Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains.

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis.

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.

Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis.

Seven dogs experimentally infected with amastigotes or culture promastigotes of Leishmania infantum MON-1 were observed for a period of up to 38 months. The course of infection was monitored by clinical and parasitological examinations, haematological and serum protein analysis, and by anti-leishmania antibody levels. Two of the three amastigote-inoculated dogs developed a symptomatic infection with haematological and protein alterations, and a strong humoral immune response. The third dog was asymptomatic with no haematological or protein alterations and developed a steady humoral response. Four promastigote-inoculated dogs remained asymptomatic throughout the observation period, with only transient antibody responses to leishmanial antigen, and no haematological or protein alterations. The detection of the parasite in biological material obtained at necropsy showed that dogs with no clinical signs or other manifestations of disease may be infected. This indicates that asymptomatic carriers may be present in the canine population, but not identifiable by the usual serological tests, and suggests that epidemiological surveys based on serology may underestimate the prevalence of canine leishmaniosis and the parasite transmission risk.

Detection of Leishmania in immunocompromised patients using peripheral blood spots on filter paper and the polymerase chain reaction.

The aim of the present study was to investigate whether the polymerase chain reaction could be used to detect Leishmania infantum in peripheral blood spots of immunocompromised patients. Although visceral leishmaniasis in immunocompromised individuals is routinely diagnosed by direct microscopy or by culture of biopsy material, both methods have disadvantages. In order to evaluate an alternative method of diagnosis, blood spots were collected on filter paper from 24 immunocompromised individuals with visceral leishmaniasis diagnosed by bone marrow microscopy or culture. The samples were tested using the polymerase chain reaction. Leishmania DNA was detected in 15 of 20 patients who had not yet begun treatment for Leishmania infection and in two of four patients undergoing treatment. Using microscopy or culture, parasites were detected in 5 of 19 and 8 of 19 fresh blood samples, respectively. The results suggest that the polymerase chain reaction can be used with blood spots on filter paper as an initial screening method for immunocompromised patients suspected to have Leishmania infection.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes.

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocytized by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Performance of immunoblotting in diagnosis of visceral Leishmaniasis in human immunodeficiency virus-Leishmania sp.-coinfected patients.

This study evaluated the performance of immunoblotting with Leishmania infantum soluble antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus (HIV)-infected and immunocompetent patients and assessed the humoral responses of patients coinfected with HIV and Leishmania. In this work, the results of the immunoblot analysis were compared to those of parasitological examination (Giemsa-stained smears and/or parasite isolation in Novy, Nicolle, and MacNeal medium from bone marrow) and indirect immunofluorescence and counterimmunoelectrophoresis techniques. Patients were considered to be infected if one or more of the comparison techniques gave a positive result. Immunoblotting was considered to be positive if at least one band was present. For 198 HIV-positive patients with a clinical suspicion of visceral leishmaniasis, immunoblot analysis had a sensitivity of 70.6%, a specificity of 73.2%, and an accuracy of 72.7%. For a separate group of 40 immunocompetent patients not infected with Leishmania, the immunoblot analysis was negative for all patients (100% specificity), and for a third group of 32 immunocompetent patients with confirmed visceral leishmaniasis, the immunoblot analysis was positive for all patients (100% sensitivity). Sera of coinfected patients recognized few bands and recognized bands at lower intensities compared with sera from immunocompetent patients. The most frequently detected band was 63 to 66 kDa (55.9%), with the difference being statistically significant compared to frequency of detection of the other bands. This study confirms that the humoral response in patients coinfected with HIV and Leishmania is much lower than that in immunocompetent patients and that the immunoblot method is a sensitive, noninvasive, and specific serological technique for the diagnosis of visceral leishmaniasis in immunocompromised patients.

[Canine leishmaniasis. New concepts of epidemiology and immunopathology: their impact in the control of human visceral leishmaniasis]. / Leishmaniose canina. Novos conceitos de epidemiologia e imunopatologia e seus reflexos no controlo da leishmaniose visceral humana.

Visceral leishmaniasis (VL) is a zoonosis in most regions where it occurs. Dogs are the most important reservoir of the disease and are mainly responsible for the persistence of VL in the Paleartic and Neotropical regions. Canine leishmaniasis (CaL) is a viscerocutaneous, chronic infection with a worse prognosis than human disease. We now know that, as in man, there are some cases of asymptomatic infection. Former studies indicated that dog cutaneous parasitism becomes infectious to the insect vector in later periods of the disease, but recent studies performed by xenodiagnosis have shown that it is possible that transmission might occur earlier. The infected animal reacts with a great production of antibodies and depression of cellular immunity. Antibodies are not protective and resistance is related with active cellular immunity. The presence of Th 1 response in asymptomatic animals, sometimes without humoral response, means that the prevalence of CaL, found in epidemiological surveys by searching for antibodies, may be underestimated.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs

A direst agglutination test (DAT) and an immunofluorescence (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrabida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres ò1:320 and ò1:128). Of 34 samples collected form apparently healthly dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25 per cent. Considering these and previous studies in the adjancent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be high magnitude.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs.

A direct agglutination test (DAT) and an immunofluorescence technique (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrábida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres > or = 1:320 and > or = 1:128). Of 34 samples collected from apparently healthy dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25%. Considering these and previous studies in the adjacent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be of high magnitude.

Comparative study of infectivity caused by promastigotes of Leishmania infantum MON-1, L. infantum MON-24 and L. donovani MON-18.

The mechanisms which permit Leishmania to survive inside macrophages are not totally understood although it is known that prolonged culture in vitro results in loss of virulence. One of the cell surface molecules often implicated in virulence mechanisms is the glycoprotein of 63 kDa (gp63). In this work we studied changes in infectivity of L. infantum promastigotes maintained in vitro by subcultures, correlated with the proteolytic activity of gp63. It was observed that L. infantum MON-1 promastigotes became unable to establish an infection after 6 subcultures in vitro independently of the size of inoculum. This corresponded to a diminution of proteolytic activity of gp63. L. infantum MON-1 promastigotes inoculated in hamsters visceralize in the mononuclear phagocytic system accompanied by an antibody response. A correlation between antibody response, inoculum size and promastigote origin was verified. L. donovani MON-18 and L. infantum MON-24 promastigotes produced a specific humoral response but failed to establish an infection in hamsters regardless of all the passages tested.

Epidemiology, diagnosis and control of leishmaniasis in the Mediterranean region.

The leishmaniases are a widespread and medically important group of parasitic diseases, some of which pose a serious health threat in communities throughout the Mediterranean basin. In 1993, a joint, collaborative study of the Mediterranean leishmaniases was initiated by scientists from Israel, Turkey, Portugal and the Netherlands. The aim of this project was the development of a multi-component approach to the successful control of all forms of leishmaniasis, with special emphasis on the more severe, visceral leishmaniasis (VL). The need for highly sensitive and accurate new tools to facilitate diagnosis and epidemiological surveys of endemic areas and for studies on the immunology of VL in laboratory models (dogs and mice) was soon recognized. It is anticipated that the development of these tools and the associated technology will provide a better understanding of the disease and improve its control.

[Quality control of the micro-ELISA technic applied to the diagnosis of human and canine visceral leishmaniasis]. / Controlo de qualidade da technica de micro-ELISA aplicada ao diagnostico da leishmaniose visceral humana e canina.

Visceral leishmaniasis (VL) or kala-azar is, in Portugal, a zoonosis with the dog as reservoir. A quality control of the technique of micro-ELISA was carried out, using as reference the technique of IFI, the most commonly used for the diagnosis of this protozoosis, both for human and canine sera. Three different methods were used to estimate the cut-off point: X + 2sd (average for negative sera plus two standard deviation), P/N and J index. As quality parameteres were used sensitivity, specificity, efficacy and positive predictive value. The cut-off point for human sera was established at 0.100 A, with 100% sensitivity, 90.5% specificity, 95.3% efficacy and 91.4% positive predictive value, and for canine sera in 0.200 A, with 80.0% sensitivity, 94.3% specificity, 87.7% efficacy and 96.6% positive predictive value. Reproducibility was not fully satisfactory and two different ways of improving it are proposed: P/N and a correction factor. A statistically significant correlation was observed between micro-ELISA's absorbances and IFI titres regarding human sera, though it was not possible to do the same for dog sera.

Leishmania-human immunodeficiency virus coinfection in the Mediterranean basin: isoenzymatic characterization of 100 isolates of the Leishmania infantum complex.

Isoenzymatic characterization was done on 100 isolates obtained from visceral leishmaniasis (VL) patients coinfected with human immunodeficiency virus (HIV); isolates had been received between 1986 and 1993 at the International Leishmania Cryobank and Identification Centre in Montpellier, France. Electrophoresis was done with 15 isoenzymes using the starch gel technique combined, where appropriate, with isoelectrofocusing. Nine Leishmania infantum zymodemes were identified; L. infantum zymodeme MON-1, the most common parasite of human VL in the Mediterranean basin, was the most frequent in coinfections. It could also occasionally be responsible for localized cutaneous leishmaniasis lesions. Several dermotropic zymodemes, which were responsible for localized cutaneous leishmaniasis in immunocompetent patients, caused VL in HIV-positive patients. In addition, in 10 patients, a second isolate obtained during relapses occurring between 1.5 and 9.0 months after treatment was identical to the original isolate.

The isolation of Leishmania donovani MON-18, from an AIDS patient in Portugal: possible needle transmission.

The spread of HIV infection into leishmaniasis endemic areas has increased the incidence of immunosupressed patients with kalaazar in Portugal. The dermotropic zymodeme MON-24 of leishmania infantum has been already isolated from a Portuguese AIDS patient, as in some other Mediterranean countries. In this paper we report the isolation of L. donovani MON-18 from a drug addicted Portuguese patient with clinical visceral leishmaniasis and AIDS, that suggests a mechanically transmitted infection by the use of a shared needle or syringe.

Serodiagnosis of canine visceral leishmaniasis in Portugal: comparison of three methods.

Sera collected in Portugal from 43 dogs were screened for specific antibodies to Leishmania donovani antigens. Three different techniques were compared: an indirect immunofluorescence assay (IFA), a direct enzyme-linked immunosorbent assay (ELISA) and a competitive-ELISA (C-ELISA) using two species-specific monoclonal antibodies, D2 and D13. By IFA, 22 of the sera examined showed positive reactions, compared with 26 by ELISA or 27 by C-ELISA. There was no direct correlation observed between the serum titre by IFA and the strength of the reaction in ELISA or inhibition in C-ELISA. However, a good correlation was observed between sera identified as positive (95.5%) by all three techniques. Western blotting on leishmanial membranes showed that common antigens with Mr of 26,000 and 70-84,000 were recognized by all infected dog sera, regardless of the serum titre. In large scale studies, ELISAs are preferred to IFA for the rapid diagnosis of canine visceral leishmaniasis because of their greater simplicity.

An experimental model for canine visceral leishmaniasis.

Seven mixed-breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post-infection. Anti-parasite specific antibody levels were measured by enzyme-linked immunosorbence, immunofluorescence, crossed-immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70-2, was also done. Mitogenic and antigen-specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen-specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 1.5 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.

Canine leishmaniasis: pathological and ecological factors influencing transmission of infection.

Canine leishmaniasis was studied in 1,823 dogs from the Lisbon metropolitan region. The breeds most affected were doberman and German shepherd, independent of sex and use. Young adult (12.2%) and older dogs (14.7%) had higher prevalences of infection. Parasitological confirmation of serological diagnosis was higher in dogs with indirect fluorescent antibody test titer greater than or equal to 1:512, indicating that parasitological patency is a late event. Exposure of Leishmania in lymph nodes is more efficient for parasitological confirmation (75.4% of cases). Frequent signs of disease were enlarged lymph nodes and onychogriphosis. However, 53.8% of the dogs with significant antibody titers (greater than or equal to 1:128) showed no symptom, suggesting that canine leishmaniasis has a prolonged asymptomatic period. This study confirmed the importance of the dog as the reservoir of visceral leishmaniasis.