|Year : 2018 | Volume
| Issue : 2 | Page : 98-108
Visceral leishmaniasis: An immunological viewpoint on asymptomatic infections and post kala azar dermal leishmaniasis
Neeraj Tiwari1, Dhiraj Kishore2, Surabhi Bajpai3, Rakesh K Singh1
1 Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi-221 005, India
2 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, India
3 Department of Bioscience and Biotechnology, Banasthali University, Banasthali-304022, Rajasthan, India
|Date of Submission||12-Oct-2017|
|Date of Acceptance||28-Dec-2017|
|Date of Web Publication||14-Feb-2018|
Rakesh K Singh
Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi-221 005
Source of Support: None, Conflict of Interest: None
Elimination of visceral leishmaniasis is a priority programme in Indian subcontinent. The World Health Organization has set a new target to eliminate kala-azar by the year 2020 as previous target elimination year (2015) has passed. The elimination programme has successfully curbed the rate of infection in endemic regions; however, there are still few challenges in its route. The current drug control regime is extremely limited and comprises only two (amphotericin B and miltefosine) drugs, which are also susceptible for parasites resistance. Moreover, these drugs do not produce sterile cure, and cured patients may develop post kala-azar dermal leishmaniasis even after a decade of cure leaving behind a potent source of parasitic reservoirs for further disease transmission. A significant proportion of endemic population remain seropositive but aymptomatic for many years without any clinical symptom that serve as latent parasitic reservoirs. The lack of tools to identify live parasites in asymptomatic infections and there association in disease transmission, parameters of sterile cure along with post kala-azar dermal leishmaniasis progression remain a major threat in its elimination. In this review, we discuss the potential of host immune inhibitory mechanisms to identify immune correlates of protective immunity to understand the mystery of asymptomatic infections, sterile cure and post kala azar dermal leishmaniasis.
Keywords: Visceral leishmaniasis, Immune response, Asymptomatic infection, Sterile cure, Post kala-azar dermal leishmaniasis
|How to cite this article:|
Tiwari N, Kishore D, Bajpai S, Singh RK. Visceral leishmaniasis: An immunological viewpoint on asymptomatic infections and post kala azar dermal leishmaniasis. Asian Pac J Trop Med 2018;11:98-108
|How to cite this URL:|
Tiwari N, Kishore D, Bajpai S, Singh RK. Visceral leishmaniasis: An immunological viewpoint on asymptomatic infections and post kala azar dermal leishmaniasis. Asian Pac J Trop Med [serial online] 2018 [cited 2019 Jul 21];11:98-108. Available from: http://www.apjtm.org/text.asp?2018/11/2/98/225016
Foundation project: A part of the grant received from Science and Engineering Research Board, Department of Science and Technology, New Delhi (SB/SO/HS/0091/2013) was utilized in this work.
| 1. Introduction|| |
A protozoan parasite of the genus Leishmania causes a vectorborne disease leishmaniasis, which is prevalent in more than 98 countries at present. Out of 53 described species of Leishmania parasites, 20 are known to cause human pathogenesis that are spread by approximately 30 species of sand flies. The infection of Leishmania in the human may produce three discrete clinical manifestations, i.e., cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL), which are caused by different species and differ in their immunopathology, degree of morbidity and mortality.
VL (kala-azar), caused by Leishmania donovani (L. donovani), is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The estimates indicate about 100 000 cases per year that includes 15 000 reported cases. Out of five VL-affected countries (India, Bangladesh, Nepal, Thailand, and Bhutan of this region), India contributes more than 80% of reported cases whereas in Bhutan and Thailand reports are sporadic. In India, Bihar is the most VL-endemic state, with 90% of the Indian VL cases reported there. Leishmania infantum causes VL in North Africa and Southern Europe while in Latin America the VL causing species is Leishmania chagasi and L.donovani is responsible for VL infections in East Africa.
The goal of elimination of VL from the Indian subcontinent by 2015, as set by World Health Assembly in 2005, has now passed. The set target was to bring one VL case in 10 000 individuals among endemic residents at district level, which is not achieved yet. The WHO road-map target has now set a new goal for regional elimination of VL by or before 2020 from five countries India, Nepal, Bangladesh, Bhutan and Thailand. The challenges in parasites and sand fly control measures along with changing epidemiology of VL in disease endemic countries are major threat in its elimination,,. Further, the migration of infected but asymptomatic individuals from endemic regions has resulted in new infections in non-endemic regions. Recently new cases have been reported from mountain areas in Nepal, which has almost eliminated the disease in Terai regions. In southern Sudan, population migration to the area of greater sand fly exposure has led to an increased incidence of VL cases. In India, new cases have been reported from various non-endemic regions, too. The risk of disease transmission is further compounded by dormant parasitic reservoirs responsible for the resurgence of VL in both endemic and non-endemic regions.
| 2. Control measures and their limitations|| |
Soon after identification of Leishmania parasite in 1901, the pentavalent antimony compounds were used to treat VL cases, which were also considered as true antileishmanials,. VL was controlled by such treatment until antimony resistant parasites appeared in early 1980s in Indian subcontinent, and treatment failure is now reached up to 65% in few endemic areas,,. In early 1980s, an antimicrobial drug pentamidine (1983) was used in antimony refractory cases and cured 99% of patients initially however, within 2 decades its efficacy declined to approximately 70% patients leading to its abandonment in VL treatment,,.
During 1990–1998, an antifungal amphotericin B and an anticancer (miltefosine) became the first choice drugs to treat VL cases,. At present, single dose AmBisome, a liposomal formulation of amphotericin B, is a drug of choice for VL elimination programme and approved by WHO as preventive measure. At present, these drugs are effective with satisfactory results but reports indicate that the parasites are developing of resistance to amphotericin B, and miltefosine,,,.
In spite of significant knowledge on host-parasite relationships and immunobiology, the accurate parameters of protective immunity are not identified; therefore, a vaccine candidate either preventive or prophylactic is far from reality. Both, native and recombinant vaccine antigens such as gp63, gp46, m2, PSA2, TSA, LACK, LmsT1, and Leish111f, have been evaluated but all have failed to achieve long lasting protective immunity,,,. Among all vaccination approaches the live attenuated parasites have shown to produce the required magnitude of protective immunity in various animal models but clinical trials on humans are awaited,.
| 3. Immunobiology of VL: Immune response and lacunae in the knowledge|| |
All type of Leishmania infections begin with the entry of flagellated metacyclic promastigotes form of parasite in the blood stream, which are transmitted by female sand flies. Leishmania has developed various evasion strategies to counter innate and adaptive immunities for its survival and proliferation in mammalian host but precise mechanisms are not identified. After infection, neutrophils and macrophages are quickly recruited under the skin, at the site of bite, to protect host in early stages,. Leishmania induces phagocytosis without eliciting oxidative burst in macrophages, which is followed by induction of disease exacerbating anti-inflammatory cytokines [like interleukin (IL-4), IL-10 and transforming growth factor (TGF)-β] production,,. Leishmania also down regulates a divalent cationic transporter; natural resistance associated with macrophage protein 1 (NRAMP1) now referred as solute carrier family 11a member 1 (SLC11A1), on phagolysosomes for its proliferation in macrophages. This transporter creates Fe2+, Mn2+, and Zn2+ deprived environment inside the phagolysosomes by pumping them out as a host protective strategy that are required for growth and proliferation.
Adaptive immunity in VL is characterized by mixed Th1/2 immune responses. The parasite alters the phenotypic differentiation of antigen experienced CD4+ T cells into Th2 phenotype. The Th2 cytokines like IL-4 and IL-10 are known to be responsible for disease outcome whereas Th1 cytokines like IL-2, IL-12, and interferon-γ (IFN-γ ) confer disease resistance,,. Some recent studies indicate the role of IL-17, IL-21, IL-22, and IL-27 in disease resistance and susceptibility,. Recently it has been observed that skin-resident CD4+ T cells also play a significant role in parasite clearance during early days of infection in a nitric oxide and reactive oxygen species dependent manner. Interestingly, these CD4+ T cells are also long lived and establish residence in the absence of persistent parasites, similar to central memory T cells hence may play substantial role in vaccine induced immunity but requires more studies. The present immunological information seems to be inadequate to classify and explain various clinical and subclinical states of VL infections. This necessitates identification of new correlates of host immunity for a better understanding on sterile cure, post kala-azar dermal leishmaniasis (PKDL) progression, asymptomatic infections and development of a vaccine candidate.
| 4. Immune inhibitory mechanisms: Perspective of immune tolerance in VL|| |
The immune suppression mechanisms are mainly controlled by two types of inhibitory processes (extrinsic and intrinsic), which are mediated by various pro-and anti-inflammatory cytokines production by activated cells. The extrinsic mechanisms involve recruitment of specialized effector cells such as T regulatory cells (Tregs), which produce inhibitory (anti-inflammatory) cytokines (mainly IL-10) to control activated immune response. The intrinsic mechanisms involve expression of specialized receptors on the surface of innate and adaptive immune cells that deliver inhibitory signals via immunoreceptor tyrosine based inhibitory motifs and non-inhibitory motifs after ligand interaction,. Some of these receptors are PD1, CTLA4, BTLA, LAG3, TIM3, CD47, CD200, CD200R, and CD300, which are known to regulate activation states of both, myeloid and lymphoid cells, and have been found to alter pathophysiology of various infectious and non-infectious diseases,,. These receptors after interaction with their ligands activate Src homology region 2 domain-containing phosphatase-1/2 (SHP1/2) and Src homology region 2 domain-containing inositol phosphatases (SHIP), which dephosphorylate activated proximal and distal kinases of activated cells in order to control their functions,.
In VL, the production of IL-10 has been found to be correlated with VL pathogenesis but its source is not identified, yet. Surprisingly the increased IL-10 production in VL is not associated with Tregs, the cells of extrinsic mechanism. Very little knowledge is available on the role of intrinsic mechanisms of immune inhibition on macrophages dysfunction, poor antigen presentation by dendritic cells, CD4+ T cells phenotypic differentiation and functionality in VL. The literature indicates (discussed later in this review) that these mechanisms either lead to total or partial exhaustion of activated T cells through specific receptor ligand mediated effector mechanisms. Activation of these receptors has been linked to alter phenotypic differentiation of activated T cells, inhibition of their multifunctional abilities and production of inhibitory cytokines. The determinants of host immunity that alter or determine active phenotypes of antigen experienced CD4+ T cells are not known. Therefore, delineation of intrinsic mechanisms may help to understand immunobiology of VL pathogenesis and to identify parameters of protective immunity. Based on nature of Leishmania parasite and its dominance over host immunity, we understand that the CD200, CD200R and CD300 linked mechanisms may help to delineate the mechanisms associated with various clinical and subclinical states of VL.
CD200 is a type Ia membrane protein with extracellular immunoglobulin superfamily domain, a single transmembrane region and a short cytoplasmic tail with no signalling motifs. It is widely expressed on myeloid, lymphoid cell lineage and non-immune cells as well. Its receptor, CD200R, is differentially expressed on T cells, B cells, NK cells and cells of myeloid origin,,. The 67 aa long cytoplasmic tail of CD200R has three tyrosine residues of which the distal residue is located in a phosphotyrosine binding domain recognition motif, NPxy. After phosphorylation by Src kinases, NPxy binds to phosphotyrosine binding domain containing downstream of tyrosine kinase 1 and 2 adaptor protein, which results in further recruitment of Src homology region 2 domain containing ionositol phosphate and RAS p21 protein activator 1. The Src homology region 2 domain-containing inositol phosphatase eventually dephosphorylate phosphotydylionositol 3 phosphate and RAS p21 protein activator 1 leading to the deactivation of Ras related kinases, which eventually inhibit Akt pathway related effector molecules required in cellular growth, differentiation and function.
The CD200–CD200R interaction may negatively or positively regulate activated cells towards an auto-regulatory process for their self-inactivation by controlling both, pro-and anti-inflammatory cytokines production, and also maintain required balance between immune response and immune tolerance,. Studies suggest that CD200 down regulates macrophage effector functions, inhibit antigen specific T cell response in various tumors,,. Recently, Rygiel et al. observed that the external and internal tumor antigens up regulate CD200–CD200R axis, which result in altered immune tolerance and increased frequency of Treg/Th17 cells. Further, the CD200 blockade results in antigen specific Th1 response and significant decrease in Tregs in various cancers that further suggest an important role of this axis in regulation of T cell functions,,. Although there is not much data on its role in pathogenic infections, studies reveal that this axis controls exacerbated inflammation during viral and bacterial infections and the lack of CD200 signal exacerbate disease pathology,,. In herpes (Kaposi's sarcoma-associated herpesvirus) infection CD200 and its viral analogue OX2 inhibit antigen specific T cells, IFN-γ production and target killing ability of the cytolytic granule component, CD107a, to cell surface along with inhibited Akt phosphorylation. During influenza infection, CD200-/- mice results in severe pathology in spite of adequate immune response that was dependent on the presence of T cells since T cell depletion yielded in resolution of pathological symptoms,. Although these studies did not analyze the functional characteristics of antigen experienced T cells, this suggested the loss of protective ability and acquisition of disease promoting ability of T cells. Further, in herpes simplex virus-I infection CD200 blockade suppressed Th1 type response and up regulated Tregs production suggesting the role of axis in T cell function and differentiation. The only study in Leishmania by Cortez et al. has shown that Leishmania amazonensis induces CD200 expression and suppresses macrophage activation via inducible nitric oxide synthase inhibition that eventually leads to increased parasite growth.
Similarly, the other immune inhibitory receptor, CD300, is known to play significant role in various diseases like cancer and sepsis. The human CD300 family is a family of seven membrane receptors and all of them have an extracellular immunoglobulin V like domain. The inhibitory receptors of this family have long immunoreceptor tyrosine-based inhibition motif for their adaptor proteins. These receptors are expressed on both lymphoid and myeloid lineages. CD300 recognizes phosphatidylserine and phosphatidylethanolamine, exposed on the outer leaflet of dead and activated cells, and delivers inhibitory signals to activated cells through SHP1/2 phosphatases. The role of CD300 receptors are not studied in any parasitic diseases yet. Since Leishmania expresses plenty of phosphatidylserine in the outer leaflet of cytoplasmic membrane, the same molecule to which CD300 is known to bind, it would be worthy to explore CD300 role in VL pathogenesis. Therefore, on these backgrounds it would be worthy to delineate their role in VL pathogenesis in the context to existing challenges in VL such as asymptomatic infections, sterile cure and PKDL progression.
| 5. Challenges in VL elimination|| |
The existing lacunae in the knowledge on VL affected subjects are a serious threat in its elimination. The drug control regime is very limited and also believed to be inefficient to produce sterile cure as successfully cured patients have shown to carry live parasites and remain seropositive for prolonged year,. The immunological information is not adequate to define parameters of protective immunity that is why a vaccine candidate is far from reality.
In VL the sterile protection or cure is a debatable issue, but there are plenty of reasons and evidences to believe that sterile protection can be achieved. After successful treatment a person develop substantial protective immunity and considered to be protected from reinfection or relapse that suggest such possibilities. In all endemic regions there are asymptomatic individuals who remain seropositive for many years without developing disease. It is not well understood whether they carry live parasites and help in disease transmission. Further, a majority of these individuals turn to seronegative in due course of time, albeit they live in endemic regions suggesting a possibility of resistant development in such individuals. The identification of immune correlates of disease resistance and susceptible will also help to monitor VL progression in non-endemic regions.
Along with altered functionality and exhaustion, the proliferation of T cells (CD4 and CD8) is also highly compromised during active VL, though the associated mechanisms are not well known. An ideal magnitude of immune response is required for generation and proliferation of effector T and B cells, which eventually leads to effective establishment of memory T and B cells. Although it is practically difficult to establish the magnitude of required immune response for sterile protection, it is achievable as this has already been established in malaria infection. The factors to establish protective threshold of immune response, either host or parasite, are needed to be identified in Leishmania infections too. Certainly, there are some factors in VL as well, which protect few endemic individuals from infections as evidenced by conversion of their seropositive to seronegative status as well as prolong onset of disease. Nevertheless, some seropositive endemic individuals develop VL, which may be due to failure of protective immune response in presence of excessive parasite load after successive sand fly bites or other unknown factors. Substantial knowledge on the mechanisms by which Leishmania modulates host immune response, as discussed above, in its favour is available but how it manipulates host immune tolerance mechanisms is still unknown. It has already been established that for a stable protection and long term immunity a perfect balance of immune response and immune tolerance mechanisms is essentially required. It is quite possible that Leishmania induces immune tolerance mechanisms to control antigen experienced T and B cells phenotypic differentiation, proliferation and functions soon after infection. Since our knowledge on these mechanisms in VL is very limited therefore, their understanding may provide an insight and can divulge to identify correlates of protective immunity.
The nature of VL pathogenesis is one of the biggest hurdles in finding such parameters but a comparative and comprehensive study on immune tolerance and immune response mechanisms in various clinical and subclinical states of VL may provide such information. We have tried to summarize the possible pathologies in VL affected subjects, which are based on both, and scientific evidences [Table 1].
|Table 1: A perceptible classification of various categories of L. donovani affected individuals in disease endemic regions.|
Click here to view
| 6. Mysteries of VL|| |
6.1. Mystery of asymptomatic infections: Do they carry live parasites?
Asymptomatic infections are those who remain seropositive for many (up to 10–12) years without developing into active disease,, and are more prevalent in VL endemic regions. Of these, 10%–20% of healthy endemic population without past history of VL, show seroreactivity with parasitic antigens, and 20%–25% display polymerase chain reaction positivity in Indian subcontinent. Reports from the other endemic regions also confirm the existence of parasitic DNA in all VL causing species in asymptomatic individuals,.,. Although majority of seropositive asymptomatic individuals convert in seronegative status in due course of time, these are known to contribute in disease progression in non-endemic regions,,,. A report from Bangladesh also confirms that approximately 80% of asymptomatic individual contribute in disease transmission as compared to 8%–10% of VL and PKDL.
The conversions of asymptomatic infections to symptomatic VL also indicate the persistence of parasites in these individuals albeit various host and parasite factors may be additional contributors. The reported ratios of conversion are 4:1 in Kenya and Bangladesh, 1.2:4 to 11:1 in Africa between 6.5:1 to 18.5:1 in brazil and 8.9:1 in India and Nepal,. Interestingly, these numbers clearly suggest that a large number of individuals do not turn into active disease and also turn to seronegative status, which is suggestive of protective immune response generation in such individuals.
The factors that play role in transition of asymptomatic to symptomatic pathologies are largely unknown. Few studies indicate that host genetic association and development of clinical symptoms is linked to NRAMP1, TNF- α, IL–4 and interferon-γ receptor (IFNGR1), TGFß 1, IL–8, C-X-C chemokine receptor 1 (CXCR1) and C-X-C chemokine receptor 2 (CXCR2), IL–2R β, Delta-like1 (DLL1), and mannan binding lectin genes.
The extrinsic factors such as host immune compromised status, age and nutritional status are also considered to play an important role in asymptomatic to symptomatic conversion,.
In addition, increased serum levels of IFN-γ , C reactive protein, nitric oxide, and IL-12 has also been found to provide resistant to asymptomatic subjects,. This indicates that disease resistant endemic subjects develop protective immune response after sand fly bite that protects them from pathogenesis. On contrary, the increased levels of IL-10, IL-4 and TNF-α in these cases have been found to be associated with the development of disease susceptibility suggesting a breach in protective immune response due to various unidentified host and parasitic factors,. However, these findings are more or less similar to VL immunology hence cannot be used to frame a clear picture of parameters of protective immunity in asymptomatic subjects.
6.2. Disease susceptible asymptomatic infections: The carrier of parasites
Among asymptomatic infections, the first group comprises those individuals who remain seropositive for many years (>1 years) without developing clinical symptoms. None of the study, so far, either provides or discusses the reason of persistent antibody in these individuals. Along with protective immune response, it seems that these individuals may either have strong pool of long lived memory B cells, which constantly secret antibodies, or they get constant Leishmania exposure but do not develop VL due to protective immune response,. In these cases, the strong generation of cell mediated immunity, sufficient number of activated T cells with retained multi-functionality to produce pro-inflammatory cytokines, specific B cells population, and a greater pool of memory T and B cells protect them from future infections and VL pathogenesis. These individuals clearly suggest that they produce required threshold of immune response, which is sufficient to counter a fresh bite of infected sand flies and prevent them from active disease for a longer duration. Probably they also do not carry live parasite due to strong immune response generation that efficiently eliminates invading infections but they show seropositivity probably because of long lived plasma cells. However, the possibility of live parasites in such cases cannot be overruled due to constant seropositive nature of such individuals. A detailed and comparative study on immune tolerance and immune response mechanisms in these individuals can provide the correlates of protective immunity, which can be further used to engineer a vaccine candidate or can be used to discriminate between clinical and subclinical states of VL.
The second group comprise those seropositive asymptomatic individuals who remain positive for certain period but develop VL in due course of time (<1 years). Studies indicate that 1.5%–23% of asymptomatic infections develop sign and symptoms of VL within 1 year,,,,,,,. The rate of asymptomatic to symptomatic VL conversion has been linked to amount of antibodies in their blood. The individuals with high (up to 40 times) antibody titre are more susceptible for asymptomatic to symptomatic conversion,,. These cases further provide evidence that they carry live parasites throughout the period before appearance of active disease. The detection of parasitic DNA in about 25% of such asymptomatic carriers who had no past history of VL further confirm such possibility,,. The low titre individuals suggest the possible existence of required immune response threshold that protects them for a quite long time from active disease as compared to those with high antibody titre. A breach in immune status either by parasitic (successive sand fly bite as they live in endemic zone) or their compromised immune status (due to other infections or poor nutritional status) may lead to appearance of VL symptoms,. Therefore, such asymptomatic cases may be classified as true asymptomatic carriers who may transmit disease. This may be due to the breach in protective immune response threshold by either parasitic or compromised host immunity. Although it will be very hard to monitor these cases, such individuals may help in identification of host immune factors responsible in disease resistance and susceptibility.
6.3. Disease resistant asymptomatic infections: An array of hope to identify parameters of sterile cure and protective immunity
The third asymptomatic category comprises those individuals of endemic regions who turn seronegative in due course of time. The spontaneous conversion of seropositive into seronegative status varies from 33%–86%,,,. However, within a year among individuals with high antibody titre, this conversion is as low as 6.3% as observed in a study conducted in Bangladesh. These individuals provide further concrete evidence that there is a requirement of threshold immune response level to protect host from parasite. These individuals probably achieve desired threshold of immune response after the parasite exposure, which protect them from future disease onset by effectively clearing live parasites. The probable reasons of their seronegativity can be understood as they either do not encounter Leishmania infected sand fly bite (not possible as they live in endemic region) or host immunity efficiently/immediately eliminates infection. They probably do not carry live parasites, and can be considered as true resistant cases. Such a condition also support that sterile cure is achievable in VL. In addition, successfully cured patients are supposed to be resistant for recurrence/reinfection, and very small percentage (about 4%) of treated patients relapse that further confirms the existence of protective immune response post cure,,. The immune biology of resistant asymptomatic infections may be associated with strong cell mediated immunity, a large repertoire of memory T and B cells along with short lived plasma cells (they do not show seropositivity for a longer period). Focussed studies on these individuals may reveal the sought parameters of protective immunity to develop a prophylactic vaccine candidate.
6.4. Active VL individuals: The sufferers
The fourth category of VL comprises those individuals who develop VL soon after infection or in due course of time. However, the current available tools are not adequate to predict the exact time line for development of VL symptoms in Leishmania infected individuals. They harbour live parasites and require medical intervention. The possible reasons of VL pathogenesis may be the insufficient generation of cell mediated immunity at the time of infection due to their compromised immune status or other unknown factors. Various socioeconomic, environmental factors and their genetic integrity may also be associated factors for breach in the immunity as discussed above,,. However, the early onset of disease in some (chronic VL cases) endemic individuals, as compared to seropositive asymptomatics, further suggests the possibility of protective host traits and hence requires more studies.
6.5. PKDL ambiguity
PKDL is an unusual presentation of VL causing Leishmania species, which is characterized by discrete clinical symptoms like hypo pigmented macular, popular and nodular lesions with presence of live parasites. Out of three VL causing species, L. donovani infection results in more PKDL cases followed by Leishmania infantum. PKDL cases are very rare in Leishmania chagasi infections, which suggest parasitic role in PKDL development albeit the host or parasitic parameters responsible in changing the properties of parasite, i.e., viscerotropic to dermatotropic are not identified, yet. An estimated 30% of Leishmania affected individuals develop signs and symptoms of PKDL without developing VL or any past history of VL,. Interestingly, about 15% of successfully cured VL cases also develop PKDL symptoms within a year in Indian subcontinent,, whereas in Sudan the conversion rate is 50%–60%. Considerable cases of PKDL resolve spontaneously suggesting protective immune response. The increasing incidence of PKDL in endemic regions, post amphotericin B era including miltefosine, warrants more studies on its etiological factors,,. In addition, in certain disease endemic regions PKDL cases (versus VL) are gradually increasing as the disease may develop even after 10 years post cure. A recent study also reports increased numbers of PKDL case in Indian states of Bihar, West Bengal and eastern Uttar Pradesh. So far the role of PKDL in disease transmission is not clear but xenodiagnostic and culture studies have demonstrated that these individuals carry live parasites and therefore may be involved in spread of the disease,,,.
The immunological presentation of PKDL is similar to VL but dissociated between the visceral organs and skin. However, dominance of Th2 cytokines especially IL-10 over Th1 is more prominent in PKDL. The host or parasite factors that enable Leishmania to appear and survive in the skin are not clear. As PKDL lesions occur in sun exposed areas, the compromised dendritic cells under UV light have been linked to Th2 type immune response,. The increased level of IL-10 secreting CD3+ CD8+ Tregs and IL-10, TGF-β by keratinocytes has also been linked to PKDL. The clinical manifestation of PKDL is also associated to host cell mediated immunity as in acute PKDL it is way higher than chronic cases suggesting its protective role in PKDL development. Few studies also indicate the role of M2 type macrophages, metalloproteinases, and Th17 response. in development and persistent of PKDL. However, it is still not clear that why there is so much variability in PKDL development among asymptomatic infections and cured VL cases; moreover, the current tools are insufficient to understand this puzzle.
| 7. Concluding remarks|| |
We have made significant progress in VL diagnostic, epidemiology, and understanding on the role of innate immune mechanisms in VL pathogenesis such as a test of cure (sterile vs. nonsterile), quantitative test to measure parasites load in different VL cases (asymptomatic, PKDL, fresh and cured cases), correlates of protective immunity and PKDL development, and prevention is still required. On the basis of current knowledge and available tools, it is very difficult to discriminate individuals among resistant, sterile, asymptomatic, who will develop active disease or develop PKDL or remain asymptomatic or become sterile. Finding such knowledge and identification of parameters of protective immunity will not only help to discriminate such individuals in disease endemic regions but also will help to design new prophylactic control measures and disease monitoring. A comprehensive and comparative study on immunological responses considering all aspects of host immune response and immune tolerance mechanisms of chronic, cured, PKDL and asymptomatic VL infections may help to identify parameters of protective immunity. However, this will be a multi-centric task, therefore focused and collaborative work is required to solve the remaining challenges of visceral leishmaniasis.
Conflict of interest statement
The authors declared that they have no conflict of interest.
A part of the grant received from Science and Engineering Research Board, Department of Science and Technology, New Delhi (SB/SO/HS/0091/2013) was utilized in this work. N.T. is thankful to University Grants Commission (UGC) for research fellowship.
| References|| |
Akhoundi M, Kuhls K, Cannet A, Votypka J, Marty P, Delaunay P, et al. A historical overview of the classification, evolution, and dispersion of Leishmania
parasites and sandflies. PLoS Negl Trop Dis
Poché DM, Grant WE, Wang H-H. Visceral leishmaniasis on the Indian Subcontinent: Modelling the dynamic relationship between vector control schemes and vector life cycles. PLoS Negl Trop Dis
Sundar S. Visceral leishmaniasis. Trop Parasitol
Alexander B, Maroli M. Control of phlebotomine sandflies. Med Vet Entomol
Quinnell RJ, Courtenay O. Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology
Claborn DM. The biology and control of leishmaniasis vectors. J Glob Infect Dis
Alvar J, Aparicio P, Aseffa A, Den Boer M, Canavate C, Dedet JP, et al. The relationship between leishmaniasis and AIDS: The second 10 years. Clin Microbiol Rev
Abubakar A, Ruiz-Postigo JA, Pita J, Lado M, Ben-Ismail R, Argaw D, et al. Visceral leishmaniasis outbreak in South Sudan 2009–2012: Epidemiological assessment and impact of a multisectoral response. PLoS Negl Trop Dis
Kumar A, Vinita R, Thapliyal N, Saxena SR. Kala-azar--a case series from non endemic area, Uttarakhand. J Commun Dis
Ready PD. Epidemiology of visceral leishmaniasis. Clin Epidemiol
Brahmachari U. A new form of cutaneous leishmaniasis-dermal leishmanoid. Indian Med Gaz
Brahmachari U. Chemotherapy of antimonial compounds in kala-azar infection. Part IV. Further observations on the therapeutic values of urea stibamine. Indian J Med Res
Peters W. The treatment of kala-azar--new approaches to an old problem. Indian J Med Res
Thakur CP, Kumar M, Singh SK, Sharma D, Prasad US, Singh RS, et al. Comparison of regimens of treatment with sodium stibogluconate in kala-azar. Br Med J (Clin Res Ed)
Sundar S. Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health
Alvar J, Croft S, Olliaro P. Chemotherapy in the treatment and control of leishmaniasis. Adv Parasitol
Chakravarty J, Sundar S. Drug resistance in leishmaniasis. J Glob Infect Dis
Jha TK. Evaluation of diamidine compound (pentamidine isethionate) in the treatment resistant cases of kala-azar occurring in North Bihar, India. Trans R Soc Trop Med Hyg
Jha SN, Singh NK, Jha TK. Changing response to diamidine compounds in cases of kala-azar unresponsive to antimonial. J Assoc Physicians India
Mishra M, Biswas UK, Jha DN, Khan AB. Amphotericin versus pentamidine in antimony-unresponsive kala-azar. Lancet
Sundar S, Rosenkaimer F, Makharia MK, Goyal AK, Mandal AK, Voss A. Trial of oral miltefosine for visceral leishmaniasis. Lancet
Maintz E-M, Hassan M, Huda MM, Ghosh D, Hossain MS, Alim A, et al. Introducing single dose liposomal amphotericin B for the treatment of visceral leishmaniasis in rural Bangladesh: Feasibility and acceptance to patients and health staff. J Trop Med
Sinha PK, Roddy P, Palma PP, Kociejowski A, Lima MA, Rabi Das VN. Effectiveness and safety of liposomal amphotericin B for visceral leishmaniasis under routine program conditions in Bihar, India. Am J Trop Med Hyg
Burza S, Sinha PK, Mahajan R, Lima MA, Mitra G, Verma N, et al. Risk factors for visceral leishmaniasis relapse in immunocompetent patients following treatment with 20 mg/kg liposomal amphotericin B (Ambisome) in Bihar, India. PLoS Negl Trop Dis
Burza S, Nabi E, Mahajan R, Mitra G, Lima MA. One-year follow-up of immunocompetent male patients treated with miltefosine for primary visceral leishmaniasis in Bihar, India: Clin Infect Dis
Garcia-Hernandez R, Manzano JI, Castanys S, Gamarro F. Leishmania donovani
develops resistance to drug combinations. PLoS Negl Trop Dis
Rai K, Cuypers B, Bhattarai NR, Uranw S, Berg M, Ostyn B. Relapse after treatment with miltefosine for visceral leishmaniasis is associated with increased infectivity of the infecting Leishmania donovani
Rijal S, Ostyn B, Uranw S, Rai K, Bhattarai NR, Dorlo TP, et al. Increasing failure of miltefosine in the treatment of Kala-azar in Nepal and the potential role of parasite drug resistance, reinfection, or noncompliance. Clin Infect Dis
Webb JR, Kaufmann D, Campos-Neto A, Reed SG. Molecular cloning of a novel protein antigen of Leishmania major
that elicits a potent immune response in experimental murine leishmaniasis. J Immunol
Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YA, Reed SG. Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major
infection in susceptible BALB/c mice. Infect Immun
Nagill R, Kaur S. Vaccine candidates for leishmaniasis: A review. Int Immunopharmacol
Singh B, Sundar S. Leishmaniasis: Vaccine candidates and perspectives. Vaccine
Dey R, Dagur PK, Selvapandiyan A, McCoy JP, Salotra P, Duncan R, et al. Live attenuated Leishmania donovani
p27 gene knockout parasites are non-pathogenic and elicit long term protective immunity in BALB/c mice. J Immunol
Gannavaram S, Dey R, Avishek K, Selvapandiyan A, Salotra P, Nakhasi HL. Biomarkers of safety and immune protection for genetically modified live attenuated Leishmania
vaccines against visceral leishmaniasis – Discovery and implications. Front Immunol
Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL. Vector transmission of Leishmania
abrogates vaccine-induced protective immunity. PLoS Pathog
Thalhofer CJ, Chen Y, Sudan B, Love-Homan L, Wilson ME. Leukocytes infiltrate the skin and draining lymph nodes in response to the protozoan Leishmania infantum
chagasi. Infect Immun
Brittingham A, Morrison CJ, McMaster WR, McGwire BS, Chang KP, Mosser DM. Role of the Leishmania
surface protease gp63 in complement fixation, cell adhesion, and resistance to complement- mediated lysis. J Immunol
Gupta G, Oghumu S, Satoskar AR. Mechanisms of immune evasion in leishmaniasis. Adv Appl Microbiol
Saha S, Mondal S, Ravindran R, Bhowmick S, Modak D, Mallick S, et al. IL-10-and TGF-beta-mediated susceptibility in kala-azar and post-kala-azar dermal leishmaniasis: The significance of amphotericin B in the control of Leishmania donovani
infection in India. J Immunol
Singh N, Bajpai S, Kumar V, Gour JK, Singh RK. Identification and functional characterization of Leishmania donovani
secretory peroxidase: Delineating its role in NRAMP1 regulation. PLoS One
Sundar S, Reed SG, Sharma S, Mehrotra A, Murray HW. Circulating T helper 1 (Th1) cell-and Th2 cell-associated cytokines in Indian patients with visceral leishmaniasis. Am J Trop Med Hyg
Nylen S, Sacks D. Interleukin-10 and the pathogenesis of human visceral leishmaniasis. Trends Immunol
Ansari NA, Kumar R, Gautam S, Nylen S, Singh OP, Sundar S, et al. IL-27 and IL-21 are associated with T cell IL-10 responses in human visceral leishmaniasis. J Immunol
Pitta MGR, Romano A, Cabantous S, Henri S, Hammad A, Kouriba B, et al. IL-17 and IL-22 are associated with protection against human kala azar caused by Leishmania donovani. J Clin Invest
Glennie ND, Volk SW, Scott P. Skin-resident CD4+ T cells protect against Leishmania major
by recruiting and activating inflammatory monocytes. PLoS Pathog
Rygiel TP, Karnam G, Goverse G, van der Marel AP, Greuter MJ, van Schaarenburg RA, et al. CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor. Oncogene
Kamphorst AO, Ahmed R. Manipulating the PD-1 pathway to improve immunity. Curr Opin Immunol
Caserta S, Nausch N, Sawtell A, Drummond R, Barr T, Macdonald AS, et al. Chronic infection drives expression of the inhibitory receptor CD200R, and its ligand CD200, by mouse and human CD4 T cells. PLoS One
Norde WJ, Hobo W, van der Voort R, Dolstra H. Coinhibitory molecules in hematologic malignancies: Targets for therapeutic intervention. Blood
Gannavaram S, Bhattacharya P, Ismail N, Kaul A, Singh R, Nakhasi HL. Modulation of innate immune mechanisms to enhance Leishmania
vaccine-induced immunity: Role of coinhibitory molecules. Front Immunol
Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol
Nylén S, Maurya R, Eidsmo L, Manandhar KD, Sundar S, Sacks D. Splenic accumulation of IL-10
mRNA in T cells distinct from CD4(+) CD25(+) (Foxp3) regulatory T cells in human visceral leishmaniasis. J Exp Med
Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol
Rijkers ES, de Ruiter T, Buitenhuis M, Veninga H, Hoek RM, Meyaard L. Ligation of CD200R by CD200 is not required for normal murine myelopoiesis. Eur J Haematol
Gorczynski RM. CD200:CD200R-mediated regulation of immunity. ISRN Immunol
Mihrshahi R, Barclay AN, Brown MH. Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. J Immunol
Jenmalm MC, Cherwinski H, Bowman EP, Phillips JH, Sedgwick JD. Regulation of myeloid cell function through the CD200 receptor. J Immunol
Minas K, Liversidge J. Is the CD200/CD200 receptor interaction more than just a myeloid cell inhibitory signal? Crit Rev Immunol
Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science
Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH, et al. Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity
Gorczynski RM, Lee L, Boudakov I. Augmented Induction of CD4+
Treg using monoclonal antibodies to CD200R. Transplantation
McWhirter JR, Kretz-Rommel A, Saven A, Maruyama T, Potter KN, Mockridge CI, et al. Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation. Proc Natl Acad Sci U S A
Siva A, Xin H, Qin F, Oltean D, Bowdish KS, Kretz-Rommel A. Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200. Cancer Immunol Immunother
Pallasch CP, Ulbrich S, Brinker R, Hallek M, Uger RA, Wendtner CM. Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leuk Res
Rygiel TP, Rijkers ES, de Ruiter T, Stolte EH, van der Valk M, Rimmelzwaan GF, et al. Lack of CD200 enhances pathological T cell responses during influenza infection. J Immunol
Mukhopadhyay S, Pluddemann A, Hoe JC, Williams KJ, Varin A, Makepeace K, et al. Immune inhibitory ligand CD200 induction by TLRs and NLRs limits macrophage activation to protect the host from meningococcal septicemia. Cell Host Microbe
Misstear K, Chanas SA, Rezaee SA, Colman R, Quinn LL, Long HM, et al. Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200. J Virol
Snelgrove RJ, Goulding J, Didierlaurent AM, Lyonga D, Vekaria S, Edwards L, et al. A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nat Immunol
Sarangi PP, Woo SR, Rouse BT. Control of viral immunoinflammatory lesions by manipulating CD200:CD200 receptor interaction. Clin Immunol
Cortez M, Huynh C, Fernandes MC, Kennedy KA, Aderem A, Andrews NW. Leishmania
promotes its own virulence by inducing expression of the host immune inhibitory ligand CD200. Cell Host Microbe
Zenarruzabeitia O, Vitallé J, García-Obregón S, Astigarraga I, Eguizabal C, Santos S, et al. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults. Sci Rep
Simhadri VR, Andersen JF, Calvo E, Choi S-C, Coligan JE, Borrego F. Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells. Blood
Martinez-Barriocanal A, Comas-Casellas E, Schwartz S, Jr. Martin M, Sayos J. CD300 heterocomplexes, a new and family-restricted mechanism for myeloid cell signaling regulation. J Biol Chem
Wanderley JL, Moreira ME, Benjamin A, Bonomo AC, Barcinski MA. Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis
in mammalian hosts. J Immunol
Dereure J, Duong Thanh H, Lavabre-Bertrand T, Cartron G, Bastides F, Richard-Lenoble D, et al. Visceral leishmaniasis. Persistence of parasites in lymph nodes after clinical cure. J Infect
Mukhopadhyay D, Dalton JE, Kaye PM, Chatterjee M. Post kala-azar dermal leishmaniasis: An unresolved mystery. Trends Parasitol
Kalia V, Sarkar S, Gourley TS, Rouse BT, Ahmed R. Differentiation of memory B and T cells. Curr Opin Immunol
Van Braeckel-Budimir N, Harty JT. CD8 T-cell-mediated protection against liver-stage malaria: Lessons from a mouse model. Front Microbiol
Vaine CA, Soberman RJ. The CD200-CD200R1 inhibitory signaling pathway: Immune regulation and host-pathogen interactions. Adv Immunol
Gidwani K, Kumar R, Rai M, Sundar S. Longitudinal seroepidemiologic study of visceral leishmaniasis in hyperendemic regions of Bihar, India. Am J Trop Med Hyg
Gidwani K, Picado A, Ostyn B, Singh SP, Kumar R, Khanal B, et al. Persistence of Leishmania donovani
antibodies in past visceral leishmaniasis cases in India. Clin Vaccine Immunol
Hirve S, Boelaert M, Matlashewski G, Mondal D, Arana B, Kroeger A, et al. Transmission dynamics of visceral leishmaniasis in the Indian Subcontinent – A systematic literature review. PLoS Negl Trop Dis
Srivastava P, Gidwani K, Picado A, Van der Auwera G, Tiwary P, Ostyn B, et al. Molecular and serological markers of Leishmania donovani
infection in healthy individuals from endemic areas of Bihar, India. Trop Med Int Health
Schaefer KU, Kurtzhals JA, Gachihi GS, Muller AS, Kager PA. A prospective sero-epidemiological study of visceral leishmaniasis in Baringo District, Rift Valley Province, Kenya. Trans R Soc Trop Med Hyg
Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, et al. Development of a species-specific PCR assay for detection of Leishmania donovani
in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol
Costa CH, Stewart JM, Gomes RB, Garcez LM, Ramos PK, Bozza M, et al. Asymptomatic human carriers of Leishmania chagasi. Am J Trop Med Hyg
Alborzi A, Rasouli M, Shamsizadeh A. Leishmania
tropica-isolated patient with visceral leishmaniasis in southern Iran. Am J Trop Med Hyg
Ostyn B, Gidwani K, Khanal B, Picado A, Chappuis F, Singh SP. Incidence of symptomatic and asymptomatic Leishmania donovani
infections in high-endemic foci in India and Nepal: A prospective study. PLoS Negl Trop Dis
Stauch A, Sarkar RR, Picado A, Ostyn B, Sundar S, Rijal S, et al. Visceral leishmaniasis in the Indian subcontinent: Modelling epidemiology and control. PLoS Negl Trop Dis
Saha P, Ganguly S, Chatterjee M, Das SB, Kundu PK, Guha SK, et al. Asymptomatic leishmaniasis in kala-azar endemic areas of Malda district, West Bengal, India. PLoS Negl Trop Dis
McCall L-I, Zhang W-W, Matlashewski G. Determinants for the development of visceral leishmaniasis disease. PLoS Pathog
Bern C, Haque R, Chowdhury R, Ali M, Kurkjian KM, Vaz L, et al. The epidemiology of visceral leishmaniasis and asymptomatic leishmanial infection in a highly endemic Bangladeshi village. Am J Trop Med Hyg
Hailu A, Gramiccia M, Kager PA. Visceral leishmaniasis in Aba-Roba, south-western Ethiopia: prevalence and incidence of active and subclinical infections. Ann Trop Med Parasitol
Evans TG, Teixeira MJ, McAuliffe IT, Vasconcelos I, Vasconcelos AW, Sousa Ade A, et al. Epidemiology of visceral leishmaniasis in northeast Brazil. J Infect Dis
Topno RK, Das VN, Ranjan A, Pandey K, Singh D, Kumar N. Asymptomatic infection with visceral leishmaniasis in a disease-endemic area in bihar. India Am J Trop Med Hyg
Bucheton B, Abel L, Kheir MM, Mirgani A, El-Safi SH, Chevillard C, et al. Genetic control of visceral leishmaniasis in a Sudanese population: candidate gene testing indicates a linkage to the NRAMP1 region. Genes Immun
Karplus TM, Jeronimo SM, Chang H, Helms BK, Burns TL, Murray JC, et al. Association between the tumor necrosis factor locus and the clinical outcome of Leishmania chagasi
infection. Infect Immun
Mohamed HS, Ibrahim ME, Miller EN, Peacock CS, Khalil EA, Cordell HJ, et al. Genetic susceptibility to visceral leishmaniasis in the Sudan: Linkage and association with IL4 and IFNGR1. Genes Immun
Frade AF, Oliveira LC, Costa DL, Costa CH, Aquino D, Van Weyenbergh J, et al. TGFB1 and IL8 gene polymorphisms and susceptibility to visceral leishmaniasis. Infect Genet Evol
Mehrotra S, Fakiola M, Oommen J, Jamieson SE, Mishra A, Sudarshan M, et al. Genetic and functional evaluation of the role of CXCR1 and CXCR2 in susceptibility to visceral leishmaniasis in north-east India. BMC Med Genet
Bucheton B, Argiro L, Chevillard C, Marquet S, Kheir MM, Mergani A, et al. Identification of a novel G245R polymorphism in the IL-2 receptor beta membrane proximal domain associated with human visceral leishmaniasis. Genes Immun
Mehrotra S, Fakiola M, Mishra A, Sudarshan M, Tiwary P, Rani DS, et al. Genetic and functional evaluation of the role of DLL1 in susceptibility to visceral leishmaniasis in India. Infect Genet Evol
Alonso DP, Ferreira AF, Ribolla PE, de Miranda Santos IK, do Socorro Pires e Cruz M, Aecio de Carvalho F, et al. Genotypes of the mannan-binding lectin gene and susceptibility to visceral leishmaniasis and clinical complications. J Infect Dis
Desjeux P, Alvar J. Leishmania/HIV
co-infections: epidemiology in Europe. Ann Trop Med Parasitol
Maciel BL, Lacerda HG, Queiroz JW, Galvao J, Pontes NN, Dimenstein R, et al. Association of nutritional status with the response to infection with Leishmania chagasi. Am J Trop Med Hyg
Ansari NA, Saluja S, Salotra P. Elevated levels of interferon-gamma, interleukin-10, and interleukin-6 during active disease in Indian kala azar. Clin Immunol
Stanley AC, Engwerda CR. Balancing immunity and pathology in visceral leishmaniasis. Immunol Cell Biol
Verma S, Kumar R, Katara GK, Singh LC, Negi NS, Ramesh V, et al. Quantification of parasite load in clinical samples of leishmaniasis patients: IL-10 level correlates with parasite load in visceral leishmaniasis. PLoS One
Pape KA, Taylor JJ, Maul RW, Gearhart PJ, Jenkins MK. Different B cell populations mediate early and late memory during an endogenous immune response. Science
Mondragon-Shem K, Al-Salem WS, Kelly-Hope L, Abdeladhim M, Al-Zahrani MH, Valenzuela JG, et al. Severity of old world cutaneous leishmaniasis is influenced by previous exposure to sandfly bites in Saudi Arabia. PLoS Negl Trop Dis
Koirala S, Karki P, Das ML, Parija SC, Karki BM. Epidemiological study of kala-azar by direct agglutination test in two rural communities of eastern Nepal. Trop Med Int Health
Das VN, Siddiqui NA, Verma RB, Topno RK, Singh D, Das S, et al. Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: A challenge to kala-azar elimination programmes. Trans R Soc Trop Med Hyg
Hasker E, Malaviya P, Gidwani K, Picado A, Ostyn B, Kansal S. Strong association between serological status and probability of progression to clinical visceral leishmaniasis in prospective cohort studies in India and Nepal. PLoS Negl Trop Dis
Vallur AC, Duthie MS, Reinhart C, Tutterrow Y, Hamano S, Bhaskar KR, et al. Biomarkers for intracellular pathogens: establishing tools as vaccine and therapeutic endpoints for visceral leishmaniasis. Clin Microbiol Infect
Sudarshan M, Singh T, Singh AK, Chourasia A, Singh B, Wilson ME, et al. Quantitative PCR in epidemiology for early detection of visceral leishmaniasis cases in India. PLoS Negl Trop Dis
Alborzi A, Pourabbas B, Shahian F, Mardaneh J, Pouladfar GR, Ziyaeyan M. Detection of Leishmania infantum
kinetoplast DNA in the whole blood of asymptomatic individuals by PCR-ELISA and comparison with other infection markers in endemic areas, southern Iran. Am J Trop Med Hyg
van Griensven J, Carrillo E, Lopez-Velez R, Lynen L, Moreno J. Leishmaniasis in immunosuppressed individuals. Clin Microbiol Infect
Hasker E, Kansal S, Malaviya P, Gidwani K, Picado A, Singh RP, et al. Latent infection with Leishmania donovani
in highly endemic villages in Bihar, India. PLoS Negl Trop Dis
Bimal S, Das VN, Sinha PK, Gupta AK, Verma N, Ranjan A, et al. Usefulness of the direct agglutination test in the early detection of subclinical Leishmania donovani
infection: A community-based study. Ann Trop Med Parasitol
Burza S, Mahajan R, Sinha PK, Griensven J, Pandey K, Lima MA. Visceral leishmaniasis and HIV co-infection in Bihar, India: Long-term effectiveness and treatment outcomes with liposomal amphotericin B (AmBisome). PLoS Negl Trop Dis
Alvar J, Yactayo S, Bern C. Leishmaniasis and poverty. Trends Parasitol
Sheets D, Mubayi A, Kojouharov HV. Impact of socio-economic conditions on the incidence of visceral leishmaniasis in Bihar, India. Int J Environ Health Res
Abdullah AYM, Dewan A, Shogib MRI, Rahman MM, Hossain MF. Environmental factors associated with the distribution of visceral leishmaniasis in endemic areas of Bangladesh: Modeling the ecological niche. Trop Med Health
Das VN, Ranjan A, Pandey K, Singh D, Verma N, Das S, et al. Clinical epidemiologic profile of a cohort of post-kala-azar dermal leishmaniasis patients in Bihar, India. Am J Trop Med Hyg
Islam S, Kenah E, Bhuiyan MA, Rahman KM, Goodhew B, Ghalib CM, et al. Clinical and immunological aspects of post-kala-azar dermal leishmaniasis in Bangladesh. Am J Trop Med Hyg
Ramesh V, Singh R, Salotra P. Short communication: Post-kala-azar dermal leishmaniasis--an appraisal. Trop Med Int Health
Zijlstra EE, Khalil EA, Kager PA, El-Hassan AM. Post-kala-azar dermal leishmaniasis in the Sudan: clinical presentation and differential diagnosis. Br J Dermatol
Kumar D, Ramesh V, Verma S, Ramam M, Salotra P. Post-kala-azar dermal leishmaniasis (PKDL) developing after treatment of visceral leishmaniasis with amphotericin B and miltefosine. Ann Trop Med Parasitol
Hasnain G, Shomik MS, Ghosh P, Rashid MO, Hossain S, Hamano S, et al. Post-kala-azar dermal leishmaniasis without previous history of visceral leishmaniasis. Am J Trop Med Hyg
Mittal R, Behl PN, Srivastava G. Post-kala-azar dermal leishmanasis occurring after 10 years of treated kala azar. Int J Dermatol
Ramesh V, Singh R, Avishek K, Verma A, Deep DK, Verma S, et al. Decline in clinical efficacy of oral miltefosine in treatment of post kala-azar dermal leishmaniasis (PKDL) in India. PLoS Negl Trop Dis
Dinesh DS, Kar SK, Kishore K, Palit A, Verma N, Gupta AK, et al. Screening sandflies for natural infection with Leishmania donovani
, using a non-radioactive probe based on the total DNA of the parasite. Ann Trop Med Parasitol
Molina R, Ghosh D, Carrillo E, Monnerat S, Bern C, Mondal D, et al. Infectivity of post-kala-azar dermal leishmaniasis patients to sand flies: Revisiting a proof of concept in the context of the Kala-azar Elimination Program in the Indian Subcontinent. Clin Infect Dis
Argov S, Jaffe C, Krupp M, Slor H, Shoenfeld Y. Autoantibody production by patients infected with Leishmania. Clin Exp Immunol
Ganguly S, Das NK, Panja M, Pal S, Modak D, Rahaman M, et al. Increased levels of interleukin-10 and IgG3 are hallmarks of Indian post-kala-azar dermal leishmaniasis. J Infect Dis
Ganguly S, Mukhopadhyay D, Das NK, Chaduvula M, Sadhu S, Chatterjee U, et al. Enhanced lesional Foxp3 expression and peripheral anergic lymphocytes indicate a role for regulatory T cells in Indian post-kala-azar dermal leishmaniasis. J Invest Dermatol