Publications by authors named "Cristina Ballart"

17 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

Evaluation of the Performance of the Loopamp Trypanosoma cruzi Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain.

J Clin Microbiol 2021 Apr 20;59(5). Epub 2021 Apr 20.

Foundation for Innovative New Diagnostics FIND, Geneva, Switzerland.

In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp detection kit (Eiken Chemical Co. Ltd., Japan) (-LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of -LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The -LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, -LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of -LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp detection kit showed good performance for the diagnosis of congenital CD. -LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection.
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http://dx.doi.org/10.1128/JCM.01860-20DOI Listing
April 2021

Clinical and immunological characteristics of tegumentary leishmaniasis cases in Bolivia.

PLoS Negl Trop Dis 2021 Mar 5;15(3):e0009223. Epub 2021 Mar 5.

ISGlobal, Hospital Clínic-Universitat de Barcelona, Barcelona, Spain.

Background: Tegumentary leishmaniasis (TL) is a parasitic disease that can present a cutaneous or mucocutaneous clinical form (CL and MCL, respectively). The disease is caused by different Leishmania species and transmitted by phlebotomine sand flies. Bolivia has one of the highest incidences of the disease in South America and the diagnosis is done by parasitological techniques. Our aim was to describe the clinical and immunological characteristics of CL and MCL patients attending the leishmaniasis reference center in Cochabamba, Bolivia, in order to gain updated clinical and epidemiological information, to evaluate the diagnostic methods used and to identify biomarkers related to clinical disease and its evolution.

Methodology/principal Findings: The study was conducted from September 2014 to November 2015 and 135 patients with lesions compatible with CL or MCL were included. Epidemiological and clinical data were collected using a semi-structured questionnaire. Two parasitological diagnostic methods were used: Giemsa-stained smears and culture of lesion aspirates. Blood samples obtained from participants were used to measure the concentrations of different cytokines. 59.2% (80/135) were leishmaniasis confirmed cases (CL: 71.3%; MCL: 28.7%). Sixty percent of the confirmed cases were positive by smears and 90.6% were positive by culture. 53.8% were primo-infections. Eotaxin and monokine induced by IFN-γ presented higher serum concentrations in the MCL clinical presentation compared to CL cases and no-cases. None of the cytokines presented different concentrations between primo-infections and secondary infections due to treatment failure.

Conclusions/significance: In Bolivia, parasitological diagnosis remains the reference standard in diagnosis of leishmaniasis because of its high specificity, whereas the sensitivity varies over a wide range leading to loss of cases. Until more accurate tools are implemented, all patients should be tested by both smears and culture of lesion aspirates to minimize the risk of false negatives. Our results showed higher concentrations of several cytokines in MCL compared to CL, but no differences were observed between CL and no-cases. In addition, none of the cytokines differed between primary and secondary infections. These results highlight the need of further research to identify biomarkers of susceptibility and disease progression, in addition to looking at the local cellular immune responses in the lesions.
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http://dx.doi.org/10.1371/journal.pntd.0009223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968743PMC
March 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

Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease.

Emerg Infect Dis 2020 08;26(8):1846-1851

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.
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http://dx.doi.org/10.3201/eid2608.191042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392439PMC
August 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

Isoenzymatic characterization of Phlebotomus ariasi and P. perniciosus of canine leishmaniasis foci from Eastern Pyrenean regions and comparison with other populations from Europe.

Parasite 2018 5;25. Epub 2018 Feb 5.

Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Av. Joan XXIII 27-31, 08028 Barcelona, Spain - ISGlobal, Barcelona Centre for International Health Research (CRESIB), Rosselló 134, 4a planta, 08036 Barcelona, Spain.

An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbour-joining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.
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http://dx.doi.org/10.1051/parasite/2018005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798223PMC
November 2018

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

Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene.

Acta Trop 2015 Dec 3;152:96-102. Epub 2015 Sep 3.

Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain. Electronic address:

Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the disease to adopt control measures. In previous studies, a polymerase chain reaction (PCR) of 359bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak in southwest Madrid, Spain, providing significant information about blood meal preferences in the focus. In this work, a PCR-restriction fragment length polymorphism (RFLP) targeting a fragment of 359bp of vertebrate cyt b gene was developed. Restriction endonucleases HaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sand flies. The protocol has been validated with twenty six engorged females collected in the field with CDC traps. Blood meals from nine vertebrates were identified based on PCR-cyt b and sequencing-human, dog, cat, horse, hare, rabbit, sheep, goat and chicken - and mixed blood meals (sheep/human; sheep/goat) - and successfully distinguished by PCR-RFLP. Therefore, this approach is an efficient and reliable alternative method to be applied in entomological surveys.
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http://dx.doi.org/10.1016/j.actatropica.2015.08.020DOI Listing
December 2015

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

Parasit Vectors 2014 Sep 4;7:421. Epub 2014 Sep 4.

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

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.
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http://dx.doi.org/10.1186/1756-3305-7-421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261246PMC
September 2014

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.

Geospat Health 2014 May;8(2):389-403

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.
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http://dx.doi.org/10.4081/gh.2014.28DOI Listing
May 2014

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.

Prev Vet Med 2013 Apr 27;109(1-2):116-27. Epub 2012 Sep 27.

Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain.

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.
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http://dx.doi.org/10.1016/j.prevetmed.2012.09.003DOI Listing
April 2013

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.

Parasitol Res 2012 Aug 2;111(2):515-23. Epub 2012 Mar 2.

Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain.

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.
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http://dx.doi.org/10.1007/s00436-012-2863-4DOI Listing
August 2012

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

Parasitology 2011 Dec 14;138(14):1878-91. Epub 2011 Sep 14.

Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.
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http://dx.doi.org/10.1017/S003118201100148XDOI Listing
December 2011

UGT1A1, UGT1A6 and UGT1A7 genetic analysis: repercussion for irinotecan pharmacogenetics in the São Miguel Island Population (Azores, Portugal).

Mol Diagn Ther 2009 ;13(4):261-8

Molecular Genetics and Pathology Unit, Hospital of Divino Espirito Santo of Ponta Delgada, São Miguel Island, Azores, Portugal.

Background: Glucuronidation reactions, catalyzed by uridine-diphosphate glucuronosyltransferase (UGT) enzymes, constitute a detoxification process that adds glucuronic acid to endogenous and exogenous compounds, aiding their excretion. UGT1A proteins have been implicated as risk factors for both the development of cancer and adverse drug effects.

Methods: Here, we assess the genome of 469 individuals from São Miguel Island (Azores, Portugal) in order to determine the frequencies of polymorphisms and haplotypes in UGT1A1, UGT1A6, and UGT1A7, the co-occurrence of reduced enzyme activity UGT1A variants related to irinotecan toxicity, and to calculate the extent of linkage disequilibrium (LD) in the genomic region encompassing these genes.

Results: Allelic analysis disclosed the presence of rare alleles - UGT1A1*36 and UGT1A1*37--only found in individuals of African descent, and UGT1A7*4. These alleles confirm our previous results on the São Miguel Island genetic background. We identified five different genotypes in UGT1A1 and UGT1A6 and nine in UGT1A7. Haplotype analysis showed that three haplotypes constituted approximately 80% of the allelic variants. Interestingly, haplotype 3 (UGT1A1*28-UGT1A6*2-UGT1A7*3), with a frequency of 0.235, gathers the three alleles encoding the low-function UGT isoforms. Additionally, LD indicates a strong interaction between functional polymorphisms related to the alteration of the UGT enzyme activity.

Conclusions: In summary, the results demonstrate a high variability of alleles and haplotypes, which have important roles in modifying expression and activity of UGTs. The data presented here could improve the understanding of the predisposition to cancers and susceptibility to the adverse effects of irinotecan in the São Miguel Island population.
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http://dx.doi.org/10.2165/11317170-000000000-00000DOI Listing
January 2010