Enrichment of invariant natural killer T cells in the bone marrow of visceral leishmaniasis patients

Lipid antigens of Leishmania donovani like lipophosphoglycans are shown as a potent ligand for the activation of invariant natural killer T (iNKT) cells. It is reported that activation of iNKT cells augments the disease pathology in experimental visceral leishmaniasis (VL). In this study, we demonstrate the enrichment of iNKT cells in the bone marrow, one of the disease sites among patients with VL.     

Protein-energy malnutrition as a risk factor for visceral leishmaniasis: a review

The protein-energy malnutrition (PEM) and visceral leishmaniasis (VL) are important problems of public health, which affect millions of people worldwide. Currently, it has been accepted that the immunity or susceptibility to infect-parasitic diseases are directly related to the nutritional status of the host. However, the mechanisms that govern the relationship between the PEM and the course of the VL are multiple and little explained. In this study the current most important aspects and the synergism between these two illnesses were presented. Bibliographic search includes empirical reports, reviews, commentaries, reports from professional associations, books, editorials and annals of congress published in diverse languages between 1960 and January 2009. As much the PEM as the infections caused by parasites of the Leishmania genus are frequent problems in the current days. As new studies are developed on the subject, it becomes essential that the society knows them.     

Brief Definitive Report: Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3+ CD4 cells in blood or spleen

Natural regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺), natural regulatory T cells (nTreg), play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavourably affect the host’s ability to clear certain infections. In human visceral leishmaniasis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3⁺ nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4⁺CD25⁺Foxp3⁺ cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of patients with VL and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3⁺ cells in patient with VL pre‐ and post‐treatment, neither were they elevated when compared to PBMC of EC. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.     

河南省1例输入性内脏利什曼病的实验室诊断

目的　分析河南省1例输入性内脏利什曼病病例,探讨内脏利什曼病的实验室诊断方法。方法　分析患者的流行病学资料和临床资料,镜检观察骨髓涂片中杜氏利什曼原虫无鞭毛体;rK39试纸条检测血清中利什曼原虫抗体;用两对引物K13A?K13B和 LITSR?L5.8S分别扩增利什曼原虫动基体DNA和核糖体DNA内转录间隔区基因片段。结果　 该患者曾去过内脏利什曼病流行区,有不规则发热、脾肿大、全血细胞减少、白蛋白/球蛋白比例倒置等症状,骨髓涂片查见杜氏利什曼原虫无鞭毛体,rK39试纸条检测阳性,两对引物K13A?K13B和 LITSR?L5.8S分别扩增出87 bp和285 bp的片段。两片段序列与杜氏利什曼原虫相应序列的相似性分别为94%和100%。结论　结合患者的流行病学资料和临床表现以及实验室检测结果,确诊该病例为内脏利什曼病病例,病原体为杜氏利什曼原虫。     

河南省1例输入性内脏利什曼病的实验室诊断

目的　分析河南省1例输入性内脏利什曼病病例，探讨内脏利什曼病的实验室诊断方法。方法　分析患者的流行病学资料和临床资料，镜检观察骨髓涂片中杜氏利什曼原虫无鞭毛体；rK39试纸条检测血清中利什曼原虫抗体；用两对引物K13A?K13B和 LITSR?L5.8S分别扩增利什曼原虫动基体DNA和核糖体DNA内转录间隔区基因片段。结果　 该患者曾去过内脏利什曼病流行区，有不规则发热、脾肿大、全血细胞减少、白蛋白/球蛋白比例倒置等症状，骨髓涂片查见杜氏利什曼原虫无鞭毛体，rK39试纸条检测阳性，两对引物K13A?K13B和 LITSR?L5.8S分别扩增出87 bp和285 bp的片段。两片段序列与杜氏利什曼原虫相应序列的相似性分别为94%和100%。结论　结合患者的流行病学资料和临床表现以及实验室检测结果，确诊该病例为内脏利什曼病病例，病原体为杜氏利什曼原虫。     

Immunohistological features of visceral leishmaniasis in BALB/c mice

It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.     

Evidence for determining parasitic factors in addition to host genetics and immune status in the outcome of murine Leishmania infantum visceral leishmaniasis

C.B-17 SCID and congenic BALB/C mice were used to examine Leishmania infantum strain pathogenicity independently of host genetic factors. While parasite loads were significantly higher in immunodeficient mice than in immunocompetent mice, the kinetics of infection during a long-term follow-up were similar, suggesting that intrinsic parasitic factors also influence the outcome of L. infantum infection.     

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0.0001), granuloma (7/25, P = 0.0102), structural disorganization (14/25, P < 0.0001), and atrophy of the lymphoid follicles (20/25, P = 0.0036) and of the marginal zone (15/25, P = 0.0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis.     

中国犬源性和野生动物源性内脏利什曼病的研究进展

我国内脏利什曼病在整个演化过程中反映出不同的流行病学特点,从原始野生动物经犬到人的三个演化阶梯,即野生动物源性内脏利什曼病、犬源性内脏利什曼病和人源性内脏利什曼病。我国陇南川北山区是内脏利什曼病自然疫源地和犬源性内脏利什曼病并存的疫区。人偶尔可直接从野生动物宿主经野生中华白蛉而感染,而更多的是野生动物宿主通过野生白蛉由犬再经白蛉传染给人。阐明这一传播关系,从理论上探讨内脏利什曼病的起源和演化规律,按其各自的特点和规律来制定计划,将对内脏利什曼病和媒介白蛉的预防与控制的实施具有重要指导意义。     

In situ immune responses to interstitial pneumonitis in human visceral leishmaniasis

Lung disease during active human visceral leishmaniasis is frequently reported. As such, studies have associated pulmonary symptoms to interstitial pneumonitis with a mononuclear infiltrate. However, the immune response in this condition has never been described before. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of lung involvement (IPL) in human visceral leishmaniasis. Quantitative methods of analysis were performed using immunohistochemistry, and were compared with a control group of normal lung. Interstitial macrophages and cd8 cells were increased in IPL, and IL-4 as well as TNF-α displayed increased expression when compared to the control group. This inflammatory process with a Th2 pattern, as suggested by increased IL-4 and low IFN-γ expression, is consistent with the immune response in other organs of visceral leishmaniasis. The microenvironment of the immune response in this condition is associated with lung disease in patients with interstitial pneumonitis related to visceral leishmaniasis, increasing the chance of bacterial infection.     

Visceral leishmaniasis in southeastern Nepal: a cross-sectional survey on Leishmania donovani infection and its risk factors

OBJECTIVE: To document the frequency of Leishmania donovani infection at community level in a highly endemic region in southeastern Nepal, and to assess socioeconomic and environmental risk factors. METHODS: A random cross-sectional population survey was held in two visceral leishmaniasis (VL) foci in Morang District in April to May 2003, enrolling individuals 2 years or older and residing in the endemic area for at least 12 months. Leishmania infection was defined as a direct agglutination test (DAT) titre equal to or higher than 1:3200. Risk factors were identified by logistic regression. RESULTS: The direct agglutination test was positive in 7.5% (95% CI: 5.1-10.8) and the leishmanin skin test (LST) in 13.2% (95% CI: 9.9-17.2) of the 373 study participants. No case of current kala-azar was found, but 5.1 % (95% CI: 3.1-7.8) reported having suffered from VL. Independent risk factors for Leishmania infection were proximity of the house to ponds [odds ratio (OR) 3.7, 95% CI: 1.6-8.5], family size (OR 4.4, 95% CI: 1.6-12.6), age > or =15 years (OR 5.5, 95% CI: 1.2-25.0) and house constructed in mud (OR 3.0, 95% CI: 1.1-7.6). Bednets, not impregnated and in poor condition, were used by 95.2% (95% CI: 92.3-97.0) of the population, but did not show any protective effect. CONCLUSION: This study shows that there is a serious problem of transmission of VL in this area of Nepal. The risk factors identified are linked with the socioeconomic level and the environment. The population would benefit from a community intervention to improve the environmental and housing conditions in the villages.     

Post‐kala‐azar dermal leishmaniasis in visceral leishmaniasis‐endemic communities in Bihar,India

We assessed the prevalence of post‐kala‐azar dermal leishmaniasis (PKDL), a late cutaneous manifestation of visceral leishmaniasis (VL), in 16 VL‐endemic communities in Bihar, India. The prevalence of confirmed PKDL cases was 4.4 per 10 000 individuals and 7.8 if probable cases were also considered. The clinical history and treatment of the post‐kala‐azar dermal leishmaniasis cases are discussed.     

The burden of the Leishmania chagasi/infantum infection in a closed rural focus of visceral leishmaniasis in Lara state, west-central Venezuela

A prospective study was conducted in El Brazilar, Curarigua, Lara State, Venezuela, a small rural focus of visceral leishmaniasis (VL) to investigate the burden and the evolution of Leishmania infection in the human and canine population. The incidence of the disease from February 1998 to February 2002 was recorded and two cross-sectional surveys using the leishmanin skin test (LST) and immunofluorescent antibody test (IFAT) were carried out. The dipstick test with recombinant r-K39 antigen was also applied in 2002. The incidence of the disease per year among the population (n=118) during the period of study was 0.004. The rate of new infections per year was 0.07 [95% confidence interval (95% CI): 0.15-1.09]. The overall prevalence of infection measured by LST was not significantly higher in 2002 (43.2%) than in 1998 (28.3%), but it was with IFAT [16.3%vs. 4.6%; odds ratio (OR): 4.01; 95% CI: 1.03-22.78; P=0.022] which would indicate an increasing transmission. The dipstick test only detected infection in children up to 10 years (19.4%). Prevalence in dogs was not significantly different in 2002 (57%) vs. 1998 (33%). The parasite was isolated from dogs and identified by a polymerase chain reaction based on telomeric sequences as Leishmania chagasi/infantum.     

Detection and characterization by immunoblot analysis of potentially diagnostic Leishmania infantum polypeptides in human visceral leishmaniasis

Humoral immune responses were studied in 53 sera from 18 patients with visceral leishmaniasis by immunoblot analysis. Sera from visceral leishmaniasis patients recognized antigens with molecular weights ranging from < 14kDa to more than 100 kDa. Bands ranging between 49 and < 14 kDa were the most specific. The 40, 33 and 17kDa antigens were recognized by 90%, 79% and 79% of the patients sera, respectively. Sera from one patient with Chagas' disease identified 8 of 11 antigens of the specific region. Treatment with periodate eliminated the cross-reaction in three of these antigens (40, 29, 26 kDa). The study of serial sera collected from the different patients showed a decrease in intensity or dissappearance in some of the diagnostic bands, particularly the 17 kDa band. The band of 17 kDa seems to be useful to study the clinical evolution, for post-treatment control and also for epidemiologic purposes. (It has been identified in 7% of control sera from endemic areas.) Immunoblot could be a valuable tool in the diagnosis of visceral leishmaniasis, being more sensitive and specific than other serologic tests.     

A recombinant chimeric protein composed of human and mice‐specific CD4+ and CD8+ T‐cell epitopes protects against visceral leishmaniasis

In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4⁺ and CD8⁺ T‐cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin‐vaccinated mice presented significantly higher levels of antileishmanial IFN‐γ, IL‐12 and GM‐CSF before and after challenge, which were associated with the reduction of IL‐4 and IL‐10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real‐time PCR techniques were used. In addition, the protected animals presented higher levels of parasite‐specific nitrite, as well as the presence of anti‐Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL.     

Retracted: Evaluating the immunomodulatory responses of LdODC-derived MHC Class-II restricted peptides against VL

In a bid to develop a novel immunoprophylactic measure against visceral leishmaniasis (VL), MHC class-II–restricted epitopes of LdODC were identified by reverse vaccinology approach. Five consensus HLA-DRB1*0101-restricted epitopes were screened. The analysis revealed that the set of epitopes was presented by at least 54 diverse MHC class-II alleles. Based on in silico screening, followed by molecular dynamics simulation, population coverage analysis, and HLA cross-presentation ability, five best epitopes were evaluated. PBMCs isolated from treated VL subjects, when stimulated with synthetic peptide alone or as a cocktail of peptides, triggered a secretory IFN-γ, but not the IL-10 level. Support in this notion came from intracellular cytokine level with a considerable up-regulated IFN-γ produced by CD4⁺ T cells. Also, the enhanced IFN-γ seemed to be augmented with the activation of macrophages with prominent IL-12 production. Therefore, it can be concluded that the screened MHC class-II–restricted epitope hotspots derived from Leishmania ODC can trigger CD4⁺ T cells, which can skew macrophage functions towards protection. However, a detailed analysis can explore its potentiality as a vaccine candidate.     

Leishmaniasis in Turkey: molecular characterization of Leishmania from human and canine clinical samples

Human leishmaniasis, both visceral and cutaneous, and canine leishmaniasis have been reported in Turkey for centuries. However, the advent of new diagnostic tools during the last 30 years has led to the recognition that leishmaniasis is an important public health problem throughout the country. In most disease foci both canine and human leishmaniases exist together and identification of parasite species causing these diseases is a pre-requisite for understanding disease epidemiology. A total of 109 samples obtained from human and canine leishmaniasis cases were examined using internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis. Our results indicate that two species, Leishmania tropica and Leishmania infantum, are primarily responsible for cutaneous and visceral leishmaniasis, respectively, in Turkey. However, a new focus of human cutaneous leishmaniasis caused by L. infantum in Hatay region is described. This finding further stresses the importance of Leishmania species molecular characterization in prescribing appropriate therapy and understanding the disease's transmission in different endemic foci.     

Drug regimens for visceral leishmaniasis in Mediterranean countries

Until the early 1990s, pentavalent antimony was the only documented first-line drug employed for the treatment of zoonotic visceral leishmaniasis (VL) in the Mediterranean, with reported cure rates exceeding 95% in immunocompetent patients. The emergence of antimony resistance in other endemic settings and the increase in drug options have stimulated re-evaluation of the current therapeutic approaches and outcomes in Mediterranean countries. A scientific consortium ('LeishMed' network) collected updated information from collaborating clinical health centres of 11 endemic countries of Southern Europe, Northern Africa and the Middle East. In contrast with the previous situation, VL is now treated differently in the region, basically through three approaches: (1) In Northern Africa and in part of the Middle East, pentavalent antimony is still the mainstay for therapy, with no alternative drug options for treating relapses; (2) In some European countries and Israel, both pentavalent antimony and lipid-associated amphotericin B (AmB) formulations are used as first-line drugs, although in different patients' categories; (3) In other countries of Europe, mainly liposomal AmB is employed. Importantly, cure rates exhibited by different drugs, including antimonials in areas where they are still in routine use, are similarly high (>/=95%) in immunocompetent patients. Our findings show that antimony resistance is not an emerging problem in the Mediterranean. A country's wealth affects the treatment choice, which represents a balance between drug efficacy, toxicity and cost, and costs associated with patient's care.     

Evaluation of T-cell subsets in the lesion infiltrates of human cutaneous and mucocutaneous leishmaniasis

We have characterized the T-lymphocytes in the skin lesions of 10 patients with cutaneous leishmaniasis and in the nasal lesions of seven patients with mucosal leishmaniasis, with the immunoperoxidase and monoclonal antibody techniques. There was predominance of cells with helper phenotype (Leu 3A+ 3B) over suppressor phenotype (Leu 2a) in the lesions of both groups. The helper/suppressor (H/S) ratio in the skin lesions was 1.6 +/- 0.5 and in the nasal lesions of mucosal leishmaniasis was 1.7 +/- 0.8. The H/S ratios in the peripheral blood of patients with cutaneous leishmaniasis (2.1 +/- 0.8) and in patients with mucosal leishmaniasis (1.6 +/- 0.8) were comparable and similar to the ratios in the skin and nasal biopsies. The percentage of T-cells and macrophages expressing the Dr antigen in the cutaneous group (69.5 +/- 13.7) was not significantly different from the mucosal patients (90.3 +/- 5.7). We conclude that the immunopathology of the skin lesions in cutaneous leishmaniasis is similar to the nasal lesions of mucosal leishmaniasis.     

The northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors

Visceral leishmaniasis (VL) incidence has been increased in Italy in humans and dogs since the 1990s, with new foci being detected within traditional boundaries of endemic transmission but also in northern regions previously regarded as non-endemic. To monitor the putative VL spreading, surveillance was implemented in northern continental Italy comprising: analysis of human cases recorded from 1990 through 2005; retrospective literature analysis of canine leishmaniasis (CanL) and phlebotomine sandfly records through 2002; prospective investigations in dogs from 2003 through 2005 and surveys on sandflies in 2003 and 2004. Two-hundred-thirty human cases (11% of Italian cases) were recorded. Their stratification by age and HIV status disclosed a sharp decrease of HIV/VL co-infections paralleled by concomitant increase of paediatric and HIV-negative adult patients during the study period. Four patients had no travel history. Seven leishmaniasis foci were retrospectively identified since 1990, whereas prospective investigations in dogs disclosed 47 autochthonous clinical cases and 106 autochthonous seropositives among 5442 dogs (2.1%) from 16 foci of six regions. Parasites were typed as Leishmania infantum MON-1. Four vector species were identified among 1696 Phlebotomus (Larroussius) collected specimens. Comparisons with historical data showed that P. perniciosus and P. neglectus have increased in density and expanded their geographic range in the study area. Northern continental Italy is now focally endemic for VL and a moderate risk for human disease does exist, although the intensity of transmission seems to be lower than in traditional settings of Mediterranean VL.