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  • 1.
    Ahlstrand, Erik
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital. Department of Medicine, Hematology, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Persson, Lennart
    Örebro University Hospital. Department of Infectious diseases, Örebro University Hospital, Örebro, Sweden.
    Mölling, Paula
    Örebro University Hospital. Department of Laboratory Medicine, Örebro University Hospital, Örebro, Sweden.
    Tidefelt, Ulf
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Söderquist, Bo
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Infectious diseases & Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Evaluation of a PCR method to determine the clinical significance of blood cultures with Staphylococcus epidermidis in patients with hematological malignancies2014In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 122, no 6, p. 539-544Article in journal (Refereed)
    Abstract [en]

    The aim was to investigate whether the detection and quantification of Staphylococcus epidermidis DNA in blood could distinguish S. epidermidis blood stream infections (BSIs) from blood culture contaminations in patients with hematological malignancies. The hld gene was chosen to identify S. epidermidis DNA and DNA in blood samples was detected by real-time PCR. Blood samples were obtained simultaneously with blood cultures positive for S. epidermidis (n = 30), during blood culture-negative episodes (n = 10) and episodes of bacteremia with other bacteria than S. epidermidis (n = 4) and from healthy blood donors (n = 10). In addition, DNA from S. epidermidis and a selection of other bacterial species were analyzed. Three different sets of criteria were used to classify episodes with positive blood cultures with S. epidermidis as BSIs or contaminations. All DNA preparations from S. epidermidis (n = 48) were hld-positive, but other bacterial species (n = 13) were negative. Sixteen (53%) of 30 blood samples from patients with blood cultures positive for S. epidermidis were hld-positive, but none of the controls. There was no clear association between a positive hld PCR and episodes interpreted as BSIs. In conclusion, hld PCR failed to distinguish S. epidermidis BSIs from blood culture contaminations in patients with hematological malignancies.

  • 2.
    Bäckman, Anders
    et al.
    Örebro University, School of Medical Sciences. Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Makdoumi, Karim
    Örebro University, School of Medical Sciences. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden; Centre for Health Care Sciences, Örebro University Hospital, Örebro, Sweden.
    Mortensen, Jes
    Department of Ophthalmology, Ryhov County Hospital, Jönköping, Sweden.
    Crafoord, Sven
    Örebro University, School of Medicine, Örebro University, Sweden. Örebro University Hospital. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    The efficiency of cross-linking methods in eradication of bacteria is influenced by the riboflavin concentration and the irradiation time of ultraviolet light2014In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 92, no 7, p. 656-661Article in journal (Refereed)
    Abstract [en]

    Purpose: To determine bacterial eradication using numerous riboflavin concentrations and different ultraviolet light A (UVA) radiant and exposure time in an experimental model.

    Methods: Dilutions of Staphylococcus epidermidis were mixed with riboflavin at varying concentrations (0.007-0.09%). Effects on bacterial growth were evaluated after 0, 3, 6, 30 and 60min of UVA exposure (irradiance 30 and 3mW/cm(2)). Standard settings of UVA were compared with high-power UVA approach. Different fluid thicknesses of the exposed dilutions were also examined to improve the model.

    Results: Bacterial eradication (%) was increased after 60 compared with 30min of UVA exposure for concentrations of 0.03-0.07% but not for 0.09% riboflavin. There was a significant difference between the efficacy between 0.03 and 0.09% and eradication dropped from 80% to 50% (p=0.01). A correlation could be calculated for the amount of riboflavin at 60min of UVA and the ability to kill bacteria (p=0.01). The antibacterial effect was more pronounced when the tested bacterial suspension thickness was reduced. High-power UVA method was less potent in microbial elimination, eradicating only 60% of bacteria after 6min versus 97-99% after 60min in the low-power setting, compared with respective controls (p=0.02).

    Conclusions: In these in vitro experiments, a longer UVA exposure time in combination with lower riboflavin levels were found to be favourable in killing bacteria as compared to the standard cross-linking settings. Further studies are needed to evaluate the clinical relevance of these findings.

  • 3.
    Cajander, Sara
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Tina, Elisabet
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital. Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Strålin, Kristoffer
    Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden; Department of Infectious Diseases, Karolinska University Hospital, Huddinge, Sweden.
    Söderquist, Bo
    Örebro University, School of Medicine, Örebro University, Sweden. Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Källman, Jan
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Preliminary results in quantitation of HLA-DRA by real-time PCR: a promising approach to identify immunosuppression in sepsis2013In: Critical Care, ISSN 1364-8535, E-ISSN 1466-609X, Vol. 17, no 5, article id R223Article in journal (Refereed)
    Abstract [en]

    Introduction: Reduced monocyte human leukocyte antigen (mHLA)-DR surface expression in the late phase of sepsis is postulated as a general biomarker of sepsis-induced immunosuppression and an independent predictor of nosocomial infections. However, traditional monitoring of mHLA-DR by flow cytometry has disadvantages due to specific laboratory requirements. An mRNA-based HLA-DR monitoring by polymerase chain reaction (PCR) would improve the clinical usage and facilitate conduction of large multicenter studies. In this study, we evaluated an mRNA-based HLA-DR monitoring by quantitative real-time PCR (qRT-PCR) as an alternative method to traditional flow cytometry.

    Methods: Fifty-nine patients with sepsis and blood culture growing pathogenic bacteria were studied. Blood samples were collected at day 1 or 2 after admission, for measurement of mHLA-DR by flow cytometry and mRNA expression of HLA-DRA and class II transactivator (CIITA) by qRT-PCR. Blood samples from blood donors were used as controls (n = 30).

    Results: A significant reduced expression of mHLA-DR, HLA-DRA, and CIITA was seen in septic patients compared with controls. HLA-DRA mRNA level in whole blood was highly correlated with surface expression of mHLA-DR.

    Conclusions: Patients with sepsis display a diminished expression of HLA-DR at the monocyte surface as well as in the gene expression at the mRNA level. The mRNA expression level of HLA-DRA monitored by qRT-PCR correlates highly with surface expression of HLA-DR and appears to be a possible future biomarker for evaluation of immunosuppression in sepsis.

  • 4.
    Cajander, Sara
    et al.
    Örebro University, School of Medical Sciences. Örebro University Hospital.
    Stammler Jaliff, B.
    Karolinska University Hospital, Stockholm, Sweden.
    Somell, A.
    Karolinska University Hospital, Stockholm, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Alpkvist, H.
    Karolinska University Hospital, Stockholm, Sweden.
    Özenci, V.
    Karolinska University Hospital, Stockholm, Sweden.
    Wernerman, J.
    Karolinska University Hospital, Stockholm, Sweden.
    Strålin, K.
    Karolinska University Hospital, Stockholm, Sweden.
    HLA-DRA and CD74 on intensive care unit admission related to outcome in sepsis2018In: Critical Care, E-ISSN 1466-609X, Vol. 22, no Suppl. 1, article id 82Article in journal (Refereed)
    Abstract [en]

    Introduction: mRNA expressions of the major histocompatibility complex class II-related genes HLA-DRA and CD74 have been found to be promising markers for sepsis-induced immunosuppression. In the present study we aimed to study how expression of HLA-DRA and CD74 on intensive care unit (ICU) admission were related to death and/or secondary infections in patients with sepsis.

    Methods: During a full year adult patients admitted to the ICU of Karolinska University Hospital Huddinge were consecutively subjected to blood sampling within 1 hour from ICU admission. Patients treated with antibiotic therapy were eligible for inclusion. The plausibility of infection (definite, probable, possible, none) was determined based on the Centers for Diseases Control (CDC) criteria. Patients with sepsis (definite/probable/possible infection and a SOFA score increase of >=2) were screened for death within 60 days and secondary infections 48 h to 60 days after ICU admission, using the CDC criteria. HLA-DRA and CD74 mRNA expressions were determined by reverse transcription quantitative PCR.

    Results: Among 579 ICU admissions, a blood sample for RNA analysis was collected in 551 cases. Two hundred fifty-seven patients met the inclusion criteria and provided written informed consent. Sepsis was noted in 134 patients. The sepsis patients experienced death in 36 cases (27%), secondary infection in 32 cases (24%), and death and/or secondary infection in 60 cases (45%). Table 1 shows the results of HLA-DRA and CD74 expression related to death and secondary infections.

    Conclusions: The mRNA expression of HLA-DRA on ICU admission was significantly decreased in patients with sepsis who died or contracted secondary infections within 60 days. CD74 expression was not significantly decreased in patients with negative outcome.

  • 5.
    Cajander, Sara
    et al.
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Tina, Elisabet
    Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory, Örebro University Hospital, Örebro, Sweden.
    Magnuson, Anders
    Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Strålin, Kristoffer
    Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden .
    Söderquist, Bo
    Örebro University, School of Medical Sciences.
    Källman, Jan
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 5, article id e0154690Article in journal (Refereed)
    Abstract [en]

    Introduction: A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).

    Aims: The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA-mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis.

    Methods and Findings: Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1-28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1-2, HLA-DRA 0.40 (0.28-0.59) p<0.001, CIITA 0.48 (0.32-0.72) p = 0.005, mHLA-DR 0.63 (0.45-1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46-0.77) p<0.001, CIITA 0.56 (0.41-0.76) p<0.001, mHLA-DR 0.81 (0.66-1.00) p = 0.28.

    Conclusion: We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.

  • 6. Falk, Lars
    et al.
    Lindberg, Margret
    Jurstrand, Margaretha
    Örebro University, Department of Clinical Medicine.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Olcén, Per
    Fredlund, Hans
    Örebro University, School of Health Sciences.
    Genotyping of Chlamydia trachomatis would improve contact tracing2003In: Sexually Transmitted Diseases, ISSN 0148-5717, E-ISSN 1537-4521, Vol. 30, no 3, p. 205-210Article in journal (Refereed)
    Abstract [en]

    Background: The reported number of genital Chlamydia trachomatis infections has increased 15% annually since 1997 in Sweden. Inaccurate partner notification might be one reason.

    Goal: The goals were to determine if genotyping of C trachomatis would improve partner notification and to study the duration of infection.

    Study Design: Sexual networks were constructed. C trachomatis isolates from 231 individuals attending the Örebro STD clinic during 1 year were typed by sequencing of the omp1 gene.

    Results: All individuals were traced and diagnoses were established in 30 of 161 networks. More than one genotype was seen in seven networks. The mean duration of C trachomatis infection in each network was calculated to be 23 weeks.

    Conclusion: Genotyping could be a useful tool in partner notification when there are discrepant or uncommon genotypes. Limited clinic catchment areas create information difficulties that obstruct accurate contact tracing.

  • 7.
    Gili, Nasser J.
    et al.
    Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Norén, Torbjörn
    Örebro University, School of Medical Sciences. Department of Laboratory Medicine.
    Törnquist, Eva
    Department of Laboratory Medicine, Örebro University Hospital, Örebro, Sweden.
    Crafoord, Sven
    Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory.
    Preoperative preparation of eye with chlorhexidine solution significantly reduces bacterial load prior to 23-gauge vitrectomy in Swedish health care2018In: BMC Ophthalmology, ISSN 1471-2415, E-ISSN 1471-2415, Vol. 18, article id 167Article in journal (Refereed)
    Abstract [en]

    Background: Bacteria in the conjunctiva present a potential risk of vitreous cavity infection during 23-gauge pars plana vitrectomy (PPV). Current preoperative procedures used in Sweden include irrigation with chlorhexidine solution (CHX) 0.05% only and no iodine solutions. We evaluated the bacterial diversity and load before and after this single antibacterial measure.

    Methods: In a prospective, consecutive cohort we investigated bacterial growth in samples from 40 eyes in 39 consecutive individuals subjected to vitrectomy. A conjunctival specimen was collected from each preoperative patient before and after irrigating of eye with CHX, 0.05% solution. Iodine was not used during any part of the surgery. One drop of chloramphenicol was administered prior to surgery. Samples from vitreous cavity were collected at the beginning and end of vitrectomy. All conjunctival specimens were cultured for different species and quantified using colony forming units (CFU).

    Results: There was a significant 82% reduction in the total number of CFUs for all bacteria in all eyes (P < 0.0001), and 90% reduction for coagulase negative staphylococci (CoNS) alone (P = 0.0002). The number of eyes with positive bacterial growth in conjunctival samples decreased from 33 to 18 after irrigation with CHX (P = 0.0023). The most common bacteria prior to surgery were CoNS (70%), Propionibacterium acnes (55%) and Corynebacterium species (36%). No case of post-vitrectomy endophthalmitis was reported during mean follow-up time, which was 4.6 +/- 2.3 (range; 1.5 to 9) months.

    Conclusions: Patients undergoing PPV harbored bacteria in conjunctiva capable of causing post-vitrectomy endophthalmitis. Preoperative preparation with CHX significantly reduced the bacterial load in the conjunctival samples subsequently leading to very low inoculation rates in recovered vitreous samples. Thus, CHX used as a single disinfectant agent might be an effective preoperative procedure for eye surgery in Sweden. This is a relatively small study but the results could be a reference for other intraocular surgeries.

  • 8.
    Jurstrand, Margaretha
    et al.
    Örebro University, Department of Clinical Medicine.
    Falk, Lars
    Fredlund, Hans
    Örebro University Hospital.
    Lindberg, Margareta
    Olcén, Per
    Andersson, Sören
    Persson, Kenneth
    Albert, Jan
    Bäckman, Anders
    Örebro University Hospital.
    Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden2001In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 39, no 11, p. 3915-3919Article in journal (Refereed)
    Abstract [en]

    A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the omp1 gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The omp1 gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (n = 6) and H (n = 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different C. trachomatis strains. This omp1 genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted Chlamydia infections.

  • 9.
    Makdoumi, Karim
    et al.
    Örebro University, School of Medical Sciences. Department of Ophthalmology , Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Photodynamic UVA-riboflavin bacterial elimination in antibiotic-resistant bacteria2016In: Clinical and Experimental Ophthalmology, ISSN 1442-6404, E-ISSN 1442-9071, Vol. 44, no 7, p. 582-586Article in journal (Refereed)
    Abstract [en]

    Background: To evaluate the bactericidal effect of clinical ultraviolet A (UVA) settings used in photoactivated chromophore for infectious keratitis (PACK)-collagen cross-linking (CXL) in antibiotic-resistant and non-resistant bacterial strains.

    Methods: Well-characterized bacterial strains from clinical isolates, without and with antibiotic resistance, were studied in a pairwise comparison. The evaluated pathogens were Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. Bacteria were dispersed in PBS and diluted to a concentration of approximately 4x10(5)/ml. Riboflavin was added to a concentration of 0.01%. By spreading the solution on a microscope slide, a fluid film layer, with a thickness of around 400mm, was formed and UVA exposure followed. Eight separate exposures were made for each strain (n=8). The degree of elimination in resistant and non-resistant pathogens was compared.

    Results: The bactericidal efficacy of exposure differed between the tested microorganisms, and the mean elimination ranged between 60 and 92%, being most extensive in both of the evaluated Pseudomonas strains and least in the E. faecalis strains. Similar reductions were seen in antibiotic-resistant and non-resistant strains, with the exception of S. aureus, in which the resistant strain metchicillin-resistant Staphylococcus aureus (MRSA) was eradicated in a greater extent than the non-resistant strain (P=0.030).

    Conclusion: UVA-riboflavin settings used in PACK-CXL are effective in reducing both antibiotic-resistant and non-resistant bacteria. Antibiotic resistance does not appear to be protective against the photooxidative exposure.

  • 10.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Crafoord, Sven
    Örebro University, School of Medical Sciences.
    Response to: Bactericidal effect of photo-activated riboflavin using UVA2010In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 248, no 5, p. 757-758Article in journal (Refereed)
  • 11.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences.
    Bäckman, Anders
    Mortensen, Jes
    Crafoord, Sven
    Evaluation of antibacterial efficacy of photo-activated riboflavin using ultraviolet light (UVA)2010In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 248, no 2, p. 207-212Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: To evaluate the antibacterial efficacy of photo-activated riboflavin using Ultraviolet A (UVA) on three bacterial strains commonly detected in keratitis. METHODS: Three bacterial strains (Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa) were cultured on blood/hematin-agar plates and dispersed in PBS. Dispersion was done of 10 microl of bacterial stock-solutions in 90 microl of RPMI, where different riboflavin molarities had been added, to achieve a bacterial concentration of 1-4 x 10 (4)/ml. Riboflavin end molarities before illumination were 0, 100, 200, 300 and 400 microM. Each solution had a negative control. The solutions were illuminated with UVA (365 nm) for 30 minutes (5.4 J/cm(2)) and then continued for a total time of 60 minutes (10.8 J/cm(2)). A count of CFU was conducted after incubation and results compared. RESULTS: In all tested strains, a slight decrease of bacteria was seen when exposed to UV for 30 minutes. A doubling of the UV dose showed a marked decrease of bacterial count in all bacteria tested. The combination of UV and riboflavin showed a more extensive reduction of CFU, confirming an interaction effect between UV and riboflavin. CONCLUSION: Riboflavin photo-activation using UVA (365 nm) can achieve an extensive eradication of bacteria, and the combination is more potent in reducing bacterial number than UV alone.

  • 12.
    Makdoumi, Karim
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Ophthalmology, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Clinical Research Centre, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Mortensen, Jes
    Dept. Ophthalmology, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Magnuson, Anders
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Clinical Epidemiology and Biostatistic Unit, Örebro University Hospital, Örebro, Sweden.
    Crafoord, Sven
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Comparison of UVA- and UVA/riboflavin-induced growth inhibition of Acanthamoeba Castellanii2013In: Graefe's Archives for Clinical and Experimental Ophthalmology, ISSN 0721-832X, E-ISSN 1435-702X, Vol. 251, no 2, p. 509-514Article in journal (Refereed)
    Abstract [en]

    Purpose To investigate whether ultraviolet light (UVA) at 365 nm can inhibit/eliminate Acanthamoeba growth and if riboflavin would potentiate such an association.

    Method: Acanthamoeba castellanii in a fluid medium with a concentration of approximately 1.7 x 10(4) protozoa/ml were prepared with (0.01 %) and without riboflavin. Exposure of UVA (dose 5.475 J/cm(2)) took place twice, with each illumination period followed by culturing of 10 mu l in peptone yeast-extract glucose (PYG) medium for 7 days. Every suspension prepared had a non-exposed control solution. Determination of Acanthamoeba was conducted daily, by count in Burker chamber days 4 through 7 after exposure. Statistical analysis was done by repeated-measurement ANOVA and post-hoc analysis for unpaired samples.

    Results: The exposure of ultraviolet light resulted in an inhibited growth of Acanthamoeba compared to the non-exposed solutions, with a statistically significant reduction over time (p = 0.0003). The addition of riboflavin did not amplify the effect, and there were no tendencies for an interaction effect between UVA and riboflavin. The antiprotozoal effect of the UVA wavelength, utilized in CXL, is solely mediated by ultraviolet light, and riboflavin does not seem to amplify the antimicrobial efficacy.

  • 13.
    Makdoumi, Karim
    et al.
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Goodrich, Ray
    Terumo BCT Inc., Lakewood CO, USA.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Örebro University Hospital. , Örebro University Hospital, Örebro, Sweden.
    Photochemical eradication of methicillin-resistant Staphylococcus aureus by blue light activation of riboflavin2017In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 95, no 5, p. 498-502Article in journal (Refereed)
    Abstract [en]

    Purpose: To compare elimination of methicillin-resistant Staphylococcus aureus (MRSA) by exposure of blue light alone and with riboflavin.

    Methods: A reference strain of MRSA was cultured and diluted in PBS with and without riboflavin (0.01%). Fifteen microlitre was added on a microscope slide, creating a fluid layer with a thickness of around 400 microns. Both of the bacterial suspensions were exposed to blue light, and the effect between exposure with and without riboflavin was compared. Evaluation involved two different wavelengths (412 and 450 nm) of blue light with a lower (5.4 J/cm(2) ) and higher dose (approximately 28.5 J/cm(2) ). The effect of 412 nm light was also evaluated for a thicker fluid layer (1.17 mm). After exposure, colony-forming units (CFUs) were determined for each solution. All measurements were repeated eight times.

    Results: The reductions in bacteria were similar for both wavelengths. With riboflavin, a statistically significant elimination was observed for both 412 and 450 nm (p < 0.001). At both dosages, the mean reduction was more pronounced with the presence of riboflavin than without it. Using the higher dose, CFU reduction was 99% and 98%, respectively, for 412 and 450 nm light. The bactericidal efficacy was high also in the deeper fluid layer (93%, higher dose).

    Conclusion: Riboflavin enhanced the antibacterial effect on the exposed MRSA strain of blue light for both 412 and 450 nm blue light. This indicates that blue light could be considered for possible implementation in deep corneal infections.

  • 14.
    Makdoumi, Karim
    et al.
    Örebro University, School of Medical Sciences. Department of Ophthalmology, Örebro University Hospital, Örebro, Sweden.
    Hedin, Marie
    Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Clinical Research Laboratory.
    Different photodynamic effects of blue light with and without riboflavin on methicillin-resistant Staphylococcus aureus (MRSA) and human keratinocytes in vitro2019In: Lasers in Medical Science, ISSN 0268-8921, E-ISSN 1435-604X, Vol. 34, no 9, p. 1799-1805Article in journal (Refereed)
    Abstract [en]

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections in humans. Photodynamic therapy using blue light (450 nm) could possibly be used to reduce MRSA on different human tissue surfaces without killing the human cells. It could be less harmful than 300–400 nm light or common disinfectants. We applied blue light ± riboflavin (RF) to MRSA and keratinocytes, in an in vitro liquid layer model, and compared the effect to elimination using common disinfection fluids. MRSA dilutions (8 × 105/mL) in wells were exposed to blue light (450 nm) ± RF at four separate doses (15, 30, 56, and 84 J/cm2). Treated samples were cultivated on blood agar plates and the colony forming units (CFU) determined. Adherent human cells were cultivated (1 × 104/mL) and treated in the same way. The cell activity was then measured by Cell Titer Blue assay after 24- and 48-h growth. The tested disinfectants were chlorhexidine and hydrogen peroxide. Blue light alone (84 J/cm2) eliminated 70% of MRSA. This dose and riboflavin eradicated 99–100% of MRSA. Keratinocytes were not affected by blue light alone at any dose. A dose of 30 J/cm2 in riboflavin solution inactivated keratinocytes completely. Disinfectants inactivated all cells. Blue light alone at 450 nm can eliminate MRSA without inactivation of human keratinocytes. Hence, a high dose of blue light could perhaps be used to treat bacterial infections without loss of human skin cells. Photodynamic therapy using riboflavin and blue light should be explored further as it may perhaps be possible to exploit in treatment of skin diseases associated with keratinocyte hyperproliferation.

  • 15.
    Ohlin, Andreas
    et al.
    Department of Pediatrics, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Ewald, Uwe
    Women’s and Children’s Health, Uppsala University, Uppsala, Sweden .
    Schollin, Jens
    Örebro University, School of Health and Medical Sciences.
    Björkqvist, Maria
    Örebro University, School of Health and Medical Sciences. Department of Pediatrics, Örebro University Hospital, Örebro, Sweden.
    Diagnosis of neonatal sepsis by broad-range 16S real-time polymerase chain reaction2012In: Neonatology, ISSN 1661-7800, E-ISSN 1661-7819, Vol. 101, no 4, p. 241-246Article in journal (Refereed)
    Abstract [en]

    Background: The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. Objectives: To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Methods: Immediately after blood culture, a study sample of 0.5–1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. Results: 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. Conclusions: This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics.

  • 16.
    Ohlin, Andreas
    et al.
    Department of Paediatrics, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Clinical Research Centre, Örebro University Hospital, Örebro, Sweden.
    Söderquist, Bo
    Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden.
    Wingren, Sten
    Örebro University, School of Health and Medical Sciences.
    Björkqvist, Maria
    Örebro University, School of Health and Medical Sciences. Department of Paediatrics, Örebro University Hospital, Örebro, Sweden.
    Rapid typing of neonatal Staphylococcus epidermidis isolates using polymerase chain reaction for repeat regions in surface protein genes2010In: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 29, no 6, p. 699-704Article in journal (Refereed)
    Abstract [en]

    Staphylococcus epidermidis is a significant pathogen in neonatal sepsis and other nosocomial infections. For further investigations of the colonisation patterns and invasive pathways, typing methods that are applicable on large populations of bacterial isolates are warranted. In the present study, a genotyping method based on polymerase chain reaction (PCR) for the repeat regions of four genes (sdrG, sdrF, aap and sesE) that encode for bacterial surface proteins was developed and applied to a sample of well-characterised neonatal blood isolates of S. epidermidis (n = 49). The PCR products were visualised on agarose gel (sdrG, sdrF and sesE) or by fragment analysis (aap). The discriminatory index (D-index) for genotyping of the different genes was compared to genotyping by pulsed-field gel electrophoresis (PFGE). The highest D-index for the PCR-based typing methods was found for the combination of sdrF, sdrG and aap (D-index 0.94), whereas the optimal two-gene combination (sdrF and aap) resulted in a D-index of 0.92. We conclude that the described method can be used for the genotyping of large populations of S. epidermidis isolates with a sufficient discriminatory capacity, and we suggest that the combination of sdrF and aap is the most suitable to use.

  • 17.
    Rasmussen, Gunlög
    et al.
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Asfaw Idosa, Berhane
    Örebro University, School of Medical Sciences.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Monecke, Stefan
    Alere Technologies, GmbH, Jena, Germany.
    Strålin, Kristoffer
    Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
    Särndahl, Eva
    Örebro University, School of Medical Sciences.
    Söderquist, Bo
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Caspase-1 Inflammasome Activity in Patients with Staphylococcus aureus BacteraemiaManuscript (preprint) (Other academic)
  • 18.
    Rasmussen, Gunlög
    et al.
    Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden; School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Asfaw Idosa, Berhane
    Örebro University, School of Medical Sciences.
    Monecke, S.
    InfectoGnostics Research Campus Jena, Jena, Germany.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Clinical Research Laboratory.
    Strålin, Kristoffer
    Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden .
    Särndahl, Eva
    Örebro University, School of Medical Sciences.
    Söderquist, Bo
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Caspase-1 Inflammasome Activity in Patients with Staphylococcus aureus Bacteremia2019In: Microbiology and immunology, ISSN 0385-5600, E-ISSN 1348-0421, Vol. 63, no 12, p. 487-499Article in journal (Refereed)
    Abstract [en]

    The inflammasome is a multiprotein complex that mediates caspase-1 activation with subsequent maturation of the pro-inflammatory cytokines IL-1β and IL-18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. The present study therefore aimed to investigate NLRP3 inflammasome activity in 20 S. aureus bacteremia patients, by repeated measurement during the first week of bacteremia, compared with controls. Caspase-1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (Fluorescent Labelled Inhibitor of Caspase-1), while IL-1β and IL-18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, mRNA expression of NLRP3, CASP1 (pro-caspase-1) and IL1B (pro-IL-1β) was analyzed by qPCR. We found induced caspase-1 activity in innate immune cells with subsequent release of IL-18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial inter-individual variation in caspase-1 activity between S. aureus bacteremia patients. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in S. aureus bacteremia could provide support in the effort to optimize management and treatment of each individual patient.

  • 19.
    Rasmussen, Gunlög
    et al.
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Cajander, Sara
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences.
    Källman, Jan
    Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden; School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Söderquist, Bo
    Örebro University, School of Medical Sciences.
    Strålin, Kristoffer
    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden; Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden .
    Expression of HLA-DRA and CD74 mRNA in whole blood during the course of complicated and uncomplicated Staphylococcus aureus bacteraemiaManuscript (preprint) (Other academic)
  • 20.
    Rasmussen, Gunlög
    et al.
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Cajander, Sara
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden.
    Bäckman, Anders
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Clinical Research Laboratory, Örebro University Hospital, Örebro, Sweden.
    Källman, Jan
    Department of Infectious Diseases, Örebro University Hospital, Örebro University, Örebro, Sweden; Faculty of Medicine and Health, School of Medical Sciences, Örebro University, Örebro, Sweden.
    Söderquist, Bo
    Örebro University, School of Medical Sciences. Department of Infectious Diseases, Faculty of Health and Medical Sciences, Örebro University, Örebro, Sweden.
    Strålin, Kristoffer
    Faculty of Medicine and Health, School of Medical Sciences, Örebro University, Örebro, Sweden; Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden; Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
    Expression of HLA-DRA and CD74 mRNA in whole blood during the course of complicated and uncomplicated Staphylococcus aureus bacteremia2017In: Microbiology and immunology, ISSN 0385-5600, E-ISSN 1348-0421, Vol. 61, no 10, p. 442-451Article in journal (Refereed)
    Abstract [en]

    To improve management of Staphylococcus aureus bacteremia (SAB), better understanding of host-pathogen interactions is needed. In vitro studies have shown that S. aureus bacteria induce dose-dependent immunosuppression that is evidenced by reduced expression of major histocompatibility complex (MHC) class II on antigen presenting cells. Thus, the aim of this study was to determine whether expression of the MHC class II-related genes HLA-DRA and CD74 is more greatly reduced in complicated SAB, with its probable higher loads of S. aureus, than in uncomplicated SAB. Adult patients with SAB were prospectively included and blood samples taken on the day of confirmation of SAB (Day 1) and on Days 2, 3, 5 and 7. HLA-DRA and CD74 mRNA expression was determined by quantitative reverse transcription PCR. Sepsis was defined according to the Sepsis-3 classification and SAB was categorized as complicated in patients with deep-seated infection and/or hematogenous seeding. Twenty patients with SAB were enrolled and samples obtained on all assessment days. HLA-DRA and CD74 expression did not differ significantly between patients with SAB and sepsis (n=13) and those without sepsis (n=7) on any assessment day. However, patients with complicated SAB (n=14) had significantly weaker HLA-DRA expression on all five assessment days than patients with uncomplicated SAB (n=6). Additionally, they tended to have weaker CD74 expressions. Neutrophil, monocyte and leukocyte counts did not differ significantly between complicated and uncomplicated SAB. In conclusion, patients with complicated SAB show weaker HLA-DRA expression than those with uncomplicated SAB during the first week of bacteremia.

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