Widespread atypical cutaneous Leishmaniasis caused by Leishmania (L.) Chagasi in Nicaragua.

Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recently been associated with atypical cutaneous leishmaniasis (ACL) in Central America; however, little comprehensive information about this disease is available. Clinical, epidemiologic, and parasitologic characteristics of 252 ACL cases and 44 VL cases in Nicaragua were analyzed. Visceral leishmaniasis is primarily associated with malnourished children less than five years of age, whereas ACL is found predominantly in children greater than five years of age and young adults. Genetically similar parasites are associated with both disease manifestations. The sand fly Lutzomyia evansi, in addition to Lu. longipalpis, may be involved in transmission of L. chagasi to humans. Our results indicate that ACL is more prevalent than previously thought, affecting up to 10% of a local population. The fact that the same parasite appears to cause both ACL and the potentially fatal visceral disease suggests that the host immune response is critical in determining the outcome of L. chagasi infection. The public health implications of the wide-spread presence of L. chagasi are discussed.     

Genetic predisposition to self-curing infection with the protozoan Leishmania chagasi: a genomewide scan

The protozoan Leishmania chagasi can cause disseminated, fatal visceral leishmaniasis (VL) or asymptomatic infection in humans. We hypothesized that host genetic factors contribute to this variable response to infection. A family study was performed in neighborhoods of endemicity for L. chagasi near Natal in northeastern Brazil. Study subjects were assessed for the presence of VL or asymptomatic infection, which was defined by a positive delayed-type hypersensitivity (DTH) skin test response to Leishmania antigen without disease symptoms. A genomewide panel of 385 autosomal microsatellite markers in 1254 subjects from 191 families was analyzed to identify regions of linkage. Regions with potential linkage to the DTH response on chromosomes 15 and 19, as well as a novel region on chromosome 9 with potential linkage to VL, were identified. Understanding the genetic factors that determine whether an individual will develop symptomatic or asymptomatic infection with L. chagasi may identify proteins essential for immune protection against this parasitic disease and reveal strategies for immunotherapy or prevention.     

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-α, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-α showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.     

Competence of the human host as a reservoir for Leishmania chagasi

The failure of control programs for visceral leishmaniasis (VL) that depend on elimination of infected dogs suggests that other reservoir hosts may participate in the transmission cycle. To determine whether persons infected with Leishmania chagasi can infect the vector sand fly, laboratory-reared Lutzomyia longipalpis were allowed to feed on Brazilian subjects with active, cured, and asymptomatic VL and on asymptomatic residents of houses of persons with active VL. Of 3747 insects that had fed, 26 acquired infection from 11 of the 44 persons with active VL, but none acquired infection from the 137 asymptomatic persons. Among persons <4 years old with active VL, a history of diarrhea and higher peripheral blood neutrophil counts were independent predictors of infectivity. Further experiments using larger numbers of insects are necessary to evaluate the reservoir competence of persons with asymptomatic infections, who represent a large segment of the population of several Brazilian cities.     

Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi.

The study attempted to identify immunologic markers for progression of Leishmania donovani chagasi infection to disease in children in an area endemic for visceral leishmaniasis (VL). [3H]thymidine uptake of lymphocytes stimulated with L. donovani chagasi antigen from children with asymptomatic infection (25,286 +/- 11,648) and from children with self-healing subclinical infection (15,511 +/- 4681) was greater (P = .001) than that observed with lymphocytes from children who progressed to classic VL (4811 +/- 2984). The interferon-gamma (IFN-gamma) levels from asymptomatic and subclinically infected children (74 +/- 90 units/ml) were higher (P = .02) than those observed in children who progressed to VL (7 +/- 8 units/ml). Absence of lymphocyte blastogenesis and IFN-gamma production were associated with progression of infection to classic VL. In the presence of these markers, children should be closely followed to identify signs and symptoms that would permit early initiation of therapy.     

Asymptomatic human carriers of Leishmania chagasi

In Brazil, programs based on elimination of infected dogs have not curtailed the spread of visceral leishmaniasis (VL), suggesting that other reservoirs of infection exist. Persons with active VL can infect the sand fly vector, but in endemic areas, persons with asymptomatic infections, whose infectivity to sand flies is unknown, are far more numerous. In this study, a polymerase chain reaction-based assay detected kinetoplast DNA of Leishmania chagasi in the blood of eight of 108 asymptomatic persons living with patients with recently diagnosed VL. These eight persons had low or unmeasurable levels of IgG antibodies to Leishmania, demonstrating the insensitivity of serology for subclinical infection. All eight persons had positive leishmanin skin test results, as did 70% of persons living in households of persons with active VL. Even if a small proportion of such asymptomatic persons are infective to sand flies, they represent a formidable reservoir of infection in endemic areas.     

Tissue cytokine responses in canine visceral leishmaniasis

To elucidate the local tissue cytokine response of dogs infected with Leishmania chagasi, cytokine mRNA levels were measured in bone marrow aspirates from 27 naturally infected dogs from Brazil and were compared with those from 5 uninfected control animals. Interferon-gamma mRNA accumulation was enhanced in infected dogs and was positively correlated with humoral (IgG1) but not with lymphoproliferative responses to Leishmania antigen in infected dogs. Increased accumulation of mRNA for interleukin (IL)-4, IL-10, and IL-18 was not observed in infected dogs, and mRNA for these cytokines did not correlate with antibody or proliferative responses. However, infected dogs with detectable IL-4 mRNA had significantly more severe symptoms. IL-13 mRNA was not detectable in either control or infected dogs. These data suggest that clinical symptoms are not due to a deficiency in interferon-gamma production. However, in contrast to its role in human visceral leishmaniasis, IL-10 may not play a key immunosuppressive role in dogs.     

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.     

Epidemiology of visceral leishmaniasis in Colombia

Epidemiologic studies were conducted during the period 1986-1988 in a small rural community in Colombia (El Callejon) where visceral leishmaniasis is highly endemic. In this community of 185 people, 14 cases of infantile visceral leishmaniasis were diagnosed in the 9 years 1981-1988. Leishmanin skin testing of a sample of the human residents showed that prevalence of Leishmania chagasi infection increased with age; overall, 51.2% of the subjects had a positive reaction. A canine surveillance program was instituted, using introduced sentinel dogs as well as the indigenous dog population. Eleven of 16 sentinel dogs were infected within 8 months of exposure; mean seroconversion time was 4.4 months. Eleven of 25 seronegative local dogs were also infected during the 26 month period; mean seroconversion time was 8 months. Parasites identified by isozyme electrophoresis as L. chagasi were recovered from 18 of 22 seropositive dogs. Collections of wild animals using baited live traps yielded mainly the neotropical opossum, Didelphis marsupialis. Leishmania chagasi was recovered from 12 of 37 (32.4%) opossums. Six of 681 female Lutzomyia longipalpis collected in the community had flagellates in their guts; cultures from 4 were identified as L. chagasi. These data confirmed that active parasite transmission occurred. The relatively high prevalence of L. chagasi infection found among D. marsupialis captured near human dwellings suggests that these animals may be an important peridomestic reservoir.     

Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity

Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgG1 and IgE anti-SGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.     

Leishmania chagasi antigens recognized in cured visceral leishmaniasis and asymptomatic infection.

Active visceral leishmaniasis is associated with antigen-specific immuno-suppression. However, cured patients develop a cellular immune response associated with resistance to reinfection. Recent studies have identified patients with asymptomatic or subclinical infections, which are also accompanied by an immune response. In order to identify subjects immune to Leishmania chagasi, we performed a skin-test survey in an endemic area in eastern Venezuela. The delayed-type hypersensitivity (DTH) response was assessed in patients cured of visceral leishmaniasis, as well as in their relatives and neighbors. Of the latter, 36 (34.2%) of 105 were positive and 26 (24.7%) of 105 gave intermediate responses. The DTH reaction correlated with age. The antigens recognized by a subgroup of cured patients, those with positive skin-test results, and controls (skin-test negative) were assessed by Western blotting with sera, and T cell immunoblotting with peripheral blood mononuclear cells. No consistent differences between the groups were noted in Western blots with L. chagasi antigens. T cell blots were performed on five patients from each group. For the cured patients and skin-test positive contacts, a significant proliferative response to fraction 12 (less than 20.5 kDa) was noted in four of five patients in each group. Cells from three of five cured patients and two of five skin-test-positive patients proliferated in response to fraction 4 (73-115 kDa). The response to other fractions was variable, with only a minority of patients responding to any one fraction. These data suggest that the antigens recognized by patients with evidence of immunity to L. chagasi are quite variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of nutritional status with the response to infection with Leishmania chagasi

Outcomes of infection with Leishmania chagasi range from self-resolving infection to visceral leishmaniasis (VL). Risk factors determining development of disease are not totally understood, but probably include environmental influences and host genetics. We assessed whether nutrition influenced the outcome of Leishmania infection by comparing relatives of children with VL with either self-resolving Leishmania spp. infection or apparently uninfected households. We observed a decrease in body mass index (P < 0.0005) and mid-upper arm circumference for age (P = 0.022) z-scores for children with VL. Levels of vitamin A were lower in active children with VL as measured by serum retinol (P = 0.035) and the modified-relative-dose-response test (P = 0.009). Higher birth weight (P = 0.047) and albumin concentrations (P = 0.040) protected against disease. Increased breastfeeding time (P = 0.036) was associated with asymptomatic infection. The results indicate that modifiable nutritional aspects are associated with the outcome of Leishmania spp. infection in humans.     

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.     

First occurrence of an autochthonous canine case of Leishmania (Leishmania) infantum chagasi in the municipality of Campinas, State of São Paulo, Brazil

An autochthonous case of visceral leishmaniasis is reported in a dog (Canis familiaris) as an apparently natural infection in a non-endemic area. DNA obtained from spleen and liver samples produced the expected fragment in a Leishmania-specific rDNA-based nested-PCR assay. The PCR product, a 490 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania) infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported in this municipality. This case suggests that natural transmission of this disease is occurring in this area.     

Use of ELISA employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.     

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta)

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.     

Kala-azar in a high transmission focus: an ethnic and geographic dimension

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.     

Central role of interleukin-15 in human immunodeficiency virus (HIV)-infected patients with visceral leishmaniasis

To evaluate clinical and immunological parameters, interleukin (IL)-15 production and outcome of patients with visceral leishmaniasis (VL), including HIV positive patients, we analyzed 48 cases of VL. Clinical manifestations and response to therapy were similar in VL/HIV- and VL/HIV+ patients. However, relapses were more frequent in patients with HIV infection. Low levels of IL-15 concentrations were found in HIV+ patients without VL. These levels were comparable to concentrations obtained in healthy donors. We found a relationship between response to therapy and IL-15 levels. We found increased levels of IL-15 in VL/HIV- and VL/HIV+ patients with clinical and parasitological response to therapy. Our data demonstrate that VL in HIV-infected patients occurs in subjects with severe immunodeficiency and presents high rate of relapses. Low levels of IL-15 in illness patients and restored production in cured persons suggest that this cytokine could play a central role in immune responses during Leishmania/HIV co-infection.     

Elevated plasma levels of interferon (IFN)-gamma, IFN-gamma inducing cytokines, and IFN-gamma inducible CXC chemokines in visceral leishmaniasis

Interferon (IFN)- gamma plays an important role during immune responses against leishmaniasis. Production of IFN-gamma is regulated by interleukin (IL)-12, IL-18, and IL-15. Interferon-gamma-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig) are CXC chemokines, the production of which, at least in part, is IFN-gamma dependent. A follow-up study of individuals infected with Leishmania donovani was undertaken in an area of Ethiopia endemic for visceral leishmaniasis (VL). Plasma levels of IFN-gamma, IL-12p40, IL-18, IL-15, IP-10, and Mig were markedly elevated in symptomatic VL patients (n = 70) compared with individuals with asymptomatic Leishmania infections (n = 39), malaria patients (n = 13), and healthy controls from the endemic area (n = 12). A significant decrease of IFN-gamma and all mediators was observed after treatment of VL patients (n = 33). These data show that increased plasma levels of IFN-gamma, as well as the mediators involved in the production and the activity of this cytokine, are characteristic of active VL in humans, and may play an important immunopathogenic role. The data also suggest that in patients with VL, the production of type 1 cytokines is not depressed, but there appears to be an unresponsiveness to the stimuli of type 1 cytokines. The underlying causes of immunologic unresponsiveness remain a subject of further investigation.     

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.