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Parasitology Jan 2020The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination...
The presence of bacterial DNA in Dientamoeba fragilis DNA extracts from culture poses a substantial challenge to sequencing the D. fragilis genome. However, elimination of bacteria from D. fragilis cultures has proven difficult in the past, presumably due to its dependence on some unknown prokaryote/s. This study explored options for removal of bacteria from D. fragilis cultures and for the generation of genome sequence data from D. fragilis. DNA was extracted from human faecal samples and xenic D. fragilis cultures. Extracts were subjected to 16S ribosomal DNA bacterial diversity profiling. Xenic D. fragilis cultures were then subject to antibiotic treatment regimens that systematically removed bacterial species depending on their membrane structure (Gram-positive or Gram-negative) and aerobic requirements. The impact of these treatments on cultures was assessed by 16S amplicon sequencing. Prior to antibiotic treatment, the cultures were dominated by Gram-negative bacteria. Addition of meropenem to cultures eliminated anaerobic Gram-negative bacteria, but it also led to protozoan death after 5 days incubation. The seeding of meropenem resistant Klebsiella pneumoniae strain KPC-2 into cultures before treatment by meropenem prevented death of D. fragilis cells beyond this 5 day period, suggesting that one or more species of Gram-negative bacteria may be an essential nutritional requirement for D. fragilis. Gram-positive cells were completely eliminated using vancomycin without affecting trophozoite growth. Finally, this study shows that genome sequencing of D. fragilis is feasible following bacterial elimination from cultures as the result of the major advances occurring in bioinformatics. We provide evidence on this fact by successfully sequencing the D. fragilis 28S large ribosomal DNA subunit gene using culture-derived DNA.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Physiological Phenomena; Culture Techniques; Dientamoeba; Genetic Variation; Genome, Protozoan; RNA, Ribosomal, 16S; RNA, Ribosomal, 28S
PubMed: 31452478
DOI: 10.1017/S0031182019001173 -
Parasitology International Feb 2024Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still...
Dientamoeba fragilis (D. fragilis) represents a common protozoan in both high and low income countries. Despite this, epidemiological data on dientamoebiasis are still limited, and it is possible that the actual prevalence rates of D. fragilis have been underestimated due to the challenges in its detection and identification. In the present study, symptomatic patients from Rome (Central Italy) were surveyed for two years to determine D. fragilis percentage of infection and genotypes. Stool samples collection was performed over 864 patients, DNA extracted, and RT-PCR performed by the SeeGene Allplex™ Gastrointestinal Parasite Panel Assays. Seventy-nine resulted positive for D. fragilis (9.1%). Co-infections were detected in 22 isolates: 21 displayed Blastocystis sp. + D. fragilis (27.8%). Based on the sequence of a central fragment of the SSU rRNA gene, only genotype 1 was identified. These findings are among the few available data regarding genetic diversity of D. fragilis in Italy. Large-scale human and animal research are required to enhance our knowledge of prevalence, host range, genetic variability and zoonotic transmission of this little-known intestinal protozoan.
Topics: Animals; Humans; Dientamoeba; Genotype; Intestinal Diseases, Parasitic; Dientamoebiasis; Feces; Italy
PubMed: 37838287
DOI: 10.1016/j.parint.2023.102816 -
PloS One 2022The increasing interest to perform and investigate the efficacy of fecal microbiota transplantation (FMT) has generated an urge for feasible donor screening. We report...
BACKGROUND
The increasing interest to perform and investigate the efficacy of fecal microbiota transplantation (FMT) has generated an urge for feasible donor screening. We report our experience with stool donor recruitment, screening, follow-up, and associated costs in the context of clinical FMT trials.
METHODS
Potential stool donors, aged between 18-65 years, underwent a stepwise screening process starting with an extensive questionnaire followed by feces and blood investigations. When eligible, donors were rescreened for MDROs and SARS-CoV-2 every 60-days, and full rescreening every 4-6 months. The costs to find and retain a stool donor were calculated.
RESULTS
From January 2018 to August 2021, 393 potential donors underwent prescreening, of which 202 (51.4%) did not proceed primarily due to loss to follow-up, medication use, or logistic reasons (e.g. COVID-19 measures). 191 potential donors filled in the questionnaire, of which 43 (22.5%) were excluded. The remaining 148 candidates underwent parasitology screening: 91 (61.5%) were excluded, mostly due to Dientamoeba fragilis and/or high amounts of Blastocystis spp. After additional feces investigations 18/57 (31.6%) potential donors were excluded (mainly for presence of Helicobacter Pylori and ESBL-producing organisms). One donor failed serum testing. Overall, 38 out of 393 (10%) potential donors were enrolled. The median participation time of active stool donors was 13 months. To recruit 38 stool donors, €64.112 was spent.
CONCLUSION
Recruitment of stool donors for FMT is challenging. In our Dutch cohort, failed eligibility of potential donors was often caused by the presence of the protozoa Dientamoeba fragilis and Blastocystis spp.. The exclusion of potential donors that carry these protozoa, especially Blastocystis spp., is questionable and deserves reconsideration. High-quality donor screening is associated with substantial costs.
Topics: Humans; Adolescent; Young Adult; Adult; Middle Aged; Aged; Fecal Microbiota Transplantation; Donor Selection; SARS-CoV-2; COVID-19; Feces; Clostridium Infections
PubMed: 36264933
DOI: 10.1371/journal.pone.0276323 -
Dientamoeba fragilis associated with microbiome diversity changes in acute gastroenteritis patients.Parasitology International Dec 2023This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were...
This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were diagnosed with gastroenteritis. In comparison to traditional microscopic diagnosis methods, Multiplexed-tandem PCR proved to be more effective in detecting intestinal parasites. Among the identified protozoans, Blastocystis sp. and Dientamoeba fragilis were the most prevalent. Notably, D. fragilis was significantly associated with an increase in the alpha-diversity of host prokaryotic microbes. Although the exact role of Blastocystis sp. and D. fragilis as the primary cause of gastroenteritis remains debatable, our data indicates a substantial correlation between these protozoans and the prokaryote microbiome of their hosts, particularly when compared to other protists or patients with gastroenteritis but no detectable parasitic cause. These findings underscore the significance of comprehending the contributions of intestinal protozoans, specifically D. fragilis, to the development of gastroenteritis and their potential implications for disease management.
Topics: Animals; Humans; Dientamoeba; Intestinal Diseases, Parasitic; Blastocystis; Gastroenteritis; Parasites; Feces
PubMed: 37482266
DOI: 10.1016/j.parint.2023.102788 -
Parasites & Vectors Nov 2023Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 positively affect the fecal bacteriome in children with celiac disease autoimmunity after 6... (Randomized Controlled Trial)
Randomized Controlled Trial
Effects of Lactiplantibacillus plantarum and Lacticaseibacillus paracasei supplementation on the single-cell fecal parasitome in children with celiac disease autoimmunity: a randomized, double-blind placebo-controlled clinical trial.
BACKGROUND
Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 positively affect the fecal bacteriome in children with celiac disease autoimmunity after 6 months of supplementation. The aim of the present investigation was to study the effects of Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 on the single-cell parasitome, with a primary focus on Blastocystis.
METHODS
Stool samples were collected from 78 Swedish children with celiac disease autoimmunity participating in a randomized, double-blind, placebo-controlled clinical trial to either receive a mixture of supplementation with L. plantarum HEAL9 and L. paracasei 8700:2 (n = 38) or placebo (n = 40). A total of 227 stool samples collected at baseline and after 3 and 6 months of intervention, respectively, were retrospectively analyzed for Blastocystis by quantitative real-time PCR and subtyped by massively parallel amplicon sequencing. Other single-cell parasites were detected by untargeted 18S rDNA amplicon sequencing and verified by real-time PCR. The relation between the parasites and the bacteriome community was characterized by using 16S rDNA profiling of the V3-V4 region.
RESULTS
Three different single-cell protists were identified, of which the highest prevalence was found for Dientamoeba fragilis (23.1%, 18/78 children), followed by Blastocystis (15.4%, 12/78) and Entamoeba spp. (2.6%, 2/78). The quantity of the protists was stable over time and not affected by probiotic intervention (P = 0.14 for Blastocystis, P = 0.10 for D. fragilis). The positivity of the protists was associated with increased bacteriome diversity (measured by multiple indices, P < 0.03). Bacterial composition was influenced by the presence of the protists: positivity of Blastocystis was inversely associated with Akkermansia (at the levels of the genus as well as its family, order, class and phylum); P < 0.002), Faecalibacterium (P = 0.003) and Romboutsia (P = 0.029); positivity of D. fragilis was inversely associated with families Enterobacteriaceae (P = 0.016) and Coriobacteriaceae (P = 0.022) and genera Flavonifractor (P < 0.001), Faecalibacterium (P = 0.009), Lachnoclostridium (P = 0.029), Ruminococcus (P < 0.001) and Granulicatella (P = 0.018).
CONCLUSIONS
The prevalence of single-cell protists is low in children with celiac disease autoimmunity. The colonization was stable regardless of the probiotic intervention and associated with increased diversity of the fecal bacteriome but inversely associated with some beneficial bacteria.
Topics: Humans; Child; Lacticaseibacillus; Lacticaseibacillus paracasei; Autoimmunity; Celiac Disease; Retrospective Studies; Feces; Blastocystis; Bacteria; Probiotics; Double-Blind Method; DNA, Ribosomal
PubMed: 37946274
DOI: 10.1186/s13071-023-06027-1 -
Parasite (Paris, France) 2014Recently, Dientamoeba fragilis has emerged as a significant and common enteropathogen. The majority of patients with dientamoebiasis present with gastrointestinal... (Comparative Study)
Comparative Study
Activity of benzimidazoles against Dientamoeba fragilis (Trichomonadida, Monocercomonadidae) in vitro and correlation of beta-tubulin sequences as an indicator of resistance.
Recently, Dientamoeba fragilis has emerged as a significant and common enteropathogen. The majority of patients with dientamoebiasis present with gastrointestinal complaints and chronic symptoms are common. Numerous studies have successfully demonstrated parasite clearance, coupled with complete resolution of clinical symptoms following treatment with various antiparasitic compounds. Despite this, there is very little in vitro susceptibility data available for the organism. Benzimidazoles are a class of antiparasitic drugs that are commonly used for the treatment of protozoan and helminthic infections. Susceptibility testing was undertaken on four D. fragilis clinical isolates against the following benzimidazoles: albendazole, flubendazole, mebendazole, nocodazole, triclabendazole and thiabendazole. The activities of the antiprotozoal compounds at concentrations ranging from 2 μg/mL to 500 μg/mL were determined via cell counts of D. fragilis grown in xenic culture. All tested drugs showed no efficacy. The beta-tubulin transcript was sequenced from two of the D. fragilis isolates and amino acid sequences predicted a susceptibility to benzimidazoles. This is the first study to report susceptibility profiles for benzimidazoles against D. fragilis, all of which were not active against the organism. This study also found that beta-tubulin sequences cannot be used as a reliable marker for resistance of benzimidazoles in D. fragilis.
Topics: Amino Acid Sequence; Antiprotozoal Agents; Benzimidazoles; Consensus Sequence; Dientamoeba; Drug Resistance; Genotype; In Vitro Techniques; Molecular Sequence Data; Protozoan Proteins; RNA, Protozoan; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Transcriptome; Tubulin
PubMed: 25148459
DOI: 10.1051/parasite/2014043 -
Clinical Microbiology and Infection :... Dec 2018Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix... (Comparative Study)
Comparative Study
Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.
OBJECTIVES
Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures.
METHODS
Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR.
RESULTS
D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27).
CONCLUSIONS
The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blastocystis hominis; Child; Child, Preschool; Clinical Laboratory Techniques; Cryptosporidium; Dientamoeba; Entamoeba histolytica; Feces; Female; Giardia lamblia; Humans; Immunoenzyme Techniques; Infant; Infant, Newborn; Intestinal Diseases, Parasitic; Male; Microscopy; Middle Aged; Multiplex Polymerase Chain Reaction; Prospective Studies; Reagent Kits, Diagnostic; Sensitivity and Specificity; Young Adult
PubMed: 29581055
DOI: 10.1016/j.cmi.2018.03.025 -
Tropical Medicine and Infectious Disease Sep 2021While the prevalence of intestinal parasitic infections (IPI) has been most commonly studied in African and Asian populations, less is known about the prevalence rates... (Review)
Review
While the prevalence of intestinal parasitic infections (IPI) has been most commonly studied in African and Asian populations, less is known about the prevalence rates of IPI in European children, as well as the potential risk factors that favor the spread of parasites. We aimed to review published evidence on the prevalence rates of IPI in children residing in Europe, and to quantitatively synthesize the results of published studies. We searched Medline from 1 January 2015 to 1 April 2021 to address the most recently published prevalence patterns of IPI in European children. Random-effects meta-analyses were performed by type of IPI infection, age group and sex, depending on data availability. Of the 967 potentially relevant articles, eight eligible cross-sectional studies were included in this analysis, yielding a sample of 3376 children (0-19 years). The overall prevalence rate was 5.9% for any IPI in children residing in European countries. was the most commonly detected parasite yielding a prevalence rate of 10.7%. Other parasites included and . Studies focusing on specific types of parasites showed prevalence rates ranging from 1.3% for to 68.3% for . Despite the scarce literature, the present review showed relatively low prevalence rates of IPI in Europe. Future studies accounting for proper diagnostic methods used for the detection of parasites and including information on potential sociodemographic factors, such as travelling history and history of immigration, are needed to guide clinicians about which children to test, as well as when and how to test children for IPI.
PubMed: 34564544
DOI: 10.3390/tropicalmed6030160 -
Pathogens (Basel, Switzerland) Jun 2023We aimed to assess the performance of the Novodiag Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections.
OBJECTIVES
We aimed to assess the performance of the Novodiag Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections.
METHODS
A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia.
RESULTS
Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for , and but was lower for (85.2%) and ≤50% for and . In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for , , spp. and Sensitivity was 100% for , but none of the five samples containing spp. were detected.
CONCLUSIONS
The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR.
PubMed: 37513736
DOI: 10.3390/pathogens12070889 -
International Maritime Health 2023Poland has experienced increased economic migration since 2021. Large waves of migrants, mostly from Asian and African countries, are trying to get into the European...
BACKGROUND
Poland has experienced increased economic migration since 2021. Large waves of migrants, mostly from Asian and African countries, are trying to get into the European Union by crossing Poland's eastern border illegally. The influx of illegal migrants into Poland is the result of a policy adopted by the Belarusian and Russian regimes that are trying to provoke another migrant crisis in Europe. In the opinion of some Polish politicians illegal migration contributes to the spread of parasitic diseases in our country as many migrants arriving into Poland carry intestinal parasites. The aim of this study was to assess the prevalence of infections with intestinal parasites in the Polish Border Guard officers safeguarding Poland's eastern borders.
MATERIALS AND METHODS
Parasitological diagnostics was carried out between April and May 2023. The study involved 218 Polish Border Guard officers from the Podlaski Border Guard Unit (PBGU) and 209 officers from the Bug Border Guard Unit (BBGU), whose task is to patrol and safeguard Poland's border with Ukraine and Belarus. Faecal examinations were performed using three different light microscopy testing methods (direct smear, decantation, flotation) at the Department of Epidemiology and Tropical Medicine at the Military Institute of Medicine - National Research Institute, Warsaw, Poland.
RESULTS
Considered to be potentially pathogenic intestinal parasites were diagnosed in 20 out of 218 officers serving in the PBGU (8.7% infected with Blastocystis spp., 0.5% with Dientamoeba fragilis) and in 9 out of 209 officers serving in the BBGU (3.8% infected with Blastocystis spp., 0.5% with Dientamoeba fragilis). There were no infections with nematodes, cestodes or trematodes in the study participants. No correlation was found between a parasitic infection and the presence of diarrhoea or other gastrointestinal symptoms within 6 months prior to the study in both groups.
CONCLUSIONS
Although Polish Border Guard officers deployed to the eastern border are exposed to difficult environmental conditions and have frequent contacts (either directly or indirectly) with migrants arriving from countries which report high incidence of parasitic infections, the rates of infections with potentially pathogenic protozoa in officers from the PBGU and BBGU are low and mainly attributable to pathogens which are widespread in the general Polish population. Low rates of parasitic infections in officers serving in the border zone suggest that the epidemiological situation of parasitic diseases in East Poland is satisfactory and that the disease prevention strategies (including the use of personal protection gear) implemented by the Polish medical services are effective.
Topics: Humans; Poland; Intestinal Diseases, Parasitic; European Union; Military Personnel; Europe
PubMed: 37781943
DOI: 10.5603/imh.97185