Reduced patient restrictions following total hip arthroplasty: study protocol for a randomized controlled trial

Stepped wedge cluster randomised trials introduce interventions to groups of clusters in a random order and have been used to evaluate interventions for health and wellbeing. Standardised guidance for reporting stepped wedge trials is currently absent, and a range of potential analytic approaches have been described. We systematically identified and reviewed recently published (2010 to 2014) analyses of stepped wedge trials. We extracted data and described the range of reporting and analysis approaches taken across all studies. We critically appraised the strategy described by three trials chosen to reflect a range of design characteristics. Ten reports of completed analyses were identified. Reporting varied: seven of the studies included a CONSORT diagram, and only five also included a diagram of the intervention rollout. Seven assessed the balance achieved by randomisation, and there was considerable heterogeneity among the approaches used. Only six reported the trend in the outcome over time. All used both ‘horizontal’ and ‘vertical’ information to estimate the intervention effect: eight adjusted for time with a fixed effect, one used time as a condition using a Cox proportional hazards model, and one did not account for time trends. The majority used simple random effects to account for clustering and repeat measures, assuming a common intervention effect across clusters. Outcome data from before and after the rollout period were often included in the primary analysis. Potential lags in the outcome response to the intervention were rarely investigated. We use three case studies to illustrate different approaches to analysis and reporting. There is considerable heterogeneity in the reporting of stepped wedge cluster randomised trials. Correct specification of the time-trend underlies the validity of the analytical approaches. The possibility that intervention effects vary by cluster or over time should be considered. Further work should be done to standardise the reporting of the design, attrition, balance, and time-trends in stepped wedge trials.     

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.Invasive aspergillosis is a fungal disease caused by Aspergillus species that primarily affects immunocompromised patients, such as those treated for hematological malignancy. Patients may become infected by inhalation of ambient air that contains fungal spores. The Aspergillus conidia can penetrate into the alveoli and if not effectively removed, may germinate, proliferate, and cause invasive aspergillosis. Mortality and morbidity due to invasive aspergillosis remain a significant problem.Triazoles, such as itraconazole (ITZ), voriconazole, and posaconazole, are used increasingly in the management of patients with this disease. Although the risk of resistance due to the increased use of triazoles is considered low (11), we recently observed ITZ resistance rapidly emerging in clinical Aspergillus fumigatus isolates (19, 22, 24, 25). Azole resistance was observed in up to 6% of patients in our hospital and in up to 14.5% of isolates sent to our laboratory from other hospitals in The Netherlands, which were obtained from patients with aspergillus disease (19). Furthermore, azole resistance has been reported in other European countries (3, 13, 19). The ITZ-resistant isolates also showed significantly reduced susceptibility to the other mold-active medical triazoles voriconazole and posaconazole (19). A substitution of leucine for histidine at codon 98 (L98H), combined with a 34-bp tandem repeat (designated TR) in the promoter region of the cyp51A gene (TR/L98H), which is the target for antifungal azoles, was found in 94% of isolates (14, 19, 24).Azole resistance can develop through the exposure of the fungus to azole compounds, which may occur in azole-treated patients or through the use of azole compounds in the environment. The dominance of a single resistance mechanism is difficult to explain by resistance development in individual azole-treated patients, as one would expect multiple resistance mechanisms to develop. Also, spread by person-to-person transmission of any Aspergillus isolate is highly unlikely. As inhalation of airborne aspergillus spores is the common route of infection for aspergillus diseases, we hypothesized that the dominance of a single resistance mechanism in clinical ITZ-resistant isolates was more consistent with acquisition from a common environmental source (19). If azole-resistant A. fumigatus is present in our environment, patients could inhale resistant spores and subsequently develop azole-resistant disease. Indeed, azole-resistant aspergillosis was reported in azole-naïve patients, indicating that resistance does not exclusively develop during azole therapy (24).Favorable conditions for resistance development are exposure to azole compounds and the presence of reproducing fungus (1). A. fumigatus is abundantly present in our environment as saprophytic, reproducing fungi, most notably in soil and compost. Furthermore, azoles are commonly used for plant protection as well as material preservation. Therefore, it appears that resistance development in A. fumigatus is feasible in the environment, and isolates that develop resistance to fungicides might be cross-resistant to medical triazoles.We investigated if A. fumigatus isolates that are present in our environment are resistant to medical triazoles and if they are cross-resistant to azole fungicides. Furthermore, we characterized the isolates by microsatellite typing in order to determine if they were genetically related to clinical A. fumigatus isolates previously obtained from patients cared for in our University Medical Center.     

A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

Background

Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.

Methodology

LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.

Principal Findings

VLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.

Conclusions

Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.     

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.     

Development of a 24 h screen to detect viable salmonellas in faeces

A 24 h screen to detect viable salmonellas in faeces was developed by studying growth dynamics of salmonellas and competing flora in combinations of enrichment media and artificially-inoculated pig faeces. Muller-Kauffmann tetrathionate (MK) broth, incubated overnight at 42°C, maintained the lowest ratio of salmonella: competing flora and identified all inoculated samples. A 4 h postenrichment in M broth plus novobiocin reduced the number of false-positive results in subsequent ELISAs. Adjusting the negative cut-off values and incubation time of the chromogenic substrate from that recommended in the ELISA instructions reduced the rate of false-positive results further and allowed the detection of 10³ salmonellas per ml in the presence of up to 10⁷ ml⁻¹ aerobic-competing cells. Suspension of faeces diluted 1 in 2 and 1 in 5, rather than 1 in 10 in MK broth did not necessitate further adjustments to the ELISA baseline values. The proposed screen protocol is an overnight incubation of faeces suspended 1 in 10 in MK broth, a 1 in 100 subculture into M broth plus 10 μg ml⁻¹ novobiocin (MbN) for 4 h, steam inactivation of MbN cultures and testing by ELISA, and can detect three salmonella cells per g faeces.     

Effect of Fluticasone propionate Aqueous Nasal Spray Treatment on Platelet Activating Factor and Eicosanoid Production By nasal Mucosa in Patients with A house Dust Mite Allergy

The relationship between the release of platelet activating factor (PAF), leukotriene C(4)/D(4)/E(E) (LTC(4)/D(4)/E(4)) and prostaglandin D(2) (PGD(2)) from nasal mucosa in vivo was examined in 24 rhinitis patients allergic to the house dust mite (HDM). During a double blind placebo controlled cross-over study 200 mug fluticasone propionate aqueous nasal spray (FPANS) was administered twice daily for two weeks. In response to allergen provocation (100, 1 000, 10 000 Bu/ml) and during the 9.5 h after this challenge the nasal fluid was obtained by washing the nose with saline and the levels of PAF, LTC(4)/D(4)/E(4) and PGD(2), as indicators of mediator release, were measured at the following time-points: baseline (t = - 1/2), allergen provocation with 10 000 Bu/ml (t = 0), 3.5 and 7.5 h (late phase). After allergen provocation the levels of the mediators increased in the nasal fluids of placebo treated patients (x-fold increase to baseline: PAF, 15; LTC(4)/D(4)/E(4), 12; PGD(2), 1.5). In fluids of patients treated with FPANS these levels tended to decrease. At the time of provocation the levels of PAF, LTC(4)/D(4)/E(4) and PGD(2) showed a significant correlation. The results indicate that these mediators can be used as markers of allergic reactions against house dust mites and that fluticasone propionate aqueous nasal spray tended to reduce the release of mediators of inflammation correlated with beneficial effects on clinical symptoms in this type of allergic reactions.     

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV⁺, its plasmidless isogenic derivative W22703 pYV^- and four recent field isolates was examined.
The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV^- strains grew consistently faster than pYV⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C.
Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1–20°C range no obvious temperature- or plasmid-related differences in protein patterns could be detected.
The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.