Introduction
Leishmaniases is a group of diseases that occur in humans and in other mammals in the tropical and subtropical regions of world (in 90 countries: www.cdc.gov/parasites/leishmaniasis), with a prevalence in countries around the Mediterranean basin, parts of Africa, Asia, and Central and South America (Ref. Reference Selvapandiyan, Croft, Rijal, Nakhasi and Ganguly1). It is caused by protozoan parasitic species of the genus Leishmania and spread by the bite of the insect sand fly, Phlebotomus spp. The disease manifests in several clinical forms, ranging from self-healing skin lesions (cutaneous leishmaniasis (CL)) to potentially fatal visceral infections (visceral leishmaniasis (VL) or kala-azar (KA)). CL typically results in ulcers on the skin, while VL affects internal organs such as the liver, spleen and bone marrow. The disease affects millions of people worldwide, particularly those living in poverty, with compromised immune systems, or in areas with inadequate healthcare infrastructure. This review article delves into the various forms of leishmaniases, including those less commonly discussed, alongside the emerging challenges we encounter in efforts towards its elimination.
Taxonomy of Leishmania
Genus Leishmania belongs to the family Trypanosomatidae in the order Kinetoplastida within the class of Euglenozoa, a phylum of Protista. The family Trypanosomatidae encompasses a diverse group of flagellated protozoan parasites, including the genera Trypanosoma and Leishmania, which cause diseases such as trypanosomiasis and leishmaniasis, impacting human and animal populations globally (Ref. Reference Rioux, Lanotte, Serres, Pratlong, Bastien and Perieres2). The order Kinetoplastida is a group of single-celled parasitic protozoa characterised by the presence of a distinctive DNA-containing structure called a kinetoplast. We present here the disease dynamics with the various species/strains of Leishmania causing several clinical manifestations in humans/mammals and the regions globally affected by the parasite variants depicted in Figure 1 and Table 1.
Figure 1. The sketch describes various forms of leishmaniases and the continents where each of the diseases is prevalent.
Table 1. Various forms of leishmaniases and their causative Leishmania species, along with the globally affected regions, symptoms and challenges in treatment/elimination strategies
Cutaneous leishmaniasis (typical)
CL, the most prevalent form of the disease, is characterised by skin lesions and ulcers caused by Leishmania species, particularly Leishmania braziliensis, L. guyanensis, L. panamensis, L. peruviana, L. mexicana and L. amazonensis in the New World and the L. tropica, L. major and L. infantum complexes in the Old World (Ref. Reference Eiras, Kirkman and Murray38). CL is traditionally known as ‘oriental sores’ and typically occurs at the site of inoculation. Transmitted through the bite of infected phlebotomine sand flies, this parasitic infection often results in self-healing lesions within 6 months without intervention. The specific ulcerating granuloma of skin is an initial papule, later transforming into an ulcer (Ref. Reference David and Craft39). The non-healing skin ulcers, especially on the face or limbs, can lead to considerable morbidity. The global burden is substantial, with an estimated 700,000 to 1 million new cases reported annually worldwide (Ref. 40). The incubation period between an infected sand fly bite and lesion development ranges from 2 weeks to 6 months. Despite the self-limiting nature of most cases, the persistence of non-healing ulcers underscores the potential for significant public health impacts, emphasising the importance of ongoing research and interventions to address this widespread and debilitating disease.
Apart from the parasite species/variant involved, the disease prognosis and outcome in terms of number of lesions, types of lesions, extent of host tissue damage and lesional parasite load depends on the host immune response to parasite specific antigens (Ref. Reference Scorza, Carvalho and Wilson41). On this line, there is a risk of dissemination in immunodeficient patients with prolonged illness and the disease may take a chronic form. Different modalities of cutaneous disease outcomes are discussed in the following sections.
Localised cutaneous leishmaniasis
Localised cutaneous leishmaniasis (LCL) may be caused by several species of Leishmania, with the lesion(s) occurring at the site of the insect bite. The incubation period for this form ranges from 1 to 4 weeks and can last for up to several years (Ref. Reference Sharma42). The affected body sites are the ears, nose, upper lip, cheeks, legs, hands and forearms.
A typical clinical manifestation of this form of disease is the appearance of an erythematous, non-itchy and painless papule. It may transform into a nodule or an ulcer with nodular or thick borders, having sharp and elevated edges. LCL can heal spontaneously within 3–9 months in the case of L. mexicana, 2–6 months for L. major and 6–15 months for infections with L. braziliensis, L. tropica or L. panamensis. There are high chances of relapse, with similar or more severe clinical manifestations than those observed at the initial episode (Ref. Reference Scorza, Carvalho and Wilson41).
Diffuse cutaneous leishmaniasis
In rare incidences of CL, diffuse cutaneous leishmaniasis (DCL) may arise, characterised by non-ulcerating nodules affecting large skin areas, causing prolonged and severe disabilities persisting for months or even years (Ref. Reference Kumari, Balai, Gupta, Khare, Mittal and Mehta43). This is characterised by an anergic of cellular immune response to parasite antigens (Ref. Reference Mehta, Balachandran, Rao, Dil and Indusri44). The disease is disseminated through tissue, lymph and blood, giving rise to widespread skin lesions. It often starts with hard, erythematous nodules and reddish-brown, infiltrative smooth or verrucous plaques. The disease phenotype is observed in Amazonian Brazil, Central America, Ethiopia, Kenya and Venezuela, and is caused by the L. mexicana complex (L. amazonensis, L. braziliensis and L. pifanoi). With a poor T cell response, lesions exhibit a larger number of parasitised macrophages in DCL (Ref. Reference Hashiguchi, Gomez, Kato, Martini, Velez and Uezato45). This clinical form is generally resistant to treatment. There is no spontaneous resolution, and a prolonged disease of up to 20 years has been observed.
Disseminated cutaneous leishmaniasis
The occurrence of multiple polymorphic cutaneous lesions distributed over more than two non-contiguous parts of the body is described as disseminated cutaneous leishmaniasis (DSL). It occurs less frequently and is mainly seen in the New World region (Ref. Reference Machado, Prates and Machado30). In almost half of the cases, an association with nasal mucosal lesions has been observed. In the phylogenetic analysis of Cytochrome b gene sequences of various species of Leishmania, the Leishmania strain that causes DSL was observed among the group responsible for mucocutaneous leishmaniasis (MCL) and more closely placed with L. guyanensis (Ref. Reference Hashiguchi, Gomez, Kato, Martini, Velez and Uezato45), which is also commonly observed in the New World. Classically CL, DSL and MCL are grouped as American tegumentary leishmaniasis in the Americas, especially due to L. braziliensis (Ref. Reference Guimaraes46).
Lupoid leishmaniasis
Lupoid leishmaniasis, also referred to as leishmaniasis rucidivans (LR), is a rare cutaneous form of leishmaniasis that occurs in patients with a strong cellular immune response (Ref. Reference Pazoki, Fakhar, Rasooli, Karamian and Nazar21). It is caused by the recurrence of cutaneous disease at the sites of previously cured CL lesions. LR detection is hard as it is different from acute lesions, owing to the absence of parasites in tissue biopsies of the lesion (Ref. Reference Oliveira-Neto, Mattos, da Silva, de Souza, Fernandes and Pirmez47). It is mostly caused by L. tropica in the Old World.
Mucocutaneous Leishmaniasis
MCL is a less common form of leishmaniasis that can result in partial or complete destruction of the mucous membranes in the nose, mouth and throat (Ref. Reference Tejura, Kim, Dever and Chew48). Clinically, there is early infiltration of the mucosa with superficial ulcerations, and the borders have a necrotic appearance, being torn and detached. The uvula, pillars of the palate roof, and tonsils are often destroyed. This condition can occur as a consequence of infection with certain species of the leishmaniasis parasite that cause CL in parts of Latin America. Some types of the parasites can spread from the skin and cause sores in the mucous membranes of the nose (most commonly), mouth or throat. MCL is a destructive form of leishmaniasis, only seen with the American species of Leishmania (Viannia subspecies), which includes L. braziliensis, L. guyanensis and L. panamensis. The enhanced co-lateral tissue damage involved is due to the elevated inflammatory immune-response with a low immune-regulatory mechanism in place (Ref. Reference Scorza, Carvalho and Wilson41). According to the Centers for Disease Control and Prevention, this condition mainly affects individuals in Bolivia, Brazil, Ethiopia and Peru, with over 90% of cases occurring in these countries (Ref. Reference Torres-Guerrero, Quintanilla-Cedillo, Ruiz-Esmenjaud and Arenas24). The diagnosis of oral leishmaniasis is challenging, primarily due to its rare occurrence in settings lacking sufficient laboratory support and appropriate testing capabilities.
Visceral leishmaniasis (typical)
The systemic VL disease form, commonly known as KA or dum-dum fever, is a life-threatening disease listed among neglected tropical diseases by the World Health Organization (WHO) (Ref. Reference Scarpini, Dondi, Totaro, Biagi, Melchionda, Zama, Pierantoni, Gennari, Campagna, Prete and Lanari49). If left untreated, VL proves fatal in over 95% of cases. This debilitating condition primarily targets the internal visceral organs, such as the liver, spleen and bone marrow. VL is known to induce hyperplasia of reticulo-endothelial cells of the organs involved. Clinical manifestation of the disease includes anorexia, lymphadenopathy, hepatomegaly, splenomegaly, pallor, anaemia, thrombocytopenia, fever, weakness, cutaneous pigmentation and weight loss, which progresses rapidly in weeks or months. The incubation period of the disease is from 3 to 8 months. Children have prominent symptoms than adults in many areas, and the disease progresses rapidly in people with a weakened immune system, particularly those with AIDS, than in people with a healthy immune system.
L. donovani is the causative agent in the Indian subcontinent, Asia and Africa, affecting both adults and children. In the Mediterranean region, southwest and central Asia and South America, particularly in young children, VL is caused by L. infantum or L. chagasi. While significant progress has been made in many regions, the disease persists as a major health concern in East Africa, Southeast Asia and Brazil (Ref. Reference Rakhshanpour50). India accounts for 18% of the global burden of VL in 2020. It is present in 54 districts across four endemic states in India: Bihar (33 out of 38 districts), Jharkhand (4 out of 24 districts), Uttar Pradesh (6 out of 75 districts) and West Bengal (11 out of 23 districts) (Ref. Reference Patil, Chatterjee, Almeida-Souza, de Oliveira Cardoso, Abreu-Silva and Calabrese51). Sporadic cases are also reported in other states, including Assam, Gujarat, Himachal Pradesh, Jammu and Kashmir, Kerala, Madhya Pradesh, Haryana, Puducherry, Sikkim, Tamil Nadu and Uttaranchal (Ref. Reference Patil, Chatterjee, Almeida-Souza, de Oliveira Cardoso, Abreu-Silva and Calabrese51).
WHO’s global leishmaniasis surveillance for 2017–2018, along with additional indicators, underscores the continued importance of monitoring and addressing VL. An estimated 50,000–90,000 new cases emerge annually worldwide (Ref. 40). This stark prevalence emphasises the urgency of sustained efforts to control the disease, particularly in regions where it continues to impact vulnerable populations. The global health community’s commitment to tackling VL remains crucial in preventing its high mortality rate and reducing the burden on affected communities (Ref. Reference Selvapandiyan, Croft, Rijal, Nakhasi and Ganguly1).
KA elimination approach and strategies have witnessed a huge upsurge. Due to intense control and elimination strategies in the country, KA cases have decreased by 98% (1,275 cases in 2021) since the start of intensified activities in 1992 (77,102 cases). To get to the 2030 Sustainable Development Goals and WHO targets for KA elimination, the block-level incidence of cases needs to be reduced to less than 1 case per 10,000 population. This target aligns with the new NTDs roadmap 2021–2030 (Refs Reference Selvapandiyan, Croft, Rijal, Nakhasi and Ganguly1, Reference Farooq, Singh, Selvapandiyan, Ganguly, Selvapandiyan, Singh, Puri and Ganguly52). By the end of 2021, 98% of blocks had achieved the WHO elimination threshold.
India is redoubling its efforts to resolve known and newer challenges of under-reporting, detection of asymptomatic cases, post-kala-azar dermal leishmaniasis (PKDL; described separately below), atypical leishmaniasis cases and emergence of newer endemic zones in the elimination of VL (Ref. 53). India has hugely expanded vector control interventions. The endemic states need to mandatorily notify cases to the National Vector Borne Disease Control Programme every month, even if there are zero cases (Ref. Reference Pilot54). In recent years of KA, India has witnessed about 97% reduction of VL cases largely due to the introduction of single-dose AmBisome. In endemic villages that have reported cases of KA over the past 3 years, two rounds of indoor residual spraying (IRS) are being applied. WHO in coordination with the Ministry of Health and Family Welfare, Government of India and NCVBDC organised a coordinated programme to assess the situation and progress of the KA elimination programme in two endemic states: West Bengal and Uttar Pradesh (Ref. 55). PKDL is a sequel of VL in certain populations following the apparent cure of VL (Ref. Reference Kumar, Chatterjee and das14). PKDL patients harbour the parasite in skin lesions and may be the source of new infection to vectors even after two decades of eliminating the disease. Focused efforts on control of PKDL cases, along with the recent challenge of cutaneous cases caused by L. donovani variants, are being recognised as an existing source of parasite in circulation that can lead to newer cases of VL upsurge.
Post-kala-azar dermal leishmaniasis
PKDL is a disease form of concern as a cutaneous sequel following VL or KA. It is a form of CL that usually occurs months to years after VL treatment (Ref. Reference Kumar, Chatterjee and das14). It typically manifests 6 months to a year or more after KA that is assumed to have been cured; however, it can happen even earlier. It typically manifests as hypopigmented macular, papular and nodular rash. People with PKDL are considered a potential source of Leishmania infection. While it typically emerges as a sequelae of VL, intriguingly, some individuals exhibit PKDL symptoms without a prior history of VL. First described by Dr. U. N. Brahmachari in 1922, the condition was termed ‘dermal leishmanoid’ (Ref. Reference Saha56). The symptoms of PKDL encompass a variable combination of hypopigmented patches, erythematous succulent papulo-plaques and nodular lesions, primarily on the face and upper body, and occasionally extending to the extremities, genitalia and tongue. Recent documentation indicates a notable decrease in the interval between VL and PKDL, with over 35% of cases presenting within just 1 year after a bout of VL, adding complexity to the understanding of the disease progression (Ref. Reference Kumar, Chatterjee and das14).
Leprosy and PKDL resemble each other closely in their clinical manifestations. A rapid accurate assay called ‘m-LAMP’ could be used for the differential diagnosis of leprosy versus PKDL (Ref. Reference Joshi57). In a comparison of treatment susceptibilities between VL and PKDL isolates, the latter displayed reduced susceptibility to miltefosine than the VL isolates (Ref. Reference Bhandari58). Towards that end, a combination therapy with liposomal amphotericin B and miltefosine displayed larger efficacy in healing of PKDL (Ref. Reference Ramesh59). Correct diagnosis and timely treatment of PKDL is the next important milestone to be achieved in the consolidation phase of VL elimination operational in South-East Asia.
Para-kala-azar dermal leishmaniasis
Para-kala-azar dermal leishmaniasis (Para-KDL) is an evolved condition associated with the presence of both PKDL and VL (Refs Reference Sarraf60, Reference Maruf61). Despite cross-sectional studies revealing only 16 cases from 2012 to 2021, these cases were successfully treated and cured using high doses of Liposomal Amphotericin B (20 mg/Kg) in Bangladesh (Ref. Reference Maruf61). Active prevalence of Para-KDL has been reported in East Africa, although rare cases have also been documented in India and Brazil (Refs Reference Kumar62, Reference Lindoso63). A study identified nine cases in India, mainly from Bihar, linked to relapse from miltefosine treatment (Ref. Reference Kumar62). The challenges of diagnosing Para-KDL contribute to poor prognoses for affected individuals, particularly as parasites show reduced susceptibility to current treatments (Ref. Reference Maruf61). To explore this issue, genome sequencing was conducted on sodium stibogluconate (SSG)-sensitive and -resistant L. donovani strains, revealing 24 unique mutations in Para-KDL strains that may contribute to their dermatotropic behaviour (Ref. Reference Sarraf60). Interestingly, three cases of human immunodeficiency virus (HIV)–Para-KDL comorbidities were also observed in Brazil (Ref. Reference Alves64). These findings highlight the need for ongoing monitoring and secondary prophylaxis in patients with VL.
Atypical leishmaniasis
The association between the infecting Leishmania species, more importantly the VL-causing L. donovani, and its clinical outcome appears to be modifying in recent years with the emergence of newer parasite variants and disease occurrence in newer regions. Such atypical forms in Sri Lanka include cutaneous lesions that exhibit unusual characteristics, as well as cases presenting with systemic symptoms not typically associated with VL (Refs Reference Karunaweera65, Reference Siriwardana66, Reference Samarasinghe67, Reference Lypaczewski and Matlashewski68, Reference Silva69). Similar cases had been reported from the Himachal regions of India and other northern neighbouring countries (Refs Reference Thakur, Singh, Shanker, Negi, Jain, Matlashewski and Jain31, Reference Lypaczewski70, Reference Pradhan71, Reference Rai72). Nevertheless, L. donovani is not the sole causative agent of CL in Sri Lanka, potentially explaining a haplotype that resulted in interspecies dermotropic hybrids of L. donovani with L. tropica (Ref. Reference Silva69). The changing disease landscape warrants detailed molecular surveillance of the heterogeneous parasite populations that emerge in new endemic sites posing challenges to the disease elimination strategies. Kinetoplast DNA-based phylogenetic analysis reveals distinct differences between VL-causing L. donovani and CL-causing L. donovani variants (Ref. Reference Kariyawasam73). Whole-genome sequence analysis has also shed considerable light on genetic variations and polymorphisms that exist between causative parasites in different regions (Ref. Reference Lypaczewski70). Interestingly, L. donovani that causes CL in Sri Lanka has been placed a considerable distance from the CL causing other Leishmania species in phylogenetic analysis (Ref. Reference Selvapandiyan74). In Himachal Pradesh, as a new endemic site for CL caused by L. donovani, parasite isolates from CL patients comprise considerable heterogeneity at the genetic level, with accumulation of wide genetic mutations in terms of ploidy changes, copy number variations, InDels and single nucleotide polymorphisms (SNPs) that are different from those detected for L. donovani CL isolates from Sri Lanka (Ref. Reference Lypaczewski70).
On a similar note, the atypical phenotype caused by L. donovani is further exemplified through reports on MCL cases in Sri Lanka and India due to L. donovani (Refs Reference Pulimood75, Reference Sethuraman76). There were also several studies in the past describing the viscerotropic (VL-causing) nature of L. tropica (that causes CL worldwide) in India (Ref. Reference Sacks77) and Bangladesh supported by subsequent molecular confirmation (Ref. Reference Thakur, Singh, Shanker, Negi, Jain, Matlashewski and Jain31). Similarly L. tropica causing VL has also been observed in U.S. soldiers of Operation Desert Storm (Ref. Reference Magill78). In addition, L. infantum, and not L. donovani infection, had been reported to have caused PKDL in an HIV-1-infected patient in Australia (Refs Reference Stark79, Reference Carnauba80).
Experiments on clinical isolates from distinct atypical VL and CL endemic regions have identified strain-specific genetic variations upon sequence analysis of targeted genes, and polymorphisms of other regions defining parasite variants compared to the standard species-specific parasite genotypes associated with classical VL and/or CL disease phenotypes. Such new genetic variants can possibly explain the emergence of atypical leishmaniasis and thus the need for more studies on genetic analysis of the clinical isolates from known and newer disease foci for an insight into unusual phenotypic outcomes.
Canine leishmaniasis
VL in domestic dogs is another notable vector-borne zoonotic disease in humans. The causative organism is L. infantum, and the disease is prevalent in Europe and South American countries (Ref. Reference Almeida81). Such VL-infested dogs/canines in these countries serve as a reservoir of VL. The key to the management of canine VL is continuous employment of prophylactic measures, through the correct use of repellents/insecticides and vaccines and prompt detection and monitoring of VL in dogs. In the middle East and in North Africa, canine CL due to L. major and L. tropica has been reported (Ref. Reference Baneth82). Furthermore, three beagle dogs displaying atypical VL due to L. infantum in Europe had been reported with rare granulomatous peritonitis (Ref. Reference Peris83). Due to the importance of canine leishmaniasis as a natural reservoir for human disease, a comprehensive plan for its control, including surveillance, phylogenetic studies and early and effective management, should be employed to minimise its spread. There are several vaccines available to cure canine leishmaniasis, which exploit various antigens such as LACK, A2, Q-protein, GP63, KMP-11 and TYRP (Refs Reference Sinha84, Reference Coelho85, Reference Chan86, Reference Carson87, Reference Basu88, Reference Carcelen89). Challenged with such antigens provides protective immunity in the canines. Few commercialised vaccines for the canines are Leishmune, whose production and marketing licence had been withdrawn in 2014, Leish-Tec, LetiFend and CaniLeish, mostly used in Brazil and European countries to treat dogs, although they do not work for humans (Refs Reference Velez and Gallego90, Reference Shital91, Reference Selvapandiyan92).
Other forms of leishmaniasis/parasites
Asymptomatic infections
A significant challenge in the parasite elimination program is that a substantial proportion of healthy people living in endemic areas with no history of VL show positivity for antibodies to Leishmania owing to asymptomatic infections. Like PKDL, asymptomatic individuals are also considered as anthropogenetic reservoirs of VL. The guidelines of the panel of the American Society of Tropical Medicine and Hygiene and Infectious Diseases Society of America suggest close monitoring of asymptomatic individuals with the initiation of treatment only upon symptom development (Ref. Reference Ready93). Interestingly, these patients have elevated CD4+ T cell counts and test positive for leishmanin skin test (Refs Reference Hailu94, Reference Mary95). In addition, a high level of IFNγ in CD8+ T cells is also observed in such individuals, with a few reported cases of elevated IL-17 and IL-22. (Ref. Reference Pitta96). These results suggest the protective role of host immune response against Leishmania infections and disease progression. Further focus on detection, understanding and tackling asymptomatic cases would be essential for effective development of strategies for the elimination of leishmaniasis.
Drug-resistant parasites
In the treatment of leishmaniasis, drug-resistant strains (DRS) of Leishmania are a concerning issue. The emergence of DRS complicates the treatment efforts and underscores the need for ongoing research and development of new therapeutic strategies. Leishmania parasites exhibit genetic diversity, allowing some strains to develop resistance to specific drugs more easily than others. Pentavalent antimonial that became popular for use during the latter half of the twentieth century has faced stiff resistance over the past decade or two, particularly in areas such as Bihar, India (Refs Reference Ponte-Sucre97, Reference Madusanka98). The increased antimonial unresponsiveness is ascribed to the inappropriate use of drug schedules, paving the way for progressive tolerance to drugs by the parasites (Ref. Reference Sundar99). The derivative of antimony, SSG also has been discouraging due to the development of resistance to SSG by the parasite (Ref. Reference Carter100). The genetically diverse Sb-resistant parasites displayed elevated thiol-synthesising and antimony transporter gene expression compared to the susceptible ones (Ref. Reference Khanra101). As a lesson, antimonials are used in combination with paromomycin as a first-line treatment for VL in East Africa to minimise the chance of resistance development (Refs 102, Reference Sundar and Singh103).
Alternatively, lipid-formulated amphotericin B deoxycholate is also being used against VL in the ISC (Indian subcontinent), instead of just amphotericin B deoxycholate, to reduce side effects. However, its high cost has become a major concern, together with a considerable number of relapses noticed in the ISC (Ref. Reference Sundar and Singh103). The other commonly known drug is miltefosine, an orally administered medication that has been in use since 2002 in the ISC. However, resistance shown against miltefosine in VL patients has raised significant concerns in recent years (Ref. Reference Singh and Selvapandiyan104). Miltefosine’s long half-life is responsible for retaining sub-therapeutic doses in circulation for an extended period, leading to exposure of surviving parasites to the drug for a longer period that is believed to result in the emergence of drug resistance (Ref. Reference Guimaraes46). Apart from such pharmacokinetics-based reasons, there could also be parasite’s own mechanisms leading to resistance. In addition to such disadvantages, its serious adverse side effects, believed to be due to immunopathological consequences, have led to the discontinuation of its use (Ref. Reference Singh and Selvapandiyan104).
Cohabitation with other animals/insects
The cohabitation of Leishmania parasites with other beings, including sandflies, reservoir hosts and humans, as well as ecological and environmental factors, plays a crucial role in the transmission dynamics and epidemiology of leishmaniasis (Ref. Reference Selvapandiyan, Croft, Rijal, Nakhasi and Ganguly1). Zoonotically, Leishmania-infected rodents or sand flies serve as reservoirs of infection for humans. In addition, PKDL and atypical VL and CL patients serve as parasite reservoirs. The relationship between sand fly species and Leishmania can be complex and may vary depending on the region, ecology and other factors. In the Old World, Phlebotomus spp. of sand flies transmit leishmaniasis, whereas in the New World, Lutzomyia spp. are the vectors. Hence, the relationship between sand fly species and Leishmania can be complex and may vary depending on the region, ecology and other factors. The majority of the cases of newly emergent foci of CL observed in recent years in the hilly regions of Himachal state in India have been reported along the Sutlej River belt. This could be due to the possible upstream migration of vectors along the rivers (Ref. Reference Sharma105).
Cohabitation of Leptomonas with Leishmania has been much debated in recent years, especially when L. donovani and Leptomonas seymouri, which look alike, were isolated in culture from VL patients (Refs Reference Ahuja106, Reference Selvapandiyan107, Reference Srivastava108). Their high similarity results in the anomalous outcomes. Additionally, myosinXXI localisation has been used as a biomarker to distinguish Leptomonas in Leishmania cultures (Ref. Reference Kajuluri109). To the best of our knowledge, the involvement of L. seymouri in VL pathogenesis has not been assessed or reported in the literature. Furthermore, Leptomonas co-infection was also reported in a fraction of atypical CL cases caused by L. donovani in newer endemic pockets of Himachal Pradesh (Ref. Reference Thakur110). Moreover, the identification of L. seymouri narna-like virus (NLV1) in serum samples of VL cases in India and its plausible role in disease progression has been reported (Refs Reference Sukla111, Reference Sukla112), adding another dimension to the research on the causes of VL in the Indian subcontinent. The detection of Leptomonas spp. with a monoxenous life cycle and considered non-pathogenic to humans implies emerging evidence on the newer parasitic capability of this group of parasites. A rapid, high-resolution melting-based discriminatory diagnostic tool has been described to identify Leptomonas contamination in the VL clinical isolates (Ref. Reference Ahuja113), which can be used for further investigations.
Comorbidity with other parasitic, bacterial and viral diseases
Leishmaniasis frequently coexists with a range of other infections, including HIV, leprosy, tuberculosis (TB), schistosomiasis, malaria and, more recently, COVID-19. These co-infections pose significant challenges due to the diverse pathological outcomes associated with varying host immune status. In many cases, co-infection exacerbates disease severity and increases mortality rates. Co-infection of VL with HIV is a life-threatening condition. This is because HIV infection and leishmaniasis together promote the replication of both causative pathogens and accelerate the progression of both VL and HIV (Refs Reference Tremblay114, Reference Mock115). The first reported case of VL/HIV co-infection in Europe was in 1980, and now it is documented in many countries, with the highest reports coming from Brazil, Ethiopia and Bihar state in India. Patients co-infected with VL/HIV have the highest relapse rate and mortality, which poses significant challenges in the prevention and control of VL (Ref. Reference Takele116). To address this issue, the WHO has recommended new guidelines to target VL in East Africa and South-East Asia based on the results of studies conducted in India by Médecins Sans Frontières and partners, and in Ethiopia by the Drugs for Neglected Diseases initiative and partners (Ref. 117). HIV-infected people contracting leishmaniasis are at a high risk of developing the full-blown disease, with high relapse and mortality rates (Refs Reference Diro118, Reference Rahman119). Antiretroviral treatment is known to reduce the development of the disease, delay relapses and increase the survival rates. As of 2021, Leishmania–HIV co-infection has been reported in 45 countries. This has intensified the burden of leishmaniasis due to the increased difficulty in clinical management and treatment of the disease.
Certainly, the interaction between leishmaniasis and COVID-19 co-infection is an emerging area of interest in the medical literature. According to a study published in 2020 (Ref. Reference Saidi and Jelassi120), three cases of Leishmania–COVID-19 co-infection have been reported, highlighting the need for further investigation into the clinical implications of such co-occurrence. Another study (Ref. Reference Pikoulas121) analysed the clinical characteristics of Leishmania–SARS-CoV-2 co-infection and suggested that the presence of COVID-19 may lead to the reactivation of previously asymptomatic leishmaniasis. This finding underscores the importance of monitoring individuals with a history of Leishmania infection or their asymptomatics, particularly in regions where both diseases are endemic.
Interestingly, there is evidence suggesting a potential protective effect of Leishmania or other neglected tropical diseases against COVID-19 (Ref. Reference Saidi and Jelassi120). This observation may be attributed to the immune response mounted against Leishmania parasites, which could confer some level of immunity or resistance to SARS-CoV-2 infection. For example, the clearance of CL involves mast cells, cytotoxic CD8+ T cells, CD4+ helper T cells and the production of IFN-γ (Refs Reference Muller122, Reference Naqvi123), which are also important in controlling COVID-19. However, it is important to note that while an effective immune response is crucial in controlling both leishmaniasis and COVID-19, the timing and specific components of the immune response may vary between the two diseases. Early Th1 type of response is critical in controlling COVID-19; failure to do so can result in viral replication, tissue damage and severe disease progression (Ref. Reference De Biasi124). Further research is needed to elucidate the complex interactions between leishmaniasis and COVID-19 co-infection, including their impact on disease severity, immunopathogenesis and treatment outcomes. This understanding will be essential for guiding clinical management and public health interventions in regions where both diseases are prevalent.
Co-infection of VL and TB/pulmonary TB is common and a significant concern in regions where both diseases are endemic, such as parts of Africa, Asia and Latin America (Refs Reference Gautam125, Reference Li and Zhou126). Second to VL, MCL can also co-exist with TB in certain parts of Asia (Refs Reference Rathnayake127, Reference Strazzulla128).
Malaria, caused by the apicomplexan protozoan parasites Plasmodium falciparum or P. vivax, co-infecting with Leishmania, has been extensively reported worldwide (Ref. Reference Ornellas-Garcia129). For instance, a case study from Malaysia documented a human infection with P. vivax (detected in a blood biofilm test) and Leishman–Donovan complex (involving L. infantum and L. chagasi) observed in bone marrow aspirate (Ref. Reference Ab Rahman and Abdullah130). The prevalence of malaria co-infection with VL varies from 7% to 18% across different geographical areas in Asia and Africa. However, further longitudinal studies would be needed to fully understand their combined impact on the host and on each other.
Despite Plasmodium and Leishmania operating in different host cells and exhibiting distinct life cycles based on their unique biology and tropism, they may employ immune evasion strategies that commonly affect the host or increase the susceptibility to infections. This suggests a potential synergistic effect in co-infection scenarios, where the presence of one parasite could potentially modulate the host immune response, leading to increased susceptibility or severity of infection by the other parasite. Further research is needed to elucidate the precise mechanisms underlying these interactions and their implications for disease outcomes.
Molecular typing and whole-genome sequencing to study genotypic variations of Leishmania spp.
The genus Leishmania encompasses a complex group of parasites with a wide range of genotypic (and phenotypic) characteristics, which are often used to divide them into species, subspecies and strains. Molecular tools developed in the field have made such classifications easy and relevant, considering the plastic nature of the parasite genome with the accumulation of newer genetic variations. Multilocus sequence typing (Refs Reference El Mazini131, Reference Banu132), randomly amplified polymorphic DNA (Refs Reference Ikram133, Reference Yazidi134), microsatellite typing (Refs Reference Karakus135, Reference Rugna136) and restriction fragment length polymorphism (Refs Reference Usmael137, Reference Koohsar138) are a few examples of such genotyping methods that addressed Leishmania variations.
In contrast to the widely used genotyping tools, whole-genome sequencing (WGS) provides detailed information on genetic variations across the entire genome, including SNPs, insertions, deletions and structural variations that may be more informative enabling studies on a range of aspects that include genetic diversity, polymorphisms, phylogeny, drug resistance and other disease aspects viz virulence factors, and epidemiological surveillance. Complete/partial WGS information with nucleotide/gene/protein annotation information on several Leishmania species/strains is already available at https://tritrypdb.org/tritrypdb/app. Such species/strains of Leishmania include L. aethiopica L147, L. amazonensis MHOM/BR/71973/M2269, L. amazonensis strain PH8, L. arabica strain LEM1108, L. braziliensis MHOM/BR/75/M2903, L. braziliensis MHOM/BR/75/M2904, L. braziliensis MHOM/BR/75/M2904 2019, L. donovani BPK282A1, L. donovani CL-SL, L. donovani HU3, L. donovani strain LV9, L. enriettii MCAV/BR/2001/CUR178, L. enriettii strain LEM3045, L. gerbilli strain LEM452, L. infantum JPCM5, L. major Friedlin 2021, L. major strain LV39c5, L. major strain SD 75.1, L. martiniquensis LEM2494, L. martiniquensis MHOM/TH/2012/LSCM1, L. mexicana MHOM/GT/2001/U1103, L. orientalis MHOM/TH/2014/LSCM4, L. panamensis MHOM/COL/81/L13, L. panamensis strain MHOM/PA/94/PSC-1, L. sp. Ghana MHOM/GH/2012/GH5, L. sp. Namibia MPRO/NA/1975/252/LV425, L. tarentolae Parrot Tar II 2019, L. tarentolae Parrot-TarII, L. tropica L590 and Leishmania turanica strain LEM423.
A recently described minicircle-based DNA footprint assay has simplified the detection and speciation of Leishmania clinical isolates (Ref. Reference Selvapandiyan74). This method has enabled the study of phylogenetic relationships and variations of many Leishmania species that have originated from different parts of the world. Parasites from CL lesions from red kangaroos of Australia (Ref. Reference Rose139) were found to be grouped into a unique cluster in the sequence-based dendrogram analysis. This method enabled the detection of strain-specific variations of L. braziliensis from Peru and Brazil that cause MCL (Ref. Reference Selvapandiyan74). Therefore, it remains a promising approach for phylogenetic analysis, including the measurement of the phylogenetic distances and the identification of parasite isolates of unknown origin.
Molecular basis of understanding parasite variants in leishmaniasis
Several molecular tools have been developed in the past to investigate the evolutionary aspects and differentiate species or strains of Leishmania. Some of these tools, mentioned above, facilitate parasite groupings based on their genetic make-up in relation to phenotypic characteristics or clinical disease manifestations. These groupings may be crucial in the detection of newer parasite variants circulating in different geographical sites and can help in aligning policymakers for evidence-driven strategies for disease diagnosis, treatment and elimination. Targeting multicopy DNA regions such as 18S rRNA, heat shock proteins or mini- or maxicircle kDNA regions for sequence-based analysis often poses challenges due to the huge heterogeneity of the said sequences in Leishmania. The sequence-specific heterogeneity complicates the construction of accurate phylogenetic trees. Homologous recombination, gene conversion and other evolutionary processes can obscure phylogenetic signals as a readout of species and strain identification. Addressing these challenges requires a multifaceted approach, combining appropriate molecular techniques, bioinformatics tools and a thorough understanding of Leishmania biology. Towards this end, we attempted to develop a dendrogram-based analysis of a single-copy gene, centrin5 (calcium binding structural protein (Ref. Reference Selvapandiyan140)), in Leishmania together with those of a few other Trypanosomatid parasites. The results are presented in Figure 2. Among Trypanosomatid members, centrin5 proteins mostly consist of 165 amino acids. The tree analysis developed from 17 such centrin5 protein sequences of Trypanosomatid genera (comprising Leishmania, Trypanosoma and Leptomonas) obtained through respective accession numbers (Figure 2 legend) displays distinct clades for typical VL, CL and MCL parasites. There is a separate group having two autochthonous (L. orientalis and L. martiniquensis) (Ref. Reference Anuntasomboon141) and one non-human parasite L. enriettii (Ref. Reference Paranaiba142) (Figure 2A). The three non-Leishmania parasites (Trypanosoma brucei, Trypanosoma cruzi and L. seymouri) formed a separate group.
Figure 2. Molecular tool to support pathogenic variations. A. Phylogenetic tree based on centrin5 proteins to infer group formations among Leishmania and other Trypanosome members. The parasite groups are described by distinct colours and labelled on their right. The parasite genera and species are described with sources indicated for some. The accession numbers of centrin5 proteins of the parasites, along with their associated serial numbers, are as follows: 1. LdBPK_366370.1.1, 2. XP_001469992.1, 3. XP_003874970.1, 4. XP_001687199.1, 5. GET93710.1, 6. KAG5465198.1, 7. KAG5465937.1, 8. XP_001569255.1, 9. KAG5464459.1, 10. KPI90528.1, 11. KAF8287755.1, 12. XP_011778227.1, 13. LTRL590_360073600.1, 14. LPAL13_350071100.1, 15. LAEL147_000875700.1, 16. LdCL_360071100-t42_1 and 17. AYU83995. The branch lengths and the bootstrap % values are also shown. The tree was constructed by the Maximum Likelihood method and JTT matrix-based model using the MEGA X program (Refs Reference Kumar, Stecher, Li, Knyaz and Tamura146, Reference Ahmad, Selvapandiyan and Bhatnagar147). B,C. Multiple-sequence alignment and % identity of centrin5 proteins of only four of the red branches ‘A’ above using the Clustal W Omega program. The amino acid sequences of 17 centrin5 proteins, their combined Clustal W alignment and percent identity are described separately in the Supplementary Material.
However, based on branch length/subgrouping, the outliers in some of these groups can be identified as distinct categories. For example, both the Sri Lankan and Himachal (India) L. donovani genetic variants that cause CL are seen closer to each other than classical VL causing L. donovani and L. infantum (Refs Reference Siriwardana143, Reference Thakur144). Their differences in amino acid percent identity are also compared in Figure 2B,C. In addition, interestingly, based on centrin5, L. tarentolae, the parasite that infects exclusively lizards (Ref. Reference Raymond145), has been grouped with CL-causing parasites (Figure 2A). Overall, this study gives us a meaningful grouping of a few of Leishmania and other genera of Trypanosomatid family that we attempted via cladogram analysis using the MEGAX program. Such genetic variations across Leishmania species/strains circulating in known and newer endemic zones need molecular surveillance for the detection and prediction of region-specific parasite variants and associated disease outcomes.
Overall challenges, solutions and conclusion
Tropical countries are continuously striving to eliminate various forms of leishmaniasis endemic to their regions. A significant focus lies on combating VL, particularly in countries like India, where it poses a grave threat due to its potentially fatal nature. Challenges persist due to the persistent forms of PKDL and ALI, which hinder the progress of elimination programs. Continuous monitoring of cases through molecular screenings in endemic regions is essential to track occurrences effectively (Ref. Reference Selvapandiyan148). Preventing the development of drug resistance is a key aspect of the elimination strategy. For pathogens that exhibit shifting clinical manifestations, such as atypical leishmaniasis, standard medications may prove ineffective. Hence, developing appropriate treatment regimens tailored to the evolving clinical nature of the disease becomes imperative in such cases.
In scenarios where Leishmania species co-infect with other pathogens, such as viruses, bacteria and parasites, they may collectively induce a synergistic immune response profile. This interaction can either enhance or limit the immune response, leading to decreased host resistance and a failure to control the infection (Ref. Reference Supali149). However, it is important to note that each pathogen manipulates different aspects of the host immune response (Ref. Reference Selvapandiyan150). Therefore, the development of a broad-spectrum therapy against these infections could potentially eliminate not only the primary Leishmania infection but also any secondary and/or co-morbid infections. This approach would target a wide range of pathogens, providing a comprehensive treatment strategy to address the complexities of co-infection scenarios.
Disease prevention remains the cornerstone of sustainable leishmaniasis elimination efforts. Currently, for efficacy, the use of effective combinations of existing drugs is recommended for VL. For example, combinations such as miltefosine–AmBisome or miltefosine–paromomycin have shown promise. These combinations also offer hope for co-infections. In Ethiopia, AmBisome plus miltefosine has proven efficacious in HIV–VL patients. Additionally, improved genetic, immunological and serological markers are needed to determine the progression from parasite infection to clinical VL. Markers for asymptomatic infections have been utilised in clinical studies. However, in the absence of specific safe drugs or markers of disease progression, further research is required to develop newer tools to address these challenges. Various vaccine strategies have also been explored, including those utilising recombinant peptide, DNA, killed whole parasite and genetically modified live-attenuated parasites (Ref. Reference Selvapandiyan92). Notably, the L. donovani and L. major centrin gene knockout strains show promise as a live attenuated vaccine against both VL and CL (Refs Reference Avishek151, Reference Bhattacharya152, Reference Zhang153). Additionally, a clinical trial utilising ChAd63-KH, an adenoviral vaccine encoding KMP-11 and HASPB, was conducted in Sudan with 24 PKDL patients, and the vaccine successfully generated a potent innate and cell-mediated immune response (Ref. Reference Younis154). The results showed that 30.4% of patients had over 90% clinical improvement, while 21.7% showed partial improvement. Following this, a Phase 2 vaccine trial with ChAd63-KH was conducted on 100 patients with persistent PKDL in Sudan. This vaccine has been proven effective in those patients (Ref. Reference Lacey155).
Periodic genome sequencing of the parasite isolates in affected regions can provide valuable insights into emerging Leishmania variants. These data serve as an alert for clinicians and researchers, prompting increased attention towards emerging parasite variants and the associated clinical manifestation in region-specific manner. Implementing effective measures to control vector populations is another crucial approach for achieving the successful elimination of leishmaniasis. Addressing gaps in our understanding of vector bionomics is essential in this regard. These gaps include screening for infected sand flies using PCR, determining sand fly biting rates, assessing parasite infection rates within the vector population and understanding the spatial and temporal variations of these parameters in response to interventions such as IRS. Bridging these knowledge gaps is paramount to achieving sustained elimination of VL and implementing an appropriate post-elimination program. Many countries are now prioritising boosting immunity to prevent infectious diseases, including leishmaniasis, either as a primary infection or as an opportunistic infection alongside other pathogens (Ref. Reference Farooq, Singh, Selvapandiyan, Ganguly, Selvapandiyan, Singh, Puri and Ganguly52). India’s significant investment in AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) as an alternative therapy approach exemplifies this direction. Other immune modulators, such as liposomal cholesterol, which have proven effective experimentally in treating VL, might need further studies. Some countries have transitioned to the post-elimination maintenance phase for leishmaniasis control, emphasising the importance of periodic screenings to detect any reemergence early and prevent resurgence. Although the march towards leishmaniasis elimination appears to be increasing, the achievement of the program remains uncertain in the light of already existing and newly emerging challenges such as sporadic outbreaks, asymptomatic infections and newer changing foci.
Funding statement
A.S. is supported by the Indian Council of Medical Research (ICMR), New Delhi, India (Grant No. GIA/2/VBD/2021/ECD/II). N.K. is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, USA, under Award Number U01AI136033. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M.J. is supported by the ICMR, India (Grant No. 6/9-7 (272/KA/2021/ECD-II)).
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/erm.2025.4.
Competing interests
The authors declare no competing interests.
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