Citation:

Nelson, S., K. Hofacre, Sanjivkumar Shah, E. Silvestri, V. Gallardo, A. Mikelonis, R. James, AND Michael Calfee. Evaluation of Sample Processing Methods to Improve Detection of Bacillus anthracis in Difficult Sample Matrices. ENVIRONMENTAL MONITORING AND ASSESSMENT. Springer, New York, NY, 194(10):789, (2022). https://doi.org/10.1007/s10661-022-10467-0

Impact/Purpose:

The purpose of this study was to evaluate various sample processing method procedural steps and their outcomes in terms of improving sample analysis sensitivity, for difficult sample matrices being analyzed for Bacillus spores. These data will help improve current sample processing and analysis methods for anthrax incidents. 

Description:

Sub-product reports the evaluation and development of sampling extraction and processing procedures for difficult matrices samples. Such samples may contain high organic and environmental material, which will be challenging to process and analyze using the existing bio agent sample processing procedures. Improved processing procedures will help lower detection limits and increase assay sensitivity.    Large area sampling approaches have been developed and implemented by the US Environmental Protection Agency (EPA) to increase sample sizes, and potentially representativeness, in outdoor urban environments (e.g., concrete, asphalt, grass/landscaping). These sampling approaches could be implemented in response to an outdoor biological contamination incident or bioterrorism attack to determine the extent of contamination and for clearance following remediation. However, sample collection over large areas often contains an extensive amount of co-collected debris and native background microorganisms that interfere with the detection of biological threat agents. Sample processing methods that utilize basic laboratory equipment amenable to field deployment were selected and applied to turbid aqueous samples (TAS) to reduce particulates and native environmental organisms prior to culture and rapid viability-polymerase chain reaction (RV-PCR) analytical methods. Bacillus anthracis Sterne (BaS) spores were spiked into TAS collected by soil grab, wet vacuum collection from an outdoor concrete surface, or storm water runoff from an urban parking lot. The implementation of a sample processing method improved the sensitivity of culture and RV-PCR analytical methods for BaS spore detection in soil and wet vacuum TAS samples compared to baseline (minimal to no field processing methods applied). For soil, when the processing method was applied, samples with 15 colony forming units (CFU)/ml (60 CFU/g) and 1.5 CFU/mL (6 CFU/g) BaS spore load were detected using culture and RV-PCR, respectively. Most notably, the processing methods greatly improved the sensitivity of the RV-PCR analytical method for the wet vacuum TAS from no detection at the 1500 CFU/mL BaS spore load level to as low as 1.5 CFU/mL BaS spore load.

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