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.

Introduction

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.

Methods

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.

Results

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).

Discussion

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.

Author Summary

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.

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.

Background

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.

Methodology/Principal Findings

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.

Conclusions

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.

Author Summary

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.

Background

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.

Conclusions

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.

Author Summary

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.

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC+ve, n = 20) or modified Quantiferon negative (EHC−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.

Author Summary

Visceral leishmaniasis is a parasitic infection that results in death in susceptible people unless they are treated. Current drugs are expensive and toxic, and there are no vaccines in use in humans. We know that it is possible to become immune to infection with this parasite because people who have been cured using drug treatment are resistant to further infection. In addition, a large percentage of people infected with the parasite remain asymptomatic and develop a specific immune response that can be measured using crude leishmanial antigens. We hypothesized that these resistant people might hold the key to understanding the kind of immune response required for protection. In this paper we compared the immune response to a series of novel vaccine candidates in people with active disease, in those drug-cured from the disease, and in the naturally resistant individuals. We show that immune individuals make strong cytokine responses to five of eleven novel vaccine candidates that were tested, making them ideal candidates to take forward in the development of a defined vaccine against leishmaniasis.

The mechanisms underlying the failure to contain the growth of Leishmania parasites in human visceral leishmaniasis (VL) are not understood. L donovani amastigotes were quantified in cultured splenic aspirate cells to assess the function of IL-10 in lesional tissue ex vivo. In 67 patients with active VL, IL-10 neutralization promoted parasite killing in 73% and complete clearance in 30%, while 18% had more parasites and 9% did not change. The splenic cells secreted increased levels of both tumor necrosis factor α (TNFα) and interferon γ (IFNγ) under IL-10-neutralizing conditions. These findings provide direct support for targeting IL-10 as an approach to therapy in human VL.

Abstract

Background

For the diagnosis of visceral leishmaniasis (VL), rK39 antigen-based rapid test is widely used. Unfortunately, up to 32% healthy individuals from endemic region test positive with this antigen. There is an urgent need to search for a more specific antigen with sensitivity similar to rK39.

Methods

We identified a Leishmania donovani-specific 12.6-kDa (BHUP3) soluble promastigote antigen through sensitive western blot technique. The identified protein was partially purified from sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the antigenic response of eluted protein was determined by western blot with different groups of individual sera. The diagnostic potential was further validated by enzyme-linked immunosorbent assay using serum of 100 VL patients, 93 nonendemic healthy control individuals, 110 endemic healthy control individuals, and 110 disease control individuals. Further, it was characterized by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time-of-flight analysis.

Results

On blotting, antibody against this protein was recognized by all (9/9) VL patient's sera, but it was absent in every control group (nonendemic healthy control and endemic healthy control). Sensitivity of the enzyme-linked immunosorbent assay was 88% (89/101), whereas the specificity for endemic healthy, nonendemic healthy, and different disease groups were 96% (106/110), 100% (93/93), and 97% (107/110), respectively. The two-dimensional gel electrophoresis showed a single spot, and matrix-assisted laser desorption/ionization-time-of-flight analysis revealed that it is a 113-amino-acid-long putative uncharacterized protein of 12.6-kDa anamorsin homolog matched completely with Leishmania major (GenBank accession number: Q4QIS1).

Conclusion

Despite marginally lower sensitivity of BHUP3, excellent specificity warrants its further development as a tool for diagnosis of VL.

To identify factors associated with incidence of visceral leishmaniasis (VL), we surveyed 13,416 households in Bihar State, India. VL was associated with socioeconomic status, type of housing, and belonging to the Musahar caste. Annual coverage of indoor residual insecticide spraying was 12%. Increasing such spraying can greatly contribute to VL control.

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9–97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

This study evaluates commercially available rK39 Immunochromatographic (ICT) strips using urine for diagnosis of visceral leishmaniasis (VL). Freshly collected urine and serum samples of 280 parasitologically confirmed VL patients and 66 endemic healthy control (EHC), 48 non-endemic healthy controls (NEHC) and 45 different diseases (DD) were tested with rK39 strips. The sensitivity of rK39 in urine were 96.4% while the specificity was low varying from 66.7% in EHC, 77.08% in NEHC to 62.2% in DD, With serum, sensitivity was 100% whereas the specificity was 100%, 92.4% and 95.55% for respective control group. In its present format, the ICT strips cannot be used for the diagnosis of VL using urine samples.

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G1 cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element–Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1–DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Accuracy of rapid diagnostic tests was high in the Indian subcontinent; however, in Brazilian and East African samples, reduced sensitivity suggests that several cannot be used alone to exclude visceral leishmaniasis. Data on ease of use and performance using whole blood and in human immunodeficiency virus coinfections is needed.

Background. Poor access to diagnosis stymies control of visceral leishmaniasis (VL). Antibody-detecting rapid diagnostic tests (RDTs) can be performed in peripheral health settings. However, there are many brands available and published reports of variable accuracy.

Methods. Commercial VL RDTs containing bound rK39 or rKE16 antigen were evaluated using archived human sera from confirmed VL cases (n = 750) and endemic non-VL controls (n = 754) in the Indian subcontinent (ISC), Brazil, and East Africa to assess sensitivity and specificity with 95% confidence intervals. A subset of RDTs were also evaluated after 60 days’ heat incubation (37°C, 45°C). Interlot and interobserver variability was assessed.

Results. All test brands performed well against ISC panels (sensitivity range, 92.8%–100%; specificity range, 96%–100%); however, sensitivity was lower against Brazil and East African panels (61.5%–91% and 36.8%–87.2%, respectively). Specificity was consistently > 95% in Brazil and ranged between 90.8% and 98% in East Africa. Performance of some products was adversely affected by high temperatures. Agreement between lots and readers was good to excellent (κ > 0.73–0.99).

Conclusions. Diagnostic accuracy of VL RDTs varies between the major endemic regions. Many tests performed well and showed good heat stability in the ISC; however, reduced sensitivity against Brazilian and East African panels suggests that in these regions, used alone, several RDTs are inadequate for excluding a VL diagnosis. More research is needed to assess ease of use and to compare performance using whole blood instead of serum and in patients coinfected with human immunodeficiency virus.

Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play significant in clinical resistance to antimonials.

Indole-3-carbinol (I3C), a natural autolysis product of a gluccosinolate present in Brassica vegetables such as broccoli and cabbage, has anti-proliferative and anti-estrogenic activities in human breast cancer cells. A new and significantly more potent I3C analogue, 1-benzyl-I3C was synthesized, and in comparison to I3C, this novel derivative displayed an approximate 1000-fold enhanced potency in suppressing the growth of both estrogen responsive (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cells (I3C IC50 of 52 μM, and 1-benzyl-I3C IC50 of 0.05 μM). At significantly lower concentrations, 1-benzyl-I3C induced a robust G1 cell cycle arrest and elicited the key I3C-specific effects on expression and activity of G1 acting cell cycle genes including the disruption of endogenous interactions of the Sp1 transcription factor with the CDK6 promoter. Furthermore, in estrogen responsive MCF-7 cells, with enhanced potency 1-benzyl-I3C down regulated production of estrogen receptor-alpha protein, acts with tamoxifen to arrest breast cancer cell growth more effectively than either compound alone, and inhibited the in vivo growth of human breast cancer cell-derived tumor xenografts in athymic mice. Our results implicate 1-benzyl-I3C as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of indole-sensitive cancers.

Background. Visceral leishmaniasis (VL) is caused by Leishmania donovani and Leishmania infantum chagasi. Genome-wide linkage studies from Sudan and Brazil identified a putative susceptibility locus on chromosome 6q27.

Methods. Twenty-two single-nucleotide polymorphisms (SNPs) at genes PHF10, C6orf70, DLL1, FAM120B, PSMB1, and TBP were genotyped in 193 VL cases from 85 Sudanese families, and 8 SNPs at genes PHF10, C6orf70, DLL1, PSMB1, and TBP were genotyped in 194 VL cases from 80 Brazilian families. Family-based association, haplotype, and linkage disequilibrium analyses were performed. Multispecies comparative sequence analysis was used to identify conserved noncoding sequences carrying putative regulatory elements. Quantitative reverse-transcription polymerase chain reaction measured expression of candidate genes in splenic aspirates from Indian patients with VL compared with that in the control spleen sample.

Results. Positive associations were observed at PHF10, C6orf70, DLL1, PSMB1, and TBP in Sudan, but only at DLL1 in Brazil (combined P = 3 × 10−4 at DLL1 across Sudan and Brazil). No functional coding region variants were observed in resequencing of 22 Sudanese VL cases. DLL1 expression was significantly (P = 2 × 10−7) reduced (mean fold change, 3.5 [SEM, 0.7]) in splenic aspirates from patients with VL, whereas other 6q27 genes showed higher levels (1.27 × 10−6 < P < .01) than did the control spleen sample. A cluster of conserved noncoding sequences with putative regulatory variants was identified in the distal promoter of DLL1.

Conclusions. DLL1, which encodes Delta-like 1, the ligand for Notch3, is strongly implicated as the chromosome 6q27 VL susceptibility gene.

Objectives

This study describes parasite kinetics in the blood of visceral leishmaniasis patients treated with liposomal amphotericin B (L-AmB) or a preformed fat emulsion of amphotericin B (ApL) using real-time quantitative PCR (qPCR).

Methods

Forty-six patients were treated with a single dose (15 mg/kg of body weight) of either L-AmB (n = 13) or ApL (n = 33). qPCR was used to estimate parasite kinetics by detection of Leishmania donovani DNA using kinetoplast DNA-specific primers in peripheral blood samples using an absolute quantification method.

Results

The mean parasite load decreased from baseline (day 0) values of 894.07 and 980.48 to 71.72 and 211.52 parasite genomes/mL at day 7 in L-AmB and ApL groups, respectively, and at day 30 these further declined to 8.30 and 133.98 parasite genomes/mL, respectively. At day 30 post-treatment evaluation, the decline in parasite load was significantly greater (P = 0.024) with L-AmB compared with ApL. Four of 33 patients in the ApL group failed treatment (1 primary failure and 3 relapses) with the presence of parasites, whereas all patients in the L-AmB group were cured at 6 month follow-up.

Conclusions

qPCR can be a tool to measure parasite dynamics accurately and provide a marker to measure the efficacy of various drugs. It can be used as a test of cure, allowing us to do away with invasive and risky methods such as splenic or bone marrow aspiration.

A patient with visceral leishmaniasis showing indequate response to amphotericin-B belonging to non-endemic region is reported, with L. donovani showing increased tolerance to amphotericin-B in vivo. Four SNPs, detected in the cysteine proteinase B gene, resulted in changes to the deduced amino acid sequence: Valine→Alanine, Arginine→Leucine. Over and under expression of protein/s was observed in 65-80 kDa range and 20 kDa, respectively.

Sol-gel synthesized nickel oxide (NiO) film deposited onto indium tin oxide (ITO) coated glass plate has been utilized for the development of sensitive and stable DNA biosensor and demonstrated for diagnosis of Visceral Leishmaniasis also known as Kala-azar. Leishmania specific sensor is developed by immobilizing 23mer DNA sequence (oligonucleotide) identified from 18S rRNA gene sequences from Leishmania donovani. Characterization studies like X-ray diffraction and Scanning Electron Microscopy revealed the formation of nano-structured NiO, while immobilization of single strand (ss)-DNA of Leishmania was supported by UV-visible, Fourier Transform Infrared Spectroscopy and Scanning Electron Microscopy techniques. Response studies of ss-DNA/NiO/ITO bioelectrode are carried out using differential pulsed voltammetry in presence of methylene blue redox dye as a redox mediator. A linear response is obtained in the wide concentration range of 2pg/ml to 2μg/ml of complementary target genomic DNA (parasite DNA) within the variation of 10% for 5 sets of studies. The observed results holds promise for not only diagnosis of Kala-azar patients but also enormous potential of the nano NiO based probe for development of stable and sensitive biosensors.

Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.

Context:

There is a complex and significant correlation between respiratory disorders and psychiatric conditions. Reliability of self-reported tobacco use has been questioned in recent times.

Aims:

The current study aims at assessment of accuracy of self-reported tobacco use (both smoked and smokeless) among psychiatric out-patients.

Settings and Design:

We recruited 131 consecutive subjects from the out-patient psychiatry department of a tertiary care hospital.

Materials and Methods:

Male patients meeting the study criteria were approached for participation in the study. They were asked about their recent tobacco use history. Those reporting recent use were assessed for severity of dependence using Fagerstrom Test for Nicotine Dependence (FTND)-smoking and FTND-smokeless scales. Quantitative urine cotinine analysis was performed using the Enzyme Linked Immunesorbant Assay (ELISA) method. Based on this method, a (50 ng/ml) cut off score for urinary cotinine level for tobacco use was set.

Statistical Analysis Used:

Concordance between the self-report of tobacco use and urinary cotinine level was assessed using the Cohen's kappa. Additionally, Pearson's correlation coefficient was used to examine the correlation between the FTND-smoking and FTND-smokeless scales and the urinary cotinine levels.

Results:

The values of Cohen's kappa suggest no significant concordance between the self-reported recent tobacco use and urinary cotinine levels for both smoking and smokeless tobacco forms. The discordance was present irrespective of a higher (550 ng/ml) or a lower (50 ng/ml) cut off score for a urinary cotinine level. Pearson's correlation coefficient failed to reveal any significant direct correlation between the FTND scores and urinary cotinine levels.

Conclusions:

It is recommended to use biological markers such as urinary cotinine levels to corroborates the information provided by the patients.

This randomized, open-label study of patients in India with visceral leishmaniasis (VL) investigated the effect of food on sitamaquine and desethyl-sitamaquine pharmacokinetics. Patients were randomized to receive oral sitamaquine, 2 mg/kg/day, once a day for 21 days across four cohorts (n = 41) (fasted/fed, fed/fasted, fed/fed, and fasted/fasted) over two periods (days 1−10 and 11−21), or intravenous amphotericin B (AmB), 1 mg/kg every other day for 30 days (n = 20). Mean day 21 pharmacokinetics across the four cohorts were sitamaquine, area under curve (AUC)(0−τ) = 6,627−8,903 ng.hr/mL, AUC(0−16) = 4,859−6,633 ng.hr/mL, maximum plasma concentration (Cmax) = 401−570 ng/mL, apparent terminal half-life (t1/2) = 18.3−22.8 hr, time to reach Cmax (tmax) = 3.5−6 hr; and desethyl-sitamaquine, AUC(0−τ) = 2,307−3,163 ng.hr/mL, Cmax = 109−154 ng/mL, t1/2 = 23.0−27.9 hr, tmax = 2−10 hr, with no significant food effect. On-therapy adverse events were observed for sitamaquine in 4 (10%) of 41 patients and for AmB in 17 (85%) of 20 patients. The final clinical cure (day 180) was 85% (95% confidence interval = 70.8–94.4%) for sitamaquine and 95% (95% confidence interval = 75.1–99.9) for AmB. Sitamaquine can be taken regardless of food intake, was generally well tolerated, and showed potential efficacy in patients with visceral leishmaniasis.