There is increasing interest in the role of asymptomatic infection in transmission of Visceral Leishmaniasis (VL). We studied the individual, household and environmental factors associated with asymptomatic Leishmania donovani infected individuals and VL. 7,538 individuals living in VL endemic villages in India and Nepal were divided into three mutually exclusive groups based on their VL history and Direct Agglutination Test (DAT) results in yearly serosurveys over a two-year period. The groups were (1) VL cases, (2) asymptomatically infected individuals (seroconverters) and (3) seronegative individuals. VL cases and seroconverters were compared to seronegative individuals in mixed logistic regression models. The risk of seroconversion and disease was significantly increased in individuals aged 14 to 24 years old and by the presence of other DAT-positive, asymptomatically infected individuals and VL cases in the house. The risk of seroconversion was higher in Indian than in Nepalese villages and it increased significantly with age, but not so for VL. This study demonstrates that, when risk factors for leishmanial infection and VL disease are evaluated in the same population, epidemiological determinants for asymptomatic infection and VL are largely similar.
Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani - Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters’ along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.
Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We assessed the risk of progression from infection to disease as a function of DAT and rK39 serological titers.
We used available data on four cohorts from villages in India and Nepal that are highly endemic for Leishmania donovani. In each cohort two serosurveys had been conducted. Based on results of initial surveys, subjects were classified as seronegative, moderately seropositive or strongly seropositive using both DAT and rK39. Based on the combination of first and second survey results we identified seroconvertors for both markers. Seroconvertors were subdivided in high and low titer convertors. Subjects were followed up for at least one year following the second survey. Incident VL cases were recorded and verified.
We assessed a total of 32,529 enrolled subjects, for a total follow-up time of 72,169 person years. Altogether 235 incident VL cases were documented. The probability of progression to disease was strongly associated with initial serostatus and with seroconversion; this was particularly the case for those with high titers and most prominently among seroconvertors. For high titer DAT convertors the hazard ratio reached as high as 97.4 when compared to non-convertors. The strengths of the associations varied between cohorts and between markers but similar trends were observed between the four cohorts and the two markers.
There is a strongly increased risk of progressing to disease among DAT and/or rK39 seropositives with high titers. The options for prophylactic treatment for this group merit further investigation, as it could be of clinical benefit if it prevents progression to disease. Prophylactic treatment might also have a public health benefit if it can be corroborated that these asymptomatically infected individuals are infectious for sand flies.
To study the association between positive serology for VL among asymptomatic persons and progression to disease, we analyzed combined data from two different cohort studies in which over 32,000 subjects were enrolled. All subjects were screened for presence of antibodies to L. donovani with DAT and rK39. After an interval of 6–12 months, a second round of surveys was conducted in which seroconvertors were identified, i.e. persons seronegative at baseline that had become seropositive in the interval between surveys. We subdivided the subjects according to serologic titers scored as negative, moderately positive or strongly positive. During follow-up we observed that the risk of progressing to disease was not very much different between seronegative and moderately seropositive subjects, but strongly increased for high titer seropositive subjects. This pattern was true for the initial serology, and even more pronounced among seroconvertors. Given the strongly increased risk of progression to disease among those with high DAT or rK39 titers, these data raise the question whether prophylactic treatment would be beneficial. The case for prophylactic treatment would be further strengthened if it could be shown that asymptomatically infected individuals are infectious to sand flies.
Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.
Methodology and principal findings
The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.
This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.
Visceral Leishmaniasis (VL) is the most fatal form in Indian subcontinent. Till last few years, the treatment of the disease was done with Sodium antimony gluconate (SAG), the first line drug against VL. This, however, was severely eroded by the resistance developed by the parasite against it. In order to understand the underlying mechanism, earlier a proteomic analysis of SAG sensitive as well as SAG resistant isolates of L.donovani (Ld) was done in which 60s ribosomal L23a (Ld60sRL23a) protein, one of the essential member of translational machinery, was found to be over-expressed. To examine its role in SAG resistance mechanism, which is hitherto not known, 60sRL23a was characterized and over-expressed in the sensitive isolate of L.donovani. The sensitivity of these transfectants, was found to be decreased towards SAG. The growth curve of transfectants clearly showed its proliferation potential in both promastigote and amastigote forms. Cell cycle analysis of the transfectants further assured its rapid progression towards the G2/M phase. The above studies, thus, indicate that 60s RL23a regulates proliferation of L.donovani parasites and represents a unique strategy to resist SAG. 60sRL23a could be further explored as a potential drug target to strengthen the chemotherapy strategy against L.donovani.
We previously established that Indole-3-Carbinol (I3C), a natural hydrolysis product of glucobrassicin in cruciferous vegetables, arrests the proliferation of estrogen-dependent human breast cancer cells and induces protein degradation of estrogen receptor-alpha (ERα). We demonstrate in human MCF-7 breast cancer cells that I3C ablates expression of Insulin-like Growth Factor Receptor-1 (IGF1R) and Insulin Receptor Substrate-1 (IRS1), downstream effectors of the IGF1 signaling pathway. Exogenous ERα reversed the I3C mediated loss of IGF1R and IRS1 gene expression demonstrating that down-regulation of ERα is functionally linked to I3C control of IGF1R and IRS1 expression. I3C disrupted binding of endogenous ERα, but not Sp1, to ERE-Sp1 composite elements within the IGF1R/IRS1 promoters. Exogenous ERα abrogated, and combined expression of IGF1R and IRS1 attenuated, the I3C mediated cell cycle arrest. Therefore, I3C inhibits proliferation of estrogen-sensitive breast cancer cells through disruption of ERα-mediated transcription of cell signaling components within the IGF1 cascade.
I3C; Indole-3-carbinol; ERα; Estrogen receptor-alpha; IRS1; insulin receptor substrate-1; IGF1R; Insulin-like Growth Factor-1 Receptor; hormone sensitive breast cancer
PCR- Restriction fragment length polymorphism (RFLP) is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis (VL) in India, various species of morphological similar sand fly exists but only female Phlebotomus argentipes is the vector for VL. In the present study primers were designed targeting the 18S rRNA encoding gene that showed amplification in all the major sand fly species found in India. The amplified fragments were further digested using the Hinf I or Hpa II restriction enzymes. Each of the restriction enzyme produced species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be employed in the identification of the species of the sand flies.
18S rRNA; Leishmaniasis; PCR; Phlebotomine; RFLP; Sand fly
Regional variations in susceptibility of Leishmania donovani clinical isolates have been reported to antimonials but not other antileishmanial drugs. Therefore, we evaluated the susceptibility of four antileishmanial drugs in clinical use in 28 clinical isolates from endemic and non-endemic regions in the J774A.1 macrophage cell line, and we found increased tolerance of miltefosine and paromomycin in isolates from a patient from a high endemic region. Effective dose for 90% killing (ED90) values were significantly higher for miltefosine (P = 0.005) and paromomycin (P = 0.02) in isolates from the high endemic region, although there were no significant differences between ED50 values for paromomycin, miltefosine, and amphotericin B in the non- versus endemic region isolates. This report is the first of higher ED90 values for miltefosine and paromomycin indicating susceptibility difference between regions for these newly introduced drugs by the parasite, and their use should be carefully monitored through directly observed therapy or multidrug treatment to preserve their efficacy for longer periods.
In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism
We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates.
Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.
Diagnosis of Visceral Leishmaniasis (VL) is a major obstacle in the control of this disease, as demonstration of parasite by splenic or bone-marrow aspiration is almost impossible in the peripheral rural areas because of lack of facilities. In order to identify a non-invasive biomarker for VL, we employed two-dimensional electrophoresis (2D-PAGE) coupled with mass spectrometry through a comparison of urinary proteome. Though, we did not find the biomarker protein for VL, our findings provide some basic insights for future development of non invasive diagnostic tool.
Visceral leishmaniasis; 2D-PAGE; urinary proteins; MALDI-TOF
Chromosome 6q26–27 is linked to susceptibility to visceral leishmaniasis (VL) in Brazil and Sudan. DLL1 encoding the Delta-like 1 ligand for Notch 3 was implicated as the etiological gene. DLL1 belongs to the family of Notch ligands known to selectively drive antigen-specific CD4 T helper 1 cell responses, which are important in protective immune response in leishmaniasis. Here we provide further genetic and functional evidence that supports a role for DLL1 in a well-powered population-based study centred in the largest global focus of VL in India. Twenty-one single nucleotide polymorphisms (SNPs) at PHF10/C6orf70/DLL1/FAM120B/PSMB1/TBP were genotyped in 941 cases and 992 controls. Logistic regression analysis under an additive model showed association between VL and variants at DLL1 and FAM120B, with top associations (rs9460106, OR=1.17, 95%CI 1.01–1.35, P=0.033; rs2103816, OR=1.16, 95%CI 1.01–1.34, P=0.039) robust to analysis using caste as a covariate to take account of population substructure. Haplotype analysis taking population substructure into account identified a common 2-SNP risk haplotype (frequency 0.43; P=0.028) at FAM120B, while the most significant protective haplotype (frequency 0.18; P=0.007) was a 5-SNP haplotype across the interval 5’ of both DLL1 (negative strand) and FAM120B (positive strand) and extending to intron 4 of DLL1. Quantitative RT/PCR was used to compare expression of 6q27 genes in paired pre- and post-treatment splenic aspirates from VL patients (N=19). DLL1 was the only gene to show differential expression that was higher (P<0.0001) in pre- compared to post-treatment samples, suggesting that regulation of gene expression was important in disease pathogenesis. This well-powered genetic and functional study in an Indian population provides evidence supporting DLL1 as the etiological gene contributing to susceptibility to VL at Chromosome 6q27, confirming the potential for polymorphism at DLL1 to act as a genetic risk factor across the epidemiological divides of geography and parasite species.
visceral leishmaniasis; DLL1; genetic association; Notch signalling
The potential to deliver nanoparticles directly into the targeted cells is important in the therapeutic applications for infectious diseases. The possibility of therapeutic agent being attached to the nanoparticles by chemical modification has provided a novel drug delivery option. Interestingly, the discovery of carbon nanotubes and graphene has given an excellent imaging and therapeutic agent for the biomedical applications. In spite of continuous advancement in pharmaceutical drug delivery viz. micelles, vesicles and liquid crystals etc. during the past decades, their prohibitive production has limited their use. Nanomaterials with their properties of biodegradation, equal biodistribution, mass production and long time storage make them attractive alternative for future biomedical applications. Nanoparticles surface functionalized with specific biomolecules based drug delivery has driven new direction for modulating the pharmacokinetics and pharmacodynamics, biorecognition; and increasing the efficacy of targeted drugs. These new strategies are likely to minimize drug degradation and loss, increase drug availability, and opens up new vistas for drug delivery.
Nanoparticle; infectious diseases; drug delivery; therapeutic; nanotherapy
Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90–17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.
Leishmania donovani; Phlebotomus argentipes; 18S rRNA; Gene; PCR; Sand fly; Visceral leishmaniasis
Visceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures.
We collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR.
Though sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.
Intracellular pathogenic protozoan infection like visceral leishmaniasis is considered in terms of the overall inflammatory response and the complex cellular interactions leading to formation of the activated macrophage. Analysis of the development of activation is facilitated when operationally defined stage of activation are characterized using a library of objective markers. There is a role of arginase in the immune response supporting its involvement in macrophage effector mechanism in vitro and in vivo. 5′-Nucleotidase a plasma membrane component has been cited as a biochemical correlate of macrophage function in an altered morphological and biochemical state of activation and stimulation. Depression in 5′-nucleotidase activity has been generally referred to as a characteristic marker of activated macrophages. Lysozyme or lysosomal enzymes are released into the endocytic or autophagic vacuole macrophage where they serve the purpose of intracellular digestion of engulfed or segregated materials. In the present study, we have studied levels of arginase and 5′-nucleotidase (marker for macrophage activation) in monocytes of active VL patients and healthy controls. Lysozyme a secretary product of macrophages was also measured in supernatants collected from monocytes of active VL patients and healthy controls. Elevated levels of 5′-nucleotidase were observed in supernatants of monocytes from active VL patients as compared to healthy controls. Low levels of arginase and lysozyme production by monocytes isolated from VL patients were observed as compared to healthy controls. Our studies suggest that low levels of arginase and elevated 5′-nucleotidase activity could be one of the mechanisms in the pathology of VL infection. Low lysozyme activity in patients may account for persistence of Leishmania parasites in VL infections.
Arginase; 5′-Nucleotidase; Lysozyme activity; Visceral leishmaniasis
Despite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need to develop a better antigen. In this study we separated crude soluble antigens of Leishmania donovani by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hybridized with pool sera from pre- and post-treated VL patients, 6 months follow-up, endemic healthy (EHC), and nonendemic healthy controls (NEHC) by Western blotting. The sensitivity of enzyme-linked immunosorbent assay with identified protein was 95% (confidence interval [CI] = 89.6–98.01%), whereas the specificity for EHC, NEHC, and different disease groups were 96.3% (CI = 89.8–98.6%), 100% (CI = 95.8–100%), and 97.4% (CI = 91.02–99.3%), respectively. This specific antigen was subjected to two-dimensional gel electrophoresis and after tryptic digestion, antigen was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Further analysis showed that it is a member of the heat shock protein family of 70 kDa, designated as BHUP1, and has great potential in the diagnosis of VL.
The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody in human saliva being noninvasive to replace the invasive procedures of diagnosis. Enzyme-linked immunosorbent assay (ELISA) and ICT assays were performed in 300 subjects: 114-confirmed VL patients, 95 and 47 healthy controls from endemic and nonendemic regions, respectively, and 44 subjects with different diseases. Sensitivity in saliva was 83.3% by ELISA and 82.5% by ICT, compared with 100% for both ICT and ELISA in serum. Specificity in saliva was 100%, 90.5%, and 88.6% with ELISA, and 91.48%, 91.57%, and 84.06% using ICT, in nonendemic, endemic, and different diseases, respectively. In serum, specificity was 97%, 88.5%, and 89% by ELISA and 100%, 94.7%, and 95.5% by ICT in nonendemic, endemic, and different diseases, respectively. Saliva is not suitable for diagnosis of VL because of low sensitivity.
We compared the two formats of rKE16 antigen-based rapid tests, a flowthrough test (KEFT) and a lateral flow test (KELF), with the rK39 rapid test for the diagnosis of visceral leishmaniasis. Sensitivities with KEFT (99%, 198/200) and rK39 (99.5%, 199/200) were comparable and higher than that with KELF (95.5%, 191/200). In the control groups comprising subjects with diseases from areas of nonendemicity or endemicity and subjects with different diseases, the specificities were comparable for all three rapid tests, except that specificity was higher with KELF in the controls from areas of endemicity.
Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.
We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.
We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).
Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.
In this study we assessed trends with age in the probability of being sero-positive or sero-converting for two serological markers of visceral leishmaniasis (VL) among asymptomatic residents of high incidence villages. As markers we used Direct Agglutination Test (DAT) and rK39 ELISA. We also compared titers among asymptomatic sero-positives with those of known recent VL cases among our study population. Infection with VL is assumed to be permanent, but sero-positivity is a temporary phenomenon. Though clinical VL was most common among children and young adults (median age 19 years), we observed a statistically significant increase with age in the probability of being sero-positive and in the probability of sero-converting. We also observed that the average antibody titers among asymptomatic sero-positives were much lower than those among recent clinical VL cases. The increase with age in the probability of being sero-positive but also in the probability of sero-conversion can be explained by subjects experiencing repeated episodes of sero-positivity. This could be due to a boosting effect upon re-exposure, or to internal release of parasites from safe target cells. The implication of our findings is that in VL endemic areas it will be difficult to reliably distinguish between infected and non-infected subjects.
Known as ‘neglected disease’ because relatively little effort has been applied to finding cures, leishmaniasis kills more than 150,000 people every year and debilitates millions more. Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Whole genome analysis presents an excellent means to identify new targets for drugs, vaccine and diagnostics development, and also provide an avenue into the biological basis of parasite virulence in the L. donovani complex prevalent in India.
In our presently described study, the next generation SOLiD™ platform was successfully utilized for the first time to carry out whole genome sequencing of L. donovani clinical isolates from India. We report the exceptional occurrence of insect trypanosomatids in clinical cases of visceral leishmaniasis (Kala Azar) patients in India. We confirm with whole genome sequencing analysis data that isolates which were sequenced from Kala Azar (visceral leishmaniasis) cases were genetically related to Leptomonas. The co-infection in splenic aspirate of these patients with a species of Leptomonas and how likely is it that the infection might be pathogenic, are key questions which need to be investigated. We discuss our results in the context of some important probable hypothesis in this article.
Our intriguing results of unusual cases of Kala Azar found to be most similar to Leptomonas species put forth important clinical implications for the treatment of Kala Azar in India. Leptomonas have been shown to be highly susceptible to several standard leishmaniacides in vitro. There is very little divergence among these two species viz. Leishmania sp. and L. seymouri, in terms of genomic sequence and organization. A more extensive perception of the phenomenon of co-infection needs to be addressed from molecular pathogenesis and eco-epidemiological standpoint.
Amphotericin B (AmB), is a highly effective antileishmanial agent used as first-line treatment in different formulations in visceral leishmaniasis endemic areas of Bihar, India. However, parenteral infusion, prolonged hospitalization, and toxicity are major hurdles. Our previous work demonstrated the efficacy and stability of functionalized carbon nanotubes as a delivery mechanism for AmB. In this study, using the hamster model, we have shown that this novel formulation of AmB can be administered orally, resulting in 99% inhibition of parasite growth following a 5-day course at 15 mg/kg body weight.
Antibody detection is a safely applied method at wide scale in diagnosis of Visceral Leishmaniasis (VL). Towards further advancement of serodiagnosis, rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated sensitivity and specificity of rK28 antigen in a micro-ELISA format in comparison to rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirm VL cases, 251 cases were reported positive by rK28 antigen yielding 99.6% sensitivity (95% CI, 0.97–0.99) which was similar to sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97–0.99). Specificity of rK28 antigen in non-endemic and endemic healthy controls was 100% (95%CI 0.96–1) and 94.17% (95% CI, 0.88–0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84–0.96). With rK39 antigen, specificity of non-endemic and endemic controls, and different diseases was 100% (95%CI 0.96–1) and 92.23% (95% CI 0.85–0.96), and 96.59% (95% CI 0.90–0.98) respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in endemic population of Bihar.
Visceral Leishmaniasis; rk-28; serodiagnosis; ELISA
In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported.
Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF β level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity.
This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.
The disease Kala-azar or visceral leishmaniasis is still a big problem in the Indian subcontinent. The antimonials were used for the chemotherapy of Kala-azar but with time its efficacy has reduced dramatically. The newer version of orally active drug miltefosine has been introduced, but its efficacy has decreased considerably as relapse cases are on the rise. Other drugs like liposomal form of amphotericin B is expensive and the patients require hospitalization. Thus there is a genuine need for an orally active antileishmanial drug. There are reports that the cationic amphiphilic molecule, imipramine, a drug used for the treatment of depression in humans, kills the promastigotes of Leishmania donovani. We tested the efficacy of imipramine in experimental infection in hamster and mouse model. Our study showed that the drug is highly effective against antimony sensitive and antimony resistant Leishmania donovani infected hamsters as well as mouse and offered almost sterile cure.
Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, but increasing failure rates under antimonial treatment have challenged further use of these drugs in the Indian subcontinent. Experimental evidence has suggested that parasites which are resistant against antimonials have superior survival skills than sensitive ones even in the absence of antimonial treatment.
Methods and Findings
We use simulation studies based on a mathematical L. donovani transmission model to identify parameters which can explain why treatment failure rates under antimonial treatment increased up to 65% in Bihar between 1980 and 1997. Model analyses suggest that resistance to treatment alone cannot explain the observed treatment failure rates. We explore two hypotheses referring to an increased fitness of antimony-resistant parasites: the additional fitness is (i) disease-related, by causing more clinical cases (higher pathogenicity) or more severe disease (higher virulence), or (ii) is transmission-related, by increasing the transmissibility from sand flies to humans or vice versa.
Both hypotheses can potentially explain the Bihar observations. However, increased transmissibility as an explanation appears more plausible because it can occur in the background of asymptomatically transmitted infection whereas disease-related factors would most probably be observable. Irrespective of the cause of fitness, parasites with a higher fitness will finally replace sensitive parasites, even if antimonials are replaced by another drug.
The protozoan flagellate Leishmania donovani causes the neglected, life-threatening disease visceral leishmaniasis. Parasites are transmitted from man to man by the bite of the sand fly Phlebotomus argentipes, the vector of the disease. Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades but rapidly increasing failure rates up to 65% observed between 1980 and 1997 in the state of Bihar, India, have challenged further use of these drugs. Comparative in vitro and in vivo experiments indicate that antimony-resistant parasites have a higher fitness than antimony-sensitive ones even in the absence of antimonial treatment. Simulation studies based on a previously published mathematical L. donovani transmission model suggest that resistance to antimonial treatment alone cannot explain the Bihar observations but that resistance together with higher fitness offers the potential to explain the data. After an antimony-resistant parasite with higher fitness has emerged, it will finally replace the antimony-sensitive ones, even in complete absence of antimonial treatment.
Depressed cell-mediated immunity in human visceral leishmaniasis (VL) (also known as kala-azar), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We recently reported the ability of a whole-blood, gamma interferon (IFN-γ) release assay to detect subclinical infections among healthy individuals living in an area where kala-azar is endemic (Bihar, India) and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested in ascertaining whether these findings would be true for a larger cohort of subjects and in employing the whole-blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) release in 35 patients with active VL, 54 patients with VL who were cured, 27 patients with other diseases, 52 healthy controls who lived in regions where VL or kala-azar is not endemic (NEHCs [for nonendemic healthy controls]), and 147 healthy controls who lived in regions where kala-azar is endemic (EHCs [for endemic healthy controls]). The cellular responses of the EHCs were correlated with their serological antibody titers against Leishmania donovani and Phlebotomus argentipes saliva. The whole-blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed subclinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the patients with active VL also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish individuals with active disease from cured or subclinically infected, immune individuals.