Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon

Lack of conservation of the Amazon tropical rainforest has imposed severe threats to its human population living in newly settled villages, resulting in outbreaks of some infectious diseases. We conducted a seroepidemiological survey of 1100 inhabitants of 15 villages of Paço do Lumiar County, Brazil. Thirty‐five (3%) individuals had been exposed to Trypanosoma cruzi (Tc), 41 (4%) to Leishmania braziliensis (Lb) and 50 (4.5%) to Leishmania chagasi (Lc) infections. Also, 35 cases had antibodies that were cross‐reactive against the heterologous kinetoplastid antigens. Amongst these, the Western blot assays revealed that 11 (1%) had Tc and Lb, that seven (0.6%) had Lc and Tc, and that 17 (1.6%) had Lb and Lc infections. All of these cases of exposures to mixed infections with Leishmania sp, and eight of 11 cases of Tc and Lb were confirmed by specific PCR assays and Southern hybridizations. Two cases had triple infections. We consider these asymptomatic cases showing phenotype and genotype markers consistent with mixed infections by two or more kinetoplastid flagellates a high risk factor for association with Psychodidae and Triatominae vectors blood feeding and transmitting these protozoa infections. This is the first publication showing human exposure to mixed asymptomatic kinetoplastid infections in the Amazon.     

甘肃省2县犬感染利什曼原虫影响因素分析

目的 了解甘肃省犬感染利什曼原虫影响因素,为探索犬源型黑热病防控新方法提供依据。方法 在甘肃省迭部县和文县犬源型黑热病流行区,通过入户问卷调查及犬血PCR检测,调查当地家犬位置、居住场所环境、犬离房屋距离、犬来源、犬主黑热病知识知晓状况和犬感染利什曼原虫现状等信息。利用IBM SPSS23.0 Pearson χ²检验和多元Logistic回归分析与犬感染利什曼原虫有关的影响因素。结果 共调查当地居民445人,家犬537只。家犬血样利什曼原虫PCR检测阳性率为41.15%(221/537),其中文县PCR检测阳性率64.63%(95/147),迭部县PCR检测阳性率32.31%(126/390)。村中央和村边缘犬的阳性率分别为33.92%(96/283)和49.21%(125/254),两者之间有统计学差异(χ²=12.923,P=0.000)。居住场所附近房屋墙面为砖混/粉刷墙壁的犬和附近为土坯/石片墙壁的犬,阳性率分别为30.99%(110/355)、60.99% (111/182),两者之间有统计学差异(χ²=44.723,P=0.000)。家庭存在阳性犬的犬主是否知晓黑热病基础知识与犬感染无统计学差异。多因素分析犬的环境位置、犬居住场所附近房屋条件显示与犬感染具有显著相关性。结论 甘肃省文县和迭部县分布在村边缘的犬、犬附近房屋墙壁土坯/石片结构是犬感染的影响因素。针对不同的影响因素应制定针对性的防治措施,减少黑热病和犬利什曼病的传播。     

Isoenzymatic variability of Leishmania infantum in Tunisia concerning 254 human strains

The different clinical forms of leishmaniasis are the result of both the immunological status of individuals and the species of the parasite causing the infection.

In Mediterranean countries, the Leishmania infantum complex groups zymodemes which are responsible for visceral, cutaneous and exceptionally cutaneomucosal or mucosal leishmaniasis.

We report in this study a synthesis concerning 254 cases of L. infantum that have been characterized at the “Laboratoire de Parasitologie” of the Rabta Hospital. The strains were isolated from human cases of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) by culture on NNN medium: 156 VL cases and 98 CL cases.

The isoenzymatic characterization revealed three zymodemes of L. infantum.

• L. infantum MON 1, a common zymodeme of VL, occurred in 154 cases (61%): 147 VL (95%) and 7 CL (5%). All CL cases were from the northern provinces, six of them occurring during an epidemic disease in 2001.

• L. infantum MON 24, a common zymodeme of CL in the north, occurred in 98 cases (38.5%): 91 CL (93%) and 7 VL (7%). The seven VL cases were immunocompetent children aged from 8 months to 9 years and native of northern Tunisia. Two of the CL cases were from central regions of the country. This is the first time that cases from these regions are reported.

• L. infantum MON 80, an uncommon zymodeme in Tunisia, occurred in two VL cases (0.5%): two children aged 7 and 5. The small number of strains of this zymodeme does not allow understanding of its epidemiological role.

The results of this study indicate a low enzymatic variability of L. infantum in the country. However, our study includes only human strains and should be extended to animal ones (dogs, rodents and sand flies). This would lead to a better understanding of the epidemiology of leishmaniasis in Tunisia.     

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.     

Characterization of Leishmania infantum thiol-dependent reductase 1 and evaluation of its potential to induce immune protection

The need to develop an effective vaccine against leishmaniasis to prevent the 2 million new cases each year led to the search for antigens able to elicit protection against infection with Leishmania. In this study, we have characterized a parasite-specific protein of Leishmania infantum named thiol-dependent reductase 1 (TDR1). The protein is present in both life cycle stages of L. infantum with a notable higher expression in the amastigote forms, suggesting a role in the interaction between the parasite and the mammalian host. Thiol-dependent reductase 1 is localized in the cytosol, although we were able to detect the protein in the culture medium of both promastigotes and axenic amastigotes, and consequently, TDR1 is considered an excreted/secreted molecule of the parasite. Therefore, we have evaluated the potential of TDR1 recombinant protein to protect against experimental challenge with L. infantum parasites using a murine model. Despite a reduction in spleen parasite load in the chronic phase of disease, TDR1 administration was not effective in the protection of Balb/c mice against visceral leishmaniasis and thus TDR1 do not have a crucial role in the modulation of mammalian host immune response, as observed with its protein counterpart Tc52 of Trypanosoma cruzi.     

婴儿利什曼原虫实验感染草原兔尾鼠的进一步观察

给草原兔尾鼠经腹腔接种不同量的婴儿利什曼原虫前鞭毛体,结果表明,不同量的原虫感染对动物体重及肝重没有明显的影响,但脾重则有一定的差异。肝脏原虫负荷随感染虫量的增加而加大,实验结果进一步证明草原兔尾鼠是一种对利什曼原虫非常敏感的实验动物,同时在用级差较小的不同量的原虫接种后,可以显感染程度的差别,这就为利什曼病的免疫学研究提供了良好的动物模型。     

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.     

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.     

The cellular and humoral immune response in subjects vaccinated against cutaneous leishmaniasis using Leishmania tropica major promastigotes

In vitro lymphocyte transformation was studied in 24 subjects 12 months after vaccination with live Leishmania tropica major vaccine, in 10 normal control subjects and in three controls residing in an endemic area. The vaccines had lesions in various stages of clinical development. Lymphocytes from all the subjects were studied for their response to stimulation with L. tropical major promastigotes. Lymphocytes of vaccinated subjects responded to low concentrations of antigen whereas the lymphocyte response of the controls tended to be depressed by the same concentration of the antigen. Serum from each subject was subjected to a study of the humoral antibody titre against L. tropica major and L. donovani using indirect immunofluorescence. A humoral response to L. donovani was present in a majority of vaccinees who had developed a positive lesion whereas no such responses was present in any of the controls. The data suggest that high humoral responses were accompanied by relatively low cell-mediated responses and vice versa. No significant humoral response to L. tropica major could be demonstrated in any of the subjects. A combination of both the cell-mediated and humoral mechanisms may participate in the immune response although their usefulness in the assessment of the protectivity of leishmania vaccines has not been established.     

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.     

Identification and Characterization of Leishmania spp. in Impreession Smears of Patients with Cutaneous Leishmaniasis

Background: Monoclonal antibodies have been employed extensively for the identification of Leishmania species, development of diagnostic tests and in the characterization of defined leishmanial antigens. Objectives: Identification and characterization of  Leishmania spp. directly from cutaneous lesions of infected individuals. Methods: An immunoperoxidase test (Avidin-Biotin technique) using monoclonal antibodies was used for this purpose. One hundred and fifty individuals referring to Dermatology Clinic or Parasitology and Mycology Department of Shiraz University of Medical Sciences were chosen of whom a total of 28 individuals whose smears showed a large number of amastigotes after staining with Giemsa were included in this study. Five monoclonal antibodies designated: D2 (against L. donovani), A11 and T10 (against L. tropica), T1 (against L. major) and T7 (against L. tropica and L. major) were used. Amastigotes were identified by Labeled Avidin Biotin (LAB) method. Results: LAB method for identification of amastigotes in impression smears of patient lesions showed that 20 out of 28 cases (71%) were positive. Among these 12 (60%) and 7(35%) were identified as L. tropica and L. major respectively. Conclusion: The results showed that immunoperoxidase is suitable for in situ identification and characterization of Leishmania spp. at the species level.     

克拉玛依地区婴儿利什曼原虫在猴体内寄生特性的研究

为了解克拉玛依地鞠婴儿利什曼原虫（Ｌｅｉｓｈｍａｎｉａｉｎｆａｎｔｕｍ）在猴体内的寄生特性，共用４只恒河猴（Ｍａｃａｃａｍｕｌａｔｔａ）进行了研究，其中３猴分别经部皮下接种从当地皮肤利会上曼病患者的皮损组织以及从硕大白蛉吴氏亚种（Ｐｈｌｅｂｏｔｏｍｕｓｍａｊｏｒｗｕｉ）胃内分离出来的婴儿利什曼原虫，另一只猴作为对照。     

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light

BACKGROUND AND OBJECTIVES: Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates. MATERIALS AND METHODS: In visceral Leishmaniasis, amastigotes are present in the blood and in the reticuloendothelial system within monocytes. For each unit of plasma or platelets treated, isolated mononuclear cells obtained from 100 ml of normal donor whole blood were incubated with 1.0 x 10(8) Leishmania donovani infantum promastigotes to produce amastigote-laden macrophages. The infected macrophages were added to 250 ml of human plasma or to 250 ml of platelet concentrates. Infected units were cultured pretreatment in 10-fold serial dilutions to determine the limits of detection. Thirty millilitres of 500 microM riboflavin was added to each unit, which was then illuminated with 5.9 J/cm2 of ultraviolet light (6.24 J/ml). After treatment and after 2 months of frozen storage, plasma units were cultured in 10-fold serial dilutions. Platelets were cultured on the day of treatment and on day 5 of storage post-illumination. RESULTS: A 5 log reduction of Leishmania was demonstrated in five of six units of plasma, and a 7 log reduction of Leishmania was demonstrated in one plasma unit. A 5 log reduction of Leishmania was demonstrated in five of six units of platelets, and a 6 log reduction of Leishmania was demonstrated in one unit. CONCLUSIONS: There is no donor screen for Leishmania and other pathogens constantly emerging in our blood supply. The Mirasol PRT System for Platelets and Plasma is an effective means of killing Leishmania and other emerging pathogens in these blood products.     

新疆克拉玛依皮肤利什曼病病原生物学的研究

从新疆克拉玛依地区皮肤利 曼病患者皮损内分离的３株婴儿利什曼原虫，接种至草原名词行鼠或背纹仓鼠的腹腔／睾丸内后，引起内脏感染。习的病理变化与内脏利什曼病人体内分离出来的婴儿利什曼原虫／杜氏利什曼原虫引起的一致。     

The primary humoral immune response of European green lizards (Lacerta viridis) to Leishmania agamae

Summary European green lizards, Lacerta viridis, produced relatively thermostable, dithiothreitol-sensitive, non-precipitating, agglutinins and complement-fixing antibodies (CFA) to Leishmania agamae administered subcutaneously (SC), intraperitoneally (IP) or orally (OR). Antibodies were also detected by the immobilization test (IMM) and by enzyme-linked immunosorbent assay (ELISA). The most sensitive method for the detection of stimulated immunoglobulins was ELISA. Antibodies were detected as early as 3 days post-infection with ELISA and between 5 and 7 for CFA, direct agglutination (DA) and indirect haemagglutination (IHA). In the case of IMM, the times of first detection varied from 14 to 28 days. Maximum CFA (2^-8), DA (2^-8), IHA (2^-11) and ELISA (2^-16) titres were reached from 42 to 49 days with significantly higher values occurring in the OR and IP groups. With IMM, maxima occurred after 5 or 6 weeks. Following exposure, two- to five-fold significant increases in serum lysozyme levels were demonstrated but the concentrations in sera following SC, IP or OR routes of antigen administration were not significantly different when the groups were compared with each other. The highest lysozyme values (approximately 12.3–12.5 μgml^-1) were found in the SC and OR groups when compared to the IP(7.40 μgml^-1).     

Th17 lymphocytes in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi in Central America

Skin lesions in nonulcerated cutaneous leishmaniasis (NUCL) caused by Leishmania (L.) infantum chagasi are characterized by a mononuclear inflammatory infiltrate in the dermis, which is composed mainly of lymphocytes, followed by macrophages, few plasma cells and epithelioid granulomas with mild tissue parasitism. Previous studies have shown that the main population of lymphocytes present in the dermal infiltrate is CD8⁺ T cells, followed by CD4⁺ T cells, which are correlated with IFN-γ⁺ cells. To improve the knowledge of cellular immune responses in NUCL, skin biopsies were submitted to immunohistochemistry using anti-ROR-γt, anti-IL-17, anti-IL-6, anti-TGF-β, and anti-IL-23 antibodies to characterize the involvement of Th17 cells in the skin lesions of patients affected by NUCL. ROR-γt⁺, IL-17⁺, IL-6⁺, TGF-β⁺ and IL-23⁺ cells were observed in the dermal inflammatory infiltrate of NUCL skin lesions. A positive correlation between CD4⁺ T-lymphocytes and ROR-γt⁺ and IL-17⁺ cells suggests that some of the CD4⁺ T-lymphocytes in NUCL could be Th17 lymphocytes. Moreover, a positive correlation between ROR-γt⁺ cells and TGF-β⁺, IL-6⁺, IL-17⁺ and IL-23⁺ cells could indicate the role of these cytokines in the differentiation and maintenance of Th17 lymphocytes. Our findings improve knowledge of the pathogenesis of this rare and atypical clinical form of leishmaniasis.     

Palestinian infantile visceral leishmaniasis caused by a genetic variant of Leishmania infantum belonging to a new zymodeme

The parasites causing a Palestinian case of infantile visceral leishmaniasis (IVL) and those from four dogs from the Jenin District were identified serologically, biochemically and molecular biologically as Leishmania infantum, showing dogs act as a reservoir. The strain from the human case was distinct because of its unique 200-bp kDNA-polymerase chain reaction (PCR) component in its restriction fragment length polymorphism (RFLP) profile after digestion with the endonuclease RsaI, and by the electrophoretic mobility of its malate dehydrogenase (MDH(140)), designating it the reference strain of a new zymodeme of L. infantum, MON-281.     

A study of cell mediated and humoral immunity in haemophilia and related disorders

Helper (OKT4) and suppressor (OKT8) T lymphocyte populations and functional assays of cellular immunity were studied in 37 patients with haemophilia and related disorders in parallel with age matched control subjects. The study included 26 patients with factor VIII (FVIII:C) deficiency, eight patients with factor IX deficiency and three patients with severe von Willebrand's disease (vWd). In patients with factor VIII deficiency low helper T lymphocyte counts, low T helper: suppressor ratios and a diminished response to the lymphocyte mitogen phytohaemagglutinin with decreased natural killer cell activity were observed. Individuals with factor IX deficiency had low absolute T lymphocyte (OKT3) counts and T helper cell counts. Patients with severe factor VIII deficiency (FVIII:C less than 1 u/dl) had lower T helper suppressor ratios and lower killer cell and natural killer cell activity in comparison to mildly affected individuals and all of the severely affected factor IX deficient cases. Studies of humoral immunity revealed a generalized increase in immunoglobulin levels in patients with coagulation disorders of any type. The total haemolytic complement activity was reduced in a significant proportion of haemophilic subjects. Levels of alpha-1-interferon were elevated in the groups of haemophilic subjects studied. The abnormalities of cellular and humoral immunity observed did not correlate with the amount or type of coagulation factor administered to individual patients in the preceding 2 years. The most marked abnormalities of immune function occurred in the one patient diagnosed as suffering from AIDS on clinical grounds.     

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas.

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modualtion of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50, 90/10, 10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12, 28, 35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime® Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote–promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.