Access to bacteriophage therapy: discouraging experiences from the human cell and tissue legal framework.

Cultures of human epithelial cells (keratinocytes) are used as an additional surgical tool to treat critically burnt patients. Initially, the production environment of keratinocyte grafts was regulated exclusively by national regulations. In 2004, the European Tissues and Cells Directive 2004/23/EC (transposed into Belgian Law) imposed requirements that resulted in increased production costs and no significant increase in quality and/or safety. In 2007, Europe published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products. Overnight, cultured keratinocytes became (arguably) 'Advanced' Therapy Medicinal Products to be produced as human medicinal products. The practical impact of these amendments was (and still is) considerable. A similar development appears imminent in bacteriophage therapy. Bacteriophages are bacterial viruses that can be used for tackling the problem of bacterial resistance development to antibiotics. Therapeutic natural bacteriophages have been in clinical use for almost 100 years. Regulators today are framing the (re-)introduction of (natural) bacteriophage therapy into 'modern western' medicine as biological medicinal products, also subject to stringent regulatory medicinal products requirements. In this paper, we look back on a century of bacteriophage therapy to make the case that therapeutic natural bacteriophages should not be classified under the medicinal product regulatory frames as they exist today. It is our call to authorities to not repeat the mistake of the past.

Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products.

Belgian and European experience with the European Homograft Bank (EHB) cryopreserved allograft valves.--assessment of a 20 year activity.

European Homograft Bank (EHB) has been selecting, preparing, storing and distributing the cryopreserved allograft valves in Belgium and some other European Countries since 1989. It was established in 1988 by a pathologist and the cardiac and vascular surgeons from Belgian and other European centres as an inter-university, international nonprofit association. Due to its neutral behavior and very high quality criteria, European Homograft Bank became one of the prominent heart valve banks in Europe and wider. It collaborates with the transplant coordination in donor selection as well as with the huge network of the implanting surgeons in Belgium and other European Countries. The EHB responsible discusses with the implanting surgeon the allograft selection on basis of the indication and the patients state of emergency. A total of 8.911 donor heart valves have been evaluated in EHB during the last 20 years. After selection, 5.258 allograft valves (1.996 aortic, 3.189 pulmonary and 73 mitral) were cryopreserved and stored in vapors of liquid nitrogen between 6 weeks and 5 years. A total of 4.516 allograft valves (1.391 aortic, 2.620 pulmonary and 48 mitral) were implanted in the left or right ventricular outflow tract for replacement of the diseased aortic or pulmonary valve and for mitral or tricuspid valve replacement or repair. In 1.380 cases the allograft valves were used for right ventricular outflow tract reconstruction as part of the Ross- procedure, whereas in 668 cases the allograft valve served for replacement of the aortic valve for endocarditis. The most important indications for use of cryopreserved allograft valves were: important cardiac and valve malformation in children, female patients of child-bearing age with diseased cardiac valves, cases with contra-indication for anti-coagulation and the patients with severe endocarditis with septal or annular abscesses. Although the number of the donation increased by year, the available allograft valves in stock are still insufficient to respond to all the surgeons' request for different indications.

European homograft bank: twenty years of cardiovascular tissue banking and collaboration with transplant coordination in Europe.

Established in 1989 in Brussels as an international nonprofit association, the European Homograft Bank (EHB) has been collaborating closely with the transplant coordination of the different centers in Belgium and other European countries. Donor selection is made after discussion of exclusion criteria with the transplant coordinator of the procurement center. EHB collaborates with 15 Belgian, 11 German, 10 French, 10 Swiss, 3 Italian, 3 Dutch, and some other procurement and/or implantation centers. Donor ages range from newborn to 65 years. Tissue preparation, morphologic evaluation, and functional testing are performed under Class A laminar flow. After decontamination in a cocktail of 3 antibiotics (lincomycin, vancomycin, and polymixin B) during 20-48 hours, the tissues cryopreserved with liquid nitrogen to -100 degrees C are stored in vapors of liquid nitrogen below -150 degrees C for a maximum of 5 years. Systematic virologic examination of donor blood is performed for HIV, HTLV, hepatitis B/C, and syphilis, as well as for enteroviruses, Q fever, malaria, and West Nile virus by indication. Bacteriologic examination for anaerobic and aerobic contamination is performed at the different steps of processing. Histologic examination for malignant disease and infection is performed systematically. Indications for implantation are discussed with the requesting surgeon. Transport to the implantation center is carried out safely in a dry shipper at -150 degrees C or in dry ice at -76 degrees C. The EHB received 4,511 hearts and 1,169 batches of arteries from January 1989 to December 2008. The 5,133 heart valves (1,974 aortic, 3,106 pulmonary, and 53 mitral) and 2,066 arterial segments have been prepared and stored; 4,600 cryopreserved valvular (2,717 pulmonary, 1,835 aortic, and 48 mitral) and 1,937 arterial allografts have been distributed for implantation in various European Cardiovascular Centers. EHB is not always able to meet the increased demand for heart valves and arterial allografts. Collaboration between the EHB and the Transplant Coordination is satisfactory. Donor selection criteria are discussed with the transplant coordinator; whereas, implantation indication, with the implanting surgeon. Because the EHB is not always able to meet demands for the cryopreserved valves and arterial segments, there is a need to increase number of procurements. Cardiovascular surgeons need to play more active roles in the resolution of this problem.

Decontamination of heart valve and arterial allografts in the European Homograft Bank (EHB): comparison of two different antibiotic cocktails in low temperature conditions.

The aim of the study was to compare the efficiency of two different antibiotic cocktails in the cardiovascular allograft decontamination. Low temperature, low-concentration antibiotic cocktail with Cefoxitin, Lincomycin, Polymixin B and Vancomycin was decontamination protocol in EHB for many years. The modified cocktail doesn't contain Cefoxitin. The study had two steps. First step: cardiovascular allografts from 80 donors are incubated in classical (group 1) or modified cocktail (group 2). Second step: 184 and 182 allografts of group 1 and group 2 are incubated in the modified and classical antibiotic cocktail, respectively. The bacteriological examination is performed in three steps: A-transport solution, B-decontamination solution and C-cryopreservation solution. During the first step 23.75% of the tissues were initially contaminated mainly with Staphylococcus (78.95%). 93.75% of the allografts of group 1 and 100% of group 2 were sterile after incubation (p = 0.058). 25.54% and 30.77% of group 1 and 2, respectively were contaminated in A-examination during the second step. Staphylococci were isolated in 82.98% and 69.64% in group 1 and 2, respectively. About 4.35% of group 1 and 5.5% of group 2 were contaminated in A, B, and C whereas 5.4% of group 1 and 4.4% of group 2 were contaminated in B or C after being sterile in A. Finally 9.78% of the tissues were rejected and 90.22% cryopreserved in the modified, whereas 9.89% rejected and 90.11% accepted in the classical group (p = 0.1). The difference was non-significant in the level of decontamination between the two cocktails. Contamination of some tissues with low growing, low-pathogen germs that appeared in B or C examination, couldn't be explained. This issue needs complementary investigation.

Banking of the human heart valves and the arteries at the European homograft bank (EHB)--overview of a 14-year activity in this International Association in Brussels.

Processing of the human heart valves and arteries has been carried out at the European Homograft Bank (EHB) in Brussels since 1989 and 1991, respectively. Heart valve donors of 0-65 years were classified in (1) Beating heart donors (BHD), of which recipients of heart transplantation (RHT) and multiorgan donors (MOD) after brain death, and (2) non-beating heart donors (NBHD) with warm ischaemic time (WIT) of less then 6 h. Past history of the donors has been checked for malignant and chronic diseases, as well as biology for transmissible and infectious diseases. Perfect collaboration has been established with the transplant coordinators and transplant teams of the implanting centres. Dissection, decontamination, cryopreservation and storing in fluid nitrogen has been carried out in accordance with the Belgian and European Standards of cardiovascular allografts. During this period, a total of 2.828 hearts, 28 predissected valves and 616 batches of arteries arrived in the EHB. 3.537 valves and 1.137 different arteries were accepted for implantation. The main reasons for tissue rejection were morphology, contamination and cuts during the tissue retrieval or dissection. A huge network of different hospitals in Belgium and elsewhere in Europe and Switzerland were included in this process. Pulmonary allografts were not sent for implantation in the left ventricular outflow tract after 1998, since the early and mid-term results after 76 implantations were disappointing. The number of implanted aortic and pulmonary allografts remains stable from year to year, however the number of the allografts used for Ross operation is still increasing. Since the results of the follow up were disappointing, we still only require the implantation and immediate postoperative results, whereas the follow-up information only for specific study purposes.

Is ABO group incompatibility really the reason of accelerated failure of cryopreserved allografts in very young patients?--Echography assessment of the European Homograft Bank (EHB) cryopreserved allografts used for reconstruction of the right ventricular outflow tract.

Right ventricular outflow tract reconstruction (RVOTR) with cryopreserved allograft for Ross operation and other congenital or acquired cardiac malformation has become a routine and currently, the procedure of choice for children and young patients. A tendency of accelerated degeneration in the youngest recipients has been reported. Some authors advocate the ABO group incompatibility as the main reason for such failure. This retrospective monocentric study presents the long-term outcome of the European Homograft Bank (EHB) cryopreserved allografts, used for RVOTR in Ross operation (group one) and other congenital heart malformation-s (group two). The evaluation of the allograft performance was done by means of echography, considering the allografts with the transvalvular gradient of > or =40 mmHg and/or regurgitation of > or =3+ as failed. Fifty-one patients of group one and 123 of group two were analysed after completed follow-up information. About 25.5% of patients of group one and 30.8% of group two had a compatible, whereas 74.5% of group one and 68.92 of group two an incompatible ABO group with the donor. The mean follow up was 45.77 and 68.88 months, respectively. In second group 22.76% received the aortic, while 77.24% pulmonary allograft. Only three cases of group one (5.88%) failed: one with a compatible (7.69%) and two with an incompatible ABO group (5.26%) (p=0.1), whereas 39 patients (29.4%) of group two failed between 20.1 and 120.2 months (29.73% with and 29.07% without ABO compatibility, p=0.03). Contrary, the age showed more importance in the allograft failure: out of 41 failed allografts, 24 (58.54%) were implanted in patients of 0-5 years (9 or 37.5% with compatible and 15 or 62.5% with incompatible ABO group). Generally, analysing both groups together, there was no influence of ABO mismatching on the allograft failure (p=0.79). Contrary, there was a significant difference in survival between Ross and non-Ross group (p=0.00082).

Processing of cardiovascular allografts: effectiveness of European Homograft Bank (EHB) antimicrobial treatment (cool decontamination protocol with low concentration of antibiotics).

To assess the effectiveness of antimicrobial treatment by using cool decontamination protocol with low concentration of antibiotics during processing of cardiovascular allografts, 948 allografts processed during a 2-year period were analysed. Five hundred and fourty one donors aged <62 years were classified in: multiorgan donors (MOD) with non-transplantable hearts; recipients of cardiac transplantation (RHT); and non-beating heart cadavers with a warm ischemic time of less than 6 h (NBHD). During processing three samples for bacteriology testing were taken A (sampling before decontamination); B (sampling after decontamination); C (sampling on the final product). Samples A were positive in 348 cases (36.4%), respectively 36% for MOD, 21.6% for RHT and 78.1% for NBHD. All the allografts were immersed in a cocktail of four antibiotics at 4 degrees C. After exposure to antibiotics the rate of decontamination of those with A positive was 90.4, 92.5, 82.5% respectively for MOD, RHT, NBHD. At the end of processing, 57 allografts (6%) were positive in B and/or C, 15 allografts remained contaminated with the same bacteria as in A, 42 were contaminated during processing. The overall rate of sterility in the end of processing is 94% and for each group this is: 95.4% for MOD, 96.8% for RHT and 86.3% for NBHD. Analysis shows that there is no influence of time of exposure in AB in the rate of decontamination for MOD and RHT. The most predominant germ in contamination is Coagulase Negative Staphylococcus (CNS) (53.4% alone, 8.9% with other bacteria). 83.3% of MOD; 88.5% of RHT were contaminated with one germ, while 40.4% of NBHD were contaminated with more than one.

High frequency percussive ventilation in burn patients: hemodynamics and gas exchange.

High frequency percussive ventilation (HFPV) is a recent ventilatory mode, which combines conventional cycles with high frequency percussions. HFPV was initially instituted as salvage therapy after acute respiratory failure following smoke inhalation injury achieving in each case a dramatic improvement of blood oxygenation, PaCO(2) and ventilatory pressures. This study investigates the influence of HFPV on hesmodynamics, blood oxygenation and ventilatory parameters in eight stable ICU burn patients requiring artificial ventilatory support during a postoperative period following traumatic injury. Periods of 2h were analysed receiving conventional ventilation and HFPV with a high frequency of 400 and 800 cycles/min. Hemodynamic data were not significantly modified; peak inspiratory pressure was significantly lower under HFPV but mean airway pressure was unchanged. Blood oxygenation and CO(2) elimination were significantly improved under HFPV. No side effects were noted. These observations suggest that HFPV could improve pulmonary gas exchanges under lower peak pressures and without hemodynamic compromise. HFPV could represent an interesting alternative open lung strategy method to improve alveolar recruitment.

High frequency percussive ventilation and conventional ventilation after smoke inhalation: a randomised study.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. Thirty-five severely burned patients were randomised on admission for conventional ventilation (CV; control group) versus high frequency percussive ventilation (HFPV; study group). HFPV is a ventilatory mode, introduced 10 years ago which combines the advantages of CV with some of those of high frequency ventilation. Arterial blood gases, ventilatory and hemodynamic variables were recorded for 5 days at 2h intervals. Incident complications were classically managed. A statistical analysis (Student's t-test and Wilcoxon signed rank test) demonstrated a significant higher PaO(2)/FiO(2) from days 0 to 3 in the HFPV group. No significant differences were observed for the other parameters. Our findings suggest that HFPV can improve blood oxygenation during the acute phase following inhalation injury allowing reduction of FiO(2). No significant differences were observed between groups for mortality nor incidence of infectious complications in this study.

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft? Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, we report an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at approximately 4 degrees C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85% glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchanged mechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 degrees C, or cryopreservation with 10% Me(2)SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85% glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time.

Psychological approaches during dressing changes of burned patients: a prospective randomised study comparing hypnosis against stress reducing strategy.

A prospective study was designed to compare two psychological support interventions in controlling peri-dressing change pain and anxiety in severely burned patients. Thirty patients with a total burned surface area of 10-25%, requiring a hospital stay of at least 14 days, were randomised to receive either hypnosis or stress reducing strategies (SRS) adjunctively to routine intramuscular pre-dressing change analgesia and anxiolytic drugs. Visual analogue scale (VAS) scores for anxiety, pain, pain control and satisfaction were recorded at 2-day intervals throughout the 14-day study period, before, during and after dressing changes. The psychological assistance was given on days 8 and 10 after hospital admission. The comparison of the two treatment groups indicated that VAS anxiety scores were significantly decreased before and during dressing changes when the hypnotic technique was used instead of SRS. No difference was observed for pain, pain control and satisfaction, although VAS scores were always better in the hypnosis group. The study also showed that, overall, psychological support interventions reduced pain and increased patient satisfaction. These results confirm the potential benefits of psychological assistance during dressing changes in burned patients.

Medical treatment of a central vein suppurative thrombosis with cerebral metastatic abscesses in a burned child.

A 2-year-old girl admitted with third degree burns (35% TBSA) received 7 weeks poly-antibiotic therapy combined with heparin for a severe Methicillin-resistant Staphylococcus aureus sepsis with multiple metastatic abscesses (lung, skin, brain), from a suppurative thrombophlebitis of the right jugularis interna, extended to the axillary and cava superior veins. Surgical treatment was contraindicated by the local extension. The child was discharged without major neurological sequelae 3 months after admission.

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction.

STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Immunoregulatory effector cells in drug-induced toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a rare drug-induced disease for which the pathomechanism remains poorly understood. The effector cells of epidermal injury in TEN were studied by taking skin biopsies of early lesions in 23 TEN patients and by performing immunohistochemical tests using antibodies to factor XIIIa (type I dendrocytes), L1-protein (mainly Mac 387+ monocytes and macrophages), UCLHI (mainly CD45R0+ T-memory lymphocytes), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Computerized image analysis was used to evaluate the cell density relative to each immunolabeling. A statistical analysis of cellular counts revealed a numeric relation between the cell types in skin with TEN. Factor XIIIa+ dendrocytes were abundant and plump in the dermis, although Mac 387+ macrophages were the most numerous inflammatory cells in the epidermis. Their numbers greatly exceeded those of CD45R0+ T lymphocytes and cells showing immunoreactivity for either IL-6 or TNFalpha. In the epidermis, IL-6+ cells were significantly less numerous than TNFalpha+ cells. No quantitative difference was found between IL-6+ and CD45R0+ cell populations. Correlations were observed between either the numbers of TNFalpha+ cells or Mac 387+ macrophages and CD45R0+ lymphocytes. In the dermis, a significant correlation was also present between the numbers of Mac 387+ and factor XIIIa+ cells. These findings highlight the complex interactions between the inflammatory cells that mediate epidermal damage in skin with TEN. The high density of factor XIIIa+ dendrocytes and Mac 387+ macrophages in lesional skin assigns these cellular populations a prominent role in the pathomechanism of TEN. Despite a lower cell density, CD45RO+ T-memory lymphocytes likely participate in TNFalpha- and IL-6-regulated processes in the epidermis of TEN. TNFalpha seems to be a major cytokine involved in TEN, although a less prominent role can be ascribed to IL-6.

The usefulness of combined high-frequency percussive ventilation during acute respiratory failure after smoke inhalation.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. We describe 11 severely burned patients with acute respiratory failure due to inhalation injury who did not respond adequately to conventional respiratory support. High-frequency percussive ventilation (HFPV) is a recent ventilatory mode, which combines the advantages of conventional ventilation with some of those of high-frequency ventilation. Seven patients developed pulmonary infection during the acute phase; one patient died of multiple organ failure on day 25. All the other patients survived; two developed bronchiolitis obliterans symptoms before discharge. No side-effects were noted and haemodynamic tolerance of HFPV was excellent. Our findings suggest that HFPV can improve pulmonary function and gas exchange in these catastrophic pulmonary failures following inhalation injury.

Cultured allogeneic keratinocyte sheets accelerate healing compared to Op-site treatment of donor sites in burns.

Donor site treatment is a crucial issue in the treatment of extensive burns. In this single-blind, randomized study treatment of donor sites with a polyurethane dressing, Op-Site (Smith & Nephew, York, U.K.) is compared to treatment with allogeneic cultured keratinocyte sheets. Results show a mean healing time of 6.7 days with use of cultured keratinocyte sheets compared to mean healing time of 13.6 days with Op-Site treatment. Also, improvement in the comfort of patients as the result of less exudate formation and pain attenuation was noted.

Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.