Molecular Detection and Genotyping of Enteric Protists in Asymptomatic Schoolchildren and Their Legal Guardians in Madrid, Spain

Asymptomatic carriage of diarrhoea-causing enteric protist parasites in the general population is poorly understood, particularly in medium- to high-income countries. This molecular epidemiological survey investigates the presence, molecular diversity, and household transmission of Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., and Enterocystozoon bieneusi in schoolchildren aged 2–13 years (n = 74) and their legal guardians (n = 6) in Madrid, Spain. Enteroparasite detection and genotyping was conducted in stool samples by molecular (PCR and Sanger sequencing) methods. Potential associations linked to infections were investigated through epidemiological questionnaires. Giardia duodenalis was the most prevalent enteric parasite found (14%, 95% CI: 7.1–23), followed by Blastocystis sp. (10%, 95% CI: 6.2–22) and Cryptosporidium spp. (3.8%, 95% CI: 0.78–11). None of the participants tested positive for E. bieneusi. Sequence analyses revealed the presence of G. duodenalis assemblage B, sub-assemblage BIV in a single child. The three Cryptosporidium isolates obtained were assigned to C. hominis, two of them belonging to the gp60 subtype IbA10G2. Four Blastocystis subtypes were identified including ST2 (38%, 3/8), ST3 (25%, 2/8), ST4 (25%, 2/8), and ST8 (12%, 1/8). All G. duodenalis and Cryptosporidium isolates were detected in children only. Blastocystis ST3 and ST4 were circulating in members of the same household. Blastocystis carriage rates increased with the age of the participants. Presence of diarrhoea-causing enteric protists was common in apparently healthy children.This research was funded by Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities, Spain, grant number PI16CIII/00024.S

Chronic Exercise Improves Mitochondrial Function and Insulin Sensitivity in Brown Adipose Tissue

The aim of the present work was to study the consequences of chronic exercise training on factors involved in the regulation of mitochondrial remodeling and biogenesis, as well as the ability to produce energy and improve insulin sensitivity and glucose uptake in rat brown adipose tissue (BAT). Male Wistar rats were divided into two groups: (1) control group (C; n = 10) and (2) exercise-trained rats (ET; n = 10) for 8 weeks on a motor treadmill (five times per week for 50 min). Exercise training reduced body weight, plasma insulin, and oxidized LDL concentrations. Protein expression of ATP-independent metalloprotease (OMA1), short optic atrophy 1 (S-OPA1), and dynamin-related protein 1 (DRP1) in BAT increased in trained rats, and long optic atrophy 1 (L-OPA1) and mitofusin 1 (MFN1) expression decreased. BAT expression of nuclear respiratory factor type 1 (NRF1) and mitochondrial transcription factor A (TFAM), the main factors involved in mitochondrial biogenesis, was higher in trained rats compared to controls. Exercise training increased protein expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and AMP-activated protein kinase (pAMPK/AMPK ratio) in BAT. In addition, training increased carnitine palmitoyltransferase II (CPT II), mitochondrial F1 ATP synthase α-chain, mitochondrial malate dehydrogenase 2 (mMDH) and uncoupling protein (UCP) 1,2,3 expression in BAT. Moreover, exercise increased insulin receptor (IR) ratio (IRA/IRB ratio), IRA-insulin-like growth factor 1 receptor (IGF-1R) hybrids and p42/44 activation, and decreased IGF-1R expression and IR substrate 1 (p-IRS-1) (S307) indicating higher insulin sensitivity and favoring glucose uptake in BAT in response to chronic exercise training. In summary, the present study indicates that chronic exercise is able to improve the energetic profile of BAT in terms of increased mitochondrial function and insulin sensitivity

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Multilocus genotyping of Giardia duodenalis in Southwestern Iran. A community survey.

Giardia duodenalis is one of the main enteric pathogens associated with diarrheal disease. In developing countries, giardiasis is a major public health concern, particularly in children under five years of age. This study aimed to evaluate the occurrence and genetic diversity of G. duodenalis causing human infections in Shushtar County, Southwestern Iran. Individual faecal specimens were collected from 1,163 individuals (male/female ratio: 0.9; age range 2-75 years) with (n = 258) and without (n = 905) gastrointestinal symptoms living in rural and urban settings during the period 2017-2018. Conventional (sucrose flotation and microscopy) methods were used for the initial detection of G. duodenalis cysts in faecal specimens. Microscopy-positive samples were confirmed by PCR amplification and sequencing of the small subunit rRNA (ssu rRNA) gene of the parasite. A multilocus genotyping (MLG) scheme targeting the triose phosphate isomerase (tpi), the glutamate dehydrogenase (gdh), and the beta-giardin (bg) genes was used for genotyping purposes. Giardia duodenalis cysts were detected in 7.7% (90/1,163) of samples by microscopy, of which 82 were confirmed by ssu-PCR. Successful amplification and sequencing results were obtained for 23.2% (19/82), 9.8% (8/82), and 8.5% (7/82) of the confirmed samples at the tpi, gdh, and bg loci, respectively. MLG data for the three loci were available for two samples only. Out of the 24 samples genotyped at any loci, 50% (12/24) were identified as assemblage A and the remaining half as assemblage B. Overall, AII was the most prevalent sub-assemblage detected (41.7%, 10/24), followed by BIII (25.0%, 6/24), discordant BIII/BIV (5/24) or AII/AIII (2/24) sequences, and BIV (1/24). No significant correlation was demonstrated between a given assemblage/sub-assemblage and the occurrence of clinical symptoms. No genotypes adapted to animal hosts other than humans (e.g. assemblages C-F) were found circulating in the investigated human population, suggesting that transmission of human giardiasis in this Iranian region is primarily of anthroponotic nature. Further molecular-based studies are needed to confirm and expand these results, and to ascertain the presence and public health relevance of the parasite in environmental (e.g. drinking water) samples.This study was funded by the Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz (Iran) under grant OG/9731 to MB. Additional funding was obtained from the Health Institute Carlos III (ISCIII), Ministry of Economy and Competitiveness (Spain), under project PI16CIII/00024 to DC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Occurrence and subtype distribution of Blastocystis sp. in humans, dogs and cats sharing household in northern Spain and assessment of zoonotic transmission risk.

Blastocystis sp. is probably the most common enteric parasite in humans globally. Although the role of Blastocystis in human disease is still controversial, epidemiological and experimental evidence suggests that pathogenicity may be associated with certain subtypes of the protist. Since the life cycle of Blastocystis is maintained through still elusive pathways, companion animals have attracted the attention of researchers as potential reservoirs of human infections. In order to evaluate the risk of zoonotic transmission of Blastocystis, we investigated the occurrence and molecular diversity of this microorganism in human, canine and feline populations sharing temporal and spatial settings in the province of Álava, northern Spain. A total of 268 (including 179 human, 55 canine and 34 feline) faecal specimens were obtained from 63 family households during February-December 2014. Detection of Blastocystis was achieved by PCR amplification and sequencing of small subunit rRNA genes. Blastocystis was found in 35.2% (95% CI: 0.29%-0.42%) of the human stool samples analysed, but not in any of the canine or feline faecal specimens investigated. Out of the 63 PCR-positive human samples, 84.1% (53/63) were successfully subtyped, allowing the identification of the subtypes ST2 (62.3%), ST3 (17.0%), ST1 (13.2%) and ST4 (7.5%). No mixed subtype infections were identified. Blastocystis carriage was independent of the gender and region of origin of the affected individuals, but children in the age groups of >5-10 years and >10-15 years were significantly more affected by the protist. None of the risk factors considered (water-use practices, contact with livestock, contact with individual undergoing diarrhoeal episodes) were associated with increased prevalence of Blastocystis. Our data demonstrate that pet dogs and cats play a negligible role as natural reservoirs of human Blastocystis infection in this geographic region, although the applicability of these results should be corroborated in future molecular epidemiological studies.Funding Information: Health Institute Carlos III, Ministry of Economy and Competitiveness. Grant Number: CP12/03081S

Prevalence of intestinal parasites, with emphasis on the molecular epidemiology of Giardia duodenalis and Blastocystis sp., in the Paranaguá Bay, Brazil: a community survey

BACKGROUND: Intestinal protozoan parasites are major contributors to the global burden of gastrointestinal disease causing significant socioeconomic consequences. Children living in resource-poor settings with restricted access to water and sanitary services are particularly at risk of these infections. METHODS: A prospective, community-based, cross-sectional survey was conducted in Paraná (southern Brazil) between May 2015 and May 2016. A total of 766 stool samples were individually collected from volunteers (male/female ratio: 0.99; age range: 0-76 years) and used for investigating the presence of intestinal helminth and protozoan species by routine microscopic procedures including the Kato-Katz and modified Ritchie concentration methods and the Ziehl-Neelsen stain technique. Quantitative real-time PCR confirmed microscopy-positive samples for Giardia duodenalis and the assemblages and sub-assemblages determined by multilocus sequence-based genotyping of the glutamate dehydrogenase (gdh) and β-giardin (bg) genes of the parasite. Identification of Blastocystis subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene (SSU rDNA) of this heterokont microorganism. RESULTS: Overall, 46.1% (353/766) of the participants were infected/colonised by at least one intestinal parasite/commensal species. Protozoan and helminth species were detected in 42.7% and 10.1% of the surveyed population, respectively. Blastocystis sp. (28.2%), Endolimax nana (14.9%), and Giardia duodenalis (11.0%) were the most prevalent species found among protozoans and Ascaris lumbricoides (5.0%), Trichuris trichiura (4.6%) and hookworms (1.0%) among helminths. A total of 38 G. duodenalis-positive samples were genotyped at gdh and bg markers, revealing the presence of the sub-assemblages AII (47.4%), AII/AIII (2.6%), BIII (5.3%), BIV (26.3%) and BIII/BIV (13.1%). Two samples (5.3%) were only identified as assemblage B. AII was predominantly found in females aged 5-9 years and was associated with a higher likelihood of reporting gastrointestinal symptoms. A total of 102 Blastocystis-positive samples were successfully subtyped at the SSU rRNA gene revealing the presence of ST1 (36.3%), ST2 (15.7%), ST3 (41.2%), ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%). CONCLUSIONS: Data presented here indicate that enteric parasites still represent a pressing health concern in Paraná, Brazil, probably due to sub-optimal water, sanitation and hygiene conditions. A mostly anthroponotic origin is suspected for G. duodenalis and Blastocystis sp. infections.This study was funded by the Health Institute Carlos III (ISCIII), Ministry of Economy and Competitiveness (Spain) - Collaborative Research Network on Tropical Diseases (RICET) and the European Regional Development Fund (ERDF) under project RD12/0018/0013. Additional funding was also provided by the ISCIII under project CP12/03081 and by the Secretária Municipal de Saúde de Paranaguá (Paraná), Brazil.S

Molecular Diversity of Giardia duodenalis, Cryptosporidium spp. and Blastocystis sp. in Asymptomatic School Children in Leganés, Madrid (Spain)

Enteric parasites including Giardia duodenalis, Cryptosporidium spp., and to a lesser extent, Blastocystis sp. and Enterocytozoon bieneusi, are major worldwide contributors to diarrhoeal disease. Assessing their molecular frequency and diversity is important to ascertain the sources of infection, transmission dynamics, and zoonotic potential. Little molecular information is available on the genotypes of these pathogens circulating in apparently healthy children. Here, we show that asymptomatic carriage of G. duodenalis (17.4%, 95% CI: 15.5‒19.4%), Blastocystis sp. (13.0%, 95% CI: 11.4‒14.8%), and Cryptosporidium spp. (0.9%, 95% CI: 0.5‒1.5%) is common in children (1‒16 years; n = 1512) from Madrid, Spain. Our genotyping data indicate that; (i) the observed frequency and diversity of parasite genetic variants are very similar to those previously identified in Spanish clinical samples, so that the genotype alone does not predict the clinical outcome of the infection, (ii) anthroponotic transmission accounts for a large proportion of the detected cases, highlighting that good personal hygiene practices are important to minimizing the risk of infection, (iii) Blastocystis ST4 may represent a subtype of the parasite with higher pathogenic potential, and (iv) Enterocytozoon bieneusi does not represent a public health concern in healthy children.This research was funded by Health Institute Carlos III (ISCIII), Ministry of Science, Innovation and Universities, Spain, grant number PI16CIII/00024.S

Risk associations for intestinal parasites in symptomatic and asymptomatic schoolchildren in central Mozambique.

Chronic infections by enteric parasites including protist and helminthic species produces long-term sequelae on the health status of infected children. This study assesses potential associations linked with enteric parasite infections in symptomatic and asymptomatic children in Zambézia province, Mozambique. In this prospective cross-sectional study, stool samples and epidemiological questionnaires on demographics and risk associations were collected from symptomatic children (n = 286) from clinical settings and asymptomatic (n = 807) children from 17 schools and creches aged 3‒14 years. We detected enteric parasites by PCR-based methods. We calculated prevalence (adjusted for age, sex, house construction, drinking water, and latrine use) and odds ratios (OR) for risk associations with logistic regression, after adjusting for district, neighbourhood, and symptoms. Numbers and adjusted prevalences (95% confidence intervals in brackets) for the symptomatic and asymptomatic populations were G. duodenalis 120, 52%(22-82), 339, 42% (25-59); followed by S. stercoralis 52, 14%(9‒20), 180, 20%(15-25). Risk associations for G. duodenalis included drinking untreated river/spring water, OR 2.91 (1.80-4.70); contact with ducks, OR 14.96 (2.93‒76.31); dogs, OR 1.92 (1.04-3.52); cats, OR 1.73 (1.16-2.59), and a relative with diarrhoea, OR 2.59 (1.54‒4.37). Risk associations for S. stercoralis included having no latrine, OR 2.41 (1.44-4.02); drinking well water, OR 1.82 (1.02-3.25), and increasing age, OR 1.11 (1.04-1.20). We found a high prevalence of intestinal parasites regardless of the children's symptoms. Drinking well or river water, domestic animals, and latrine absence were contributing factors of human infections.This study was funded by the Health Institute Carlos III (ISCIII), Ministry of 273 Economy and Competitiveness (Spain), under project PI16CIII/00024. Additional funds 274 were provided by the Spanish Tropical Diseases Research Network (RICET, ISCIII) under 275 project RD16CIII/0003/0004 for the testing of Entamoeba histolytica and E. dispar.S

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.Sin financiación1.790 JCR (2016) Q3, 55/78 Biochemical Research Methods, 86/125 MicrobiologyUE

Occurrence and molecular epidemiology of Giardia duodenalis infection in dog populations in eastern Spain

Abstract Background Giardia duodenalis is one of the most common enteric parasites in domestic animals including dogs. Young animals are more prone to the infection, with clinical manifestations ranging from asymptomatic to acute or chronic diarrhoea. Dogs are primarily infected by canine-specific (C-D) assemblages of G. duodenalis. However, zoonotic assemblages A and B have been increasingly documented in canine isolates, raising the question of whether and to which extent dogs can act as natural reservoirs of human giardiosis. Methods In this cross-sectional epidemiological survey we assessed the molecular diversity of G. duodenalis in dogs in the province of Castellón, Eastern Spain. A total of 348 individual faecal samples from sheltered (n = 218), breeding (n = 24), hunting (n = 68), shepherd (n = 24), and pet (n = 14) dogs were collected between 2014 and 2016. Detection of G. duodenalis cysts in faecal material was carried out by direct fluorescence microscopy as a screening test, whereas a qPCR targeting the small subunit ribosomal RNA gene of the parasite was subsequently used as a confirmatory method. Results Giardia duodenalis was detected in 36.5% (95% CI: 31.6–41.7%) of dogs. No significant differences in prevalence rates could be demonstrated among dogs according to their sex and geographical origin, but breeding (45.8%; 95% CI: 27.9–64.9%) and sheltered (40.4%; 95% CI: 34.1–47.0%) dogs harboured significantly higher proportions of G. duodenalis. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of G. duodenalis allowed the characterization of 35 canine isolates that were unambiguously assigned to assemblages A (14.3%), B (22.9%), C (5.7%), and D (37.1%). A number of inter-assemblage mixed infections including A + B (11.4%), A + D (2.9%), and A + B + D (5.7%) were also identified. Conclusions Data presented here are strongly indicative of high infection pressures in kennelled animals. Zoonotic sub-assemblages AII, BIII, and BIV were responsible for a considerable proportion of the G. duodenalis infections detected, but very few of the genotypes identified have been previously documented in Spanish human populations. Although possible, zoonotic transmission between dogs and humans seems an infrequent event in this Spanish region