Guidelines for the statistical analysis of a collaborative study of a laboratory method for testing disinfectant product performance.

This paper presents statistical techniques suitable for analyzing a collaborative study (multilaboratory study or ring trial) of a laboratory disinfectant product performance test (DPPT) method. Emphasis is on the assessment of the repeatability, reproducibility, resemblance, and responsiveness of the DPPT method. The suggested statistical techniques are easily modified for application to a single laboratory study. The presentation includes descriptions of the plots and tables that should be constructed during initial examination of the data, including a discussion of outliers and QA checks. The statistical recommendations deal with evaluations of prevailing types of DPPTs, including both quantitative and semiquantitative tests. The presentation emphasizes tests in which the disinfectant treatment is applied to surface-associated microbes and the outcome is a viable cell count; however, the statistical guidelines are appropriate for suspension tests and other test systems. The recommendations also are suitable for disinfectant tests using any microbe (vegetative bacteria, virus, spores, etc.) or any disinfectant treatment. The descriptions of the statistical techniques include either examples of calculations based on published data or citations to published calculations. Computer code is provided in an appendix.

Report from the Latin American Spondyloarthritis Society for Education and Research in Immunology and Medicine organization 2012 workshop.

The first annual meeting of the Latin American Spondyloarthritis Society for Education and Research in Immunology and Medicine (LASSERIM) was held in Bogotá, Colombia, in September 2012 and was attended by key opinion leaders, researchers, and rheumatologists. The meeting included presentations and discussions from renowned speakers during 2 days and a coaching leadership exercise led by an expert in the field followed by an open forum. Two groups defined a priori discussed the establishment of a professional network and organization to be involved in the identification, assessment, and effective resolution of health care issues in Latin America.A broad spectrum of topics were discussed but focused on the following: pharmacoeconomics in general rheumatology, spondyloarthritis and chronic back pain, therapeutic interventions in rheumatoid arthritis, ultrasonography in spondyloarthritis, impact of social media in medicine and global trends in leadership, quality of life, and innovation. A special workshop on coaching in health care and coaching as a tool to implement LASSERIM goals was part of the 2-day conference.LASSERIM will be working in the future on education, research, and innovation in the field of rheumatology and immunology. A special focus will be on spondyloarthritis, by promoting research, open discussions, and by conducting carefully planned research studies to impact on the quality of life of patients and doctors from Latin American countries.

Quantifying signal dispersion in a hybrid ice core melting system.

We describe a microcontroller-based ice core melting and data logging system allowing simultaneous depth coregistration of a continuous flow analysis (CFA) system (for microparticle and conductivity measurement) and a discrete sample analysis system (for geochemistry and microparticles), both supplied from the same melted ice core section. This hybrid melting system employs an ice parcel tracking algorithm which calculates real-time sample transport through all portions of the meltwater handling system, enabling accurate (1 mm) depth coregistration of all measurements. Signal dispersion is analyzed using residence time theory, experimental results of tracer injection tests and antiparallel melting of replicate cores to rigorously quantify the signal dispersion in our system. Our dispersion-limited resolution is 1.0 cm in ice and ~2 cm in firn. We experimentally observe the peak lead phenomenon, where signal dispersion causes the measured CFA peak associated with a given event to be depth assigned ~1 cm shallower than the true event depth. Dispersion effects on resolution and signal depth assignment are discussed in detail. Our results have implications for comparisons of chemistry and physical properties data recorded using multiple instruments and for deconvolution methods of enhancing CFA depth resolution.

Procedural revision to the use-dilution methods: establishment of maximum log density value for test microbes on inoculated carriers.

(Staphylococcus aureus) and 964.02 (Pseudomonas aeruginosa), were revised in 2009 to include a standardized procedure to measure the log density of the test microbe and to establish a minimum mean log density value of 6.0 (geometric mean of 1.0 x 10(6) CFU/carrier) to qualify the test results. This report proposes setting a maximum mean log density value of 7.0 (geometric mean of 1.0 x 10(7) CFU/carrier) to further standardize the procedure. The minimum value was based on carrier count data collected by four laboratories over an 8-year period (1999-2006). The data have been updated to include an additional 4 years' worth of data (2006-2010) collected by the same laboratories. A total of 512 tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed in each test, for a total of 3072 carriers. Mean log densities for each of the 512 tests were at least 6.0. With the exception of two tests, one for P. aeruginosa without OSL and one for S. aureus with OSL, the mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). Across microbes and OSL treatments, the mean log density (+/- SEM) was 6.80 (+/- 0.07) per carrier (a geometric mean of 6.32 x 10(6) CFUlcarrier) and acceptable repeatability (0.28) and reproducibility (0.31) SDs were exhibited. A maximum mean log density per carrier of 7.0 is being proposed here as a validity requirement for S. aureus and P. aeruginosa. A modification to the method to allow for dilution of the final test cultures to achieve carrier counts within 6.0-7.0 logs is also being proposed. Establishing a range of 6.0-7.0 logs will help improve the reliability of the method and should allow for more consistent results within and among laboratories.

Performance of the AOAC use-dilution method with targeted modifications: collaborative study.

The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.

Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.

Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.

Semi-automated open water iceberg detection from Landsat applied to Disko Bay, West Greenland.

Changes in Greenland's marine-terminating outlet glaciers have led to changes in the flux of icebergs into Greenland's coastal waters, yet icebergs remain a relatively understudied component of the ice-ocean system. We developed a simple iceberg delineation algorithm for Landsat imagery. A machine learning-based cloud mask incorporated into the algorithm enables us to extract iceberg size distributions from open water even in partially cloudy scenes. We applied the algorithm to the Landsat archive covering Disko Bay, West Greenland, to derive a time series of iceberg size distributions from 2000-02 and 2013-15. The time series captures a change in iceberg size distributions, which we interpret as a result of changes in the calving regime of the parent glacier, Sermeq Kujalleq (Jakobshavn Isbræ). The change in calving style associated with the disintegration and disappearance of Sermeq Kujalleq's floating ice tongue resulted in the production of more small icebergs. The increased number of small icebergs resulted in increasingly negative power law slopes fit to iceberg size distributions in Disko Bay, suggesting that iceberg size distribution time series provide useful insights into changes in calving dynamics.

First-Order Estimates of Coastal Bathymetry in Ilulissat and Naajarsuit Fjords, Greenland, from Remotely Sensed Iceberg Observations.

Warm water masses circulating at depth off the coast of Greenland play an important role in controlling rates of mass loss from the Greenland Ice Sheet through feedbacks associated with the melting of marine glacier termini. The ability of these warm waters to reach glacier termini is strongly controlled by fjord bathymetry, which was unmapped for the majority of Greenland's fjords until recently. In response to the need for bathymetric measurements in previously uncharted areas, we developed two companion methods to infer fjord bathymetry using icebergs as depth sounders. The main premise of our methods centers around the idea that deep-drafted icebergs will become stranded in shallow water such that estimates of iceberg surface elevation can be used to infer draft, and thus water depth, under the assumption of hydrostatic equilibrium. When and where available, surface elevations of icebergs stranded on bathymetric highs were extracted from digital elevation models (DEMs) and converted to estimates of iceberg draft. To expand the spatial coverage of our inferred water depths beyond the DEM footprints, we used the DEMs to construct characteristic depth-width ratios and then inferred depths from satellite imagery-derived iceberg widths. We tested and applied the methods in two fjord systems in western Greenland with partially constrained bathymetry, Ilulissat Isfjord and Naajarsuit Fjord, to demonstrate their utility for inferring bathymetry using remote sensing datasets. Our results show that while the uncertainties associated with the methods are high (up to ±93 m), they provide critical first-order constraints on fjord bathymetry.

Measurement of recent exposure to Phlebotomus argentipes, the vector of Indian visceral Leishmaniasis, by using human antibody responses to sand fly saliva.

Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs.

Statistical quantification of detachment rates and size distributions of cell clumps from wild-type (PAO1) and cell signaling mutant (JP1) Pseudomonas aeruginosa biofilms.

The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon. Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics. In manufacturing plants detached clumps entering a process stream decrease product quality. Two strains of Pseudomonas aeruginosa, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment. We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis. The two strains were similar in terms of both biofilm structure and detachment patterns. Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells. The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data. For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm(-2) min(-1) for PAO1 and 4.3 events mm(-2) min(-1) for JP1. These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure.