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  • 1.
    Allard, A
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Albinsson, B
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Wadell, G
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis.2001In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 39, no 2, p. 498-505Article in journal (Refereed)
    Abstract [en]

    We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.

  • 2.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Enteric adenovirus type 41: genome organization and specific detection procedures1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enteric adenoviruses (EAd) types 40 and 41 (Ad40 and Ad41) representing subgenus F, are primary pathogens of children being second only to rotaviruses as the most important cause of infantile diarrhea.

    The EAds differ from all other adenoviruses in their inability to grow in most conventional established cell lines and have been suggested to be deficient in some early gene functions since they could be complemented by Ad 5 early regions EIA and E1B. In order to search for differences that could explain its characteristic growth restriction, the early regions EIA and E1B of Ad41 (strain D389) were sequenced, analysed and compared with the corresponding regions of Adl2, Ad7, Ad2, and Ad4. As revealed by the analysis of Ad2, three major mRNAs of 9S, 12S and 13S are generated from region EIA. The EIA region of Ad41 encodes two mRNAs corresponding to the 12S and 13S mRNAs. Only the 13S mRNA is transcribed at detectable levels. This mRNA can be translated into a 251 aa putative protein that contains the three highly conserved domains found in all other human adenoviruses and shown to be responsible for many important regulatory functions during infection.

    The E1B region of Ad41 encodes three transcripts that correspond to 22S, 14S and 9S mRNA of Ad2. No equivalent to the 13S mRNA of Ad2 E1B is found. In addition the Ad41 14S mRNA exhibits an additional exon of 23 bp created by a donor and an acceptor splice sites not desribed for other adenovirus E1B sequences.

    Due to their growth restriction in conventional cultures, rapid diagnostic procedures developed for the enteric adenovirus infections have mainly been aimed at the detection of viral antigens or nucleic acids. This thesis also describes several procedures developed for the general detection of adenoviruses and specific detection of the enteric types in stools specimens. General and specific hybridization assays were developed by use of two BamHI clones obtained from the EIA region of Ad41. One- and two-step PCR procedures were also developed for the general detection of adenoviruses using primers corresponding to highly conserved sequences within the hexon gene. Subgenus F specific one- and two-step PCRs were developed by using primers located in the Ad41 E1B region.

    The one-step PCR systems were tested and validated against isolation in tissue culture, DNA restriction enzyme analysis and a commercial latex agglutination test in the study of 60 specimens obtained from children with rotavirus negative diarrhea. The asymptomatic fecal excretion of adenoviruses was evaluated by two-step PCR amplifications on samples from 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea.

    Finally, a simplified procedure for detection, discrimination and typing of EAd was also designed by combining the one-step PCR amplification of the hexon region with the restriction of the 300 bp product.

  • 3.
    Andersson, Emma K
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Strand, Mårten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Edlund, Karin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Lindman, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Enquist, Per-Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Spjut, Sara
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mei, Ya-Fang
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Wadell, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Small molecule screening using a whole cell viral replication reporter gene assay identifies 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid as a novel anti-adenoviral compound2010In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 54, no 9, p. 3871-3877Article in journal (Refereed)
    Abstract [en]

    Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their anti-adenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential anti-adenoviral compounds. The assay is unique in that it is based on a replication competent adenovirus type 11p GFP-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by >/= 80% but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.

  • 4.
    Bergh, Johanna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Marklund, Ingrid
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Gustavsson, C
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Wiklund, Fredrik
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Grönberg, Henrik
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Alexeyev, Olog
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Elgh, Fredrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    No link between viral findings in the prostate and subsequent cancer development2007In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 96, no 1, p. 137-139Article in journal (Refereed)
    Abstract [en]

    In an investigation of 201 prostate tissue samples from patients with benign prostate hyperplasia that later progressed to prostate cancer and 201 matched controls that did not, there were no differences in the prevalence of adenovirus, herpesvirus, papilloma virus, polyoma virus and Candida albicans DNA.

  • 5.
    Edin, Alicia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Granholm, Susanne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Koskiniemi, Satu
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia2015In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 3, p. 315-324Article in journal (Refereed)
    Abstract [en]

    Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and Lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.

  • 6.
    Evander, Magnus
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Eriksson, Irene
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Pettersson, Lisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Juto, Per
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Olsson, Gert E
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bucht, Göran
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome.2007In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 45, no 8, p. 2491-7Article in journal (Refereed)
    Abstract [en]

    Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 10(6) virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.

  • 7. Formiga-Cruz, M
    et al.
    Allard, Annika K
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Conden-Hansson, A-C
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Henshilwood, K
    Hernroth, B E
    Jofre, J
    Lees, D N
    Lucena, F
    Papapetropoulou, M
    Rangdale, R E
    Tsibouxi, A
    Vantarakis, A
    Girones, R
    Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 3, p. 1556-1563Article in journal (Refereed)
    Abstract [en]

    The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

  • 8. Formiga-Cruz, M
    et al.
    Tofiño-Quesada, G
    Bofill-Mas, S
    Lees, D N
    Henshilwood, K
    Allard, A K
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Conden-Hansson, A-C
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Hernroth, B E
    Vantarakis, A
    Tsibouxi, A
    Papapetropoulou, M
    Furones, M D
    Girones, R
    Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.2002In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 68, no 12, p. 5990-8Article in journal (Refereed)
    Abstract [en]

    Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

  • 9. Hernroth, Bodil E
    et al.
    Conden-Hansson, Ann-Christine
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Rehnstam-Holm, Ann-Sofi
    Girones, Rosina
    Allard, Annika K
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report.2002In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 68, no 9, p. 4523-33Article in journal (Refereed)
    Abstract [en]

    This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of "clean" mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.

  • 10.
    Holm, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Eriksson, Irene
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Laurell, Göran
    Department of Surgical Sciences, Division of Otorhinolaryngology, Uppsala University.
    Nylander, Karin
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Olofsson, Katarina
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papillomaManuscript (preprint) (Other academic)
    Abstract [en]

    Objectives: Sinonasal inverted papilloma (SIP) is a relatively rare disease, and its etiology is not understood. It is characterized by locally aggressive growth and a strong tendency to recur despite its benign histology.

    Aims: The aim of this study was to identify the presence of human papilloma virus (HPV) and its surrogate marker p16 in SIP tissue samples from a regional cohort.

    Material and Methods: Subjects were identified from our regional center cohort of 88 SIP patients treated between 1984-2014. From these subjects, 54 were included in this study.  Of these, 53 biopsies were analyzed with PCR, and 54 samples were immunohistochemically stained for p16. DNA was extracted from histopathologically verified SIP.  Genotype screening for 13 high risk-, 5 oncogenic and 6 low risk HPV types was performed using the PapilloCheck® HPV-screening test.

    Results: HPV analysis was successful for 38 of 53 samples. Of the 38 successfully analyzed samples, only 2 samples were positive for HPV 11.  Notably, p16 was present in the epithelia in all samples, and in the papilloma lesions in 37 samples.

    Conclusion: Since only 2 out of 38 SIPs were positive for HPV (type 11), and at the same time p16 was positive in epithelia in all samples and in 37 of 38 papilloma lesions of the samples, it is concluded that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP. We are currently planning a prospective, multicenter study in order to increase the study power and in order to be able to better evaluate the clinical implications of HPV-and p16 in SIP.

  • 11.
    Holm, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Schindele, Alexandra
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology. Östersunds hospital, Sweden.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Eriksson, Irene
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Sandström, Karl
    Laurell, Göran
    Nylander, Karin
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Olofsson, Katarina
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Mapping of Human Papilloma Virus, p16, and Epstein-Barr Virusin Non-Malignant Tonsillar Disease2019In: Laryngoscope Investigative Otolaryngology, E-ISSN 2378-8038, Vol. 4, no 3, p. 285-291Article in journal (Refereed)
    Abstract [en]

    Objectives: Due to their location in the entrance of the aero‐digestive tract, tonsils are steadily exposed to viruses. Human papilloma virus (HPV) and Epstein‐Barr virus (EBV) are two potentially oncogenic viruses that tonsils encounter. The incidence of HPV positive tonsillar cancer is on the rise and it is unknown when infection with HPV occurs.

    Aim: To investigate if tonsils are infected with HPV and EBV, to study the co‐expression of HPV and its surrogate marker p16, and to evaluate the number of EBV positive cells in benign tonsillar disease.

    Materials and Methods: Tonsils from 40 patients in a university hospital were removed due to hypertrophy, chronic or recurrent infection. These were analyzed for presence of HPV, its surrogate marker p16, and EBV. HPV was studied using PapilloCheck (a PCR method), while p16 was identified in epithelial and lymphoid tissue with immunohistochemistry and EBV using EBER‐ISH (Epstein‐Barr encoding region–in situ hybridization).

    Results: HPV was not detected, and p16 was present at low numbers in all epithelial samples as well as in 92.5% of the lymphoid tonsillar samples. At least one EBER‐positive cell was seen in 65% of cases. Larger numbers of EBER‐expressing cells were only seen in two cases.

    Conclusion: These findings demonstrate that EBV and HPV infect tonsils independently, but further studies are warranted to confirm their infectious relationship.

    Level of Evidence: Cross‐sectional study

  • 12. Johansen, Kari
    et al.
    Mannerqvist, Kerstin
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Andersson, Yvonne
    Burman, Lars G
    Dillner, Lena
    Hedlund, Kjell-Olof
    Jönsson, Klas
    Kumlin, Urban
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Leitner, Thomas
    Lysén, Maria
    Thorhagen, Margareta
    Tiveljung-Lindell, Annika
    Wahlström, Cecilia
    Zweygberg-Wirgart, Benita
    Widell, Anders
    Norovirus strains belonging to the GII.4 genotype dominate as a cause of nosocomial outbreaks of viral gastroenteritis in Sweden 1997--2005: Arrival of new variants is associated with large nation-wide epidemics2008In: Journal of Clinical Virology, ISSN 1386-6532, E-ISSN 1873-5967, Vol. 42, no 2, p. 129-134Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: In recent years an increase of the incidence of nosocomial outbreaks caused by noroviruses has been observed throughout Sweden, with high peaks noted in the winter seasons 2002/2003 and 2004/2005, respectively. OBJECTIVES: To phylogenetically characterize norovirus strains causing nosocomial outbreaks from 1997 to 2005 and estimate the impact of norovirus-like disease on the Swedish health care system during the peak season 2002/2003 when a new variant of norovirus occurred. STUDY DESIGN: Stool samples from 115 randomly selected nosocomial outbreaks occurring during 1997--2005 throughout Sweden were studied by RT-PCR and sequencing. In addition, to investigate the impact on the health-care system, a questionnaire was distributed to infection control units (n=90) serving all Swedish hospitals, nursing homes and other health-care institutions during the largest epidemic of nosocomial outbreaks. RESULTS: Sequencing of 279 nucleotides of the norovirus RNA polymerase gene in stools containing norovirus RNA showed that strains belonging to the GII.4 genotype dominated. Each of the two large epidemics was due to a new variant within this cluster. The questionnaire revealed that 30,000-35,000 episodes of nosocomial norovirus-like infections occurred in 80 of 82 major Swedish hospitals affected in 2002/2003. CONCLUSION: New norovirus variants within the cluster GGII.4 may have a major impact on the health-care system.

  • 13.
    Kaján, Gyõzõ L.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary.
    Lipiec, Agnieszka
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bartha, Daniel
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    A multigene typing system for human adenoviruses reveals a new genotype in a collection of Swedish clinical isolates2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 12, article id e0209038Article in journal (Refereed)
    Abstract [en]

    Human adenoviruses (HAdVs) are common pathogens that can cause respiratory, gastrointestinal, urogenital, and ocular infections. They are divided into seven species containing 85 genotypes. Straightforward typing systems might help epidemiological investigations. As homologous recombination frequently shapes the evolution of HAdVs, information on a single gene is seldom sufficient to allow accurate and precise typing, and complete genome-based methods are recommended. Even so, complete genome analyses are not always easy to perform for practical reasons, and in such cases a multigene system can provide considerably more information about the strain under investigation than single-gene-based methods. Here we present a rapid, generic, multigene typing system for HAdVs based on three main deterministic regions of these viruses. Three PCR systems were used to amplify the genes encoding the DNA polymerase, the penton base hypervariable Arg-Gly-Asp-containing loop, and the hexon loop 1 (hypervariable region 1–6). Using this system, we typed 281 clinical isolates, detected members of six out of seven HAdV species (Human mastadenovirus AF), and could also detect not only divergent strains of established types but also a new recombinant strain with a previously unpublished combination of adenovirus genomes. This strain was accepted by the Human Adenovirus Working Group as a novel genotype: HAdV-86. Seven strains that could not be typed with sufficient accuracy were also investigated using a PCR based on part of the fiber gene. By analysis of corresponding sequences of the 86 known HAdV genotypes, we determined that the proposed typing system should be able to distinguish all non-recombinant types, and with additional fiber information, all known HAdV genotypes.

  • 14.
    Kuoppa, Yvonne
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Boman, Jens
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Scott, Lena
    Kumlin, Urban
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Eriksson, Iréne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR2002In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 6, p. 2273-2274Article in journal (Refereed)
    Abstract [en]

    Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.

  • 15.
    Olofsson, Katarina
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Sjöstedt, M
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Hellström, S
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    The cytokine profile of T cells in the pharyngeal tonsil of childrenManuscript (preprint) (Other academic)
  • 16. Rusiol, Marta
    et al.
    Fernandez-Cassi, Xavier
    Hundesa, Ayalkibet
    Vieira, Carmen
    Kern, Anita
    Eriksson, Irene
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Ziros, Panos
    Kay, David
    Miagostovich, Marize
    Vargha, Marta
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Vantarakis, Apostolos
    Wyn-Jones, Peter
    Bofill-Mas, Silvia
    Girones, Rosina
    Application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas2014In: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 59, p. 119-129Article in journal (Refereed)
    Abstract [en]

    Integrated river basin management planning to mitigate the impacts of economic, demographic and climate change is an important issue for the future protection of water resources. Identifying sources of microbial contamination via the emerging science of Microbial Source Tracking (MST) plays a key role in risk assessment and the design of remediation strategies. Following an 18-month surveillance program within the EU-FP7-funded VIROCLIME project, specific MST tools were used to assess human markers such as adenoviruses (HAdV) and JC polyomaviruses (JCPyV) and porcine and bovine markers such as porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) via quantification with real-time PCR to analyze surface water collected from five sites within different climatic zones: the Negro River (Brazil), Glafkos River (Greece), Tisza River (Hungary), Llobregat River (Spain) and Umealven River (Sweden). The utility of the viral MST tools and the prevalence and abundance of specific human and animal viruses in the five river catchments and adjacent seawater, which is impacted by riverine contributions from the upstream catchments, were examined. In areas where no sanitation systems have been implemented, sewage can directly enter surface waters, and river water exhibited high viral loads; HAdV and JCPyV could be detected at mean concentrations of 10(5) and 10(4) Genome Copies/Liter (GC/L), respectively. In general, river water samples upstream of urban discharges presented lower human viral loads than downstream sampling sites, and those differences appeared to increase with urban populations but decrease in response to high river flow, as the elevated river water volume dilutes microbial loads. During dry seasons, river water flow decreases dramatically, and secondary effluents can represent the bulk of the riverine discharge. We also observed that ice cover that formed over the river during the winter in the studied areas in North Europe could preserve viral stability due to the low temperatures and/or the lack of solar inactivation. Porcine and bovine markers were detected where intensive livestock and agricultural activities were present; mean concentration values of 10(3) GC/L indicated that farms were sometimes unexpected and important sources of fecal contamination in water. During spring and summer, when livestock is outdoors and river flows are low, animal pollution increases due to diffuse contamination and direct voiding of feces onto the catchment surface. The field studies described here demonstrate the dynamics of fecal contamination in all catchments studied, and the data obtained is currently being used to develop dissemination models of fecal contamination in water with respect to future climate change scenarios. The results concerning human and animal targets presented in this study demonstrate the specificity and applicability Of the viral quantitative parameters developed to widely divergent geographical areas and their high interest as new indicators of human and animal fecal contamination in water and as MST tools.

  • 17.
    Segerman, Anna
    et al.
    Umeå University, Faculty of Medicine, Clinical Microbiology.
    Lindman, Kristina
    Mei, Ya-Fang
    Allard, Annika
    Wadell, Göran
    In contrast to adenovirus types 3p, 7p (species B1) and type 5p (species C), adenovirus types 11p and 35 (species B2) bind to and infect primary lymphocytes and monocytes efficiently.Manuscript (Other academic)
  • 18.
    Strand, Mårten
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Islam, Koushikul
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Edlund, Karin
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Öberg, Christopher T
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bergström, Tomas
    Univ Gothenburg, Sahlgrenska Acad, Dept Virol, Gothenburg, Sweden.
    Mei, Ya-Fang
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wadell, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, an antiviral compound with activity against acyclovir-resistant isolates of herpes simplex virus type 1 and 22012In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 56, no 11, p. 5735-5743Article in journal (Refereed)
    Abstract [en]

    Herpes simplex viruses (HSV-1 and HSV-2) are responsible for life-long latent infections in humans, with periods of viral reactivation associated with recurring ulcerations in the orofacial and genital tract. In immunosuppressed patients and neonates, HSV infections are associated with severe morbidity, and in some cases even mortality. Today, acyclovir is the standard therapy for management of HSV infections. However, the need for novel antiviral agents is apparent since HSV isolates resistant to acyclovir therapy are frequently isolated in immunosuppressed patients. In this study, we assessed the anti-HSV activity of the anti-adenoviral compounds 2-[2-(2-benzoylamino)-benzoylamino]benzoic acid, (Benzavir-1) and 2-[4,5-difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid, (Benzavir-2) on HSV-1 and HSV-2. Both compounds were active against both viruses. Importantly, Benzavir-2 had similar potency to acyclovir against both HSV types and it was active against clinical acyclovir-resistant HSV isolates.

  • 19. Swartling, L.
    et al.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Torlen, J.
    Ljungman, P.
    Mattsson, J.
    Sparrelid, E.
    Prolonged outbreak of adenovirus A31 in allogeneic stem cell transplant recipients2015In: Transplant Infectious Disease, ISSN 1398-2273, E-ISSN 1399-3062, Vol. 17, no 6, p. 785-794Article in journal (Refereed)
    Abstract [en]

    Background: An outbreak of human adenovirus (HAdV) A31 occurred from December 2011 to March 2012 at the Center for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital in Sweden. We analyzed the outbreak, the routes of transmission, and report the medical consequences.

    Methods: The medical records of all patients admitted to CAST during the outbreak period were studied. Phylogenetic analysis of the patient HAdV strains was performed by sequencing the hexon gene and the more variable E3 gene.

    Results: We identified 9 cases of HAdV A31. Hygiene measures were implemented, but transmission continued for 2 months. All 9 patients had been admitted to the ward, but 2 had no connection in time to other known HAdV A31 cases. DNA sequencing of the patient strains strongly suggested nosocomial transmission. Transplantation was postponed and then cancelled in 1 patient, and 5 patients were treated with cidofovir because of high levels of viremia. In 7 patients, concomitant graft-versus-host disease (GVHD) grade II–V complicated the clinical picture, as it was difficult to distinguish symptoms of GVHD from those of HAdV infection.

    Conclusion: An outbreak of HAdV in HSCT recipients can be difficult to control. Although none of the patients had severe disease, the medical consequences were significant. It is possible that unidentified cases with mild symptoms may have caused continuous transmission at the unit. Regular testing of all patients several weeks beyond the last case identified may be an important measure to control transmission.

  • 20. Swartling, L. P.
    et al.
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Torlen, J.
    Ljungman, P.
    Mattsson, J.
    Sparrelid, E.
    Prolonged Outbreak of Adenovirus A31 Among Allogeneic Stem Cell Transplant Recipients2015In: Bone Marrow Transplantation, ISSN 0268-3369, E-ISSN 1476-5365, Vol. 50 1, p. S77-S77Article in journal (Other academic)
  • 21.
    Westerberg, Sonja
    et al.
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Hagbom, Marie
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Rajan, Anandi
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Loitto, Vesa
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Persson, David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nordgren, Johan
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Sharma, Sumit
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Magnusson, Karl-Eric
    Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Svensson, Lennart
    Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Medicine, Karolinska Institute, Stockholm, Sweden.
    Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells2018In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, no 7, article id e00026-18Article in journal (Refereed)
    Abstract [en]

    Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells.

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