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  • 1.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Stralin, Kristoffer
    Olcen, Per
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Molling, Paula
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia2013In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 76, no 2, p. 141-146Article in journal (Refereed)
    Abstract [en]

    A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions.

  • 2.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Strålin, Kristoffer
    Department of Infectious Diseases, Örebro University Hospital.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Olcén, Per
    Department of Clinical Microbiology, Örebro University Hospital.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction2009In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, no 4, p. 366-373Article in journal (Refereed)
    Abstract [en]

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  • 3.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Strålin, Kristoffer
    Olcén, Per
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Toward a quantitative DNA-based definition of pneumococcal pneumonia: a comparison of Streptococcus pneumoniae target genes, with special reference to the Spn9802 fragment2008In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 60, no 2, p. 143-150Article in journal (Refereed)
    Abstract [en]

    The current shift from phenotypically toward genotypically based microbial diagnosis is not unproblematic. A novel quantitative real-time polymerase chain reaction (PCR) assay based on the Spn9802 DNA fragment was therefore developed for detection of Streptococcus pneumoniae. Out of 44 bacterial species, only S. pneumoniae and Streptococcus pseudopneumoniae were positive in Spn9802 PCR. In an evaluation on nasopharyngeal aspirates from 166 patients with community-acquired pneumonia, the assay was positive in 49 of 50 culture-positive cases. Of 19 culture-negative but Spn9802 PCR-positive cases, 12 were confirmed as S. pneumoniae by rnpB sequence analysis. With an expanded reference standard, including culture and rnpB sequencing, Spn9802 had a sensitivity of 94% and a specificity of 98%. A cutoff for clinically significant positivity was 10(4) DNA copies/mL, giving 71% sensitivity and 100% specificity. In conclusion, Spn9802 real-time PCR is highly sensitive and specific. The quantification it provides enables differentiation between pneumococcal pathogenicity and commensalism.

  • 4. Barisic, Ivan
    et al.
    Schoenthaler, Silvia
    Ke, Rongqin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Noehammer, Christa
    Wiesinger-Mayr, Herbert
    Multiplex detection of antibiotic resistance genes using padlock probes2013In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 77, no 2, p. 118-125Article in journal (Refereed)
    Abstract [en]

    The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse beta-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the beta-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of beta-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.

  • 5.
    Dumke, Roger
    et al.
    Tech Univ Dresden, Dresden, Germany.
    Benitez, Alvaro J.
    Ctr Dis Control & Prevent, Atlanta, GA USA.
    Chalker, Victoria
    Publ Hlth England, London, England.
    Gullsby, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Centre for Research and Development, Gävleborg.
    Henrich, Birgit
    Univ Dusseldorf, Dusseldorf, Germany.
    Hidalgo-Grass, Carlos
    HGC Biomed Res Diagnost SL, Huelva, Spain.
    Hoogenboezem, Theo
    Sophia Childrens Univ Hosp, Erasmus MC Univ Med Ctr, Dept Pediat, Pediat Lab, Rotterdam, Netherlands.
    Kese, Darja
    Univ Ljubljana, Ljubljana, Slovenia.
    Loens, Katherine
    Univ Ziekenhuis Antwerpen, Edegem, Belgium.
    Maaskant, Jolanda
    Erasmus MC Univ Med Ctr, Virosci, Rotterdam, Netherlands.
    Michael-Gayego, Ayelet
    Hadassah Hebrew Univ, Med Ctr, Jerusalem, Israel.
    Moses, Allon E.
    Hadassah Hebrew Univ, Med Ctr, Jerusalem, Israel.
    Nir-Paz, Ran
    Hadassah Hebrew Univ, Med Ctr, Jerusalem, Israel.
    Pas, Suzan D.
    Erasmus MC Univ Med Ctr, Virosci, Rotterdam, Netherlands.
    Pereyre, Sabine
    Univ Bordeaux, Bordeaux, France.
    Petersen, Randi F.
    Statens Serum Inst, Copenhagen, Denmark.
    Rosenblatt, Meike
    Univ Dusseldorf, Dusseldorf, Germany.
    van Rossum, Annemarie M. C.
    Sophia Childrens Univ Hosp, Erasmus MC Univ Med Ctr, Dept Pediat, Div Infect Dis & Immunol, Rotterdam, Netherlands.
    Uldum, Soren A.
    Statens Serum Inst, Copenhagen, Denmark.
    Unger, Wendy W. J.
    Sophia Childrens Univ Hosp, Erasmus MC Univ Med Ctr, Dept Pediat, Pediat Lab, Rotterdam, Netherlands.
    Ursi, Dominique
    Univ Ziekenhuis Antwerpen, Edegem, Belgium.
    Winchell, Jonas M.
    Ctr Dis Control & Prevent, Atlanta, GA USA.
    Bebear, Cecile
    Univ Bordeaux, Bordeaux, France.
    Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae2017In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 88, no 2, p. 111-114Article in journal (Refereed)
    Abstract [en]

    Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20 M. pneumoniae genomes per reaction.

  • 6.
    Isaksson, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Rasmussen, Magnus
    Nilson, Bo
    Stadler, Liselott Svensson
    Kurland, Siri
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Olaison, Lars
    Ek, Elisabeth
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems2015In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 81, no 4, p. 240-245Article in journal (Refereed)
    Abstract [en]

    Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp.

  • 7. Karah, Nabil
    et al.
    Poirel, Laurent
    Bengtsson, Stina
    Sundqvist, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Nordmann, Patrice
    Sundsfjord, Arnfinn
    Samuelsen, Ørjan
    Plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in Escherichia coli and Klebsiella spp. from Norway and Sweden2010In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 66, no 4, p. 425-431Article in journal (Refereed)
    Abstract [en]

    The prevalence of the plasmid-mediated quinolone resistance genes qnr and aac(6')-Ib-cr was investigated among clinical isolates of Escherichia coli and Klebsiella spp. selected from 2 collections of consecutive isolates collected in 2004 to 2005 in Norway (n = 2479) and Sweden (n = 2980) and 1 group of extended-spectrum beta-lactamase (ESBL)-producing isolates collected in 2003 in Norway (n = 71). A total of 414 isolates was selected for screening based on resistance to nalidixic acid and/or reduced susceptibility/resistance to ciprofloxacin. The prevalence of both qnr and aac(6')-Ib-cr was higher among the ESBL producers (9.1% and 52.3%, respectively) than in the consecutive isolates (1.1% and 3.2%, respectively). qnrS1 was detected in 6 isolates, whereas qnrB1 and qnrB7 were detected in 2 isolates. The genetic structure surrounding qnrS1 was similar to previously described structures. In 2 isolates, qnrS1 was located on conjugative IncN-type plasmids of approximately 140 kb.

  • 8.
    Pettersson, John
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Piorkowski, Geraldine
    UVE Aix Marseille Univ, Inserm 1207, IRD 190, IHU Mediterranee Infect,Unite Virus Emergents, Marseille, France.
    Mayxay, Mayfong
    Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos;Univ Hlth Sci, IRED, Viangchan, Laos;Univ Oxford, Ctr Trop Med & Global Hlth, Churchill Hosp, Nuffield Dept Med, Oxford, England.
    Rattanavong, Sayaphet
    Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos.
    Vongsouvath, Manivanh
    Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos.
    Davong, Viengmon
    Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos.
    Alfsnes, Kristian
    Norwegian Inst Publ Hlth, Infect Dis & Environm Hlth, Lovisenberggata 8, N-0456 Oslo, Norway.
    Eldholm, Vegard
    Norwegian Inst Publ Hlth, Infect Dis & Environm Hlth, Lovisenberggata 8, N-0456 Oslo, Norway.
    de Lamballerie, Xavier
    UVE Aix Marseille Univ, Inserm 1207, IRD 190, IHU Mediterranee Infect,Unite Virus Emergents, Marseille, France.
    Holmes, Edward C.
    Univ Sydney, Charles Perkins Ctr, Sch Life & Environm Sci, Marie Bashir Inst Infect Dis & Biosecur, Sydney, NSW, Australia;Univ Sydney, Sydney Med Sch, Sydney, NSW, Australia.
    Newton, Paul N.
    Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos;Univ Oxford, Ctr Trop Med & Global Hlth, Churchill Hosp, Nuffield Dept Med, Oxford, England.
    Dubot-Peres, Audrey
    UVE Aix Marseille Univ, Inserm 1207, IRD 190, IHU Mediterranee Infect,Unite Virus Emergents, Marseille, France;Mahosot Hosp, Lao Oxford Mahosot Hosp, Microbiol Lab, Wellcome Trust Res Unit LOMWRU, Viangchan, Laos;Univ Oxford, Ctr Trop Med & Global Hlth, Churchill Hosp, Nuffield Dept Med, Oxford, England.
    Meta-transcriptomic identification of hepatitis B virus in cerebrospinal fluid in patients with central nervous system disease2019In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 95, no 4, article id UNSP 114878Article in journal (Refereed)
    Abstract [en]

    Determining the etiological basis of central nervous system (CNS) infections is inherently challenging, primarily due to the multi-etiological nature. Using RNA sequencing, we aimed to identify microbes present in cerebrospinal fluid (CSF) of two patients suffering CNS infection, previously diagnosed with Cryptococcus sp. and Streptococcus pneumoniae infection, respectively. After meta-transcriptomic analysis, and confirmation with real-time PCR, hepatitis B virus (HBV) was detected in the CSF of two patients diagnosed with CNS syndrome. Phylogenetic analysis of the partial HBV genomes from these patients showed that they belonged to genotypes B and C and clustered with other viruses of Asian origin. In countries with high levels of HBV endemicity, the virus is likely to be found in patients diagnosed with CNS infections, although whether it contributes to symptoms and pathology, or is simply a coincidental infection, is unknown and merits further investigation. (C) 2019 The Authors. Published by Elsevier Inc.

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