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  • 1.
    Karlsson, Anneli
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Dehnoei, Marjan Nosouhi
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken US.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden2012Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, nr 129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UNLABELLED: ABSTRACT:

    BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.

    RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.

    CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

  • 2.
    Karlsson, Anneli
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Nosouhi Dehnoei, Marjan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken i Östergötland.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Variation in number of cagA EPIYA-C phosphorylation motifs between cultured Helicobacter pylori and biopsy strain DNA2012Ingår i: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, nr 1, s. 175-179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

  • 3.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Karlsson, Anneli
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs2010Ingår i: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 3, nr 35Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs.

    Findings

    MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7- sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy.

    Conclusion

    Automated capillary electrophoresis and amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.

  • 4.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Karlsson, Anneli
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Assessment of the mosaic structure in the Helicobacter pylori cagA gene 3 -region using an improved polymerase chain reaction-based assay2012Ingår i: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 73, nr 3, s. 281-283Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mosaic structure of the cagA gene has been suggested to affect Helicobacter pylori CagA-associated pathogenesis. An improved polymerase chain reaction assay allowed for a rapid and detailed molecular analysis of the cagA gene 3-region in a single amplification step, followed by amplicon sequencing using universal M13 and T7 sequencing primers.

  • 5.
    Monstein, Hans-Jurg
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Molekylärbiologiska tekniklaboratoriet.
    Truedsson, M.
    Department of Clinical Sciences Malmö University.
    Ryberg, Anna
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin.
    Ohlsson, M.
    Department of Clinical Sciences Malmö University.
    Vasopressin receptor mRNA expression in the human gastrointestinal tract2008Ingår i: European Surgical Research, ISSN 0014-312X, E-ISSN 1421-9921, Vol. 40, nr 1, s. 34-40Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background/Aim: Vasopressin and oxytocin are closely related peptides, and both exert effects on the gastrointestinal function. In the present study, we wanted to map the expression of vasopressin receptor mRNAs (V1a, V1b/V3, and V2) in nontumorous tissue biopsy specimens of human gastrointestinal tract and surrounding tissues. Methods: Total and polyA+ RNAs were isolated from human tissue biopsy specimens using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA. Seminested PCR amplifications were carried out, using gene-specific V1a, V1b/V3, and V2 receptor primers. The PCR amplicons were partially sequenced to confirm their identity. Results: The present study demonstrated the expression of vasopressin receptor mRNAs in human gastrointestinal tract, pancreas, kidney, lung, brain, and ovary. The expression pattern varied between different parts of the gastrointestinal tract. In the colon ascendens, V1a receptor mRNA expression could not be detected in 3 out of 4 analyzed tissue biopsy specimens. On the other hand, all the vasopressin receptor mRNAs were expressed in all colon transversum biopsy samples. Conclusions: V1a, V1b/V3, and V2 receptor mRNAs are widely expressed throughout human gastrointestinal tract and surrounding tissues. The data obtained provide information for further mapping and determination of the physiological role of the vasopressin receptor mRNA expression in normal and tumorous tissues.

  • 6.
    Redéen, Stefan
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Petersson, Fredrik
    Ryhov Hospital.
    Eriksson, Olle
    Linköpings universitet, Institutionen för datavetenskap, Statistik. Linköpings universitet, Filosofiska fakulteten.
    Nägga, Katarina
    Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Geriatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Närsjukvården i centrala Östergötland, Geriatriska kliniken.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Homocysteine Levels in Chronic Gastritis and Other Conditions: Relations to Incident Cardiovascular Disease and Dementia2010Ingår i: DIGESTIVE DISEASES AND SCIENCES, ISSN 0163-2116, Vol. 55, nr 2, s. 351-358Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background Homocysteine levels in circulation are determined by several factors and hyperhomocysteinemia is reportedly associated with cardiovascular diseases and dementia. The aim of this study is to determine the relation of chronic gastritis and other conditions to homocysteine levels and their relation to incident cardiovascular diseases and dementia. Methods An adult population-based cohort (N = 488) was screened for H. pylori infection, gastro-duodenitis ( endoscopic biopsies), disease history, and lifestyle factors. Blood samples were analyzed for pepsinogen I and II ( gastric function), vitamin B12, folate, homocysteine, and cystatin C ( renal function). The methylenetetrahydrofolate reductase C677T polymorphism reportedly associated with hyperhomocysteinemia was analyzed by pyrosequencing. Incident cardiovascular diseases and dementia were monitored during a median follow-up interval of 10 years. Results At baseline, there was a positive relation of S-homocysteine to male gender, age, S-cystatin C, methylenetetrahydrofolate reductase 677TT genotype and atrophic gastritis. During follow-up, cardiovascular diseases occurred in 101/438 and dementia in 25/488 participants, respectively. Logistic regression analysis ( adjusting for gender, age at baseline, follow-up interval, BMI, smoking, alcohol consumption, NSAID use, P-cholesterol, and P-triglycerides) showed an association of S-homocysteine higher than 14.5 mu mol/l to cardiovascular diseases (OR 2.05 [95% c.i. 1.14-3.70]), but not to dementia overall. Conclusions Gender, age, vitamin B12, folate, renal function, atrophic gastritis and the methylenetetrahydrofolate 677TT genotype were significant determinants of homocysteine levels, which were positively related to incident cardiovascular diseases.

  • 7.
    Ryberg, Anna
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi.
    Borch, Kurt
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Centrum för kirurgi, ortopedi och cancervård, Kirurgiska kliniken i Östergötland.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinoma2011Ingår i: BMC research notes, ISSN 1756-0500, Vol. 4, s. 131-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer.

    FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas.

    CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.

  • 8.
    Ryberg, Anna
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Borch, Kurt
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Kirurgiska kliniken i Östergötland med verksamhet i Linköping, Norrköping och Motala.
    Sun, Yi-Qian
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
    Monstein, Hans-Jürg
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi.
    Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA2008Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, s. 175-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods.

    Results: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions.

    Conclusion: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.

  • 9.
    Ryberg, Anna
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Medicinsk mikrobiologi.
    Olsson, Crister
    Malmö University Hospital.
    Ahrné, Siv
    Lund University.
    Monstein, Hans-Jürg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk mikrobiologi.
    Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates2011Ingår i: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, nr 2, s. 183-188Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)5- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)5 and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)5 and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)5 and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.

  • 10.
    Woksepp, Hanna
    et al.
    Kalmar County Hospital.
    Jernberg, Cecilia
    Swedish Institute for Communicable Disease Control, Solna.
    Tärnberg, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Brolund, Alma
    Swedish Institute for Communicable Disease Control, Solna.
    Nordvall, Michaela
    Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Olsson-Liljequist, Barbro
    Swedish Institute for Communicable Disease Control, Solna.
    Tegmark Wisell, Karin
    Swedish Institute for Communicable Disease Control, Solna.
    Monstein, Hans-Jurg
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk mikrobiologi.
    Nilsson, Lennart E
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk mikrobiologi. Linköpings universitet, Hälsouniversitetet.
    Schön, Thomas
    Kalmar County Hospital.
    High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli2011Ingår i: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 49, nr 12, s. 4032-4039Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

  • 11.
    Woksepp, Hanna
    et al.
    Kalmar County Hospital, Sweden; Linnaeus University, Sweden.
    Ryberg, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Billstrom, Hanna
    Public Health Agency Sweden, Sweden.
    Hällgren, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Infektionskliniken i Östergötland.
    Nilsson, Lennart E
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet.
    Marklund, Britt-Inger
    Linnaeus University, Sweden.
    Olsson-Liljequist, Barbro
    Public Health Agency Sweden, Sweden.
    Schön, Thomas
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Hälsouniversitetet. Kalmar County Hospital, Sweden.
    Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens2014Ingår i: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 52, nr 12, s. 4339-4342Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.

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