Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype.

An in-field demonstration of the true relationship between skin infections and their sources in occupational diving systems in the North Sea.

INTRODUCTION: Skin infections in saturation diving are caused by microbes that flourish in saturation environments. Improvements in the prevention of infections must therefore be based on environmental control and elimination. Furthermore, only a few genotypes seem to be responsible for the majority of infections in the Norwegian sector of the North Sea, and these have all been demonstrated in saturation systems for many years. Although reservoirs of infectious genotypes have been identified, their true sources have not been identified. OBJECTIVES: The purpose of this field study was to log the contamination by Pseudomonas aeruginosa of the saturation system throughout a diving operation. MATERIALS AND METHODS: Daily water samples from the vessels drinking water system and from the heated seawater systems to divers suits were taken throughout the diving period of 1 month in the summer of 2001. All P.aeruginosa isolates were genotyped by pulsed field gel electrophoresis. RESULTS: A total of 17 P.aeruginosa genotypes were identified in the course of this field study. None of the most common infectious genotypes previously observed in the Norwegian sector were among these strains. Two genotypes were involved in skin infections during the period of operation: TP2 and TP12. TP2 was shown to be an inhabitant of the diving systems throughout the investigation period, while TP12 was introduced from seawater in the course of the operation and rapidly spread and established itself throughout the diving system. CONCLUSIONS: The study has demonstrated seawater as a true source of an infectious P.aeruginosa genotype in occupational diving systems.

The impact of environmental Pseudomonas aeruginosa genotypes on skin infections in occupational saturation diving systems.

Skin infection caused by Pseudomonas aeruginosa is the most frequent health problem associated with occupational saturation diving on the Norwegian continental shelf. In the course of 14-y surveillance of infection and environmental control in occupational offshore saturation diving systems, a collection of approximately 1000 P. aeruginosa isolates has been amassed. Retrospective genomic analyses using restriction enzyme fragmentation and pulsed-field gel electrophoresis have identified 24 of 76 environmental P. aeruginosa genotypes as being of significance for single infections, outbreaks of infections and recurrent skin infections in occupational diving systems. In addition, these genomic analyses have made it possible to separate outbreaks of infection into outbreaks with 1 single genotype and clusters of infections where different genotypes are involved. We conclude that the established, assumed diver-to-diver contagion vector ought to be replaced by a environmental contagion vector as the most likely vector within these specific occupational environments. Furthermore, consecutive presence of the frequent environmental/infectious genotypes demands specific improvement of infection prevention and control in these systems.

Survival of infectious Pseudomonas aeruginosa genotypes in occupational saturation diving environment and the significance of these genotypes for recurrent skin infections.

BACKGROUND: Occupational saturation divers suffer from various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated. METHODS: P. aeruginosa isolates from 292 skin infections in operational saturation divers and about 800 isolates from occupational saturation diving systems have been collected during the period 1986 to 1998. Genotyping of the isolates has been performed by using restriction enzyme fragmentation and pulsed field gel electrophoresis. RESULTS: Four hundred and seventy-two P. aeruginosa isolates have been analyzed, of which 181 originate from skin infections in divers. Ninety-seven significantly different P. aeruginosa genotypes have been defined. Some of these genotypes are solely found from skin infections, some solely from the saturation environment and about 25% were found both from infections and from the saturation environment. Eight frequent infectious genotypes have been identified, and these are shown to be present over several years, both in infections and in the saturation environment. CONCLUSIONS: The study suggests that skin infections in occupational saturation divers are commonly caused by environmental strains.

Expression of the psoriasis-associated antigen, Pso p27, is inhibited by cyclosporin A.

The psoriasis-associated antigen, pso p27, can be isolated from psoriasis scale and is present in complement-activating immune complexes in psoriatic scale, and in serum from patients with psoriasis. The antigen is produced by tryptase-positive cells in the skin lesions and is shown to be a major antigen in the immune reactions in psoriasis. The synthesis of this particular antigen is reduced with the remission of inflammation in the skin lesions. In this study we followed 3 patients with severe plaque psoriasis during treatment with cyclosporin A. In all patients we observed a decrease in the expression of the antigen pso p27 during the therapy. The effectiveness of the therapy varied among the patients, but there was a clear correlation between disease activity and expression of the antigen pso p27 as demonstrated by immunofluorescence in biopsies from selected skin lesions. This observation strengthens our hypothesis that the pso p27 antigen plays an important role in the inflammation in psoriasis.

Higher levels of antibodies against the psoriasis-associated antigen pso p27 in cerebrospinal fluid from patients with low back pain and sciatica.

STUDY DESIGN: A prospective study comparing the presence of antibodies against the psoriasis-associated antigen pso p27 in pain-free control subjects and patients with low back pain and/or sciatica. OBJECTIVES: To analyze the amount of local inflammation present in human lumbar disc disorders, using anti-pso p27 antibodies in the cerebrospinal fluid as a marker and to analyze whether pain intensity correlates with this marker of inflammation. SUMMARY OF BACKGROUND DATA: Pso p27 is a major antigen in psoriasis that is also present, mostly locally, in other inflammatory disorders, such as sarcoidosis, inflammatory bowel disease, and ankylosing spondylitis, inflammation is also thought to play a major role in the generation of lumbar and radicular pain in degenerative disc disorders. METHODS: Anti-pso p27 antibodies in cerebrospinal fluid were quantified using an indirect enzyme-linked immunosorbent assay with pso p27 obtained from patients with psoriasis for use as an antigen. Fifteen patients with spinal stenosis, 11 patients without myelographic disc herniation, 17 patients with disc herniation, and 24 pain-free patient control subjects were studied. RESULTS: Significantly higher levels of anti-pso p27 antibodies were found in patients with myelographic signs of disc herniation than in with patients with no signs of herniation, patients with spinal stenosis, and control subjects. Patients with no known signs of disc herniation and patients with myelographic signs of spinal stenosis (< 10 mm in diameter) caused by degenerative changes, had higher levels of anti-pso p27 antibodies than did control subjects. However, these differences reached only borderline statistical significance. CONCLUSIONS: The results support those in previous reports, that inflammation probably plays an important role in degenerative disk disorders, particularly in disk herniations. That there was no correlation between pain intensity and anti-pso p27 activity indicates that the antigen is probably not essential in pain generation per se. The results may indicate that pso p27 is expressed secondary to, not as an initiator of, inflammation.

Identification of infectious Pseudomonas aeruginosa strains in an occupational saturation diving environment.

OBJECTIVES: Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe. RESULTS: During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver. CONCLUSIONS: The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.

Diver's hand: a skin disorder common in occupational saturation diving.

Saturation divers regularly inspect North Sea installations, working at depth for periods of 12-16 days. Diver's hand is a particular problem in saturation diving, and there is no effective protection or treatment available. This paper presents the occurrence of diver's hand and describes the disease in clinical and epidemiological terms. Three studies of diver's hand have been carried out, in 1990, 1994, and 1995. Most long term saturation divers have had diver's hand at some time in their professional career. Diver's hand seems able to occur without any previous skin symptoms, and divers without diver's hand can have several other skin symptoms during a saturation period. It is likely that diver's hand is a specific phenomenon associated with saturation diving.

Effects of cascade apheresis in patients with psoriasis and psoriatic arthropathy.

Eight patients with psoriasis, all with skin scales and 7 with disabling psoriatic arthritis, were subjected to cascade apheresis starting with three treatments per week for 2 weeks, followed by one treatment a week, comprising ten treatments in all. Six out of 7 patients (86%) with arthropathy and 3 out of 8 patients (38%) with scales experienced a beneficial effect. There was a large drop in the levels of circulating immune complexes (CIC) due to the treatment, and the removal of CIC was followed by reduced inflammatory activity in skin lesions and joints as evaluated by pain, morning stiffness, grip strength, plaque score, and PASI index. However, there was no correlation between the level of CIC, disease activity, or treatment response. From the present results it is concluded that CIC may play a more significant role regarding psoriatic arthropathy than in skin manifestations, and apheresis may be beneficial in patients not responding to conventional therapy.

Detection of psoriasis-associated antigen pso p27 in sarcoidosis bronchoalveolar lavage fluid using monoclonal antibodies.

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology, predominantly affecting the lung. The increased concentration of pulmonary lymphocytes with specific receptors in subgroups of sarcoidosis patients suggests a local specific immune response. pso p27, a psoriatic scale antigen linked to the pathogenesis of psoriasis, was previously found in BAL cells, serum, and Kveim-Siltzback test in sarcoidosis. With an enzyme-linked immunoassay based on murine monoclonal antibodies, we analyzed BAL fluid from 21 patients with pulmonary sarcoidosis. Eleven (52%) of the patients have detectable levels of pso p27 antigen. No antigen is detected in the BAL fluid from five healthy, nonsmoking controls. Serum concentrations of pso p27 shows no significant difference between the two groups, but three of the sarcoidosis patients have detectable levels of the antigen. Mean concentration of pso p27 is >100 fold higher in BAL fluid than in serum from the sarcoidosis patients. This strongly suggests local pulmonary production of pso p27 antigen.

Complement activation in divers after repeated air/heliox dives and its possible relevance to DCS.

Plasma levels of the anaphylatoxin C5a were measured in 19 divers performing repeated air dives. Blood samples were collected immediately before the first dive and 2 h after the first and the second or third dive. Serum obtained at the same times was subjected to complement activation in vitro by air bubbles. Six divers developed symptoms of decompression sickness (DCS). Most intravascular bubbles were observed in divers with the lowest plasma levels of C5a. Postdive plasma levels of C5a did not increase compared with predive levels, nor were postdive levels significantly different after two or three dives compared with the first dive. Repeated dives did not influence the amounts of C5a generated in vitro. Neither plasma levels of C5a nor C5a generated in vitro were significantly different in divers who experienced symptoms of DCS vs. divers without symptoms of DCS. We conclude that plasma level of C5a and measurement of C5a generation in vitro cannot be used to predict DCS.

Activation of the complement system and adverse effects of biodegradable pins of polylactic acid (Biofix) in osteochondritis dissecans.

Biodegradable pins of polyglycolic acid (PGA) or polylactic acid (PLA) have been used in the treatment of fractures and osteotomies during the past 5 years. Adverse effects reported have included swelling at the implantation site and sinus formation, considered to represent nonspecific foreign-body reactions. Recent reports, however, have shown severe reactions after intraarticular fracture fixation. Reactions in 2 patients, treated with polylactic pins for osteochondritis dissecans (OCD) in our hospital, prompted the present clinical investigation and further evaluation of the complement-activating potential of polylactic pins. 10 knees underwent arthroscopic fixation of an OCD-lesion with Biofix (PLA) pins. Clinical follow-ups were carried out at 2, 6, and 12 weeks and at 6 and 12 months. Blood samples were collected from 5 patients 9-24 months postoperatively for biocompatibility tests. Quantification of human C5a des Arg was performed with a recently developed sandwich ELISA technique, using neoepitope-specific monoclonal antibodies. 6 knees developed diffuse swelling and a prolonged postoperative course. 2 patients had a particularly prolonged course which could not be attributed to infection. Levels of C5a des Arg in plasma incubated in the presence of polylactic acid were higher than in plasma incubated in the absence of PLA. The high frequency of long-term postoperative inflammatory signs in these knees treated for OCD and the demonstration of a complement activation potential of PLA pins warrant further studies on the biocompatibility of this material. Until more information is available, we do not recommend intraarticular use of PLA pins.

Heparin-coated circuit during cardiopulmonary bypass. A clinical study using closed circuit, centrifugal pump and reduced heparinization.

A prospective randomized study was performed to investigate the effect of surface coating with covalently endpoint-attached heparin (Carmeda Bio Active Surface) and reduced general heparinization on haematological indices and complement C5 activation. Care was taken to optimize the rheological design of the system using centrifugal pump and a closed system without venting or machine suction. Twenty patients scheduled for aortocoronary bypass grafting (EF > 0.5) participated in the study. Ten patients were randomized to be treated with heparin-coated equipment (CBAS) and reduced i.v. heparin (1.5 mg.kg-1) while 10 patients treated with identical but noncoated equipment and full heparinization (3 mg.kg-1) served in a Control group. A vacuum suction was used to collect the blood from the operating field and it was autotransfused at weaning from extracorporeal circulation (ECC). Blood samples were obtained from the venous (precircuit) and arterial (postcircuit) side. We used a new and very specific method for detection of C5a based on monoclonal antibodies. The concentration of C5a was low in both groups during the operation but a significant increase was seen on days 1 and 2. In the Control group there was an increase from 10.2 ng.ml-1 +/- 1.2 to 27.5 ng.ml-1 +/- 4.8 on day 2 and in the CBAS group from 10.7 ng.ml-1 +/- 1.2 to 35.6 ng.ml-1 +/- 11.6 on day 2 (NS between groups). The granulocytes and total leukocyte count increased at the end of ECC and was maintained at the elevated level throughout the study period. The amount of free haemoglobin was high in the autotransfused blood in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

[The new medical curriculum in Trondheim. Organization and planning of a new curriculum]. / Det nye medisinstudiet i Trondheim. Organisering og planlegging av et nytt studium.

In autumn 1993, the Faculty of Medicine in Trondheim will be able to offer complete undergraduate medical education to 60 students per year, after 18 years of 3 1/2 years' education, based only on the clinical part of undergraduate medical school. The entire new curriculum is based on problem-based learning with the preclinical and the clinical part of the study totally integrated for 5 1/2 years. Throughout the curriculum any health care problem will be analyzed in terms of three perspectives: the biological, the environmental and the behavioural. The new medical curriculum was planned as a project during which both teachers and students took part in planning groups organized on several levels. Through clinical demonstrations and a course on the doctor-patient-relationship, the 60 students will meet patients as a natural part of their study from day one. In the Family Practice and in the Skills Lab they will acquire clinical skills during the first two years. In the fourth and fifth years the students spend two 8-week clerkship periods in community hospitals and community health practices.

Variability over time of complement activation induced by air bubbles in human and rabbit sera.

Complement activation induced by air bubbles in rabbit and human sera was studied by measuring the generation of anaphylatoxin des-Arg-C5a. des-Arg-C5a was quantified by sandwich enzyme-linked immunosorbent assays based on neoepitope-specific anti-des-Arg-C5a monoclonal antibodies. Air bubbles were continuously introduced to serum via a calibrated microflowmeter, and the serum was incubated at 37 degrees C for 30 min. Air bubbles clearly generated increased amounts of des-Arg-C5a compared with corresponding levels in control serum, and a dose-dependent effect was also noted. Strong positive correlations between des-Arg-C5a concentrations in control sera and sera incubated with air bubbles at a flow of 0.5 ml/min were found. To study variation over time, serum was obtained at regular intervals from six rabbits and from six healthy humans during 66- and 196-day periods, respectively. A pronounced intraindividual variability over time was thus observed. The reason for the large variability is at present unknown. We conclude that the sensitivity of complement to activation by air bubbles is not an inherent, static feature of the complement system of an individual. Therefore single-point analysis of complement activation by air bubbles appears to be an inappropriate parameter by which to differentiate a "sensitive" or "insensitive" complement system between individuals.

Use of scale antibodies for the detection of antigens in psoriatic lesions.

Extractable IgG from psoriatic scale was purified, labelled with biotin and used in ELISA and immunofluorescence (IF) in an attempt to detect and localize prominent antigens in psoriatic scale extracts and in psoriatic lesions, respectively. Biotinylated immunoglobulins isolated from psoriatic scale from each of 5 patients were used. Scale extracts were fractionated on a Sephacryl S-300 column, and antigens detected by scale IgG were eluted in the void volume and at a Kav 0.55. The profile was very similar for each antibody preparation. Antigens recognized by serum IgGs from both healthy controls and psoriatic patients were detected in the void volume only. Antigens recognized by a rabbit antiserum against the psoriasis-associated antigen, pso p27 (6), were restricted to the fractions eluted at a Kav 0.55. Furthermore, the binding of scale IgG to the antigens eluted at Kav 0.55 was inhibited by purified pso p27 antigen. Two of the scale antibody preparations gave rise to a distinct fluorescence on skin biopsies from psoriatic lesions in indirect immunofluorescence. The antigens recognized were localized to a subfraction of dermal cells and in the endothelial lining of some of the dermal vessels. Double labelling with these scale antibodies and a rabbit anti-pso p27 antiserum showed that both antibody preparations bound to the same cells in the psoriatic lesions, while only a minority of these cells were recognized by a murine monoclonal antibody against human IgG. The observations described indicate that the pso p27 is a major antigen in the immune reactions in psoriasis.