Indian Visceral Leishmaniasis Research Articles

Susceptibility Status ofPhlebotomus Argentipes, to Synthetic Pyrethroid Insecticides in Kala Azar Endemic Parts of Bihar, India in Relation to Elimination of Visceral Leishmaniasis

Since 1976, DDT @1gm/sq meter being sprayed to control Kala azar in India. Government of India along with Government of Bihar have decided to use synthetic pyrethroid insecticides in order to eliminate kala azar from India by 2016. A study was therefore conducted in 2015 in Bihar, selecting 13 villages of four highly kala azar endemic districts like Siwan, Samastipore, Muzaffarpore and Katihar in order to note baseline susceptibility status of commonly used synthetic pyrethroids like Deltamethrin, Lambda-cyhalothrin, Alpha-cypermethrin to Phlebotomus argentipes, the vector of Kala azar in India. The test was conducted as per standard WHO technique using WHO test kits. Adult Phlebotomus argentipes were found highly susceptible to those insecticides.

Short-Course Treatment Regimen of Indian Visceral Leishmaniasis with an Indian Liposomal Amphotericin B Preparation (Fungisome™)

India bears the burden of about half of global visceral leishmaniasis (VL) cases with emerging problems of stibanate resistance. Liposomal preparations have improved treatment outcome through shorter duration of therapy and lower toxicity compared with conventional amphotericin B. We report the efficacy of two short-course regimens of an Indian preparation of liposomal amphotericin B (Fungisome™) for VL caused by Leishmania donovani in India. An open-label, randomized, single-center comparative study was undertaken from 2008 to 2011, involving 120 treatment naive non-human immunodeficiency virus VL patients randomly allocated to two groups. Fungisome™ was given, in groups A (N = 60), 5 mg/kg daily for 2 days and B (N = 60), 7.5 mg/kg daily for 2 days, as intravenous infusion. Initial cure rate was 100% in both the groups after 1 month posttreatment. At 6 months after completion of treatment, definitive cure rate was group A 90% (54/60, 95% confidence interval (CI): 80.55-95.72%); group B: 100% (95% CI: 95.92-100%); (P = 0.027). No serious adverse events occurred in either group. The short-course, 2-day regimen of 15 mg/kg Fungisome™ infusion is easy to administer, effective, and safe for treatment of VL caused by L. donovani in India.

Mannose-binding Lectin (MBL) as a susceptible host factor influencing Indian Visceral Leishmaniasis

Visceral Leishmaniasis (VL), caused by Leishmania donovani is endemic in the Indian sub-continent. Mannose-binding Lectin (MBL) is a complement lectin protein that binds to the surface of Leishmania promastigotes and results in activation of the complement lectin cascade. We utilized samples of 218 VL patients and 215 healthy controls from an Indian population. MBL2 functional variants were genotyped and the circulating MBL serum levels were measured. MBL serum levels were elevated in patients compared to the healthy controls (adjusted P=0.007). The MBL2 promoter variants −78C/T and +4P/Q were significantly associated with relative protection to VL (−78C/T, OR=0.7, 95% CI=0.5–0.96, adjusted P=0.026 and +4P/Q, OR=0.66, 95% CI=0.48–0.9, adjusted P=0.012). MBL2*LYQA haplotypes occurred frequently among controls (OR=0.69, 95% CI=0.5–0.97, adjusted P=0.034). MBL recognizes Leishmania and plays a relative role in establishing L. donovani infection and subsequent disease progression. In conclusion, MBL2 functional variants were associated with VL.

Association of Ficolin-2 Serum Levels and FCN2 Genetic Variants with Indian Visceral Leishmaniasis.

BackgroundVisceral leishmaniasis (VL), one of the neglected tropical diseases, is endemic in the Indian subcontinent. Ficolins are circulating serum proteins of the lectin complement system and involved in innate immunity.MethodsWe have estimated ficolin-2 serum levels and analyzed the functional variants of the encoding gene FCN2 in 218 cases of VL and in 225 controls from an endemic region of India.ResultsElevated levels of serum ficolin-2 were observed in VL cases compared to the controls (adjusted P<0.0001). The genetic analysis revealed that the FCN2 structural variant +6359 C>T (p.T236M) was associated with VL (OR=2.2, 95% CI=1.23-7.25, P=0.008) and with high ficolin-2 serum levels. We also found that the FCN2*AAAC haplotype occurred more frequently among healthy controls when compared to cases (OR=0.59, 95%CI=0.37-0.94, P=0.023).ConclusionsOur findings indicate that the FCN2 variant +6359C>T is associated with the occurrence of VL and that ficolin-2 serum levels are elevated in Leishmania infections.

IL10 Variant g.5311A Is Associated with Visceral Leishmaniasis in Indian Population.

BackgroundVisceral leishmaniasis (VL) is a multifactorial disease, where the host genetics play a significant role in determining the disease outcome. The immunological role of anti-inflammatory cytokine, Interleukin 10 (IL10), has been well-documented in parasite infections and considered as a key regulatory cytokine for VL. Although VL patients in India display high level of IL10 in blood serum, no genetic study has been conducted to assess the VL susceptibility / resistance. Therefore, the aim of this study is to investigate the role of IL10 variations in Indian VL; and to estimate the distribution of disease associated allele in diverse Indian populations.MethodologyAll the exons and exon-intron boundaries of IL10 were sequenced in 184 VL patients along with 172 ethnically matched controls from VL endemic region of India.Result and DiscussionOur analysis revealed four variations; rs1518111 (2195 A>G, intron), rs1554286 (2607 C>T, intron), rs3024496 (4976 T>C, 3’ UTR) and rs3024498 (5311 A>G, 3’ UTR). Of these, a variant g.5311A is significantly associated with VL (χ2=18.87; p =0.00001). In silico approaches have shown that a putative micro RNA binding site (miR-4321) is lost in rs3024498 mRNA. Further, analysis of the above four variations in 1138 individuals from 34 ethnic populations, representing different social and linguistic groups who are inhabited in different geographical regions of India, showed variable frequency. Interestingly, we have found, majority of the tribal populations have low frequency of VL (‘A’ of rs3024498); and high frequency of leprosy (‘T’ of rs1554286), and Behcet’s (‘A’ of rs1518111) associated alleles, whereas these were vice versa in castes. Our findings suggest that majority of tribal populations of India carry the protected / less severe allele against VL, while risk / more severe allele for leprosy and Behcet’s disease. This study has potential implications in counseling and management of VL and other infectious diseases.

Generation of luciferase-expressing Leishmania infantum chagasi and assessment of miltefosine efficacy in infected hamsters through bioimaging.

BackgroundThe only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters.Methodology/Principal FindingsA transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival.Conclusions/SignificanceLuciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.

Monitoring Susceptibility Status of Phlebotomus argentipes (Diptera:Psychodidae) at Bihar (India) for the Procurement of Homozygous DDT Resistant Colony

Brisk and continuous application of routine insecticide i.e., dichlorodiphenyltrichloroethane (DDT) for long had evoked the problem of resistance among Indian Visceral Leishmaniasis (VL) vector Phlebotomus argentipes (Diptera: Psychodidae) in Bihar, affecting DDT based strategies for controlling vector population. In this way, strong resistant colony of P. argentipes would provide fundamental platform to reveal the mechanistic reason behind the arousal of DDT resistance among sand flies. Present report summarizes our experiences with baseline establishment and maintenance of DDT resistant colony of P. argentipes after studying susceptibility profile for districts of Bihar. The district with lowest susceptibility or highest resistance against DDT would inculcate the collection of resistant sand flies for procuring resistant colony of P. argentipes, the foremost objective of the present study. For this, DDT susceptibility test were carried out at seven districts of Bihar (India), followed by the selection of Vaishali district as a ‘perfect site for resistant sand fly collection’. Here Percentage Mortality Range (PMR) and Corrected Mortality Rate (CMR) i.e., 41.00-52.73 and 44.83 % respectively were recorded to be lowest indicating the presence of sand flies highly resistant to DDT. Single blood-fed female and a male P. argentipes, collected from the selected site were confined together for oviposition and rearing of developmental stages for obtaining its next generation.. Inherited DDT resistant character of progenies of highly resistant parent was estimated and compared by quantifying Mortality Frequency Rate and DDT Resistant Rate through the insecticide susceptibility test conducted at its subsequent generations. Significant fluctuation within DRR (with 67.29 ± 17.28; 95% C.I=54.49-80.09) was observed throughout the 7th generation establishing intrusion of susceptibility factor in homozygous resistant colony of P. argentipes after achieving absolute resistant in F4 generation of P. argentipes. Though invaded with insecticide susceptibility, resistance dominate the colony. So far, colony has reached its 7th generation and culturing well in the laboratory.

The overexpression of genes of thiol metabolism contribute to drug resistance in clinical isolates of visceral leishmaniasis (kala azar) in India.

BackgroundVisceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Chemotherapy remains one of the most important tools in the control of VL. Current chemotherapy for Kala Azar in India relies on a rather limited arsenal of drugs including sodium antimony gluconate and amphotericin B in addition to the very expensive drug miltefosine. Pentavalent antimonials have been used for more than half a century in the therapy of leishmaniasis as it is relatively safe and inexpensive, however, the spread of resistance to this drug is forcing clinicians in India to abandon this treatment. Consequently, improvement of antimonial chemotherapy has become a major challenging area of study by leishmaniacs worldwide. The alarming emergence of resistance to the commonly used antleishmanial drug, sodium antimony gluconate, in India, has led us to elucidate the resistance mechanism(s) in clinical isolates. Studies on laboratory mutants have shown that resistance to antimonials is highly dependent on thiol levels. The parasite evades cytotoxic effects of antimonial therapy by enhanced efflux of drug upon conjugation with thiols, through overexpressed membrane proteins belonging to the superfamily of ABC transporters.MethodsWe have carried out functional studies to determine the activity of the efflux pumps in antimonial resistant clinical isolates collected from disease endemic areas in India and also carried out molecular characterization of thiol levels in these parasites.ResultsOverexpression of the gene coding for γ glutamylcysteine synthetase was observed in these resistant clinical isolates thereby establishing that thiols represent the key determinants of antimonial resistance. The SbIII/thiol conjugates can be sequestered by ABC transporter multidrug resistance protein A (MRPA) into intracellular organelles or can be directly pumped out by an uncharacterized transporter.ConclusionsOur studies investigating antimonial resistance in different L. donovani clinical isolates suggest that over functioning of MRP plays a role in generation of antimony resistance phenotype in some L. donovani clinical isolates.

Susceptibility of the sandfly Phlebotomus argentipes Annandale and Brunetti (Diptera: Psychodidae) to insecticides in endemic areas of visceral leishmaniasis in Bihar, India.

We present the results of susceptibility tests conducted on the sandfly Phlebotomus argentipes, the vector of visceral leishmaniasis in India. Adult P. argentipes insects were collected from 42 villages in 6 districts of the state of Bihar, India, as follows: Patna, Vaishali, Muzaffarpur, Samastipur, Sheohar, and Sitamarhi. These adult insects were exposed to 4% DDT-, 5% malathion-, and 0.05% deltamethrin-impregnated papers using a WHO test kit by following the standard procedures. In 16 (38.1%) of 42 villages surveyed, the P. argentipes populations developed resistance to DDT. Susceptibility tests using the organophosphate malathion in 22 villages revealed that in 1 (4.5%) village, the species developed resistance to this insecticide. P. argentipes was, however, highly susceptible to the synthetic pyrethroid deltamethrin. For long-term vector control of P. argentipes, it will be necessary to overcome the threat of insecticide resistance in this species.

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

BackgroundVisceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care.Methodology/Principal FindingsAll IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001).Conclusions/SignificanceSix months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.

Analysis of genetic variants in the IL4 promoter and VNTR loci in Indian patients with Visceral Leishmaniasis

Visceral Leishmaniasis (VL) is the most severest form of Leishmaniasis and resistance to infection is mediated by cellular immune responses. Interleukin 4 (IL-4) orchestrates of Th2 and Th1 immune responses during infections. In this study, we aimed to investigate possible association between three functional IL-4 polymorphisms −590C/T (rs2243250), −34C/T (rs2070874) and 70bp VNTR (rs79071878 in intron3) with VL in an Indian cohort comprising of 197 VL patients and 193 healthy controls. The three investigated IL-4 polymorphisms were in strong linkage disequilibrium. The investigated IL-4 alleles, genotypes and the reconstructed haplotypes were not significantly distributed between the VL patients and healthy controls. Our study signifies no possible association of functional IL-4 polymorphisms with Indian VL and postulate other vital genes involved in the IL-4 pathway may provide genetic clues to elucidate of IL-4 regulation and immune-pathogenesis during VL.

Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis

BackgroundVisceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.ResultIn this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA.ConclusionsThese proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-014-0048-z) contains supplementary material, which is available to authorized users.

Parasite load estimation by qPCR differentiates between asymptomatic and symptomatic infection in Indian visceral leishmaniasis

Using quantitative PCR (qPCR), we differentiated asymptomatic and symptomatic Indian Leishmania donovani infection. qPCR on blood of 40 visceral leishmaniasis, 130 endemic, and 40 non-endemic healthy controls showed 500 times less (P < .0001) parasitemia in asymptomatic compared to the symptomatic ones and threshold of 5 parasite genome/mL for the clinical disease.

Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis

BackgroundVisceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response.Methodology/Principal FindingsWe determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log10 50% end-point titres (1/log10t50) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8–61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log10t50: 3.60–4.15) versus Sudan (mean 1/log10t50: 1.88–2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log10t50: 4.15 versus 1.99 = 144-fold (p<0.0001).Conclusions/SignificanceAnti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn2+ deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa.

In vitro Susceptibility of Leishmania donovani to Miltefosine in Indian Visceral Leishmaniasis

Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM = 3.74 ± 0.38 μM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 μM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM = 5.58 ± 0.56 μM) and those who failed treatment (N = 28, mean IC50 ± SEM = 4.53 ± 0.47 μM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible for MIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs.

Sand Fly (Diptera: Psychodidae) Abundance and Species Diversity in Relation to Environmental Factors in Parts of Coastal Plains of Southern India

Abundance pattern of sand flies in relation to several environmental factors, such as type of areas, dwellings, landforms, land usage pattern, and surface soil pH, was assessed in 81 areas or villages of Puducherry district, Puducherry Union Territory, located on the coastal plain of southern India, for three seasons, between November 2006 and October 2008, adopting hand-catch method. In total, 1,319 sand fly specimens comprising 12 species under two genera, viz., Phlebotomus and Sergentomyia, were collected. Among them, Phlebotomus (Euphlebotomus) argentipes Annandale & Brunetti, the vector of visceral leishmaniasis in India, was the predominant species in all habitats surveyed. The hierarchical cluster analysis showed that the density of sand flies was 10-fold higher in high-density group and fivefold higher in medium-density group, compared with the no or low-density group. Sand fly density was found to be influenced significantly with the type of areas, dwellings, landforms, land usage pattern, and surface soil pH in different groups. Rural areas located on fluvial landform with alkaline surface soil pH, supporting rice cultivation and luxuriant vegetation, are the most influencing factors that favor sand fly abundance and diversity in this district.

Visceral and post-Kala-Azar dermal leishmaniasis isolates show significant difference in their in vitro drug susceptibility pattern

Visceral leishmaniasis (VL) remains a major health problem in old world, and India accounts for half of the world burden. The widespread emergence of resistance to standard drug in India poses a major obstacle in the control of leishmaniasis. Post-Kala-Azar dermal leishmaniasis (PKDL) is considered as main source of drug resistance. Experimental data indicate that resistance against newer drugs is also imminent. Therefore, in vitro studies were carried out to test minimum parasiticidal concentration of five conventional and newly introduced anti-leishmanial drugs against 20 field isolates of Leishmania donovani obtained from visceral and post-Kala-Azar dermal leishmaniasis patients of India. Study revealed wide range of variation in minimum inhibitory concentration of sodium antimony gluconate (SAG). PKDL isolates displayed significantly lower susceptibility to SAG and miltefosine than VL isolates with P value of 0.0006 and 0.0243, respectively. All clinical isolates had higher IC(50) value for paromomycin and miltefosine as compared to reference strain indicating their vulnerability to develop unresponsiveness. However, isolates were uniformly susceptible to pentamidine and amphotericin B. The results of gene expression analysis of AQP1 were largely in agreement with phenotypic drug sensitivity results. Interestingly, significant down-regulation of AQP1 was observed in PKDL isolates as compared to VL isolates indicating their increased propensity for drug unresponsiveness. However, no significant difference in mRNA expression of LdMT and LdRos3 gene was found for two groups. The present study unravels valuable baseline scientific data showing variation in the drug susceptibility pattern in the L. donovani isolates. The information might have impact on the management and control of Indian visceral leishmaniasis.

Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

The Burden of Visceral Leishmaniasis in India: Challenges in Using Remote Sensing and GIS to Understand and Control

Visceral leishmaniasis (VL) continues to constitute immense public health problems and be an obstacle to socioeconomic development in India. The scrutiny of this disease remains a necessary step in its control, eradication, and prevention. Space technologies proffer new opportunities for rapid appraisal of endemic areas, stipulation of trustworthy estimation of populations at risk, prediction of disease distributions in areas that lack baseline data and are difficult to access, and guiding intervention strategies. Here, we focus on the epidemiology and control of VL in India and review remote sensing (RS) and GIS applications to date. For the original evidenced report, a search was done on electronic databases of PubMed, ERMSS, EQUEST and from online Internet searching to come across the recent research problems. We discuss the previous work conducted in this field, outlined potential new applications of GIS and RS techniques, and utilization of emerging satellite information, as they hold a promising area to further enhance VL risk mapping and prediction. Finally, we concluded with a discussion of strategies to overcome some of the perspective challenges of GIS and RS applications for VL control, so that supplementary and continued progress can be made to control this disease in India and elsewhere.

PCR-RFLP Based Method for Molecular Differentiation of Sand Fly Species &lt;I&gt;Phlebotomus argentipes&lt;/I&gt;, &lt;I&gt;Phlebotomus papatasi&lt;/I&gt;, and &lt;I&gt;Sergentomyia babu&lt;/I&gt; Found in India

PCR-Restriction fragment length polymorphism is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis in India, various species of morphological similar sand fly exist but only female Phlebotomus argentipes is the vector for visceral leishmaniasis. In the current 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 HinfI or HpaII restriction enzymes. Each of the restriction enzyme produced a species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be used in the identification of sand fly species.