Marta Mateo

Detection of Toxoplasma gondii in cats by comparing bioassay in mice and polymerase chain reaction (PCR).

Feline toxoplasmosis can usually be diagnosed by serological and coprological methods. However, when the interpretation of results is difficult, it becomes necessary to rely on the direct detection of the parasite. In this study, samples of brain from 64 Toxoplasma-seropositive cats were subjected to direct detection of Toxoplasma gondii by bioassay in mice and nested-PCR. T. gondii was isolated by bioassay in mice in 41 (64%) cases, and nested-PCR was positive in 37 (57.8%) cases (p>0.05). The results therefore suggest that nested-PCR could be a rapid technique for diagnosing toxoplasmosis in cats.

Detection and molecular characterization of Giardia duodenalis in children attending day care centers in Majadahonda, Madrid, Central Spain.

AbstractInfections by the protozoan enteroparasites Giardia duodenalis and Cryptosporidium spp are a major cause of morbidity in children attending day care facilities in developed countries. In this cross-sectional study, we aimed to estimate the occurrence and genotype frequencies of these pathogens in children attending day care centers in Majadahonda, Central Spain. To do so, single stool samples were obtained from 90 children and tested for the presence of G duodenalis and Cryptosporidium spp by conventional microscopy and immunochromatography. Positive results by these techniques were subsequently confirmed by immunofluorescence microscopy. G duodenalis-positive samples were subjected to molecular characterization studies by multilocus sequence-based genotyping of the glutamate dehydrogenase and &bgr;-giardin genes of the parasite. G duodenalis assemblages were confirmed by restriction fragment length polymorphism analyses and sequencing. A socioepidemiological questionnaire was used to identify variables potentially associated with giardiasis/cryptosporidiosis in the population of children under investigation. Overall, G duodenalis and Cryptosporidium spp were detected in 15.5% and 3.3% of stool samples, respectively. Giardiasis and cryptosporidiosis were found in 3/3 and 2/3 day care centers, respectively, affecting mainly infants aged 13 to 24 months. A total of 8 G duodenalis isolates were confirmed as subassemblage BIV, all of them belonging to asymptomatic children. Attempts to genotype Cryptosporidium isolates failed. None of the variables considered could be associated with higher risk of infection with giardiasis or cryptosporidiosis. These results clearly indicate that asymptomatic infections with G duodenalis and Cryptosporidium spp are frequent in <3-year-old children in Central Spain.

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in a cohort of 1,000 neonates in Madrid (Spain).

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1–2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8–18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK

Abstract Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennetts wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennetts wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.

A Novel Mutation of the α2-Globin causing α+-Thalassemia: Hb Plasencia [α125(H8)Leu→Arg (α2)

We describe, in a Spanish family with moderate microcytosis and hypochromia, a novel nondeletional α-thalassemia (thal) mutation localized on the α2-globin gene. DNA sequencing revealed a point mutation at codon 125 (CTG→CGG) in the heterozygous state, that was confirmed by restriction analysis. The resulting variant, which causes a nondeletional α-thal, was named Hb Plasencia [α125(H8)Leu→Arg (α2)] after the place of residence of the affected family.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.

Incidence of the HFE gene mutations in a cohort of non-Spanish origin neonates in Madrid

Dear Editor, Hereditary Hemochromatosis (HH) is a potentially serious disease due to the build up of iron excess and attendant cells damage in several organs [4] that issues from the irongreedy bowels. Unless it is early diagnosed and treated, HH can lead to liver cirrhosis and hepatocellular carcinoma, diabetes mellitus, infertility, heart disease, and arthropathy [8]. However, an early diagnosis, before irreversible injuries become established, can prevent all associated disorders and re-establish a life expectancy similar to that of the general population [4, 8]. The disease is passed on as an autosomal recessive trait associated to C282Y (Cys282Tyr) and H63D (His63Asp) mutations of the hemochromatosis (HFE) gene, located on the short arm of chromosome 6 [5, 9]. Geographical distribution of HH is quite irregular. Both mutations are frequent in Europe. However, H63D has also been found in northern Africa, the Middle East, and Asia, whereas C282Y seems to affect, only, European populations, particularly in the Scandinavian countries, where seemingly this mutation on the HFE gene was originated and spread with the Vikings [14]. In these countries, the prevalence ranges from 5 to 10%, and the mutation has been found to be homozygous in 1:100 to 1:400 of the overall population [12, 15]. In recent years, migratory movements have changed the demographic, cultural, and social characteristics of Spanish cities. At this time, 12.1% of the Madrid population was not born in Spain. And even more significant, in 2005, the increase in children born to non-Spanish parents reached 17.37% [10]. Given this demographic shift, the aim of this study is to determine the incidence of C282Y and H63D mutations in a cohort of neonates born from non-Spanish parents in the Madrid Autonomous Community territory. And in this way, the knowledge of the genotype of these new populations could be helpful for planning future health policy strategies related to HH. In a cross-sectional study, parents of all 1,000 neonates born in the Hospital Clínico San Carlos of Madrid, from February 1st to May 31st, 2006, were asked to answer a socio-demographic questionnaire, but the study was limited to 449 neonates who issued from non-Spanish parents. The study was submitted to the Institutional Ethics and Research Committee for approval, and an informed consent was secured, always, from the parents. The HFE gene was studied using polymerase chain reaction amplification primed with previously described oligonucleotides [2, 9, 13]. Data on genotype and allele frequency, in all neonates, are shown with the respective count, percentage, and 95% confidence interval. The Hardy–Weinberg test was applied to determine the population genetics equilibrium. Allelic frequency was 0.9% for the C282Y mutation and 11.5% in the H63D variant. None of the neonates presented homozygosity for the C282Y mutation, whereas in eight neonates, the mutation was present in a single allele (1.8%). As for mutation H63D, homozygosity was identified in ten neonates (2.2%), and heterozygosity was found in 83 neonates (18.5%). Double heterozygosity was not identified. Ann Hematol (2007) 86:459–462 DOI 10.1007/s00277-007-0264-z

Hb LA Coruña [β38(C4)Thr→Ile]: A New Hemoglobin Variant Leading to Familial Polycythemia

Hb La Coruña [β38(C4)Thr → Ile] is a new hemoglobin (Hb) variant that has an increased oxygen affinity. Clinically, this Hb leads to erythrocytosis. Hb La Coruña is an electrophoretically silent variant that can be detected by reversed phase high performance liquid chromatography (HPLC) and characterized by DNA sequencing. The patient was a 22-year-old Spanish male whose family lived in La Coruña, in the northwest of Spain. His mother was also a carrier.

Hb Santander [β34(B16)Val→Asp (GTC→GAC)]: A New Unstable Variant Found as a De Novo Mutation in a Spanish Patient

A careful analysis of hematological data obtained from patients with mild hemolytic anemia may suggest the presence of an unstable hemoglobin (Hb) variant. This diagnosis needs to be confirmed by appropriate laboratory procedures (1,2). This was the case for a 22-year-old Spanish male patient presenting with jaundice and suffering from hemolytic crises during infections. In this patient, we found a new unstable Hb variant, in which the valine residue at position 34 of the b-globin chain was replaced by an aspartic acid. This variant has been characterized at the gene level by DNA sequencing, revealing a GTC!GAC mutation at codon 34 of the b-globin gene. It was named Hb Santander after the city where the proband lives. Hematological data were obtained with a Coulter GENS (Coulter Electronics, Ltd, Luton, UK). Hb A2 was measured by anion exchange chromatography (3) and Hb F according to the method described by Betke et al. (4). Hemoglobin was studied by electrophoresis on cellulose acetate at pH 8.6, electrophoresis on citrate agar gel (pH 6.0), isoelectrofocusing (IEF) on polyacrylamide gel (pH 5.5‐8.5) and by ion exchange high performance liquid chromatography (HPLC) using a TSK-CM-2-SW column (Tosohaas, Montgomeryville, PA, USA) and a linear gradient from 100% buffer A to 100% buffer B