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.

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.

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.

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.

Diagnostic potential of Western blot analysis of sera from dogs with leishmaniasis in endemic areas and significance of the pattern.

Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.

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.

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.

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.

Serologic diagnosis of canine leishmaniasis by dot-ELISA.

A dot-enzyme-linked immunosorbent assay (ELISA) using protein A-peroxidase was evaluated as a diagnostic test for canine leishmaniasis. The test results were in agreement with parasitologic diagnosis and indirect immunofluorescence assay results. The sensitivity of the test calculated on 31 dogs with positive parasitologic examination was 90% when a titer of 1/800 was established as a cutoff and 100% when a titer of 1/400 was established. The specificity calculated on the canine population from nonendemic areas was 100% when both titers were established. Nevertheless, in endemic areas titers near the cutoff need careful interpretation. The results of this study demonstrate that dot-ELISA protein A using a bio-dot apparatus is highly suitable for seroepidemiologic field work.

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.

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.

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.

Gynandromorphism in a population of Sergentomyia minuta (Rondani, 1843) in the northeast of Spain.

A study carried out, from July 1990 until April 1991, in a small village situated in the northeast of Spain (Pradell de la Teixeta) has allowed the discovery of three sandflies species: Phlebotomus ariasi, P. perniciosus and Sergentomyia minuta. P. ariasi was present from the initial data of capture to the end of October, disappearing the two others at the first half of November. The population of S. minuta furnished gynandromorphic individuals (1.6%) being the female-like specimens most commonly found (37/45, 82.2%). The different degrees of development of the genital region are discussed.

First cases of gynandromorphism in Phlebotomus perniciosus Newstead 1911 (Diptera, Psychodidae, Phlebotominae).

Two gynandromorphic specimens of Phlebotomus perniciosus Newstead, 1911 are described and illustrated for the first time. The specimens were collected in the Northeast of the Iberian Peninsula (Spain).

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.

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.