Isabel Fuentes

Genotypic Characterization of Toxoplasma gondii Strains Associated with Human Toxoplasmosis in Spain: Direct Analysis from Clinical Samples

ABSTRACT Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.

Detection of Cryptosporidium parvum and Cryptosporidium hominis in human patients in Cairo, Egypt

Cryptosporidium is a significant cause of diarrheal disease in developing and industrialized nations. Cryptosporidium hominis and Cryptosporidium parvum are the main agents of cryptosporidiosis in humans. In Egypt, very little is known about genetic structure of Cryptosporidium spp. Therefore, this study was designed to examine samples from sporadic cases of cryptosporidiosis in Egyptians in order to identify the species involved in infection as well as the transmission dynamics and distribution of the parasite in the Great Cairo area. A total of 391 human faecal samples were collected, between May 2008 and March 2009, from ten public hospitals in Great Cairo. Initial screening by immunochromatographic detection kit “the Stick Crypto-Giardia; Operon” showed 23 possible positive cases. Twenty of them were confirmed by microscopic examination. PCR was performed by amplification of the oocyst wall protein (COWP) gene where 18 out of 23 samples were positive, one not detected by microscopy. Cryptosporidium genotyping was performed by RFLP analysis of PCR products of the diagnosis PCR. Only 15 samples rendered a digestion pattern. The genotyping distribution was nine cases showing C. hominis genotype, three showing C. parvum genotype and three showing mixed infection by C. hominis and C. parvum. The data showed an elevated prevalence of C. hominis (80.0%), the most anthroponotic species, suggesting a human–human transmission. Furthermore, the presence of up to 40% of samples infected with C. parvum shows that further investigations are required to determine the subgenotypes of C. parvum to clarify the mode of transmission in order to improve the control measures.

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.

Diagnóstico de laboratorio de la enfermedad de Chagas importada

La enfermedad de Chagas, o tripanosomiasis americana, es una parasitosis generalmente silenciosa y de evolucion lenta. En Espana, como consecuencia de la creciente inmigracion, se ha constituido en una infeccion importada y emergente. Por ello, el diagnostico de laboratorio, parasitologico y serologico requiere una evaluacion critica dentro del marco de la informacion clinica y epidemiologica. Aunque en nuestro entorno ya se han descrito casos agudos, la mayoria de los afectados se diagnostican en la etapa cronica de la infeccion. Como no existe una tecnica de referencia o “patron de oro”, el diagnostico serologico se basa en la concordancia entre diferentes pruebas de principios y antigenos distintos, que requieren un adecuado control de calidad para conseguir resultados optimos. En esta revision se describen las herramientas parasitologicas y serologicas utilizadas en el diagnostico de la enfermedad de Chagas, teniendo en cuenta los factores que afectan a sus ventajas y sus limitaciones.

Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid, Spain

Background The flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country. Methods In this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013–January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed. Principal findings Of 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study. Conclusions Human giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.

Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea.

The aim of the study was to perform a molecular characterization of clinical isolates of Cryptosporidium species from Equatorial Guinea. Standard laboratory methods were used to identify 35 cryptosporidiosis cases among 185 patients. PCR-RFLP successfully identified 34 Cryptosporidium species from these 35 cases, comprising C. parvum (52.9%), C. hominis (44.1%) and C. meleagridis (2.9%); over 90% of the species were isolated from HIV-positive patients. This is the first report of the molecular characterization of Cryptosporidium species isolated from humans in Equatorial Guinea and shows that zoonotic and anthroponotic transmission is present in this country.

Detection and molecular characterisation of Giardia duodenalis, Cryptosporidium spp. and Entamoeba spp. among patients with gastrointestinal symptoms in Gambo Hospital, Oromia Region, southern Ethiopia

To assess the prevalence and genetic diversity of the enteric protozoa species G. duodenalis, Cryptosporidium spp. and Entamoeba histolytica in individuals with gastrointestinal symptoms compatible with infections by these pathogens seeking medical attention in a rural area in southern Ethiopia.

Prevalence and Genetic Diversity of Giardia duodenalis and Cryptosporidium spp. among School Children in a Rural Area of the Amhara Region, North-West Ethiopia.

Backgroud Giardia duodenalis and Cryptosporidium spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries. Methods We report here the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. G. duodenalis was detected by real-time PCR, and the assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of Cryptosporidium species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene. Principal Findings The PCR-based prevalences of G. duodenalis and Cryptosporidium spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 G. duodenalis isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. Cryptosporidium diversity was demonstrated by the occurrence of C. hominis, C. parvum, and C. viatorum in the population under study. Conclusions Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of G. duodenalis genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in G. duodenalis.

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.