Distinct pattern of immunophenotypic features of innate and adaptive immunity as a putative signature of clinical and laboratorial status of patients with localized cutaneous leishmaniasis.

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2­4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT−;MT+). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA-DR+ neutrophils; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio; ↑HLA-DR in B lymphocytes, ↑%CD23+ neutrophils, monocytes and B cells; ↑α-Leishmania IgG and ↑serum NO2⁻ + NO3⁻). Selective changes were observed in L1 (↑%HLA-DR+ neutrophils, ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑serum NO2⁻ + NO3⁻) as compared to L2­4 (↑%CD5− B cells; ↑CD23+ B cells and ↑α-Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28+ neutrophils and ↑%CD4+ T cells), >60 showed a well-known leishmanicidal events (↑CD8+ T cells; ↑serum NO2⁻ + NO3⁻ and ↑α-Leishmania IgG). MT+ patients showed increased putative leishmanicidal capacity (↑%HLA-DR+ neutrophils; ↑%CD23+ monocytes; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑ serum NO2⁻ + NO3⁻). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs.

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.

Contrasting human cytokine responses to promastigote whole-cell extract and the Leishmania analogue receptor for activated C kinase antigen of L. amazonensis in natural infection versus immunization.

It is known that the same antigen can induce different immune responses, depending upon the way that it is presented to the immune system. The objective of this study was to compare cytokine responses of peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients and subjects immunized with a first-generation candidate vaccine composed of killed Leishmania amazonensis promastigotes to a whole-cell promastigote antigen extract (La) and to the recombinant protein LACK (Leishmania analogue receptor for activated C kinase), both from L. amazonensis. Thirty-two patients, 35 vaccinees and 13 healthy subjects without exposure to Leishmania, were studied. Cytokine production was assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. The interferon (IFN)-gamma levels stimulated by La were significantly higher and the levels of interleukin (IL)-10 significantly lower than those stimulated by LACK in the patient group, while LACK induced a significantly higher IFN-gamma production and a significantly lower IL-10 production compared with those induced by La in the vaccinated group. LACK also induced a significantly higher frequency of IFN-gamma-producing cells than did La in the vaccinated group. The contrast in the cytokine responses stimulated by LACK and La in PBMC cultures from vaccinated subjects versus patients indicates that the human immune response to crude and defined Leishmania antigens as a consequence of immunization differs from that induced by natural infection.

Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi.

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8(+) T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8(+) lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8(+) T cells re-emphasizes the role of the T cell-mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4(+) and CD8(+) T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years.

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

Clinical value of anti-live Leishmania (Viannia) braziliensis immunoglobulin G subclasses, detected by flow cytometry, for diagnosing active localized cutaneous leishmaniasis.

OBJECTIVE: To evaluate the clinical value of flow cytometry anti-live promastigate antibody (FC-ALPA), for diagnosing active cutaneous leishmaniasis. METHOD: Serum samples from 145 individuals living in endemic areas for localized cutaneous leishmaniasis (population 1) were classified as having the disease or not and then tested for their IgG reactivity by indirect immunofluorescence assay and FC-ALPA-IgG. The results of FC-ALPA-IgG were expressed as percentage of positive fluorescent parasite. Both tests were also evaluated in serum samples of people with visceral leishmaniasis and Chagas disease (population 1A). RESULTS: In population 1, FC-ALPA-IgG performed better than the immunofluorescence assay regarding sensitivity, specificity and predictive values. Analysis of the results according to the likelihood ratios indicated that a percentage of positive fluorescent parasite 60% it reinforces diagnosis of the disease (likelihood ratio = 7.0). Immunofluorescent assay is of little value (likelihood ratio=2.04). In population 1A, both tests performed worse, but FC-ALPA-IgG achieved better statistical indexes than immunofluorescent assay. CONCLUSION: The FC-ALPA-IgG is a valuable method for serological diagnosis of localized cutaneous leishmaniasis. FC-ALPA-IgG1/ALPA-IgG2 combined analysis is an additional serological tool for discriminating localized visceral leishmaniasis, Chagas disease and visceral leishmaniasis in areas where these infections co-exist.

Flow cytometric assay in peripheral blood of dogs--reference values for leukocytes from Brazilian beagles.

Use of domestic reference values in the flow cytometry analysis is known to improve its accuracy by integrating local variations as gender, race and age. Up to date application of flow cytometry in veterinary medicine has been limited to describe the percentual values just for peripheral lymphocytes subsets of blood. We now report establishment of reference values for a wide range of proportional and absolute numbers of peripheral blood leukocytes, including T cells subsets, B cells, monocytes and eosinophils, applicable to the healthy population of Beagles in Brazil and other regions with similar demographic characteristics. Normal reference values were also established to estimate the gender-related differences. This information will provide clinical aid in the evaluation of immunologic status as well as standard values for experimental animals of dogs from Brazil and other similar regions.

Randomized, double-blind, placebo-controlled study on the immunogenicity of the leishmanin skin test.

A positive reaction to the leishmanin skin test (LST) indicates previous contact with Leishmania antigens and is a useful criterion for the diagnosis of cutaneous leishmaniasis. In leishmaniasis vaccine trials, selection of volunteers has always been based on skin testing. During 1999 we performed a randomized controlled study in order to evaluate the immunogenicity of the LST. Fifty-nine (29 male and 30 female) healthy volunteer undergraduate students from the Medical School of Volta Redonda, Rio de Janeiro State, Brazil, with no evidence of previous infection with Leishmania, were randomly assigned into 2 groups: 29 subjects received LST and 30 received a placebo (merthiolate-phosphate-buffered saline). All volunteers received LST 41 d after the first injection of LST or placebo. Blood samples were taken immediately before the applications of LST or placebo for the assessment of Leishmania antigen-induced proliferation and cytokine production in peripheral blood mononuclear cell cultures. A significant increase in proliferative responses to L. braziliensis (P < 0.005) and L. amazonensis (P = 0.01) antigens as well as in L. braziliensis antigen-induced interferon-gamma production (P < 0.01) followed the application of LST but not the administration of the placebo. A single LST application is therefore able to induce Leishmania-specific cell-mediated immune responses. This observation should be considered in human trials of candidate vaccines against leishmaniasis.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

Impact of canine control on the epidemiology of canine and human visceral leishmaniasis in Brazil.

Brazil is the only country endemic for zoonotic visceral leishmaniasis (ZVL) that regularly conducts epidemiologic and prophylactic control programs that involve the treatment of human cases, insect vector control, and the removal of seropositive infected dogs. This report reviews 60 studies reporting data on the efficacy of these recommended control tools and concludes that in Brazil 1) eradication of the disease in Minas Gerais was achieved by the concomitant use of the three control methods, 2) although seropositivity by an immunofluorescent assay is not completely related to infectiousness, the removal of seropositive dogs leads to a significant reduction of canine and human incidence, 3) improvement of the sensitivity of the diagnostic tool used for canine control should optimize the efficacy of control, and 4) although difficult and expensive, the public health dog control campaigns performed in Brazil reduced the incidence of ZVL and should be maintained since treatment of dogs is an unrealistic intervention, both because of its prohibitive cost and relatively poor effectiveness.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis.

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

American cutaneous leishmaniasis in Southeast Brazil: space-time clustering.

BACKGROUND: American cutaneous leishmaniasis (ACL) is endemic in many rural areas of Brazil where different transmission patterns of the disease have been described. This ecological study was carried out in a municipality located in Southeast Brazil and aimed to investigate the space-temporal patterns of the disease and environmental risk factors from 1966 to 1996. METHODS: Incident ACL cases were defined by clinical diagnosis, confirmed by a positive skin test and/or parasitological examination. Age-adjusted morbidity rate of ACL was calculated by year for this municipality and their different census enumeration districts. The homogeneity chi2 test, Moran and empirical Bayes index and Knox procedure were employed for testing the significance of clusters in time, space and in time-space, respectively. A Poisson regression model was used to identify environmental factors related to rate variability. RESULTS: A total of 1712 new ACL cases were reported with a yearly incidence rate of 48/100000 inhabitants. Higher incidence rates were detected in 1968, 1974, and 1988 (100, 160, and 190 cases/100000, respectively) with evidence of spatial clustering from 1986 to 1993. Significant space-time clustering with epidemic peaks followed by low incidence in subsequent periods was observed. The incidence rates of ACL were independently associated with rural areas; areas lacking sanitary installations and with higher proportion of exposed garbage (P < 0.01). CONCLUSIONS: This study suggests that ACL rates vary across space and time. Rural areas and some environmental factors could explain part of this variation. Environmental modifications in the vicinity of households over time and accumulation of susceptible individuals are discussed as possible factors responsible for variability.

PIP: This paper presents the results of an ecological study on American cutaneous leishmaniasis (ACL) cases carried out in Caratinga municipality, Southeast Brazil. The study aimed to estimate the incidence rates of ACL from 1966 to 1996, to test for space and temporal patterns in the rates and to correlate them to sociogeographic factors. Findings of the study showed an increase in the incidence rates of reported ACL cases. A total of 1712 ACL cases were reported in Caratinga during 1966-96, with a yearly incidence rate of 48/100,000 inhabitants. Evidence of spatial clustering was noted during 1986-93. Higher incidence rates were detected in 1968 (100 cases/100,000 inhabitants), 1974 (160/100,000), and 1988 (190/100,000). In addition, an observation noted that explosive outbreaks of ACL are followed with very low incident rate cases in the subsequent period. This observation indicates a lifelong resistance for treated individuals following clinically apparent infection. Moreover, prevalence of ACL infection was associated with rural areas, lack of sanitary disposal, and exposed garbage.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacinregister mark or target. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 microg of each protein at 15 days interval. One hundred microg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 10(5) infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57. 14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7. 60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

Short report: evaluation of the potency and stability of a candidate vaccine against American cutaneous leishmaniasis.

Availability of a safe, immunogenic, and affordable vaccine would represent the best strategy for control of cutaneous leishmaniasis (CL). Stability in field conditions is a essential property for any candidate vaccine. The stability and immunogenicity of three different preparations (thimerosal-preserved, autoclaved, and lyophilized) of a killed Leishmania amazonensis vaccine were assessed using fresh products and after 12 months of storage at 4 degrees C. Autoclaving was associated with a time-dependent decrease in the immunogenicity of the vaccine, as measured by the leishmanin skin test and production of interferon-gamma. These findings are of importance in the decision of which preparation of candidate killed CL vaccines should move to phase III trials.

The fucose-mannose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brazil.

The fucose-mannose ligand (FML)-ELISA assay showed a sensitivity of 100% and a specificity of 100% in diagnosis of canine visceral leishmaniasis (CVL) (kala-azar) in sera from naturally infected dogs from São Gonçalo do Amaranto, Rio Grande de Norte, Brazil. The overall prevalence of antibodies to Leishmania in the endemic area was 23% (79 of 343). Seroreactivity detected by a Leishmania chagasi immunofluorescent (IF) assay was much lower (2.9%) and similar to the percentage of dogs with kala-azar symptoms (2.6%). Twenty-one of 21 asymptomatic, FML-seropositive animals died of kala-azar in a period ranging from 0 to 6 months after diagnosis. The predictive value was 100% for the FML-ELISA, 43% for an L. mexicana ELISA, and 24% for the L. mexicana and L. chagasi IF assays, respectively. In experimentally infected dogs, all assays detected seropositivity between 90 and 120 days after infection. Since the current strategy for control of CVL is based on detection and destruction of infected dogs, the highly predictive, sensitive, and specific FML-ELISA represents a useful tool for field control of the disease.

Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis.

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.