of FeSODA in L. infantum was paradoxically associated with increased resistance, attributed to compensatory induction of alternative antioxidant pathways, including ascorbate peroxidase [160]. Activation of the pentose phosphate pathway in L. donovani, with overexpression of glucose-6-phosphate dehydrogenase and transaldolase, increases NADPH availability and confers resistance to multiple drugs, including miltefosine, amphotericin B, and antimony, though not paromomycin [111]. Stress-response and chaperone proteins further modulate mitochondrial integrity: HSP83 enrichment in an antimony-resistant L. donovani clinical isolate prevented mitochondrial membrane depolarization, and HSP83 transfection conferred miltefosine resistance in otherwise susceptible parasites [161]. Overexpression of LdTCP1γ increased the miltefosine IC50 approximately 1.8-fold through increased thiol buffering and induction of tryparedoxin peroxidase [162]. Differential abundance of these proteins in resistant clinical isolates does not by itself establish causal contribution. Comparable calcium responses, mitochondrial vulnerabilities, redox-buffering pathways, and resistance-associated proteomic adaptations have not been defined in L. martiniquensis or L. orientalis.
5.2.4 Cell death-associated phenotypes and survival adaptation.
In susceptible L. donovani and L. amazonensis, miltefosine exposure induces phosphatidylserine externalization with preserved plasma membrane integrity, accompanied by cell shrinkage, oligonucleosomal DNA fragmentation, and accumulation of parasites in the sub-G0/G1 population [163–166] (Fig 3). These features have often been interpreted as apoptosis-like cell death [165], but the classification of programmed cell death in protozoa remains debated because several hallmarks may also occur during incidental or non-regulated death pathways [153]. Canonical caspases are absent in Leishmania, although metacaspases undergo stimulus-dependent auto-processing during oxidative stress and drug exposure, and endonuclease G translocates from the mitochondrion to the nucleus to mediate oligonucleosomal DNA fragmentation [167–169]. In L. donovani, methionine aminopeptidase 2 inhibition prevents ΔΨm collapse, cytosolic Ca2+ accumulation, and DNA fragmentation during miltefosine-induced stress, but does not ultimately prevent parasite death [170].
Schematic representation of major phenotypic hallmarks associated with miltefosine-induced apoptosis-like death. (A) Metacaspase activation associated with Ca2+ and ROS stress, contributing to proteolytic processing of nuclear, cytoskeletal, and membrane-associated proteins. (B) DNA fragmentation through endonuclease-mediated cleavage accompanied by nuclear condensation. (C) Loss of phospholipid asymmetry resulting in phosphatidylserine externalization while plasma membrane integrity is preserved. (D) Cytoskeletal remodeling and progressive cell disassembly culminating in parasite death. These features resemble metazoan apoptosis phenotypically but are mediated through parasite-specific, non-canonical molecular machinery and should not be interpreted as evidence of canonical caspase-dependent apoptosis. Created in BioRender. Lerona, P.G. (2026) https://BioRender.com/q3d7unm.
kDNA disruption during miltefosine exposure is best interpreted as a downstream mitochondrial injury phenotype rather than a primary drug target. In L. donovani, dyskinetoplastidy (loss or disintegration of the kinetoplast) follows mitochondrial membrane potential collapse, and kDNA loss is attributed to mitochondrial permeabilization and nuclease access rather than direct inhibition of kDNA maintenance [142]. Drug-induced stress can also activate autophagy as an early survival response, although prolonged activation may converge with apoptosis-like pathways and promote parasite death [171].
Resistant L. donovani lines preserve mitochondrial function through upregulation of antioxidative enzymes that suppress oxidative stress-associated apoptosis-like death [109,110,112]. Evidence from L. infantum indicates mechanistic heterogeneity: an in vitro-selected resistant line sustained bioenergetics through metabolic remodeling rather than canonical ROS detoxification [113], whereas a refractory clinical isolate showed increased abundance of antioxidative enzymes and stress-response proteins, including HSP60, STI1, and PCNA [117]. Resistant L. tropica similarly shows induction of stress-response and DNA repair proteins, including HSP60, HSP70, HSP83, and PCNA, alongside reprogramming of energy metabolism [96]. Kinetoplastid calpain-related proteins further illustrate context-dependent regulation: SKCRP14.1 transfection in L. donovani increased miltefosine tolerance while sensitizing parasites to antimony [161]. Functional evidence supports causal roles for selected effectors such as SKCRP14.1 and HSP83 [161], but many reported stress-response signatures derive from comparative omics analyses of clinical isolates or laboratory-selected lines. Differential protein abundance should therefore be interpreted as associative unless supported by direct perturbation experiments. Comparable cell death-associated phenotypes, kDNA disruption, autophagic responses, and survival adaptations under miltefosine pressure have not been characterized in L. martiniquensis or L. orientalis.
5.2.5 Host immunomodulation and host-parasite determinants of treatment outcome.
Miltefosine immunomodulation has been systematically reviewed by Palić and colleagues [172], and only host-mediated effects directly relevant to treatment outcome and resistance interpretation are considered here. In L. donovani-infected macrophages, miltefosine restores IFN-γ responsiveness by increasing IFN-γ receptor expression, promoting STAT1 phosphorylation, and counteracting SHP-1 phosphatase activity [173]. The same study showed induction of inducible nitric oxide synthase and nitric oxide production through PKC- and PI3K-dependent p38 MAPK signaling [173]. Miltefosine also upregulates TLR4 and TLR9 in L. donovani-infected macrophages, amplifying IL-12, TNF-α, nitric oxide, and downstream IFN-γ production [174]. At the parasitophorous vacuole membrane, miltefosine disrupts host Akt activation that normally supports intracellular persistence in L. donovani and L. amazonensis infection models [175].
Miltefosine enhances nitric oxide production in L. amazonensis-infected macrophages but reduces nitric oxide production in uninfected macrophages, indicating that host immune activation depends on the infected cellular state rather than reflecting uniform pro-inflammatory stimulation [176]. In human PBMCs infected with L. panamensis, miltefosine suppresses IL-10 and IL-13 without significantly altering IFN-γ or TNF-α production [177]. This context dependence is clinically relevant in immunosuppressed patients, including those with HIV co-infection, where impaired cellular immunity may reduce the contribution of host-mediated parasite clearance and partly explain heterogeneous outcomes and the need for combination regimens to achieve durable cure [73] (Section 4.1).
Miltefosine-resistant parasites may modify macrophage infection and cytokine balance through resistance-associated metabolic adaptations. In L. donovani, altered MAPK expression has been associated with miltefosine resistance and may influence stress adaptation and life-cycle regulation [178]. A lipase precursor-like protein associated with miltefosine tolerance enhances macrophage infectivity and skews cytokine responses toward anti-inflammatory profiles, linking metabolic adaptation with host-cell persistence [118]. In a murine model, an experimentally selected miltefosine-resistant L. infantum strain triggered enhanced early innate immune activation in the liver, with increased NK and NKT cell activity and higher systemic IFN-γ; miltefosine treatment partially restored parasite fitness in vivo, indicating a drug-dependent rather than constitutive immune-evasion phenotype [179]. Comparable resistance-associated host-parasite fitness effects have not been characterized in L. martiniquensis or L. orientalis under miltefosine pressure.
In L. guyanensis, the endosymbiotic Leishmania RNA virus 1 (LRV1) is detected by host TLR3 and triggers proinflammatory signaling that supports parasite persistence and metastatic spread [180]. Clinical studies associate LRV1-positive infections with treatment failure or relapse in L. guyanensis [124], L. braziliensis [121], and L. naiffi [129] despite preserved in vitro drug susceptibility, although this association is not universal and one cohort of L. guyanensis isolates showed no correlation between LRV1 status and pentamidine treatment failure [125], indicating that viral effects may modulate therapeutic outcome independently of intrinsic parasite resistance without uniformly determining it. LRV1 suppresses inflammasome activation by inhibiting NLRP3 through TLR3-mediated autophagic degradation [181] and by interfering with non-canonical caspase-11 activation [182,183], reducing effective microbicidal responses despite increased inflammation, while packaging within parasite-derived extracellular vesicles promotes viral persistence [122]. In contrast, the immunomodulatory effects of Leishmania RNA virus 2 (LRV2) in L. major appear distinct; while LRV2 is associated with reduced IL-1β expression, it significantly upregulates NLRP3 gene expression at later infection stages in vitro [127], indicating divergent or species-specific viral-host interactions. The leishbunyavirus LmarLBV1, the first non-LRV RNA virus identified in Leishmania, was detected in a Martinique isolate of L. martiniquensis [126]. Experimental viral depletion in this isolate reduces parasite infectivity in murine macrophages, supporting a direct role for viral endosymbionts in host-parasite interactions, although effects on antileishmanial drug susceptibility remain untested [126]. Viral endosymbionts should therefore be interpreted as host-parasite modifiers of treatment outcome rather than proven miltefosine resistance mechanisms. The distribution, functional effects, and clinical relevance of viral endosymbionts in Southeast Asian Mundinia infections require direct investigation.
5.3 Cross-resistance with amphotericin B in L. martiniquensis
Both an in vitro-selected amphotericin B-resistant line and a clinical relapse isolate of L. martiniquensis exhibit elevated amphotericin B IC50 values together with reduced miltefosine susceptibility, indicating a cross-resistance phenotype between these membrane-targeting drugs under experimental and relapse-associated contexts [37]. Genome analysis of resistant lines identified a stop-gained mutation in sterol C-24 reductase, disrupting the terminal step of ergosterol synthesis (S2 Fig), alongside additional mutations affecting an ABC transporter-like protein, LSCM1_01856 [90]. These findings make sterol remodeling and altered membrane organization plausible contributors to reduced miltefosine susceptibility, potentially through mechanisms independent of canonical MT/Ros3 transporter disruption.
Amphotericin B formulations have been the therapeutic mainstay for autochthonous leishmaniasis in Thailand [24,32], and sustained amphotericin B exposure could plausibly select parasite phenotypes with reduced miltefosine susceptibility before wider miltefosine deployment. Current evidence remains restricted to in vitro selection and one relapse isolate, and clinical cross-resistance has not been demonstrated in patient cohorts. The convergence of amphotericin B reliance, emerging Mundinia infection, and experimentally observed reduced miltefosine susceptibility supports prospective phenotypic and genotypic monitoring of clinical isolates from amphotericin B-treated patients in Thailand.
5.4 Knowledge gaps in Mundinia
Miltefosine resistance models remain insufficiently defined for Mundinia in Southeast Asia. The major mechanistic axes described in this section derive largely from L. donovani, L. infantum, L. major, L. mexicana, or other non-Mundinia species. Direct Mundinia evidence is limited to reduced miltefosine susceptibility in amphotericin B-resistant L. martiniquensis [37,90] and the identification of LmarLBV1 as a host-parasite modifier in L. martiniquensis [126]. These observations support focused mechanistic studies of Mundinia, but they do not establish which resistance pathways will emerge under miltefosine exposure in regional clinical isolates.
Mechanistic interpretation of miltefosine response in Southeast Asia requires baselines that are currently unavailable for Mundinia. Species-resolved susceptibility surveys are needed for L. martiniquensis and L. orientalis to distinguish natural variation from emerging reduced susceptibility. Functional studies should test whether resistance axes defined in other Leishmania species, including MT/Ros3 transport, ABC efflux, lipid and sterol remodeling, mitochondrial and redox adaptation, cell death tolerance, and host-parasite fitness effects, operate in Mundinia. The amphotericin B-associated cross-resistance signal described above should be incorporated into future Mundinia susceptibility baselines. These unresolved questions define the stewardship conditions needed to introduce miltefosine as a useful but carefully monitored therapeutic option in Southeast Asia.
6. Diagnostics, surveillance, and resistance preparedness
Resistance preparedness for miltefosine in Southeast Asia requires two technical foundations: species-level diagnosis and longitudinal susceptibility surveillance. In Thailand, current diagnostic capacity supports case confirmation but remains insufficient for establishing the species-resolved baseline needed to interpret future shifts in drug response.
6.1 Diagnostic capacity in Thailand
Diagnosis of leishmaniasis in Thailand relies on parasitological, serological, and molecular approaches, each constrained by sensitivity, species resolution, or operational feasibility. Microscopic examination of stained smears and parasite culture retains high specificity but is limited by low sensitivity, prolonged turnaround times, and dependence on specialized expertise, with culture capacity restricted to a small number of laboratories [50,184]. Serological assays widely deployed in other endemic regions, including enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody testing (IFAT), direct agglutination test (DAT), and the rK39 rapid test, remain poorly validated for autochthonous Thai Leishmania species and exhibit uncertain diagnostic performance in local settings [50,184]. These constraints have driven increased reliance on PCR-based diagnostics, which combine high analytical sensitivity and specificity with the capacity to detect parasite DNA from non-invasive samples such as saliva and buccal swabs, supporting earlier identification of infection in immunocompromised patients [50,185,186].
Species-level identification is particularly important in Thailand because L. martiniquensis and L. orientalis are associated with distinct clinical manifestations and may require different treatment approaches [186]. Conventional PCR assays targeting internal transcribed spacer 1 (ITS1) and heat shock protein 70 (HSP70) loci are widely applied for species identification, and a duplex TaqMan-based quantitative PCR has been developed to permit simultaneous detection and quantification of L. martiniquensis and L. orientalis in clinical samples [186,187]. Point-of-care platforms, including loop-mediated isothermal amplification and colorimetric assays, have been evaluated as rapid screening tools, although definitive species assignment continues to depend on sequencing-based confirmation [184,188]. Diagnostic infrastructure for asymptomatic carriage screening, vector-host investigation, and population-level surveillance remains unevenly distributed across Thai endemic areas, and several routinely available platforms have not been validated for the Mundinia species relevant to autochthonous transmission.
6.2 Genomic surveillance and parasitomics
Parasitomic approaches extend molecular diagnostics into surveillance by combining species identification with population-level genetic characterization [189]. Amplicon-based sequencing methods, ranging from Sanger sequencing to nanopore-enabled metabarcoding, have been applied to characterize Leishmania haplotype diversity, vector blood-meal sources, and transmission ecology in Thailand, supporting the integration of human, animal, and entomological surveillance [26,28–30]. These platforms generate species-resolved genomic data that can be used to track parasite population structure over time, identify cross-border movement of strains, and provide reference data against which future susceptibility studies can be calibrated.
Genomic surveillance does not directly detect emerging clinical resistance, but species-resolved sequencing enables monitoring of molecular markers associated with reduced susceptibility in well-characterized species, which may inform treatment decisions and stewardship policy when interpreted alongside phenotypic susceptibility data. Resistance-associated variants reported in L. donovani and L. infantum (Section 5.2.1), including loss-of-function changes in MT and Ros3 and aneuploidy at chromosome 13, provide candidate markers for surveillance in Mundinia species, although their predictive value in L. martiniquensis and L. orientalis has not been established and requires direct validation. Phenotypic susceptibility testing performed in conjunction with sequencing has revealed reduced amphotericin B sensitivity in relapsed L. martiniquensis isolates [37], demonstrating that integrated phenotype-genotype surveillance is technically feasible in regional reference laboratories. Whether the same framework can detect early shifts in miltefosine susceptibility before they translate into clinical failure depends on the prior establishment of Mundinia-specific susceptibility baselines and on systematic phenotypic testing of clinical isolates over time, neither of which is currently in place.
The integration of parasitomic platforms with clinical surveillance is most useful for characterizing baseline parasite populations, identifying species-level shifts in incident cases, and detecting genomic features that warrant phenotypic follow-up. Direct detection of clinically meaningful resistance in patients before treatment failure occurs remains a longer-term aspiration that depends on validated species-specific molecular markers, regional reference panels of phenotypically characterized isolates, and surveillance capacity sustained across the human, animal, and vector interfaces addressed in Section 7.
7. Strategic implementation: A One Health framework for miltefosine stewardship in Southeast Asia
The therapeutic case for miltefosine in Southeast Asia rests not only on the available clinical and mechanistic evidence reviewed in Sections 4 and 5 but on the conditions under which the drug would be deployed. The Indian subcontinent experience shows that pharmacokinetic vulnerability, adherence barriers, and inadequately monitored selective pressure must be anticipated before wider deployment [9,88]. For Mundinia infections in Thailand and the broader region, where parasite populations remain largely naïve to alkylphospholipid exposure and where epidemiological transmission cycles are incompletely characterized, evidence-based introduction must be framed within a coordinated implementation strategy that integrates clinical use, surveillance, and ecological context.
7.1 Principles for evidence-based introduction
Stewardship principles for miltefosine introduction should align with the resistance-aware framing established in Section 5. Combination therapy with liposomal amphotericin B substantially improves cure rates in HIV-coinfected VL patients and reduces relapse, providing the strongest empirical support for combination-based regimens in immunocompromised cohorts [73,83]. The Thai compassionate-use case (Section 4.2) demonstrated that combination induction with miltefosine and liposomal amphotericin B can achieve sustained remission of refractory L. martiniquensis infection, where amphotericin B-based regimens alone had failed, while miltefosine monotherapy was insufficient to maintain remission in the context of advanced immunosuppression. This pattern supports combination-based induction as the operational framework for the use of miltefosine in immunocompromised patients with Mundinia infections in the region.
Adherence support is equally important given miltefosine’s pharmacokinetic profile, i.e., the terminal elimination half-life of approximately 150–200 hours produces prolonged subtherapeutic exposure when treatment courses are incomplete, generating selection pressure favorable to the emergence of reduced susceptibility [87,88]. Programmatic safeguards used elsewhere in the Indian subcontinent, including supervised drug distribution [11], pregnancy testing and contraception counseling because of teratogenic risk, and structured adherence monitoring across the 28-day oral regimen, are directly transferable to Thai clinical practice.
Pre-deployment isolate monitoring should therefore include the amphotericin B-associated cross-resistance concern described in Section 5.3.
7.2 Integrated surveillance across human, animal, and vector interfaces
Thailand already has precedents for multisectoral infectious disease control that could support leishmaniasis stewardship. The Lawa model for opisthorchiasis reduced human infection prevalence from approximately 60% to below 5% through coordinated interventions spanning human health, animal reservoirs, environmental management, and sustained community engagement [190]. Multisectoral surveillance principles have also been applied in Thailand for avian influenza, linking human, animal, and environmental monitoring systems to support early detection and response [191]. These examples indicate that One Health surveillance frameworks are operationally feasible in the Thai public health context, although leishmaniasis implementation remains constrained by fragmented data systems, limited veterinary and entomological capacity, and legal or administrative barriers to data sharing across sectors [191,192].
For Mundinia leishmaniasis, this model would require embedding the diagnostic and susceptibility-surveillance workflow described in Section 6 within entomological monitoring and targeted reservoir investigation. The transmission ecology of L. martiniquensis and L. orientalis in Thailand remains incompletely characterized, with vector competence, reservoir hosts, land-use change, and human mobility remaining active areas of investigation. A One Health stewardship system should therefore prioritize shared reporting structures across clinical, laboratory, veterinary, and entomological sectors rather than treating these activities as separate programs.
7.3 Regional implications beyond Thailand
Autochthonous Mundinia transmission has been documented across mainland Southeast Asia, making Thailand’s diagnostic, surveillance, and stewardship framework relevant to neighboring settings with similar epidemiological and therapeutic constraints. Regional coordination is important because parasite movement, shared vector ecologies, and treatment-policy differences may allow resistance signals detected in one national context to affect neighboring programmes.
Despite frequent reference to Southeast Asia in regional treatment frameworks, the evidence base informing antileishmanial policy derives substantially from clinical and programmatic data generated in the Indian subcontinent, with limited representation of autochthonous Mundinia transmission settings in mainland Southeast Asia. Filling this evidence gap requires regional investment in susceptibility surveillance, harmonized diagnostic standards, and shared platforms for genomic monitoring of resistance-associated markers. Coordinated stewardship across countries with shared Mundinia transmission cycles would strengthen the regional capacity to detect epidemiological shifts, respond to therapeutic challenges, and preserve the clinical utility of the limited treatment options currently available.
8. Conclusions
Miltefosine represents a valuable oral therapeutic addition for emerging L. martiniquensis and L. orientalis infections in Southeast Asia, where current parenteral therapies remain limited by toxicity, relapse, and access constraints. The Thai compassionate-use case supports the clinical plausibility of combined miltefosine and liposomal amphotericin B for refractory L. martiniquensis infection, but it does not establish generalizable efficacy, optimal dosing, or durable monotherapy. Mechanistic evidence from non-Mundinia species and cross-resistance observations in L. martiniquensis indicate that regional deployment should proceed with species-resolved diagnosis, susceptibility baselines, relapse monitoring, and phenotype-genotype surveillance. Under these conditions, miltefosine can be framed neither as an unqualified solution nor as a drug to avoid, but as a rational therapeutic option whose durability depends on resistance-aware stewardship.
Supporting information
S1 Table. WHO 2022 guidelines for treatment and secondary prophylaxis of visceral leishmaniasis in HIV co-infected patients in East Africa and South-East Asia.
https://doi.org/10.1371/journal.pntd.0014555.s001
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S1 Fig. Phosphatidylcholine biosynthesis through the Kennedy and salvage pathways.
The Kennedy pathway comprises two parallel branches operating across cytosolic and endoplasmic reticulum (ER)-associated steps: the CDP-choline branch, initiated by cytosolic choline phosphorylation and culminating in phosphatidylcholine (PC) formation via condensation of CDP-choline with diacylglycerol (DAG); and the CDP-ethanolamine branch, which generates phosphatidylethanolamine (PE). PE may undergo stepwise methylation by phosphatidylethanolamine N-methyltransferase activity (PEMT; mediated by sequential methyltransferases in Leishmania) to yield PC, with the relative contribution of this route varying among kinetoplastids. The salvage pathway originates at the mitochondrial inner membrane, where phosphatidylserine (PS) is decarboxylated by phosphatidylserine decarboxylase (PSD) to form PE, which is subsequently transported to ER-associated membranes for further modification. Cofactors and reaction byproducts, including ATP, CTP, S-adenosylmethionine (SAM), ADP, pyrophosphate (PPi), and S-adenosylhomocysteine (SAH), are shown to indicate the energetic and methyl-donor requirements of PC biosynthesis. (!) Circled exclamation marks indicate pathway steps or lipid pools reported to be perturbed during miltefosine exposure, including choline availability, CTP:phosphocholine cytidylyltransferase activity, PEMT-associated methylation, PC depletion, and altered PE abundance. (*) Circled asterisks indicate resistance-associated metabolic features reported in reduced-susceptibility parasites, including altered phosphocholine or methyl-donor metabolism and remodeled baseline PC and PE pools. This schematic is intended as generalized biochemical context and does not imply that all steps are equally active across parasite stages, species, or resistant backgrounds. Created in BioRender. Lerona, P.G. (2026) https://BioRender.com/cv5zdyq.
https://doi.org/10.1371/journal.pntd.0014555.s002
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S2 Fig. Sterol biosynthesis and compositional remodeling through the mevalonate pathway.
Acetyl-CoA is condensed to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), reduced to mevalonate, and phosphorylated to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These intermediates are sequentially condensed to form geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and squalene, which is oxidized to 2,3-oxidosqualene and cyclized to lanosterol. In the endoplasmic reticulum, successive demethylation, methylation, isomerization, and desaturation reactions generate ergosterol and related C24-alkylated sterols. Key enzymes, including sterol 14α-demethylase (CYP51), sterol C24-methyltransferase (SMT), and Δ5-sterol desaturase, are shown for pathway context. Miltefosine does not have a functionally validated direct target within the sterol biosynthesis pathway, but drug exposure is associated with acute disruption of sterol homeostasis, whereas resistant parasites exhibit adaptive remodeling of sterol composition. Circled exclamation marks indicate nodes or steps reported to be perturbed during miltefosine exposure. Circled asterisks indicate resistance-associated alterations reported in reduced-susceptibility parasites. These annotations reflect predominantly omics-based and compositional evidence from non-Mundinia species and should not be interpreted as direct proof of enzyme inhibition. Created in BioRender. Lerona, P.G. (2026) https://BioRender.com/3xds2ca.
https://doi.org/10.1371/journal.pntd.0014555.s003
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Acknowledgments
We sincerely thank the staff of the Center of Excellence in Vector Biology and Vector-Borne Disease, Department of Parasitology, Faculty of Medicine, Bangkok, Thailand, for their invaluable inspiration and technical support throughout the preparation of this review.
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