Statens Serum Institut Research Articles

Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells

Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Evaluation of an immunochromatographic assay for rapid identificationof Mycobacterium tuberculosis complex in clinical isolates

Identification of Mycobacterium tuberculosis complex (MTC) remains slow. Over the years, several new technologies have been proposed to accelerate and simplify the detection of MTC. In this context, we evaluated an immunochromatographic assay (ICA) (BIO-LINE SD Ag MPT64 TB) for rapid identification of MTC, based on detection of a specific MPT64 antigen of MTC. We have tested it on i) mycobacterial cultures: 210 MTC strains and 28 nontuberculous mycobacteria; ii) M. bovis bacille Calmette–Guérin strain SSI (Statens Serum Institut, Denmark); and iii) 22 microorganisms other than mycobacteria, isolated from cultures. We concluded that this kit has an excellent specificity (100%) and sensitivity (99%) from isolated cultures. The ICA (BIO-LINE SD Ag MPT64 TB) allows excellent MTC identification from clinical isolates. It is a rapid, simple, and inexpensive test, and has a definite contribution in the rapid laboratory diagnosis of tuberculosis.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFLP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Evaluation of three swab transport systems for the maintenance of clinically important bacteria in simulated mono- and polymicrobial samples

In this study, three swab transport systems were evaluated: M40 Transystem, Amies broth with a relatively new type of swab (both Copan Diagnostics, Corona, CA, USA), and SSI transportmedium (Statens Serum Institut, Copenhagen Denmark). The CLSI M40-A standard procedures and 11 culture collection strains were used. The transport systems were tested at room temperature for holding times of 0, 24, and 48 h, and both mono- and polymicrobial samples were included. After 24 h of simulated transportation, all systems were able to maintain the viability of all organisms tested. SSI transportmedium exhibited the lowest maintaining ability, whereas the two Copan systems were the most growth-promoting system. In polymicrobial samples, this latter feature was a problem. At 48 h, no transport system could maintain the viability of all strains, and the recovery rates differed depending on organism and device. The species most difficult to recover in all the three systems was Neisseria gonorrhoeae. When selecting a swab transport system, consideration must be given to the sample type, the conditions that prevail locally, and the performance in the clinical setting.

A specific skin test: the best for both worlds?

For a new tuberculin skin test (TST) to be considered useful, it must meet several requirements including safety, a readable skin response, affordability, a high predictive value, and it cannot easily sensitize, depend on patient characteristics, nor depend on the Mycobacterium tuberculosis isolate. After review of previously conducted animal model studies, we designed a double-blind randomized Phase 1 study comparing recombinant dimer ESAT-6 (rdESAT-6, Statens Serum Institut, Copenhagen, Denmark) to tuberculin as a skin test reagent in the diagnosis of TB. The goals of the study were to assess the safety of an intradermal method of administering the test and to determine the appropriate human dose. The study compared the administration of 2.0 TU PPD (standard dose of RT 23) to four doses of rdESAT-6 (0.01, 0.1, 1, and 10μg). The protocol also called for intra-subject randomization of the left and right forearm. Participants were enrolled in eight different groups. Four groups comprised five healthy controls each for the purpose of testing safety and sensitization issues. The other four groups comprised patients who had been treated for TB in order to determine safety and optimal dose. Several exclusion criteria were applied, including individuals who had received a TST test within the past year or had a known immune deficiency. During the 28-day trial, the guidelines called for 2 hours of close observation after administering the test on Day 0; clinical parameters, photography on Days 1 to 4 with quantiFERON testing of control subjects on Day 2 and lab and urine tests on Day 4; diary keeping on Days 5 to 28 with physical exam, lab and urine tests on Day 28 and quantiFERON testing of control groups. In control groups, all four dosages of rdESAT-6 were administered with no serious side effects; transient redness occurred at 24 hours at the 10 μg dose; and no indication of sensitization was measured in vitro. In the treated TB group, only doses of 0.01 and 0.1 of rdESAT-6 were well-tolerated; equivalent responses to PPD and 0.1 μg rdESAT-6 were observed. Participants reported significant local side effects at 1 μg of rdESAT-6. Results indicate that the new skin test is safe, produced a readable skin test response, did not easily sensitize, and is robust in all practical aspects. Further studies must be conducted on subjects ranging from individuals with active and latent TB, children, pregnant women, and patients who are immunocompromised.

The International Circumpolar Surveillance Interlaboratory Quality Control Program for Streptococcus pneumoniae , 1999 to 2008

The International Circumpolar Surveillance (ICS) Program was initiated in 1999 to conduct population-based surveillance for invasive pneumococcal disease in select regions of the Arctic. An interlaboratory quality control (QC) program for pneumococcal serotyping and antibiotic susceptibility testing was incorporated into ICS by reference laboratories in northern Canada (Laboratoire de Santé Publique du Québec [LSPQ] in Sainte-Anne de Bellevue, Québec; National Centre for Streptococcus [NCS] in Edmonton, Alberta) and Alaska (Arctic Investigations Program [AIP]). The World Health Organization's Collaborating Centre for Reference and Research on Pneumococci at the Statens Serum Institute (SSI) in Copenhagen, Denmark, joined the QC program in 2004. The Iceland Reference Laboratory (IRL) in Reykjavik, Iceland, joined the QC program in 2006, but due to small sample sizes, data from IRL are not included in this report. From 1999 through 2008, 190 isolates were distributed among four laboratories (AIP, NCS, LSPQ, and SSI). The overall serotype concordance was 95.8%, and the overall serogroup concordance was 97.4%. The overall modal MIC concordance for testing by broth microdilution (BMD) and agar dilution was >96% for all the antibiotics except erythromycin (92.1%) and clindamycin (89.5%). MIC comparisons between the Etest and BMD resulted in lower concordance for erythromycin (73.9%), clindamycin (65.5%), and trimethoprim-sulfamethoxazole (80%); however, categorical concordance (susceptible, resistant) remained high at 98.6%, 89.1%, and 90.9%, respectively. Our data demonstrate a high degree of correlation of serotyping and antimicrobial susceptibility testing results between four participating laboratories.

Practical usage of computer-supported outbreak detection in five European countries

This paper discusses computer-supported outbreak detection using routine surveillance data, as implemented at six institutes for infectious disease control in five European countries. We give an overview of the systems used at the Statens Serum Institut (Denmark), Health Protection Agency (England, Wales and Northern Ireland), Robert Koch Institute (Germany), Governmental Institute of Public Health of Lower Saxony (Germany), National Institute for Public Health and the Environment (the Netherlands) and Swedish Institute for Infectious Disease Control (Sweden). Despite the usefulness of the algorithms or the outbreak detection procedure itself, all institutes have experienced certain limitations of the systems. The paper therefore concludes with a list of recommendations for institutes planning to introduce computer-supported outbreak detection, based on experiences on the practical usage of the systems. This list--which concerns usability, standard operating procedures and evaluation--might also inspire improvements of systems in use today.

How Many Factor Antisera Can Be Found for Serogroup 6 Streptococcus pneumoniae ?

We read the article of Jacobs et al. (3) with interest. The authors reported they could identify serotype 6C Streptococcus pneumoniae using factor 6d, which was developed by Statens Serum Institut, Copenhagen, Denmark. We immunized a rabbit with one serotype 6C isolate identified with a monoclonal antibody (MAb6C) in the U.S. lab in which the new type was found. Based on absorption with serotypes 6A and 6B and the Quellung test (6), we got the specific antiserum for MAb6C after absorption with 6A and 6B. The antiserum identified 10 serotype MAb6C isolates among 91 traditional serotype 6A isolates, just like the multibead assay. We concluded that serotype 6C could be set up in the Denmark typing system because it could have a new factor antigen, 6X1 (5). More importantly, it must have a new antigenic formula, 6a, 6b, 6X1, which showed it must be a new serotype (2, 5). But we found the antiserum was not specific for serotype 6C in further study. The antiserum could be used to distinguish new type 6D from traditional 6B isolates. Six such isolates were identified among 111 isolates typed as 6B before (unpublished data). Type 6D was then confirmed using a PCR method that was described previously (4). The antiserum should be specific for serotypes 6C and 6D, and it was reported that the two serotypes have replacements similar to those in the capsular genes of serotype 6A and 6B (1). It was not unexpected that the two new types have the same antigen. We named the new factor antiserum 6X1 instead of 6d in our publication (5) because we did not determine the antiserum with all known serotype antigens (7). Meanwhile, we found that factor 6b, after absorption completely by serotype 6C, was still against serotype 6A, which means factor 6b might be divided into two factors. The specific antigen for 6A had already been shown by the multibead assay, which showed that serotype 6A bound to both monoclonal antibodies 6Aα and 6Aβ whereas 6C bound to only one monoclonal antibody, 6Aβ (8). The multibead assay based on monoclonal antibodies is an effective method to identify the serotypes of S. pneumoniae, but it could cause discrepancy because the monoclonal antibodies were developed from mice, whereas the traditional diagnostic antisera were developed from rabbits. We recommend, therefore, that the names of serotypes based on monoclonal antibodies contain special added markers, for example, MAb6A, MAb6B or m6A, m6B, etc., instead of being simply 6A, 6B, and 6C.

Antinuclear antibodies: A contemporary nomenclature using HEp-2 cells

The choice of terms used to describe indirect immunofluorescence (IIF) staining patterns of autoantibodies binding to HEp-2 cells is at present quite varied and disordered because no accurate consensus on names and descriptions exist. The aim of our study was to propose a logical and ordered IIF classification taxonomy based on 29 different selected IIF patterns. In a preliminary project carried out at Statens Serum Institut it was first shown by use of a software programme named DOORS developed by Percepton Ltd, that reading of digitized images of HEp-2 patterns on an LCD monitor could be used instead of traditional microscopy. Digitized images of HEp-2 patterns were then used in the EU supported project named CANTOR (June 1998–July 2000) aiming to reach consensus among three clinical immunology expert centres and collaborating to attain a classification version that could be used to qualitatively and quantitatively test and train image recognitions skills of laboratory technicians against expert consensus. The usability of this classification version was then tested in a course consisting of training and certification. The conclusion was that participants in the training programme clearly increased their perceptive skills using images, terms, descriptions and the graphic and statistic tools in the self-administered DOORS programme and that software-assisted training could achieve a common and accurate level of visual pattern interpretation. All results from this project were reported to the European Commission but have not previously been published in scientific literature. This communication presents the final results of agreed image classifications.

Congenital toxoplasmosis—a report on the Danish neonatal screening programme 1999–2007

This paper reports on the national neonatal screening programme for congenital toxoplasmosis (CT) in Denmark conducted from 1999 to 2007, including background, basis for initiation of screening, methods, results, and finally reasons for the discontinuation of the screening. A nationwide screening was conducted at Statens Serum Institut, including >98% newborns, and using filter paper eluates (Guthrie card, PKU card) obtained from newborns 5-10days old. These were analysed for Toxoplasma gondii-specific antibodies (IgM), and if positive, then IgM (ISAGA). Confirmatory serology was performed on children and their mothers (IgM, IgG, IgA, dye test) where infection was suspected, and children with suspected or confirmed CT initiated a 3-month treatment regimen with pyrimethamine, sulfadiazine and folinic acid supplements. Selective cohorts were followed with regard to developmental and clinical outcome. A total of 100 children were diagnosed with CT in the screening period, and only 2 cases were detected outside of the screening programme. CT prevalence was 1.6 per 10,000 live-born infants. Follow-up studies showed new retinochoroidal lesions in affected children despite treatment. Screening was terminated August 2007, after it became apparent that no benefit of treatment could be shown. CT was evaluated using a Danish adaptation of the Uniform Screening Panel (ACMG), showing CT as an unlikely candidate for screening today. Whereas results might be comparable with other low-endemic countries with similar strains of T. gondii, neonatal screening and treatment might offer different results in regions with either high prevalence or different strains of T. gondii.

Cross-Reactivity of Current Serogroup 6 Factor Sera from Statens Serum Institut with the Recently Described Pneumococcal Serotype 6D

A recent study by Jacobs et al. (3) examined the specificity of a newly introduced pneumococcal capsule-specific antiserum (factor serum 6d) manufactured by Statens Serum Institut (SSI) in Denmark. The factor serum 6d was designed to be specific for serotype 6C, a newly described pneumococcal serotype (5), and was to complement factor sera 6b and 6c, which are designed to be specific for pneumococcal serotypes 6A and 6B, respectively. While Jacobs et al. confirmed the designed specificity of the factor serum 6d for serotype 6C using the standard Quellung reaction, the study did not examine the specificity of the factor serum for the novel 6D serotype, which was previously predicted (2) but was only recently found in nature (1, 4). We have therefore investigated the specificity of the currently available serogroup 6 factor sera with serotype 6D using the standard Quellung reaction, an agglutination assay, and a flow cytometric assay. The three SSI serogroup 6 rabbit factor sera (6b [lot L6b22A1], 6c [lot H6c11E1], and 6d [lot L6d11C1]) were purchased from Mira Vista Diagnostics in March of 2010. The 6b factor serum was manufactured using the protocol revised in January 2009 and was absorbed with serotype 6C according to SSI (personal communication). Initially, we tested the factor sera using the Quellung reaction and four pneumococcal strains, TIGR6A, -B, -C, and -D, which are artificially created pneumococcal strains in the TIGR4 background that express pneumococcal capsule types 6A, 6B, 6C, and 6D, respectively (2). Using the Quellung reaction, we found that factor sera 6b, 6c, and 6d reacted with serotypes 6A, 6B, and 6C, respectively, while both factor sera 6c and 6d also reacted with serotype 6D (Table ​(Table1).1). To further investigate the seroreactivity of these factor sera, we tested the sera with flow cytometry and agglutination assays. The agglutination assay was independently performed by three individuals who were blinded to the identities of the three factor sera and eight bacterial strains (Table ​(Table2).2). The bacterial strains consisted of four clinical isolates representing the four serotypes and the TIGR6A, -B, -C, and -D strains. Again, we found that factor sera 6b, 6c, and 6d reacted with serotypes 6A, 6B, and 6C, respectively (Table ​(Table2),2), although one operator scored the reaction of TIGR6C with factor serum 6c as positive. As a result of this discrepancy, 12 additional clinical 6C strains were tested with factor serum 6c. Eleven of these were scored as negative, and one was scored as weakly positive. We also found that factor sera 6c and 6d reacted with serotype 6D, although there was some disagreement with the TIGR6D strain and factor serum 6c. Overall, the agglutination test results closely resembled the Quellung reaction results, and serotype 6D seems to react positively with both factor sera 6c and 6d. TABLE 1. Specificity of the serogroup 6 factor sera in the Quellung test TABLE 2. Specificity of serogroup 6 factor sera in the agglutination test Flow cytometry was performed as previously described (1), using the factor sera at a 1:50 dilution and goat anti-rabbit R-phycoerythrin conjugate (Southern Biotech). We considered staining under 30 fluorescence units to be negative, and there is moderate staining of some factor sera for multiple serotypes. However, the moderate staining may not be meaningful since these factor sera are not meant for use in flow cytometry. The more meaningful results may be the strong staining (more than 300 fluorescence units) produced by the factor sera (Fig. ​(Fig.1).1). With strong staining as the selection criterion, the factor sera bound to the expected serotypes: factor serum 6d bound to 6C, and factor sera 6c and 6d reacted with serotype 6D, similar to the Quellung reaction results. Taken together, our results confirm and extend the findings of Jacobs et al. by showing that the currently available factor sera 6c and 6d may cross-react with serotype 6D. Furthermore, their cross-reaction may be useful in the serological identification of serotype 6D. However, this cross-reaction with 6D may vary among reagent lots since the currently available factor sera may not be tested for cross-reaction with serotype 6D. FIG. 1. Results of flow cytometry performed with pneumococcal strains TIGR6A, -B, -C, and -D. Controls included are None (no primary antibody) and NRS (normal, unimmunized rabbit serum). 6b, 6c, and 6d represent factor sera 6b, 6c, and 6d, respectively.

Serotype-Specific Typing Antisera for Pneumococcal Serogroup 6 Serotypes 6A, 6B, and 6C

Streptococcus pneumoniae strains can express one of at least 92 capsular serotypes. To our knowledge, our laboratory is one of two that maintain antisera for resolution of the first 90 discovered pneumococcal serotypes (4; unpublished data). Recently, the evaluation of serogroup 6 isolates using monoclonal antibodies led to the discovery of the 91st serotype, 6C (7), which has become the prevalent invasive serogroup 6 serotype within the United States (2, 9). The pneumococcal 7-valent conjugate vaccine (PCV7) does not protect against it, and it is not included within the newly licensed 13-valent conjugate vaccine. In addition, serotype 6C carriage may also be common within PCV7-vaccinated populations (3). Prior to the discovery of serotype 6C, serotypes 6A and 6B were the 2 known serogroup 6 serotypes. CDC antisera for quellung-based resolution of serotypes 6A and 6B are designated as Danish factors 6b and 6c (DF-6b and DF-6c), respectively. Our original DF-6b antiserum was positive for both 6A and 6C serotypes, preventing their resolution (Table ​(Table11). TABLE 1. DF-6b, DF-6c, and DF-6d quellung results We produced DF-6d antiserum specific to serotype 6C and DF-6b antiserum specific to 6A as follows. New Zealand White female rabbits were inoculated with S. pneumoniae serotypes 6C or 6A formalin-fixed whole cells. The inocula consisted of serotype 6C or 6A cell suspensions (108 to 109 cells per ml in phosphate-buffered saline), which were administered intravenously via the marginal ear vein (Table ​(Table2).2). Blood was drawn after week 4 of dosing, and serum was evaluated for antibody titer to serotype 6C or 6A and cross-reactivity to serotypes 6A, 6B, and 6C by the quellung reaction. Cross-reactivity was removed from DF-6d by absorbing the serum with formalin-fixed cells of serotypes 6A and 6B. DF-6b antiserum was absorbed with formalin-fixed cells of serotypes 6B and 6C. The resulting DF-6d antiserum was specific for serotype 6C only, and DF-6b was specific to 6A only (Table ​(Table11). TABLE 2. Dosing schedule for preparation of antisera The specificity of DF-6b, DF-6c, and DF-6d antisera was evaluated with stock strains and 159 clinical isolates, each representing one of the serotypes 6A, 6B, or 6C. For all strains, Neufeld quellung results were compared to PCR testing specific for the serotype 6C wicN6C locus (2, 7, 8) with no inconsistencies observed. We have replaced our previous methods with these procedures for producing specific factor sera against serotypes 6A and 6C. We have now learned that Statens Serum Institute (SSI) has also recently produced factor 6d against serotype 6C (5). Our DF-6b, unlike the SSI factor 6b, is now serotype 6A specific and does not cross-react against serotype 6C. Serotype 6C-specific antiserum will be useful for laboratories which utilize quellung testing. Recently a fourth serogroup 6 serotype, serotype 6D, has been discovered in nature (1, 6) that is recognizable by a positive quellung test for serotype 6B combined with a positive PCR test for wciN6C. Despite extensive screening, we have not yet encountered this serotype (2). We do not yet know if our DF-6d antiserum would cross-react with serotype 6D. Consequently we screen all quellung test 6B strains by PCR for the presence of wciN6C (2, 8).

Detection and Identification of Staphylococcus lugdunensis Are Not Hampered by Use of Defibrinated Horse Blood in Blood Agar Plates

Staphylococcus lugdunensis is a coagulase-negative staphylococcus with pathogenic properties similar to those of Staphylococcus aureus (4). Identification of S. lugdunensis is not necessarily straightforward, resulting in considerable variation in detection rates in different laboratories. Bocher and coworkers recently published data to increase awareness of this pathogen and described some basic criteria for identification to the species level (2). These criteria were (i) Eikenella corrodens-like odor, (ii) colony pleomorphism, (iii) prominent β-hemolysis after 2 days of incubation, and (iv) ID32Staph identification (bioMerieux). These criteria seemed, however, to be accurate only when the samples were cultured on Columbia sheep blood agar and not on agar containing 5% horse blood. They reported less-prominent odor and hemolysis on horse blood agar than on sheep blood agar. The identification problems led to differences in detection rates, and they reported an incidence (or in our view detection rate) of 53 infections per 100,000 inhabitants per year in a county in Denmark using Colombia sheep blood agar and zero to four S. lugdunensis infections per 100,000 habitants per year in neighboring counties using 5% horse blood. At the Department of Clinical Microbiology, Central Hospital, Vaxjo, Sweden, we have identified between 40 and 90 cases of S. lugdunensis per year over the last 10 years in our laboratory (Table ​(Table1).1). We have used Eikenella corrodens-like (bleach-like) odor, colony pleomorphism, and prominent β-hemolysis after 2 days of incubation combined with negative tube coagulase (with horse plasma; Statens Serum Institut, Copenhagen, Denmark), positive reactions with ornithine decarboxylase and pyrrolidonyl aminopeptidase (1, 3), and resistance to deferoxamine (250 μg) (ROSCO; A/S Rosco, Taastrup, Denmark) (1) as standard criteria for identification of S. lugdunensis during this time. This simple strategy was validated by comparison to results obtained with API-Staph (bioMerieux) and Staph-Zym (Rosco) in 1996 (G. Kahlmeter, L. Bieber, and R. Smyth, unpublished data). TABLE 1. Summary of results of finding S. lugdunensis and S. aureus in cultures from skin and soft tissue infections (SSTI) at the Department of Clinical Microbiology, Vaxjo, Sweden, from February through October 2001 to 2009 In February 2009 we changed the composition of standard blood agar plate (blood agar base [Merck]) with 5% human blood to 5% defibrinated horse blood (National Veterinary Institute, Uppsala, Sweden) at our clinical laboratory. We validated the “new” plate when the change was made but were now concerned by the statement of Bocher et al. (2) Using the same criteria for the identification of S. lugdunensis before and after the change of medium, we revalidated the medium (Table ​(Table1).1). The detection rates of S. aureus and S. lugdunensis in samples from skin and soft tissue infections (SSTI) were calculated. Although the β-hemolysis and odor were slightly less distinct, there was no change in the isolation rate of S. lugdunensis or S. aureus or in the ratio between the two bacteria (20 to 40 times as many S. aureus were seen per year) after the shift to 5% defibrinated horse blood in the agar. The detection rate the last year in our laboratory was almost identical to the level in the recent Danish study using Columbia sheep blood agar, 55 isolates per 100,000 inhabitants per year. The true incidence of S. lugdunensis infections may be higher, since not all wound infections are cultured. Our isolates were not genotyped, and we cannot exclude minor outbreaks. On the other hand, the number of cultures and number of positive patients in relation to cultures have been very stable over many years (Table ​(Table11). Thus, in contrast to Bocher et al., we did not find that the use of defibrinated horse blood influenced the identification rate of S. lugdunensis. However, all horse blood is not necessarily the same, but then neither is all sheep blood. Other factors influencing the isolation rate of S. lugdunensis are probably more important than the use of human, sheep, or horse blood in the agar. In our experience it is important to educate staff on the clinical relevance and importance of this rather unobtrusive coagulase-negative staphylococcus and not to ignore “coagulase-negative staphylococci” as probable skin contaminants. It is important to stress that S. lugdunensis looks decidedly more “impressive” with larger colony size, more β-hemolysis, and with a more distinct odor after 36 to 48 h of incubation.

Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca(2+) concentrations ([Ca( 2+)](i)) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca(2+)](i) and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 microM increased [Ca( 2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Carvedilol induced Mn(2+) quench of fura-2 fluorescence implicating Ca(2+) influx. The Ca(2+) influx was inhibited by suppression of protein kinase C activity. In Ca(2+)-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca( 2+) pump inhibitor), carvedilol-induced [Ca(2+)](i) rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca( 2+)](i) rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 microM) did not change carvedilol-induced [Ca(2+)](i) rise. At concentrations between 5 and 70 microM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM carvedilol was not reversed by prechelating cytosolic Ca(2+) with BAPTA/AM. Apoptosis was induced by 5-70 microM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca( 2+) influx via protein kinase C-regulated Ca(2+) channels. Carvedilol-caused cytotoxicity was mediated by Ca(2+)-independent apoptosis in a concentration-dependent manner.

Leisure-Time Physical Activity in Pregnancy and Risk of Postpartum Depression

To explore the association between physical activity during pregnancy and postpartum depression (PPD) in a large, prospective cohort. Exposure information from the Danish National Birth Cohort, a large, prospective cohort with information on more than 100,000 pregnancies (1996-2002), was linked to the Danish Psychiatric Central Register and the Danish Register for Medicinal Product Statistics for data on clinically identified cases of depression up to 1 year postpartum. A total of 70,866 women from the Danish National Birth Cohort were included in the analyses. Duration, frequency, and type of physical activity were assessed by a telephone interview at approximately week 12 of gestation. Admission to hospital due to depression (PPD-admission) and prescription of an antidepressant (PPD-prescription) were treated as separate outcomes. Through linkage to national registers, we identified 157 cases of PPD-admission and 1,305 cases of PPD-prescription. Women engaging in vigorous physical activity during pregnancy had a lower risk of PPD-prescription compared to women who were not physically active (adjusted odds ratio, 0.81; 95% CI, 0.66-0.99). No association was observed between physical activity and PPD-admission; but, in women who were underweight prior to pregnancy, physical activity was associated with increased risk of PPD-admission. Our data are compatible with a protective effect of vigorous physical activity, but not for other measures of physical activity, against postpartum depression requiring antidepressant therapy. No protective effect could be detected on PPD leading to hospitalization.

Direct Serogrouping of Streptococcus pneumoniae Strains in Clinical Samples by Use of a Latex Agglutination Test

Pneumotest-Latex (Statens Seruminstitut) was evaluated for direct serogrouping of Streptococcus pneumoniae strains in clinical samples from patients with invasive disease. The technique was accurate to its level of discrimination for 62 of 67 clinical samples (92.5%). Pneumotest-Latex would be a useful alternative for direct serogrouping of pneumococci in clinical samples.

Response Is Seen to Conjugate Vaccine but Not to Polysaccharide Vaccine Twelve Months After Rituximab.

Abstract 2728Poster Board II-704 Background:After therapy with the monoclonal antibody anti-CD20 rituximab ® (R), normal B-cells are depleted and their recovery in blood is delayed until six-twelve months in patients with relapsed low-grade lymphoma treated with four infusions of R. In patients treated with R-chemotherapy or high-dose therapy after R-purged autologous graft, the B-cell recovery is postponed further. The efficacy of active immunization against various bacterial and viral infections of R- treated patients has been debated as total level of immunoglobulin (Ig) M has been shown to decrease during prolonged treatment with R and humoral T cell-independent response might be defect due to lack of B-cells. Aim:To evaluate antibody-response to two vaccines after two timepoints of vaccination. Methods:An open randomized study of patients with B-cells lymphoma vaccinated six or twelve month after last treatment with R (Mabthera® Hoffman-LaRoche, Basel, Switzerland) with or without chemotherapy. Pneumococcal polysaccharide vaccine, Pneumo23® (Sanofi Pasteur MSD), assessed T-cell–independent antibody response and conjugated H. influenza type b-vaccine, Act-Hib® (Sanofi Pasteur MSD), evaluated combined B-cell/T-cell pathway. The patients randomized to vaccination at six months and without response, were revaccinated after further six months. Blood samples were collected immediately before vaccination and four weeks after. Serum was separated, frozen to −20 degrees C, sent to the Division of Microbiology, Statens Seruminstitut, Copenhagen, Denmark for analyses. A modified enzyme-linked assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies for 6 pneumococcal serotypes (1, 4, 7F, 14, 18C, 19F) was used and a geometrical mean value for the total titer of antibody was calculated. The definition of protective response was determined to >39 arbitrary units/ml. Total Ig antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HbPs) was measured by ELISA. An anti-HbPs antibody titer of >1 μg/ml was recorded as a protective response after immunization. Blood samples for flow cytometry analysis, including CD19+ and CD20+ B-cell counts, were drawn at the time of vaccination and four weeks after. Results:Nine patients, age 56–73 years, six females and three males, were randomized to vaccination; four at six months and five at twelve months after last rituximab treatment. Five patients with follicular lymphoma (FL) grade 2 and one with marginalzone lymphoma (MZL) had received single R, one of these in combination with alfa-interferon in a clinical phase II trial. One patient with mantle cell lymphoma (MCL) and one with follicular centroblastic lymphoma were treated with R-CHOP-21 × 8 prior to vaccination. One female patient with MZL randomized to vaccination at twelve months, dropped out because of progression of disease. Vaccination with Pneumo23® resulted in a protective response of pneumococcal antibodies in only one out of eight (12,5 %) patients. This responding patient had MZL treated with R 375 mg weekly x4 during 2 courses, and was randomized to vaccination 6 months after last R and also showed a response to Act-Hib vaccine. Further four patients had an immunization response to act Hib vaccine, one with FL randomized to vaccination six months after R and three after twelve months, two with FL and one with MCL. One patient was revaccinated after twelve months and had no response neither with Pneumo23® or Act-Hib® vaccine. The percentage CD19+ and CD20 + B-cells in the lymphocyte gate (by flow cytometry) was low in all patients at the time of vaccination and four weeks after, but showed a slight increase with time (0,04, 0,26, 0,42, 0,54 % and 0,24, 0,6, 0,6, 1,26, 1,0 % at six and twelve months after R, respectively). Conclusion:Conjugate vaccine is more potent to give immune response than polysaccharide vaccine after R-therapy in patients with B cell lymphoma. Despite deficient recovery of B-cells cells, five of eight (62,5 %) patients responded to the conjugate vaccine Act-Hib®. The longstanding effect on the immune system of antibody therapy has to be further studied. Disclosures:No relevant conflicts of interest to declare.

Vulnerability to Heat-Related Morbidity and Mortality in Copenhagen, Denmark

ISEE-0231 Background and Objectives: Climate change has potentially grave implications for human health. The association between the daily 3-hr maximum apparent temperature (Tappmax) and respiratory, cardio- or cerebrovascular (RD, CVD, CBD) hospital admissions/non-accidental deaths was investigated during the period 1 Jan 2002 to 31 Dec 2006. Methods: Health outcome data for Greater Copenhagen were retrieved from the Danish Hospital and Cause of Death registers. The meteorological (temperature and relative humidity) and air pollution (CO, NO2, PM10) data were collected at a fixed urban background monitor by the Danish National Environmental Research Institute. Data on % weekly general practitioner (GP) visits due to influenza were provided by the Statens Serum Institute. The associations between Tappmax and health outcomes were studied in a case-crossover design, with every 2nd day as a control day, in the same month and year as the case day. Associations are expressed as hazard ratios (HR) and 95 confidence intervals (CI), per inter-quartile range (IQR) increase in Tappmax. Models were adjusted for day of the week, public holidays, % weekly GP visits due to influenza. The admission models were also adjusted for PM10. Different lags and moving averages of Tappmax, NO2, CO and PM10 were investigated. Effect modification was investigated by stratification and interaction terms: Warm/cold periods, age, socio-economic status, existing diabetes and/or hypertension, previous RD and CVD hospital admissions and distance between household and fixed urban background air pollution monitor. Various sensitivity analyses were conducted. Results and Conclusion: For an IQR (8°C) increase in the 5-day lag of Tappmax, a 20% increase in the RD mortality rate was observed in the warm period (Apr-Sep) (HR 1.201, 95% CI 1.059-1.361). Significant associations were observed between CVD hospital admissions and Tappmax in the cold (HR 1.048, 95% CI 1.025-1.073) and warm periods (HR 0.950, 95% CI 0.920-0.981).

A virally inactivated functional growth factor preparation from human platelet concentrates

Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet. Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines. The GF preparation contained a mean of 16.66, 2.04, 1.53, 72.19, 0.33, 48.59 and 0.44 ng/ml of PDGF-AB, -BB, -AA, TGF-beta1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0.1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates. STANDARDIZED and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.