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Background Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. Methods We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. Results MPN patients had a higher observed richness (median, 245 [range, 49–659]) compared with HCs (191.5 [range, 111–300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. Conclusion The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more., Background: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. Methods: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. Results: MPN patients had a higher observed richness (median, 245 [range, 49–659]) compared with HCs (191.5 [range, 111–300; p =.003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p <.001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. Conclusion: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Background Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. Methods We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. Results MPN patients had a higher observed richness (median, 245 [range, 49–659]) compared with HCs (191.5 [range, 111–300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. Conclusion The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more., Background: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. Methods: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. Results: MPN patients had a higher observed richness (median, 245 [range, 49–659]) compared with HCs (191.5 [range, 111–300; p =.003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p <.001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. Conclusion: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Background: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. Methods: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation.Results: MPN patients had a higher observed richness (median, 245 [range, 49–659]) compared with HCs (191.5 [range, 111–300; p =.003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p <.001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. Conclusion: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3–V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75–535) compared with HCs (median-observed richness, 191.5; range, 111–300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut micr

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3–V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75–535) compared with HCs (median-observed richness, 191.5; range, 111–300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated., Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3-V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75-535) compared with HCs (median-observed richness, 191.5; range, 111-300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut micr

Nasopharyngeal swabs (NPS) are considered the gold standard for SARS-CoV-2 testing but are technically challenging to perform and associated with discomfort. Alternative specimens for viral testing, such as oropharyngeal swabs (OPS) and nasal swabs, may be preferable, but strong evidence regarding their diagnostic sensitivity for SARS-CoV-2 testing is still missing. We conducted a head-to-head prospective study to compare the sensitivity of NPS, OPS and nasal swabs specimens for SARS-CoV-2 molecular testing. Adults with an initial positive SARS-CoV-2 test were invited to participate. All participants had OPS, NPS and nasal swab performed by an otorhinolaryngologist. We included 51 confirmed SARS-CoV-2-positive participants in the study. The sensitivity was highest for OPS at 94.1% (95% CI, 87 to 100%) compared to NPS at 92.5% (95% CI, 85 to 99%) (p = 1.00) and lowest for nasal swabs at 82.4% (95% CI, 72 to 93%) (p = 0.07). Combined OPS/NPS was detected in 100% of cases, while the combined OPS/nasal swab increased the sensitivity significantly to 96.1% (95% CI, 90 to 100%) compared to that of the nasal swab alone (p = 0.03). The mean Ct value for NPS was 24.98 compared to 26.63 for OPS (p = 0.084) and 30.60 for nasal swab (p = 0.002). OPS achieved a sensitivity comparable to NPS and should be considered an equivalent alternative for SARS-CoV-2 testing.

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3–V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75–535) compared with HCs (median-observed richness, 191.5; range, 111–300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated., Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3-V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75-535) compared with HCs (median-observed richness, 191.5; range, 111-300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut micr

Nasopharyngeal swabs (NPS) are considered the gold standard for SARS-CoV-2 testing but are technically challenging to perform and associated with discomfort. Alternative specimens for viral testing, such as oropharyngeal swabs (OPS) and nasal swabs, may be preferable, but strong evidence regarding their diagnostic sensitivity for SARS-CoV-2 testing is still missing. We conducted a head-to-head prospective study to compare the sensitivity of NPS, OPS and nasal swabs specimens for SARS-CoV-2 molecular testing. Adults with an initial positive SARS-CoV-2 test were invited to participate. All participants had OPS, NPS and nasal swab performed by an otorhinolaryngologist. We included 51 confirmed SARS-CoV-2-positive participants in the study. The sensitivity was highest for OPS at 94.1% (95% CI, 87 to 100%) compared to NPS at 92.5% (95% CI, 85 to 99%) (p = 1.00) and lowest for nasal swabs at 82.4% (95% CI, 72 to 93%) (p = 0.07). Combined OPS/NPS was detected in 100% of cases, while the combined OPS/nasal swab increased the sensitivity significantly to 96.1% (95% CI, 90 to 100%) compared to that of the nasal swab alone (p = 0.03). The mean Ct value for NPS was 24.98 compared to 26.63 for OPS (p = 0.084) and 30.60 for nasal swab (p = 0.002). OPS achieved a sensitivity comparable to NPS and should be considered an equivalent alternative for SARS-CoV-2 testing.

Background Olfactory and gustatory dysfunctions are early symptoms of SARS-CoV-2 infection. Between 20–80% of infected individuals report subjective altered sense of smell and/or taste during infection. Up to 2/3 of previously infected experience persistent olfactory and/or gustatory dysfunction after 6 months. The aim of this study was to examine subjective and psychophysical olfactory and gustatory function in non-hospitalized individuals with acute COVID-19 up to 6 months after infection. Methods Individuals aged 18-80-years with a positive SARS-CoV-2 PCR test no older than 10 days, were eligible. Only individuals able to visit the outpatient examination facilities were included. Gustatory function was tested with the Burgharts Taste Strips and olfactory function was examined with the Brief Smell Identifications test (Danish version). Subjective symptoms were examined through an online questionnaire at inclusion, day 30, 90 and 180 after inclusion. Results Fifty-eight SARS-CoV-2 positive and 56 negative controls were included. 58.6% (34/58) of SARS-CoV-2 positive individuals vs. 8.9% (5/56) of negative controls reported subjective olfactory dysfunction at inclusion. For gustatory dysfunction, 46.5% (27/58) of positive individuals reported impairment compared to 8.9% (5/56) of negative controls. In psychophysical tests, 75.9% (46/58) had olfactory dysfunction and 43.1% (25/58) had gustatory dysfunction among the SARS-CoV-2 positive individuals at inclusion. Compared to negative controls, SARS-CoV-2 infected had significantly reduced olfaction and gustation. Previously infected individuals continued to report lower subjective sense of smell 30 days after inclusion, whereafter the difference between the groups diminished. However, after 180 days, 20.7% (12/58) positive individuals still reported reduced sense of smell and taste. Conclusion Olfactory and gustatory dysfunctions are prevalent symptoms of SARS-CoV-2 infection, but there is inconsistency between subje

Background Olfactory and gustatory dysfunctions are early symptoms of SARS-CoV-2 infection. Between 20–80% of infected individuals report subjective altered sense of smell and/or taste during infection. Up to 2/3 of previously infected experience persistent olfactory and/or gustatory dysfunction after 6 months. The aim of this study was to examine subjective and psychophysical olfactory and gustatory function in non-hospitalized individuals with acute COVID-19 up to 6 months after infection. Methods Individuals aged 18-80-years with a positive SARS-CoV-2 PCR test no older than 10 days, were eligible. Only individuals able to visit the outpatient examination facilities were included. Gustatory function was tested with the Burgharts Taste Strips and olfactory function was examined with the Brief Smell Identifications test (Danish version). Subjective symptoms were examined through an online questionnaire at inclusion, day 30, 90 and 180 after inclusion. Results Fifty-eight SARS-CoV-2 positive and 56 negative controls were included. 58.6% (34/58) of SARS-CoV-2 positive individuals vs. 8.9% (5/56) of negative controls reported subjective olfactory dysfunction at inclusion. For gustatory dysfunction, 46.5% (27/58) of positive individuals reported impairment compared to 8.9% (5/56) of negative controls. In psychophysical tests, 75.9% (46/58) had olfactory dysfunction and 43.1% (25/58) had gustatory dysfunction among the SARS-CoV-2 positive individuals at inclusion. Compared to negative controls, SARS-CoV-2 infected had significantly reduced olfaction and gustation. Previously infected individuals continued to report lower subjective sense of smell 30 days after inclusion, whereafter the difference between the groups diminished. However, after 180 days, 20.7% (12/58) positive individuals still reported reduced sense of smell and taste. Conclusion Olfactory and gustatory dysfunctions are prevalent symptoms of SARS-CoV-2 infection, but there is inconsistency between subje

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for im

A correct identification of Streptococcus pseudopneumoniae is a prerequisite for investigating the clinical impact of the bacterium. The identification has traditionally relied on phenotypic methods. However, these phenotypic traits have been shown to be unreliable, with some S. pseudopneumoniae strains giving conflicting results. Therefore, sequence-based identification methods have increasingly been used for identification of S. pseudopneumoniae. In this study, we used 64 S. pseudopneumoniae strains, 59 S. pneumoniae strains, 22 S. mitis strains, 24 S. oralis strains, 6 S. infantis strains, and 1 S. peroris strain to test the capability of three single genes (rpoB, gyrB, and recA), two multilocus sequence analysis (MLSA) schemes, the single nucleotide polymorphism (SNP)-based phylogeny tool CSI phylogeny, a k-mer-based identification method (KmerFinder), average nucleotide identity (ANI) using fastANI, and core genome analysis to identify S. pseudopneumoniae. Core genome analysis and CSI phylogeny were able to cluster all strains into distinct clusters related to their respective species. It was not possible to identify all S. pseudopneumoniae strains correctly using only one of the single genes. The MLSA schemes were unable to identify some of the S. pseudopneumoniae strains, which could be misidentified. KmerFinder identified all S. pseudopneumoniae strains but misidentified one S. mitis strain as S. pseudopneumoniae, and fastANI differentiated between S. pseudopneumoniae and S. pneumoniae using an ANI cutoff of 96%.

OBJECTIVE To determine whether positive expiratory pressure (PEP) by PEP flute self-care is effective in reducing respiratory symptoms among community dwelling adults with SARS-CoV-2 infection and early stage covid-19. DESIGN Non-drug, open label, randomised controlled trial. SETTING Capital Region and Region Zealand in Denmark from 6 October 2020 to 26 February 2021. PARTICIPANTS Community dwelling adults, able to perform self-care, with a new SARS-CoV-2 infection (verified by reverse transcription polymerase chain reaction tests) and symptoms of covid-19. INTERVENTION Participants were randomised to use PEP flute self-care in addition to usual care or have usual care only. Randomisation was based on permuted random blocks in a 1:1 ratio, stratified for sex and age (<60 or ≥60 years). The PEP self-care group was instructed to use a PEP flute three times per day during the 30 day intervention. MAIN OUTCOME MEASURES Primary outcome was a change in symptom severity from baseline to day 30, as assessed by the self-reported COPD (chronic obstructive pulmonary disease) assessment test (CAT), which was adjusted for baseline values and stratification factors. Participants completed the CAT test questionnaire every day online. Secondary outcomes were self-reported urgent care visits due to covid-19, number of covid-19 related symptoms, and change in self-rated health, all within 30-days’ follow-up. RESULTS 378 participants were assigned to the PEP flute self-care intervention (n=190) or usual care only (n=188). In the PEP self-care group, the median number of days with PEP flute use was 21 days (interquartile range 13-25). For the intention-to-treat population, a group difference was observed in changes from baseline in CAT scores of −1.2 points (95% confidence interval −2.1 to −0.2; P=0.017) in favour of the PEP flute self-care group. At day 30, the PEP flute self-care group also reported less chest tightness, less dyspnoea, more vigour, and higher level of daily act

This study evaluated the ability of the MALDI-ToF MS from Bruker Daltonics to identify clinical Mitis-Group-Streptococcus isolates with a focus on Streptococcus pseudopneumoniae. The results were analyzed using the standard log(score) and the previously published list(score). Importantly, using the log(score) no misidentifications occurred and 27 of 29 (93%) S. pneumoniae and 27 of 30 (90%) S. oralis strains were identified, but only 1 of 31 (3%) S. pseudopneumoniae and 1 of 13 (8%) S. mitis strains were identified. However, our results show that 30 of 31 S. pseudopneumoniae strains had a S. pseudopneumoniae Main Spectral Profiles within the 3 best matches. Using the list(score) all S. oralis and S. pneumoniae strains were identified correctly, but list(score) misidentified 10 S. pseudopneumoniae and 5 S. mitis. We propose to use the log(score) for identification of S. pneumoniae, S. pseudopneumoniae, S. mitis and S. oralis, but for some strains additional testing may be needed.

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.

A correct identification of Streptococcus pseudopneumoniae is a prerequisite for investigating the clinical impact of the bacterium. The identification has traditionally relied on phenotypic methods. However, these phenotypic traits have been shown to be unreliable, with some S. pseudopneumoniae strains giving conflicting results. Therefore, sequence-based identification methods have increasingly been used for identification of S. pseudopneumoniae. In this study, we used 64 S. pseudopneumoniae strains, 59 S. pneumoniae strains, 22 S. mitis strains, 24 S. oralis strains, 6 S. infantis strains, and 1 S. peroris strain to test the capability of three single genes (rpoB, gyrB, and recA), two multilocus sequence analysis (MLSA) schemes, the single nucleotide polymorphism (SNP)-based phylogeny tool CSI phylogeny, a k-mer-based identification method (KmerFinder), average nucleotide identity (ANI) using fastANI, and core genome analysis to identify S. pseudopneumoniae. Core genome analysis and CSI phylogeny were able to cluster all strains into distinct clusters related to their respective species. It was not possible to identify all S. pseudopneumoniae strains correctly using only one of the single genes. The MLSA schemes were unable to identify some of the S. pseudopneumoniae strains, which could be misidentified. KmerFinder identified all S. pseudopneumoniae strains but misidentified one S. mitis strain as S. pseudopneumoniae, and fastANI differentiated between S. pseudopneumoniae and S. pneumoniae using an ANI cutoff of 96%.

OBJECTIVE To determine whether positive expiratory pressure (PEP) by PEP flute self-care is effective in reducing respiratory symptoms among community dwelling adults with SARS-CoV-2 infection and early stage covid-19. DESIGN Non-drug, open label, randomised controlled trial. SETTING Capital Region and Region Zealand in Denmark from 6 October 2020 to 26 February 2021. PARTICIPANTS Community dwelling adults, able to perform self-care, with a new SARS-CoV-2 infection (verified by reverse transcription polymerase chain reaction tests) and symptoms of covid-19. INTERVENTION Participants were randomised to use PEP flute self-care in addition to usual care or have usual care only. Randomisation was based on permuted random blocks in a 1:1 ratio, stratified for sex and age (<60 or ≥60 years). The PEP self-care group was instructed to use a PEP flute three times per day during the 30 day intervention. MAIN OUTCOME MEASURES Primary outcome was a change in symptom severity from baseline to day 30, as assessed by the self-reported COPD (chronic obstructive pulmonary disease) assessment test (CAT), which was adjusted for baseline values and stratification factors. Participants completed the CAT test questionnaire every day online. Secondary outcomes were self-reported urgent care visits due to covid-19, number of covid-19 related symptoms, and change in self-rated health, all within 30-days’ follow-up. RESULTS 378 participants were assigned to the PEP flute self-care intervention (n=190) or usual care only (n=188). In the PEP self-care group, the median number of days with PEP flute use was 21 days (interquartile range 13-25). For the intention-to-treat population, a group difference was observed in changes from baseline in CAT scores of −1.2 points (95% confidence interval −2.1 to −0.2; P=0.017) in favour of the PEP flute self-care group. At day 30, the PEP flute self-care group also reported less chest tightness, less dyspnoea, more vigour, and higher level of daily act

Streptococcus gordonii and Streptococcus sanguinis belong to the Mitis group streptococci, which mostly are commensals in the human oral cavity. Though they are oral commensals, they can escape their niche and cause infective endocarditis, a severe infection with high mortality. Several virulence factors important for the development of infective endocarditis have been described in these two species. However, the background for how the commensal bacteria, in some cases, become pathogenic is still not known. To gain a greater understanding of the mechanisms of the pathogenic potential, we performed a comparative analysis of 38 blood culture strains, S. sanguinis (n = 20) and S. gordonii (n = 18) from patients with verified infective endocarditis, along with 21 publicly available oral isolates from healthy individuals, S. sanguinis (n = 12) and S. gordonii (n = 9). Using whole genome sequencing data of the 59 streptococci genomes, functional profiles were constructed, using protein domain predictions based on the translated genes. These functional profiles were used for clustering, phylogenetics and machine learning. A clear separation could be made between the two species. No clear differences between oral isolates and clinical infective endocarditis isolates were found in any of the 675 translated core-genes. Additionally, random forest-based machine learning and clustering of the pan-genome data as well as amino acid variations in the core-genome could not separate the clinical and oral isolates. A total of 151 different virulence genes was identified in the 59 genomes. Among these homologs of genes important for adhesion and evasion of the immune system were found in all of the strains. Based on the functional profiles and virulence gene content of the genomes, we believe that all analysed strains had the ability to become pathogenic.

The case presented here illustrates a sometimes complex disease presentation where interdisciplinary teamwork is essential. A 74-years-old woman with stable chronic obstructive pulmonary lung disease and a composite graft aortic valve implant was admitted to hospital with an episode of acute lower back pain combined with long-term fever. The final diagnoses of a left-sided, infected and radiologically evident kidney cyst caused by the urinary tract pathogens Actinotignum schaalii and Aerococcus urinae was established by the use of partial 16S/18S rRNA gene sequence analysis on kidney cyst drainage material taken after four weeks of relevant antibiotic therapy.

Streptococcus gordonii and Streptococcus sanguinis belong to the Mitis group streptococci, which mostly are commensals in the human oral cavity. Though they are oral commensals, they can escape their niche and cause infective endocarditis, a severe infection with high mortality. Several virulence factors important for the development of infective endocarditis have been described in these two species. However, the background for how the commensal bacteria, in some cases, become pathogenic is still not known. To gain a greater understanding of the mechanisms of the pathogenic potential, we performed a comparative analysis of 38 blood culture strains, S. sanguinis (n = 20) and S. gordonii (n = 18) from patients with verified infective endocarditis, along with 21 publicly available oral isolates from healthy individuals, S. sanguinis (n = 12) and S. gordonii (n = 9). Using whole genome sequencing data of the 59 streptococci genomes, functional profiles were constructed, using protein domain predictions based on the translated genes. These functional profiles were used for clustering, phylogenetics and machine learning. A clear separation could be made between the two species. No clear differences between oral isolates and clinical infective endocarditis isolates were found in any of the 675 translated core-genes. Additionally, random forest-based machine learning and clustering of the pan-genome data as well as amino acid variations in the core-genome could not separate the clinical and oral isolates. A total of 151 different virulence genes was identified in the 59 genomes. Among these homologs of genes important for adhesion and evasion of the immune system were found in all of the strains. Based on the functional profiles and virulence gene content of the genomes, we believe that all analysed strains had the ability to become pathogenic.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

The case presented here illustrates a sometimes complex disease presentation where interdisciplinary teamwork is essential. A 74-years-old woman with stable chronic obstructive pulmonary lung disease and a composite graft aortic valve implant was admitted to hospital with an episode of acute lower back pain combined with long-term fever. The final diagnoses of a left-sided, infected and radiologically evident kidney cyst caused by the urinary tract pathogens Actinotignum schaalii and Aerococcus urinae was established by the use of partial 16S/18S rRNA gene sequence analysis on kidney cyst drainage material taken after four weeks of relevant antibiotic therapy.

Streptococcus gordonii and Streptococcus sanguinis belong to the Mitis group streptococci, which mostly are commensals in the human oral cavity. Though they are oral commensals, they can escape their niche and cause infective endocarditis, a severe infection with high mortality. Several virulence factors important for the development of infective endocarditis have been described in these two species. However, the background for how the commensal bacteria, in some cases, become pathogenic is still not known. To gain a greater understanding of the mechanisms of the pathogenic potential, we performed a comparative analysis of 38 blood culture strains, S. sanguinis (n = 20) and S. gordonii (n = 18) from patients with verified infective endocarditis, along with 21 publicly available oral isolates from healthy individuals, S. sanguinis (n = 12) and S. gordonii (n = 9). Using whole genome sequencing data of the 59 streptococci genomes, functional profiles were constructed, using protein domain predictions based on the translated genes. These functional profiles were used for clustering, phylogenetics and machine learning. A clear separation could be made between the two species. No clear differences between oral isolates and clinical infective endocarditis isolates were found in any of the 675 translated core-genes. Additionally, random forest-based machine learning and clustering of the pan-genome data as well as amino acid variations in the core-genome could not separate the clinical and oral isolates. A total of 151 different virulence genes was identified in the 59 genomes. Among these homologs of genes important for adhesion and evasion of the immune system were found in all of the strains. Based on the functional profiles and virulence gene content of the genomes, we believe that all analysed strains had the ability to become pathogenic.

Aerococcus sanguinicola and Aerococcus urinae are emerging pathogens in clinical settings mostly being causative agents of urinary tract infections (UTIs), urogenic sepsis and more seldomly complicated infective endocarditis (IE). Limited knowledge exists concerning the pathogenicity of these two species. Eight clinical A. sanguinicola (isolated from 2009 to 2015) and 40 clinical A. urinae (isolated from 1984 to 2015) strains from episodes of UTIs, bacteremia, and IE were whole-genome sequenced (WGS) to analyze genomic diversity and characterization of virulence genes involved in the bacterial pathogenicity. A. sanguinicola genome sizes were 2.06-2.12 Mb with 47.4-47.6% GC-contents, and 1783-1905 genes were predicted whereof 1170 were core-genes. In case of A. urinae strains, the genome sizes were 1.93-2.44 Mb with 41.6-42.6% GC-contents, and 1708-2256 genes of which 907 were core-genes. Marked differences were observed within A. urinae strains with respect to the average genome sizes, number and sequence identity of core-genes, proteome conservations, phylogenetic analysis, and putative capsular polysaccharide (CPS) loci sequences. Strains of A. sanguinicola showed high degree of homology. Phylogenetic analyses showed the 40 A. urinae strains formed two clusters according to two time periods: 1984-2004 strains and 2010-2015 strains. Genes that were homologs to virulence genes associated with bacterial adhesion and antiphagocytosis were identified by aligning A. sanguinicola and A. urinae pan- and core-genes against Virulence Factors of Bacterial Pathogens (VFDB). Bacterial adherence associated gene homologs were present in genomes of A. sanguinicola (htpB, fbpA, lmb, and ilpA) and A. urinae (htpB, lap, lmb, fbp54, and ilpA). Fifteen and 11-16 CPS gene homologs were identified in genomes of A. sanguinicola and A. urinae strains, respectively. Analysis of these genes identified one type of putative CPS locus within all A. sanguinicola strains. In A. urinae genomes

Aerococcus sanguinicola and Aerococcus urinae are emerging pathogens in clinical settings mostly being causative agents of urinary tract infections (UTIs), urogenic sepsis and more seldomly complicated infective endocarditis (IE). Limited knowledge exists concerning the pathogenicity of these two species. Eight clinical A. sanguinicola (isolated from 2009 to 2015) and 40 clinical A. urinae (isolated from 1984 to 2015) strains from episodes of UTIs, bacteremia, and IE were whole-genome sequenced (WGS) to analyze genomic diversity and characterization of virulence genes involved in the bacterial pathogenicity. A. sanguinicola genome sizes were 2.06-2.12 Mb with 47.4-47.6% GC-contents, and 1783-1905 genes were predicted whereof 1170 were core-genes. In case of A. urinae strains, the genome sizes were 1.93-2.44 Mb with 41.6-42.6% GC-contents, and 1708-2256 genes of which 907 were core-genes. Marked differences were observed within A. urinae strains with respect to the average genome sizes, number and sequence identity of core-genes, proteome conservations, phylogenetic analysis, and putative capsular polysaccharide (CPS) loci sequences. Strains of A. sanguinicola showed high degree of homology. Phylogenetic analyses showed the 40 A. urinae strains formed two clusters according to two time periods: 1984-2004 strains and 2010-2015 strains. Genes that were homologs to virulence genes associated with bacterial adhesion and antiphagocytosis were identified by aligning A. sanguinicola and A. urinae pan- and core-genes against Virulence Factors of Bacterial Pathogens (VFDB). Bacterial adherence associated gene homologs were present in genomes of A. sanguinicola (htpB, fbpA, lmb, and ilpA) and A. urinae (htpB, lap, lmb, fbp54, and ilpA). Fifteen and 11-16 CPS gene homologs were identified in genomes of A. sanguinicola and A. urinae strains, respectively. Analysis of these genes identified one type of putative CPS locus within all A. sanguinicola strains. In A. urinae genomes

Aerococcus sanguinicola and Aerococcus urinae are emerging pathogens in clinical settings mostly being causative agents of urinary tract infections (UTIs), urogenic sepsis and more seldomly complicated infective endocarditis (IE). Limited knowledge exists concerning the pathogenicity of these two species. Eight clinical A. sanguinicola (isolated from 2009 to 2015) and 40 clinical A. urinae (isolated from 1984 to 2015) strains from episodes of UTIs, bacteremia, and IE were whole-genome sequenced (WGS) to analyze genomic diversity and characterization of virulence genes involved in the bacterial pathogenicity. A. sanguinicola genome sizes were 2.06–2.12 Mb with a 47.4–47.6% GC-contents, and 1783–1905 genes were predicted whereof 1170 were core-genes. In case of A. urinae strains, the genome sizes were 1.93–2.44 Mb with 41.6–42.6% GC-contents, and 1708–2256 genes of which 907 were core-genes. Marked differences were observed within A. urinae strains with respect to the average genome sizes, number and sequence identity of core-genes, proteome conservations, phylogenetic analysis, and putative capsular polysaccharide (CPS) loci sequences. Strains of A. sanguinicola showed high degree of homology. Phylogenetic analyses showed the 40 A. urinae strains formed two clusters according to two time periods: 1984–2004 strains and 2010–2015 strains. Genes that were homologs to virulence genes associated with bacterial adhesion and antiphagocytosis were identified by aligning A. sanguinicola and A. urinae pan- and core-genes against Virulence Factors of Bacterial Pathogens (VFDB). Bacterial adherence associated gene homologs were present in genomes of A. sanguinicola (htpB, fbpA, lmb, and ilpA) and A. urinae (htpB, lap, lmb, fbp54, and ilpA). Fifteen and 11–16 CPS gene homologs were identified in genomes of A. sanguinicola and A. urinae strains, respectively. Analysis of these genes identified one type of putative CPS locus within all A. sanguinicola strains. In A. uri

Strains belonging to the genus Aerococcus are causative agents of human and animal infections, including urogenital infections, bacteremia/septicemia, and infective endocarditis. This study reports the first fully closed and complete genome sequences of six type strains belonging to the genus Aerococcus using a combination of Illumina HiSeq and PacBio sequencing technologies.

Introduction: Cardiobacterium hominis and Cardiobacterium valvarum are well known, though rare, etiologic agents of infective endocarditis. Cardiac devices are increasingly implanted. Case Reports: Two cases of infective episodes in pacemaker (PM) treated patients with respectively C. hominis and C. valvarum are presented. In one case blood-culture bottles yielded growth of C. hominis at two episodes with two years apart. At the second episode a vegetation was recognized at the PM lead and the PM device and lead was removed. In the C. valvarum case, echocardiography revealed a bicuspid aortic valve with severe regurgitation and a more than 1 cm sized vegetation. Conclusion: The cases illustrate the diversity in disease severity by Cardiobacterium species. Careful follow up has to be performed in order not to overlook a relatively silent relapsing infection.

Introduction: Cardiobacterium hominis and Cardiobacterium valvarum are well known, though rare, etiologic agents of infective endocarditis. Cardiac devices are increasingly implanted. Case Reports: Two cases of infective episodes in pacemaker (PM) treated patients with respectively C. hominis and C. valvarum are presented. In one case blood-culture bottles yielded growth of C. hominis at two episodes with two years apart. At the second episode a vegetation was recognized at the PM lead and the PM device and lead was removed. In the C. valvarum case, echocardiography revealed a bicuspid aortic valve with severe regurgitation and a more than 1 cm sized vegetation. Conclusion: The cases illustrate the diversity in disease severity by Cardiobacterium species. Careful follow up has to be performed in order not to overlook a relatively silent relapsing infection.

We present the first case of intracerebral abscess after blood-borne infection with non-toxigenic Vibrio cholerae (non-01). The patient was a 66 year-old woman who was infected after swimming in Danish seawater during an unusually hot summer. She had predisposing haemochromatosis and a skin lesion on the ankle. We treated the patient with meropenem and ciprofloxacin for six weeks followed by ciprofloxacin for four weeks, and she recovered with hemiparesis and speech impairment. Marine Vibrio species may produce intracranial infection in predisposed individuals, even in temperate climate zones.

We present the first case of intracerebral abscess after blood-borne infection with non-toxigenic Vibrio cholerae (non-01). The patient was a 66 year-old woman who was infected after swimming in Danish seawater during an unusually hot summer. She had predisposing haemochromatosis and a skin lesion on the ankle. We treated the patient with meropenem and ciprofloxacin for six weeks followed by ciprofloxacin for four weeks, and she recovered with hemiparesis and speech impairment. Marine Vibrio species may produce intracranial infection in predisposed individuals, even in temperate climate zones.

Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch’s criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients., Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch's criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients.

Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch’s criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients., Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch's criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients.

Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch’s criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients., Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch's criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF patients.

Udgivelsesdato: 2009

Udgivelsesdato: 2009-Apr, Nonhemolytic streptococci (NHS) cause serious infections, such as endocarditis and septicemia. Many conventional phenotypic methods are insufficient for the identification of bacteria in this group to the species level. Genetic analysis has revealed that single-gene analysis is insufficient for the identification of all species in this group of bacteria. The aim of the present study was to establish a method based on sequence analysis of the 16S-23S intergenic spacer (ITS) region and the partial gdh gene to identify clinical relevant NHS to the species level. Sequence analysis of the ITS region was performed with 57 NHS reference or clinical strains. Satisfactory identification to the species level was achieved for 14/19 NHS species included in this study on the basis of sequence analysis of the ITS region. Streptococcus salivarius and Streptococcus vestibularis obtained the expected taxon as the best taxon match, but there was a short maximum score distance to the next best match (distance, <10). Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae could not be unambiguously discriminated by sequence analysis of the ITS region, as was also proven by phylogenetic analysis. These five species could be identified to the group level only by ITS sequence analysis. Partial gdh sequence analysis was applied to the 11 S. oralis strains, the 11 S. mitis strains, and the 17 S. pneumoniae strains. All except one strain achieved a satisfactory identification to the species level. A phylogenetic algorithm based on the analysis of partial gdh gene sequences revealed three distinct clusters. We suggest that sequence analysis of the combination of the ITS region and the partial gdh gene can be used in the reference laboratory for the species-level identification of NHS.

Udgivelsesdato: 2009

Udgivelsesdato: 2009-Apr, Nonhemolytic streptococci (NHS) cause serious infections, such as endocarditis and septicemia. Many conventional phenotypic methods are insufficient for the identification of bacteria in this group to the species level. Genetic analysis has revealed that single-gene analysis is insufficient for the identification of all species in this group of bacteria. The aim of the present study was to establish a method based on sequence analysis of the 16S-23S intergenic spacer (ITS) region and the partial gdh gene to identify clinical relevant NHS to the species level. Sequence analysis of the ITS region was performed with 57 NHS reference or clinical strains. Satisfactory identification to the species level was achieved for 14/19 NHS species included in this study on the basis of sequence analysis of the ITS region. Streptococcus salivarius and Streptococcus vestibularis obtained the expected taxon as the best taxon match, but there was a short maximum score distance to the next best match (distance, <10). Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae could not be unambiguously discriminated by sequence analysis of the ITS region, as was also proven by phylogenetic analysis. These five species could be identified to the group level only by ITS sequence analysis. Partial gdh sequence analysis was applied to the 11 S. oralis strains, the 11 S. mitis strains, and the 17 S. pneumoniae strains. All except one strain achieved a satisfactory identification to the species level. A phylogenetic algorithm based on the analysis of partial gdh gene sequences revealed three distinct clusters. We suggest that sequence analysis of the combination of the ITS region and the partial gdh gene can be used in the reference laboratory for the species-level identification of NHS.