Publications by authors named "Silvia Tebar"

11 Publications

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Autochthonous and imported tegumentary leishmaniasis in Catalonia (Spain): Aetiological evolution in the last four decades and usefulness of different typing approaches based on biochemical, molecular and proteomic markers.

Transbound Emerg Dis 2021 Apr 17. Epub 2021 Apr 17.

Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain.

Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is endemic for both visceral and cutaneous leishmaniasis, the autochthonous aetiological agent being Leishmania infantum. Around the world, the L. donovani complex is associated with visceral symptoms, while any species of the Leishmania or Viannia subgenera affecting human can produce tegumentary forms. In a context of growing numbers of imported cases, associated with globalisation, the aim of this study was to analyse the aetiological evolution of human tegumentary leishmaniasis in a region of Spain (Catalonia). Fifty-six Leishmania strains, isolated from 1981 to 2018, were analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI-TOF. The utility of these different analytical methods was compared. The results showed an increase in leishmaniasis over the two last decades, particularly imported cases, which represented 39% of all cases studied. Leishmania infantum, L. major, L. tropica, L. braziliensis, L. guyanensis and L. panamensis were identified. The combination of molecular and enzymatic methods allowed the identification of 29 different strain types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L. major types were relatable with geo-temporal data. Among the autochthonous cases, type C prevailed throughout the studied period (39%). Minor types generally appeared within a short time interval. While all the techniques provided identical identification at the species complex level, MALDI-TOF and rpoIILS or fh sequencing would be the most suitable identification tools for clinical practice, and the tandem hsp70-ITS2 could substitute MLEE in the epidemiological field.
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http://dx.doi.org/10.1111/tbed.14107DOI Listing
April 2021

The Leishmania donovani species complex: A new insight into taxonomy.

Int J Parasitol 2020 Nov 1;50(13):1079-1088. Epub 2020 Sep 1.

Institut de Recerca Biomèdica Sant Pau, Barcelona, Spain; Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau Barcelona, Spain & Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain. Electronic address:

Among the 20 or so Leishmania spp. described as pathogenic for humans, those of the Leishmania donovani complex are the exclusive causative agents of systemic and fatal visceral leishmaniasis. Although well studied, the complex is taxonomically controversial, which hampers clinical and epidemiological research. In this work, we analysed 56 Leishmania strains previously identified as L. donovani, Leishmania archibaldi or Leishmania infantum, isolated from humans, dogs and sandfly vectors throughout their distribution area. The strains were submitted to biochemical and genetic analyses and the resulting data were compared for congruence. Our results show: i) a partial concordance between biochemical and genetic-based data, ii) very limited genetic variability within the L. donovani complex, iii) footprints of frequent genetic exchange along an east-west gradient, marked by a widespread diffusion of alleles across the geographical range, and iv) a large-scale geographical spreading of a few genotypes. From a taxonomic point of view, considering the absence of relevant terminology in existing classes, the L. donovani complex could be treated as a single entity.
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http://dx.doi.org/10.1016/j.ijpara.2020.06.013DOI Listing
November 2020

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PLoS One 2018 17;13(4):e0195738. Epub 2018 Apr 17.

Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Background: Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.

Methodology/principal Findings: We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.

Conclusions/significance: This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195738PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903661PMC
July 2018

Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

J Clin Microbiol 2017 10 19;55(10):2924-2933. Epub 2017 Jul 19.

Laboratoire de Parasitologie-Mycologie, CHU Timone, Université d'Aix-Marseille, Marseille, France

Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the genus. species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were strains. All strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the , , and complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers.
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http://dx.doi.org/10.1128/JCM.00845-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625378PMC
October 2017

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

J Clin Microbiol 2017 05 15;55(5):1396-1407. Epub 2017 Feb 15.

Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain.

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 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 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 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.
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http://dx.doi.org/10.1128/JCM.02248-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405257PMC
May 2017

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

Parasitol Int 2017 Apr 7;66(2):83-88. Epub 2016 Dec 7.

Laboratorio de Biología Molecular de la Enfermedad de Chagas (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" (INGEBI-CONICET), Vuelta de Obligado 2490-2°, C1428ADN Ciudad Autónoma de Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1016/j.parint.2016.12.003DOI Listing
April 2017

Serological Diagnosis of Chronic Chagas Disease: Is It Time for a Change?

J Clin Microbiol 2016 06 6;54(6):1566-1572. Epub 2016 Apr 6.

Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Chagas disease has spread to areas that are nonendemic for the disease with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New-generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new-generation kit, the Architect Chagas (cutoff, ≥1 sample relative light units/cutoff value [S/CO]), as a single technique for the diagnosis of chronic Chagas disease. The Architect Chagas showed a sensitivity of 100% (95% confidence interval [CI], 99.5 to 100%) and a specificity of 97.6% (95% CI, 95.2 to 99.9%). Five out of six false-positive serum samples were a consequence of cross-reactivity with Leishmania spp., and all of them achieved results of <5 S/CO. We propose the Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only gray-zone and positive sera with a result of ≤6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity with Leishmania species. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease.
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http://dx.doi.org/10.1128/JCM.00142-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879299PMC
June 2016

Altered Hypercoagulability Factors in Patients with Chronic Chagas Disease: Potential Biomarkers of Therapeutic Response.

PLoS Negl Trop Dis 2016 Jan 4;10(1):e0004269. Epub 2016 Jan 4.

ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic- Universitat de Barcelona, Barcelona, Spain.

Thromboembolic events were described in patients with Chagas disease without cardiomyopathy. We aim to confirm if there is a hypercoagulable state in these patients and to determine if there is an early normalization of hemostasis factors after antiparasitic treatment. Ninety-nine individuals from Chagas disease-endemic areas were classified in two groups: G1, with T.cruzi infection (n = 56); G2, healthy individuals (n = 43). Twenty-four hemostasis factors were measured at baseline. G1 patients treated with benznidazole were followed for 36 months, recording clinical parameters and performance of conventional serology, chemiluminescent enzyme-linked immunosorbent assay (trypomastigote-derived glycosylphosphatidylinositol-anchored mucins), quantitative polymerase chain reaction, and hemostasis tests every 6-month visits. Prothrombin fragment 1+2 (F1+2) and endogenous thrombin potential (ETP) were abnormally expressed in 77% and 50% of infected patients at baseline but returned to and remained at normal levels shortly after treatment in 76% and 96% of cases, respectively. Plasmin-antiplasmin complexes (PAP) were altered before treatment in 32% of G1 patients but normalized in 94% of cases several months after treatment. None of the patients with normal F1+2 values during follow-up had a positive qRT-PCR result, but 3/24 patients (13%) with normal ETP values did. In a percentage of chronic T. cruzi infected patients treated with benznidazole, altered coagulation markers returned into normal levels. F1+2, ETP and PAP could be useful markers for assessing sustained response to benznidazole.
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http://dx.doi.org/10.1371/journal.pntd.0004269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700971PMC
January 2016

Evaluation of a chemiluminescent enzyme-linked immunosorbent assay for the diagnosis of Trypanosoma cruzi infection in a nonendemic setting.

Mem Inst Oswaldo Cruz 2013 Nov;108(7):928-31

Barcelona Centre for International Health Research, Hospital Clinic.

The disappearance of lytic, protective antibodies (Abs) from the serum of patients with Chagas disease is accepted as a reliable indicator of parasitological cure. The efficiency of a chemiluminescent enzyme-linked immunosorbent assay based on a purified, trypomastigote-derived glycosylphosphatidylinositol-anchored mucin antigen for the serologic detection of lytic Abs against Trypanosoma cruzi was evaluated in a nonendemic setting using a panel of 92 positive and 58 negative human sera. The technique proved to be highly sensitive {100%; 95% confidence interval (CI) = 96-100} and specific (98.3%; 95% CI = 90.7-99.7), with a kappa score of 0.99. Therefore, this assay can be used to detect active T. cruzi infection and to monitor trypanosomicidal treatment.
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http://dx.doi.org/10.1590/0074-0276130112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970649PMC
November 2013

Ultrasensitive real-time PCR for the clinical management of visceral leishmaniasis in HIV-Infected patients.

Am J Trop Med Hyg 2013 Jul 29;89(1):105-10. Epub 2013 Apr 29.

Infectious Disease Department, Universitat Autònoma de Barcelona, Barcelona, Spain.

Molecular methods have been proposed as an alternative tool for the diagnosis of visceral leishmaniasis (VL), but no data are available regarding use for monitoring clinical outcome. A prospective cohort study of human immunodeficiency virus-(HIV) and VL-coinfected patients was conducted in a university-affiliated hospital in Barcelona, Spain. Leishmania parasite load was monitored using a real-time polymerase chain reaction (PCR) at baseline and every 3 months. Cutoff values for PCR were determined using receiver operating characteristic (ROC) curves. Overall, 37 episodes were analyzed, and 25 of these episodes were considered as relapsing episodes. A significant decrease of parasite load measured 3 months after treatment could predict the clinical evolution of VL. A parasite load over 0.9 parasites/mL measured 12 months after treatment could predicts relapse with a sensitivity of 100% and a specificity of 90.9%. Monitoring parasite load by an ultrasensitive quantitative Leishmania PCR is useful to predict the risk of relapse after a VL episode in HIV-infected patients.
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http://dx.doi.org/10.4269/ajtmh.12-0527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748463PMC
July 2013

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

Am J Trop Med Hyg 2012 Mar;86(3):412-6

Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Spain.

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.
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http://dx.doi.org/10.4269/ajtmh.2012.11-0111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284355PMC
March 2012