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Title: Evaluation of PCR Systems for Field Screening of Bacillus anthracis

Abstract

There is little published data on the performance of hand-portable polymerase chain reaction (PCR) instruments that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated five commercially available hand-portable PCR instruments for detection of Bacillus anthracis (Ba). We designed a cost-effective, statistically-based test plan that allows instruments to be evaluated at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) on the probability of detection (POD) at confidence levels of 80-95%. We assessed specificity using purified genomic DNA from 13 Ba strains and 18 Bacillus near neighbors, interference with 22 common hoax powders encountered in the field, and PCR inhibition when Ba spores were spiked into these powders. Our results indicated that three of the five instruments achieved >0.95 LCB on the POD with 95% confidence at test concentrations of 2,000 genome equivalents/mL (comparable to 2,000 spores/mL), displaying more than sufficient sensitivity for screening suspicious powders. These instruments exhibited no false positive results or PCR inhibition with common hoax powders, and reliably detected Ba spores spiked into common hoax powders, though some issues with instrument controls were observed. Our testing approach enables efficient instrument performance testing to a statistically rigorousmore » and cost-effective test plan to generate performance data that will allow users to make informed decisions regarding the purchase and use of biodetection equipment in the field.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1344637
Report Number(s):
PNNL-SA-115228
Journal ID: ISSN 2326-5094; 400904120
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Health Security; Journal Volume: 15; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Ozanich, Richard M., Colburn, Heather A., Victry, Kristin D., Bartholomew, Rachel A., Arce, Jennifer S., Heredia-Langner, Alejandro, Jarman, Kristin, Kreuzer, Helen W., and Bruckner-Lea, Cynthia J.. Evaluation of PCR Systems for Field Screening of Bacillus anthracis. United States: N. p., 2017. Web. doi:10.1089/hs.2016.0043.
Ozanich, Richard M., Colburn, Heather A., Victry, Kristin D., Bartholomew, Rachel A., Arce, Jennifer S., Heredia-Langner, Alejandro, Jarman, Kristin, Kreuzer, Helen W., & Bruckner-Lea, Cynthia J.. Evaluation of PCR Systems for Field Screening of Bacillus anthracis. United States. doi:10.1089/hs.2016.0043.
Ozanich, Richard M., Colburn, Heather A., Victry, Kristin D., Bartholomew, Rachel A., Arce, Jennifer S., Heredia-Langner, Alejandro, Jarman, Kristin, Kreuzer, Helen W., and Bruckner-Lea, Cynthia J.. Wed . "Evaluation of PCR Systems for Field Screening of Bacillus anthracis". United States. doi:10.1089/hs.2016.0043.
@article{osti_1344637,
title = {Evaluation of PCR Systems for Field Screening of Bacillus anthracis},
author = {Ozanich, Richard M. and Colburn, Heather A. and Victry, Kristin D. and Bartholomew, Rachel A. and Arce, Jennifer S. and Heredia-Langner, Alejandro and Jarman, Kristin and Kreuzer, Helen W. and Bruckner-Lea, Cynthia J.},
abstractNote = {There is little published data on the performance of hand-portable polymerase chain reaction (PCR) instruments that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated five commercially available hand-portable PCR instruments for detection of Bacillus anthracis (Ba). We designed a cost-effective, statistically-based test plan that allows instruments to be evaluated at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) on the probability of detection (POD) at confidence levels of 80-95%. We assessed specificity using purified genomic DNA from 13 Ba strains and 18 Bacillus near neighbors, interference with 22 common hoax powders encountered in the field, and PCR inhibition when Ba spores were spiked into these powders. Our results indicated that three of the five instruments achieved >0.95 LCB on the POD with 95% confidence at test concentrations of 2,000 genome equivalents/mL (comparable to 2,000 spores/mL), displaying more than sufficient sensitivity for screening suspicious powders. These instruments exhibited no false positive results or PCR inhibition with common hoax powders, and reliably detected Ba spores spiked into common hoax powders, though some issues with instrument controls were observed. Our testing approach enables efficient instrument performance testing to a statistically rigorous and cost-effective test plan to generate performance data that will allow users to make informed decisions regarding the purchase and use of biodetection equipment in the field.},
doi = {10.1089/hs.2016.0043},
journal = {Health Security},
number = 1,
volume = 15,
place = {United States},
year = {Wed Feb 01 00:00:00 EST 2017},
month = {Wed Feb 01 00:00:00 EST 2017}
}
  • The goal of this testing was to evaluate the ability of currently available commercial off-the-shelf (COTS) biological indicator tests and immunoassays to detect Bacillus anthracis (Ba) spores and ricin. In general, immunoassays provide more specific identification of biological threats as compared to indicator tests [3]. Many of these detection products are widely used by first responders and other end users. In most cases, performance data for these instruments are supplied directly from the manufacturer, but have not been verified by an external, independent assessment [1]. Our test plan modules included assessments of inclusivity (ability to generate true positive results), commonlymore » encountered hoax powders (which can cause potential interferences or false positives), and estimation of limit of detection (LOD) (sensitivity) testing.« less
  • This is a response to the Letter on Immunoassays for Field Screening of Bacillus anthracis and ricin.
  • 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 coupon-1) 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 tomore » 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.« less
  • This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence ofmore » dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.« less
  • 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 amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » 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 ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG; values increased as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent article.« less