Subclinical form of the American visceral leishmaniasis

The subclinical form of visceral leishmaniasis (VL) shows nonspecific clinical manifestations, with difficulties being frequently met in its clinical characterization and diagnostic confirmation. Thus, the objective of the present study was to define the clinical-laboratory profile of this clinical form. A cohort study was conducted in the state of Maranh?o, Brazil, from January/1998 to December/2000, with monthly follow-up of 784 children aged 0-5 years. Based on the clinical-laboratory parameters reported in the literature, four categories were established, with the children being classified (according to their clinical-evolutive behavior) as asymptomatic (N = 144), as having the subclinical form (N = 33) or the acute form (N = 12) or as subjects "without VL" (N = 595). Multiple discriminant analysis demonstrated that the combination of fever, hepatomegaly, hyperglobulinemia, and increased blood sedimentation rate (BSR) can predict the subclinical form of VL as long as it is not associated with splenomegaly or leukopenia. Subjects with the subclinical form did not show prolonged or intermittent evolution or progression to the acute form of VL. Subclinical cases have a profile differing from the remaining clinical forms of VL, being best characterized by the combination of fever, hepatomegaly, hyperglobulinemia, and increased BSR.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

The use of immunoblot analysis in the diagnosis of canine visceral leishmaniasis in an endemic area of Rio de Janeiro

In this article, we describe the findings obtained using immunoblot analysis in the diagnosis of canine visceral leishmaniasis (VL) and its correlation with serological titer and clinical status. We found that all animals bearing amastigote forms recognized antigens with 29 and 32 kDa and that this pattern can be exploited for diagnostic and epidemiological purposes. The recognition of the 29- and 32-kDa antigens was verified even in seronegative dogs and preceded seroconversion in periods ranging from several months to 2 yr. We found a correlation between serological titer and parasite burden. Although no correlation between antigenic recognition pattern and clinical status was observed, immunoblot analysis proved to be a reliable test to detect antibodies against Leishmania sp. antigens in dogs from areas with endemic VL.     

Analysis by enzyme-linked immunosorbent assay and immunoblot of the antibody responses of patients infected with Mycobacterium marinum

The serum antibody responses of a total of 14 patients with active or recently cured Mycobacterium marinum infections were analysed via a combination of enzyme-linked immunosorbent assay (ELISA) and the immunodevelopment of Western blots of M. marinum antigen. Normal human sera and sera from patients with active pulmonary tuberculosis were also analysed as controls. The detectable IgG response of M. marinum patients, as demonstrated by ELISA, was highly variable and did not differ significantly from normal controls. IgA and IgM levels were generally low in the M. marinum patients and were not significantly different from normal controls. Immunodevelopment of Western blots of M. marinum antigen with the sera of patients with M. marinum infections revealed that a number of antigens were recognised. Of particular note was an 18-kDa species that was recognised by 11 out of 14 patients (and by none of the normal controls). The 18-kDa antigen may be a useful serodiagnostic marker in the identification of M. marinum infections.     

Remote Sensing and Geographic Information Systems and risk of American visceral leishmaniasis in Bahia, Brazil

The spatial distribution of American visceral leishmaniasis (VL) was studied within the context of the environmental characteristics of northwest Bahia State in Brazil during an epidemic year. Geographic Information Systems (GIS) and Remote Sensing (RS) were used to characterize the landscape epidemiology of VL in order to identify and map high risk areas and endemic zones in a northwestern Bahia study area. Normalized Difference Vegetation Index (NDVI) was shown to be one of the most important risk factors in the area of study. Low NDVI values were related to high numbers of sand flies and high numbers of human and canine VL positive cases. Caatinga vegetation type was the dominant vegetation type in the endemic area. The use of RS and GIS allowed the identification of classes of VL risk that may be useful information to guide control program interventions.     

Balancing immunity and pathology in visceral leishmaniasis

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ-specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell-mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)-10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38(+) stromal cells, and IL-10 promotes impaired DC migration into T-cell areas with consequent ineffective T-cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL-10-producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.     

Induction of cellular and humoral responses by autoclaved and heat-killed antigen of Leishmania donovani in experimental visceral leishmaniasis

The potential of autoclaved and heat-killed antigen of Leishmania donovani to induce cell-mediated and humoral response has been evaluated in the present study. The vaccines were delivered thrice subcutaneously at an interval of 2 weeks. Two weeks after second booster, BALB/c mice were challenged with 10⁷ stationary phase promastigotes of L. donovani. Significant protection was achieved in immunized mice against L. donovani challenge with 69% to 76% and 59% to 64% reduction in parasite load in the liver and spleen respectively. Immunization induced significantly higher level of delayed type hypersensitivity (DTH) response in mice immunized with heat-killed antigen followed by autoclaved antigen. The immune response was assessed by quantifying Leishmania-specific antibodies and cytokine production. The antibody response was predominantly of IgG type with increased IgG2a production and lesser amount of IgM. The immunization preferentially stimulates the production of IFN-γ and IL-2 in splenocytes which suggests a Th1 type response with a concomitant down-regulation of IL-10 and IL-4. These results indicate a potential for the heat-killed and autoclaved antigen as a vaccine which could trigger cell-mediated immune response.     

First visceral leishmaniasis focus in Argentina

An eight-year old boy from Posadas (27 masculine 23'S, 55 masculine 54'W) was diagnosed with visceral leishmaniasis (VL) during 2006. Lutzomyia longipalpis was discovered in the backyard of his house, while the spread of canine visceral leishmaniasis was confirmed in Posadas. This is the southernmost report of a VL transmission focus and the first in Argentina.     

Experimental visceral leishmaniasis in golden hamsters

As a result of a long passage of L. donovani isolate on golden hamsters (21 passages were observed), in transplanting the agent from animals with a distinct clinical picture there was formed a highly virulent strain "G" of L. donovani for this species of animals. The weight arrest and then body mass losses were the most early signs of the disease. Parasites were regularly accumulated in spleen and liver and to a less extent in bone marrow. The main manifestations of visceral leishmaniasis in hamsters are cachexia, lienal syndrome, polyglandular deficiency on the background of hypoplasia of lymphoid tissue and defects of the system of monocytic phagocytes. Such symptom-complex can be a result of neuroendocrine deficiency during visceral leishmaniasis. Pathohistological picture of experimental visceral leishmaniasis is similar to that of man, so L. donovani infection in hamsters can serve as a model for studies of different medical and biological aspects of leishmaniasis.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

Immune responses and protection induced in mice by an industrialized vaccine against American cutaneous leishmaniasis.

An industrialized vaccine against American cutaneous leishmaniasis was compared to a laboratory made vaccine in its ability to induce cellular and humoral immune responses in mice. No differences were observed between seric IgG levels or lymphoblastic proliferation response of mice immunized with either vaccine. Antigenic composition, evaluated by SDS-PAGE, was identical in both preparations. Protection induced in mice against a challenge with infective parasites was also compared. The level of protection obtained with the industrialized vaccine was comparable to that induced by the laboratory made preparation. The results showed that the industrialization process did not alter the efficacy of the vaccine.     

Present situation of visceral leishmaniasis in China

Visceral leishmaniasis or kala azar is a disease that is distributed world-wide from countries around the Mediterranean Sea to Africa, the Middle East, Asia and South America (Fig. 1). Visceral leishmaniasis was highly prevalent in China but since 1958, after a nationwide campaign, it has been brought under control. Only sporadic cases occur in the hilly and newly reclaimed desert areas in NW China.     

Antibody changes in paragonimiasis patients after praziquantel treatment as observed by ELISA and immunoblot

To observe antibody changes after praziquantel treatment in paragonimiasis, a total of 46 serum samples from 13 serologically diagnosed patients was collected for 4-28 months. The specific antibody (IgG) levels were measured by enzyme-linked immunosorbent assay (ELISA). All but one patient who needed retreatment became symptom-free within a week. Antibody levels were dropped near to or below a cut-off absorbance (abs.) of 0.25 in varying intervals from 4 to 18 months. Of 9 patients who were retested within 3 months, 5 revealed temporary elevation of antibody level. After the elevation, the levels began to decline slowly to negative ranges. If treated earlier after symptoms developed, the temporary elevation did not occur and intervals to negative conversion were shorter. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot, antigen-antibody reactions in individual patient faded gradually without significant changes in reacting antigen bands.     

Genes and environment in susceptibility to visceral leishmaniasis

Kala azar (KA) is a lethal disease caused by Leishmania parasites (Leishmania donovani s.l.) that multiply in large numbers in deep organs such as spleen and liver. The host immunological response to these organisms is complex and experimental studies in animals have detected a large number of genetic loci involved in the control of infection and disease. We report here on a study in a human population of Sudan carried out during an outbreak of KA. The following conclusions are presented: (1) environmental factors that could have affected the distribution of the insect vector, influenced progression of KA in the initial phase of the epidemics - but they became less important later at the peak of transmission, probably after infected phlebotomies had spread to all parts of the village -; (2) Leishmania population during the epidemics was heterogeneous, suggesting a possible parasite evolution during the outbreak; (3) the incidence of KA varied markedly among age groups, families and ethnic groups. Susceptibility to KA was shown to depend on a locus on chromosomes 22q12 and on NRAMP1 on chromosome 2q35; the data also suggested a third locus in the region 2q23-q24. Overall, this study indicates complex interactions between host genes and environment in the spreading of KA in that population. It is also suspected that the large parasite diversity observed in the outbreak has contributed to disease spreading across host genetic barriers.     

Urban parasitology: visceral leishmaniasis in Brazil

Since the early 1980s, visceral leishmaniasis (VL) which is, in general, a rural zoonotic disease, has spread to the urban centers of the north, and now the south and west of Brazil. The principal drivers differ between cities, though human migration, large urban canid populations (animal reservoir), and a decidedly peripatetic and adaptable sand fly vector are the primary forces. The exact number of urban cases remains unclear as a result of challenges with surveillance. However, the number of urban cases registered continues to increase annually. Most control initiatives (e.g. culling infected dogs and household spraying to kill the sand fly) could be effective, but have proven hard to maintain at large scales due to logistical, financial and other reasons. In this article, the urbanization of VL in Brazil is reviewed, touching on these and other topics related to controlling VL within and outside Brazil.     

Bacterial infection in patients with visceral leishmaniasis

In an analysis of 63 hospitalized cases with visceral leishmaniasis, the clinical or post-mortem diagnosis of bacterial infection was performed in 33; 13 (39.3%) patients had respiratory infection, 4 (12.1%) had skin infection, 4 had urinary tract infection, 3 (9.0%) showed ear infection and 2 (6.6%) had infection of the oral cavity. It is worth mentioning that in 7 (21%) cases there was infection in multiple sites. Gram positive and/or Gram negative organisms were isolated from 10 patients. In only two (autopsied) cases, infection with less common organisms was recorded, one with disseminated candidiasis and another with disseminated tuberculosis. Death occurred in 9 of the 63 cases, and in 8 of these, concomitant bacterial infection of importance was documented. Patients who had serum globulins lower than 4 g% had significantly more infection (p less than 0.05) than patients with globulin levels higher than 4 g%; there was no significant difference when the number of leucocytes and neutrophils in patients with associated infection was compared with those in patients without bacterial infection. The present study demonstrates that bacterial infection frequently occurs in patients with visceral leishmaniasis, and indicates an unfavourable prognosis. Even though the mechanism of increased susceptibility to infection in this condition was unclear, the widespread range of infections and of infective agents, suggests a multifactorial process.     

Evaluation of immunoglobin isotype specific to Leishmania in tegumentary American leishmaniasis

Leishmania-specific immunoglobulin subclass response was evaluated in 133 patients infected with Leishmania braziliensis. The indirect immunofluorescent antibody test (IFAT) was employed with amastigotes of L. mexicana amazonensis as antigen. Among the 133 sera obtained at consultation for diagnosis of active lesions, IgM was detected in 54 following absorption with Staphylococcus aureus Cowan strain I, and in 5 sera prior to absorption. IgM reactive with Leishmania antigen was only found in sera from patients whose lesions had evolved over the past two months or less. Leishmania-specific IgG was detected in all sera prior to absorption. Sera obtained at the time of recurrence or after complete healing of lesions presented only specific IgG. The combined use of the Montenegro skin test and specific IgM increased the sensitivity of immunodiagnostic methods in patients with lesions of less than 2 months duration. Normal control volunteers were negative for specific IgM and unreactive to Montenegro skin testing. Among 16 patients with non-leishmanial lesions, 3 with sporotrichosis showed IgG reactive with Leishmania; none, including 4 with lesions of less than two months duration, showed specific IgM. We conclude that in patients infected with L. braziliensis the presence of specific IgG and IgM is associated with the time of lesion evolution and the primary or recurrent nature of the lesions. In addition, the combined use of IgM titer and Montenegro reactivity is of potential utility in the diagnosis of early lesions.     

Evaluation of the histopathological classifications of American cutaneous and mucocutaneous leishmaniasis

In order to evaluate the reliability of histopathological classifications of cutaneous and mucocutaneous leishmaniasis the authors compared the histopathological patterns of two biopsies taken simultaneously from the same patient, and classified the material according to Ridley et al. (1980), to Magalh?es et al. (1986a), and to a more simplified classification with only three patterns. Distinct histopathological aspects were observed in different lesions or even in the same lesion. The authors concluded that histopathological patterns do not represent a stage of tegumentary leishmaniasis, thus they can not be correlated with prognosis and therapeutical response as suggested in the literature.     

Evaluation of nephroprotective and immunomodulatory activities of antioxidants in combination with cisplatin against murine visceral leishmaniasis

Background

Most available drugs against visceral leishmaniasis are toxic, and growing limitations in available chemotherapeutic strategies due to emerging resistant strains and lack of an effective vaccine against visceral leishmaniasis deepens the crisis. Antineoplastic drugs like miltefosine have in the past been effective against the parasitic infections. An antineoplastic drug, cisplatin (cis-diamminedichloroplatinum II; CDDP), is recognized as a DNA-damaging drug which also induces alteration of cell-cycle in both promastigotes and amastigotes leading to cell death. First in vivo reports from our laboratory revealed the leishmanicidal potential of cisplatin. However, high doses of cisplatin produce impairment of kidney, which can be reduced by the administration of antioxidants.

Methodology/Principal Findings

The present study was designed to evaluate the antileishmanial effect of cisplatin at higher doses (5 mg and 2.5 mg/kg body weight) and its combination with different antioxidants (vitamin C, vitamin E and silibinin) so as to eliminate the parasite completely and reduce the toxicity. In addition, various immunological, hematological and biochemical changes induced by it in uninfected and Leishmania donovani infected BALB/c mice were investigated.

Conclusion/Significance

A significant reduction in parasite load, higher IgG2a and lower IgG1 levels, enhanced DTH responses, and greater concentration of Th1 cytokines (IFN-γ, IL-2) with a concomitant down regulation of IL-10 and IL-4 pointed towards the generation of the protective Th1 type of immune response. A combination of cisplatin with antioxidants resulted in successful reduction of nephrotoxicity by normalizing the enzymatic levels of various liver and kidney function tests. Reduction in parasite load, increase in Th1 type of immune responses, and normalization of various biochemical parameters occurred in animals treated with cisplatin in combination with various antioxidants as compared to those treated with the drug only. The above results are promising as antioxidants reduced the potential toxicity of high doses of cisplatin, making the combination a potential anti-leishmanial therapy, especially in resistant cases.