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With the ever-increasing complexity of dynamic radiotherapy treatments, dose calculation algorithms are challenged to accurately calculate the dose resulting from small, on- and off-axis multileaf collimator (MLC) aperture movements. Although the currently available Eclipse (Varian Medical Systems, Palo Alto) dose calculation algorithms still use a simplified, binary MLC model, a more advanced and detailed modeling of the MLC could be beneficial for the dose calculation precision of high-end treatments.To improve the modeling of the MLC in the dose calculation algorithms of the Eclipse treatment planning system, an enhanced MLC attenuation model was constructed through ray tracing through the actual leaf designs for the most commonly used Varian MLC types. The enhanced leaf model (ELM) thus includes the rounded leaf tip shape, the drive screw cutout, and the leaf body thickness. The purpose of this work is to test out this new model and explore possible improvements compared to the previous model.Dose calculations were performed in a research Eclipse environment equipped with the original and enhanced MLC model. Measurements were performed on TrueBeam and on Halcyon dual MLC treatment units. Dedicated static and dynamic MLC test plans were designed to challenge the dose calculation and highlight differences between both models while keeping the experimental setup simple in order to minimize measurement uncertainties. Measurements were performed with single ion chambers, 2D ion chamber arrays and film.The improved MLC model considerably improves the accuracy of the dose calculation for the test fields used in this study. For the TrueBeam MLC, improvements are most prominent for off-axis dose delivery through narrow (static or dynamic) MLC gaps. For 3 mm narrow sweeping gap deliveries at 12 cm off-axis, the advanced model agrees within 2% with the measurement, in contrast to the 12% deviation observed with the original MLC model. For the Halcyon MLC, improvements are especially prominent when the leaves of both MLC stacks are aligned, regardless of their position in the field. Sweeping gap measurements improve from a 7%-10% deviation with the original model to within 2% with the new model.Although test fields designed in this study emphasize the flaws in the original MLC dose calculation model, the enhanced MLC model resolves all of the observed discrepancies, showing excellent on- and off-axis agreements with all of the performed measurements.

Purpose This manuscript describes the experience of two institutions in commissioning the new HalcyonTM platform. Its purpose is to: (a) validate the pre-defined beam data, (b) compare relevant commissioning data acquired independently by two separate institutions, and (c) report on any significant differences in commissioning between the Halcyon linear accelerator and other medical linear accelerators. Methods Extensive beam measurements, testing of mechanical and imaging systems, including the multi-leaf collimator (MLC), were performed at the two institutions independently. The results were compared with published recommendations as well. When changes in standard practice were necessitated by the design of the new system, the efficacy of such changes was evaluated as compared to published approaches (guidelines or vendor documentation). Results Given the proper choice of detectors, good agreement was found between the respective experimental data and the treatment planning system calculations, and between independent measurements by the two institutions. MLC testing, MV imaging, and mechanical system showed unique characteristics that are different from the traditional C-arm linacs. Although the same methodologies and physics equipment can generally be used for commissioning the Halcyon, some adaptation of previous practices and development of new methods were also necessary. Conclusions We have shown that the vendor pre-loaded data agree well with the independent measured ones during the commission process. This verifies that a data validation instead of a full-data commissioning process may be a more efficient approach for the Halcyon. Measurement results could be used as a reference for future Halcyon users.

Rapid diagnosis is critical to minimize morbidity and mortality associated with infections of the central nervous system (CNS). In this study, we evaluated the performance of a multiplex polymerase chain reaction (PCR) and microarray-based method, Prove-it™ Herpes, in a routine clinical laboratory setting for the diagnostics of 7 herpesviruses in viral CNS infections. Cerebrospinal fluid samples (n = 495), which had arrived for diagnostics in the 5 participating laboratories, were analyzed for herpesvirus DNA both by the current PCR-based method of the laboratory and by the microarray assay. The sensitivity and specificity for the microarray assay were 93% and 99%, respectively. The microarray assay was considered as a rapid and robust diagnostic platform that was easily implemented into the laboratory workflow. The broad herpesvirus coverage and the small sample volume required by the assay could benefit the diagnostics and thus the treatment of life-threatening infections of the CNS, especially among immunocompromised patients.

After a nosocomial outbreak caused by Legionella pneumophila serogroup 5, the hospital water distribution system, which was found to be colonized by L. pneumophila serogroups 5 and 6, was decontaminated by the superheat and flush method and by installing an additional heat-shock unit in one of the hot water circuits. This unit exposed the recirculated water to a temperature of 80 degrees C. The efficacy of the decontamination measures was evaluated by monitoring the temperatures and legionella concentrations at different parts of the hot water distribution system. The genetic diversity of the colonizing legionella flora was examined using two genotyping methods: amplified fragment length polymorphism analysis (AFLP) and random amplified polymorphic DNA (RAPD) analysis. Selected serogroup 6 strains were also analyzed by sequence-based typing (SBT). The results indicated that long-term eradication of serogroup 5 strains was never achieved. Only one serogroup 6 strain was never isolated after the superheat and flush. In all, according to genetic fingerprints, the diversity of Legionella strains in a hospital water system remains stable over the years regardless of the use of recommended disinfection procedures.

The mechanisms of aposematism (unprofitability of prey combined with a conspicuous signal) have mainly been studied with reference to vertebrate predators, especially birds. We investigated whether dragonflies, Aeshna grandis, avoid attacking wasps, Vespula norwegica, which are an unprofitable group of prey for most predators. As a control we used flies that were painted either black or with yellow and black stripes. The dragonflies showed greater aversion to wasps than to flies. Black-and-yellow-striped flies were avoided more than black ones, suggesting that aposematic coloration on a harmless fly provides a selective advantage against invertebrate predators. There was no significant difference in reactions to black-painted and black-and-yellow wasps, indicating that, in addition to coloration, some other feature in wasps might deter predators. In further experiments we offered dragonflies artificial prey items in which the candidate warning signals (coloration, odour and shape) were tested separately while other confounding factors were kept constant. The dragonflies avoided more black-and-yellow prey items than solid black or solid yellow ones. However, we found no influence of wasp odour on dragonfly hunting. Dragonflies were slightly, but not significantly, more reluctant to attack wasp-shaped prey items than fly-shaped ones. Our results suggest that the typical black-and-yellow stripes of wasps, possibly combined with their unique shape, make dragonflies avoid wasps. Since black-and-yellow stripes alone significantly decreased attack rate, we conclude that even profitable prey species (i.e. Batesian mimics) are able to exploit the dragonflies' avoidance of wasps. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.

Background Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. Objective The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule. Methods Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones. Results In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15–38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones. Conclusions The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.

Uropathogenicity of Escherichia coli has a strong association with P fimbriae. At the tip of P fimbriae are located G adhesins which mediate binding to glycolipid receptors. The G adhesins are encoded by pap or prs gene clusters. The three receptor binding variants of G adhesins (Classes I to III) recognized so far, differ in their binding to different isoreceptors of the Galα1–4gal-containing glycolipid family. Lately, the genetic detection of G adhesins by colony hybridization or polymerase chain reaction (PCR) has been used as an alternative to previous phenotypic P fimbria detection methods. In the present study, 257 E. coli urine isolates from clinically properly characterized patients with either acute cystitis or recurrent E. coli infections were examined for the three G adhesin classes with a PCR-based method. The PCR reaction was carried out with four oligonucleotide primers in a single reaction tube. Sixty-five isolates (25%) had genes for Class II G adhesin, 31 (12%) for Class III and two additional isolates had genes for both Class II and III G adhesins. Of the total of 98 strains possessing genes for P fimbriae, 26 (27%) were undetectable with the previous P-specific latex agglutination test and 7 (7%) strains with the mannose-resistant heme agglutination method. The new PCR method presented in this study allows an easy, fast, cheap, sensitive and reliable detection method for P fimbriae as well as the specific G adhesin type. Thus, it allows further evaluation of the clinical significance of these G adhesins in strains causing other types of urinary tract infections (UTI) other than cystitis.

To examine the intraspecies variation of Mycobacterium malmoense, 29 clinical isolates, chemotyped by thin-layer chromatography of their surface glycolipids into five subgroups, were typed using RFLP analysis. Two 32P-labelled ribosomal RNA (rRNA) gene fragments were used as probes for chromosomal DNA digested with BanI. By comparison of the fingerprint patterns, the strains could be divided into five ribotypes, two major and three minor ones. The chemotypes were not connected to any of the ribotypes. Combination of the results of the two techniques produced a considerable resolution. This method is a useful additional tool when studying the epidemiological behaviour of M. malmoense.

Nosocomial acquisition ofMycobacterium fortuitumled to a disseminated infection in a leukemia patient. A linkage to shower-head water was supported by molecular typing of clinical and environmental isolates. Contamination of the hospital water system with microbes that are relatively resistant to common sanitation processes poses an increased risk of infection to neutropenic patients.

Cervical lymphadenitis is the main manifestation of Mycobacterium avium infection in immunocompetent children. Exposure to birds has been discussed as a source of infection. To clarify from where children acquire the infection, M. avium isolates from different origins were analysed with restriction fragment length polymorphism (RFLP) on insertion sequence IS1245, and compared by computer cluster correlation analysis. This molecular epidemiological tool has previously revealed a distinction between multiband profiles found mainly in strains from humans, and a 3-band/bird type profile in strains isolated mainly from birds. 32 isolates from children were compared with 28 isolates from adults and 45 isolates from animals. We found that 67% of the animal isolates had the bird type profile, also found in 1 sputum isolate from an adult. Strains from children showed only multiband profiles that did not differ significantly from profiles of isolates from adults. All but 2 bird isolates showed the bird type profile. Neither of the remaining 2, which had multiband profiles, clustered with the isolates from children. Our results indicate that the true reservoir of M. avium is unknown. Thus the question of whether or not M. avium can be incriminated as a zoonotic disease remains unanswered.

An outbreak of infections caused by Legionella pneumophila serogroup 5 was detected in a university hospital, and nosocomial reservoirs of the legionella epidemic were examined. Clinical isolates from two patients who had been affected by the L. pneumophila serogroup 5 outbreak, and from another patient with a legionella infection caused by the same serogroup 3 years later, were compared to L. pneumophila serogroup 5 isolates from the hospital water supply by two molecular methods, amplified fragment length polymorphism (AFLP) analysis and random amplified polymorphic DNA analysis (RAPD). Genotyping confirmed the epidemiological linkage of the first two patients, and linked their infections with the hospital water supply. The third clinical strain, which was also linked to the hospital water, was very similar to the epidemic strain. Even though the water distribution system was sanitized (superheat and flush sanitation), the epidemic strain was shown to be persisting in the hospital water outlets several years after its initial discovery.

A cluster of septicaemias due to several water-related species occurred in a haematological unit of a university hospital. In recurrent septicaemias of a leukaemic patient caused by Sphingomonas paucimobilis, genotyping of the blood isolates by use of random amplified polymorphic DNA-analysis verified the presence of two distinct S. paucimobilis strains during two of the separate episodes. A strain of S. paucimobilis identical to one of the patient's was isolated from tap water collected in the haematological unit. Thus S. paucimobilis present in blood cultures was directly linked to bacterial colonization of the hospital water system. Heterogeneous finger-printing patterns among the clinical and environmental isolates indicated the distribution of a variety of S. paucimobilis clones in the hospital environment. This link also explained the multi-microbial nature of the outbreak.

Mycobacterium avium is an important veterinary pathogen causing avian tuberculosis in birds. The aim of the study was to evaluate the genetic relatedness in M. avium isolates from deep tissues of farmed lesser white-fronted geese with avian tuberculosis and in samples from the farm environment. The strains were analyzed by two PCR-based typing methods, inverted repeat (IR) typing and random amplified polymorphic DNA (RAPD) analysis. The primers for the inverted repeats of the insertion sequences IS1245 and IS1311 were used in IR typing, and the RAPD analysis was performed with six primers. Seven of the nine avian strains yielded an identical pattern in the IR typing, but they could be divided into two groups in the RAPD analysis. The remaining two bird isolates had an identical IR pattern (IR cluster II) which they shared with two environmental isolates. However, the RAPD analysis revealed that these environmental isolates had a RAPD pattern (RAPD cluster VI) distinct and different from either of the bird isolates (RAPD clusters II and IV). In all, four M. avium strains were verified as being inducers of avian tuberculosis in birds, and all were distinct from the three environmental strains identified. Thus, the results did not confirm the preliminary idea that a single strain had caused the epidemic. The polymorphism among M. avium strains highlighted the great biodiversity among an M. avium population even in a limited environmental setting during a short time span, and indicated the high susceptibility to avian tuberculosis of lesser white-fronted geese.

The relationship between the molecular structure of allergenic proteins and the allergenic determinants is one of the central issues in allergology. We report here that the natural preparation of Bos d 2, a mammalian lipocalin allergen, comprises three molecular variant proteins of 17,829, 17,781, and 17,800 Da. When cDNA of Bos d 2 (Genome Sequence Data Base No. L42867) was recloned and expressed in Pichia pastoris, two proteins were produced. One of the proteins (17,831 Da) and the proteins in the natural preparation had pyroglutamate as the N-terminal residue; in the other (17,849 Da) the N-terminal residue was glutamine. Recombinant Bos d 2 protein was crystallized and the native data set was collected at 1.8 A resolution.

The discriminatory power of random amplified polymorphic DNA (RAPD) analysis was assessed for detection of intraspecies variation in Escherichia coli strains of clinical origin. Three primers (OPF 5, OPF 7 and OPF 8) were preselected from commercial 10-mer primers by the number of distinct bands obtained. These primers were used in testing 26 urinary and 13 blood isolates from 26 patients and E. coli ATCC 25922, OPF 5, OPF 7 or OPF 8 alone separated the strains into 15 to 21 RAPD types. A combination of the results of the three primers gave 25 RAPD types. When blood and urine isolates of each patient were analysed in parallel, all blood-urine pairs were found identical, and with one exception they were also unique. RAPD analysis had a high discriminatory power. It separated the strains equally well or better than ribotyping, and obviously better than serotyping which grouped the urine strains into 8 serogropus leaving 18 strains untypable or incompletely typed. Thus, to verify the identity or non-identity of isolated E. coli strains, RAPD analysis was shown to be a sensitive and reproducible technique which is technically less demanding, more rapid and more economical than either serotyping or ribotyping. However, in its present application, this technique cannot fully replace determination of the serotype or virulence factors which may show correlations with different manifestations of infection.

The suitability of random amplified polymorphic DNA-PCR for the detection of differences between Mycobacterium malmoense strains was evaluated. With two of the 32 tested primers seven fingerprint patterns which proved excellent in distinguishing intraspecies variations of M. malmoense were obtained. The combination of the results obtained with the two primers permitted a clear separation of the strains. This technique is useful for analyzing species whose DNA sequences are not known. It can easily be adapted to test any mycobacterial species.