V. N. R. Das

Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: a challenge to kala-azar elimination programmes

A cohort of 91 asymptomatic individuals with visceral leishmaniasis (VL) were identified during base line screening using recombinant 39-aminoacid antigen (rk-39) and polymerase chain reaction (PCR) conducted from December 2005 to June 2006 involving 997 individuals of two highly endemic villages of Vaishali district, Bihar. The point prevalence of asymptomatic infection was 98 per 1000 persons at baseline. There was no statistically significant difference between rk-39 and PCR positivity rate (P>0.05), even though PCR positivity alone was found significantly higher (4.2%) than rk-39 positivity alone (2.6%). The monthly follow-up of the asymptomatic cohort revealed a disease conversion rate of 23.1 per 100 persons within a year. There was a statistically significant difference in conversion of disease when individuals were positive by both tests as compared to single tests by rk-39 and PCR (P<0.01). Disease conversion rate in the subjects residing in households with a history of VL (62%, 13/21) was higher than those residing in the households without a history of VL (38%, 8/21). Most of the identified asymptomatic individuals were from low socio-economic strata similar to that of VL cases in general. Apart from rk-39, PCR may be considered for screening of asymptomatic Leishmania donovani infection in large-scale epidemiological studies. Screening of asymptomatic cases and their close follow-up to ascertain early detection and treatment of VL may be considered in addition to the existing VL control strategies.

Oral miltefosine for Indian post-kala-azar dermal leishmaniasis: a randomised trial

Standard treatment of Indian post‐kala‐azar dermal leishmaniasis (PKDL) is unsatisfactory because to achieve therapeutic effectiveness, heroic courses of parenteral and toxic agents have to be administered. Our objective was to evaluate oral miltefosine for its potential to provide effective as well as tolerable treatment for this disease.

Designing Therapies against Experimental Visceral Leishmaniasis by Modulating the Membrane Fluidity of Antigen-Presenting Cells

ABSTRACT The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-γ signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.

Human visceral leishmaniasis: decrease in serum cholesterol as a function of splenic parasite load

Kala azar or human visceral leishmaniasis (VL) is a debilitating disease associated with hepato–splenomegaly, anaemia, thrombocytopaenia and immunosuppression (Pearson et al., 1983). The predominant causative agent, Leishmania donovani, replicates within the reticulo-endothelial system of the liver, and the liver parenchyma, although initially unaffected, is slowly damaged as the disease progresses (Alsaffar and Al Mudhaffar, 1979). Consequently, hepatic dysfunction — showing as coagulation defects and changes in the serum concentrations of several liver-specific enzymes — is typical of VL (Chakroborty et al., 1949). As the liver is the main source of cholesterol biosynthesis in mammals (Tennent et al., 1957), hepatic dysfunction may lead to low serum concentrations of cholesterol and this may lead to further morbidity. Hypocholesterolaemic men tend to have significantly fewer circulating lymphocytes, total T cells, helper T-cells and CD8+ cells than hypercholesterolaemic men (Muldoon et al., 1997). In their meta-analysis of 19 cohort studies covering 68,406 deaths, Jacobs et al. (1992) found an inverse correlation between blood concentrations of cholesterol and mortality from respiratory and gastro-intestinal diseases (most of which are of infectious origin). Subsequently, in a 15-year follow-up study of >120,000 individuals, Iribarren et al. (1998) found a strong inverse association between blood concentrations of cholesterol and the risk of being admitted to hospital because of an infectious disease. It appears that hypercholesterolaemia may confer a survival advantage in many, if not all, infectious diseases. In an experimental study, Netea et al. (1996) showed that mice deficient in receptors for low-density lipoprotein (LDL) and with endogenous hypercholesterolaemia were protected against infection with Gram-negative micro-organisms, the lower cholesterol levels observed being associated with increased mortality. In tuberculosis, serum concentrations of cholesterol, high-density lipoprotein and LDL can be used as indirect markers of disease severity, with relatively low levels indicative of advanced disease (Rao, 2009). Akerlund et al. (1986) described how, in patients with severe bacterial infections, total serum cholesterol concentrations were lowered during the acute stage of their disease. Decreased serum cholesterol has already been reported in patients with VL (Lal et al., 2007). In experimentally infected hamsters, Banerjee et al. (2009) not only demonstrated a significant decrease in membrane cholesterol during the active stage of L. donovani infection but also found that the liposomal delivery of cholesterol offered significant protection. These observations led to the present study, which was focused on cholesterol and not other lipids. In this study, since cellular cholesterol and serum cholesterol are in dynamic equilibrium (Chobanian et al., 1962), serum concentrations of cholesterol in Indian patients with VL were determined as surrogate markers of cellular cholesterol. Subsequently, the relationship between the observed serum concentrations of cholesterol and the parasite burdens in the patients’ spleens was explored.

Usefulness of the direct agglutination test in the early detection of subclinical Leishmania donovani infection: a community-based study

Abstract The value of a direct agglutination test (DAT) in the detection of subclinical infections with Leishmania donovani has recently been investigated in the Indian state of Bihar, after the sensitivity and specificity of the test had been determined. When used to screen sera from 108 parasitologically confirmed cases of visceral leishmaniasis, 50 patients with active, non-leishmanial infection, and 641 healthy controls living close to, or distant from, an endemic area, the test was found to be 91.7% sensitive and 100% specific if a titre of 1 : 800 was used as the threshold for seropositivity. During a longitudinal clinical study in a rural, VL-endemic area of the Indian state of Bihar, the test was used, with 1 : 800 set as the threshold titre, to determine the baseline prevalence of infection with L. donovani among villagers who, though showing no symptoms of VL, had recently been febrile for at least 2 weeks. The 234 subjects of this study were either VL-case contacts [i.e. members of households in which there were active or cured VL cases (N=78)] or the members of control households with no cases or history of the disease (N=156). The results of DAT at the start of the study indicated that 49 (20.9%) of the subjects — 29 (37.2%) of the VL-case contacts and 20 (12.8%) of the other subjects — were seropositive and therefore probably had subclinical infections with L. donovani. During the subsequent 9 months of follow-up, however, only eight of the subjects found seropositive at the start of the study — seven (24.1%) of the seropositive case contacts but only one (5.0%) of the other seropositives — developed symptomatic VL, all by month 6 of the follow-up. Compared with their neighbours, therefore, individuals who shared households with active or cured cases of VL appeared at greater risk not only of L. donovani infection (indicating focal transmission) but also of developing symptomatic disease once infected. Curiously, among the seropositive case contacts, those from the households that harboured active cases of VL at the baseline survey were less likely to develop symptomatic VL during the 9 months of follow-up than those from households that harboured only cured cases (18.8% v. 30.8%). The wide-spread use of DAT could allow the detection and early treatment of latent L. donovani infections and so contribute to the elimination of VL, at least as a public-health problem, from India.

Novel Noninvasive Method for Diagnosis of Visceral Leishmaniasis by rK39 Testing of Sputum Samples

Visceral leishmaniasis (VL) is a major public health problem in the eastern states in India, namely, Bihar, West Bengal, Jharkhand, and Eastern Uttar Pradesh. Bihar alone accounts for >90% of the total number of cases of kala-azar reported in India. The disease is predominantly found in poor and malnourished people (9). The development of a simple, noninvasive, cheap, and reliable diagnostic tool has been suggested as a prerequisite to controlling the disease (3, 4). Ideally, diagnosis of kala-azar is done by direct demonstration of the parasite in splenic or bone marrow aspirates under the microscope (1, 5). Detection of the parasite in splenic aspirate is sensitive, but splenic aspiration is invasive and painful and carries the risk of serious or fatal hemorrhage, whereas bone marrow aspiration is safer and relatively easy, but detection of the parasite in bone marrow aspirate is less sensitive (60 to 85%), and both methods of detection demand technical support. In vitro cultivation of parasites is expensive and time consuming and requires expertise, thus severely restricting its use in routine diagnosis. Serological tests, though good, have their own limitations, viz., they cannot differentiate between present and past infection, asymptomatic cases versus clinical cures, etc. Sensitivity and specificity also vary from test to test, and some serological tests are not user friendly and not suitable for field conditions (2). In the last few years, a serum immunoglobulin G (IgG)-based detection system using rK39 antigen has been found to be useful in diagnosing VL. The test was found to be highly sensitive, specific, simple, and reproducible, and results can be obtained within 10 to 15 min (7). To the best of our knowledge, the use of rK39 to detect VL infection in sputum samples has not been reported. This is perhaps the first early report of the use of rK39 strips (InBios, Seattle, WA) to detect active VL cases using sputum samples. The assay was performed according to the manufacturers protocol. Briefly, the bottom of the absorbent pad of an rK39 strip was dipped in a freshly collected sputum sample for 5 min. Two drops of the chase buffer provided with the kit was added to the pad and was allowed to migrate up to the strip by capillary action. The results were read after 10 min. The appearance of a red upper (control) band indicated the proper functioning of the test, and that of a red lower (test) band suggested the presence of anti-rK39 IgG in the sputum. The test was considered positive if two bands, viz., upper and lower, were present, whereas the test was considered negative if only the upper, control band appeared. A total of 505 sputum samples from confirmed VL patients and controls were screened with the rK39 strip test. Of the 126 microscopically confirmed VL cases, 125 (99.2%) were found positive by rK39 in sputum samples and all by rK39 in blood samples (100%) (Table ​(Table1).1). However, in controls from areas where the disease is endemic and where it is not endemic, the rK39 test was found to be more specific for sputum than for blood because sputum did not show any positive reaction, whereas 2.5% positivity in controls from areas of endemicity and no positivity in controls from areas of nonendemicity were observed using blood samples. This test was also found to be more specific in samples from the control group of patients with other diseases, because none of the sputum samples cross-reacted with the rK39 strip, whereas the blood samples of seven (5.73%) of these patients showed a positive reaction. The 2.5% and 5.73% positivities by rK39 strip test for blood samples from healthy controls from areas of endemicity and the control group with other diseases, respectively, were false positives because the positive samples in both groups did not show a Leishmania donovani-positive reaction by PCR (6). Moreover, these subjects did not exhibit clinical symptoms of VL, such as fever, pancytopenia, and hepatosplenomegaly. Even the follow-up over more than 6 months did not show conversion of any of these cases to symptomatic kala-azar cases. Furthermore, the PCR results (6) for sputum samples from both these groups were also negative for L. donovani infection. Lastly, it was not possible ethically to subject these cases to splenic or bone marrow aspiration. TABLE 1. Comparison of sensitivities and specificities of rK39 strip test in diagnosis of kala-azar using sputum samples versus serum samples from L. donovani body-positive VL patients and controls The present data clearly demonstrate that though the newly developed sputum-based rK39 test is only 0.80% less sensitive, it is much more specific than the rK39 test for diagnosis of VL using blood samples. This test can be easily adapted for routine diagnosis of VL using sputum samples. This will be highly beneficial for diagnosis of VL in difficult field conditions, as the test is purely noninvasive, uses easily collectable samples, and needs no special equipment or sophisticated technology, and the results can be read visually. Finally, this diagnostic tool can be useful for the kala-azar elimination program, which is scheduled to end by 2015 in the Indian subcontinent (8).

Leishmania donovani: CD2 biased immune response skews the SAG mediated therapy for a predominant Th1 response in experimental infection.

We have evaluated the effect of combining CD2 with conventional antimonial (sb) therapy in protection in BALB/c mice infected with either drug sensitive or resistant strain of Leishmania donovani with 3×10(7) parasites via-intra-cardiac route. Mice were treated with anti CD2 adjunct SAG sub-cutaneously twice a week for 4 weeks. Assessment for measurement of weight, spleen size, anti-Leishmania antibody titer, T cell and anti-leishmanial macrophage function was carried out day 0, 10, 22 and 34 post treatments. The combination therapy was shown boosting significant proportion of T cells to express CD25 compared to SAG monotherapy. Although, the level of IFN-γ was not statistically different between combination vs monotherapy (p=0.298) but CD2 treatment even alone significantly influenced IFN-γ production than either SAG treatment (p=0.045) or with CD2 adjunct SAG treatment (p=0.005) in Ld-S strain as well as in Ld-R strain. The influence of CD2 adjunct treatment was also documented in anti-leishmanial functions in macrophages. As shown, the super-oxide generation began enhancing very early on day 10 after SAG treatment with CD2 during which SAG action was at minimum. Interestingly, the super-oxide generation ability remained intact in macrophage after treatment with immuno-chemotherapy even in mice infected with Leishmania resistant strain. Unlike SAG treatment, treatment of SAG with CD2 also led to production of nitric oxide and TNF-α, resulting in resulting in most effective clearance of L. donovani from infected macrophages. Our results indicate that CD2, which can boost up a protective Th1 response, might also be beneficial to enable SAG to induce Macrophages to produce Leishmanicidal molecules and hence control the infection in clinical situation like Kala-azar. Drug resistance is the major impedance for disease control but the encouraging results obtained after infecting mice with resistant strain of the parasite strongly imply that this drug can be effective even in treating resistant cases of Kala-azar.

Post-Kala-Azar Dermal Leishmaniasis in a Patient Treated with Injectable Paromomycin for Visceral Leishmaniasis in India

ABSTRACT Post kala-azar dermal leishmaniasis (PKDL) is a skin manifestation that usually develops after treatment of visceral leishmaniasis (VL), a major public health problem in India. The diagnosis and management of PKDL is complex. This is the first case report from India in which PKDL occurred after paromomycin treatment for VL in an Indian patient.

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.

Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes

Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.