Leishmania infantum chagasi in northeastern Brazil: asymptomatic infection at the urban perimeter

Visceral leishmaniasis (VL) is endemic in large cities in Brazil, including Natal. We determined the prevalence of asymptomatic human infection with Leishmania infantum chagasi and associated environmental risks around Natal. Infection was detected by Leishmania skin test (LST) and anti-leishmanial antibodies in humans and anti-leishmanial antibodies in dogs. Amongst 345 humans, 24.6% were seropositive, and 38.6% were LST-positive. Prevalence of positive serology was similar in both sexes and across all ages. However, positive LST responses increased with age, suggesting that LST is long-lasting and cumulative. Multinomial logistic analysis showed that LST response varied with location (P = 0.007) and that males were more frequently LST-positive (P = 0.027). Indicators of lower socioeconomic status associated significantly with human infection. Furthermore, there was geographic coincidence of seropositive humans and dogs (r = 0.7926, P = 0.011). These data suggest that dog and human L. i. chagasi infection are intimately interrelated in environmental conditions associated with low income.     

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 microL and photomicrographed. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10(2) promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.     

Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis

We analyzed the development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. In addition, we compared sandfly infections initiated with axenic amastigotes or promastigotes. Our data showed no important difference between Lu. longipalpis infection rates resulting from either type of infections. Furthermore, development of infection was equivalent in both cases. All promastigote forms were found inside the sandfly and, after blood digestion, most of the population consisted of procyclics and nectomonads. A low percentage of metacyclic forms was coincident with a high number of nectomonads during late stages of infection, but which form gives rise to metacyclic forms in L. infantum chagasi is unknown. These results also show that the promastigote infection model, at least for this situation, is suitable for obtaining of infected sandflies because it is easier and less laborious.     

Importance of worldwide asymptomatic carriers of Leishmania infantum (L. chagasi) in human

Leishmaniasis due to Leishmania infantum (syn. L. chagasi) infection is a zoonotic disease present mainly in Mediterranean basin, central Asia and Brazil. Besides a limited number of human cases of clinical visceral leishmaniasis, a great number of infections remains asymptomatic. In this review, the prevalence of asymptomatic carriers of L. infantum was evaluated worldwide using parasitological methods or indirect testing such as a skin test or serology. The consequences of the presence of asymptomatic carriers on parasite transmission by blood donation or the development of clinical visceral leishmaniasis in immunocompromised individuals and its possible role as reservoir are discussed.     

CTL Responses to DCs Stimulated with Leishmania Antigens Detected by DCs Expressing Leishmania gp63

Background: Leishmania is a pathogenic parasite which infects mononuclear cells in vertebrate hosts. Different strategies have been taken to develop immunity against Leishmania . DCs loaded with immunogenic antigen have resulted in different levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity. Objective: To evaluate the potency of DCs primed with soluble Leishmania mexicana antigens (SLA) in developing CTL activity. Methods: DCs were loaded with SLA and injected to Balb/c mice. After two weeks the mice were sacrificed and their splenocytes were used as effector cells in a standard 4-hour cytotoxicity assay against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. Results: Immunization of Balb/c mice with DCs loaded with SLA resulted in high levels of CTL activity against DCs transfected with pcDNA3 containing L. mexicana gp63 gene. Conclusions: The results indicate a high potency for DCs primed with Leishmania antigens in inducing CTL activity, which can be used for developing an immunogenic vaccine against Leishmania.     

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.     

几种利什曼抗原对草原兔尾鼠婴儿利什曼原虫实验感染的保护作用

本项研究旨在确定几种利什曼表现分子抗原对婴儿利什曼原虫实验感染的免疫保护作用。用重组的利什曼原虫细胞表现糖蛋白ＧＰ６３和脂磷酸聚糖（ＬＰＧ）以短小棒状杆菌菌苗为佐剂免疫草原兔尾鼠。用婴儿利什曼原母亲朱前鞭毛体攻击感染,测定其免疫保护作用。经ｒＧＰ６３＋ＬＰＧ＋ＣＰ免疫的动物,在用２×１０＾２前鞭毛体攻击时,其肝脏的利什曼原虫数比对照动物降低８９．７９％。ＬＰＧ＋ＣＰ免疫组降低６０．６％,ｒＧＰ６３     

The burden of Leishmania chagasi infection during an urban outbreak of visceral leishmaniasis in Brazil

First noted in the city of Teresina in 1981, the last decades have witnessed a remarkable increase in urban transmission of American visceral leishmaniasis (VL) in many Brazilian cities. Teresina, the site of this study, has faced two large outbreaks of VL. The first occurred from 1981-1985 when almost 1000 new cases were reported. The second started in the 1990s, and between 1993 and 1996 more than 1200 new cases were detected. This report describes the prevalence of infection with Leishmania chagasi in Teresina at the end of the second outbreak and gives estimates of the number of people who became infected during the epidemic. Between June 1995 and May 1996, 200 households were chosen at random from a list of addresses covering about 93% of Teresina's urban households. In each household, one person over the age of 1 year was screened for Leishmania antibodies and skin-tested. Nearly 50% of persons had a positive leishmanin reaction, but only 13.9% had detectable antibodies to L. chagasi. While prevalence estimates based on the leishmanin skin-test increased with age (P<0.001), those based on serological tests showed a lesser, and non significant, variation with age (P=0.31). Using a geometric growth equation, and assuming that the annual distribution of clinical cases may serve as an approximation to what would have been the distribution of infections by year, we estimated that over 320000 persons were infected during the epidemic. Little is known about the epidemiology of VL in urban areas, where social networks, population density, and relationships of housing with the natural environment are more varied and complex than in the rural scene. In those areas, control interventions have failed to eliminate transmission of the parasite and prevent new epidemics. Further epidemiological studies of VL in urban areas might be needed to inform control actions.     

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP‐HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients’ urine. Specifically, these proteins were the following: Li‐isd1 ( XP_001467866.1 ), Li‐txn1 ( XP_001466642.1 ) and Li‐ntf2 ( XP_001463738.1 ). Initial vaccine validation studies were performed with the rLi‐ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA‐SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li‐ntf2+ BpMPLA‐SE had a marked parasite burden reduction in spleens at 40 days post‐challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.     

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.     

An assessment of the genetic diversity of Leishmania infantum isolates from infected dogs in Brazil

Correlations between the genetic diversity of Leishmania infantum (syn. L. chagasi) isolates and their respective geographic origins support the theoretic assumption that visceral leishmaniasis probably originated in the Old World. Because dogs are widely considered to be the main reservoir of this disease, the present study aimed to investigate the degree of genetic divergence among 44 leishmanial canine isolates from two Brazilian cities, Jequié and Campo Grande, located approximately 2,028 km from each other. We hypothesized that a low degree of genetic divergence would be observed among these isolates. In fact, statistical analyses found no significant differences between the isolates using both random amplified polymorphic DNA and multilocus microsatellite typing genotyping techniques with three and seven markers, respectively. These findings provide support for the recent introduction of L. infantum into the New World.     

Use of parasitological culture to detect Leishmania (Leishmania) chagasi in naturally infected dogs

In Brazil, although the domestic dog is a major target for the control actions for visceral leishmaniasis, knowledge gaps of the Leishmania species present in those animals still exist in many endemic areas. The objective of this study was the use of parasitological culture as a diagnosis tool and identification of species of Leishmania and other trypanosomatids in the canine population in the city of Cuiaba/Mato Grosso. Biological samples such as blood, intact skin fragments, cutaneous ulcers, and bone marrow were collected during a cross-sectional study and cultured on biphasic medium (Novy-MacNeil-Nicolle [NNN]/Schneider's). Leishmania isolates were characterized through isoenzyme electrophoresis. Isolates were obtained from 11.2% (n=54) of the 482 animals studied considering the different anatomical sites investigated. Leishmania chagasi was confirmed in 8.3% (n=40) dogs and Trypanosoma caninum in 2.9% (n=14). The sample of intact skin presented a higher chance of isolation of L. chagasi in symptomatic dogs and bone marrow in asymptomatic dogs (p<0.05). The results presented in this study emphasize the value of culture and confirm, for the first time, the circulation of L. chagasi in the canine population in different neighborhoods of the city of Cuiaba and broaden the knowledge of the geographical distribution of T. caninum in Brazil.     

Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.     

An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n = 135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n = 390); Ab +: individuals with negative DTH response and seropositive (n = 21); DTH -: individuals with negative DTH and seronegative (n = 560). The mean +/-SD age of VL was 9.3+/-12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.