Selective decontamination and the anaerobic faecal flora

Selective decontamination with non-absorbable antibiotics of the gastrointestinal tract of patients with impaired host defense is increasingly applied to prevent infections with gram-negative facultatively anaerobic rods. In vivo experiments on the effect of antibiotics on the human intestinal flora were performed with (ex-germ-free) mice. The experiments have shown that the human obligately anaerobic flora did not change during selective decontamination with polymyxin B, but Enterobacteriaceae were eliminated. The magnitude of differences between the sensitivity to antibiotics of the human obligately anaerobic flora and the Enterobacteriaceae in vitro may be used as an index for the usefulness of the antibiotic for selective decontamination. Binding of the antibiotics to intestinal contents has been found to be important for the estimation of the daily dose. The release of the bound antibiotic could contribute to the maintenance of equable concentrations in the intestine.     

Public Experiences of Mass Casualty Decontamination

In this article, we analyze feedback from simulated casualties who took part in field exercises involving mass decontamination, to gain an understanding of how responder communication can affect people's experiences of and compliance with decontamination. We analyzed questionnaire data gathered from 402 volunteers using the framework approach, to provide an insight into the public's experiences of decontamination and how these experiences are shaped by the actions of emergency responders. Factors that affected casualties' experiences of the decontamination process included the need for greater practical information and better communication from responders, and the need for privacy. Results support previous findings from small-scale incidents that involved decontamination in showing that participants wanted better communication from responders during the process of decontamination, including more practical information, and that the failure of responders to communicate effectively with members of the public led to anxiety about the decontamination process. The similarity between the findings from the exercises described in this article and previous research into real incidents involving decontamination suggests that field exercises provide a useful way to examine the effect of responder communication strategies on the public's experiences of decontamination. Future exercises should examine in more detail the effect of various communication strategies on the public's experiences of decontamination. This will facilitate the development of evidence-based communication strategies intended to reduce anxiety about decontamination and increase compliance among members of the public during real-life incidents that involve mass decontamination.     

Evaluation of microwave steam bags for the decontamination of filtering facepiece respirators

Reusing filtering facepiece respirators (FFRs) has been suggested as a strategy to conserve available supplies for home and healthcare environments during an influenza pandemic. For reuse to be possible, used FFRs must be decontaminated before redonning to reduce the risk of virus transmission; however, there are no approved methods for FFR decontamination. An effective method must reduce the microbial threat, maintain the function of the FFR, and present no residual chemical hazard. The method should be readily available, inexpensive and easily implemented by healthcare workers and the general public. Many of the general decontamination protocols used in healthcare and home settings are unable to address all of the desired qualities of an efficient FFR decontamination protocol. The goal of this study is to evaluate the use of two commercially available steam bags, marketed to the public for disinfecting infant feeding equipment, for FFR decontamination. The FFRs were decontaminated with microwave generated steam following the manufacturers' instructions then evaluated for water absorption and filtration efficiency for up to three steam exposures. Water absorption of the FFR was found to be model specific as FFRs constructed with hydrophilic materials absorbed more water. The steam had little effect on FFR performance as filtration efficiency of the treated FFRs remained above 95%. The decontamination efficacy of the steam bag was assessed using bacteriophage MS2 as a surrogate for a pathogenic virus. The tested steam bags were found to be 99.9% effective for inactivating MS2 on FFRs; however, more research is required to determine the effectiveness against respiratory pathogens.     

Limited effects of among-lineage rate variation on the phylogenetic performance of molecular markers

Variation in substitution rates among evolutionary lineages (among-lineage rate variation or ALRV) has been reported to negatively affect the estimation of phylogenies. When the substitution processes underlying ALRV are modeled inadequately, non-sister taxa with similar substitution rates are estimated incorrectly as sister species due to long-branch attraction. Recent advances in modeling site-specific rate variation (heterotachy) have reduced the impacts of ALRV on phylogeny estimation in several empirical and simulated datasets. However, the addition of parameters to the substitution model reduces power to estimate each parameter correctly, which can also lead to incorrect phylogeny estimation. A potential solution to this problem is to identify the levels of ALRV that negatively impact phylogeny estimation such that molecular markers with non-deleterious levels of ALRV can be identified. To this end, we used analyses of empirical and simulated gene datasets to evaluate whether levels of ALRV identified in a mitochondrial genomic dataset for salamanders negatively impacted phylogeny estimation. We simulated data with and without ALRV, holding all other evolutionary parameters constant, and compared the phylogenetic performance of both simulated and empirical datasets. Overall, we found limited, positive effects of ALRV on phylogeny estimation in this dataset, the majority of which resulted from an increase in substitution rate on short branches. We conclude that ALRV does not always negatively impact phylogeny estimation. Therefore, ALRV can likely be disregarded as a criterion for marker selection in comparable phylogenetic studies.     

Substitution rate variation among sites in mitochondrial hypervariable region I of humans and chimpanzees

Mitochondrial D-loop hypervariable region I (HVI) sequences are widely used in human molecular evolutionary studies, and therefore accurate assessment of rate heterogeneity among sites is essential. We used the maximum-likelihood method to estimate the gamma shape parameter alpha for variable substitution rates among sites for HVI from humans and chimpanzees to provide estimates for future studies. The complete data of 839 humans and 224 chimpanzees, as well as many subsets of these data, were analyzed to examine the effect of sequence sampling. The effects of the genealogical tree and the nucleotide substitution model were also examined. The transition/transversion rate ratio (kappa) is estimated to be about 25, although much larger and biased estimates were also obtained from small data sets at low divergences. Estimates of alpha were 0.28-0.39 for human data sets of different sizes and 0.20-0.39 for data sets including different chimpanzee subspecies. The combined data set of both species gave estimates of 0.42-0.45. While all those estimates suggest highly variable substitution rates among sites, smaller samples tend to give smaller estimates of alpha. Possible causes for this pattern were examined, such as biases in the estimation procedure and shifts in the rate distribution along certain lineages. Computer simulations suggest that the estimation procedure is quite reliable for large trees but can be biased for small samples at low divergences. Thus, an alpha of 0.4 appears suitable for both humans and chimpanzees. Estimates of alpha can be affected by the nucleotide sites included in the data, the overall tree length (the amount of sequence divergence), the number of rate classes used for the estimation, and to a lesser extent, the included sequences. The genealogical tree, the substitution model, and demographic processes such as population expansion do not have much effect.     

A preliminary study on viral decontamination of chicken serum using gamma-irradiation

A preliminary experiment was carried out to determine whether a decontamination procedure using gamma irradiation, similar to that adopted in the European guideline for bovine serum contaminated by pestivirus, could be applied to chicken serum. Chicken sera spiked with known amounts of enveloped and non-enveloped chicken viruses were gamma irradiated. The remaining live viruses were then measured by titration and the virus reduction capacity of the irradiation process was established for both enveloped and non-enveloped virus models. In parallel with the irradiation procedure, a classical in vivo extraneous agent test was also evaluated in order to see if it has the capacity to detect low enough levels of live viruses to be used for testing irradiated serum. The results suggest that the principles of the bovine serum decontamination procedure may be applied to chicken serum. Further studies are required to determine if this process would provide an acceptable solution for the viral ‘decontamination’ of chicken serum.     

Intraspecific DNA contamination distorts subtle population structure in a marine fish: Decontamination of herring samples before restriction‐site associated sequencing and its effects on population genetic statistics

Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (a) the efficacy of bleach decontamination, (b) the effect of decontamination on RAD genotypes and (c) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and nonspawning (uncontaminated) herring. Samples were soaked for 10 min in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction site‐associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high‐quality sequences, reproducible genotypes and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high‐throughput sequencing data and can impact downstream analyses.     

Performance of a divergence time estimation method under a probabilistic model of rate evolution

Rates of molecular evolution vary over time and, hence, among lineages. In contrast, widely used methods for estimating divergence times from molecular sequence data assume constancy of rates. Therefore, methods for estimation of divergence times that incorporate rate variation are attractive. Improvements on a previously proposed Bayesian technique for divergence time estimation are described. New parameterization more effectively captures the phylogenetic structure of rate evolution on a tree. Fossil information and other evidence can now be included in Bayesian analyses in the form of constraints on divergence times. Simulation results demonstrate that the accuracy of divergence time estimation is substantially enhanced when constraints are included.     

A fast and reliable computational method for estimating population genetic parameters

The estimation of ancestral and current effective population sizes in expanding populations is a fundamental problem in population genetics. Recently it has become possible to scan entire genomes of several individuals within a population. These genomic data sets can be used to estimate basic population parameters such as the effective population size and population growth rate. Full-data-likelihood methods potentially offer a powerful statistical framework for inferring population genetic parameters. However, for large data sets, computationally intensive methods based upon full-likelihood estimates may encounter difficulties. First, the computational method may be prohibitively slow or difficult to implement for large data. Second, estimation bias may markedly affect the accuracy and reliability of parameter estimates, as suggested from past work on coalescent methods. To address these problems, a fast and computationally efficient least-squares method for estimating population parameters from genomic data is presented here. Instead of modeling genomic data using a full likelihood, this new approach uses an analogous function, in which the full data are replaced with a vector of summary statistics. Furthermore, these least-squares estimators may show significantly less estimation bias for growth rate and genetic diversity than a corresponding maximum-likelihood estimator for the same coalescent process. The least-squares statistics also scale up to genome-sized data sets with many nucleotides and loci. These results demonstrate that least-squares statistics will likely prove useful for nonlinear parameter estimation when the underlying population genomic processes have complex evolutionary dynamics involving interactions between mutation, selection, demography, and recombination.     

Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR

Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.     

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

Evaluation of decontamination process of heart valve and artery tissues in European Homograft Bank (EHB): a retrospective study of 1,055 cases

To evaluate the efficiency of decontamination practice in European Homograft Bank (EHB), the data of the cardiovascular tissues received during recent 2?years were retrospectively analysed in this study. After initial assessment, the tissues were incubated in a 3-antibiotics?? cocktail at 4°C for 20?C48?h. The states of contamination were evaluated before and after incubation with the focus on the differences in donor type, tissue type, germ type and incubation time. Amongst 1,055 eligible tissues, 77.2% were hearts and 22.8% were arteries. 82.2% of the tissues were retrieved from the multi-organ donors (MOD), 15.4% from the recipients of heart transplantation (RHT) and 2.4% from the non-heart beating donors (NHBD). The initial contamination rate was 27.4% with a significantly higher incidence in arteries. The RHT tissues had the lowest contamination rate comparing to that of MOD and NHBD. Staphylococcus species was the major source of contamination. After antibiotic incubation, 76.8% of the contaminated tissues were disinfected, which was significantly higher for the hearts than the arteries. The RHT tissues had the highest decontamination rate than that of MOD and NHBD tissues. Propionibacterium acnes was detected in 48.1% of the remaining contaminated cases. The average incubation time of the Propionibacterium-positive tissues was significantly shorter than that of decontaminated tissues. In conclusion, the current decontamination protocol of EHB is sufficient for most of the initially contaminated bacteria, whereas it is inadequate for Propionibacterium acnes. This may be related to the slow-growing nature of this bacterium and thereby the relative shorter antibiotic incubation time.     

Estimation of Respiratory Nasal Pressure and Flow Rate Signals Using Different Respiratory Sound Features

BackgroundRespiratory sounds are associated with the flow rate, nasal flow pressure, and physical characteristics of airways. In this study, we aimed to develop the flow rate and nasal flow pressure estimation models for the clinical application, and find out the optimal feature set for estimation to achieve the optimal model performance.MethodsRespiratory sounds and flow rate were acquired from nine healthy volunteers. Respiratory sounds and nasal flow pressure were acquired from twenty-three healthy volunteers. Four types of respiratory sound features were extracted for flow rate and nasal flow pressure estimation using different estimation models. Effects of estimations using these features were evaluated using Bland-Altman analysis, estimation error, and respiratory sound feature calculation time. Besides, expiratory and inspiratory phases divided estimation errors were compared with united estimation errors.ResultsThe personalized logarithm model was selected as the optimal flow rate estimation model. Respiratory nasal flow pressure estimation based on this model was also performed. For the four different respiratory sound features, there is no statistically significant difference in flow rate and pressure estimation errors. LogEnvelope was, therefore, chosen as the optimal feature because of the lowest computational cost. In addition, for any type of respiratory sound feature, no statistically significant difference was observed between divided and united estimation errors (flow rate and pressure).ConclusionRespiratory flow rate and nasal flow pressure can be estimated accurately using respiratory sound features. Expiratory and inspiratory phases united estimation using respiratory sounds is a more reasonable estimation method than divided estimation. LogEnvelope can be used for this united respiratory flow rate and nasal flow pressure estimation with minimum computational cost and acceptable estimation error.     

Temperature dependence of F-, D- and z-values used in steam sterilization processes

Aims:  To develop a model, based on microbiological principles, to safely optimize steam sterilization processes.
Methods and Results:  The minimum exposure time F for a decontamination process at a certain temperature is usually calculated from an empirical model with the decimal reduction time D and the temperature resistance coefficient z as parameters. These are implicitly assumed to be independent of temperature. Using a microbiological approach, it is shown that also D and z depend on temperature, indicating that the usual models provide only reliable results in a limited temperature region. The temperature dependence of F resulting from this approach describes the available experimental data very well. Safety margins to assure sterility can be included in a straightforward way.
Conclusions:  The results from the present approach can be used to safely optimize decontamination processes. The corresponding mathematical model can be implemented rather directly in process control systems.
Significance and Impact of the Study:  Our results show that for steam sterilization and disinfection processes the values of F predicted by the usual models largely underestimate the required minimum exposure times at temperatures below 120°C. This has important consequences for the optimization of such processes.     

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Background

PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents.

Methodology/Principal Findings

Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein.

Conclusions/Significance

There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.     

BioNano engineered hybrids for hypochlorous acid generation

Enzyme-based systems represent a user- and environmentally-friendly alternative to current corrosive and/or toxic decontamination technologies used for microbial decontamination. Herein an easily deployable enzyme-nanosupport hybrid system was developed for in situ generation of hypochlorous acid (HOCl), a strong decontaminant. The user-controlled strategy allowed co-immobilization of two different enzymes at a nanosupport interface and decontaminant generation through a chain reaction. For this, glucose oxidase was used as the working enzyme and co-immobilized onto multi-walled carbon nanotubes along with chloroperoxidase. Our hypothesis was that hydrogen peroxide produced at the nanosupport interface through the glucose oxidase enzymatic reaction can further be used as substrate by the co-immobilized CPO to convert (Cl^?) into HOCl. The chemistry of the immobilization method, as well as the enzyme loading, activity, kinetics and enzyme stability at the nanointerface were evaluated. The multi-enzyme system was found to be able to initiate and propagate the chain reaction resulting in decontaminant production. The strong capability of HOCl generation can be viewed as an important first step toward creating self-sustainable microbial decontamination coatings to be used against various pathogens such as bacteria and spores.     

Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores

The aim of this study was to evaluate the efficacy of hydrogen peroxide vapour (HPV) against spores of Clostridium botulinum, for use as a method for decontaminating environments where this pathogen has been handled. Spores were dried onto stainless steel slides and exposed to HPV in a sealed glovebox enclosure, transferred to a quenching agent at timed intervals during the exposure period, before survivors were cultured and enumerated. D-values were calculated from graphs of log₁₀ survivors plotted against time and were found to range from 1.41 to 4.38 min. HPV was found to be effective at deactivating spores of toxigenic Cl. botulinum, non-toxigenic Clostridium spp. and Geobacillus stearothermophilus dried onto stainless steel surfaces. HPV could be used to decontaminate cabinets and rooms where Cl. botulinum has been handled. The cycle parameters should be based on studies carried out with relevant spores of this organism, rather than based on inactivation data for G. stearothermophilus spores, which have been used in the past as a standard biological challenge for disinfection and sterilisation procedures. HPV could provide an attractive alternative to other decontamination methods, as it was rapid, residue-free and did not give rise to the health and safety concerns associated with other gaseous decontamination systems.     

Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus (“Bacillus subtilis subsp. niger,” also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10⁵ to 10⁶ CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

Antimicrobial efficiency and stability of two decontamination solutions

Two decontamination solutions, commercially produced BASE?128 and laboratory decontamination solution (LDS), with analogous content of antibiotic and antimycotic agents, were compared in their antimicrobial efficiency and stability (pH and osmolarity). Both solutions were compared immediately after thawing aliquots frozen for 1, 3 or 6 months. Agar well diffusion method was used to test their antimicrobial efficiency against five human pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Enterococcus faecalis. The difference in the inhibition of growth between the two decontamination solutions was mostly not statistically significant, with few exceptions. The most pronounced difference between the LDS and BASE?128 was observed in their decontamination efficacy against E. coli and E. faecalis, where the LDS showed to be more efficient than BASE?128. The osmolarity value of LDS decreased with cold-storage, the osmolarity values of the BASE?128 could not be measured as they were below the range of the osmometer. Slight changes were found in pH of the less stable LDS solution, whose pH increased from initial value 7.36?±?0.07 to 7.72?±?0.19 after 6 m-storage. We verified that BASE?128 and LDS are similarly efficient in elimination of possible placental bacterial contaminants and may be used for decontamination of various tissues.