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BackgroundNeoehrlichia mikurensis (N. mikurensis) is a newly discovered tick-borne pathogen that can inflict life-threatening illness in immunocompromised patients. N. mikurensis infection is only detectable by polymerase chain reaction (PCR)-based methodologies. We describe three distinct clinical manifestations of N. mikurensis infection (neoehrlichiosis) in Danish patients receiving B-lymphocyte-depleting therapy, rituximab, for underlying hematological, rheumatological, or neurological disorders. All three patients went through a protracted pre-diagnostic period.MethodsN. mikurensis DNA was detected and confirmed using two methods. Blood was tested by specific real-time PCR targeting the groEL gene and by 16S and 18S profiling followed by sequencing. Bone marrow was analyzed by 16S and 18S profiling.ResultsN. mikurensis was detected in blood samples in all three cases and in bone marrow from one of the three. The severity of the symptoms ranged from prolonged fever lasting more than 6 months to life-threatening hyperinflammation in the form of hemophagocytic lymphohistiocytosis (HLH). Interestingly, all patients presented with splenomegaly and two with hepatomegaly. After starting doxycycline therapy, symptoms were relieved within a few days, and biochemistry and organomegaly quickly normalized.ConclusionWe present three Danish patients recognized by the same clinician over a period of 6 months, strongly suggesting that many cases are going unrecognized. Second, we describe the first case of N. mikurensis-induced HLH and emphasize the potential severity of undetected neoehrlichiosis. Background: Neoehrlichia mikurensis (N. mikurensis) is a newly discovered tick-borne pathogen that can inflict life-threatening illness in immunocompromised patients. N. mikurensis infection is only detectable by polymerase chain reaction (PCR)-based methodologies. We describe three distinct clinical manifestations of N. mikurensis infection (neoehrlichiosis) in Danish patients receiving B-lymphocyte-depleting therapy, rituximab, for underlying hematological, rheumatological, or neurological disorders. All three patients went through a protracted pre-diagnostic period. Methods: N. mikurensis DNA was detected and confirmed using two methods. Blood was tested by specific real-time PCR targeting the groEL gene and by 16S and 18S profiling followed by sequencing. Bone marrow was analyzed by 16S and 18S profiling. Results: N. mikurensis was detected in blood samples in all three cases and in bone marrow from one of the three. The severity of the symptoms ranged from prolonged fever lasting more than 6 months to life-threatening hyperinflammation in the form of hemophagocytic lymphohistiocytosis (HLH). Interestingly, all patients presented with splenomegaly and two with hepatomegaly. After starting doxycycline therapy, symptoms were relieved within a few days, and biochemistry and organomegaly quickly normalized. Conclusion: We present three Danish patients recognized by the same clinician over a period of 6 months, strongly suggesting that many cases are going unrecognized. Second, we describe the first case of N. mikurensis-induced HLH and emphasize the potential severity of undetected neoehrlichiosis.

Background Microsporidia are rarely reported to cause outbreaks of diarrhea. We describe a foodborne outbreak of microsporidiosis from a workplace canteen in November 2020 in Denmark. Methods A probable case was defined as any person using the canteen between 4 November and 13 December 2020, reporting at least one gastrointestinal symptom, whereas a confirmed case also had an Enterocytozoon bieneusi positive stool sample. A web-based questionnaire was used to collect clinical, epidemiological, and food exposure data. We performed a retrospective cohort study and tested stool samples from affected individuals for bacterial, viral, and parasitic pathogens, including E. bieneusi. Results Altogether, 195 individuals completed the questionnaire. We identified 52 cases (65% male; median age 45 years [range 25–65]). Diarrhea (90%), fatigue (83%), and abdominal pain (79%) were the most commonly reported symptoms. Eight cases were laboratory-confirmed and had E. bieneusi genotype C. The incubation period was between 5 and 12 days, and polymerase chain reaction (PCR)-detectable spore shedding occurred up to 43 days after symptom onset. Disease was associated with consuming food from the workplace canteen on 4 November 2020 (relative risk [RR[, 2.8 [95% confidence interval [CI]: 1.4 – 5.4]) and lunchboxes containing open sandwiches (RR, 3.2 [95% CI: 1.4 – 7.2]) served that day. Conclusions This is the second documented foodborne outbreak of E. bieneusi genotype C-associated diarrhea worldwide. Epidemiological findings advocated an open sandwiches lunchbox from 4 November 2020, as a likely source. E. bieneusi may be an under-reported cause of outbreaks of diarrhea, and testing for it might be useful in foodborne outbreak investigations.

Optimising the production of insects for food and feed and ensuring their health are growing concerns for producers. Insects suffer from a range of insect pathogenic microorganisms, and the management of such diseases is essential. One solution is the introduction of beneficial probiotic bacteria into the diet of the insects. Here, we show that a lactic acid bacterial strain, Pediococcus pentosaceus, isolated from the gut of the mealworm, Tenebrio molitor, was able to inhibit the growth of selected insect pathogens in vitro. Using in vivo assessments of the host’s fitness benefits conferred by the lactic bacterium we show a significant effect of P. pentosaceus on larval growth rate and survival into adulthood. Gut microbiota analysis focusing on bacterial composition based on 16S rRNA gene amplicon sequencing suggests that P. pentosaceus could have successfully colonised the guts, or altered their bacteria, of the larvae that received it. Finally, we discuss our results in the context of mass insect production systems and outline the remaining work needed to explore and secure the role of beneficial bacterial additives in the field.

The enteric protozoan parasites

Chronic inflammation is believed to play an important role in the development and disease progression of polycythemia vera (PV). Since an association between gut microbiota, hematopoiesis, and inflammation is well established we hypothesized that patients with PV have a gut microbiota distinct from healthy controls. Recombinant interferon-α2 (IFN) treatment of patients with PV has been shown to be disease-modifying in terms of normalization of elevated blood cell counts in concert with a reduction in the JAK2V617F allelic burden. Therefore, we hypothesized that patients treated with IFN might have a composition of the gut microbiota towards normalization. Herein, via amplicon-based next generation sequencing of the V3-V4 regions of the 16S rRNA gene, we report on an abnormal gut microbiota in 102 patients with PV as compared with 42 healthy controls (HCs). Patients with PV had a lower alpha diversity and a lower relative abundance of several taxa belonging to Firmicutes (45%) compared with HCs (59%, p

Background: Blastocystis has been associated with various symptoms of the gastrointestinal tract. We aimed to investigate the prevalence of Blastocystis in children with celiac disease (CeD) or functional abdominal pain (FAP) and to evaluate its subtypes (STs) with respect to demographic, socioeconomic and epidemiological factors. Methods: Overall, 161 fecal samples were collected from healthy children and patients with FAP or CeD in Hitit University Erol Olçok Research and Training Hospital, Corum, Turkey between 2016-2018. Samples were examined using both native-Lugol (NL) and trichrome-stained (TS) smears, and further analyses by PCR and Sanger sequencing were performed. A standard questionnaire was applied to obtain demographic, socioeconomic, epidemiological data. Results: Blastocystis was found in 10.6% of the total study population. Neither bacteria nor any other parasites were found, except for one Giardia (0.6%) in the CeD group. The presence/absence of the parasite was not found to be associated with demographic, socioeconomic and epidemiological factors. Blastocysis was detected in 11.5% (6/52) of the CeD, 7.7% (4/52) of the FAP, and 12.3% (7/57) of the healthy group. Diagnostic methods were similar in terms of Blastocystis detection (P= 0.671), and there was fair agreement between the NL, TS and PCR (Fleiss' Kappa=0.847, P=0.001). ST2 (42.8%) and ST3 (35.7%) were the predominant STs followed by ST1 (21.4%). Conclusion: We observed no difference between study groups in terms of Blastocystis prevalence. ST1, ST2 and ST3 subtypes were detected. Blastocystis prevalence and STs were not related to any of the demographic, socioeconomic and epidemiological factors.

Blastocystis is a common protist colonizing the gastrointestinal tract of humans and various animals. The first subtyping of Blastocystis isolates in pigs and humans in Serbia revealed unusual avian-specific subtype ST6 in humans. In total, 48 pig faecal specimens collected on seven pig farms and 50 human faecal specimens positive to Blastocystis by microscopic examination were selected for the study. Eleven randomly selected PCR-positive pig samples and 10 samples from human patients (with gastrointestinal complaints) were subjected to SSU rDNA sequencing. Three subtypes were identified (ST3, ST5 and ST6) by phylogenetic analysis. ST5 was found in all pig samples; while in human samples, we detected ST3 and ST6. The latter subtype is relatively uncommon in Europe and highly adapted to avian hosts; therefore, the possibility of sporadic zoonotic transmission to human patients should not be ignored.

Objectives The intestinal parasite Dientamoeba fragilis (D. fragilis) is a common colonizer of children in Denmark. Metronidazole has been used to reduce gastrointestinal symptoms in children colonized with D. fragilis. We aimed to identify gut microbiota changes associated with i) D. fragilis carrier status and ii) metronidazole treatment of D. fragilis-positive children. Methods The fecal microbiota of 275 fecal samples from children treated with metronidazole (n = 48) or placebo (n = 48) were characterized by ribosomal DNA sequencing. Samples collected before (T1), 2 weeks after (T2), and 8 weeks (T5) after treatment were included. Seventy fecal samples from 70 age-matched parasite-negative children served as controls. Results The abundance of 24 bacterial genera differed significantly according to D. fragilis carrier status, with Flavonifractor being remarkably more abundant in children testing negative for D. fragilis. Eight bacterial genera changed significantly in abundance in children losing vs. keeping D. fragilis after metronidazole treatment. Of these, seven returned to pre-treatment (T1) levels at T5. Meanwhile, the abundance of Flavonifractor continued to differ at T5, whereas for Ruminococcus the abundance only remained high in children who were D. fragilis-negative at T2 and T5. Increases in Hungatella, Sutterella, and Streptococcus abundances observed at T2 were specific to metronidazole exposure and hence independent of D. fragilis colonization. Conclusions This study revealed that specific bacterial genera were associated with D. fragilis colonization. Metronidazole treatment had a short-term impact on the abundance of some bacterial genera, with most of these reverting to pre-treatment levels eight weeks after completed treatment.

A 46-year-old woman presented with facial pain and discomfort. Diagnosis of subcutaneous dirofilariasis was reached after several months from symptom onset. Dirofilariasis should be suspected, also in non-endemic areas, in patients with a migrating subcutaneous nodule. Plastic surgery is preferred, as the face is often involved.

Blastocystis is a unicellular eukaryote found in the gastrointestinal tract of both human and other animal hosts. The clinical significance of colonic Blastocystis colonization remains obscure. In this study, we used metabarcoding and bioinformatics analyses to identify differences in stool microbiota diversity between Blastocystis-positive and Blastocystis-negative individuals (n = 1285). Alpha diversity was significantly higher in Blastocystis carriers. At phylum level, Firmicutes and Bacteroidetes were enriched in carriers, while Proteobacteria were enriched in non-carriers. The genera Prevotella, Faecalibacterium, Flavonifracter, Clostridium, Succinivibrio, and Oscillibacter were enriched in carriers, whereas Escherichia, Bacteroides, Klebsiella, and Pseudomonas were enriched in non-carriers. No difference in beta diversity was observed. Individuals with Blastocystis-positive stools appear to have gut microbiomes associated with eubiosis unlike those with Blastocystis-negative stools, whose gut microbiomes are similar to those associated with dysbiosis. The role of Blastocystis as an indicator organism and potential modulator of the gut microbiota warrants further scrutiny.

Overall, 161 fecal samples were collected from healthy children and patients with FAP or CeD in Hitit University Erol Olçok Research and Training Hospital, Corum, Turkey between 2016-2018. Samples were examined using both native-Lugol (NL) and trichrome-stained (TS) smears, and further analyses by PCR and Sanger sequencing were performed. A standard questionnaire was applied to obtain demographic, socioeconomic, epidemiological data.We observed no difference between study groups in terms of

While some single-celled intestinal parasites are direct causes of diarrhoea and other types of intestinal pathology, the impact of other gut micro-eukaryotes on human health remains elusive. The fact that some common luminal intestinal parasitic protists (CLIPPs) have lately been found more often in healthy than in diseased individuals has fuelled the hypothesis that some parasites might in fact be protective against disease. To this end, the use of new DNA technologies has helped us investigate trans-kingdom relationships in the gut. However, research into these relationships is currently hampered by the limited data available on the genetic diversity within the CLIPPs genera, which results in limited efficacy of publicly available DNA sequence databases for taxonomic annotation of sequences belonging to the eukaryotic component of the gut microbiota. In this paper, I give a brief overview of the status on CLIPPs in human health and disease and challenges related to the mapping of intestinal eukaryotic diversity of the human gut.

The enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis and Dientamoeba fragilis are—to various extents—contributors to the burden of gastrointestinal illness in high-income countries. Detection of these pathogens by microscopy examination is challenging because of the limited sensitivity and need for specific staining procedures. We developed and optimised a new multiplex real-time PCR assay for the simultaneous detection of Cryptosporidium spp., G. duodenalis and D. fragilis in clinical (stool) samples. The diagnostic performance of the assay was evaluated against a large panel of well-characterised DNA samples positive for Cryptosporidium spp. (n = 126), G. duodenalis (n = 132) and D. fragilis (n = 49). The specificity of the test was assessed against a DNA panel from other intestinal or phylogenetically related parasites (n = 105) and faecal DNA from individuals without clinical manifestations (n = 12). The assay exhibited a diagnostic sensitivity of 0.90–0.97 and a diagnostic specificity of 1. The limit of detection was estimated for Cryptosporidium (1 oocyst) and G. duodenalis (5 × 10−4 cysts). The method allowed the detection of four Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. cuniculus) and five G. duodenalis assemblages (A–E) without cross-reacting with other parasites belonging to the phyla Amoebozoa, Apicomplexa, Euglenozoa, Microsporidia, Nematoda and Platyhelminthes. This newly developed multiplex real-time PCR assay represents a novel alternative for the rapid and accurate detection of Cryptosporidium, G. duodenalis and D. fragilis in clinical settings.

Strongyloidiasis is a disease caused by the intestinal helminth Strongyloides stercoralis. When the immune system of infected individuals is compromised, larvae may migrate from the gastrointestinal tract to other tissues, causing S. stercoralis hyperinfection syndrome, which has a reported mortality of 71%. In this case, we report a patient with S. stercoralis hyperinfection syndrome with central nervous system (CNS) involvement. An elderly South East Asian male tourist presented with pulmonary symptoms, fever and infiltrates on chest X-ray. He later developed symptoms of CNS infection. S. stercoralis larvae were found in a stool sample. Microbiological examination of cerebrospinal fluid revealed S. stercoralis-specific DNA. The patient was treated with oral and rectal ivermectin and albendazole. The condition was complicated by sepsis, bacteraemia and hypereosinophilia. Unfortunately, the patient eventually died from pulmonary oedema and insufficiency. This case highlights the global importance of Strongyloides CNS infection in endemic and non-endemic regions.

The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), C. ubiquitum (n = 2), and one each of C. suis, C. viatorum, C. ditrichi, and Cryptosporidium horse genotype. One patient was co-infected with C. parvum and C. hominis. Subtyping was successful for all species/genotypes, except for C. ditrichi, and revealed large diversity, with 29 subtype families (including 4 novel ones: C. parvum IIr, IIs, IIt, and Cryptosporidium horse genotype VIc) and 81 different subtypes. The most common subtype families were IIa (n = 164) and IId (n = 118) for C. parvum and Ib (n = 26) and Ia (n = 12) for C. hominis. Infections caused by the zoonotic C. parvum subtype families IIa and IId dominated both in patients infected in Sweden and abroad, while most C. hominis cases were travel-related. Infections caused by non-hominis and non-parvum species were quite common (8%) and equally represented in cases infected in Sweden and abroad.

Blastocystis is the most common intestinal parasite found in epidemiological studies in Iran. The genus Blastocystis comprises multiple subtypes (ST), evincing the existence of extensive intrageneric diversity. Meanwhile, information on Blastocystis STs in domestic animals in Iran is limited. This study was designed to identify the prevalence and subtype distribution of Blastocystis in domestic animals and to evaluate the extent of zoonotic origin of human Blastocystis carriage. A total of 395 faecal samples from farm animals (220 from poultry, 100 from sheep and 75 from cattle) and 23 Blastocystis positive samples from humans (available from the previous report of this study and included for reference) from the Khuzestan province, southwestern Iran, were analysed. Standard parasitological methods and PCR-based analysis of the SSU-rRNA gene were used. Overall, 115 (29.1%; 95% CI, 26.5%-31.6%) of the animals were positive for Blastocystis sp. by parasitological methods. The highest colonization rate was found in cattle (50.6%, 38/75), followed by sheep (32.0%, 32/100) and poultry (20.4%, 45/220). Eight Blastocystis STs were identified among 55 sequenced isolates from animals (n = 32) and humans (n = 23): ST3 (27.3%), ST14 (25.4%), ST1 (12.7%), ST7 (12.7%), ST2 (9.1%), ST6 (7.3%), ST5 (3.6%) and ST10 (1.8%). Blastocystis ST1 and ST3 were found in both human and non-human hosts, and examples of shared alleles (strains) were observed. Meanwhile, ST2 was observed only in humans, ST5 only in sheep, ST6 only in poultry and ST10 in only cattle. This is the most comprehensive report of Blastocystis STs in farm animals in Iran.

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including Acanthamoeba.

Giardia intestinalis is one of the most common causes of parasite-induced diarrhea, abdominal pain, flatulence, and malabsorption. Yet, data on the epidemiology of G. intestinalis infections in North Africa are limited. The purpose of this study was to carry out a retrospective survey on the level of intestinal parasitism with a particular emphasis on G. intestinalis in children and adults in Algiers, Algeria. A total of 2,054 individuals from outpatient clinics or hospitalized at Beni-Messous University Hospital of Algiers undergoing stool microscopy for ova and parasites were included. The overall parasite infection rate was 28%. In the 567 parasite-positive samples, Blastocystis was found most frequently (57.3%), followed in frequency by Endolimax nana (41.0%), Entamoeba histolytica/dispar (19.6%), G. intestinalis (17.1%), Entamoeba coli (13.9%), Chilomastix mesnili (1.0%), Iodamoeba bütschlii (0.7%), Entamoeba hartmanni (0.5%), and Cryptosporidium spp. (0.2%). Intestinal parasites were generally more common in adults than in children, except for Giardia, which was more common in children (P = 0.0001). Giardia infection was independent of gender (P = 0.94). Compared with other intestinal parasitic infections, clinical manifestations, such as abdominal pain (P = 0.28) and diarrhea (P = 0.82), were found not to be significantly linked to Giardia infection. In conclusion, G. intestinalis is common in individuals referred to the University Hospital of Beni-Messous with digestive symptoms, particularly so in children. However, in our study, intestinal symptoms appeared not to be more linked to Giardia than to other intestinal parasites.

Herbivores can be accidental hosts for the zoonotic nematode parasites Trichinella spp., which are endemic at high prevalence in wildlife in northeastern Europe. Using direct and indirect detection methods for Trichinella spp., we investigated samples from 463 wild moose (Alces alces) harvested by hunters in Estonia in 2015. A total of 460 moose were tested directly by artificial digestion of diaphragm muscle, 463 moose were tested indirectly by enzyme-linked immunosorbent assay (ELISA), and 34 moose also by western blot. Positive-control reference sera were from other host species. Nematode larvae were found in six muscle samples; five of which were pooled samples. None of the larvae were identified as Trichinella spp., based on their morphology and molecular analyses. Twelve moose (2.6%) were positive by ELISA, but none were positive by the western blot test. Trichinella spp. infection was not detected, but ELISA results may suggest Trichinella spp. exposure in a small proportion of moose in Estonia.

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses., Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including Acanthamoeba.

An outbreak of cryptosporidiosis among veterinary students performing fetotomy exercises on euthanized calves took place in September 2018 in Denmark. A prospective cohort investigation was performed to identify risk factors and provide guidance for preventing outbreaks of cryptosporidiosis in this setting. Ninety-seven students attended the fetotomy exercises and completed a questionnaire about symptoms and potential risk behavior. Real-time PCR was used to detect

Blastocystis is a common inhabitant of the human gut, colonizing at least one billion people at a prevalence ranging from

The development and integration of DNA-based methods in research and clinical microbiology laboratories have enabled standardised and comprehensive detection and differentiation of the microbes colonising our guts. For instance, the single-celled parasites Blastocystis and Dientamoeba appear to be much more common than previously thought, especially so in healthy individuals. While increasing evidence appears to suggest limited pathogenicity of these parasites, next-generation-sequencing-based studies have helped us to appreciate links between parasite colonisation and certain host phenotypical characteristics and gut microbial profiles. The fundamental question remains as to whether such parasites are merely indicators or active manipulators of gut microbiota structure and function. In this article, we collate existing evidence that these parasites are, at minimum, indicators of intestinal microbiota structure.

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

Despite the species' wide distribution, studies of the genetic diversity within Entamoeba coli and Entamoeba hartmanni remain limited. In the present study, we provide further insight into the genetic diversity of both species based on analysis of partial nuclear small subunit ribosomal DNA sequences generated from human faecal DNAs from samples collected in Africa, South America and Europe. Reinforcing the previous recognition that E. coli is a species complex, our data confirm the existence of the two subtypes, ST1 and ST2, previously identified plus, potentially, a new subtype, ST3. While ST1 appears to be genetically quite homogenous, ST2 shows a substantial degree of intra-subtype diversity. ST2 was more common in samples collected outside Europe, whereas ST1 showed no geographical restriction. The potentially novel subtype is represented to date exclusively by sequences from South American and African samples. In contrast to previous reports, our new data also indicate substantial variation in E. hartmanni that could also support the establishment of subtypes within this species. Here, however, no links were identified between subtype and geographical origin.

Background Application of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants. Materials and methods Influent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis. Results A total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n = 20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba bütschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1–4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples. Conclusions Entamoeba and Blastocystis were identified as universal members of the “sewage microbiome”. Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax.

Blastocystis is a genetically diverse intestinal protist colonizing both human and non-human hosts. By 2013, 17 subtypes had been acknowledged. Since then, nine more subtypes have been proposed. We argue that several recently proposed subtypes are invalid. We also revisit recommendations regarding the requirements for annotating sequences as new subtypes.

The contribution of Blastocystis from non-human hosts to zoonotic transmission is only partly known. The objective of this study was to determine the distribution of Blastocystis genetic subtypes in different animal species in United Arab Emirates. A total of 114 stool samples were tested using PCR of the small subunit (SSU) rRNA gene and sequence analysis. Twenty-three Blastocystis-positive samples were identified. The following detection rates were observed: cattle, 22.7%; sheep, 63.6%; rabbits, 33.3%; rodents, 37.5%; reptiles, 21.2%. Four subtypes were identified in this study; ST4, ST10, ST14, and ST17; ST10 was isolated from sheep and cattle, corroborating previous data indicating that these are natural hosts for this subtype. Cases of mixed subtype colonization were also detected. Conspicuously, we found ST14 in rabbits. The discovery of ST17 in a squirrel indicates a novel host for this subtype. Furthermore, the discovery of ST4 in rodents suggests that these may serve as reservoir for human Blastocystis ST4 colonization. Six tortoises and one iguana were positive for Blastocystis. In conclusion, this is the first report of Blastocystis infection in various animals in the UAE. Apart from ST4, no potentially zoonotic subtypes were detected.

This study aimed to elucidate aspects of the epidemiology of Blastocystis in Nigerian school children, including the distribution of subtypes (STs) and ST alleles. A total of 199 genomic DNAs extracted from fecal samples from 199 Nigerian children aged 2–14 years were tested by real-time polymerase chain reaction for Blastocystis. Positive DNAs were submitted to barcoding by PCR and sequencing to obtain information on STs and ST alleles. A total of 167 (84%) samples were positive for Blastocystis, with prevalence increasing by age. No association between Blastocystis colonization and gender (P = 0.51) or type/presence of toilet facilities (P = 0.21) was observed. Blastocystis carriers were more prone to using water collected from wells than from sachets (P = 0.0044). Moreover, Blastocystis positivity was associated with positivity for fecal-orally transmitted protozoa (P = 0.018) and helminths (P < 0.0001). A clear inverse association of Blastocystis colonization and malaria infection was observed (P < 0.0001); however, malaria-positive children being younger than malaria-negative children, this finding was attributed to the age effect of Blastocystis colonization. ST data were available for 127/167 (76%) samples. Fifty-one children were positive for ST1, while 42 and 33 children were colonized with ST2 and ST3, respectively; a single case of ST7 was observed. By and large, the ST alleles identified for ST1 and ST2 did not differ from those observed in humans in other regions of the world; meanwhile, the distribution of ST3 alleles was remarkably distinct and potentially specific to humans in sub-Saharan Africa.

Blastocystis is a genus of common single-celled intestinal parasitic protists with an unsettled role in human health and disease. Being a stable component of intestinal microbiota, once established, the Blastocystis parasite appears more common in healthy individuals than in patients with infectious, functional, or inflammatory bowel disease. Recent data suggest that the parasite is associated with certain gut microbiota profiles and health indices. Convincing data and tools differentiating asymptomatic colonization from infection are yet to be demonstrated. Although the parasite may elicit disease under certain circumstances, the focus on Blastocystis may be shifting from a clinical to a public health perspective.

Background Application of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants. Materials and methods Influent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis. Results A total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n = 20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba butschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1–4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples. Conclusions Entamoeba and Blastocystis were identified as universal members of the “sewage microbiome”. Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax.

IntroductionBy end of 2018, the European Union countries hosted approximately 2.5 million refugees and Lebanon alone hosted more than 1 million. The majority of refugees worldwide came from Syria. The prevailing study design in published studies on asylum seekers’ and refugees’ health leaves a number of fundamental research questions unanswerable. In the Asylum seekers’ and Refugees’ Changing Health (ARCH) study, we examine the health of a homogeneous group of refugees and asylum seekers in two very different host countries with very different migration histories. We aim to study the health impact of the migration process, living conditions, access to healthcare, gene–environment interactions and the health transition.Methods and analysisARCH is an international multisite study of the health of adult (>18 years old) Syrian refugees and asylum seekers in Lebanon and Denmark. Using a standardised framework, we collect information on mental and physical health using validated scales and biological samples. We aim to include 220 participants in Danish asylum centres and 1100 participants in Lebanese refugee camps and settlements. We will use propensity score weights to control for confounding and multiple imputation to handle missing data.Ethics and disseminationEthical approval has been obtained in Lebanon and Denmark. In the short term, we will present the cross-sectional association between long-distance migration and the results of the throat and wound swab, blood and faeces samples and mental health screenings. In the longer term, we are planning to follow the refugees in Denmark with collection of dried blood spots, mental health screenings and semistructured qualitative interviews on the participant’s health and access to healthcare in the time lived in Denmark. Here, we present an overview of the background for the ARCH study as well as a thorough description of the methodology.

Background We performed a 12-month cohort study of the stability and resilience of the intestinal microbiota of healthy children in daycare in Denmark in relation to diarrheal events and exposure to known risk factors for gastrointestinal health such as travelling and antibiotic use. In addition, we analyzed how gut microbiota recover from such exposures. Results We monitored 32 children in daycare aged 1–6 years. Fecal samples were submitted every second month during a one-year observational period. Information regarding exposures and diarrheal episodes was obtained through questionnaires. Bacterial communities were identified using 16S rRNA gene sequencing. The core microbiota (mean abundance > 95%) dominated the intestinal microbiota, and none of the tested exposures (diarrheal events, travel, antibiotic use) were associated with decreases in the relative abundance of the core microbiota. Samples exhibited lower intra-individual variation than inter-individual variation. Half of all the variation between samples was explained by which child a sample originated from. Age explained 7.6–9.6% of the variation, while traveling, diarrheal events, and antibiotic use explained minor parts of the beta diversity. We found an age-dependent increase of alpha diversity in children aged 1–3 years, and while diarrheal events caused a decrease in alpha diversity, a recovery time of 40–45 days was observed. Among children having had a diarrheal event, we observed a 10x higher relative abundance of Prevotella. After travelling, a higher abundance of two Bacteroides species and 40% less Lachnospiraceae were seen. Antibiotic use did not correlate with changes in the abundance of any bacteria. Conclusion We present data showing that Danish children in daycare have stable intestinal microbiota, resilient to the exposures investigated. An early age-dependent increase in the diversity was demonstrated. Diarrheal episodes decreased alpha diversity with an estimated recovery time of 40–45 days. Electronic supplementary material The online version of this article (10.1186/s12866-018-1367-5) contains supplementary material, which is available to authorized users.

In this case report, a 64-year-old woman with symptoms of lethargy, nausea, fluctuating temperature, and weight loss was suspected of a disease of cardiological or haematological aetiology. She was diagnosed with liver fluke infection based on serological evidence, eosinophilia, relevant exposure, and clinical presentation. High-dose triclabendazole led to symptom resolution. On several occasions, she had been eating watercress from a meadow with grazing sheep. The tendency of eating raw watercress and other green plants collected in nature confers an increased risk of developing liver fluke infections.

Blastocystis is estimated to be one of the most common parasites of the intestinal tract of humans, comprising multiple subtypes (ST). Meanwhile, the distribution of Blastocystis ST in many communities and countries remains unknown. In the present work, we aimed to identify the prevalence of Blastocystis and the ST distribution in human stool samples collected from healthy expatriates from different geographical regions and residing in Sharjah, United Arabian Emirates (UAE). A total of 133 samples were screened and subtyped using partial small subunit ribosomal RNA gene sequencing. Fifty-nine (44.4%) samples were identified as positive. Among these, 39 were successfully sequenced and subtyped. The ST distribution was as follows: ST3, 58.9% (23/39); ST1, 28.2% (11/39); and ST2, 7.6% (3/39). No correlation between geographic origin and infection (χ(2)=11.006; P=0.528) nor gender and infection (χ(2)=1.264; P=0.261) was observed. The data were compared with those available for other Middle Eastern and North African neighboring countries. This study is the first to provide data concerning the prevalence of Blastocystis and the frequency of various STs in the UAE, confirming the absence of ST4 and the commonness of ST1, ST2, and ST3 in this geographical region.

Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in co-abundance gene groups, which are groups of genes that co-vary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD), and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis- positive stool (Matthew's correlation coefficient = -0.25, P < 0.0001) than individuals with Ruminococcus - and Prevotella -driven enterotypes. This is the first study to investigate the relationship between Blastocystis and communities of gut bacteria using a metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities.

The human gut is host to a diversity of microorganisms, including the single-celled microbial eukaryote Blastocystis. Research has shown that most carriers host a single Blastocystis subtype (ST), which is unusual given the considerable within-host species diversity observed for other microbial genera in this ecosystem. However, our limited knowledge of both the incidence and biological significance of Blastocystis diversity within hosts (i.e., so-called mixed infections) is likely due to problems with existing methodologies. Here, we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes of Blastocystis (ST1 to ST4) to a healthy human cohort ( n = 50). We detected mixed infections in 22% of the cases, all of which had been identified as single-ST infections in a previous study using state-of-the-art methods. Our results show that certain STs occur predominantly as either single (ST3 and 4) or mixed (ST1) infections, which may reflect inter alia transient colonization patterns and/or cooperative or competitive interactions between different STs. Comparative analyses with other primers that have been used extensively for ST-specific analysis found them unsuitable for detection of mixed- and, in some cases, single-ST infections. Collectively, our data shed new light on the diversity of Blastocystis within and between human hosts. Moreover, the development of these PCR assays will facilitate future work on the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.

The relationship between intestinal helminth infection and susceptibility to malaria remains unclear. We studied the relationship between these infections. Seven schools in Ilero, Nigeria referred all pupils with febrile illness to our study center for free malaria treatment during a 3-month study period. At the end, all pupils submitted a stool sample for microscopic investigation for helminth eggs. We used an unmatched case-control design to calculate the odds ratios for helminth infection in children with at least one attack of malaria (cases) and children with no malaria episodes during the study (controls). We recorded 115 malaria cases in 82 of 354 (23.2%), 16 of 736 (2.2%), and 17 of 348 (4.7%) children ages ≤ 5, 6–10, and 11–15 years old, respectively (P = 0.001). Helminth infection rate in cases was 21 of 115 (18.3%) compared with 456 of 1,327 (34.4%) in controls. Weighted odds ratio stratified by age group for helminth infection in cases versus controls was 0.50 (95% confidence interval = 0.2–0.84, P < 0.01). Ascaris and hookworm were the most common helminths detected, with prevalence rates of 14 (12.2%) and 6 (5.2%) among cases compared with 333 (25.1%) and 132 (10.0%) in controls, respectively (P = 0.001). The negative association between helminth infection and malaria may be of importance in the design of deworming programs.

Entamoeba polecki is a parasite of human and nonhuman primates, other mammals, and birds. Due to overlapping morphological features, cysts of E. polecki may be confused with those of other Entamoeba species commonly found in human fecal samples, including immature cysts of Entamoeba histolytica . Although the presence of E. polecki in human Entamoeba -positive stool samples may be rare, its prevalence is likely underestimated due to such confusion. Here, we give examples of diagnostic approaches applied so far and summarize data on the molecular epidemiology of E. polecki , including host specificity and phylogeography. Moreover, we evaluate a novel diagnostic conventional PCR developed for the screening of fecal samples for E. polecki . The assay was highly sensitive and specific when used on genomic DNA extracted directly from stool and Swedish wastewater samples. The PCR enabled the identification of all four subtypes (ST1 to ST4) of E. polecki by PCR product sequencing. Most (23/28) subtyped E. polecki- positive samples detected in patients in Sweden between 2002 and 2015 reflected colonization by ST4 and were seen in travelers/foreigners. Two and three human cases of ST2 and ST3, respectively, were also detected. Subtypes 1, 2, and 3 were detected in 3/21 wastewater samples, suggesting local endemicity of these E. polecki subtypes; interestingly, ST4 was not detected in wastewater. In conclusion, the current PCR assay enables simple and cost-effective screening of fecal and wastewater samples for E. polecki . Human cases of E. polecki appear to involve primarily ST4, while E. polecki detected in wastewater may be primarily of animal origin.

Blastocystis is an enteric parasitic protist with a widespread distribution in a variety of human and non-human hosts and supposedly a source of zoonotic transmission. This study aimed to determine the frequency and distribution of Blastocystis subtypes in free-ranging Macaca fascicularis (long-tailed macaques, LTMs) in Thailand. A total of 628 faecal samples were collected from free-ranging LTMs inhabiting fourteen tourist attraction sites in Thailand. Fresh faecal samples were individually cultured in Jones' medium, and Blastocystis-positive samples were subtyped using nuclear small subunit ribosomal DNA (SSU rDNA) sequencing. Two hundred and sixty-three (41.87%) samples were positive by culture, and 197 and 154 were successfully SSU rDNA-amplified and sequenced, respectively. Three subtypes (ST1, ST2, and ST3) comprising 19 alleles were observed. ST3 was the most common subtype detected (36.55%), followed by ST2 and ST1 (24.37% and 17.26%, respectively). Some subtype alleles not previously observed were identified. A couple of strains appeared similar to those found in humans as evidenced by SSU rDNA allelic profiles, while further evidence of cryptic host specificity emerged. This study provides the first data on Blastocystis subtypes in non-human primates in Thailand and confirms the trend observed in other Old-World countries with regard to the colonization rate and subtype distribution.

Species of Sarcocystis are Apicomplexan parasites requiring intermediate and definitive hosts to complete their life cycle. Humans are one of many natural host species and may serve as both intermediate and definitive hosts. However, the extent and public health significance of human Sarcocystis infection are incompletely known. In this minireview, we provide an update on the epidemiology and diagnosis of human sarcocystosis and propose some tools that could contribute to a better understanding of the clinical significance and epidemiology of Sarcocystis infections.