First report on natural infection with Leishmania infantum in a domestic ferret (Mustela putorius furo) in Spain.

A pet domestic ferret (Mustela putorius furo) with a papular lesion involving the right pinna was diagnosed with chronic pyogranulomatous dermatitis by histopathologic examination. Intralesional, intracytoplasmic oval microorganisms compatible with Leishmania spp. or Histoplasma spp. were observed in macrophages and multinucleate giant cells. Leishmania infantum (L. infantum) infection was diagnosed by PCR, culture in Novy-MacNeal-Nicolle medium, and immunohistochemistry. Abnormal clinicopathological results included increased alanine transferase, alkaline phosphatase, serum gamma glutamyl transferase and polyclonal gammpathy. Anti-Leishmania antibodies were detected by enzyme-linked immunosorbent assay, immunofluorescence antibody test and western blot using L. infantum antigen. Immunoreactivity against the 16 kDa specific L. infantum antigen fraction was observed by western blot. PCR performed in blood samples obtained from this patient after positive parasite isolation detected L. infantum DNA. To the authors' knowledge, this is the first diagnosis and isolation of L. infantum in a domestic ferret naturally infected in an endemic region (Spain) where canine and feline leishmaniosis is frequently detected. According to these findings, ferrets should be included as potential reservoir hosts of L. infantum. Future investigations should analyze the epidemiological role of ferrets in L. infantum infection including the prevalence of infection.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors.

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donor's residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donor's residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain.

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n=53), 29% of foxes (n=48), 29% of stone martens (n=21) and in 25-50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.

Parasitic genotypes appear to differ in leishmaniasis patients compared with asymptomatic related carriers.

For numerous infectious diseases affecting humans, clinical manifestations range from asymptomatic forms to severe pathologies. The originality of this study was its focus on asymptomatic carriers of Leishmania infantum in southern France. The fundamental interest in these asymptomatic carriers is that they can be a reservoir of potentially pathogenic microorganisms. It remains to be established whether the parasitic genomes from asymptomatic carriers differ from those of patients. Multilocus microsatellite typing was used to investigate the genetic variation among 36 French strains of L. infantum. Nine Leishmania strains isolated from blood donors (asymptomatic carriers) were compared with 27 strains of L. infantum belonging to zymodemes, MON-1, -33 and -183. These strains were isolated from HIV positive or negative patients with visceral leishmaniasis, cutaneous leishmaniasis, from canine leishmaniasis or from phlebotomine sandflies. Multilocus microsatellite typing data generated using 33 loci were analyzed by a Bayesian model-based clustering algorithm and construction of a phylogenetic tree based on genetic distances. Both analyses structured the MON-1 sample into two main clusters. Furthermore, genetic analysis demonstrated that these nine asymptomatic carrier strains are divided into two clusters grouped with the MON-1 strains. One cluster with seven strains is related to, but different from, human symptomatic strains from the Alpes-Maritimes region whereas the other cluster has the two remaining strains together with canine leishmaniasis strains as well as one strain from a visceral leishmaniasis patient. Genetic diversity among asymptomatic carrier was very weak since the nine Leishmania strains belong to only two genotypes. Genetic differentiations were evidenced between asymptomatic carrier strains and non-asymptomatic carrier strains and especially between asymptomatic carrier and HIV+ populations, although these findings require confirmation with a larger sample size. We believe that our data explore for the first time, the genetic diversity among L. infantum from asymptomatic human carriers and reveal a weak polymorphism compared with Leishmania parasites isolated from human patients.

Comparative value of microscopy, serology and real time pcr in the diagnosis of asymptomatic canine Leishmania infantum infection

Se investigó la sensibilidad (SE) del examen citológico mediante microscopia óptica (MO) de improntas de bazo y linfonodo, de la prueba de anticuerpos ELISA (inmuno-ensayo ligado a enzima) y de la PCR (reacción en cadena de la polimerasa) a tiempo real (tr), para diagnosticar la infección asintomática por Leishmania infantum en 110 perros aparentemente sanos, del sureste de España. El porcentaje de perros positivos a MO, ELISA y PCRtr fue 2% (2/110), 27% (26/97) y 67% (39/58), respectivamente, aunque el porcentaje de PCR-positivos osciló entre 35-41% para cada tejido individualmente y 9% en sangre. La SE estimada (intervalos de confianza del 95%) de la MO en relación a la PCRtr y al ELISA fue 5% (0-12) y 8% (0-18), respectivamente. Estos resultados confirman que la mayoría de perros aparentemente sanos de una población endémica de L. infantum están infectados, que aproximadamente solo la tercera parte de éstos desarrolla anticuerpos frente al parásito y solo unos pocos tienen suficiente carga parasitaria en tejido linfoide como para ser detectada mediante MO. Consecuentemente, el grado de concordancia de la PCRtr, el ELISA y la MO en el diagnóstico de leishmaniosis canina asintomática es escaso (AU)

The sensitivity (SE) of cytological examination of spleen and lymphnode smears by optical microscopy (OM), antibody-ELISA (enzyme-linked immunosorbent assays) and real-time (rt) PCR (polymerase chain reaction), for diagnosing asymptomatic canine Leishmania infantum infection was investigated in 110 apparently healthy dogs from southeast Spain. The percentage of OM, ELISA and rtPCR positive dogs were 2% (2/110), 27% (26/97) y 67% (39/58), respectively, although the percentage of rtPCR-positive dogs were 35-41% in individual tissues and 9% in blood. The estimated SE (95% confidence interval) of OM relative to the rtPCR and ELISA tests was 5% (0-12) and 8% (0-18), respectively. Results confirm that most apparently healthy dogs from L. infantum endemic areas are infected, that approximately only one third of these infected dogs develop antibodies and that very few have parasite loads that are high enough to allow detection by OM. As a result, the degree of agreement between rtPCR, ELISA and OM for L. infantum diagnosis in subclinnically infected dogs is low (AU)

Pathogen inactivation technology applied to a blood component collected from an asymptomatic carrier of Leishmania infantum: a case report.

Asymptomatic Leishmania infections have been the main cause of transfusion transmission in endemic areas. Polymerase chain reaction has been used to detect L. infantum DNA in the peripheral blood of asymptomatic Leishmania carriers. In our region, the prevalence of asymptomatic L. infantum infection in donors is markedly high (5·9% of donors studied). We investigated the ability of pathogen inactivation technology, using amotosalen and UVA illumination, to eliminate L. infantum in a blood component collected from an asymptomatic L. infantum infected donor. This is the first report of the INTERCEPT system being used to eliminate a parasite from a component collected from a donor.

Clinical pleiomorphism in human leishmaniases, with special mention of asymptomatic infection.

This review gives an update of current knowledge on the clinical pleiomorphism of Leishmania, with a special emphasis on the case of asymptomatic carriage. The first part describes the numerous unusual expressions of the disease that occur besides the classic (visceral, cutaneous, and mucocutaneous) forms of leishmaniases. The second part deals with progress in the understanding of disease outcome in humans, and the possible future approaches to improve our knowledge in the field. The third part highlights the role of the too often neglected asymptomatic carrier compartment. This group could be key to understanding infraspecific differences in virulence and pathogenicity of the parasite, as well as identifying the genetic determinants involved in the expression of the disease.

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain.

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.

Role of Leishmania spp. infestation in nondiagnostic cutaneous granulomatous lesions: report of a series of patients from a Western Mediterranean area.

BACKGROUND: Leishmaniasis is a parasitic disease prevalent in countries of the Mediterranean area. OBJECTIVES: The potential role of Leishmania as the aetiological factor for cutaneous granulomatous lesions in a series of patients from a Western Mediterranean area was evaluated. The practical usefulness of Leishmania-specific polymerase chain reaction (PCR) amplification and immunohistochemical techniques in skin biopsy specimens was assessed. METHODS: Twenty-five skin biopsies diagnosed as nonspecific granulomatous dermatoses were included in the study. A panel of histopathological features was blindly evaluated by two independent observers. Only those cases showing nondiagnostic clinicopathological features and lacking demonstrable microorganisms after bacteriological, mycological or mycobacteriological cultures and specific stains (Ziehl-Neelsen, Giemsa, Gram, periodic acid-Schiff stains) were finally selected. Quantitative real-time PCR was performed in all selected samples. In available samples, immunohistochemical detection of specific Leishmania spp. antigens was also performed. RESULTS: From the selected 25 biopsies, Leishmania spp. DNA was detected by real-time PCR in 13 cases. In seven of eight PCR-positive cases the presence of a varying density of amastigotes could also be demonstrated immunohistochemically. CONCLUSIONS: Leishmania infection seems to be an important aetiological factor in cutaneous granulomatous lesions showing nondiagnostic features in endemic areas. In such areas, Leishmania-specific PCR amplification and/or immunohistochemical studies may be useful diagnostic tools. These techniques may be specifically indicated in the evaluation of patients showing nonspecific granulomatous inflammatory infiltrates of unknown aetiology lacking the histopathological evidence of parasites.

Efficacy of liposomal amphotericin B for secondary prophylaxis of visceral leishmaniasis in HIV-infected patients.

BACKGROUND AND OBJECTIVES: Visceral leishmaniasis (VL) is characterized by frequent relapses in HIV-infected patients, even in those who receive secondary prophylaxis. The aim of our study was to evaluate the efficacy of liposomal amphotericin B (L-AMB) for secondary prophylaxis of VL in HIV-infected patients. METHODS: From January 2001 to December 2005, 17 HIV patients, with at least one previous episode of VL who received L-AMB as secondary prophylaxis for VL, were included in the study. Efficacy was measured as the proportion of patients remaining free (non-relapse) of VL at different time points. Relapses were analysed as time-to-relapse distribution and were evaluated by survival analysis using the Kaplan-Meier method. RESULTS: Twenty-one episodes of VL were diagnosed and nine relapsed. The median follow-up time was 14 (5-44) months. The probability of remaining free of relapse at 6 months was 89.7% (95% CI, 76.2-100); at 12 months, the probability was 79.1% (95% CI, 61-97.2) and at 24 and 36 months, the probability was 55.9% (95% CI, 30.5-81.3). In the non-relapsing group, patients had a significant increase in CD4 cell levels of 102 (10-174) and 126 (4-159) cells/mm(3) at 12 and 24 months, respectively (P = 0.037), whereas in the relapsing group, no significant increase was observed. Prophylaxis with L-AMB was well tolerated and only three patients had a mild impairment of renal function without requiring any change in treatment. CONCLUSIONS: L-AMB is well tolerated and useful for secondary prophylaxis of VL.

Evaluation of a latex agglutination test (KAtex) for detection of Leishmania antigen in urine of patients with HIV-Leishmania coinfection: value in diagnosis and post-treatment follow-up.

The usefulness of antigen detection in urine as an alternative tool for diagnosis of leishmaniasis and post-treatment follow-up in patients with Leishmania-HIV coinfection was evaluated with a latex agglutination test (KAtex; Kalon Biological, UK). Forty-nine HIV-infected patients with visceral leishmaniasis were included in the study. Antigen detection in urine (ADU) was positive in 42 of 49 (sensitivity, 85.7%) samples obtained during a primary episode. After treatment, a follow-up study in 23 patients was performed by simultaneous ADU and culture of peripheral blood mononuclear cells in 148 determinations. The two methods gave concordant results in 94 cases, 38 of which were positive and 56 negative. In five cases, ADU was negative and culture of peripheral blood mononuclear cells was positive: two of these cases corresponded to clinical relapses. In 49 cases, culture of peripheral blood mononuclear cells was negative and ADU was positive. In the absence of clinical symptoms, the detection of parasite antigens in 71 of 130 (54.6%) urine samples was not associated with clinical disease. The Kaplan-Meier estimates of the probability of relapse at 6, 12, 18, and 24 months were 16% (95%CI, 15-17%), 20% (95%CI, 18-22%), 31% (95%CI, 27-35%), and 71% (95%CI, 52-89%), respectively, in patients with a positive ADU result. In contrast, when ADU was negative, the probability of relapse was 5% at 6 months (95%CI, 2-8%) (only 2 of 11 patients who relapsed had a negative test). ADU by KAtex is appropriate for primary diagnosis of visceral leishmaniasis, for monitoring the efficacy of treatment, and for detection of subclinical infection.

Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods.

The extent of cryptic leishmaniasis in blood donors from a Spanish endemic area, (Eivissa Island) was studied using various immunological and parasitological methods. Sera from 656 blood donors were analysed: 16 (2.4%) were positive by ELISA and 50 (7.6%) by Western blot. Peripheral blood mononuclear cells (PBMC) and buffy coat (BC) samples, were analyzed by culture and nested-PCR. DNA of L. infantum was amplified in 27 (22.1%) of 122 PBMC. Parasites were isolated in 3 (4.5%) of 67 BC cultures and the strains were identified as L. infantum zymodeme MON-28. No parasites were isolated in PBMC culture. After 12 months, a second blood sample was obtained from 18 blood donors who were positive by nested-PCR in the first extraction; nine of them remained positive. Delayed type hypersensitivity (DTH) tests on 15/67 donors (22.3%) were positive. Comparison of results obtained by ELISA, WB and DTH; ELISA, WB and nested-PCR and nested-PCR and BC culture showed a significant association (Pearson test, P < 0.05). L. infantum zyodeme MON-28 was identified in three strains isolated from asymptomatic donors, which suggests a low virulence capacity of these strains. The detection of Leishmania DNA in a high number of asymptomatic subjects supports the need to monitor it in blood donors endemic areas.

A nested polymerase chain reaction for diagnosis and follow-up of human visceral leishmaniasis patients using blood samples.

A nested polymerase chain reaction (PCR) assay for the diagnosis of human visceral leishmaniasis (VL) due to Leishmania infantum infection was developed using primers selected from the parasite's genomic deoxyribonucleic acid (DNA). The assay, which is based on the use of leucocytes separated from blood samples by Ficoll-Paque centrifugation, was compared with culture in vitro. Blood samples were collected from 17 patients in Spain with a history of clinical VL, 15 of whom were also infected with human immunodeficiency virus (HIV) (13 samples during the VL episode and 31 samples during post-treatment monitoring) and one sample was collected from each of 28 patients with HIV infection and fever but no history of VL. The nested PCR using blood detected all the cases of parasitologically confirmed, clinically active VL, while culture detected 92%. The nested PCR detected Leishmania DNA in 18% of the HIV-infected patients with fever and no history of VL, none of whom gave a positive culture. Follow-up examination of the VL patients by nested PCR and culture demonstrated the persistence of L. infantum in blood for a long time after treatment.

Isoenzymatic identification of Leishmania isolates from repeated clinical human leishmaniasis episodes in Catalonia (Spain).

Forty human strains of Leishmania infantum isolated in 1985-99 from 17 patients with repeated cutaneous, mucosal or visceral leishmaniasis episodes in Catalonia (Spain) were examined by isoenzyme electrophoresis. Six zymodemes were revealed: MON-1, MON-24, MON-28, MON-29, MON-33 and MON-34. In 2 patients 2 different zymodemes were identified in consecutive episodes.

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates.

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment.

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.

Epidemiology of canine leishmaniosis in Catalonia (Spain) the example of the Priorat focus.

An epidemiological survey of canine leishmaniosis was conducted in the Priorat, a rural region in the Northeast of Spain, for 10 years (1985-1994). Seroprevalence throughout the region, determined by dot-ELISA and IFI, was 10.2% (8-12%). Forty percent of the dogs studied had a low level of anti-Leishmania antibodies, whereas only 50% were seronegative. Only one-third of the seropositive dogs had evident symptoms of the disease. Annual incidence of the disease was 5.7% and the level of endemicity was stable during the study. Four Leishmania zymodemes (MON-1, MON-29, MON-77, MON-105) were present in the focus, and their distribution in the different hosts is discussed. Apart from dogs and foxes, no other reservoir host has been found in the region.