Teresa Gárate

Transfusional Chagas Disease: Parasitological and Serological Monitoring of an Infected Recipient and Blood Donor

Chagas disease is endemic to Latin America, but human migration is extending its distribution. This report describes the parasitological and serological course of disease in a Spanish patient fatally infected via a blood product transfusion, as well as the monitoring of the donor. Before undergoing immunosuppression, multitransfused patients should be screened for anti-Trypanosoma cruzi antibodies.

Further molecular discrimination of Spanish strains of Echinococcus granulosus.

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.

PCR tools for the differential diagnosis of Taenia saginata and Taenia solium taeniasis/cysticercosis from different geographical locations

The potential value of PCRs in the species-specific diagnosis of have been investigated, using samples of T. saginata and T. solium from different geographical areas. The PCRs examining inter-species differences were based on the sequence of the HDP2 DNA fragment, specific for T. saginata/T. solium, and the sequence of the rDNA internal transcribed spacer 1 and spacer 2 (ITS-1 and ITS-2). This PCR analysis of DNA isolates confirmed morphologic diagnosis and allowed the speciation of samples too small or fragmented for morphologic identification, with clear and consistent inter-species differences between T. saginata (twenty-two) and T. solium (three) geographical isolates. Possible intra-species genomic variability, within these species, was similarly studied through analysis of PCR amplification products (PCR-RFLP) and only encountered one exceptional T. saginata isolate from Kenya, which yielded a unique PCR-RFLP pattern, different from T. saginata DNA of Mexican (one sample) and Spanish (seven samples) origin.

Using entropy of drug and protein graphs to predict FDA drug-target network: Theoretic-experimental study of MAO inhibitors and hemoglobin peptides from Fasciola hepatica

There are many drugs described with very different affinity to a large number of receptors. In this work, we selected Drug-Target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets like proteins. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately, most QSAR models predict activity against only one protein. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 32:32-15-1:1. This MLP classifies correctly 623 out of 678 DTPs (Sensitivity = 91.89%) and 2995 out of 3234 nDTPs (Specificity = 92.61%), corresponding to training Accuracy = 92.48%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 313 out of 338 DTPs (Sensitivity = 92.60%) and 1411 out of 1534 nDTP (Specificity = 91.98%) in validation series, corresponding to total Accuracy = 92.09% for validation series (Predictability). This model favorably compares with other LDA and ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. These mt-QSARs offer also a good opportunity to construct drug-protein Complex Networks (CNs) that can be used to explore large and complex drug-protein receptors databases. Finally, we illustrated two practical uses of this model with two different experiments. In experiment 1, we report prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of 10 rasagiline derivatives promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, SEC and 1DE sample preparation, MALDI-TOF MS and MS/MS analysis, MASCOT search, MM/MD 3D structure modeling, and QSAR prediction for different peptides of hemoglobin found in the proteome of the human parasite Fasciola hepatica; which is promising for anti-parasite drug targets discovery.

The Anisakis simplex Ani s 7 major allergen as an indicator of true Anisakis infections

Ani s 7 is currently the most important excretory/secretory (ES) Anisakis simplex allergen, as it is the only one recognized by 100% of infected patients. The allergenicity of this molecule is due mainly to the presence of a novel CX17–25CX9–22CX8CX6 tandem repeat motif not seen in any previously reported protein. In this study we used this allergen as a model to investigate how ES allergens are recognized during Anisakis infections, and the usefulness of a recombinant fragment of Ani s 7 allergen (t‐Ani s 7) as a marker of true Anisakis infections. The possible antigenic relationship between native Ani s 7 (nAni s 7) from Anisakis and Pseudoterranova decipens antigens was also investigated. Our results demonstrate that nAni s 7 is secreted and recognized by the immune system of rats only when the larvae are alive (i.e. during the acute phase of infection), and that this molecule is not present in, or is antigenically different from, Pseudoterranova allergens. The t‐Ani s 7 polypeptide is a useful target for differentiating immunoglobulin E antibodies induced by true Anisakis infections from those induced by other antigens that may cross‐react with Anisakis allergens, including P. decipiens. The results also support the hypothesis that the Ani s 7 major allergen does not participate in maintaining the antigenic stimulus during chronic infections.

Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen.

Background:  Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O‐deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3‐ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis.

Thelaziosis in Humans, a Zoonotic Infection, Spain, 2011

After Thelazia callipaeda infection in dogs and cats were reported in Spain, a human case of thelaziosis in this country was reported, suggesting zoonotic transmission. The active reproductive status of this nematode in situ indicates that humans are competent hosts for this parasite.

Assessing the validity of an ELISA test for the serological diagnosis of human fascioliasis in different epidemiological situations

Objectives  To improve the diagnosis of human fascioliasis caused by Fasciola hepatica and Fasciola gigantica, we evaluated the diagnostic accuracy of an enzyme‐linked immunosorbent assay (ELISA), with Fasciola antigen from the adult liver fluke, for the detection of IgG against fascioliasis in human sera.

Ag-ELISA and PCR for monitoring the vaccination of cattle against Taenia saginata cysticercosis using an oncospheral adhesion protein (HP6) with surface and secreted localization.

ATaenia saginata oncosphere-derived adhesion protein (HP6) with surface and secreted localization was used to successfully vaccinate calves against oral challenge withT. saginata eggs. In contrast, vaccination using a combination ofT. saginata oncosphere-derived peptides, selected on the basis of their antigenic index, and including three derived from the HP6 molecule (HP6-1, HP6-2 and HP6-3), was unsuccessful. This either indicated that the wrong peptides were selected or, in the case of the HP6 protein, that the protective epitope is conformational in nature. The protection experiments were monitored using a parasite antigen detection ELISA (HP10 Ag-ELISA), which allowed the early determination of the success of the vaccination protocol, subsequently confirmed at autopsy. PCR assays were used for the first time to confirm the presence ofT. saginata DNA in lesions recovered at autopsy and thus verify the parasite origin of the lesions.

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.