Xiaohui Chen Nielsen

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful.

Identification of Clinically Relevant Nonhemolytic Streptococci on the Basis of Sequence Analysis of 16S-23S Intergenic Spacer Region and Partial gdh Gene

ABSTRACT 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.

Unusual Distribution of Burkholderia cepacia Complex Species in Danish Cystic Fibrosis Clinics May Stem from Restricted Transmission between Patients

ABSTRACT Forty-four of 48 Burkholderia cepacia complex strains cultured from Danish cystic fibrosis patients were Burkholderia multivorans, a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species distribution may represent the sporadic acquisition of bacteria from the environment.

Influenza vaccine effectiveness in adults 65 years and older, Denmark, 2015/16 - a rapid epidemiological and virological assessment.

In Denmark, both influenza A(H1N1)pdm09 and influenza B co-circulated in the 2015/16 season. We estimated the vaccine effectiveness (VE) of the trivalent influenza vaccine in patientsu200965 years and older using the test-negative case-control design. The adjusted VE against influenza A(H1N1)pdm09 was 35.0% (95% confidence interval (CI): 11.1-52.4) and against influenza B 4.1% (95% CI: -22.0 to 24.7). The majority of influenza A(H1N1)pdm09 circulating in 2015/16 belonged to the new genetic subgroup subclade 6B.1.

Changes in genetically drifted H3N2 influenza A viruses and vaccine effectiveness in adults 65 years and older during the 2016/17 season in Denmark

BACKGROUNDnIn Denmark, influenza A virus of the subtype H3N2 has been dominating the 2016/17 season, as in most countries of the Northern Hemisphere.nnnOBJECTIVESnThis study was conducted as part of the Danish seasonal influenza surveillance programme to genetically characterize circulating H3N2 viruses and determine the seasonal vaccine effectiveness (VE) overall in the Danish population and further on the virus cluster level.nnnSTUDY DESIGNnInfluenza virus positive samples submitted for the national surveillance programme were genetically characterized by sequencing. VE estimates against influenza A and the circulating virus clusters were determined in patients above 65 years using the test-negative case-control design.nnnRESULTSnThe genetic characterization revealed several genetically drifted viruses, which could be divided into four main clusters by the defining amino acid substitutions: 3C.2a/N121K/S144K, 3C.2a/T131K/R142K, 3C.2a1, and 3C.2a1/N121K. Some of the drifted viruses appeared to be more prominent in vaccinated or non-vaccinated individuals, respectively. Overall the adjusted VE was 7.4% (95% confidence interval (CI): -6.0-19.2) among inpatients and 19.3% (95% CI: -5.7-38.4) among outpatients, respectively. VE for the four main virus clusters was; cluster 3C.2a1: 38.8% (95% CI: -29.8-71.1), cluster 3C.2a/N121K/S144K: 9.2% (95% CI: -63.0-49.4), cluster 3C.2a/T131K/R142K: 19.0% (95% CI: -85.3-64.6), and cluster 3C.2a1/N121K: -12.2% (95%CI: -129.7-45.2).nnnCONCLUSIONSnSeveral genetically drifted H3N2 viruses have been circulating in Denmark in the 2016-17 influenza season. An overall low VE was estimated and VE for the four main virus cluster indicate different VEs between the circulating drifted H3N2 viruses.

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5noncoding regions (5NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.

Methods to Classify Bacterial Pathogens in Cystic Fibrosis

Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Kochs 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.

Report of the First Human Case of Caulobacter sp. Infection

ABSTRACT A Caulobacter sp. isolate was recovered from the dialysis fluid of a patient undergoing peritoneal dialysis. Bacterial identification included electron microscopy and 16S rDNA sequencing. To our knowledge, this is the first report of human Caulobacter infection. Special growth requirements suggest that Caulobacter spp. may be overlooked in the clinical microbiology laboratory.

Bacteremia with the bovis group streptococci: species identification and association with infective endocarditis and with gastrointestinal disease

DNA sequencing of the intergenic spacer (ITS) region was used to identify 53 blood culture isolates that had previously been designated to the bovis group streptococci and clinical data was collected retrospectively from patients records using a standardized protocol. ITS sequencing identified 19 (35.8%) isolates as Streptococcus gallolyticus subsp. gallolyticus, 12 (22.6%) as S. gallolyticus subsp. pasteurianus, two (3.8%) as S. gallolyticus subsp. macedonicus, seven (13.2%) as S. infantarius subsp. infantarius, 12 (22.6%) as S. lutetiensis and one (1.9%) as S. equinus. The association of S. gallolyticus subsp. gallolyticus with colorectal neoplasia and with infective endocarditis and the association between S. gallolyticus subsp. pasteurianus and pancreatic cancer were found to be clinically important. Also, a very high 1-year mortality rate with S. lutetiensis (66.7%) and S. gallolyticus subsp. pasteurianus (58.7%) bacteremia calls for intensive investigation for underlying disease focusing on the pancreas and the hepatobiliary system.

Whole genome sequencing as a tool for phylogenetic analysis of clinical strains of Mitis group streptococci.

Identification of Mitis group streptococci (MGS) to the species level is challenging for routine microbiology laboratories. Correct identification is crucial for the diagnosis of infective endocarditis, identification of treatment failure, and/or infection relapse. Eighty MGS from Danish patients with infective endocarditis were whole genome sequenced. We compared the phylogenetic analyses based on single genes (recA, sodA, gdh), multigene (MLSA), SNPs, and core-genome sequences. The six phylogenetic analyses generally showed a similar pattern of six monophyletic clusters, though a few differences were observed in single gene analyses. Species identification based on single gene analysis showed their limitations when more strains were included. In contrast, analyses incorporating more sequence data, like MLSA, SNPs and core-genome analyses, provided more distinct clustering. The core-genome tree showed the most distinct clustering.