Leishmania donovani complex (Kinetoplastida, Trypanosomatidae): Comparison of deoxyribonucleic acid based techniques for typing of isolates from Ethiopia

In Ethiopia, visceral leishmaniasis (VL) is an increasing public health concern. Recently, a new outbreak of VL claimed the lives of hundreds of Ethiopians. Mapping its distribution and the identification of the causative Leishmania species is important for proper use of resources and for control planning. The choice of appropriate typing technique is the key for determining the infecting species. Here we compared three deoxyribonucleic acid (DNA) based markers. We used, for the first time, cpbE and cpbF (cpbE/F) PCR-RFLP and demonstrated that it clearly differentiates Leishmania donovani from Leishmania infantum. The cpbE/F PCR-RFLP gave identical banding pattern for all L. donovani strains irrespective of their geographic origin. With the K26 (primers) PCR-RFLP, the L. donovani strains gave a banding pattern different from L. infantum and showed variation with geographic origin. The Ethiopian isolates typed as L. donovani by the PCR-RFLP of the cpbE/F (gene) and K26 (primers) showed two types of patterns with the T2/B4 (primers) PCR-RFLP; one group with L. infantum-like and the other L. donovani-like pattern. Phylogenetic analysis using cpbE/F sequences showed variation with geographic origin of strains and the African strains of L. donovani are more distantly related to L. infantum. Moreover, the Ethiopian isolates were seen to be closely related to the Sudanese, Kenyan and Indian strains. Thus, we recommend the use of more than one marker to study the population genetics of L. donovani complex.     

Therapy with sodium stibogluconate in stearylamine-bearing liposomes confers cure against SSG-resistant Leishmania donovani in BALB/c mice

Background

Resistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life.

Methodology/Principal Findings

We investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)–stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-β, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG.

Conclusions/Significance

The design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.     

Multilocus microsatellite typing (MLMT) of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group

Background

New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.

Methodology/Principal Findings

The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.

Conclusion

The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.     

Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas

Background

Parasites'' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease.

Methodology/Principal Findings

Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system.

Conclusions/Significance

This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated.     

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background

Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Methodology/Principal Findings

We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain.

Conclusions/Significance

The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.     

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

Background

Visceral 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 Findings

We 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/Significance

Anti-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.     

Multilocus sequence and microsatellite identification of intra-specific hybrids and ancestor-like donors among natural Ethiopian isolates of Leishmania donovani

Protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis (hybrid vigour), spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. Here we analyse 11 natural Ethiopian Leishmania donovani isolates consisting of four putative hybrids, seven parent-like isolates and over 90 derived biological clones. We apply a novel combination of high resolution multilocus microsatellite typing (five loci) and multilocus sequence typing (four loci) that together distinguish parent-like and hybrid L. donovani strains. Results indicate that the four isolates (and their associated biological clones) are genetic hybrids, not the results of mixed infections, each possessing heterozygous markers consistent with inheritance of divergent alleles from genetically distinct Ethiopian L. donovani lineages. The allelic profiles of the putative hybrids may have arisen from a single hybridisation event followed by inbreeding or gene conversion, or alternatively from two or more hybridisation events. Mitochondrial sequencing showed uniparental maxicircle inheritance for all of the hybrids, each possessing a single mitochondrial genotype. Fluorescence activated cell sorting analysis of DNA content demonstrated that all hybrids and their associated clones were diploid. Together the data imply that intra-specific genetic exchange is a recurrent feature of natural L. donovani populations, with substantial implications for the phyloepidemiology of Leishmania.     

Comparison of Visceral Leishmaniasis Diagnostic Antigens in African and Asian Leishmania donovani Reveals Extensive Diversity and Region-specific Polymorphisms

Background

Visceral leishmaniasis (VL), caused by infection with Leishmania donovani complex, remains a major public health problem in endemic regions of South Asia, East Africa, and Brazil. If untreated, symptomatic VL is usually fatal. Rapid field diagnosis relies principally on demonstration of anti-Leishmania antibodies in clinically suspect cases. The rK39 immunochromatographic rapid diagnostic test (RDT) is based on rK39, encoded by a fragment of a kinesin-related gene derived from a Brazilian L. chagasi, now recognised as L. infantum, originating from Europe. Despite its reliability in South Asia, the rK39 test is reported to have lower sensitivity in East Africa. A reason for this differential response may reside in the molecular diversity of the rK39 homologous sequences among East African L. donovani strains.

Methodology/Principal Findings

Coding sequences of rK39 homologues from East African L. donovani strains were amplified from genomic DNA, analysed for diversity from the rK39 sequence, and compared to South Asian sequences. East African sequences were revealed to display significant diversity from rK39. Most coding changes in the 5′ half of repeats were non-conservative, with multiple substitutions involving charge changes, whereas amino acid substitutions in the 3′ half of repeats were conservative. Specific polymorphisms were found between South Asian and East African strains. Diversity of HASPB1 and HASPB2 gene repeat sequences, used to flank sequences of a kinesin homologue in the synthetic antigen rK28 designed to reduce variable RDT performance, was also investigated. Non-canonical combination repeat arrangements were revealed for HASPB1 and HASPB2 gene products in strains producing unpredicted size amplicons.

Conclusions/Significance

We demonstrate that there is extensive kinesin genetic diversity among strains in East Africa and between East Africa and South Asia, with ample scope for influencing performance of rK39 diagnostic assays. We also show the importance of targeted comparative genomics in guiding optimisation of recombinant/synthetic diagnostic antigens.     

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.     

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Background

For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.

Methodology and Principle Findings

We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients'' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.

Conclusion

The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.     

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

Background

Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region.

Principal Findings

KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World.

Conclusions

LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.     

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.     

Visceral Leishmaniasis Relapse in Southern Sudan (1999–2007): A Retrospective Study of Risk Factors and Trends

Background

Risk factors associated with L. donovani visceral leishmaniasis (VL; kala azar) relapse are poorly characterized.

Methods

We investigated patient characteristics and drug regimens associated with VL relapse using data from Médecins Sans Frontières - Holland (MSF) treatment centres in Southern Sudan. We used MSF operational data to investigate trends in VL relapse and associated risk factors.

Results

We obtained data for 8,800 primary VL and 621 relapse VL patients treated between 1999 and 2007. Records of previous treatment for 166 VL relapse patients (26.7%) were compared with 7,924 primary VL patients who had no record of subsequent relapse. Primary VL patients who relapsed had larger spleens on admission (Hackett grade ≥3 vs0, odds ratio (OR) for relapse = 3.62 (95% CI 1.08, 12.12)) and on discharge (Hackett grade ≥3 vs 0, OR = 5.50 (1.84, 16.49)). Age, sex, malnutrition, mobility, and complications of treatment were not associated with risk of relapse, nor was there any trend over time. Treatment with 17-day sodium stibogluconate/paromomycin (SSG/PM) combination therapy vs 30-day SSG monotherapy was associated with increased risk of relapse (OR = 2.08 (1.21, 3.58)) but reduced risk of death (OR = 0.27 (0.20, 0.37)), although these estimates are likely to be residually confounded. MSF operational data showed a crude upward trend in the proportion of VL relapse patients (annual percentage change (APC) = 11.4% (−3.4%, 28.5%)) and a downward trend in deaths (APC = −18.1% (−22.5%, −13.4%)).

Conclusions

Splenomegaly and 17-day SSG/PM vs 30-day SSG were associated with increased risk of VL relapse. The crude upward trend in VL relapses in Southern Sudan may be attributable to improved access to treatment and reduced mortality due to SSG/PM combination therapy.     

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination

Discrimination of Leishmaniainfantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteineproteaseB (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3’ end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.     

Polymorphism in the HASPB Repeat Region of East African Leishmania donovani Strains

Background/Objectives

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.

Methodology/Principal Findings

Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient''s geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions.

Conclusions/Significance

HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.     

Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

Background

Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.

Methodology and principal findings

The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.

Conclusion/Significance

This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.     

Microsatellite based molecular epidemiology of Leishmania infantum from re-emerging foci of visceral leishmaniasis in Armenia and pilot risk assessment by ecological niche modeling

BackgroundVisceral leishmaniasis (VL) is re-emerging in Armenia since 1999 with 167 cases recorded until 2019. The objectives of this study were (i) to determine for the first time the genetic diversity and population structure of the causative agent of VL in Armenia; (ii) to compare these genotypes with those from most endemic regions worldwide; (iii) to monitor the diversity of vectors in Armenia; (iv) to predict the distribution of the vectors and VL in time and space by ecological niche modeling.Methodology/Principal findingsHuman samples from different parts of Armenia previously identified by ITS-1-RFLP as L. infantum were studied by Multilocus Microsatellite Typing (MLMT). These data were combined with previously typed L. infantum strains from the main global endemic regions for population structure analysis. Within the 23 Armenian L. infantum strains 22 different genotypes were identified. The combined analysis revealed that all strains belong to the worldwide predominating MON1-population, however most closely related to a subpopulation from Southeastern Europe, Maghreb, Middle East and Central Asia. The three observed Armenian clusters grouped within this subpopulation with strains from Greece/Turkey, and from Central Asia, respectively. Ecological niche modeling based on VL cases and collected proven vectors (P. balcanicus, P. kandelakii) identified Yerevan and districts Lori, Tavush, Syunik, Armavir, Ararat bordering Georgia, Turkey, Iran and Azerbaijan as most suitable for the vectors and with the highest risk for VL transmission. Due to climate change the suitable habitat for VL transmission will expand in future all over Armenia.ConclusionsGenetic diversity and population structure of the causative agent of VL in Armenia were addressed for the first time. Further genotyping studies should be performed with samples from infected humans, animals and sand flies from all active foci including the neighboring countries to understand transmission cycles, re-emergence, spread, and epidemiology of VL in Armenia and the entire Transcaucasus enabling epidemiological monitoring.     

Species identification and phylogenetic analysis of Leishmania isolated from patients,vectors and hares in the Xinjiang Autonomous Region,The People’s Republic of China

BackgroundVisceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics.Methodology/Principal findingsHere, an eleven-year survey (2004–2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship.Conclusions/SignificanceThe above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.     

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.     

Interferon-gamma release assay (modified QuantiFERON) as a potential marker of infection for Leishmania donovani, a proof of concept study

Background

In areas endemic for visceral leishmaniasis (VL), a large number of infected individuals mount a protective cellular immune response and remain asymptomatic carriers. We propose an interferon-gamma release assay (IFN-γRA) as a novel marker for latent L. donovani infection.

Methods and Findings

We modified a commercial kit (QuantiFERON) evaluating five different leishmania-specific antigens; H2B, H2B-PSA2, H2B-Lepp12, crude soluble antigen (CSA) and soluble leishmania antigen (SLA) from L. donovani with the aim to detect the cell-mediated immune response in VL. We evaluated the assay on venous blood samples of active VL patients (n = 13), cured VL patients (n = 15), non-endemic healthy controls (n = 11) and healthy endemic controls (n = 19). The assay based on SLA had a sensitivity of 80% (95% CI = 54.81–92.95) and specificity of 100% (95% CI = 74.12–100).

Conclusion

Our findings suggest that a whole-blood SLA-based QuantiFERON assay can be used to measure the cell-mediated immune response in L. donovani infection. The positive IFN-γ response to stimulation with leishmania antigen in patients with active VL was contradictory to the conventional finding of a non-proliferative antigen-specific response of peripheral blood mononuclear cells and needs further research.