Protozoan infections are under-recognized in Swedish patients with gastrointestinal symptoms.

In acute gastroenteritis (GE), identification of the infectious agent is important for patient management and surveillance. The prevalence of GE caused by protozoa may be underestimated in Swedish patients. The purpose was to compare the prevalence of E. histolytica, Cryptosporidium spp., G. intestinalis, and C. cayetanensis in samples from patients where the clinician had requested testing for gastrointestinal parasites only (n = 758) to where testing for bacterial GE only (n = 803) or where both parasite and bacterial testing (n = 1259) was requested and a healthy control group (n = 197). This prospective cohort study was conducted in Region Jönköping County, Sweden (October 2018-March 2019). Fecal samples were analyzed with microscopy and real-time PCR. Cryptosporidium spp. was detected in 16 patients in the bacterial GE group and in 13 in the both bacterial and parasite group; no cases were detected in the group were only parasite infection was suspected. C. cayetanensis was detected in two patients in the bacterial GE group. One case of E. histolytica was detected in the bacterial group and one in the both bacterial and parasite group. G. intestinalis was detected in 14 patients in the parasite only group, 12 in the both parasite and bacterial group, three in the bacterial GE group, and one in the control group. Diarrhea caused by protozoa, especially Cryptosporidium was under-recognized by clinicians and is likely more common than hitherto estimated in Sweden. A more symptom-based diagnostic algorithm may increase detection and knowledge about protozoan infections.

Significant Differences in the Bacterial Microbiome of the Pharynx and Skin in Patients with Psoriasis Compared with Healthy Controls.

Studies have shown differences in the skin and gut bacterial microbiomes in patients with psoriasis, but the pharyngeal microbiome has not been studied previously. The aim of this study was to investigate differences in the bacterial microbiome of the pharynx and skin of patients with psoriasis compared with healthy controls. Swabs were taken from the pharynx and elbow skin of 39 patients with psoriasis and 70 controls. Microbiomes were characterized by sequencing 16S rRNA genes on the Illumina MiSeq platform. Significant differences were found in alpha and beta diversity in the skin, but not in the pharynx. Significant differences were also found between several phyla and genera in both skin and pharynx. The severity of psoriasis did not correlate with any genera in the pharynx, but with Capnocytophaga, Leptotrichia, Abiotrophia and Tannerella in the skin. The composition of the pharyn-geal and skin microbiome may be of importance in the patho-genesis of psoriasis.

Significant Changes in the Skin Microbiome in Patients with Chronic Plaque Psoriasis after Treatment with Narrowband Ultraviolet B.

Changes in the skin microbiome have been shown to promote cutaneous inflammation. The skin microbiome of patients with chronic plaque type psoriasis was analysed before and after treatment with narrowband ultraviolet B (UVB). Swab samples of the microbiome were taken from lesional and non-lesional skin of 26 patients. Microbiotas were characterized by sequencing 16S rRNA bacterial genes on the Illumina MiSeq platform. Lesional skin microbiome diversity correlated with psoriasis severity (measured with the Psoriasis Area and Severity Index; PASI). There was a significantly lower abundance of the phylum Firmicutes and the genus Staphylococcus in lesional skin compared with non-lesional skin before UVB treatment. Responders (> 75% target Psoriasis Severity Index (PSI) improvement) had significantly lower abundance of the phyla Firmicutes in lesional and non-lesional skin and lower abundance of the genera Staphylococcus, Finegoldia, Anaerococcus, Peptoniphilus, Gardnerella, Prevotella and Clostridium in lesional skin after UVB treatment. Pseudomonas significantly decreased in lesional and non-lesional skin of treatment responders. These results suggest that skin microbiome alterations after UVB treatment could be related to treatment and treatment response.

Expression of low-density lipoprotein-related receptors 5 and 6 (LRP5/6) in psoriasis skin.

Low-density lipoprotein-related receptors 5 and 6 (LRP5/6) are transmembrane receptors with key functions in canonical Wnt signalling. Wnt ligands are thought to play an important role in innate immunity and psoriasis, and recent studies assigned LRP5/6 anti-inflammatory properties. The objective of this study was to investigate the expression of LRP5 and LRP6 in lesional and non-lesional skin in peripheral blood and in mononuclear cells of patients with chronic plaque type psoriasis compared with control individuals. To investigate the effect of UV-B radiation, LRP5/6 skin gene expression was analysed before and after narrowband UV-B treatment. Our results showed significantly decreased gene expression of LRP5 and LRP6 in lesional skin and in peripheral blood from patients with psoriasis compared with non-lesional skin and healthy control skin. Immunohistochemistry did not reveal differences in protein expression of LRP5/6. Narrowband UV-B treatment induced a significant increase in LRP5 and LRP6 gene expression in lesional skin. Decreased gene expression of LRP5/6 in lesional skin and upregulation after nb UV-B treatment suggest a possible role for LRP5/6 in psoriasis.

Norovirus Dynamics in Wastewater Discharges and in the Recipient Drinking Water Source: Long-Term Monitoring and Hydrodynamic Modeling.

Norovirus (NoV) that enters drinking water sources with wastewater discharges is a common cause of waterborne outbreaks. The impact of wastewater treatment plants (WWTPs) on the river Göta älv (Sweden) was studied using monitoring and hydrodynamic modeling. The concentrations of NoV genogroups (GG) I and II in samples collected at WWTPs and drinking water intakes (source water) during one year were quantified using duplex real-time reverse-transcription polymerase chain reaction. The mean (standard deviation) NoV GGI and GGII genome concentrations were 6.2 (1.4) and 6.8 (1.8) in incoming wastewater and 5.3 (1.4) and 5.9 (1.4) log10 genome equivalents (g.e.) L-1 in treated wastewater, respectively. The reduction at the WWTPs varied between 0.4 and 1.1 log10 units. In source water, the concentration ranged from below the detection limit to 3.8 log10 g.e. L-1. NoV GGII was detected in both wastewater and source water more frequently during the cold than the warm period of the year. The spread of NoV in the river was simulated using a three-dimensional hydrodynamic model. The modeling results indicated that the NoV GGI and GGII genome concentrations in source water may occasionally be up to 2.8 and 1.9 log10 units higher, respectively, than the concentrations measured during the monitoring project.

Increased expression of the Wnt signalling inhibitor Dkk-1 in non-lesional skin and peripheral blood mononuclear cells of patients with plaque psoriasis.

Psoriasis is a common inflammatory skin disease characterised by abnormal keratinocyte proliferation, increased dermal angiogenesis and systemic inflammation. The cell signalling cascades provoked by Wnt proteins and their inhibitors, such as Dickkopf-1, play crucial roles to maintain homeostasis of a variety of tissues, including skin, and are also involved in angiogenesis and innate immunity. This study was designed to investigate the distribution of Dickkopf-1, in lesional and non-lesional skin, in serum and in peripheral blood mononuclear cell (PBMCs) of patients with psoriasis compared with healthy controls. Our results showed significantly increased mRNA and protein expression of Dickkopf-1 in non-lesional compared with lesional skin and healthy control skin. No significant differences of Dickkopf-1 serum levels were observed, but Dickkopf-1 protein expression was significantly increased in patients' PBMC. Increased levels of Dickkopf-1 in PBMC, suggest a possible role of Dickkopf-1 in the chronic systemic inflammation of psoriasis. Increased levels of Dickkopf-1 in non-lesional psoriasis skin offers new insights in the local inflammatory processes in psoriasis skin since Wnt signalling regulates angiogenesis. In conclusion, Dickkopf-1 may be a possible target for future treatment options.

Analyzing multiclonality of Staphylococcus aureus in clinical diagnostics using spa-based denaturing gradient gel electrophoresis.

We present a novel denaturing gradient gel electrophoresis (DGGE) method which characterizes multiclonal communities of Staphylococcus aureus. The spa PCR-based DGGE method simultaneously separates strains that differ in only one base, thereby revealing multiclonal colonization and infections.

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden.

Background: Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or Cryptosporidium spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of Giardia intestinalis, Dientamoeba fragilis, Cryptosporidium spp., Entamoeba histolytica and Entamoeba dispar from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results.Methods: SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region Jönköping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl-Neelsen staining on patients with certain symptoms and real-time PCR.Results: We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR.Conclusion: In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and Cryptosporidium spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.

Novel light-upon-extension real-time PCR assays for detection and quantification of genogroup I and II noroviruses in clinical specimens.

Norovirus is now recognized as the leading cause of nonbacterial acute gastroenteritis in adults, causing numerous outbreaks worldwide. We have developed two novel light-upon-extension (LUX) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assays were evaluated against clinical stool specimens (n = 103) from Sweden and Nicaragua and compared to established methods. The norovirus assay detected more positive stool specimens (47/103) than conventional PCR (39/103) and corresponded to a TaqMan real-time PCR, with the exception of one specimen. Furthermore, the assays correctly identified all (n = 11) coded control specimens in a reference panel containing various genogroups and genotypes. Both LUX real-time PCR assays had a wide dynamic range, detecting from < or = 10(1) to 10(7) genes per reaction, resulting in a theoretical lower limit of < or = approximately 20,000 viruses per gram of stool. No cross-reactivity was noticed with specimens containing other enteric viruses, and by using melting curve analysis we could differentiate between norovirus genogroups I and II.

Expression and gene polymorphisms of the chemokine CXCL5 in colorectal cancer patients.

Several studies indicate that chemokines play important roles in colorectal mucosal immunity by recruiting leukocytes into and out of the lamina propria adjacent to the epithelium. The chemokine CXCL5 which is expressed by epithelial cells within colorectal mucosa is a chemoattractant for neutrophils and has been implicated in Crohn's disease and ulcerative colitis. In addition, CXCL5 is one chemokine which promote angiogenesis related to cancer. The objective of this study was to determine by ELISA assay whether CXCL5 protein level is altered in colorectal cancer (CRC) tissues (n=80) compared with paired normal mucosa. Furthermore, the plasma CXCL5 levels from CRC patients (n=62) compared with controls (n=71) were also examined. Using a TaqMan system we screened for -156G--> C and +398G-->A CXCL5 gene variants in CRC patients (n=228) and a control group (n=231) to assess the role of CXCL5 genotype in CRC. The analyses showed that CXCL5 protein level in colorectal tumours was significantly (P<0.0001) higher than in normal tissue and was lower in plasma in CRC patients compared with controls (P=0.026). Immunohistochemistry revealed CXCL5 immunoreactivity mainly in epithelial cells of the colorectal carcinoma and in normal epithelial cells. Furthermore, patients who were -156C carriers had higher CXCL5 protein concentration compared with -156G carriers in normal tissue (P=0.027) and CXCL5 protein levels in cancerous tissue tended to be higher for the patient -156C carriers (P=0.059). To our knowledge this is the first report on the influence of CXCL5 gene variants and their relation to expression of CXCL5 protein in human CRC.

Analysis of single nucleotide polymorphism in the promoter and protein expression of the chemokine eotaxin-1 in colorectal cancer patients.

BACKGROUND: Previous studies suggest that chemokines (chemotactic cytokines) promote and regulate neoplastic progression including metastasis and angiogenesis. The chemokine eotaxin-1 is a powerful eosinophil attractant but also exerts chemotaxis of other leukocytes. Eotaxin-1 has been implicated in gastrointestinal disorders and may play an important role in colorectal mucosal immunity. PATIENTS AND METHODS: The objective of this study was to assess the role of eotaxin-1 in colorectal cancer (CRC). Levels of eotaxin-1 protein in CRC tissues (n = 86) and paired normal mucosa were compared after determination by ELISA. Plasma eotaxin-1 levels from CRC patients (n = 67) were also compared with controls (n = 103) using the same method. Moreover, a TaqMan system was used to evaluate the -384A>G eotaxin-1 gene variant in CRC patients (n = 241) and in a control group (n = 253). RESULTS: Eotaxin-1 protein levels in colorectal tumours were significantly (P < 0.0001) higher than in normal tissue. Immunohistochemistry revealed eotaxin-1 expression in stromal cells such as fibroblasts and leukocytes of the CRC tissue. The plasma eotaxin-1 level in CRC patients was lower compared with controls (P < 0.0001). Patients with tumours classified as Dukes' stage B and C had lower levels than patients with tumours in Dukes' stage A. We found no difference in genotype distribution but noted a difference regarding allele distribution (P = 0.036) and a dominance of allele G in rectal cancer patients. CONCLUSION: The up-regulated eotaxin-1 protein expression in cancer tissue may reflect an eotaxin-1 mediated angiogenesis and/or a recruitment of leukocytes with potential antitumourigenic role. We noticed a dominance of the G allele in rectal cancer patients compared with colon cancer patients that was independent of eotaxin-1 expression.

Occurrence and removal efficiency of parasitic protozoa in Swedish wastewater treatment plants.

Giardia intestinalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis are parasitic protozoa and causative agents of gastroenteritis in humans. G. intestinalis and Cryptosporidium spp. in particular are the most common protozoa associated with waterborne outbreaks in high-income countries. Surveillance of protozoan prevalence in wastewater and evaluation of wastewater treatment removal efficiencies of protozoan pathogens is therefore imperative for assessment of human health risk. In this study, influent and effluent wastewater samples from three wastewater treatment plants in Sweden were collected over nearly one year and assessed for prevalence of parasitic protozoa. Quantitative real-time PCR using primers specific for the selected protozoa Cryptosporidium spp., G. intestinalis, E. histolytica, Entamoeba dispar and D. fragilis was used for protozoan DNA detection and assessment of wastewater treatment removal efficiencies. Occurrence of G. intestinalis, E. dispar and D. fragilis DNA was assessed in both influent (44, 30 and 39 out of 51 samples respectively) and effluent wastewater (14, 9 and 33 out of 51 samples respectively) in all three wastewater treatment plants. Mean removal efficiencies of G. intestinalis, E. dispar and D. fragilis DNA quantities, based on all three wastewater treatment plants studied varied between 67 and 87%, 37-75% and 20-34% respectively. Neither E. histolytica nor Cryptosporidium spp. were detected in any samples. Overall, higher quantities of protozoan DNA were observed from February to June 2012. The high prevalence of protozoa in influent wastewater indicates the need for continued monitoring of these pathogens in wastewater-associated aquatic environments to minimise the potential risk for human infection.

Molecular characterization and genetic susceptibility of sapovirus in children with diarrhea in Burkina Faso.

Sapoviruses (SaVs) are a common cause of gastroenteritis in children. In sub-Saharan Africa, there is a scarcity of information regarding SaV as an etiological agent of diarrhea. Here, we investigated the prevalence, molecular characterization and clinico-epidemiological features of SaV infections in children less than 5years of age with diarrhea in Burkina Faso. We further investigated the role of type 1 histo blood group antigens as susceptibility factors. In total, 309 fecal and 208 saliva samples from diarrheal children in Ouagadougou, Burkina Faso, were collected between May 2009 and March 2010. SaV was detected using real-time PCR, and genogrouped/genotyped by PCR or sequencing. Saliva samples were ABO, Lewis and secretor phenotyped using in house ELISA assays. We found a high prevalence (18%) and large genetic diversity with all 4 human genogroups, and 9 genotypes/genoclusters circulating during the study period. The SaV infections were generally associated with milder symptoms, and neither ABH, Lewis or secretor phenotypes affected susceptibility to SaV infections.

Microbial risk assessment of drinking water based on hydrodynamic modelling of pathogen concentrations in source water.

Norovirus contamination of drinking water sources is an important cause of waterborne disease outbreaks. Knowledge on pathogen concentrations in source water is needed to assess the ability of a drinking water treatment plant (DWTP) to provide safe drinking water. However, pathogen enumeration in source water samples is often not sufficient to describe the source water quality. In this study, the norovirus concentrations were characterised at the contamination source, i.e. in sewage discharges. Then, the transport of norovirus within the water source (the river Göta älv in Sweden) under different loading conditions was simulated using a hydrodynamic model. Based on the estimated concentrations in source water, the required reduction of norovirus at the DWTP was calculated using quantitative microbial risk assessment (QMRA). The required reduction was compared with the estimated treatment performance at the DWTP. The average estimated concentration in source water varied between 4.8×10(2) and 7.5×10(3) genome equivalents L(-1); and the average required reduction by treatment was between 7.6 and 8.8 Log10. The treatment performance at the DWTP was estimated to be adequate to deal with all tested loading conditions, but was heavily dependent on chlorine disinfection, with the risk of poor reduction by conventional treatment and slow sand filtration. To our knowledge, this is the first article to employ discharge-based QMRA, combined with hydrodynamic modelling, in the context of drinking water.

Ixodes ricinus and Borrelia prevalence at the Arctic Circle in Norway.

The distribution limit of Ixodes ricinus ticks in northwestern Europe (Brønnøy, Norway, 1° south of the Arctic Circle), has been known since the 1930s. To reconfirm this finding and extend studies in the areas adjacent to the Arctic Circle (66°33' N), ticks were collected from dogs and cats in 8 districts in northern Norway from 64°56' N to 68°48' N. We detected 549 I. ricinus, 244 (44%) of them in Brønnøy district, and 305 (range 6-87 ticks) in 7 districts in the northern part of the study area. The prevalence of Borrelia in these ticks was determined by real-time PCR. In the Brønnøy district (65°28' N, 12°12' E), 29% of the I. ricinus were Borrelia spp.-positive, and the species B. afzelii was nearly twice as prevalent as B. garinii and/or B. valaisiana. In the study area north of Brønnøy district, only 12 (4%) of the collected ticks contained Borrelia spp. In conclusion, tick occurrence and Borrelia prevalence are high in the Brønnøy district. In contrast, I. ricinus occurrence and Borrelia prevalence are low further north across the Arctic Circle in Norway.

Dientamoeba fragilis DNA detection in Enterobius vermicularis eggs.

Dientamoeba fragilis is an intestinal protozoan suspected of causing gastrointestinal symptoms, and its mode of transmission is unknown, although first described almost a century ago. A hypothesis is that Enterobius vermicularis is a vector for D. fragilis, and recently, D. fragilis DNA was detected within surface-sterilized eggs of E. vermicularis. Using real-time PCR, we detected D. fragilis DNA in 18 (85%) of 21 samples of E. vermicularis eggs collected from patients harbouring D. fragilis in faeces. This finding supports the hypothesis that E. vermicularis may have an important role in the transmission of D. fragilis.

Real-time multiplex PCR for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture.

A real-time multiplex PCR using the orfX and staphylococcal cassette chromosome (SCC) mec of Staphylococcus aureus was developed. The aim was to achieve a rapid and sensitive high-throughput method for direct detection of heterogeneous methicillin-resistant S. aureus (MRSA) in clinical samples, present in a low-endemic population, such as in Sweden. Consecutive broth enriched pooled clinical screening samples (nares, throat and/or perineum/groin) (n = 541 pools), broth enriched clinical samples showing growth of methicillin-sensitive S. aureus (MSSA) (n = 95 pools), clinical MRSA isolates (n = 173), MRSA reference strains (n = 43) and various coagulase-negative staphylococcal isolates (n = 33) were analyzed. The multiplex PCR detected all heterogeneous MRSA strains (n = 173) obtained in our area as well as all pooled consecutive broth enriched clinical samples with MRSA, i. e. 36 of 541 pools. None of the CoNS were positive. However, 18 out of 541 pools (3.3%) were positive in the multiplex PCR but no growth of MRSA could be detected by subculture and were regarded as false positive. Furthermore, the assay is rapid and reliable negative results can be delivered to the clinician within 18 h that will facilitate the infection control management of patients and hospital staff.

Quantification of the chemokines CCL17 and CCL22 in human colorectal adenocarcinomas.

Chemokines are believed to play a crucial role in local immunoresponse by regulating leukocyte movement in various tissues, including the intestinal mucosa. It has been suggested that they are key players in cancer biology, and several studies have identified leukocyte infiltration as a hallmark of most cancers. The chemokines CCL17 and CCL22 attract CCR4-bearing cells, which are especially polarised to Th2-type cells and regulatory T cells (Treg). Recent studies have revealed the participation of the CCL17 and CCL22 proteins in diseases such as atopic dermatitis and lymphoma. The purpose of this study was to assess the role of CCL17 and CCL22 protein expression in colorectal cancer (CRC) and to ascertain whether an association exists between promoter -431C>T CCL17 and -961G>A CCL22 gene polymorphisms in CRC versus non-CRC subjects. Using the ELISA assay, we noted a significantly higher expression of CCL22 in tumour tissue with a 2.3-fold up-regulation (tumour vs. paired normal tissue, n=78) but no significant difference in CCL17 protein expression. Immunohistochemistry revealed protein expression of CCL22 and CCL17 in the epithelial compartment of cancer tissue, in epithelial cells at the resection border that reflects normal tissue, and in some stromal cells such as lymphocytes, macrophages, and fibroblasts. Using a TaqMan system we screened for -431C>T CCL17 and -961G>A CCL22 gene variants in 245 CRC patients and 256 controls, but could not find any significant difference in genotype distribution or in allelic frequencies between the two groups. The genotype and allelic distributions of CRC patients were not related to tissue levels of CCL17 and CCL22 protein, and none of the variables were associated with plasma levels or clinical characteristics. To ascertain whether the tissue expression of CCL17 and CCL22 exerts an influence on the pathogenesis of CRC, a forthcoming study on the 5-year survival rate of CRC patients will be conducted.

Polymorphisms of Fractalkine receptor CX3CR1 and plasma levels of its ligand CX3CL1 in colorectal cancer patients.

BACKGROUND AND AIMS: The chemokine Fractalkine/CX3CL1, which is expressed by epithelial cells within normal colorectal mucosa and in colorectal cancer (CRC), is thought to have a crucial role in colorectal mucosal immunity by recruiting leucocytes via the receptor CX3CR1. The purpose of this study was to investigate two single-nucleotide polymorphisms of the Fractalkine receptor/CX3CR1 gene, V249I and T280M, in CRC to find out whether they occur more often in patients with CRC than in non-CRC individuals. In the search for tumour markers, we also intended to determine whether plasma levels of Fractalkine were correlated with parameters such as Dukes' stage, tumour localisation, gender and age in CRC patients. MATERIALS AND METHODS: Genomic deoxyribonucleic acid from 223 CRC patients and 229 controls was amplified by polymerase chain reaction, and the polymorphisms were detected by the restriction fragment length polymorphism analysis. Fractalkine/CX3CL1 was analysed in plasma from 62 CRC patients and 78 controls using enzyme-linked immunosorbent assay. RESULTS: The variant V249I was significantly different in genotype and allelic distribution between CRC patients and control subjects, P = 0.028 and P = 0.048, respectively. We also found that individuals with the I249 allele in homozygote state were less frequent in the CRC group (3.1%) compared with controls (9.2%; P = 0.008). No significant difference was observed regarding Fractalkine/CX3CL1 levels in plasma between patients and the control group. CONCLUSION: Our results suggest that the lack of the allele I249 of the CX3CR1 gene may play a partial or minor role in CRC and that plasma Fractalkine/CX3CL1 does not seem to be a useful tumour marker that reflects the disease outcome of CRC.