Leishmania infantum DNA detection in Phlebotomus tobbi in a new northern focus of visceral leishmaniasis in Iran

Objective

To identify the vector(s), the parasite and the species composition of sand flies in the district during May-October 2012.

Methods

For reaching our objectives we used polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism.

Results

Two species of Phlebotomus sergenti and Phlebotomus tobbi were the most prevalent among 8 species identified comprising 51.1% and 32.9% respectively. Among the 160 specimens of female sand flies tested by polymerase chain reaction of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphisms, only 1 out of 80 Phlebotomus tobbi (1.25%) were positive to Leishmania infantum parasites.

Conclusions

Our finding showed that Phlebotomus tobbi may play as a vector to circulate the parasite of Leishmania infantum among reservoir(s) and human.     

Enzyme immunoassay using Leishmania (Viannia) braziliensis antigens for laboratorial diagnosis of American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) has a wide geographical distribution, including all the states of Brazil. Parasitological and immunological tests are the most commonly used methods for the laboratorial diagnosis of ACL. The objective of this study was to find a specific and sensitive antigen to the diagnosis of ACL. To this end, promastigotes of Leishmania (Viannia) braziliensis were submitted to gel-filtration chromatography (Sephadex G100) and to ionic-change chromatography (DEAE-Sepharose Fast-Flow), and the antigen reactivity was evaluated by enzyme immunoassay (EIA-IgG). The results showed that the peak II of fraction 8 distinguished ACL patient sera from those of healthy individuals and individuals with other diseases (P<0.0001), presenting 85.41% sensitivity and 91.22% specificity. False-positive results were found for sera from Chagas disease patients (16.67%) and healthy individuals (10.42%). False-positive results were not detected for sera from patients with toxoplasmosis or paracoccidioidomycosis. The fraction obtained shows good sensitivity and specificity for ACL diagnosis and opens up new possibilities for the use of serology in laboratorial diagnosis, seroepidemiological studies and in the follow up of ACL treatment.     

Experimental canine leishmaniasis: evolution of infection following re-challenge with Leishmania infantum

The aim of the study was to assess the clinical, parasitological and immunological effect of a second inoculation of amastigotes in dogs previously inoculated with Leishmania infantum. Three dogs primarily inoculated with amastigotes (Group I) and four with cultured virulent stationary phase promastigotes (Group II) were afterwards re-inoculated with 2x10(9) amastigotes per kg. Three other groups of dogs were used as controls: Group III was infected only once with amastigotes, Group IV only once with promastigotes and Group V was non infected. The animals were followed up by clinical and parasitological examinations, hematological and serum protein analysis, anti-leishmanial antibody levels and proliferative assays of specific peripheral blood mononuclear cells over a period up to 50 months. Parasites were isolated from lymph node of three animals during primary amastigote infection and in five animals (Group I and II) after re-challenge. Group I dogs presented a strong increase of the humoral immune response while Group II animals displayed no significant or significantly low antileishmanial antibodies titres, after re-challenge. The detection, only after challenge, of positive specific lymphoproliferation in two animals of Group II that had the longest primary infection interval (more than 26 months), indicates the requirement of a long time interval to obtain specific lymphocyte sensitization. A previous exposure to virulent cultured L. infantum promastigotes seems to confer some degree of resistance against an amastigote infection.     

Differences in transmission seasons as an epidemiological tool for characterization of anthroponotic and zoonotic cutaneous leishmaniasis in northern Afghanistan

Regional epidemiological data, when available from Afghan or international health authorities, usually include cutaneous leishmaniasis cases without further elaboration. Scientific reports from Afghanistan mainly focus on the current status of war and refugee-related anthroponotic cutaneous leishmaniasis (ACL), but little is known about zoonotic cutaneous leishmaniasis (ZCL), its regional and seasonal distribution, or other disease characteristics. Multiple field investigations revealed that both ACL and ZCL are widespread in Afghanistan and may show sharp differences in specific epidemiology and incubation periods. The previously unknown transmission dynamics and differing seasonality of ZCL, with maximum clinical cases in September and October, as opposed to the ACL peak in March and April, are here described, thus permitting for the first time prediction of the causative Leishmania species in undiscriminated CL reports. Results show that epidemiological differences may serve as a convenient tool for discriminating between ACL and ZCL, at least in northern and central Afghanistan, which can be important because specific treatment and control measures may be different.     

Treatment of mucosal leishmaniasis (L. infantum) with miltefosine in a patient with Good syndrome

We report on a 76-year old patient with recurrent mucosal leishmaniasis. Multiple treatment regimens were administered. After the second relapse, immunologic workup and review of the patient's history revealed the presence of Good syndrome, characterized by immunodeficiency in patients with thymoma. The third relapse was treated with oral miltefosine with complete resolution of the lesions. Miltefosine is an option for treating Old World leishmaniasis (Leishmania infantum) and immunodeficiency should be considered in patients with recurrent leishmaniasis.     

Systemic therapy of New World cutaneous leishmaniasis: A case report and review article

Cutaneous leishmaniasis is a disease endemic to Central and South America, Mexico and the Caribbean, and affects millions of people. As travel to these regions becomes more common, cutaneous leishmaniasis is becoming a disease of increasing importance in the developed world. However, disease recognition and access to appropriate therapy for cutaneous leishmaniasis remains a challenge in North America. The present article reports a case of cutaneous leishmaniasis in a Canadian man following a trip to Costa Rica. Species-specific diagnosis was confirmed by polymerase chain reaction analysis of a skin biopsy, which was positive for Leishmania panamensis. After failing a course of itraconazole, the patient was successfully treated with sodium stibogluconate, despite significant barriers to administering this therapy, and the paucity of data regarding its efficacy and tolerability. The pathophysiology, diagnosis and systemic treatment of cutaneous leishmaniasis, as well as its emerging presence in the developed world, are reviewed.     

Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil

The antibody response against Leishmania (Leishmania) amazonensis crude antigen was measured through the indirect immunofluorescent assay (IFA) and the immunoenzymatic assay (ELISA) in 114 patients with cutaneous leishmaniasis (CL) in Brazil. Fifty-four patients were infected by Leishmania (Viannia) braziliensis, and 60 patients had L. (V.) guyanensis infection. Patients were comparable by age, sex, disease duration and the Montenegro skin test diameter. L. (V.) braziliensis-infected patients showed significant lower number of ulcerated lesions, greater ulcerated area and higher proportion of lymph node enlargement. Sensitivity of IFA was 79.6% (95% CI 66.1-88.9) and 71.7% (95% CI 58.4-82.2) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.324). Sensitivity of ELISA was 98.2% (95% CI 88.8-99.9) and 85.0% (95% CI 72.9-92.5) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.018). Significant differences were observed in the magnitude of the antibody response before treatment with higher levels detected in L. (V.) braziliensis-infected patients by both serologic techniques. Eighty-four patients had serologic evaluations before and 12 weeks after treatment with meglumine antimoniate, 20 mg/kg/day for 20 days. Significant lower optic density values were observed after treatment with both species independent of cure or failure. Our data showed that L. (V.) braziliensis induces a higher antibody response against L. (L.) amazonensis antigens than L. (V.) guyanensis and that down-modulation of the antibody response occurs shortly during disease evolution after treatment. Moreover the data support the use of ELISA as a better tool for detection of antibodies in CL.     

PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis

A PCR-RFLP based method was developed to diagnose and identify the Leishmania species causing American cutaneous leishmaniasis (ACL) in a panel of clinical samples obtained from an endemic region of Brazil. The comparison of the results obtained by PCR-RFLP and PCR-hybridization in the identification of Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis were highly concordant (kappa=91.5%). The PCR-RFLP method was reliable, fast and easy to conduct on biopsies and presents potential value of utmost importance for the diagnosis and identification of Leishmania in clinical specimens, infected reservoirs and vectors.     

Comparison of miltefosine and meglumine antimoniate for the treatment of zoonotic cutaneous leishmaniasis (ZCL) by a randomized clinical trial in Iran

This study was a randomized, open label comparison that was designed to determine efficacy and safety of miltefosine as the first oral drug for the treatment of zoonotic cutaneous leishmaniasis caused by Leishmania major in comparison with meglumine antimoniate. Complete clinical response was defined as 100% re-epithelialization of the lesion. Definitions of lesion cure and failure were based on both clinical and parasitological criteria two weeks after the end of treatment and clinical recovery three months after this period. Of 32 patients enrolled for miltefosine treatment 28 patients completed treatment, of which 26 were cured at three months, corresponding to a cure rate of 92.9% on a per protocol analysis, and 81.3% according to intention to treat analysis. There was one failure (3.1%), one relapse (3.1%) and four dropouts due to lack of tolerability (12.5%) during the first week of treatment. Of 31 patients who received intramuscular meglumine antimoniate (20mgSb(5)/kg body weight daily for 14 days) 25 were cured (83.3% on a per protocol basis, 80.6% on intention to treat basis), five failed (16.1%) and one was lost (3.2%) at 3-month follow-up. At 6-month follow-up after the end of treatment, no relapse was observed. Both regimens were tolerated but averages of nausea (32.2%) and vomiting (21.5%) were observed in patients during two weeks after initiation of miltefosine treatment. Other gastrointestinal, musculoskeletal, and total adverse events were not statistically different in the two groups during one to four weeks after therapy initiation. No relevant changes were observed in levels of liver enzymes, creatinine and hematological tests before and after end of treatment in both groups. In conclusion, miltefosine is apparently at least as good as meglumine antimoniate for the treatment of cutaneous leishmaniasis caused by L. major in Iran, based on parasitological as well as clinical criteria two weeks, three months, and six months after end of treatment.     

Leishmania infantum MON-98: infection in a dog from Alto Douro,Portugal

We report the isolation of Leishmania infantum zymodeme MON-98 in Portugal, its first known occurrence outside the focus of El Agamy, Egypt. One dog found to be infected with Acanthocheilonema dracunculoides during a survey of canine filariosis in the Alto Douro region, north-east Portugal, was also discovered to have Leishmania in bone marrow. The isolated strain was identified by isoenzyme analysis. A search for other possible cases of L. infantum MON-98 infection in dogs, vectors and particularly humans is necessary to establish the real epidemiological importance of this zymodeme in the endemic region of Alto Douro.     

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.     

Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in São Vicente Férrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.     

Follow-up of Leishmania infantum naturally infected dogs treated with allopurinol: immunofluorescence antibody test,ELISA and Western blot

Fourteen dogs naturally infected with Leishmania infantum and treated with allopurinol were monitored clinically and serologically with immunofluorescent antibody test (IFAT, amastigotes and promastigotes), enzyme linked-immunosorbent-assay (ELISA, IgG1 and IgG2) and Western blotting (WB). In all dogs therapy lead to clinical improvement together with decreasing specific antibodies in IFAT, ELISA and WB, demonstrating the usefulness of serology for follow-up. Although IgG1 and IgG2 varied considerably between individual animals, IgG2 of all dogs was predominantly in both ELISA and WB. This suggests the value of monitoring the IgG2 response (especially against 29 and 67 kDa antigens) in the follow-up of treated dogs.     

Canine Leishmania infantum enzymatic polymorphism: a review including 1023 strains of the Mediterranean area, with special reference to Algeria

This bibliographic review reports the isoenzyme polymorphism of 1023 Leishmania infantum strains isolated from dogs that have been characterized by multilocus enzyme electrophoresis in the Leishmania Reference Centre of Montpellier, or in other laboratories, to which this typification technique has already been transferred. Between 1981 and 2010, a total of 12 zymodemes were identified around the Mediterranean basin: MON-1, MON-24, MON-34, MON-72, MON-77, MON-80, MON-98, MON-105, MON-108, MON-199, MON-199 var NP1130 and MON-281, of which 6 were present in Algeria. The zymodeme MON-1 was predominant (86.5% of the strains). The dog was confirmed as the main reservoir of L. infantum MON-1, while the reservoir of the other zymodemes has not yet been identified. The enzymatic polymorphism is relatively high in Algeria and in Spain in contrast to other Mediterranean countries. The reasons for this polymorphism 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.     

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。     

Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens’ sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.     

我国不同流行区婴儿利什曼原虫前鞭毛体比较蛋白质组学分析

目的 应用比较蛋白质组学方法分析两株来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白表达差异,为筛选和鉴定我国不同流行区利什曼原虫致病差异相关分子奠定基础。方法 分别培养两株分离于我国四川九寨沟县（SC6株）和新疆伽师县内脏利什曼病流行区（JIASHI?5株）的婴儿利什曼原虫前鞭毛体,经酶解后进行液相色谱串联质谱（Liquid chromatography?tandem mass spectrometry, LC?MS/MS）非标记（Label?free）定量分析,利用MaxQuant软件（版本号1.3.0.5）查库,进行非标定量（Label?free quantitation,LFQ）分析。结果 成功鉴定蛋白4 274个,筛选出差异蛋白1 219个（差异倍数＞2.0 或 ＜0.5,P < 0.05）,其中JIASHI?5株前鞭毛体特有蛋白550个,SC6 株前鞭毛体特有蛋白174个。SC6 株和 JIASHI?5 株间差异表达蛋白495个,其中SC6株上调蛋白（高表达）167个,JIASHI?5株上调蛋白328个。这些差异表达蛋白主要涉及能量代谢、应激反应、延长感染宿主细胞的寿命以及利什曼原虫存活与增殖。结论　来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白质表达存在差异。     

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.