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.

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.

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.

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.

Cross-sectional serosurvey of feline leishmaniasis in ecoregions around the Northwestern Mediterranean.

A cross-sectional serosurvey using Leishmania infantum ELISA was performed on 445 cats living in ecoregions around the Northwestern Mediterranean basin; 58 cats from an area of the US where leishmaniasis is not endemic were used as negative controls. ELISA results were further confirmed in 69 cats by Western blot (WB). Finally, 76 of them were also tested for FeLV and FIV. Seroprevalence by ELISA-prot A was 6.29%, and that by ELISA-IgG was 5.25%. Positive cat sera recognized patterns of polypeptides in WB, including L. infantum-specific antigenic fractions. There was no association with retroviruses. Leishmania-specific antibodies are prevalent in cats living in ecoregions around the Northwestern Mediterranean basin; thus, leishmaniasis must be included in the differential diagnosis of diseases in cats living in these ecoregions. Their role as peridomestic reservoirs for L. infantum needs further characterization, but it could be hypothesized that the cat is a secondary reservoir host, rather than an accidental one.

Application of microsatellite genotyping to the study of a restricted Leishmania infantum focus: different genotype compositions in isolates from dogs and sand flies.

Leishmania infantum polymorphism was studied by DNA microsatellite analysis of 110 L. infantum stocks (94 from dogs, 15 from sand flies, and 1 from a human visceral case) from a rural leishmaniasis-endemic area (Priorat) in northeastern Spain. Three microsatellites of the eight present in three fragments (internal transcribed spacer, Lm4, and Lm2) of L. infantum nuclear DNA are polymorphic inside the focus, resulting in 17 genotypes. Isolates from dogs and sand flies had different allelic compositions and shared only four genotypes. Microsatellite analysis is useful for L. infantum genotyping and epidemiologic tracking. Its application with strains from dogs and vectors in an area endemic for leishmaniasis shows the heterogeneous distribution of L. infantum in hosts living in sympatric conditions.

Congenital Trypanosoma cruzi infection in a non-endemic area.

In May 2004, after launching a screening programme to detect Trypanosoma cruzi infection in the Latin American population, a case of congenital infection in a 2-year-old boy was discovered in Barcelona. Few cases of congenital transmission have been described in non-endemic areas and little is known about the epidemiological and clinical features of congenital Chagas disease in this context. The increase in Latin American immigrants in Europe and the USA requires greater epidemiological surveillance and appropriate diagnostic techniques for managing T. cruzi infections.

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.

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.

Congenital transmission of Trypanosoma cruzi in Europe (Spain): a case report.

Here we report a documented case of congenital transmission of Trypanosoma cruzi from a Bolivian mother with chronic Chagas disease living in Spain. The serology and blood nested polymerase chain reaction (PCR) were positive for the mother, and amastigote forms were observed in histopathological study of the placenta and umbilical cord. Direct examination, culture, and nested PCR were positive in the blood of the neonate. At the age of 8 days, the neonate began treatment with 5-7.5 mg/kg/day of benznidazol, which was continued for 60 days. Direct examination, blood culture, and nested PCR were negative to T. cruzi 20 days after the start of treatment and remained negative 4 and 7 months thereafter. Serological tests were negative at 4 months. To detect congenital infection and initiate early treatment of infected newborns, protocols are required to detect Chagas disease in pregnant women who migrate from endemic to non-endemic areas.

Dynamics of Leishmania-specific immunoglobulin isotypes in dogs with clinical leishmaniasis before and after treatment.

Concentrations of Leishmania-specific immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) isotypes were analyzed by enzyme-linked immunosorbent assay (ELISA) in 23 dogs naturally infected with Leishmania infantum before and 1 year after initiating drug therapy. Results showed a high expression and prevalence of Leishmania-specific IgG (176.4 +/- 89 ELISA units [EU]), IgM (105.3 +/- 95.5 EU), and IgA (153.6 +/- 98 EU) in dogs before treatment (median +/- interquartile range EU). One year after treatment was started, dogs were classified as responsive dogs (RDs; n = 13) or unresponsive dogs (UDs; n = 10) based on clinicopathologic findings. Both groups of dogs experienced a statistically significant decrease (P < .05) in Leishmania-specific IgG (RDs = 27%, UDs = 41%), IgM (RDs = 42%, UDs = 29%), and IgA (RDs = 56%, UDs = 46%). Concentrations of specific IgG and IgM were not different at diagnosis or after treatment between the 2 groups. However, the median value for Leishmania-specific IgA 1 year after treatment was significantly lower (P < .05) in RDs (60.8 +/- 67 EU) than in UDs (117 +/- 54 EU). Examination of our data indicates that both the IgA isotype, which is mostly produced by mucosal plasma cells, and the IgM isotype are increased in infected symptomatic dogs, as previously reported for IgG. These 3 isotypes decreased significantly 1 year after initiation of medical treatment.

Value of culture and nested polymerase chain reaction of blood in the prediction of relapses in patients co-infected with leishmania and human immunodeficiency virus.

The use of culture and a nested polymerase chain reaction (PCR) of blood in predicting the probability of relapse was evaluated in 20 patients co-infected with Leishmania and human immunodeficiency virus (HIV). Fourteen of 20 patients relapsed, with 24 clinical relapses diagnosed. During clinical relapse, the parasite was detected by culture in 21 of 24 blood samples and by nested PCR in 23 of 24 blood samples. After treatment and during asymptomatic periods, the parasite was detected by culture in 18 (19.1%) of 94 blood samples and by nested PCR in 58 (61.7%) of 94 blood samples. For positive blood cultures, the Kaplan-Meier probability estimates for relapse at 6, 12, 18, and 24 months were 44%, 68%, 76%, and 76%, respectively, while for positive nested PCRs, the estimates were 20%, 33%, 45%, and 50%, respectively. For negative blood cultures, relapse probabilities for the same time points were 7%, 12%, 12%, and 12%, while for negative nested PCRs, these probabilities were 8%, 14%, 21%, and 26%. Nested PCR-positive results in asymptomatic periods indicated presence of the parasite, but not necessarily relapse. However, the presence of viable parasites during post-treatment follow-up increased the probability of relapse and showed that culture positivity could be a good relapse marker.

Immunoglobulin G and E responses in various stages of canine leishmaniosis.

Pathogenesis in visceral leishmaniosis is associated with depressed cellular immunity and a significant rise of antileishmanial antibodies. We assessed the relative levels of immunoglobulin E anti-Leishmania infantum, together with those of IgG, IgG1 and IgG2, using the enzyme-linked immunosorbent assay (ELISA) test, in non-infected and infected dogs with or without symptoms, and their association with symptoms to differentiate the stages of the infection. The expression of all immunoglobulins (IgG, IgG1, IgG2 and IgE) was higher in symptomatic dogs than in all other categories. IgG and IgG2 expression was higher in the infected asymptomatic group than in the non-infected group, whereas IgG1 and IgE expression was only higher in symptomatic animals. This correlation between the expression of IgG1 and IgE and the pathology of leishmaniosis points to their potential role as markers of the active disease.

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.

In vitro susceptibility to pentavalent antimony in Leishmania infantum strains is not modified during in vitro or in vivo passages but is modified after host treatment with meglumine antimoniate.

BACKGROUND: Leishmaniasis is a common parasitic disease in Southern Europe, caused by Leishmania infantum. The failures of current treatment with pentavalent antimonials are partially attributable to the emergence of antimony-resistant Leishmania strains. This study analyses the in vitro susceptibility to pentavalent antimony of intracellular amastigotes from a range of L. infantum strains, derived from the same infected animal, during in vitro and in vivo passages and after host treatment with meglumine antimoniate. RESULTS: SbV-IC50 values for strains from two distinct isolates from the same host and one stock after two years of culture in NNN medium and posterior passage to hamster were similar (5.0 +/- 0.2; 4.9 +/- 0.2 and 4.4 +/- 0.1 mgSbV/L, respectively). In contrast, a significant difference (P < 0.01, t test) was observed between the mean SbV-IC50 values in the stocks obtained before and after treatment of hosts with meglumine antimoniate (4.7 +/- 0.4 mgSbV/L vs. 7.7 +/- 1.5 mgSbV/L). Drug-resistance after drug pressure in experimentally infected dogs increased over repeated drug administration (6.4 +/- 0.5 mgSbV/L after first treatment vs. 8.6 +/- 1.4 mgSbV/L after the second) (P < 0.01, t test). CONCLUSIONS: These results confirm previous observations on strains from Leishmania/HIV co-infected patients and indicate the effect of the increasing use of antimony derivatives for treatment of canine leishmaniasis in endemic areas on the emergence of Leishmania antimony-resistant strains.

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.