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  • 1.
    Edberg, Andreas
    et al.
    Central Hospital, Karlstad.
    Jurstrand, Margaretha
    University Hospital, Örebro.
    Johansson, Eva
    Central Hospital, Karlstad.
    Wikander, Elisabeth
    Central Hospital, Karlstad.
    Höög, Anna
    Central Hospital, Karlstad.
    Ahlqvist, Thomas
    Central Hospital, Karlstad.
    Falk, Lars
    Linköping University, Department of Biomedicine and Surgery, Division of dermatology and venereology. Linköping University, Faculty of Health Sciences.
    Skov Jensen, Jørgen
    Statens Serum Institute, Denmark.
    Fredlund, Hans
    University Hospital, Örebro.
    A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women2008In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 57, p. 304-309Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M. genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3 %) of true-positive specimens by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.

  • 2. Grahn, Niclas
    et al.
    Hmani-Aifa, Mounira
    Fransén, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis2005In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 54, no 11, p. 1031-1035Article in journal (Refereed)
    Abstract [en]

    Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. © 2005 SGM.

  • 3.
    Jurstrand, Margaretha
    et al.
    Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Jensen, Jørgen Skov
    Mycoplasma Laboratory, Department of Respiratory Infections, Meningitis and Sexually Transmitted Infections, Statens Serum Institut, Copenhagen, Denmark .
    Fredlund, Hans
    Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Falk, Lars
    Outpatient Sexually Transmitted Disease Clinic, Department of Dermatovenereology, Örebro University Hospital, Örebro, Sweden.
    Mölling, Paula
    Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay2005In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 54, no 1, p. 23-29Article in journal (Refereed)
    Abstract [en]

    A real-time LightCycler PCR (LC-PCR) with hybridization probes for detection of Mycoplasma genitalium in endocervical and first void urine specimens was developed and compared to a conventional PCR. The primers for both assays were identical and designed to amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium. The LC-PCR assay had a detection limit of < 5 bacterial genomes per reaction when dilutions of genomic DNA from a type strain of M. genitalium were tested. First void urine from 398 men and first void urine and endocervical specimens from 301 women attending an STD clinic were analysed by LC-PCR and by the conventional PCR. Using the conventional PCR as reference, the LC-PCR had a specificity of 99.7 % and a sensitivity of 72.2 % for the detection of M. genitalium in first void urine samples from men. There was no significant difference in the performance of the LC-PCR assay compared to the conventional PCR when endocervical swabs were considered (58 and 65 %, respectively) or with a set of endocervical swab/urine specimens for which the LC-PCR assay detected 73 % of the infections (specificity = 98.6 % and sensitivity = 68.2 %) while the conventional PCR detected 85 % of the infections. With female urine specimens there was a significant difference between the two assays (38 and 73 %, respectively; P = 0.01 McNemar's test). This illustrates the need to analyse both endocervical and urine specimens, because M. genitalium DNA was detected in only one of the two specimens in a great number of the M. genitalium-infected women. The lower sensitivity of the LC-PCR assay was probably caused by a combination of inhibition and limitations regarding the amount of template DNA. The LC-PCR assay was easy to perform and the simultaneous amplification and detection eliminated the need for further handling of PCR products. With improvement in sample preparation methods and increased volumes of the template DNA, the LC-PCR assay could be a useful routine diagnostic method.

  • 4.
    Monstein, Hans-Jurg
    et al.
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Tiveljung, Annika
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Kraft, C. H.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Borch, Kurt
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Jonasson, Jon
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis2000In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 49, no 9, p. 817-822Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.

  • 5.
    Tiveljung, Annika
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Borch, Kurt
    Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Surgery in Östergötland.
    Jonasson, Jon
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Microbiology.
    Mårdh, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Petersson, Fredrik
    Ryhov Hospital, Jönköping.
    Monstein, Hans-Jürg
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Microbiology.
    Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?1998In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 47, no 8, p. 695-704Article in journal (Refereed)
    Abstract [en]

    The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.

  • 6.
    Tiveljung, Annika
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Söderholm, Johan D.
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Olaison, Gunnar
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Jonasson, Jon
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Presence of eubacteria in biopsies from Crohn's disease inflammatory lesions as determined by 16S rRNA gene-based PCR1999In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 48, no 3, p. 263-268Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

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