Towards a New Strategy for Diagnosis of Congenital Trypanosoma cruzi Infection.

The immigration of Latin American women of childbearing age has spread the congenital transmission of Chagas disease to areas of nonendemicity, and the disease is now a worldwide problem. Some European health authorities have implemented screening programs to prevent vertical transmission, but the lack of a uniform protocol calls for the urgent establishment of a new strategy common to all laboratories. Our aims were to (i) analyze the trend of passive IgG antibodies in the newborn by means of five serological tests for the diagnosis and follow-up of congenital Trypanosoma cruzi infection, (ii) assess the utility of these techniques for diagnosing a congenital transmission, and (iii) propose a strategy for a prompt, efficient, and cost-effective diagnosis of T. cruzi infection. In noninfected newborns, a continuous decreasing trend of passive IgG antibodies was observed, but none of the serological assays seroreverted in any the infants before 12 months. From 12 months onwards, serological tests achieved negative results in all the samples analyzed, with the exception of the highly sensitive chemiluminescent microparticle immunoassay (CMIA). In contrast, in congenitally infected infants, the antibody decline was detected only after treatment initiation. In order to improve the diagnosis of congenital T. cruzi infection, we propose a new strategy involving fewer tests that allows significant cost savings. The protocol could start 1 month after birth with a parasitological test and/or a PCR. If negative, a serological test would be carried out at 9 months, which if positive, would be followed by another at around 12 months for confirmation.

Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) in Latin-American migrants in Barcelona (Spain).

Trypanosoma cruzi, the causative agent of Chagas disease, is divided into six Discrete Typing Units (DTUs): TcI-TcVI. We aimed to identify T. cruzi DTUs in Latin-American migrants in the Barcelona area (Spain) and to assess different molecular typing approaches for the characterization of T. cruzi genotypes. Seventy-five peripheral blood samples were analyzed by two real-time PCR methods (qPCR) based on satellite DNA (SatDNA) and kinetoplastid DNA (kDNA). The 20 samples testing positive in both methods, all belonging to Bolivian individuals, were submitted to DTU characterization using two PCR-based flowcharts: multiplex qPCR using TaqMan probes (MTq-PCR), and conventional PCR. These samples were also studied by sequencing the SatDNA and classified as type I (TcI/III), type II (TcII/IV) and type I/II hybrid (TcV/VI). Ten out of the 20 samples gave positive results in the flowcharts: TcV (5 samples), TcII/V/VI (3) and mixed infections by TcV plus TcII (1) and TcV plus TcII/VI (1). By SatDNA sequencing, we classified the 20 samples, 19 as type I/II and one as type I. The most frequent DTU identified by both flowcharts, and suggested by SatDNA sequencing in the remaining samples with low parasitic loads, TcV, is common in Bolivia and predominant in peripheral blood. The mixed infection by TcV-TcII was detected for the first time simultaneously in Bolivian migrants. PCR-based flowcharts are very useful to characterize DTUs during acute infection. SatDNA sequence analysis cannot discriminate T. cruzi populations at the level of a single DTU but it enabled us to increase the number of characterized cases in chronically infected patients.

Multilocus microsatellite typing of Leishmania infantum isolates in monitored Leishmania/HIV coinfected patients.

BACKGROUND: Leishmania infantum is the main etiological agent of both visceral and cutaneous clinical forms of leishmaniasis in the Mediterranean area. Leishmania/HIV coinfection in this area is characterized by a chronic course and frequent recurrences of clinical episodes. The present study using Multilocus Microsatellite Typing (MLMT) analysis, a highly discriminative tool, aimed to genetically characterize L. infantum isolates taken from monitored Leishmania/HIV coinfected patients presenting successive clinical episodes. METHODS: In this study, by the analysis of 20 microsatellite loci, we studied the MLMT profiles of 25 L. infantum isolates from 8 Leishmania/HIV coinfected patients who had experienced several clinical episodes. Two to seven isolates per patient were taken before and after treatment, during clinical and non-clinical episodes, with time intervals of 6 days to 29 months. Genetic diversity, clustering and phenetic analyses were performed. RESULTS: MLMT enabled us to study the genetic characteristics of the 25 L. infantum isolates, differentiating 18 genotypes, corresponding to a genotypic diversity of 0.72. Fifteen genotypes were unique in the total sample set and only 3 were repeated, 2 of which were detected in different patients. Both clustering and phylogenetic analyses provided insights into the genetic links between the isolates; in five patients isolates showed clear genetic links: either the genotype was exactly the same or only slightly different. In contrast, the isolates of the other three patients were dispersed in different clusters and some could be the result of mixing between populations. CONCLUSIONS: Our data indicated a great MLMT variability between isolates from coinfected patients and no predominant genotype was observed. Despite this, almost all clinical episodes could be interpreted as a relapse rather than a reinfection. The results showed that diverse factors like an intrapatient evolution over time or culture bias could influence the parasite population detected in the patient, making it difficult to differentiate between relapse and reinfection.

Factors influencing the presence of sand flies in Majorca (Balearic Islands, Spain) with special reference to Phlebotomus pernicious, vector of Leishmania infantum.

BACKGROUND: Although the Mediterranean island of Majorca is an endemic area of leishmaniosis, there is a lack of up-to-date data on its sand fly fauna, the last report dating from 1989. The aim of the present study was to provide information on the current sand fly distribution, the potential environmental factors favoring the presence of Phlebotomus perniciosus and which areas are at risk of leishmaniosis. METHODS: In July 2008 sand fly captures were carried out in Majorca with sticky castor oil interception traps. The capture stations were distributed in 77 grids (5x5 km2) covering the entire island. A total of 1,882 sticky traps were set among 111 stations. The characteristics of the stations were recorded and maps were designed using ArcGIS 9.2 software. The statistical analysis was carried out using a bivariate and multivariate logistic regression model. RESULTS: The sand fly fauna of Majorca is composed of 4 species: Phlebotomus perniciosus, P sergenti, P. papatasi and Sergentomyia minuta. P. perniciosus, responsible for Leishmania infantum transmission, was captured throughout the island (frequency 69.4 %), from 6 to 772 m above sea level. Through logistic regression we estimated the probability of P. perniciosus presence at each sampling site as a function of environmental and meteorological factors. Although in the initial univariate analyses the probability of P. perniciosus presence appeared to be associated with a wide variety of factors, in the multivariate logistic regression model only altitude, settlement, aspect, drainage hole construction, adjacent flora and the proximity of a sheep farm were retained as positive predictors of the distribution of this species. CONCLUSIONS: P. perniciosus was present throughout the island, and thereby the risk of leishmaniosis transmission. The probability of finding P. perniciosus was higher at altitudes ranging from 51 to 150 m.a.s.l., with adjacent garrigue shrub vegetation, at the edge of or between settlements, and in proximity to a sheep farm.

Importance of individual analysis of environmental and climatic factors affecting the density of Leishmania vectors living in the same geographical area: the example of Phlebotomus ariasi and P. perniciosus in northeast Spain.

The aim of the present study was to determine the role of specific environmental and climatic factors affecting the distribution and density of Phlebotomus ariasi and P. perniciosus , the proven vectors for Leishmania infantum in Spain. An entomological study was carried out in July 2006 in the province of Lleida with sticky traps set in their diurnal resting places at altitudes ranging from 86 to 1,755 m above the mean sea level (339 sites were sampled). Bivariate analysis revealed that factors such as altitude, bioclimatic zone, temperature, precipitation, sampling site (site relative to settlement, site situation, site category), wall vegetation, particular environment (in this case a natural park), general environment, adjacent natural vegetation and land cover were significantly associated with sand fly densities. The multivariate model for P. perniciosus revealed that its density was affected by site and land cover. Specifically, paved driveways correlated negatively with vector density (Incidence Risk Ratio (IRR): 0.41) and arable land cover correlated positively (IRR: 4.59). In the case of P. ariasi, a significant correlation was observed with the altitude and bioclimatic zone, with density increasing at >800 m above the mean sea level (IRR: 3.40) and decreasing in the meso-Mediterranean bioclimatic zone (IRR: 0.08). Both species were mostly found in agricultural and forest areas far from domestic environments. However, the two species correlated differently with altitude, bio-climate, vegetation, temperature and precipitation, which emphasises the importance of their individual analysis in studies regarding risk of leishmaniasis transmission.

First survey on canine leishmaniasis in a non classical area of the disease in Spain (Lleida, Catalonia) based on a veterinary questionnaire and a cross-sectional study.

The Spanish distribution of canine leishmaniasis (CanL) is heterogeneous and very few data are available for the north of the country, including the province of Lleida (Catalonia, Spain). This work describes the results obtained from a questionnaire sent to veterinarians throughout the province of Lleida. The majority of veterinarians (25/32, 78.1%) believed CanL cases were increasing and that the dogs had been infected locally (30/32, 93.8%). Also, a cross-sectional study was performed on the seroprevalence of CanL in kennel dogs, with and without compatible clinical signs, in the county of Pallars Sobirà (Pyrenees of Lleida), where an autochthonous case of CanL had been previously detected. Four serological tests were used (IFAT, ELISA, Western blot, ICF) and dogs that tested positive with at least two immunological methods were considered seropositive and probably infected. 33.1% (48/145) of the dogs were seropositive. The results of a mixed logistic regression model showed that the risk of seropositivity increased with age (OR=1.35, p-value=0.002), among dogs living in the southern part of Pallars Sobirà (OR=6.20, p-value=0.025) and among dogs whose owners considered their animals to be at risk of leishmaniasis infection (OR=1.26, p-value=0.024) and who were unaware of anti-sand fly preventive methods (OR=11.6, p-value=0.009). The risk decreased when dogs lived in an urban-periurban habitat (OR=0.17, p-value=0.002). The information gathered in the veterinary questionnaires helped us to define the knowledge, perception and awareness of the disease in a naïve region, supporting the hypothesis of an existing CanL focus in Pallars Sobirà, which was confirmed by the seroepidemiological survey. The seroprevalence study carried out on kennel dogs of local origin proved useful for detecting an autochthonous focus of leishmaniasis through the analysis of a small number of animals.

Identification of a Western blot pattern for the specific diagnosis of Trypanosoma cruzi infection in human sera.

A Western blot (WB) method using a lysate from Trypanosoma cruzi (Maracay strain) epimastigotes was evaluated. Serum samples from 37 patients with confirmed Chagas disease (cohort I), 27 Spanish patients with visceral leishmaniasis caused by Leishmania infantum (cohort II), and 28 Colombian patients with cutaneous leishmaniasis caused by L. panamensis and negative serology for Chagas disease (cohort III) were tested. The negative controls were 55 healthy seronegative subjects for T. cruzi and Leishmania; 28 of the negative controls were from a region endemic for Chagas disease and Leishmania (cohort IV), and 27 of the negative controls were from a non-endemic area for Leishmania and T. cruzi (cohort V). A homogeneous standard band pattern consisting of six antigenic bands corresponding to 28, 32, 38, 39, 40, and 48 kDa was recognized simultaneously for all Chagasic patients' sera. Sera from Leishmania-infected patients showed a heterogeneous band pattern that was easily differentiated from the pattern of patients with Chagas disease. WB with T. cruzi epimastigote antigen is an efficient method for diagnosis and may be used as an alternative to confirm T. cruzi and detect cross-reactivity with Leishmania.

Application of molecular techniques in the study of natural infection of Leishmania infantum vectors and utility of sandfly blood meal digestion for epidemiological surveys of leishmaniasis.

Epidemiological studies on the distribution of leishmaniasis caused by Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatidae) have been based principally on serological surveys of the canine reservoir. This methodology is useful due to the facility of sampling, the rapidity in obtaining results, its consistency and because it allows the detection of heterogeneous foci of canine leishmaniasis (CanL) even in small areas. Other investigations have analysed Leishmania parasitism in sandflies (Diptera: Psychodidae: Phlebotominae) by using classical dissection techniques. These techniques allow the vector species to be incriminated in different foci, although they suffer from being very time consuming. Lately, studies in this field are increasingly using molecular techniques, which are faster and easier to perform. In the present work, we applied a nested-PCR in a study of natural infection of sandflies by Leishmania in three isolated farms where serological data on canine leishmaniasis of local dogs were also obtained. The analysis allowed the detection of 38.7% of females with positive nested-PCR (78%, 18% and 0%, respectively, in the different isolated farms). The positive Leishmania DNA samples were genotyped and identified as L. infantum. The results of this work provide new data for the vectorial capacity of Phlebotomus ariasi in a Pyrenean area, which can be considered at risk of becoming a new focus of CanL. The females with positive nested-PCR displayed blood in the midgut at different degrees of digestion, and/or were gravid. According to the multivariate logistic regression analysis, the risk of nested-PCR-positivity increased significantly with the degree of blood digestion (OR = 1.3; P value = 0.025). The Phlebotomus species and the presence of eggs were not statistically associated with nested-PCR positivity (P value of >0.05). The correlation of positive nested-PCR results with the presence of seropositive dogs in the farm confirms the utility of this technique in the study of the distribution and intensity of leishmaniasis foci. Also, the importance of sandfly blood-meal digestion for epidemiological surveys of leishmaniasis foci has been demonstrated.

Chagas disease among the Latin American adult population attending in a primary care center in Barcelona, Spain.

BACKGROUND/AIMS: The epidemiology of Chagas disease, until recently confined to areas of continental Latin America, has undergone considerable changes in recent decades due to migration to other parts of the world, including Spain. We studied the prevalence of Chagas disease in Latin American patients treated at a health center in Barcelona and evaluated its clinical phase. We make some recommendations for screening for the disease. METHODOLOGY/PRINCIPAL FINDINGS: We performed an observational, cross-sectional prevalence study by means of an immunochromatographic test screening of all continental Latin American patients over the age of 14 years visiting the health centre from October 2007 to October 2009. The diagnosis was confirmed by serological methods: conventional in-house ELISA (cELISA), a commercial kit (rELISA) and ELISA using T cruzi lysate (Ortho-Clinical Diagnostics) (oELISA). Of 766 patients studied, 22 were diagnosed with T. cruzi infection, showing a prevalence of 2.87% (95% CI, 1.6-4.12%). Of the infected patients, 45.45% men and 54.55% women, 21 were from Bolivia, showing a prevalence in the Bolivian subgroup (n=127) of 16.53% (95% CI, 9.6-23.39%). ALL THE INFECTED PATIENTS WERE IN A CHRONIC PHASE OF CHAGAS DISEASE: 81% with the indeterminate form, 9.5% with the cardiac form and 9.5% with the cardiodigestive form. All patients infected with T. cruzi had heard of Chagas disease in their country of origin, 82% knew someone affected, and 77% had a significant history of living in adobe houses in rural areas. CONCLUSIONS: We found a high prevalence of T. cruzi infection in immigrants from Bolivia. Detection of T. cruzi-infected persons by screening programs in non-endemic countries would control non-vectorial transmission and would benefit the persons affected, public health and national health systems.

Clinical profile of Trypanosoma cruzi infection in a non-endemic setting: immigration and Chagas disease in Barcelona (Spain).

BACKGROUND: Chagas disease is no longer limited to Latin America and is becoming frequent in industrialised countries in Europe and United States. METHODS: A descriptive study of Latin American immigrants in Barcelona attending two centres for imported diseases during a period of 3 years. The main outcome was the identification of Trypanosoma cruzi-infected individuals in a non-endemic country and the characterization of their clinical and epidemiological features. RESULTS: A total of 489 Latin American patients participated in the study. Forty-one percent were infected by T. cruzi, and the most frequent country of origin was Bolivia. All T. cruzi infected patients were in chronic stages of infection. 19% of cases had cardiac disorders and 9% had digestive disorders. CONCLUSIONS: A high percentage of participants in this study were infected by T. cruzi and various factors were found to be associated to the infection. It is important to improve clinical and epidemiological knowledge of T. cruzi infection in non-endemic countries and to develop appropriate screening and treatment protocols in these settings.

Prevalence and vertical transmission of Trypanosoma cruzi infection among pregnant Latin American women attending 2 maternity clinics in Barcelona, Spain.

We performed a prospective screening for Trypanosoma cruzi infection in 1350 Latin American pregnant women and their offspring in Barcelona, Spain. The rate of seroprevalence was 3.4%, and 7.3% of the newborns were infected. Routine screening and management programs in maternity wards may be warranted.

Desarrollo y evaluación de una técnica ELISA con antígeno crudo de Trypanosoma cruzi para el diagnóstico de la Enfermedad de Chagas / No disponible

Fundamentos: La creciente inmigración procedente de América Central y del Sur ha convertido el diagnóstico de la enfermedad de Chagas en un proceso habitual en los laboratorios españoles. El trabajo describe el desarrollo y evaluación de una técnica ELISA para el diagnóstico serológico de dicha enfermedad. Métodos: El antígeno se obtuvo por sonicación de epimastigotes de Trypanosoma cruzi y se fijó a la placa de microtitulación a una concentración proteica de 20 (..) (AU)

Introduction: The diagnosis of Chagas disease is now a routine procedure in Spanish laboratories due to the increasing immigration from Latin-American countries. The paper describes the development and evaluation of an ELISA technique for the serologic diagnosis of Chagas disease. Methods: Antigen was obtained by sonication of Trypanosoma cruzi epimastigotes and was used at a protein concentration of 20 (..) (AU)

Seroprevalence of Trypanosoma cruzi infection in at-risk blood donors in Catalonia (Spain).

BACKGROUND: The increasing arrival of Latin Americans to Europe and, particularly, to Spain has led to the appearance of new pathologies, such as Chagas disease, a zoonotic infection endemic to rural areas of Central and South America. In the absence of the triatomid vector, one of the main modes of transmission of Chagas disease in nonendemic regions is through blood transfusion. STUDY DESIGN AND METHODS: The Catalonian Blood Bank has implemented a screening program for Chagas disease in at-risk blood donors and has performed a study to determine the seroprevalence of Trypanosoma cruzi infection in the donor population. The two commercial tests used in all samples were the ID-PaGIA Chagas antibody test (DiaMed) and the bioelisa Chagas assay (Biokit). RESULTS: Overall seroprevalence was 0.62 percent, with 11 donors confirmed positive among the 1770 at-risk donors studied; the highest rate (10.2%) was in Bolivian donors. Interestingly, 1 of the 11 positive donors was a Spaniard who had resided various years in a Chagas disease endemic area. Furthermore, 1 of the positive donors presented detectable parasitemia. CONCLUSION: The results of this study emphasize the need for T. cruzi screening in at-risk blood donors in nonendemic countries. An important finding is the relevance of including in the at-risk category persons who have resided in, but were not necessarily born in, an endemic region. If T. cruzi screening is not routinely performed in all donations, it remains highly dependent on proper identification of at-risk donors during the predonation interview.

Leishmania infantum DNA detection in urine from patients with visceral leishmaniasis and after treatment control.

A urine-polymerase chain reaction (PCR) assay was validated for diagnosis of human visceral leishmaniasis (VL), taking advantage of the accessibility of urine samples. Leishmania infantum DNA presence was examined in 17 urine samples from 17 patients with VL during a clinical episode and in 55 urine samples from 17 patients with VL monitored after treatment at different intervals. Fifty-nine urine samples from 59 controls with no history of VL were also studied. The urine-PCR test was positive in 15/17 samples obtained during the episode (sensitivity, 88%). None of the controls tested were urine-PCR positive (specificity, 100%). During the monitoring period, 25% of the samples gave a positive urine-PCR. Results were compared with other diagnostic methods, such as urine antigen detection and peripheral blood-PCR and culture, with good concordance during the clinical episode and differences in the follow-up period. This study suggests that urine-PCR is sensitive for diagnosis and may be useful to monitor treatment efficacy.

Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain).

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania infantum is a zoonotic disease endemic throughout the Mediterranean basin. The existence of asymptomatic human infection entails the risk of transmission by blood transfusion. STUDY DESIGN AND METHODS: The prevalence of Leishmania infection was studied in 1437 blood donors from the Balearic Islands (Majorca, Formentera, and Minorca) using immunologic (Western blot [WB] and delayed-type hypersensitivity [DTH]), parasitologic (culture), and molecular (nested polymerase chain reaction [PCR]) methods. In addition, the efficiency of leukoreduction by filtration to remove the parasite was tested by nested PCR in the red blood cell (RBC) units. RESULTS: Leishmania antibodies were detected in 44 of the 1437 blood donors tested (3.1%). A sample of 304 donors from Majorca was selected at random. L. infantum DNA was amplified in peripheral blood mononuclear cells (PBMNCs) in 18 of the 304 (5.9%), and cultures were positive in 2 of the 304 (0.6%). DTH was performed on 73 of the 304 donors and was positive for 8 of them (11%). Of the 18 donors with positive L. infantum nested PCR, only 2 were seropositive. All the RBC samples tested (13 of 18) from donors with a positive PBMNC nested PCR yielded negative nested PCR results after leukodepletion. CONCLUSIONS: Cryptic Leishmania infection is highly prevalent in blood donors from the Balearic Islands. DTH and L. infantum nested PCR appear to be more sensitive to detect asymptomatic infection than the serology. The use of leukodepletion filters appears to remove parasites from RBC units efficiently.

Prevalencia de la infección por Trypanosoma cruzi en la población infantil y en mujeres en edad fértilinmigrantes procedentes de América Central y del Sur (Resultados preliminares) / No disponible

El número de inmigrantes procedentes de áreas endémicas para la enfermedad de Chagas ha experimentado un gran aumento en los últimos años. Hasta el momento, no se ha realizado ningún estudio de prevalencia de infección chagásica en los niños procedentes de esas áreas ni en mujeres en edad fértil antes de hallarse en período de gestación. El objetivo general del estudio es conocer la prevalencia de la enfermedad de Chagas en esos dos grupos poblacionales. Se analizan muestras de sangre de estos dos grupos de personas que son atendidas en el Consorci d’Atenció Primària de Salut de l´Eixample (CAPse).La duración del estudio es de un año (marzo del 2006 hasta febrero del 2007).Se realiza un cribado serológico mediante inmunocromatografía (IC) (Stat-Pak®Chagas de Chembio®) y confirmación de los casos positivos mediante un ELISAcon antígeno total de T. cruzi y un ELISA con antígeno recombinante (Biokit®ELISA) para Chagas. Se analizaron, hasta septiembre del 2006, 70 muestras de niños de 0 a 14 años y 98 de mujeres en edad fértil. Según la inmunocromatografía hallamos 17/168 (10,11%) resultados positivos, de los cuales 9 (5 niños y 4 mujeres) fueron falsos positivos. En el grupo de mujeres en edad fértil hallamos una prevalencia del 4,25%.Esteestudio aporta resultados que pueden aumentar la sensibilización del pediatrao médico de familia hacia esta enfermedad (AU)

Recently, there has been a large increase in immigration from areas with endemic Chagas disease. Until now, there has been no study of the prevalence of Chagas infection in children and non-pregnant women of childbearing age immigrating from South and Central America. This study aimed to measure the prevalence of Chagas disease in these two populations. The study began in March 2006 and is expected to conclude in Februaryof 2007. Blood samples from children and women attending the Consorcid´Atenció Primària de Salut of l´Eixample (CAPse) were analyzed. An analysis of immunocromatography (IC) (Stat-Pak® Chagas de Chembio®) for the detection of the IgG antibodies was used. Confirmation of positive cases was made using conventional ELISA and recombinant ELISA (Biokit® ELISA)for Chagas. As of September 2006, 70 samples of children aged 0 to 14years and 98 samples of women of reproductive age were analyzed. The ICindicated 18/168 (10.65%) positive results, of which 9 (5 children and 4women) were false positives. In women of childbearing age the prevalence was 4,25%. These results suggest that pediatricians and family medicine doctors should increase awareness and attention to Chagas disease in the seat-risk populations (AU)

Resultados preliminares del estudio en Banco de Sangre, Cataluña / No disponible

El aumento de población inmigrante procedente de zonas endémicas de Chagas ha creado la necesidad de una actuación en los bancos de sangre de nuestro país. Además, el reciente real decreto sobre hemodonación permite aceptar las donaciones de riesgo siempre que se realice el cribado para marcadores de T. cruzi. El objetivo principal del estudio iniciado en septiembre de 2005 en el Banc de Sang i Teixits es determinar la seroprevalenciade la infección por Trypanosoma cruzi en la población de donantes de riesgo en Cataluña. Se incluyeron en el estudio los donantes de riesgo de Chagas (nacidos, transfundidos o hijos de madre procedente de área endémica) y viajeros/residentes en estancias superiores a un mes. El test de cribado utilizado fue el ID-PaGIA Chagas antibody test (DiaMed). Como test suplementarios se utilizaron el método Bioelisa Chagas (Biokit) y un ELISA in-house con antígenos nativos. Entre Septiembre 2005 y Septiembre 2006,se han analizado 1770 donantes. Por grupos de riesgo, hubo 1524 (86%)donantes nacidos en área endémica, 37 españoles de madre originaria dezona endémica (2%) y 209 (12%) viajeros. Hubo 21 donantes inicialmente positivos en el cribado, de los cuales 11 se confirmaron positivos (0,62%).La mayoría de donantes positivos fueron bolivianos (AU)

The increasing population coming from Chagasic areas has forced blood banksin Spain to take preventive measures. The recent Blood Donation regulation provides with the guidelines to screen blood donations at risk for anti-T. cruziantibodies. The aim of the present study was to determine the prevalence of infection by Trypanosoma cruzi in blood donors at risk in Catalonia. The risk groups included were donors born or transfused in endemic areas and donors whose mother was born in endemic area. Travellers or residents in endemic areas for more than one month were also included. The screening method was ID-PaGIA Chagas antibody test (DiaMed). As supplementary test, Bioelisa Chagas (Biokit) and an in-house ELISA test with native antigens were used. Between September 2005 and September 2006, 1770 donor sat risk were screened. According to risk groups, 1524 (86%) were donorsborn in endemic areas, 37 (2%) were donors whose mother was born in an endemic area and 209 (12%) donors had travelled or lived in endemic areas. Twenty-one donors were initially positive in the screening, of which11 were confirmed positive for the presence of T. cruzi antibodies (0.62%).The majority of T. cruzi positive donors were Bolivians (AU)

Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples.

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.

Idiotype expression of IgG1 and IgG2 in dogs naturally infected with Leishmania infantum.

Here we analyzed, by Western blot analysis, the idiotype expression of IgG1 and IgG2 in 109 canine sera corresponding to 50 dogs from endemic areas of leishmaniosis in order to detect markers related to Leishmania infantum infection and clinical condition (asymptomatic or symptomatic). Twenty-four dogs from an area free of leishmaniosis were used as controls. IgG1 and IgG2 responses in symptomatic and asymptomatic L. infantum infections differed mainly in subclass production (ELISA values), with higher IgG2 production occurring particularly in symptomatic dogs. Nevertheless, we observed little difference in the idiotype expression of these IgG subclasses, which, in general, recognized the same antigenic fractions. While early L. infantum infection was characterized by recognition of polypeptide fractions of low molecular weight, mainly fractions of 14, 16 and 18 kDa by IgG1 and 14 and 16 kDa by IgG2, symptomatology was associated with recognition by both IgG subclasses of a 24 kDa fraction and other antigens belonging to the AG24 family.