Ecological interactions of visceral leishmaniasis in the state of Bahia, Brazil

The laboratory and field observations summarized in this paper on visceral leishmaniasis ecology in the State of Bahia, Brazil are based on the author's observations over the past 35 years in a number of state's foci, public health records and literature citations. The disease is endemic with epidemic outbreaks occurring every ten years and its geographical distribution is expanding rapidly in the last years. Leishmania chagasi is the main ethiologic agent of the visceral leishmaniasis but Le. amazonensis s. lato was the only leishmania isolated by other authors from some visceral leishmaniasis human cases in the state. Lutzomyia longipalpis (with one or two spots on tergites III and IV and two sized different populations) was epidemiologically incriminated as the main vector. It was found naturally infected with promastigotes, and it was infected with four species of leishmanias in the laboratory. Although the experimental transmission of Le. amazonensis by the bite of Lu. longipalpis to hamsters was performed, the author was not successful in transmitting Le. chagasi in the same way. The dog is the most important domestic source for infection of the vector, however it is not a primary reservoir. The opossum Didelphis albiventris was found naturally infected with Le. chagasi but its role as reservoir is unknown. Foxes and rodents were not found infected with leishmanias in Bahia.

Asymptomatic Leishmania chagasi Infection in Relatives and Neighbors of Patients with Visceral Leishmaniasis

The frequency of asymptomatic infection among relatives and neighbors of cases of visceral leishmaniasis (VL) was compared and characterization of the immunological response in these subjects was performed. Cases were from a new endemic area, close to the beach and near Salvador, capital of the State of Bahia, Brazil. The characterization of asymptomatic infection was made using a skin reaction test and detection of antibody to Leishmania chagasi by the ELISA test. To characterize the immunological response of these subjects with asymptomatic L. chagasi infection the cytokines profile and the lymphoproliferative response were determined after stimulation of lymphocytes by L. chagasi antigen. There was no difference in the frequency of L. chagasi infection in relatives (45%) and in neighbors (27%) of cases of VL (P>0.05). The immunological response from these subjects was characterized by high production of IFN-g and a low production of IL-10 and a good lymphoproliferative response to L. chagasi antigen

Rearing and Colonization of Lutzomyia evansi (Diptera: Psychodidae), a Vector of Visceral Leishmaniasis in Colombia

The sandfly Lutzomyia evansi from a focus of visceral leishmaniasis in northern Colombia was reared and maintained under laboratory conditions for five generations. The average time for total development was 41.8 days (range = 35.1- 49.6) at 25 oC and 89-95% of relative humidity. The mean number of eggs laid was lower in laboratory bred females either in pots (13.2 eggs/female) or vials (29.9 eggs/female) than in wild caught females (33.4 eggs/female). Immature mortality, mainly due to fungal and mite contamination, was higher during the first two instars than in the remaining immature stages. Adults were robust and healthy although difficult to feed on hamster or chick skin membrane. In summary, Lu. evansi is a colonizable species but requires specific conditions.

Immunotherapy for Visceral Leishmaniasis: Ability of Factors Produced during Anti-leishmania Responses of Skin Test Positive Adults to Inhibit Peripheral Blood Mononuclear Cell Activities Associated with Visceral Leishmaniasis

The course of human Leishmania chagasi infections appears to be determined by the balance between type 1 (T1) CD4+ and CD8+ T suppressor (Ts) cell activities. Skin test positive adults living in hyperendemic areas who have no history of visceral leishmaniasis (VL) have T1 CD4+ T cell immunodominant responses against L. chagasi. The cytokines they secrete during anti-leishmania responses are a probable source of cytokines which inhibit the CD8+ Ts cells associated with VL. The ability of supernatants generated from peripheral blood mononuclear cells derived from skin test positive adults to reverse immune responses which appear to be mediated by CD8+ Ts cells was assessed in three sets of screening assays. The supernatants displayed three candidate factors. One, which could be explained by Leishmania antigens in the supernatant, decreased high endogenous IL-10 secretion characteristic of one class of VL patients. A second activity decreased high endogenous proliferation characteristic of the same class of patients without decreasing antigen specific proliferation. The third activity inhibited or killed CD8+ T cells but not CD4+ T cells. These activities might be useful in treating VL.

Density of Sand Flies (Diptera: Psychodidae) in Domestic and Wild Animal Shelters in an Area of Visceral Leishmaniasis in the State of Rio Grande do Norte, Brazil

The objective of the present study was to determine the association of sand flies with the presence of domestic and wild animals in the peridomiciliary area. The sand flies were collected using direct aspiration and CDC light traps placed in animal shelters. The results suggest that different sand flies species have different behavioral characteristics in an apparent preference for animal baits and that Lutzomyia longipalpis and Lu. evandroi were the most eclectic species regarding their biotope choice. Lu. longipalpis showed a distinct preference for horses and Lu. evandroi for armadillos.

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

Role of CD8+ T cells in endogenous interleukin-10 secretion associated with visceral leishmaniasis

This study examined the role and source of endogenous interleukin-10 (IL) secretion in visceral leishmaniasis (VL). The amounts of endogenous and Leishmania specific IL-10 and interferon-gamma (IFN) secreted by peripheral blood mononuclear cells (PBMC) from VL patients were compared. The correlation coefficient between endogenous IL-10 secretion and Leishmania specific IFN-gamma was -0.77, suggesting a major role for endogenous IL-10 secretion in VL. The effects of CD4+ and CD8+ T cell clones, isolated from a treated VL patient, on IL-10 secretion were assayed by mixing the clones with autologous, inactivated PBMC. The CD8+ clones mediated increased levels of IL-10 secretion in the presence of PBMC alone suggesting that CD8+ T cells may mediate endogenous IL-10 secretion.

Visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, State of Minas Gerais, Brazil

In the last few years the number of human cases of American visceral leishmaniasis in the Metropolitan Region of Belo Horizonte (MRBH), Minas Gerais, Brazil has increased, indicating an elevation in the transmission rate of the disease. The total number of notified human cases in the MRBH since 1994, when the first case was identified, up to 1999 was 345 of which 223 (65%) were from the city itself, indicating an urbanization of the disease in this region of Minas Gerais. The age distribution of visceral leishmaniasis cases in the MRBH shows a higher prevalence in children from 0-4 years old, responsible for 28.9% of the notifications. Clinical and immunological findings from dogs infected with Leishmania chagasi are described. The majority of these animals showed no sign of the disease. Sera from all infected dogs showed detectable Leishmania-induced high titles of antibodies based on the results of an indirect fluorescent antibody test. Samples of isolated Leishmania from human and dogs were characterized as L. (L.) chagasi by biochemical and molecular techniques.

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

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.

Concurrent cutaneous, visceral and ocular leishmaniasis caused by Leishmania (Viannia) braziliensis in a kidney transplant patient

Although cases of leishmaniasis co-infection have been described in acquired immunodeficiency syndrome patients as well as those who have undergone organ transplants, to our knowledge, the present report is the first documented case of simultaneous cutaneous, visceral and ocular leishmaniasis due to Leishmania (Viannia) braziliensis in a transplant patient. The patient had been using immunosuppressive drugs since receiving a transplanted kidney. The first clinical signs of leishmaniasis included fever, thoracic pain, hepatosplenomegaly, leucopenia and anemia. The cutaneous disease was revealed by the presence of amastigotes in the skin biopsy. After three months, the patient presented fever with conjunctive hyperemia, intense ocular pain and low visual acuity. Parasites isolated from iliac crest, aqueous humor and vitreous body were examined using a range of molecular techniques. The same strain of L. (V.) braziliensis was responsible for the different clinical manifestations. The immunosuppressive drugs probably contributed to the dissemination of Leishmania.

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4ºC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.

Epidemiological and immunological aspects of human visceral leishmaniasis on Margarita Island, Venezuela

Sixty-five patients were diagnosed with visceral leishmaniasis (VL) on Margarita Island in the decade from 1990 to1999; 86.2% were <= 3 years old. All were leishmanin-negative at diagnosis. Evaluation of 23 cured patients in 1999 revealed that 22/23 had converted to leishmanin-positive; five had persisting antibodies to rK39 antigen, with no clinical evidence of disease. Leishmanin tests were positive in 20.2% of 1,643 healthy individuals from 417 households in endemic areas. Of the positive reactors, 39.8% were identified in 35 (8.4%) of the households, 15 of which had an antecedent case of VL, a serologically positive dog or both. Weak serological activity to rK39 antigen was detected in 3 of 488 human sera from the endemic areas. The presence of micro-foci of intense peri-urban transmission and the apparent absence of other Trypanosomatidae causing human disease offer a unique opportunity for the study of reservoirs, alternative vectors and evaluation of control measures on the Island.

Phlebotomine sand flies in Porteirinha, an area of American visceral leishmaniasis transmission in the State of Minas Gerais, Brazil

A study of the phlebotomine sand fly fauna was carried out in an endemic area of American visceral leishmaniasis (AVL) in the municipality of Porteirinha, in the Brazilian state of Minas Gerais. Captures were performed with CDC light traps in 7 districts, 5 days per month, during 2 consecutive years (January 2000 to December 2001). A total of 3240 sand flies were captured and identified. Sixteen species were found, among which 15 belonged to the genus Lutzomyia and one to the genus Brumptomyia. Lutzomyia longipalpis, a proven vector of AVL, was the predominant species (71.85%) throughout the time period. The interference of climatic factors (temperature, humidity, and rainfall) over the populational dynamics of the sand flies was determined. Statistical analysis of the data showed a significant correlation among the number of phlebotomine sand flies collected, rainfall, and humidity, whereas the effect of temperature was negligible, in that particular region. The amount of collected phlebotomine, the number of human cases, and the prevalence of canine AVL in the districts of Porteirinha are discussed.