Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD.

BACKGROUND: Leishmaniasis is one of the most neglected tropical diseases in the world and remains endemic in some underdeveloped regions, including western China. The phylogeny and classification of Chinese Leishmania has not been completely clarified to date, especially within the Leishmania (L.) donovani complex, although phylogenetic analyses based on a series of gene markers have been performed. More analytic methods and data are still needed. Random amplified polymorphic DNA (RAPD) technology can sensitively identify slight intraspecific differences, and it is a powerful tool to seek species-specific markers. This work attempted to identify Chinese Leishmania isolates from diverse geographic regions at the genomic level. Meanwhile, specific markers of the L. donovani complex were also developed by RAPD. METHODS: RAPD was applied to 14 Chinese Leishmania isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into a data matrix, based on which genetic similarity was calculated, and a UPGMA dendrogram was constructed to analyse the genetic diversity of these Leishmania isolates. Meanwhile, the specific amplification loci of the L. donovani complex were TA-cloned, sequenced and converted into sequence characterized amplified region (SCAR) markers, which were validated preliminarily in 17 available Leishmania strains in this study and analysed by bioinformatics. RESULTS: The cluster analyses showed that the three Leishmania sp. isolates SC10H2, SD and GL clustered together and apart from others, the strains of the L. donovani complex clearly divided into two clades, and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of the L. donovani complex, i.e., 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on 17 available Leishmania strains in this study. Through bioinformatic analyses, Marker 1-AD17 may have more specificity for PCR detection of VL, and Marker 3-O13 has the potential to encode a protein. CONCLUSIONS: The RAPD results verified that the undescribed Leishmania species causing visceral leishmaniasis (VL) in China was a unique clade distinguished from L. donovani and revealed that there was genetic differentiation among Chinese L. donovani. The identification of L. donovani-specific markers may help to provide a foundation for future research attempting to develop new specific diagnostic markers of VL and identify specific gene functions.

The Leishmania donovani species complex: A new insight into taxonomy☆.

Among the 20 or so Leishmania spp. described as pathogenic for humans, those of the Leishmania donovani complex are the exclusive causative agents of systemic and fatal visceral leishmaniasis. Although well studied, the complex is taxonomically controversial, which hampers clinical and epidemiological research. In this work, we analysed 56 Leishmania strains previously identified as L. donovani, Leishmania archibaldi or Leishmania infantum, isolated from humans, dogs and sandfly vectors throughout their distribution area. The strains were submitted to biochemical and genetic analyses and the resulting data were compared for congruence. Our results show: i) a partial concordance between biochemical and genetic-based data, ii) very limited genetic variability within the L. donovani complex, iii) footprints of frequent genetic exchange along an east-west gradient, marked by a widespread diffusion of alleles across the geographical range, and iv) a large-scale geographical spreading of a few genotypes. From a taxonomic point of view, considering the absence of relevant terminology in existing classes, the L. donovani complex could be treated as a single entity.

A Comprehensive Review of Cutaneous Leishmaniasis in Sri Lanka and Identification of Existing Knowledge Gaps.

PURPOSE: Sri Lanka is endemic to cutaneous leishmaniasis and reported as the latest focus of leishmaniasis in the Asian subcontinent. Annually, the number of leishmaniasis cases is increasing; therefore, more efficient diagnostic tools, treatment methods and effective prevention measures are indispensable. For this reason, many studies were conducted regarding leishmaniasis infections in Sri Lanka; however, some areas need more attention. Thus, in this review, we comprehensively discussed the studies on leishmaniasis carried out in Sri Lanka. METHODS: Published articles on leishmaniasis in Sri Lanka were searched on PubMed, Google Scholar and ResearchGate databases. Inclusion criteria for the articles were based on keyword searches including 'Leishmaniasis in Sri Lanka', 'Leishmaniasis vector in Sri Lanka', 'Sandfly species in Sri Lanka', 'Leishmaniasis epidemiology in Sri Lanka' which are publicly accessible as of 15th July 2019. RESULTS: In this study, we evaluated and summarized the leishmaniasis reports in Sri Lanka and mainly focused on clinical presentation of leishmaniasis infection, genetic characteristics of Leishmania donovani Sri Lankan strain, geographical distribution and associated environmental factors, immunological aspects of the infection, vector, reservoir host, risk factors, diagnosis and treatment, and prevention and control. Furthermore, we identified the areas where further research is needed to fill the essential knowledge gaps. CONCLUSIONS: Leishmaniasis has become a critically important parasitic infection in Sri Lanka, whereas the significant clinical form is cutaneous leishmaniasis. Prevalence of the leishmaniasis infections is reported from all the districts of the country. Therefore, more studies are essential to be carried out to fill the existing knowledge gaps emphasized in this review.

ISC1, a new Leishmania donovani population emerging in the Indian sub-continent: Vector competence of Phlebotomus argentipes.

Visceral leishmaniasis (VL), the most severe form of the disease, is caused by Leishmania donovani in the Indian sub-continent (ISC). Whole genome sequencing studies revealed that two parasite populations exist in the ISC: a main population named the Core Group (CG) found mostly in the lowlands, and a new, genetically different subpopulation called ISC1. Parasites belonging to the CG were shown to be responsible for the recent epidemics, while the ISC1 variant was originally identified in hilly districts of Nepal and was later on increasingly found in the lowlands. Importantly, the ISC1 and CG isolates differ in their drug susceptibility and virulence signatures, suggesting that ISC1 constitutes an emerging and functionally different variant of L. donovani. In present study we aimed to address the potential of ISC1 transmission by the natural vector of L. donovani in the lowlands, Phlebotomus argentipes. By experimental infection of sand flies with parasites of the different genotypes, we demonstrate that ISC1 and CG strains are developing similarly in P. argentipes, suggesting that P. argentipes is a fully competent vector for ISC1 parasites. Integration of previous and current findings shows thus that ISC1 is a new and different variant of L. donovani, fully adapted to spread in the ISC through the main vector. This information is directly useful for managers of the elimination program. Furthermore, integration of our successive studies (genotyping, phenotyping and vector competence) demonstrates the relevance of molecular surveillance and should be of interest for scientists working on vector borne diseases and control managers.

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent.

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.

Molecular evidence supports the expansion of visceral leishmaniasis towards non-program districts of Nepal.

BACKGROUND: Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020. METHODS: This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients' blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts. RESULTS: Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients' samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal. CONCLUSION: The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

Identification of Leishmania Species in Naturally Infected Sand Flies from Refugee Camps, Greece.

High infection rates of Leishmania donovani and L. tropica were detected in Phlebotomus spp. sand flies collected from refugee camps in Greece, indicating increased risk of infection among local populations. Detection and treatment of leishmaniasis, community education, and vector control are essential measures to prevent pathogen transmission and protect public health.

Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka.

BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.

The Leishmania donovani IMPACT-like protein possesses non-specific nuclease activity.

IMPACT (Imprinted and Ancient)-like proteins are known to be regulators of GCN2 (General control non-derepressible 2) kinases involved in translation regulation. Here, we report on cloning and characterization of an IMPACT-like protein, LdIMPACT from Leishmania donovani which harbours two domains. 'RWD domain' at the N-terminal end that mediates GCN2 regulation, while a conserved 'ancient domain' lies at the C-terminal end whose function remains elusive. Interestingly, our observations indicated that LdIMPACT has a novel non-specific nuclease activity. In silico analysis further revealed the resemblance of ancient domain of LdIMPACT to RNase PH domain (known to bind to nucleic acids). The recombinant LdIMPACT exhibited a Mg2+-dependent nuclease activity. Moreover, thermostability and pH stability assays of the protein suggest it to be a stress-responsive protein. Circular dichroism studies elucidated the conformational transitions of the enzyme in response to various temperature and pH conditions which correlated well with the activity profiles. Thus, the current study highlights the structural and functional characteristics of LdIMPACT which interestingly also possesses a novel nuclease activity. With its physiological relevance unresolved, the multifaceted LdIMPACT might therefore lie in a hitherto unknown network, whose perturbation could be an attractive therapeutic approach for treating leishmaniasis.

Pyridoxal kinase: A vitamin B6 salvage pathway enzyme from Leishmania donovani.

Pyridoxal kinase (PdxK, EC 2.7.1.35) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33 kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.

Molecular Diagnosis of Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a deadly parasitic disease, is a major public health concern globally. Countries affected by VL have signed the London Declaration on Neglected Tropical Diseases and committed to eliminate VL as a public health problem by 2020. To achieve and sustain VL elimination, it will become progressively important not to miss any remaining cases in the community who can maintain transmission. This requires accurate identification of symptomatic and asymptomatic carriers using highly sensitive diagnostic tools at the primary health service setting. The rK39 rapid diagnostic test (RDT) is the most widely used tool and with its good sensitivity and specificity is the first choice for decentralized diagnosis of VL in endemic areas. However, this test cannot discriminate between current, subclinical, or past infections and is useless for diagnosis of relapses and as a prognostic (cure) test. Importantly, as the goal of elimination of VL as a public health problem is approaching, the number of people susceptible to infection will increase. Therefore, correct diagnosis using a highly sensitive diagnostic test is crucial for applying appropriate treatment and management of cases. Recent advances in molecular techniques have improved Leishmania detection and quantification, and therefore this technology has  become increasingly relevant due to its possible application in a variety of clinical sample types. Most importantly, given current problems in identifying asymptomatic individuals because of poor correlation between the main methods of detection, molecular tests are valuable for VL elimination programs, especially to monitor changes in burden of infection in specific communities. This review provides a comprehensive overview of the available VL diagnostics and discusses the usefulness of molecular methods in the diagnosis, quantification, and species differentiation as well as their clinical applications.

Description of Leishmania species among dogs and humans in Colombian Visceral Leishmaniasis outbreaks.

We report the species detected in dogs and humans from outbreaks of visceral leishmaniasis in Colombia. In this study, 91 sera from patients (n = 38) and dogs (n = 53) diagnosed with visceral leishmaniasis using IFAT were analyzed to determine the causative species. DNA extraction, PCR amplification, DNA sequencing and species identification was performed. Results were obtained with 13 of the sera. A phylogenetic tree and a network of haplotypes were constructed. Leishmania infantum chagasi (11/13), Leishmania braziliensis (1/13) and Leishmania amazonensis (1/13) were identified as the circulating species and genetic variability in one of the L. infantum chagasi strains was demonstrated. This is the first study describing Leishmania species in outbreaks of visceral leishmaniasis in Colombia.

Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival.

Leishmania donovani is the main cause of visceral leishmaniasis (VL) in East Africa. Differences between northern Ethiopia/Sudan (NE) and southern Ethiopia (SE) in ecology, vectors, and patient sensitivity to drug treatment have been described, however the relationship between differences in parasite genotype between these two foci and phenotype is unknown. Whole genomic sequencing (WGS) was carried out for 41 L. donovani strains and clones from VL and VL/HIV co-infected patients in NE (n = 28) and SE (n = 13). Chromosome aneuploidy was observed in all parasites examined with each isolate exhibiting a unique karyotype. Differences in chromosome ploidy or karyotype were not correlated with the geographic origin of the parasites. However, correlation between single nucleotide polymorphism (SNP) and geographic origin was seen for 38/41 isolates, separating the NE and SE parasites into two large groups. SNP restricted to NE and SE groups were associated with genes involved in viability and parasite resistance to drugs. Unique copy number variation (CNV) were also associated with NE and SE parasites, respectively. One striking example is the folate transporter (FT) family genes (LdBPK_100390, LdBPK_100400 and LdBPK_100410) on chromosome 10 that are single copy in all 13 SE isolates, but either double copy or higher in 39/41 NE isolates (copy number 2-4). High copy number (= 4) was also found for one Sudanese strain examined. This was confirmed by quantitative polymerase chain reaction for LdBPK_100400, the L. donovani FT1 transporter homolog. Good correlation (p = 0.005) between FT copy number and resistance to methotrexate (0.5 mg/ml MTX) was also observed with the haploid SE strains examined showing higher viability than the NE strains at this concentration. Our results emphasize the advantages of whole genome analysis to shed light on vital parasite processes in Leishmania.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Miltefosine Resistant Field Isolate From Indian Kala-Azar Patient Shows Similar Phenotype in Experimental Infection.

Emergence of resistance to drugs used to treat the Indian Kala-azar patients makes control strategy shattered. In this bleak situation, Miltefosine (MIL) was introduced to treat mainly antimonial unresponsive cases. Within years, resistance to MIL has been reported. While checking the MIL sensitivity of the recent KA clinical isolates (n = 26), we came across one isolate which showed four times more EC50 for MIL than that of MIL-Sensitive (MIL-S) isolates and considered as putative MIL-Resistant (MIL-R). The expressions of LdMT and LdRos3 genes of this isolate were found down regulated. Th1/Th2 cytokines, ROS and NO, FACS dot plots and mitochondrial trans membrane potential measurement were performed. In vivo hamster model with this MIL-R isolate showed much lesser reduction in liver weight (17.5%) compared to average reduction in liver weight (40.2%) of the animals infected with MIL-S isolates. The splenic and hepatic stamps smears of MIL-R infected hamsters revealed the retention of parasite load of about 51.45%. The splenocytes of these animals failed to proliferate anti leishmanial T-cells and lack of cell mediated immunity hampered recovery. Thus, these phenotypic expressions of experimental model may be considered similar to that of the MIL unresponsive patients. This is first such kind of report.

Single locus genotyping to track Leishmania donovani in the Indian subcontinent: Application in Nepal.

BACKGROUND: We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010. METHODOLOGY / PRINCIPLE FINDINGS: As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002-2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution. CONCLUSIONS / SIGNIFICANCE: Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region.

Buparvaquone nanostructured lipid carrier development: physicochemical and in vitro leishmanicidal performances / Desenvolvimento de carreadores lipídicos nanoestruturados contendo buparvaquona: caracterização físico-química e avaliação da atividade leishmanicida in vitro

Leishmaniases is a group of diseases caused by parasites of the genus Leishmania. The estimated number of deaths from visceral leishmaniases ranges from 20,000 to 50,000 annually. The most common treatment over the past 60 years has been pentavalent antimonials. Besides the doubtful effectiveness, they present several disadvantages such as the need for parenteral administration, large doses, long treatment, severe toxicity and parasite resistance. Buparvaquone (BPQ), a drug used for veterinary treatment of theileriosis, showed promising activity against Leishmania spp. However, due to its low aqueous solubility and bioavailability, it has failed in in vivo tests. The use of nanotechnologies has the potential to overcome these drawbacks due to the following advantages: increase in drug water-solubility, increase in therapeutic efficacy and treatment toxicity reduction. Therefore, the present work aimed the development, optimization, physical-chemical evaluation and in vitro performances of nanostructured lipid carriers (NLC) for BPQ encapsulation. The NLC preparation was performed by high pressure homogenization, and surface response and factorial design were applied to formulation optimization. In vitro dissolution profiles were evaluated in phosphate buffer pH 7.4 with tween 80 0.07% w/v or sodium dodecyl sulfate 1% w/v and simulated body fluid pH 7.4. Cytotoxicity was evaluated in mouse peritoneal macrophages and leishmanicidal activity in L. infantum amastigotes. Six optimized NCL were prepared and they showed solubility improvement from 1.5- fold to 611-fold when compared with free BPQ, depending on the formulation and medium. Dissolution profiles showed the NLC formulation suitability for BPQ regarding oral administration, the release could reach 83.29% of a 4mg dose in 30 minutes for formulation of 175.1 nm, while the free drug could be dissolved only 2.89% of the same dose after 4 hours. Moreover, formulation of 230.7 nm showed 81.42% of drug release in in phosphate buffer pH 7.4 with dodecyl sulfate 1.0% w/v after 30 minutes, while BPQ did not dissolved. Cytotoxicity assay showed the safety of all formulations. The iv CC50 values were close to 500 µM, while the IC50 against amastigotes was only 456.5 nM for free BPQ. Developed NLCs showed an increase in IC50 from 2.0 to 3.1-fold when compared to free drug in the in vitro leishmanicidal evaluation. Therefore, the NLC containing BPQ are a promising alternative for the treatment of leishmaniases as oral and parenteral drug dosage forms. Additionally, they have a potential use for lymphatic targeted drug delivery, which can be an innovative approach for this neglected disease.

Leishmanioses são um grupo de doenças causadas por parasitas do gênero Leishmania. O número estimado de óbitos por leishmaniose visceral varia entre 20.000 e 50.000 por ano. O tratamento mais comum nos últimos 60 anos tem sido os antimônios pentavalentes. Além da eficácia duvidosa, eles apresentam várias desvantagens, como a necessidade de administração parenteral, altas doses, tratamento prolongado, toxicidade severa e resistência parasitária. Buparvaquona (BPQ), um fármaco usado para tratamento veterinário da teileriose, mostrou atividade promissora contra Leishmania donovani. No entanto, devido à sua baixa solubilidade e biodisponibilidade aquosa, falhou em testes in vivo. O uso das nanotecnologias tem o potencial de superar esses obstáculos devido às seguintes vantagens: aumento da solubilidade em água, aumento da eficácia terapêutica e redução da toxicidade do tratamento. Portanto, o presente trabalho objetivou o desenvolvimento, otimização, avaliação físico-química e avaliação do desempenho in vitro de carreadores lipídicos nanoestruturados (NLC) para o encapsulação da BPQ. A preparação do NLC foi realizada por homogeneização de alta pressão e superfície de resposta e planejamento fatorial foram aplicados à otimização das formulações. Os perfis de dissolução in vitro foram avaliados em tampão fosfato pH 7.4 com tween 80 a 0.07% p/v ou dodecilsulfato de sódio 1.0% p/v e fluido corporal simulado pH 7.4. A citotoxicidade foi avaliada em macrófagos peritoneais de camundongos e atividade leishmanicida em amastigotas de L. infantum. Foram preparados quatro NCL otimizados e mostraram melhora da solubilidade de 1,5 a 611 vezes quando comparado com a BPQ livre, dependendo da formulação e do meio. Os perfis de dissolução mostraram a adequação da formulação NLC para BPQ em relação à administração oral. A dissolução pode atingir 83,29% de uma dose de 4.0 mg em 30 minutos para a formulação de 175,1 nm, enquanto o fármaco livre dissolveu apenas vi 2,89% da mesma dose após 4 horas. Além disso, a formulação de 230,7 nm mostrou 81,42% de liberação do fármaco em tampão fosfato pH 7.4 com dodecil sulfato de sódio 1.0% p/v após 30 minutos, enquanto o BPQ não se dissolveu. O teste de citotoxicidade mostrou a segurança de todas as formulações. Os valores CC50 foram próximos de 500 µM, enquanto o IC50 em amastigotas foi de apenas 456,5 nM para BPQ livre. Os NLC desenvolvidos mostraram um aumento no IC50 de 2,0 a 3,1 vezes quando comparado ao;fármaco livre na avaliação leishmanicida in vitro. Logo, as NLC contendo BPQ são uma alternativa promissora para o tratamento de leishmanioses como formas farmacêuticas oral e parenteral. Além disso, eles têm um uso potencial para a sítio-específico ao sistema linfático, o que pode ser uma abordagem inovadora para esta doença negligenciada.

Genetic markers for antimony resistant clinical isolates differentiation from Indian Kala-azar.

Visceral Leishmaniasis or Kala-azar is caused by the protozoan parasites belonging to the Genus Leishmania. Once thought eradicated from the Indian subcontinent, the disease came back with drug resistance to almost all prevalent drugs. Molecular epidemiological studies revealed the polymorphic nature of the population of the main player of the disease, Leishmania donovani and involvement of other species (L. tropica) and other genus (Leptomonas) with the disease. This makes control measures almost futile. It also strongly demands the characterization of each and every isolate mandatory which is not done. In this background, the present study has been carried out to assess the genetic attributes of each clinical isolates (n=26) of KA and PKDL patients from India and Bangladesh. All the isolates were characterized through Restriction Fragment Length Polymorphism (RFLP) analysis to ascertain their species identity. 46.2% of the isolates were found to be Sodium Stibogluconate (SSG) resistant by amastigote-macrophage model. When the clinical isolates were subjected to Single Stranded Conformation Polymorphism (SSCP) of Internal Transcribed Spacer 1 (ITS1), Internal Transcribed Spacer 2 (ITS2) and some anonymous markers, the drug resistant Leishmania isolates of SSG can be distinguished from the sensitive isolates distinctly. This study showed for the first time, the genetic markers for SSG drug resistance of Indian Kala-azar clinical isolates.

Seroepidemiology and molecular diversity of Leishmania donovani complex in Georgia.

BACKGROUND: Leishmaniasis includes multiple clinical syndromes, most notably visceral, cutaneous, and mucosal forms. Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease endemic to large parts of Africa and Asia, and in South-Eastern Europe (Greece, Turkey, Georgia). Visceral leishmaniasis is a parasitic zoonosis caused by species of the L. donovani complex. In the classical epidemiological model the main reservoir for VL are canines. METHODS: The study included a cohort of 513 individuals of both genders (190 males and 323 females) from the ages of 1 to 70 years that were screened in ten villages across two districts in Kakheti using the Kalazar Detect™ rK39 rapid diagnostic test. The phylogenetic diversity patterns of local strains, based on the rDNA internal transcribed spacer (ITS) sequences, were assessed for samples obtained from patients with suspected L. donovani infection, from canine reservoirs and from Phlebotomus sand flies obtained from different geographical areas of Georgia and from Azerbaijan. RESULTS: Out of a total of 600 domestic dog blood samples 95 (15.8 %) were positive by rK39 rapid diagnostic tests. For symptomatic domestic dogs, the testing of conjunctival swabs or bone marrow aspirates revealed a higher VL incidence in Kvareli District (Kvareli; 19.4 %, n = 329) compared with that observed for Sagarejo District (Sagarejo; 11.4 %, n = 271). A total of 231 sand flies of both genders were collected during the 2-month period; of the 114 females, 1.75 % were PCR positive for the presence of Leishmania spp. CONCLUSIONS: VL infection rates remain high in both canines and humans in Georgia, with disease in several known natural foci. The genetic relationships derived from rDNA internal transcribed spacer (ITS) sequence comparisons identified genetic subgroups, revealing preliminary insights into the genetic structure of L. donovani complex members currently circulating in the South Caucasus and demonstrates the utility of ITS-based genotyping in the resource-limited country of Georgia.

In vivo characterization of two additional Leishmania donovani strains using the murine and hamster model.

Leishmania donovani is a protozoan parasite causing the neglected tropical disease visceral leishmaniasis. One difficulty to study the immunopathology upon L. donovani infection is the limited adaptability of the strains to experimental mammalian hosts. Our knowledge about L. donovani infections relies on a restricted number of East African strains (LV9, 1S). Isolated from patients in the 1960s, these strains were described extensively in mice and Syrian hamsters and have consequently become 'reference' laboratory strains. L. donovani strains from the Indian continent display distinct clinical features compared to East African strains. Some reports describing the in vivo immunopathology of strains from the Indian continent exist. This study comprises a comprehensive immunopathological characterization upon infection with two additional strains, the Ethiopian L. donovani L82 strain and the Nepalese L. donovani BPK282 strain in both Syrian hamsters and C57BL/6 mice. Parameters that include parasitaemia levels, weight loss, hepatosplenomegaly and alterations in cellular composition of the spleen and liver, showed that the L82 strain generated an overall more virulent infection compared to the BPK282 strain. Altogether, both L. donovani strains are suitable and interesting for subsequent in vivo investigation of visceral leishmaniasis in the Syrian hamster and the C57BL/6 mouse model.