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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.

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.

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

BackgroundImmune checkpoint inhibitors (ICIs) can induce a wide range of immune-related adverse events (irAEs), potentially affecting any organ. ICI-induced colitis is a frequently reported irAE, whereas enteritis is rare and not well documented.Case presentationWe are presenting a patient with metastatic melanoma who developed severe ICI-induced enterocolitis multirefractory for glucocorticoids, infliximab and vedolizumab, partially responding to faecal microbiota transplantation and final complete response to tofacitinib.ConclusionThis case supports that tofacitinib may be an(other) effective agent in managing multirefractory ICI-induced diarrhoea caused by colitis and/or enteritis.

Aims Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. Methods and Results Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%–103% and detection limits of 100–1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. Conclusion The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. Significance and impact of study In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Microbial co‐infections may contribute to the pulmonary deterioration in COVID‐19 patients needing intensive care treatment. The present study portrays the extent of co‐infections in COVID‐19 ICU patients. Conventional culture, molecular detections for atypical aetiologies, QiaStat‐Dx® respiratory panel V2 detecting 21 respiratory pathogens and ribosomal DNA genes 16S/18S amplicon‐based microbiome analyses were performed on respiratory samples from 34 COVID‐19 patients admitted to the ICU. Potential pathogens were detected in seven patients (21%) by culturing, in four patients (12%) by microbiome analysis and in one patient (3%) by respiratory panel. Among 20 patients receiving antibiotics prior to ICU admission, fungi (3 Candida albicans, 1 C. tropicalis, 1 C. dubliniensis) were cultured in 5 (15%) endotracheal aspirates. Among 14 patients who were antibiotic‐naive at ICU admission, two patients (6%) had bacterial respiratory pathogens (Staphylococcus aureus, Streptococcus pseudopneumoniae) cultured in their endotracheal aspirates. Microbiome analysis recognized four potential respiratory pathogens (3 Haemophilus influenza, 1 Fusobacterium necrophorum) isolated in samples from four other patients (12%). QiaStat‐Dx® respiratory panel V2 detected adenovirus in one patient (3%). The prevalence of pulmonary microbial co‐infections is modest among COVID‐19 patients upon admission to ICU. Microbiome analysis complements conventional microbial diagnostics in characterization of respiratory co‐infections.

We report human infection with simian Plasmodium cynomolgi in a tourist from Denmark who had visited forested areas in peninsular Malaysia and Thailand in August and September 2018. Because P. cynomolgi may go unnoticed by standard malaria diagnostics, this malaria species may be more common in humans than was previously thought.

Serological tests are routinely used to detect Toxoplasma gondii specific IgG antibodies in serum. Serological surveys of T. gondii show a medium to high prevalence in Danish indoor sows at the time of slaughter. However, little is known about when sows acquire T. gondii, and for how long they remain seropositive. Our focus was on quantifying the incidence rates in different age-cohorts and on investigating the T. gondii IgG antibody dynamics in sows. Four large Danish sow farms were longitudinally surveyed for 1 year. A total of 320 animals from 6, 12, 18, and 24-months age-cohorts were sampled at 5-week intervals. In total, 2989 plasma samples were tested using commercial enzyme linked immunosorbent assay (ELISA). The incidence rate in each of the four age-cohorts was calculated, and a time-to-event analysis was applied to the interval censored data to investigate the relationship between age and probability of T. gondii seroconversion. In the initial ELISA testing, eight sows tested positive for T. gondii at first survey, of which seven remained seropositive throughout the follow-up period. Additionally, 16 sows seroconverted, but only five of these remained seropositive. Weekly incidence rates in the 6, 12, 18, and 24-month age-cohorts were 0.0017 (95% CI = 0.0008 - 0.0037), 0.0009 (95% CI = 0.0003 - 0.0027), 0.0003 (95% CI = 0.0000 - 0.0018), and 0.0023 (95% CI = 0.0010 - 0.0051), respectively. Time-to-event analysis suggested that the incidence rate increased with age but could not conclude this definitively. The retesting of a subsample of the sows (n = 200) with the same ELISA and with modified direct agglutination test (MAT), and western blot (WB) assays suggested that 12 out of the 24 initial ELISA seropositive sows may have been false positive. These 12 sows also showed fluctuating antibody dynamics in ELISA. Hence, the unstable antibody dynamics in ELISA may pose a challenge for serological surveys of T. gondii in sows.

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.

Background Entamoeba gingivalis has been associated with periodontal diseases. Baseline data from the background population, which could help delimit the role of the parasite in health and disease, remain limited. Objective To describe epidemiological features, genetic diversity, and associations with oral microbiome signatures of E. gingivalis colonisation in Tanzanians with non-oral/non-dental diseases. Methods DNAs from 92 oral washings from 52 participants were subject to metabarcoding of ribosomal genes. DNA sequences were identified to genus level and submitted to oral microbiota diversity analyses. Results Sixteen (31%) of the 52 study participants were E. gingivalis-positive, with no difference in positivity rate according to gender or age. Only one subtype (ST1) was found. Individuals testing positive for E. gingivalis had higher oral microbiota alpha diversity than those testing negative (P = 0.03). Eight of the top-ten most common bacterial genera were shared between the two groups (Alloprevotella, Fusobacterium, Gemella, Haemophilus, Neisseria, Porphyromonas, Prevotella, Streptococcus, and Veillonella). Meanwhile, E. gingivalis carriers and non-carriers were more likely to have Aggregatibacter and Rothia, respectively, among the top-ten most common genera. Conclusion About one third of the cohort carried E. gingivalis ST1, and carriers had higher oral microbiome diversity and were more predominantly colonized by Aggregatibacter.

The present study aimed to estimate the prevalence of zoonotic pathogens Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Erysipelothrix in muskoxen (Ovibos moschatus) and sheep (Ovis aries) from Greenland. In 2017 and 2018, faecal samples were collected from wild muskoxen from three distinct populations (Zackenberg, Kangerlussuaq, and Ivittuut) and from domestic sheep from southwest Greenland. Blood samples were collected from muskoxen from Kangerlussuaq and Ivittuut and from sheep. Faecal samples were tested for specific DNA of G. duodenalis and Cryptosporidium spp., and blood samples were tested for antibodies against T. gondii and Erysipelothrix. The estimated prevalence of G. duodenalis was 0% (0/58), 17% (7/41) and 0% (0/55) in muskoxen from Zackenberg, Kangerlussuaq and Ivittuut, respectively, and 37% (16/43) in sheep. The estimated prevalence of Cryptosporidium was 0% (0/58), 2% (1/41), 7% (4/55) in muskoxen from Zackenberg, Kangerlussuaq, Ivittuut, respectively, and 2% (1/43) in sheep. Neither Giardia nor Cryptosporidium were detected in winter samples (0/78). Of the positive samples, Giardia from one muskox sample only was successfully typed as G. duodenalis assemblage A, and Cryptosporidium from two muskoxen was successfully typed as C. parvum, subtype IIdA20G1e. The estimated T. gondii seroprevalence was 2% (1/44) and 0% (0/8) in muskoxen from Kangerlussuaq and Ivittuut, respectively, and 1% (1/155) in sheep. The estimated Erysipelothrix seroprevalence was 2% (1/45) and 13% (1/8) in muskoxen from Kangerlussuaq and Ivittuut, respectively, and 7% (10/150) in sheep. The results of this study add to the scarce knowledge on zoonotic pathogens in the Arctic., We estimated the prevalence of four zoonotic agents – Giardia duodenalis, Cryptosporidium, Toxoplasma gondii and Erysipelothrix – in muskoxen (Ovibos moschatus) and sheep (Ovis aries) from Greenland and reported the first molecular detection of G. duodenalis and C. parvum in Greenland. The results add to the scarce knowledge on zoonotic pathogens in the Arctic.

Toxoplasma gondii infection in pigs is commonly diagnosed using serological tests that detect IgG antibodies targeted against the parasite. Such tests include enzyme-linked immunosorbent assay (ELISA), modified agglutination test (MAT), and western blot (WB), which are commercially available as rapid test kits. In this study, we evaluated the manufacturer recommended cut-off of ELISA-PrioCHECK test kit and determined a new optimal cut-off for identifying T. gondii infections in pigs. Assessment of the commercial ELISA kit was done by including data from two additional serological tests, MAT, and WB, applied to seven pig population categories with varying prevalences. A total of 233 plasma samples that were previously used in other studies for investigating T. gondii seroprevalence in pigs in Denmark were randomly selected for inclusion, including 95 samples that had previously been analysed with all three tests and an additional 138 samples that were analysed using the three serological tests for this study. In the absence of a gold standard test, a latent class model was fit to the data to obtain estimates of sensitivity and specificity for each of the tests along with prevalence in each of the populations. A cut-off that maximized the sensitivity and specificity of the ELISA test was then selected. The optimal cut-off value for percent of positive control (PP) in ELISA-PrioCHECK was estimated to be 27.7 PP, which is higher than the cut-off value of 20 PP that is recommended by the manufacturer. At this cut-off, the estimated sensitivities of ELISA, MAT and WB were 99.2% (96.3–100.0%), 96.3% (88.0–100.0%), and 89.8% (80.0–98.0%), respectively. The estimated specificities of ELISA, MAT and WB were 95.2% (92.5–97.6%), 99.6% (97.5–100.0%), and 98.2% (95.9–100.0%), respectively. Our findings have broad relevance to the use of the ELISA-PrioCHECK test kit for detecting Toxoplasma gondii infection in pigs.

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.

Pigs are one of several host species for Toxoplasma gondii parasites, and consumption of infected pork may lead to toxoplasmosis in humans. We estimated seroprevalence in sows and finishers from conventional and organic herds in Denmark and discussed the strategies for reducing the risk from pork. We collected 447 blood samples from 59 herds, and additional meat-juice samples from 212 of the same pigs. Using a T. gondii IgG commercial ELISA test, we found 2% (CI95% = 0.4% – 5%) apparent seroprevalence of T. gondii in conventional finishers, 11% (CI95% = 6% – 17%) in organic finishers, 19% (CI95% = 11% – 30%) in conventional sows and 60% (CI95% = 47% – 72%) in organic sows. The odds of an animal testing positive for T. gondii was 16 times higher (CI95% = 4.6 – 74.3) in organic compared to conventional herds. The odds were 22 times higher (CI95% = 6.5 – 88.3) if the animal was a sow compared to a finisher. Meat-juice ELISA values were significantly correlated with plasma results (P

Highlights • High prevalence of intestinal parasite colonisation; all were asymptomatic. • High prevalence of MRSA; low prevalence of ESBL and CPO. • Recommend attention to antimicrobial resistance when attending to Syrian refugees. • Recommend no routine screening for parasites in asymptomatic adult Syrian refugees., Introduction Concerns have been raised regarding the emergence of antimicrobial-resistance and parasitic infections in the European refugee population. Here, we estimated the prevalence of intestinal parasites and selected antimicrobial-resistant bacteria in newly arrived asylum seekers in Denmark. Materials and methods Using a cross-sectional one-stage cluster sample design, adult Syrian asylum seekers were included upon arrival in Denmark. Faecal samples were collected and tested for ova and parasites, extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) and carbapenemase-producing organisms (CPO). Throat swabs were collected and analysed for methicillin-resistant Staphylococcus aureus (MRSA) and Corynebacterium diphtheriae. Results We invited 121 eligible individuals (20% of the source population) from six different asylum centres to participate, of whom 113 agreed. Throat swabs and faecal samples were received from 104 and 48 participants, respectively. Seven individuals did not provide enough material for the entire panel of faecal analyses. Three individuals (7.3%, 95%CI: 2.5–19.4%) were colonised with Giardia intestinalis and 28 (68.3%, 95%CI: 53.0–80.4%) with Blastocystis sp. (subtypes 1 [n = 5], 2 [n = 9] and 3 [n = 14]). Seven individuals (6.7%, 95%CI: 3.3–13.3%) were colonised with MRSA and one with ESBL-E. None had CPO or Corynebacterium diphtheriae and none reported any gastro-intestinal symptoms. Discussion Even with the most conservative estimates, the prevalence of Giardia intestinalis, Blastocystis sp. and MRSA was high in this asymptomatic refugee population. Conclusions We highlight the importance of raised awareness of antimicrobial-resistant bacteria when attending to newly arrived Syrian refugees. Meanwhile, our data suggest that routine screening for intestinal parasites in this population is of limited clinical relevance.

Background: Maternal obesity is associated with adverse pregnancy outcomes. Probiotic supplementation during pregnancy may have positive effects on blood glucose, gestational weight gain (GWG), and the risk of gestational diabetes mellitus (GDM). The primary aim was to determine the feasibility of probiotic intervention in obese pregnant women from the early second trimester until delivery. The secondary aim was to investigate the effect of daily probiotic supplementation on GWG, maternal glucose homeostasis, infant birthweight, and maternal gut microbiota. We carried out a randomized double-blinded placebo-controlled study in 50 obese pregnant women. Participants were randomly allocated to two treatment groups, multi-strain probiotic [Vivomixx®] or placebo at 14–20 weeks of gestation until delivery. Participants were followed with two pre-delivery visits at gestational week 27-30 and 36-37 and with one post-delivery visit 2-3 days after birth. All visits included blood and fecal sampling. An oral glucose tolerance test was performed at inclusion and gestational week 27-30. Results: Forty-nine participants completed the study. Thirty-eight participants took more than 80% of the Vivomixx® capsules (n=21), placebo (n=17). There was no significant difference in HbA1c levels and the occurrence of GDM between groups. There was no significant difference in GWG and infant birth weight between groups in intention to treat analysis. There was, however, a lower mean GWG (11.9 vs 13.0 kg) and lower mean infant birthweight (3554 vs 3658 g) in the probiotic group in the per protocol analysis, due to sample size this difference did not reach statistical significance. Fecal microbiota analyses showed an overall increase in α-diversity over time in the Vivomixx® group only (p=0.016). Conclusions: Administration of probiotics during pregnancy is feasible in obese women. Multi-strain probiotic can modulate the gut microbiota in obese women during pregnancy. A larger study population is needed to uncover whether the results regarding lower GWG and infant birth weight after probiotic supplementation are significant. Trial registration: ClincalTrials.gov Identifier: NCT02508844, registered on May 11, 2015.

Background: The human gut microbiota (the population of microorganisms present) is important for digestion of food but also for development of the host immune system and protection against pathogens. Changes in the gut microbiota are linked to several inflammatory diseases such as diabetes, atopic diseases and Alzheimer's disease. Polycythemia vera (PV) is one of the Philadelphia chromosome negative classical myeloproliferative neoplasms (MPNs), which also include essential thrombocythemia (ET) and primary myelofibrosis (PMF). MPNs are increasingly recognized as inflammatory driven diseases. The role of the gut microbiota in patients with MPNs is largely unknown. In a small study (n=25) the microbiota of MPN patients had higher levels of Prevotellaceae compared to healthy controls, and differed significantly in composition between patients treated with hydroxyurea and ruxolitinib. Since MPNs are likely to be driven by chronic inflammation and the gut microbiota influences the immune system, investigations of the PV-microbiota are highly relevant. We compared the microbiota in a cross-sectional study of patients with PV stratified into five different treatment groups. Method and Material: Patients above 18 years diagnosed with PV, according to the 2016 World Health Organization (WHO) classification, were invited to participate in the study. The exclusion criteria were: pregnancy, use of antibiotics within the last 2 months, change in treatment within the last 3 months or inability to understand the oral and written information. Clinical and biochemical data for each patient were collected retrospectively and included co-morbidities, smoking status, anti-inflammatory treatment, hypertension, haematological parameters, haematological treatment, body mass index (BMI), among others. Stool samples, no more than 6 hours old were stored at -80°C. DNA was extracted by using the EMAG® Nucleic acid extraction system, [bioMérieux] according to the manufacturer's instructions. The bacterial microbiota was characterized by amplicon-based next generation sequencing of the V3-V4 region of the 16S ribosomal unit using a MiSeq instrument, Illumina. BION was used to assign taxonomic classification. The patients were divided into 5 groups according to treatment: no treatment (n=18), hydroxyurea (n=33), PEG-interferon-α2 (IFN) (n=23), IFN combined with ruxolitinib (COMBI) (n= 21) and patients treated with other combinations e.g. ruxolitinib, anagrelide, hydroxyurea combined with IFN hydroxyurea combined anagrelide, or hydroxyurea combined with ruxolitinib, (n=11). The alpha diversity was measured using the Shannon diversity index, and compared with a pairwise Wilcoxon test adjusted for multiple testing. Beta diversity (difference between the samples) was visualised by a PCoA plot, and compared using an ANOSIM test. Differential abundance analysis was performed by Linear discriminant analysis Effect Size (LEfSe). Results: In total, 116 patients with PV were included. Of these, 106 fulfilled the inclusion criteria (49 women and 57 men) and had a median age of 68 years (range: 31 - 85). The five treatment groups did not differ in alpha diversity. The bacterial composition differed slightly between IFN group and no treatment group (p=0.032, R=0.075), and between IFN and COMBI group (p= 0.031, R=0.054). Patients from the no treatment group had a higher relative abundance of the Bacteroides genus (39%) compared to the IFN group (14.7%) (p=0.003) and hydroxyurea group (23.1%) (p=0.047), but not the COMBI group (30.1%). A lower abundance of the Bacteroides genus was found in the IFN group compared to the COMBI group (p=0.004) and compared to the hydroxyurea group (p=0.04). Due to the small number of patients treated with other combinations, these were not included in the differential abundance analysis. Conclusions: Among the five treatment groups in patients with PV, the alpha diversity of the microbiota were similar, but the relative abundance of the Bacteroides genus in patients not receiving any treatment compared to patients treated with IFN and hydroxyurea was higher. A lower abundance of Bacteroides genus was seen in the IFN group compared to the COMBI group and compared to the hydroxyurea group. Whether these differences are explained as a treatment response to IFN or clinical parameters, such as comorbidities, age JAK2 allele burden or BMI need further investigations. Disclosures Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board.

Genetic variation within Toxoplasma gondii can have both clinical and epidemiological significance, while the genotypes circulating in many parts of the world, including the Nordic country Denmark, are still unknown. We genetically characterized T. gondii strains that had been detected in human clinical samples in Denmark in 2011-2016. Samples that had tested positive for T. gondii DNA and had a quantification cycle value

Despite their ubiquity, in most cases little is known about the impact of eukaryotic parasites on their mammalian hosts. Comparative approaches provide a powerful method to investigate the impact of parasites on host ecology and evolution, though two issues are critical for such efforts: controlling for variation in methods of identifying parasites and incorporating heterogeneity in sampling effort across host species. To address these issues, there is a need for standardized methods to catalogue eukaryotic parasite diversity across broad phylogenetic host ranges. We demonstrate the feasibility of a metabarcoding approach for describing parasite communities by analysing faecal samples from 11 nonhuman primate species representing divergent lineages of the primate phylogeny and the full range of sampling effort (i.e. from no parasites reported in the literature to the best‐studied primates). We detected a number of parasite families and regardless of prior sampling effort, metabarcoding of only ten faecal samples identified parasite families previously undescribed in each host (x̅ = 8.5 new families per species). We found more overlap between parasite families detected with metabarcoding and published literature when more research effort—measured as the number of publications—had been conducted on the host species' parasites. More closely related primates and those from the same continent had more similar parasite communities, highlighting the biological relevance of sampling even a small number of hosts. Collectively, results demonstrate that metabarcoding methods are sensitive and powerful enough to standardize studies of eukaryotic parasite communities across host species, providing essential new tools for macroecological studies of parasitism.

Anti-Leishmania antibodies may be detectable in patients with leishmaniasis. Here, we compared a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Leishmania antibodies, with an immunofluorescence antibody test (IFAT) that is no longer commercially available. Eighty-six serum samples from 73 patients were tested. The results obtained by the NovaLisa&trade, Leishmania infantum IgG ELISA, interpreted according to the instructions of the manufacturer, but with a modified cut-off for borderline positive values, were compared with the IFAT results that were already available. Moreover, Leishmania Western blot IgG results were available for 43 of the samples. The overall concordance of ELISA and IFAT was 67%. The ELISA and IFAT tests scored as 24% and 15% of the samples being positive, respectively, while 13% and 33% scored as borderline-positive, respectively. Using a Western blot (WB) as the reference, the sensitivities and specificities for the positive plus borderline-positive samples combined was 95.5% (95% confidence interval (CI), 77.2&ndash, 99.9%) and 81.0% (95% CI, 58.1&ndash, 94.6%) for ELISA, and 95.5% (95% CI, 77.2&ndash, 99.9%) and 42.9% (95% CI, 21.8&ndash, 66.0%) for IFAT, respectively. Overall, the ELISA proved to be a cost-effective alternative to the IFAT, due to its higher accuracy and specificity, and with a consequently lower number of confirmatory WB tests being required. Lastly, we also present data on the associations between seroconversion and the type of leishmaniasis.

Invasive mucormycosis in immunocompromised children is a life-threatening fungal infection. We report a case of a 7-year-old girl treated for acute lymphoblastic leukaemia complicated by disseminated mucormycosis during induction therapy. Microscopic examination of surgically removed lung tissue revealed wide, pauci-septate hyphae suggesting a Mucorales infection. This diagnosis was confirmed immunohistochemically and by PCR analysis followed by a final identification of Cunninghamella sp. The patient was treated successfully with surgical debridement and antifungal combination therapy with amphotericin B, caspofungin and isavuconazole. The use of isavuconazole in a child was not previously reported. Additionally, case reports concerning pulmonary mucormycoses in paediatric population published after 2010 were reviewed. Nineteen out of 26 identified patients suffered from haematological diseases. Reported mortality reached 38.5%. By the fact of rising morbidity, unsatisfactory results of treatment and remaining high mortality of mucormycoses in immunocompromised patients, new therapeutic options are warrant. Isavuconazole, with its broad-spectrum activity, good safety profile and favourable pharmacokinetics, is a promising drug. However, further studies are necessary to confirm positive impact of isavuconazole on mucormycosis treatment in children.

Introduction The world's malaria map is constantly changing and with it the risk for travelers to contract malaria. While some efforts to appreciate the malaria situation for indigenous populations in Indonesia have been made recently, there is only sparse data in the literature on the risk for travelers to Indonesia. Methods Data were collected from the Indonesian Ministry of Health (MoH), the World Health Organization (WHO), the Indonesian official statistics website Badan Pusat Statistik (BPS), and from the different European national surveillance bodies. Finally, a comparison between recent official guidelines for prevention of malaria in travelers from Germany, the United States, the UK, and from WHO was done. Results Data from Denmark, Germany, Sweden and Switzerland show a steady decline of imported cases of malaria from Indonesia from 1997 to 2013, with a leveling off during the last few years. In our study material, the Plasmodium falciparum incidence 2009 to 2013 was 0.35 cases per 100,000 visits and the Plasmodium vivax incidence 1.3 cases per 100,000 visits, with a 95% confidence interval of 0.1–0.9 and 0.7–2.2, respectively. Indonesian data also show a decline of malaria cases—the Annual Parasite Index (API) for all species of malaria has decreased from 4.68 cases per 1,000 inhabitants in 1990 to 1.38 cases per 1,000 inhabitants in 2013. Conclusion Based on these data updated recommendations for malaria prophylaxis in travelers to Indonesia are suggested.

The risk of contracting babesiosis after a tick bite in Sweden and on the Aland Islands, Finland, is unknown. We investigated clinical and serological outcomes in people bitten by Ixodes ricinus ticks positive for Babesia species. Ticks, blood and questionnaires were obtained from study participants in Sweden and on the Aland Islands. Sixty-five of 2098 (3.1 %) ticks were positive by real-time PCR. Three Babesia species were detected, Babesia microti (n = 33), B. venatorum (n = 27) and B. capreoli (n = 5), the latter species not known to cause human infection. Half (46 %) of the Babesia PCR-positive ticks also contained Borrelia spp. Fifty-three participants bitten by a Babesia PCR-positive tick and a control group bitten by a Babesia PCR-negative tick were tested for B. microti IgG antibodies by IFA. The overall seroprevalence was 4.4 %, but there was no significant difference between the groups. None of the participants seroconverted and no participant with a Babesia PCR-positive tick sought medical care or reported symptoms suggestive of babesiosis. Given the prevalence of Babesia in I. ricinus ticks in southern Sweden and on the Aland Islands, babesiosis should be considered a possible diagnosis in symptomatic residents who seek medical care following tick exposure.

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.

In 2008–2009 a tick-borne encephalitis virus (TBEV) micro-focus was detected in Northern Zealand, Denmark. No new cases of TBE with an epidemiological link to Northern Zealand has been reported since. Here we undertook to investigate Ixodes ricinus ticks from this endemic micro-focus in 2016 and 2017. In addition to TBEV, I. ricinus ticks may host other pathogens that include Borrelia spp., Babesia spp., Rickettsia spp. and Neoehrlichia mikurensis, together with various endosymbiont microorganisms. To detect multiple organisms we used a metagenomics PanVirus microarray and next-generation sequencing to examine the persistence and evolution of other emerging viruses, bacteria and parasites. Here we report the rise and fall of the Danish TBEV micro-focus in Northern Zealand. However, we identify for the first time in Danish I. ricinus ticks the presence of Uukuniemi virus in addition to a tick-borne phlebovirus and a range of bacteria.

Background Congenital toxoplasmosis (CT) causes a substantial disease burden worldwide. The aim of this study was to estimate the disease burden of CT in Denmark, a developed country with free public healthcare and nationwide data available. Methods Using data primarily from two public health surveillance programmes conducted between 1992 and 2007, we estimated the incidence, occurrence of sequelae, mortality and the burden of disease in terms of disability-adjusted life years (DALYs) of CT in Denmark in 2014. Findings We estimated that 14 children were born with CT in 2014, of which six will have developed sequelae by the age of 12. CT resulted in a total disease burden of 123 DALYs (95% uncertainty interval [UI], 100–148), of which 78 (95% UI, 64–94) were due to foetal loss and 2 (95% UI, 1–3) were due to neonatal death; the remaining burden was due to moderate to severe life-long sequelae. A comparison of the estimated incidence of CT with the number of reported CT cases in 2008–2014 indicated that for each reported CT case, at least five other CT cases could be expected to have occurred and gone unreported. Interpretation Early onset, severity, and life-long duration of sequelae have a major effect on the disease burden of CT. Our data suggest that CT is under-diagnosed or under-reported in Denmark. The estimated disease burden and public health impact in Denmark is lower than in other European countries, highlighting the need for country-specific studies.

Dientamoeba fragilis is an intestinal protozoan of debated clinical significance. Here, we present cross-sectional and longitudinal observations on D. fragilis in children aged 0 to 6 years from a 1-year multi-day-care-center cohort study set in Copenhagen, Denmark. The inclusion period for the cohort was 2009 through 2012. Stool samples collected from the children were accompanied by questionnaires completed by the parents or guardians of the children. Using real-time PCR, D. fragilis was detected in the first stool sample from 97 of 142 (68.3%) children. We evaluated the associations between seven plausible risk factors (age, sex, having siblings, having domestic animals at home, having had infant colic, recent history of intake of antibiotics, and recent history of travel abroad) as well as six reported symptoms (lack of appetite, nausea, vomiting, abdominal pain, weight loss, and diarrhea) and testing positive for D. fragilis . The final multivariable model identified being >3 years old and having a history of recent travel abroad as risk factors for testing positive for D. fragilis . Moreover, univariable analyses indicated that having siblings was a risk factor. There was no statistical association between a recent history of gastrointestinal symptoms and testing positive for D. fragilis . Among the 108 children who were represented by ≥2 samples and thus included in the longitudinal analysis, 32 tested negative on the first sample and positive later, and the last sample from each of the 108 children was positive. The results are in support of D. fragilis being a common enteric commensal in this population.

We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essential (within ±0, ±1, and ±2 twofold dilutions) and categorical agreements were best with the FIE (for R1/R2, 42%/41%, 75%/73%, and 90%/89% for essential agreement, and 91.1%/92.4% categorical agreement, with 6.3/8.9% very major errors and 0/1.3% major errors, respectively). The MIC Strip Isavuconazole test with the FIE appears to be useful.

Introduction: Retrospective molecular identification of Leishmania parasites in two patients with visceral leishmaniasis (VL) previously treated in Serbia was carried out. DNA was isolated from unstained bone marrow smears (BMSs) kept for 11 and 8 years. Genus-specific real-time PCR was combined with conventional PCR and sequencing for detection and species identification. Case presentation: In 2003, a 40-year-old Serbian male was admitted to the Clinical Centre of Serbia (CCS) with fever, sweating, fatigue and splenomegaly, which developed over a period of 7 weeks. He had frequently travelled around Europe. VL was confirmed by microscopy of Giemsa-stained BMS. Treatment by pentavalent antimonials was successfully completed. Two years later, the patient developed post-kala-azar dermal leishmaniasis. Treatment resulted in symptom resolution. Later on, Leishmania infantum was identified as the causative agent of the VL by sequencing of the ITS (internal transcribed spacer) region; mixed Leishmania spp. infection could not be excluded. In 2006, a 33-year-old female from Vojvodina, Serbia, with pre-existing diabetes mellitus and chronic meningoencephalitis and a history of frequent visits to the Montenegrin seacoast, was admitted to the CCS with fever, pancytopenia and moderate hepatosplenomegaly. A stained BMS revealed abundant Leishmania amastigotes. Indirect haemagglutination analysis was positive with a titre of 1 : 2048, and a rapid dipstick rK39 test was also positive. Treatment by liposomal amphotericin B was successful; however, shortly after, the patient developed neural infection and pneumonia and died. The causative agent was identified as L. infantum. Conclusion: Molecular diagnosis of VL and species delineation using DNA from unstained BMSs stored for several years is possible.