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1.
Eur J Clin Microbiol Infect Dis ; 36(12): 2335-2342, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28741097

ABSTRACT

Blastocystis is a single-celled intestinal parasite commonly found in humans and a broad range of animals all over the world. In humans, its role in health and disease remains unsettled. The aim of our study was to investigate the distribution of Blastocystis and Blastocystis subtypes (ST) in patients with inflammatory bowel disease (IBD) and control subjects. A total of 71 stool samples were collected from IBD patients, 69 and 2 of whom had ulcerative colitis (UC) and Crohn's Disease (CD), respectively. Moreover, 166 stool samples from healthy subjects were included as control samples. All stool samples were cultivated, and 550-bp fragments of the small subunit ribosomal RNA gene was amplified from Blastocystis-positive cultures. All PCR-positive samples were sequenced. Blastocystis was observed in 9 (12.67%) and 35 (21.1%) IBD patients and healthy controls, respectively. There was no statistically significant correlation between IBD and presence of Blastocystis (P = 0.147). There was a statistically significant correlation between age and Blastocystis colonization in the IBD group (P < 0.05), but not among healthy controls. No significant correlation between gender and colonization was observed. ST1 and ST3 were obtained from 1 (12.5%) and 7 (87.5%) IBD patients, respectively, while in the healthy control group, subtypes 1, 2, and 3 were found in 14 (40%), 12 (34.28%), and 9 (25.72%), respectively. Phylogenetic analysis showed no variation in the distribution of subtypes nor intra-subtype genetic diversity between samples acquired from IBD patients and healthy controls. This study showed a trend towards a lower prevalence of Blastocystis in IBD patients than in control subjects. ST3 sequences isolated from IBD patients and control individuals did not appear to differ genetically.


Subject(s)
Blastocystis/classification , Blastocystis/genetics , Inflammatory Bowel Diseases/microbiology , Phylogeny , Adult , Blastocystis/isolation & purification , Genetic Variation , Healthy Volunteers , Humans , Inflammatory Bowel Diseases/diagnosis , Iran , Middle Aged , RNA, Ribosomal, 18S/genetics , Young Adult
2.
Eur J Clin Microbiol Infect Dis ; 35(9): 1427-31, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27230509

ABSTRACT

Several studies have shown associations between groups of intestinal bacterial or specific ratios between bacterial groups and various disease traits. Meanwhile, little is known about interactions and associations between eukaryotic and prokaryotic microorganisms in the human gut. In this work, we set out to investigate potential associations between common single-celled parasites such as Blastocystis spp. and Dientamoeba fragilis and intestinal bacteria. Stool DNA from patients with intestinal symptoms were selected based on being Blastocystis spp.-positive (B+)/negative (B-) and D. fragilis-positive (D+)/negative (D-), and split into four groups of 21 samples (B+ D+, B+ D-, B- D+, and B- D-). Quantitative PCR targeting the six bacterial taxa Bacteroides, Prevotella, the butyrate-producing clostridial clusters IV and XIVa, the mucin-degrading Akkermansia muciniphila, and the indigenous group of Bifidobacterium was subsequently performed, and the relative abundance of these bacteria across the four groups was compared. The relative abundance of Bacteroides in B- D- samples was significantly higher compared with B+ D- and B+ D+ samples (P < 0.05 and P < 0.01, respectively), and this association was even more significant when comparing all parasite-positive samples with parasite-negative samples (P < 0.001). Additionally, our data revealed that a low abundance of Prevotella and a higher abundance of Clostridial cluster XIVa was associated with parasite-negative samples (P < 0.05 and P < 0.01, respectively). Our data support the theory that Blastocystis alone or combined with D. fragilis is associated with gut microbiota characterized by low relative abundances of Bacteroides and Clostridial cluster XIVa and high levels of Prevotella.


Subject(s)
Bacteria/classification , Bacteria/genetics , Blastocystis Infections/microbiology , Dientamoebiasis/microbiology , Gastrointestinal Microbiome , Real-Time Polymerase Chain Reaction , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Bacterial Load , Blastocystis/isolation & purification , Blastocystis Infections/parasitology , Child , Dientamoeba/isolation & purification , Dientamoebiasis/parasitology , Feces/microbiology , Feces/parasitology , Female , Humans , Male , Middle Aged , Young Adult
3.
J Clin Microbiol ; 53(6): 1891-7, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25832304

ABSTRACT

The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.


Subject(s)
Cryptosporidiosis/parasitology , Cryptosporidium/genetics , Genes, Protozoan/genetics , Molecular Typing/methods , Adolescent , Adult , Aged , Amino Acid Sequence , Animals , Child , Child, Preschool , Feces/parasitology , Female , Humans , Infant , Male , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Alignment , Young Adult
4.
Eur J Clin Microbiol Infect Dis ; 34(6): 1145-51, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25663130

ABSTRACT

Associations between antimicrobial use and risk of enteric infection with intestinal protozoa are scarcely studied. The aim of this study was to quantify the risk of Dientamoeba fragilis infection conferred by exposure to antimicrobials. We conducted a registry-based retrospective cohort study of 9,945 Danish patients investigated for D. fragilis infection between 2008 and 2011, using data from the Danish Register of Medicinal Product Statistics, and calculating relative risks (RR) for D. fragilis infection by stratified binary regression. Furthermore, we conducted a population based case-control study using controls sampled from the Danish Civil Registration System, calculating hazard ratios (HR) for D. fragilis infection by conditional logistic regression. Exposure to metronidazole was found to confer decreased risk of D. fragilis infection; however, similar associations were found for antimicrobials not commonly used to treat D. fragilis, such as broad-spectrum penicillin, fluoroquinolones, and macrolides. In contrast, mebendazole exposure was associated with increased risk. The intake of antimicrobials influences the risk of D. fragilis.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Dientamoeba/isolation & purification , Dientamoebiasis/epidemiology , Drug Utilization , Enteritis/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Child, Preschool , Cohort Studies , Denmark/epidemiology , Female , Humans , Infant , Male , Middle Aged , Retrospective Studies , Risk Assessment , Young Adult
5.
Eur J Clin Microbiol Infect Dis ; 32(10): 1303-10, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23609513

ABSTRACT

The intestinal protozoon Dientamoeba fragilis remains a clinical entity of dubious significance. While several previous studies address questions of epidemiology, only a handful have systematically employed and reported on the results from real-time polymerase chain reaction (qPCR), the best currently available diagnostic modality, and the comparison of results from different studies is, therefore, difficult. Since 2007, Statens Serum Institut (Denmark) has utilised qPCR for D. fragilis as routine diagnostic work-up for intestinal parasitosis, testing more than 22,000 samples from 2008 through 2011, and the aim of this study was to report on the results and experiences gained in the process. We demonstrate a staggeringly high proportion (43%) of investigated patients positive for D. fragilis, ranging from 12 to 71% depending on age group, showing a bimodal age distribution peaking in children and adults of parental age, as well as a clear association between exposure to children and risk of D. fragilis infection. We discuss these findings in light of the pinworm egg vector hypothesis and substantiate further our knowledge of risk factors pertaining to D. fragilis carriage.


Subject(s)
Dientamoeba/isolation & purification , Dientamoebiasis/epidemiology , Real-Time Polymerase Chain Reaction , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Denmark/epidemiology , Dientamoeba/genetics , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Prevalence , Young Adult
6.
Exp Parasitol ; 134(2): 155-9, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23499880

ABSTRACT

Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels.


Subject(s)
DNA, Helminth/isolation & purification , DNA, Mitochondrial/isolation & purification , DNA, Ribosomal/isolation & purification , Toxascaris/genetics , Toxocara/genetics , Animals , Cats , Dogs , Electrophoresis, Agar Gel , Polymerase Chain Reaction , Toxocara canis/genetics
7.
Water Res ; 46(18): 5935-40, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22985523

ABSTRACT

Invasive aspergillosis is the second most common cause of nosocomial fungal infections and occurring mainly by Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. There is evidence that nosocomial aspergillosis may be waterborne. This study was conducted to evaluate the ultraviolet (UV) irradiation efficiency in terms of inactivating the most important Aspergillus species in water since these are potential sources for nosocomial aspergillosis. A continuous flow UV reactor which could be used as a point-of-use (POU) system was used to survey Aspergillus inactivation by UV irradiation. The inactivation efficiency of UV fluence (4.15-25 mJ/cm(2)) was measured by determination of fungal density in water before and after radiation. Because turbidity and iron concentration are two major water quality factors impacting UV disinfection effectiveness, the potential influence of these factors on UV inactivation of Aspergillus spp. was also measured. The 4 log inactivation for A. fumigatus, A. niger and A. flavus at a density of 1000 cfu/ml was achieved at UV fluences of 12.45 mJ/cm(2), 16.6 mJ/cm(2) and 20.75 mJ/cm(2), respectively. The inactivation efficiency for lower density (100 cfu/ml) was the same as for the higher density except for A. flavus. The removal efficiency of Aspergillus spp. was decreased by increasing the turbidity and iron concentration. UV disinfection could effectively inactivate Aspergillus spores from water and eliminate potential exposure of high-risk patients to fungal aerosols by installation of POU UV systems.


Subject(s)
Aspergillus/radiation effects , Disinfection/methods , Ultraviolet Rays , Aspergillosis/prevention & control , Water Microbiology , Water Purification
8.
Eur J Clin Microbiol Infect Dis ; 31(7): 1689-96, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22350386

ABSTRACT

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P=0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.


Subject(s)
Blastocystis Infections/epidemiology , Blastocystis Infections/parasitology , Blastocystis/classification , Blastocystis/genetics , Genetic Variation , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Blastocystis/isolation & purification , Child , Child, Preschool , Cluster Analysis , Feces/parasitology , Female , Genotype , Humans , Infant , Male , Middle Aged , Molecular Epidemiology , Molecular Sequence Data , Prevalence , RNA, Ribosomal/genetics , Sequence Analysis, DNA , Sweden/epidemiology , Young Adult
9.
Intern Med J ; 42(11): 1187-95, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22032439

ABSTRACT

BACKGROUND: Blastocystis is a common, enteric parasite. The pathogenicity of the organism is uncertain, but subtypes (ST) 1 and 3 have been reported more likely to cause irritable bowel-like symptoms. AIMS: We treated symptomatic patients positive for Blastocystis with conventional therapy and analysed 16 small-subunit (SSU) rDNA to assess clearance and carriage rates and ST prevalence of the parasite in the asymptomatic household members. METHODS: In a longitudinal, prospective case study, 11 symptomatic patients positive for Blastocystis underwent outpatient clinical assessment to exclude other diagnoses before 14 days of either metronidazole 400 mg three times daily or trimethoprim/sulfamethoxazole 160/800 mg twice-daily therapy. Faecal specimens were collected from patients at baseline, day 15, 28 and 56 after therapy and from 17 family members and eight pets at day 15. Specimens were analysed using faecal smear, culture and polymerase chain reaction analysis of 16SSU rDNA. RESULTS: No patient cleared the organism following therapy. ST 1 (45%), 3 (36%), 4 (36%) and 6 (9%) were found in the symptomatic Blastocystis patients, and ST identified before and after therapy were identical in each individual. All household contacts were positive for Blastocystis and 16/17 (94%) contacts showed identical Blastocystis ST to the symptomatic family member. All pets were positive for Blastocystis with polymerase chain reaction testing, 7/8 (88%) demonstrating ST concordance with the symptomatic Blastocystis patients. CONCLUSIONS: Conventional therapy is ineffective for symptomatic Blastocystis infection. The high prevalence of Blastocystis infection within households suggested transmission between humans and their pets. Subtyping analysis of SSU rDNA alone in Blastocystis does not appear to predict pathogenicity.


Subject(s)
Blastocystis Infections/parasitology , Blastocystis/classification , Cat Diseases/parasitology , Dog Diseases/parasitology , Adult , Aged , Animals , Antiprotozoal Agents/therapeutic use , Asymptomatic Diseases , Biopsy , Blastocystis/isolation & purification , Blastocystis/pathogenicity , Blastocystis Infections/drug therapy , Blastocystis Infections/transmission , Blastocystis Infections/veterinary , Carrier State/drug therapy , Carrier State/parasitology , Cat Diseases/drug therapy , Cats , Disease Reservoirs , Dog Diseases/drug therapy , Dogs , Family Health , Female , Humans , Intestinal Mucosa/parasitology , Intestinal Mucosa/pathology , Male , Metronidazole/therapeutic use , Middle Aged , Prospective Studies , Ribotyping , Treatment Failure , Trimethoprim, Sulfamethoxazole Drug Combination/therapeutic use , Young Adult
10.
Epidemiol Infect ; 137(11): 1655-63, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19393117

ABSTRACT

Two independent studies were conducted to describe symptoms and potential risk factors associated with Blastocystis infection. Isolates were subtyped by molecular analysis. In the NORMAT study (126 individuals randomly sampled from the general population) 24 (19%) were positive for Blastocystis. Blastocystis was associated with irritable bowel syndrome (P=0.04), contact with pigs (P<0.01) and poultry (P=0.03). In the Follow-up (FU) study (follow-up of 92 Blastocystis-positive patients), reports on bloating were associated with subtype (ST) 2 (P<0.01), and blood in stool to mixed subtype infection (P=0.06). ST1 was more common in FU individuals (32%) than in NORMAT individuals (8%), whereas single subtype infections due to ST3 or ST4 were seen in 63% of the NORMAT cases and 28% of the FU cases. Only FU individuals hosted ST7, and ST6/7 infections due to ST7 or ST9 were characterized by multiple intestinal symptoms. The data indicate subtype-dependent differences in the clinical significance of Blastocystis.


Subject(s)
Blastocystis Infections/epidemiology , Dientamoebiasis/epidemiology , Population Surveillance , Adolescent , Adult , Aged , Animals , Antiparasitic Agents/therapeutic use , Blastocystis/classification , Blastocystis/genetics , Blastocystis/isolation & purification , Blastocystis Infections/complications , Blastocystis Infections/drug therapy , Child , Child, Preschool , Denmark/epidemiology , Dientamoeba/isolation & purification , Dientamoebiasis/complications , Dientamoebiasis/drug therapy , Female , Follow-Up Studies , Genotype , Humans , Infant , Irritable Bowel Syndrome/parasitology , Male , Metronidazole/therapeutic use , Middle Aged , Prevalence , Treatment Failure , Young Adult
11.
Epidemiol Infect ; 137(3): 348-56, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19134228

ABSTRACT

Foodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive for Cryptosporidium hominis infection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4.1, 95% confidence interval (CI) 2.1-8.3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2.1, 95% CI 1.1-4.0; grated carrots: OR 2.1, 95% CI 1.2-3.9; peppers: OR 3.3, 95% CI 1.7-6.6). We speculate that a person excreting the parasite may have contaminated the salad buffet.


Subject(s)
Cryptosporidiosis/epidemiology , Cryptosporidium/isolation & purification , Disease Outbreaks , Food Microbiology , Foodborne Diseases/epidemiology , Foodborne Diseases/parasitology , Adult , Animals , Case-Control Studies , Chi-Square Distribution , Denmark/epidemiology , Female , Food Contamination , Food Handling , Fruit/parasitology , Humans , Logistic Models , Male , Polymerase Chain Reaction , Surveys and Questionnaires , Vegetables/parasitology
14.
Clin Microbiol Infect ; 13(8): 839-42, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17610603

ABSTRACT

The prevalence of Dientamoeba fragilis in patients from a metropolitan area in Denmark was determined by examination of paired stool samples using two techniques: a formol ethyl-acetate concentration technique with unpreserved faeces and a permanent staining technique on faeces preserved with sodium acetate-acetic acid-formalin (SAF). Using the SAF permanent staining technique and the formol ethyl-acetate concentration technique, 25% and 15% of the specimens, respectively, were parasite-positive. D. fragilis was detected in 12 of the 103 patients, only two of whom harboured other recognised pathogenic parasites. Overall, D. fragilis had a remarkably high prevalence in the metropolitan area of Denmark investigated.


Subject(s)
Dientamoebiasis/epidemiology , Adolescent , Adult , Aged , Animals , Child , Child, Preschool , Denmark/epidemiology , Dientamoeba/isolation & purification , Dientamoeba/pathogenicity , Feces/parasitology , Female , Humans , Infant , Male , Microbiological Techniques , Middle Aged , Prevalence , Urban Population
15.
Exp Parasitol ; 116(2): 111-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17266951

ABSTRACT

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.


Subject(s)
Blastocystis Infections/parasitology , Blastocystis/classification , DNA, Protozoan/chemistry , Sequence Analysis, DNA/methods , Animals , Base Sequence , Blastocystis/genetics , Cost-Benefit Analysis , Genotype , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Protozoan/genetics , RNA, Ribosomal, 18S/genetics , Sequence Analysis, DNA/economics , Time Factors
16.
Acta Trop ; 97(1): 26-30, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16253202

ABSTRACT

In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals.


Subject(s)
Feces/parasitology , Opisthorchiasis/diagnosis , Opisthorchiasis/parasitology , Opisthorchis/isolation & purification , Animals , DNA, Helminth/analysis , DNA, Helminth/isolation & purification , Humans , Opisthorchis/genetics , Opisthorchis/growth & development , Parasite Egg Count , Polymerase Chain Reaction , Sensitivity and Specificity
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