Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

AIMS: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. METHODS AND RESULTS: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. CONCLUSIONS: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. SIGNIFICANCE AND IMPACT OF STUDY: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

False-negative rate, limit of detection and recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne and Bacillus atrophaeus spores.

AIMS: We sought to evaluate the effects of Bacillus species, low surface concentrations, and surface material on recovery efficiency (RE), false-negative rate (FNR) and limit of detection for recovering Bacillus spores using a validated macrofoam-swab sampling procedure. METHODS AND RESULTS: The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target surface concentrations (2 to 500 CFU per plate or coupon) to positive-control plates and test coupons (25·8064 cm(2) ) of four surface materials (glass, stainless steel, vinyl tile and plastic). The Bacillus species and surface material had statistically significant effects on RE, but surface concentration did not. Mean REs were the lowest for vinyl tile (50·8% with BAS and 40·2% with BG) and the highest for glass (92·8% with BAS and 71·4% with BG). FNR values (which ranged from 0 to 0·833 for BAS and from 0 to 0·806 for BG) increased as surface concentration decreased in the range tested. Surface material also had a statistically significant effect on FNR, with FNR the lowest for glass and highest for vinyl tile. Finally, FNR tended to be higher for BG than for BAS at lower surface concentrations, especially for glass. CONCLUSIONS: Concentration and surface material had significant effects on FNR, with Bacillus species having a small effect. Species and surface material had significant effects on RE, with surface concentration having a nonsignificant effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide valuable information on the performance of the macrofoam-swab method for low surface concentrations of Bacillus spores, which can be adapted to assess the likelihood that there is no contamination when all macrofoam-swab samples fail to detect B. anthracis.

Laboratory studies on surface sampling of Bacillus anthracis contamination: summary, gaps and recommendations.

This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing and analysing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (i) estimates of B. anthracis contamination, as well as the bias and uncertainties in the estimates and (ii) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Additional work is needed to quantify (i) the false-negative rates of surface-sampling methods with lower concentrations on various surfaces and (ii) the effects on performance characteristics of: aerosol vs liquid deposition of spores, using surrogates instead of B. anthracis, real-world vs laboratory conditions and storage and transportation conditions. Recommendations are given for future evaluations of data from existing studies and possible new studies.

Mixture-process variable approach to optimize a microemulsion electrokinetic chromatography method for the quality control of a nutraceutical based on coenzyme Q10.

In recent years, multivariate optimization has played an increasing role in analytical method development. ICH guidelines recommend using statistical design of experiments to identify the design space, in which multivariate combinations of composition variables and process variables have been demonstrated to provide quality results. Considering a microemulsion electrokinetic chromatography method (MEEKC), the performance of the electrophoretic run depends on the proportions of mixture components (MCs) of the microemulsion and on the values of process variables (PVs). In the present work, for the first time in the literature, a mixture-process variable (MPV) approach was applied to optimize a MEEKC method for the analysis of coenzyme Q10 (Q10), ascorbic acid (AA), and folic acid (FA) contained in nutraceuticals. The MCs (buffer, surfactant-cosurfactant, oil) and the PVs (voltage, buffer concentration, buffer pH) were simultaneously changed according to a MPV experimental design. A 62-run MPV design was generated using the I-optimality criterion, assuming a 46-term MPV model allowing for special-cubic blending of the MCs, quadratic effects of the PVs, and some MC-PV interactions. The obtained data were used to develop MPV models that express the performance of an electrophoretic run (measured as peak efficiencies of Q10, AA, and FA) in terms of the MCs and PVs. Contour and perturbation plots were drawn for each of the responses. Finally, the MPV models and criteria for the peak efficiencies were used to develop the design space and an optimal subregion (i.e., the settings of the mixture MCs and PVs that satisfy the respective criteria), as well as a unique optimal combination of MCs and PVs.

Mixture experiment methods in the development and optimization of microemulsion formulations.

Microemulsion formulations represent an interesting delivery vehicle for lipophilic drugs, allowing for improving their solubility and dissolution properties. This work developed effective microemulsion formulations using glyburide (a very poorly-water-soluble hypoglycaemic agent) as a model drug. First, the area of stable microemulsion (ME) formations was identified using a new approach based on mixture experiment methods. A 13-run mixture design was carried out in an experimental region defined by constraints on three components: aqueous, oil and surfactant/cosurfactant. The transmittance percentage (at 550 nm) of ME formulations (indicative of their transparency and thus of their stability) was chosen as the response variable. The results obtained using the mixture experiment approach corresponded well with those obtained using the traditional approach based on pseudo-ternary phase diagrams. However, the mixture experiment approach required far less experimental effort than the traditional approach. A subsequent 13-run mixture experiment, in the region of stable MEs, was then performed to identify the optimal formulation (i.e., having the best glyburide dissolution properties). Percent drug dissolved and dissolution efficiency were selected as the responses to be maximized. The ME formulation optimized via the mixture experiment approach consisted of 78% surfactant/cosurfacant (a mixture of Tween 20 and Transcutol, 1:1, v/v), 5% oil (Labrafac Hydro) and 17% aqueous phase (water). The stable region of MEs was identified using mixture experiment methods for the first time.

Modeling methods for mixture-of-mixtures experiments applied to a tablet formulation problem.

During the past few years, statistical methods for the experimental design, modeling, and optimization of mixture experiments have been widely applied to drug formulation problems. Different methods are required for mixture-of-mixtures (MoM) experiments in which a formulation is a mixture of two or more "major" components, each of which is a mixture of one or more "minor" components. Two types of MoM experiments are briefly described. A tablet formulation optimization example from a 1997 article in this journal is used to illustrate one type of MoM experiment and corresponding empirical modeling methods. Literature references that discuss other methods for MoM experiments are also provided.