Sensitivity Comparison of the BIOFIRE® BioThreat Panel v2.5 on FILMARRAY® 2.0 and SPOTFIRE® Systems

Sensitivity Comparison of the BIOFIRE® BioThreat Panel v2.5 on FILMARRAY® 2.0 and SPOTFIRE® Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sensitivity Comparison of the BIOFIRE ® BioThreat Panel v2.5 on FILMARRAY ® 2.0 and SPOTFIRE ® Systems Jason Nielson, Karol Wright, Katie Poloncic, Nicholas Duclos, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7716200/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Rapid and sensitive detection of biothreat agents represents a critical capability for first responders, federal agencies, and militaries around the world. For nearly a decade, the BIOFIRE® BioThreat Panel v2.5 has been an easy-to use, automated, “lab in a pouch” detection tool utilizing qualitative, multiplexed polymerase chain reaction (PCR) with the BIOFIRE FILMARRAY® 2.0 instrument to detect biothreat agents directly from environmental samples in approximately one hour. The BIOFIRE BioThreat Panel v2.5 detects Bacillus anthracis, Brucella melitensis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, Rickettsia prowazekii, Yersinia pestis, Eastern equine encephalitis virus, Orthomarburgvirus marburgense, Orthopoxvirus spp., variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Orthoebolavirus zairense, and toxin-encoding genes from Clostridium botulinum (botulinum toxin) and Ricinus communis (Ricin toxin). Herein we establish performance of the BioThreat Panel v2.5 on the next-in-line system, the BIOFIRE SPOTFIRE®. Sensitivity of the BioThreat Panel v2.5 was compared between the FILMARRAY 2.0 and SPOTFIRE instruments in parallel by comparing the limit of detection (LoD) for each of the 16 biothreat targets on the panel, including seven bacterial pathogens, seven viral pathogens, and two toxin-encoding genes. The BioThreat Panel v2.5 exhibited equivalent or better sensitivity on SPOTFIRE compared to FILMARRAY 2.0 for all targets, except for Yersinia pestis, which displayed reduced sensitivity due to a modified calling scheme on SPOTFIRE. This study demonstrates that the BioThreat Panel v2.5 can provide sensitive biothreat detection across both BIOFIRE FILMARRAY 2.0 and SPOTFIRE automated PCR instruments. Figures Figure 1 Figure 2 Introduction Biothreat Agents Biological threat agents, commonly referred to as biothreats or bioagents, are a diverse group of viruses, bacteria, and toxins from biological sources that pose a substantial threat to human health. This group of pathogens and pathogen byproducts are diverse and highly variable in terms of transmissibility, infectivity, and lethality. Highly lethal biothreat pathogens have emerged naturally while others have been developed with the aid of genetic engineering 1,2 . Bioagent threats may arise from natural, accidental, or intentional exposure. Furthermore, humans are not the exclusive target of naturally occurring outbreaks or intentionally targeted attacks. Biothreats may target humans, livestock, crops, water supplies, and more, resulting in substantial physical, social, economic, political, and security consequences 3 . Examples of Biothreat Attacks and Outbreaks While this publication is not intended to provide a comprehensive overview of the history of biothreat agents and associated threat to human health, noteworthy examples illustrate the devastating effects of intentional attacks with biothreat agents, as well as the damage caused by naturally occurring outbreaks. The deliberate use of biothreat agents in warfare can be observed as early as the 14 th century BC, when Hittite armies sent diseased rams, likely infected with tularemia, to their enemies to weaken opposing forces 4 . In the 4 th century BC, according to the Greek historian Herodotus, archers dipped arrows into a mixture of decomposing cadavers of humans and venomous snakes, thereby coating the arrows with Clostridium perfringens, Clostridium tetani, and snake venom 5,6 . In Europe, between 1346 and 1352, outbreaks of Yersinia pestis are estimated to have killed 25−40% of the continent’s population. It is believed that this pandemic was a result of biological warfare in which a Mongol army laying siege to the Crimean city of Kaffa flung corpses infected with Yersinia pestis over the city walls 6–10 . This technique of using infected corpses as biological weapons can be found in the centuries that followed. Variola virus, the causative agent of smallpox, was similarly used as a biological weapon following the colonization of the Americas. During the French and Indian Wars, between the years 1754 to 1767, soldiers distributed blankets from a smallpox hospital to Native Americans, killing more than 50% of the infected tribes 11,12 . The Confederate army sold clothing from yellow fever and smallpox patients to Union troops during the United States Civil War 9,13 . Due to the use of smallpox as a biological weapon and the endemic nature of smallpox at the time of the Civil War, smallpox spread throughout the Union troops, Confederate troops, and civilians on both sides 12 . Smallpox has had catastrophic effects on world populations even outside the targeted uses of variola virus as a biological weapon. Historically, endemic smallpox was responsible for as much as 10% of annual deaths, in locations with sufficient population to support its endemicity 9 . Even following the introduction of the smallpox vaccine, the devastating effects of smallpox continued for decades; approximately 50 million smallpox cases were observed annually in the 1950’s, resulting in tens of millions of fatalities 8,9 . These historical examples of smallpox help illustrate the catastrophic capabilities of bioagents; they can be invisible, indiscriminate killers infecting susceptible hosts, whether they are ally or enemy. There are many other examples of the intentional use of biothreat agents in warfare and terror, including: Napoleon flooded the plains around Mantua, Italy in 1797 to enhance the spread of malaria 14 ; during World War I, horses, cattle, and sheep inoculated with Bacillus anthracis and Burkholderia mallei were shipped to the United States, Russia, and other countries 8 ; water supplies in Oregon were intentionally contaminated with Salmonella by a religious cult in 1984 15 ; anthrax spores were sent through the United States Postal Service in 2001 10,16 ; and many more 2,7,9,11 . In addition to the intentional spread of biothreat agents through warfare or terrorism, natural outbreaks and epidemics can pose a severe threat to human health. One example is the Ebola outbreak in West Africa between the years 2014-2016. Over 28,000 cases of infection with Orthoebolavirus zairense were suspected during this time and over 11,000 associated deaths, a nearly 40% fatality rate 17 . Additional outbreaks of Orthoebolavirus zairense, Orthoebolavirus sudanese, and Orthomarburgvirus marburgense have occurred within the last decade, with fatality rates between 25% and 90% 18,19 , highlighting the severe effects that naturally occurring outbreaks of biothreats can have. BIOFIRE Technology Whether the target of a biological threat is a human, an animal, water supply, food supply, or something else, early detection of the causative biothreat agent is the key step to prevention, protection, prophylaxis, post-exposure treatment, and mitigation 9 . BioFire Defense has provided PCR-based testing capabilities for the detection of biothreat pathogens to military personnel and first-responders for over 20 years, including the Joint Biological Agent Identification and Diagnostic System (JBAIDS), RAZOR ® Mk II, and Next Generation Diagnostic System (NGDS) FILMARRAY 2.0 platforms 20 . These portable systems have provided timely and reliable answers to support critical decision making and countermeasures related to biological warfare agents. BioFire Defense developed the BioThreat Panel v2.5 for use on the FILMARRAY 2.0 instrument for the qualitative detection of a range of biothreat analytes from environmental samples, including: Bacillus anthracis, Brucella melitensis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, Rickettsia prowazekii, Yersinia pestis , Eastern equine encephalitis virus, Orthomarburgvirus marburgense, Orthopoxvirus spp., variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Orthoebolavirus zairense , and toxin-encoding genes from Clostridium botulinum (botulinum toxin) and Ricinus communis (Ricin toxin). The BioThreat Panel v2.5 for the BIOFIRE FILMARRAY 2.0 has been adopted for environmental surveillance because of its broad capabilities, ease-of-use, speed, and low logistics burden (e.g., no cold chain storage required). The BIOFIRE SPOTFIRE instrument dimensions and high-level specifications. The BIOFIRE SPOTFIRE instrument represents the next generation of biothreat detection capabilities. Compared with the FILMARRAY 2.0 21 , the SPOTFIRE 22 instrument requires less benchtop space and can be scaled vertically, up to four modules, enabling customizable throughput. In contrast to requiring an external computer to control FILMARRAY 2.0 instruments, the SPOTFIRE instrument contains an integrated, intuitive system interface, making it easy-to-use for a variety of applications (Figure 1). Like the FILMARRAY 2.0, the SPOTFIRE instrument automatically executes all steps of a PCR test, including nucleic acid purification, reverse transcription, PCR amplification, amplicon melt analysis, and report generation. Within a BIOFIRE pouch consumable, FILMARRAY 2.0 or SPOTFIRE instruments execute automated sample extraction (A), nucleic acid purification (A-D), reverse-transcription and multiplex first-stage PCR (PCR1) (E) and nested individual second-stage PCR (PCR2) reactions (G) in a disposable pouch to evaluate multiple RNA and DNA targets in a single run. The BioThreat Panel v2.5 consumable with its current chemistry configuration (Figure 2) is compatible with both the FILMARRAY 2.0 and SPOTFIRE instruments. With the corresponding BioThreat Panel v2.5 software package specific to each instrument, BioThreat Panel v2.5 consumables can be tested with both instrument platforms. Each consumable pouch is manufactured with all reagents necessary for nucleic acid purification, reverse transcription, and PCR. Each BIOFIRE pouch consumable contains built-in controls used to demonstrate that the instrument and pouch are performing correctly: 1) internal process controls made up of lyophilized cells from the yeast Schizosaccharomyces pombe that are subject to all sample preparation, reverse transcription, and PCR amplification steps, and 2) a PCR control comprised of a synthetic PCR template and corresponding primers manufactured directly into the final PCR stage of each consumable. With minimal hands-on time, and a run time of approximately one hour on both instrument platforms, the combination of instrument and consumable pouch are used to execute the following steps: nucleic acid purification from the sample (Figure 2A-D), reverse transcription, and amplification in a first-stage PCR reaction, referred to as PCR1 (Figure 2E). Following PCR1, the reaction is diluted (Figure 2F) and flooded across a 102-well array where the primers for the second stage PCR reaction, referred to as PCR2, are pre-spotted (Figure 2G). Each assay is spotted in triplicate on the PCR2 array. After completion of the nested PCR reaction, the BIOFIRE FILMARRAY 2.0 and SPOTFIRE instruments perform a DNA amplicon melting analysis on the PCR products and measure the fluorescence signal generated in each well. The software then performs several analyses, including analysis of the melt curves and analysis of amplification curves, and assigns a final, qualitative assay result for every well. The BioThreat Panel v2.5 software automatically interprets and returns a result of “Detected”, “Not Detected”, or “Possible Detection” for each pathogen. The interpretation is based on the results of one or more assays for each target pathogen as shown in Table 1 and Supplemental Tables S1-S4. Analysis of Melt Curves : The BioThreat Panel v2.5 software evaluates the DNA amplicon melt curves from each well of the PCR2 array. In wells where PCR amplification occurs, the analysis software calculates the melting temperature (Tm) of the DNA melt curve and compares it against a temperature range where Tm values would be expected if the PCR amplicon came from target pathogen nucleic acid. If the Tm falls inside this assay-specific temperature range, the melt curve is called positive. If the software determines that the melt curve is not in the appropriate temperature range, the melt curve is called negative. For an assay to be called positive, at least two out of three possible array wells must contain positive melt curves with similar Tm values. Assays that do not meet these criteria are called negative. Table 1. Assay Interpretation Scheme for the BIOFIRE BioThreat Panel v2.5 Analyte Number of Assays Assay Detection Requirements for Analyte Detection BACTERIA Bacillus anthracis/Bacillus species 3 See Table S1 Brucella melitensis/Brucella species 2 See Table S2 Burkholderia mallei/pseudomallei 1 2 Any Positive Assay = "Detected" Coxiella burnetii 2 Any Positive Assay = "Detected" Francisella tularensis 2 Any Positive Assay = "Detected" Rickettsia prowazekii/Rickettsia species 2 See Table S3 Yersinia pestis 2 See Table S4 VIRUSES Orthoebolavirus zairense 1 Positive Assay = "Detected" Orthomarburgvirus marburgense 2 Any Positive Assay = "Detected" Orthopoxvirus spp. 2 Any Positive Assay = "Detected" Variola virus 4 All Positive Assays = "Detected" Eastern equine encephalitis virus 1 Positive Assay = "Detected" Venezuelan equine encephalitis virus 2 Any Positive Assay = "Detected" Western equine encephalitis virus 1 Positive Assay = "Detected" TOXIN-ENCODING GENES 2 Clostridium botulinum (Botulinum toxin) 1 Positive Assay = "Detected" Ricinus communis (Ricin toxin) 1 Positive Assay = "Detected" 1 Cross-reactivity with other Burkholderia spp. may be observed 2 The BioThreat Panel v2.5 does not detect toxins directly. Assays on the Panel are designed to detect genomic regions that encode these toxins. It is assumed that crude toxin extracts may contain target pathogen nucleic acids. The BioThreat Panel v2.5 automatically interprets and returns results of “Detected” or “Not Detected” for each pathogen. The interpretation is based on the results of one or more assays for each pathogen. This table illustrates how many target assays need to be called positive to produce a “Detected” result for the target analyte. Study Design The investigation described herein is composed of two sequential phases. First, the limit of detection (LoD) was estimated for each BioThreat Panel v2.5 target on both the FILMARRAY 2.0 and SPOTFIRE instruments. Second, the LoD was confirmed for each Panel target on both instrument types. All testing was performed with contrived samples, wherein analytes were spiked in a phosphate-buffered saline (PBS) matrix. All work was performed in a certified BSL-2 laboratory following institutional biosafety guidelines. Each sample was tested in parallel on FILMARRAY 2.0 and SPOTFIRE instruments. LoD determination for each analyte was based on the analyses performed by the BioThreat Panel v2.5 panel software. Analyte detection refers to the qualitative software interpretation from a FILMARRAY 2.0 or SPOTFIRE pouch run. For some analyte targets, the BioThreat Panel v2.5 software integrates the results of multiple assays to determine whether an analyte is “Detected”, “Not Detected”, or “Possible Detection”. For example, to determine whether Francisella tularensis is “Detected” or “Not Detected,” the results of two assays are considered: FTT2 and FTT3 (see Table 1). Individual assays may have different specificity, inclusivity, and sensitivity towards a given analyte. The algorithms contained in the BioThreat Panel v2.5 software convert the results of individual assays into a “Detected”, “Not Detected”, or “Possible Detection” call for each analyte. The relationships between BioThreat Panel v2.5 assays and overall interpretations are described in Tables 1 and S1-S4. Estimated LoD (eLoD) The LoD estimation phase of this study utilized historical data from initial assay design and development to establish a starting concentration for each analyte. From this starting concentration, five-fold dilutions were tested in four BioThreat Panel v2.5 pouches at each dilution on each instrument type. The eLoD was determined based on the rate of detection at each concentration; the lowest concentration with analyte detection in all four pouches was designated the 1× eLoD. LoD Confirmation The LoD was confirmed for each analyte by testing 20 pouches with samples contrived at the 1× eLoD and 20 pouches contrived at a 10-fold dilution lower. Each sample at 1× eLoD and each sample at 0.1× eLoD was prepared independently and tested on both FILMARRAY 2.0 and SPOTFIRE instruments, in parallel. The LoD for each analyte was confirmed if, at the 1× concentration, a “Detected” result was produced in at least 19/20 pouches and a “Detected” result was produced in fewer than 19/20 pouches at the 10-fold lower concentration. If either concentration failed to meet these criteria, additional concentrations were tested until the criteria were met. For this study, a “Possible Detection” interpretation was considered the same as a “Not Detected” interpretation. Analyte selection Testing was performed for all 16 targets detected by the BioThreat v2.5 Panel with a total of 18 representative analytes used in this study (Table 2). All analytes were quantified in-house using digital PCR (dPCR) prior to use in this study. For this study, live or inactivated organisms suitable for use in a BioSafety Level 2 facility were the preferred analyte type. Where live or inactivated organisms were unavailable, genomic nucleic acid was the next preferred analyte type. For four analyte targets, Eastern equine encephalitis (EEE) virus, variola virus, Clostridium botulinum, and Coxiella burnetii , neither intact organism nor genomic nucleic suitable for use in a BioSafety Level 2 facility were available; synthetic templates were used in place of organism or genomic nucleic acid for these target analytes. A single analyte was tested for most BioThreat Panel v2.5 targets as part of this study. Exceptions to this include Bacillus anthracis and Orthomarburgvirus marburgense . For these exceptions where multiple isolates of an organism were tested, the LoD was determined for each strain or virus individually. Bacillus anthracis was tested with both the Ames and Sterne 34F2 strains. The Ames strain is inactivated and expected to produce a positive assay detection for all three assays associated with Bacillus anthracis (Chromosome Element, pXO1, pXO2), thereby returning a “ Bacillus anthracis Detected” interpretation. The Sterne 34F2 strain is missing the pXO2 plasmid and is expected to produce a positive assay detection call for only two of the assays associated with Bacillus anthracis . This will result in a “Bacillus species Detected” interpretation for this analyte. Two virus types of Orthomarburgvirus marburgense were tested: Ravn and Voege. Both viruses trigger both Marburg assays (Marb2 and Marb3) and produce a “Marburg virus Detected” interpretation. All analytes were tested individually for LoD estimation and LoD confirmation, except those templates used as a surrogate for variola virus. Variola virus requires four target assays to be positive to return a “Variola virus Detected” interpretation. Three separate nucleic acid templates were loaded at equivalent concentrations into the pouch together to simulate variola virus; one template is complementary to two assays, and each of the other two templates is complementary to one of the other two target assays. The presence of these nucleic acid templates complementary to the four target assays, enables all assays to be detected and for the panel software to produce a “Variola virus Detected” interpretation. Table 2. Analytes Tested for Each Target on the BioThreat v2.5 Panel Target Analyte Analyte Strain 1 Vendor ID 2 Type of Analyte Targeted Assays Expected Interpretation Bacteria Bacillus anthracis Ames AGD 1331 Inactivated Chromosome Element, pXO1, pXO2 Bacillus anthracis Detected Sterne 34F2 NR-1400 Live Chromosome Element, pXO1 Bacillus species Detected Brucella melitensis 16M AGD 0074 Inactivated (Formalin) BRT2, BRT4 Brucella melitensis Detected Burkholderia pseudomallei MSHR 146 AGD 1595 Inactivated (Formalin) Burk2, Burk8 Burkholderia mallei/pseudomallei Detected Coxiella burnetii N/A CHEM-GBK-0485 Synthetic Nucleic Acid CBT1 Coxiella burnetii Detected Francisella tularensis SCHU S4 NR-15753 Inactivated (Formalin) FTT2, FTT3 Francisella tularensis Detected Rickettsia prowazekii Brienl AGD 0079 Inactivated (Formalin) RIK2 Rickettsia prowazekii Detected Yersinia pestis CO92 NR-2717 Genomic DNA YPT1, YPT3 Yersinia pestis Detected Viruses Orthoebolavirus zairense Mayinga NR-31807 Inactivated (Gamma-irradiated) EB2 Ebola Zaire Detected Eastern equine encephalitis virus N/A CHEM-RNA-0039 Synthetic Nucleic Acid EEE01 EEE Virus Detected Orthomarburgvirus marburgense German Voege NR-31816 Inactivated (Gamma-irradiated) Marb2, Marb3 Marburg virus Detected Ravn NR-31819 Inactivated (Gamma-irradiated) Marb2, Marb3 Orthopoxvirus spp. Modified vaccinia ankara (MVA) virus NR-1 Attenuated Live OPX2, Var3 Orthopox genus virus Detected Variola virus N/A CHEM-GBK-0752 Synthetic Nucleic Acid OPX2 Variola virus Detected N/A Var1_BTNIVD_g1QC Synthetic Nucleic Acid Var1a, Var1b N/A Var3_BTNIVD_g1QC Synthetic Nucleic Acid Var3 Venezuelan equine encephalitis virus Trinidad AGD 0108 Inactivated (Formalin) VEE-MP2, VEE-RC3 VEE virus Detected Western equine encephalitis virus CBA87 Alpha 025 AGD 0110 Inactivated (Gamma-irradiated) WEE01 WEE virus Detected Toxin Clostridium botulinum (Botulinum toxin) N/A CHEM-RNA-0031 Synthetic Nucleic Acid BoNTA Clostridium botulinum Detected Ricinus communis (Ricin toxin) Castor Bean gDNA NR-44091 Genomic DNA RCN2 Ricinus communis Detected 1 Analyte strain listed for organism isolates; N/A listed for synthetic nucleic acid analytes. 2 Vendor ID or BioFire Defense part number, for synthetic nucleic acid templates. For each target analyte tested as part of the investigation described herein, additional characteristics of the analyte are listed, including the strain and vendor identification number. The column “Type of Analyte” refers to whether the analyte is a live or inactivated analyte along with the method of inactivation, genomic nucleic acid, or synthetic nucleic acid. The final two columns indicate the assays on the BioThreat Panel v2.5 that are expected to be positive and the expected interpretation of the panel software when testing the indicated analyte. Results Summary of LoD Estimation Testing Each analyte was tested on FILMARRAY 2.0 and SPOTFIRE instruments over a range of concentrations, with four pouches tested on each instrument platform at each concentration. The lowest concentration where the analyte was detected in 4/4 pouches tested was designated as the estimated LoD, unless it was determined through review of amplification curves, that it was unlikely that LoD would be confirmed at that concentration (Table 3). The bolded values in Table 3 represent the initial concentrations used in LoD confirmation testing. Summary of LoD Confirmation Testing Table 4 lists the confirmed LoD determined for the analytes tested in this study on both FILMARRAY 2.0 and SPOTFIRE instruments. Figures S1-S18 present the Cp and Tm values from individual pouch runs used to confirm the LoD values presented in Table 4. Seven analytes demonstrated the same LoD on FILMARRAY 2.0 and SPOTFIRE : Bacillus anthracis Ames, Bacillus anthracis Sterne 34F2, Brucella melitensis, Burkholderia pseudomallei, Coxiella burnetii, Franciscella tularensis, and Orthomarburgvirus marburgense German Voege. An additional eight analytes demonstrated similar sensitivity (within 5-fold) on FILMARRAY 2.0 and SPOTFIRE : Rickettsia prowazekii, Orthoebolavirus zairense, Orthomarburgvirus marburgense Ravn, modified Vaccinia Ankara virus, variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Ricinus communis . Two analytes demonstrated a greater than 5-fold improvement in sensitivity on the SPOTFIRE relative to the FILMARRAY 2.0: Eastern equine encephalitis virus, and Clostridium botulinum. A single target analyte demonstrated reduced sensitivity on the SPOTFIRE compared with the FILMARRAY 2.0: Yersinia pestis. A reduction in sensitivity towards Yersinia pestis was expected due to changes in the calling scheme designed to increase the stringency and specificity of the BioThreat Panel v2.5 towards Yersinia pestis (see Discussion). Table 3. Confirmed LoD Values for Analytes on the BioThreat v2.5 Panel on FILMARRAY 2.0 and SPOTFIRE FILMARRAY 2.0 SPOTFIRE Bacillus anthracis Ames Concentration (copies/mL) 4.0E+04 8.0E+03 1.6E+03 N/A N/A 4.0E+04 8.0E+03 1.6E+03 N/A N/A Detections 4/4 4/4 3/4 N/A N/A 4/4 4/4 2/4 N/A N/A Sterne 34F2 Concentration (copies/mL) 4.0E+03 8.0E+02 1.6E+02 N/A N/A 4.0E+03 8.0E+02 1.6E+02 N/A N/A Detections 4/4 3/4 1/4 N/A N/A 4/4 3/4 1/4 N/A N/A Brucella melitensis 16M Concentration (copies/mL) 2.5E+03 5.0E+02 1.0E+02 2.0E+01 4.0E+00 2.5E+03 5.0E+02 1.0E+02 2.0E+01 4.0E+00 Detections 4/4 4/4 4/4 1/4 0/4 4/4 4/4 4/4 2/4 1/4 Burkholderia pseudomallei 1 MSHR 146 Concentration (copies/mL) 2.4E+03 4.8E+02 9.6E+01 N/A N/A 2.4E+03 4.8E+02 9.6E+01 1.9E+01 3.8E+00 Detections 4/4 4/4 1/4 N/A N/A 4/4 4/4 4/4 1/4 1/4 Coxiella burnetii 2 N/A Concentration (copies/mL) 7.4E+05 5.9E+03 1.2E+03 2.7E+02 4.7E+02 7.4E+05 5.9E+03 1.2E+03 2.7E+02 4.7E+02 Detections 4/4 4/4 4/4 0/4 0/4 4/4 4/4 4/4 2/4 2/4 Francisella tularensis 3 SCHU S4 Concentration (copies/mL) 1.2E+03 2.4E+02 2.4E+01 9.4E+00 N/A 1.2E+03 2.4E+02 2.4E+01 9.4E+00 N/A Detections 4/4 4/4 3/4 3/4 N/A 4/4 4/4 4/4 0/4 N/A Rickettsia prowazekii 4 Brienl Concentration (copies/mL) 1.7E+03 5.0E+00 N/A N/A N/A 1.7E+03 5.0E+00 N/A N/A N/A Detections 4/4 0/4 N/A N/A N/A 7/8 0/4 N/A N/A N/A Yersinia pestis CO92 Concentration (copies/mL) 1.1E+03 2.2E+02 4.4E+01 8.8E+00 1.8E+00 1.1E+03 2.2E+02 4.4E+01 8.8E+00 1.8E+00 Detections 4/4 4/4 4/4 2/4 0/4 4/4 4/4 0/4 N/A N/A Orthoebolavirus zairense Mayinga Concentration (copies/mL) 3.7E+04 7.4E+03 1.5E+03 N/A N/A 3.7E+04 7.4E+03 1.5E+03 N/A N/A Detections 4/4 4/4 0/4 N/A N/A 4/4 4/4 2/4 N/A N/A Eastern equine encephalitis virus 5 N/A Concentration (copies/mL) 7.4E+05 1.5E+05 3.0E+04 5.9E+03 1.2E+03 7.4E+05 N/A N/A 5.9E+03 1.2E+03 Detections 4/4 3/4 1/4 2/4 0/4 4/4 N/A N/A 4/4 1/4 Orthomarburgvirus marburgense German Voege 6 Concentration (copies/mL) 1.4E+04 2.9E+03 5.7E+02 N/A N/A 1.4E+04 2.9E+03 5.7E+02 N/A N/A Detections 4/4 4/4 0/4 N/A N/A 4/4 3/4 1/4 N/A N/A Ravn Concentration (copies/mL) 6.5E+03 1.3E+03 2.6E+02 N/A N/A 6.5E+03 1.3E+03 2.6E+02 N/A N/A Detections 4/4 3/4 1/4 N/A N/A 4/4 4/4 2/4 N/A N/A Orthopoxvirus spp. MVA virus Concentration (copies/mL) 3.0E+03 6.0E+02 1.2E+02 N/A N/A 3.0E+03 6.0E+02 1.2E+02 N/A N/A Detections 4/4 4/4 2/4 N/A N/A 4/4 4/4 3/4 N/A N/A Variola virus N/A Concentration (copies/mL) 1.0E+05 2.0E+04 4.0E+03 8.0E+02 1.6E+02 1.0E+05 2.0E+04 4.0E+03 8.0E+02 1.6E+02 Detections 4/4 4/4 4/4 3/4 0/4 4/4 4/4 4/4 4/4 0/4 Venezuelan equine encephalitis virus Trinidad Concentration (copies/mL) 1.2E+04 2.4E+03 4.7E+02 N/A N/A 1.2E+04 2.4E+03 4.7E+02 N/A N/A Detections 4/4 3/4 2/4 N/A N/A 4/4 4/4 2/4 N/A N/A Western equine encephalitis virus CBA87 Alpha 025 Concentration (copies/mL) 1.2E+04 2.4E+03 4.8E+02 9.6E+01 N/A 1.2E+04 2.4E+03 4.8E+02 9.6E+01 N/A Detections 4/4 4/4 4/4 2/4 N/A 4/4 4/4 4/4 2/4 N/A Clostridium botulinum (Botulinum toxin) N/A Concentration (copies/mL) 1.6E+07 3.1E+04 6.2E+03 1.2E+03 2.5E+02 1.6E+07 3.1E+04 6.2E+03 1.2E+03 2.5E+02 Detections 4/4 4/4 2/4 0/4 0/4 4/4 4/4 4/4 2/4 0/4 Ricinus communis (Ricin toxin) Castor Bean gDNA Concentration (copies/mL) 2.0E+05 4.0E+02 8.0E+01 1.6E+01 N/A 2.0E+05 4.0E+02 8.0E+01 1.6E+01 N/A Detections 4/4 3/4 4/4 0/4 N/A 4/4 4/4 1/4 0/4 N/A 1 Delayed amplification was observed when testing at 9.6E+01 copies/mL on SPOTFIRE, so it was decided to confirm at the next highest dilution. 2 Confirmation testing was performed at 3.0E+04 copies/mL due to a clerical error. 3 Delayed amplification was observed when testing at 2.4E+01 copies/mL on SPOTFIRE, so it was decided to confirm at the next highest dilution. 4 Eight pouches were tested on SPOTFIRE at 1.7E+03 copies/mL. Since 7/8 pouches demonstrated analyte detection, a concentration of 1.7E+03 copies/mL was used for confirmation testing. 5 Confirmation testing was initiated at 5.9E+03 copies/mL on FILMARRAY 2.0 and at 7.4E+04 copies/mL on SPOTFIRE due to a clerical error. 6 Delayed amplification was observed when testing at 2.9E+03 copies/mL on FILMARRAY 2.0, so it was decided to confirm at the next highest dilution. For each analyte, the concentrations (in units of copies/mL) tested are indicated, as well as the number of pouches producing the expected interpretation. Cells representing analyte concentrations where all pouches produced the expected interpretation are highlighted in green. The bolded concentrations indicate the concentrations that were used for initial testing during the LoD confirmation phase; assay detection results and expert review of Cp values led to these determinations. Cells shaded in gray with N/A indicate that additional concentrations were not tested. Table 4. Confirmed LoD Values for Analytes on the BioThreat v2.5 Panel on FILMARRAY 2.0 and SPOTFIRE Target Analyte Analyte Strain 1 Confirmed LoD (copies/mL) FILMARRAY SPOTFIRE Bacteria Bacillus anthracis Ames 8.0E+03 8.0E+03 Sterne 34F2 4.0E+03 4.0E+03 Brucella melitensis 16M 1.0E+02 1.0E+02 Burkholderia pseudomallei MSHR 146 4.8E+02 4.8E+02 Coxiella burnetii N/A 3.0E+04 3.0E+04 Francisella tularensis SCHU S4 2.4E+02 2.4E+02 Rickettsia prowazekii Brienl 1.7E+03 8.7E+03 Yersinia pestis CO92 4.4E+01 2.2E+03 Viruses Orthoebolavirus zairense Mayinga 1.8E+05 3.7E+04 Eastern equine encephalitis virus N/A 5.9E+05 7.4E+03 Orthomarburgvirus marburgense German Voege 1.4E+04 1.4E+04 Ravn 6.5E+03 3.2E+04 Orthopoxvirus spp. MVA poxvirus 6.0E+02 3.0E+03 Variola virus N/A 4.0E+03 8.0E+03 Venezuelan equine encephalitis virus Trinidad 1.2E+04 2.4E+03 Western equine encephalitis virus CBA87 Alpha 025 2.4E+03 4.8E+02 Toxin Clostridium botulinum (Botulinum toxin) N/A 3.1E+06 3.1E+05 Ricinus communis (Ricin toxin) Castor Bean gDNA 8.0E+02 4.0E+02 1 Analyte strain listed for organism isolates; N/A listed for synthetic nucleic acid analytes The confirmed LoD in units of copies/mL are listed for each analyte on each instrument platform, FILMARRAY 2.0 and SPOTFIRE. LoD values shaded in blue indicate reduced sensitivity on the SPOTFIRE instrument relative to the FILMARRAY 2.0. LoD values shaded in orange indicate increased sensitivity on the SPOTFIRE instrument relative to the FILMARRAY 2.0. Bold text indicates that differences in confirmed LoD values between the two instrument platforms are greater than 5-fold. Discussion The sensitivity, or limit of detection (LoD), of the FILMARRAY 2.0 and SPOTFIRE instruments for 18 analytes targeted by the BioThreat v2.5 Panel was determined. Seven analytes demonstrated the same LoD on FILMARRAY 2.0 and SPOTFIRE: Bacillus anthracis Ames, Bacillus anthracis Sterne 34F2, Brucella melitensis, Burkholderia pseudomallei, Coxiella burnetii, Franciscella tularensis, and Orthomarburgvirus marburgense German Voege. An additional eight analytes demonstrated similar sensitivity (within 5-fold) on FILMARRAY 2.0 and SPOTFIRE: Rickettsia prowazekii, Orthoebolavirus zairense, Orthomarburgvirus marburgense Ravn, modified Vaccinia Ankara virus, variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Ricinus communis . Two analytes, Eastern equine encephalitis virus and Clostridium botulinum, demonstrated a greater than 5-fold improvement in sensitivity on the SPOTFIRE relative to the FILMARRAY 2.0. A single target analyte, Yersinia pestis, demonstrated an expected reduction in sensitivity on the SPOTFIRE compared with the FILMARRAY 2.0 . Overall, the SPOTFIRE demonstrated similar (within 5-fold) or improved sensitivity towards all analytes tested, except for Yersinia pestis. A decrease in sensitivity towards Yersinia pestis when testing on the SPOTFIRE was expected due to changes to the calling scheme being used on the SPOTFIRE compared with the calling scheme on FILMARRAY 2.0 (Table S4). The BioThreat Panel v2.5 contains two assays, YPT1 and YPT3, for the detection of Yersinia pestis. When testing on the FILMARRAY 2.0, if either YPT1 or YPT3 assays are positive, the software returns a “ Yersinia pestis Detected” interpretation; however, on SPOTFIRE, the YPT3 assay must be positive to produce a “ Yersinia pestis Detected” interpretation. The YPT1 and YPT3 assays target distinct extrachromosomal plasmids; the YPT1 assay targets the pPCP1 plasmid which is generally more abundant than the pMT1 plasmid targeted by the YPT3 assay 23–25 . The genetic loci targeted by the YPT1 assay, the pla gene, previously thought to be specific to Yersinia pestis, has been detected in other bacterial species 26 . Due to the potential presence of the pla gene in bacterial species other than Yersinia pestis , the calling scheme was updated for the SPOTFIRE instrument to reflect the inherent uncertainty whether nucleic acid specific to Yersinia pestis is present when only the YPT1 assay is positive. As the YPT1 assay targets the more abundant pPCP1 plasmid compared to the target of the YPT3 assay, the pMT1 plasmid, reduced sensitivity of the BioThreat v2.5 Panel on SPOTFIRE towards Yersinia pestis with this modified calling scheme is expected. BIOFIRE systems have been adopted for environmental surveillance and infectious disease testing by the US Department of Defense and others because of their broad capability, ease-of-use, speed, and low logistics burden (e.g., no cold chain storage required). Earlier generations of BIOFIRE platforms (BIOFIRE FILMARRAY 2.0 and BIOFIRE Torch) have FDA-cleared IVD syndromic panels with more than 170 pathogen target assays and non-IVD panels with more than 100 pathogen target assays. These earlier generation instruments fill a testing need for the most common infections, including respiratory, gastrointestinal, and blood infections, as well as biothreat testing capabilities. The BIOFIRE SPOTFIRE is the next-in-line system of BIOFIRE instrument 22 . SPOTFIRE panels for the detection of respiratory pathogens have been cleared by the FDA; other SPOTFIRE panels are in development to provide the same testing capabilities available with earlier generations of BIOFIRE platforms. Based on the data presented herein, it is anticipated that users of the BioThreat Panel v2.5 will observe similar levels of sensitivity and functionality upon both the legacy FILMARRAY 2.0 and next-in-line SPOTFIRE instruments. Declarations Funding Statement: The work presented in this manuscript was completed without external funding. Competing Interests Statement: All authors are employees of BioFire Defense LLC. Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics, Consent to Participate, and Consent to Publish declarations: Not applicable Clinical Trial Number: Not applicable Author Contributions Statement: Study design and conceptualization: J.N., K.W., and M.K. Study execution: K.P., N.D., D.S., S.P., R.P., C.G., E.K., A.B., W.B., and K.L. Data analysis: J.N., K.W., K.P., and N.D. Manuscript drafting: J.N. and M.K. All authors have read and agreed to the submitted version of the manuscript. References Roffey, R., Tegnell, A. & Elgh, F. Biological warfare in a historical perspective. Clin. Microbiol. Infect. 8 , 450–454 (2002). van Aken, J. & Hammond, E. Genetic engineering and biological weapons. New technologies, desires and threats from biological research. EMBO Rep. 4 Spec No , S57-60 (2003). Committee on Enhancing Global Health Security through International Biosecurity and Health Engagement Programs, Committee on International Security and Arms Control, Policy and Global Affairs, & National Academies of Sciences, Engineering, and Medicine. A Strategic Vision for Biological Threat Reduction: The U.S. Department of Defense and Beyond . 25681 (National Academies Press, Washington, D.C., 2020). doi:10.17226/25681. Trevisanato, S. I. The ‘Hittite plague’, an epidemic of tularemia and the first record of biological warfare. Med. Hypotheses 69 , 1371–1374 (2007). Maves, R. C. & Berjohn, C. M. Zoonotic Infections and Biowarfare Agents in Critical Care: Anthrax, Plague, and Tularemia. in Highly Infectious Diseases in Critical Care (eds Hidalgo, J. & Woc-Colburn, L.) 97–118 (Springer International Publishing, Cham, 2020). doi:10.1007/978-3-030-33803-9_6. Oliveira, M., Mason-Buck, G., Ballard, D., Branicki, W. & Amorim, A. Biowarfare, bioterrorism and biocrime: A historical overview on microbial harmful applications. Forensic Sci. Int. 314 , 110366 (2020). Carus, W. S. A Short History of Biological Warfare: From Pre-History to the 21st Century . (National Defense University Press, Washington, D.C., 2017). Riedel, S. Biological warfare and bioterrorism: a historical review. Proc. Bayl. Univ. Med. Cent. 17 , 400–406 (2004). Walper, S. A. et al. Detecting Biothreat Agents: From Current Diagnostics to Developing Sensor Technologies. ACS Sens. 3 , 1894–2024 (2018). Szinicz, L. History of chemical and biological warfare agents. Toxicology 214 , 167–181 (2005). Alibek, K. Smallpox: a disease and a weapon. Int. J. Infect. Dis. 8 , 3–8 (2004). Smallpox and Its Eradication . (WHO, Geneva, 1988). Riedel, S. Smallpox and biological warfare: a disease revisited. Proc. Bayl. Univ. Med. Cent. 18 , 13–20 (2005). Barras, V. & Greub, G. History of biological warfare and bioterrorism. Clin. Microbiol. Infect. 20 , 497–502 (2014). Gleick, P. H. Water and terrorism. Water Policy 8 , 481–503 (2006). Ashraf, H. Europe’s response to bioterrorism starts slowly but gathers pace. The Lancet 360 , 733–734 (2002). Centers for Disease Control and Prevention. 2014-2016 Ebola Outbreak in West Africa. https://web.archive.org/web/20170806135144/https:/www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html (2025). Centers for Disease Control and Prevention. History of Marburg Outbreaks. https://www.cdc.gov/marburg/outbreaks/index.html (2025). Centers for Disease Control and Prevention. Ebola Disease Outbreaks by Species and Size, Since 1976. https://www.cdc.gov/ebola/outbreaks/index.html (2025). BioFire Defense. Product History. https://www.biofiredefense.com/about-us/product-history/ (2025). BIOFIRE FILMARRAY 2.0 Operator’s Manual. https://www.biomerieux.com/content/dam/biomerieux-com/service-support/support-documents/instructions-for-use-and-manuals/FLM2-PRT-0002-FilmArray-2.0-Operators-Manual-IVD-EN.pdf (2025). BIOFIRE SPOTFIRE System Operator’s Manual. https://www.biomerieux.com/content/dam/biomerieux-com/service-support/support-documents/instructions-for-use-and-manuals/BFR0001-1641-BIOFIRE-SPOTFIRE-System-Operator-Manual-IVD-EN.pdf (2025). Schuenemann, V. J. et al. Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Proc. Natl. Acad. Sci. U. S. A. 108 , E746-752 (2011). Sidhu, R. K. et al. Attenuation of virulence in Yersinia pestis across three plague pandemics. Science 388 , eadt3880 (2025). Parkhill, J. et al. Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413 , 523–527 (2001). Hänsch, S. et al. The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis. BMC Res. Notes 8 , 535 (2015). Additional Declarations Competing interest reported. All authors are employees of BioFire Defense LLC. 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14:31:47","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154515,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7716200/v1/e341e6168b1428095e2e5b12.html"},{"id":98423630,"identity":"4cfe8ce8-995e-46d0-b281-286a54925d72","added_by":"auto","created_at":"2025-12-17 16:32:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":598351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe BIOFIRE SPOTFIRE instrument\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BIOFIRE SPOTFIRE instrument dimensions and high-level specifications.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7716200/v1/2b6fd0d30c80a8ace9a66085.png"},{"id":98425055,"identity":"c4860456-2b20-4e93-b1dc-2fa20cce5365","added_by":"auto","created_at":"2025-12-17 16:34:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":706329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe BIOFIRE pouch consumable\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWithin a BIOFIRE pouch consumable, FILMARRAY 2.0 or SPOTFIRE instruments execute automated sample extraction (A), nucleic acid purification (A-D), reverse-transcription and multiplex first-stage PCR (PCR1) (E) and nested individual second-stage PCR (PCR2) reactions (G) in a disposable pouch to evaluate multiple RNA and DNA targets in a single run.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7716200/v1/1341e331c1792b4acd2e7a1c.png"},{"id":98444033,"identity":"59f97b1d-4d5d-4c9e-ae8f-344ed6475c75","added_by":"auto","created_at":"2025-12-17 17:14:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2829479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7716200/v1/2fb8807e-1e2e-491e-9211-1f17d9cf0ff4.pdf"},{"id":98424414,"identity":"cc736498-e739-4997-8d2d-87397f73ce88","added_by":"auto","created_at":"2025-12-17 16:33:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4397865,"visible":true,"origin":"","legend":"","description":"","filename":"SensitivityComparisonoftheBIOFIREBioThreatPanelv2.5onFILMARRAYandSPOTFIRESystemsSUPPLEMENTARYMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-7716200/v1/8554e43a1b5ac432fe9ba500.docx"}],"financialInterests":"Competing interest reported. All authors are employees of BioFire Defense LLC.","formattedTitle":"\u003cp\u003eSensitivity Comparison of the BIOFIRE\u003csup\u003e®\u003c/sup\u003e BioThreat Panel v2.5 on FILMARRAY\u003csup\u003e®\u003c/sup\u003e 2.0 and SPOTFIRE\u003csup\u003e®\u003c/sup\u003e Systems\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBiothreat Agents\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiological threat agents, commonly referred to as biothreats or bioagents, are a diverse group of viruses, bacteria, and toxins from biological sources that pose a substantial threat to human health. This group of pathogens and pathogen byproducts are diverse and highly variable in terms of transmissibility, infectivity, and lethality. Highly lethal biothreat pathogens have emerged naturally while others have been developed with the aid of genetic engineering\u003csup\u003e1,2\u003c/sup\u003e. Bioagent threats may arise from natural, accidental, or intentional exposure. Furthermore, humans are not the exclusive target of naturally occurring outbreaks or intentionally targeted attacks. Biothreats may target humans, livestock, crops, water supplies, and more, resulting in substantial physical, social, economic, political, and security consequences\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExamples of Biothreat Attacks and Outbreaks\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile this publication is not intended to provide a comprehensive overview of the history of biothreat agents and associated threat to human health, noteworthy examples illustrate the devastating effects of intentional attacks with biothreat agents, as well as the damage caused by naturally occurring outbreaks.\u003c/p\u003e\n\u003cp\u003eThe deliberate use of biothreat agents in warfare can be observed as early as the 14\u003csup\u003eth\u003c/sup\u003e century BC, when Hittite armies sent diseased rams, likely infected with tularemia, to their enemies to weaken opposing forces\u003csup\u003e4\u003c/sup\u003e. In the 4\u003csup\u003eth\u003c/sup\u003e century BC, according to the Greek historian Herodotus, archers dipped arrows into a mixture of decomposing cadavers of humans and venomous snakes, thereby coating the arrows with \u003cem\u003eClostridium perfringens, Clostridium tetani,\u0026nbsp;\u003c/em\u003eand snake venom\u003csup\u003e5,6\u003c/sup\u003e. In Europe, between 1346 and 1352, outbreaks of \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eare estimated to have killed 25\u0026minus;40% of the continent\u0026rsquo;s population. It is believed that this pandemic was a result of biological warfare in which a Mongol army laying siege to the Crimean city of Kaffa flung corpses infected with \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eover the city walls\u003csup\u003e6\u0026ndash;10\u003c/sup\u003e. This technique of using infected corpses as biological weapons can be found in the centuries that followed.\u003c/p\u003e\n\u003cp\u003eVariola virus, the causative agent of smallpox, was similarly used as a biological weapon following the colonization of the Americas. During the French and Indian Wars, between the years 1754 to 1767, soldiers distributed blankets from a smallpox hospital to Native Americans, killing more than 50% of the infected tribes\u003csup\u003e11,12\u003c/sup\u003e. The Confederate army sold clothing from yellow fever and smallpox patients to Union troops during the United States Civil War\u003csup\u003e9,13\u003c/sup\u003e. Due to the use of smallpox as a biological weapon and the endemic nature of smallpox at the time of the Civil War, smallpox spread throughout the Union troops, Confederate troops, and civilians on both sides\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSmallpox has had catastrophic effects on world populations even outside the targeted uses of variola virus as a biological weapon. Historically, endemic smallpox was responsible for as much as 10% of annual deaths, in locations with sufficient population to support its endemicity\u003csup\u003e9\u003c/sup\u003e. Even following the introduction of the smallpox vaccine, the devastating effects of smallpox continued for decades; approximately 50 million smallpox cases were observed annually in the 1950\u0026rsquo;s, resulting in tens of millions of fatalities\u003csup\u003e8,9\u003c/sup\u003e. These historical examples of smallpox help illustrate the catastrophic capabilities of bioagents; they can be invisible, indiscriminate killers infecting susceptible hosts, whether they are ally or enemy.\u003c/p\u003e\n\u003cp\u003eThere are many other examples of the intentional use of biothreat agents in warfare and terror, including: Napoleon flooded the plains around Mantua, Italy in 1797 to enhance the spread of malaria\u003csup\u003e14\u003c/sup\u003e; during World War I, horses, cattle, and sheep inoculated with \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBurkholderia mallei\u003c/em\u003e were shipped\u003cem\u003e\u0026nbsp;\u003c/em\u003eto the United States, Russia, and other countries\u003csup\u003e8\u003c/sup\u003e; water supplies in Oregon were intentionally contaminated with \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003eby a religious cult in 1984\u003csup\u003e15\u003c/sup\u003e; anthrax spores were sent through the United States Postal Service in 2001\u003csup\u003e10,16\u003c/sup\u003e; and many more\u003csup\u003e2,7,9,11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to the intentional spread of biothreat agents through warfare or terrorism, natural outbreaks and epidemics can pose a severe threat to human health. One example is the Ebola outbreak in West Africa between the years 2014-2016. Over 28,000 cases of infection with \u003cem\u003eOrthoebolavirus zairense\u0026nbsp;\u003c/em\u003ewere suspected during this time and over 11,000 associated deaths, a nearly 40% fatality rate\u003csup\u003e17\u003c/sup\u003e. Additional outbreaks of \u003cem\u003eOrthoebolavirus zairense, Orthoebolavirus sudanese,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e have occurred within the last decade, with fatality rates between 25% and 90%\u003csup\u003e18,19\u003c/sup\u003e, highlighting the severe effects that naturally occurring outbreaks of biothreats can have.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBIOFIRE Technology\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhether the target of a biological threat is a human, an animal, water supply, food supply, or something else, early detection of the causative biothreat agent is the key step to prevention, protection, prophylaxis, post-exposure treatment, and mitigation\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBioFire Defense has provided PCR-based testing capabilities for the detection of biothreat pathogens to military personnel and first-responders for over 20 years, including the Joint Biological Agent Identification and Diagnostic System (JBAIDS), RAZOR\u003cstrong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/strong\u003e Mk II, and Next Generation Diagnostic System (NGDS) FILMARRAY\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e2.0 platforms\u003csup\u003e20\u003c/sup\u003e. These portable systems have provided timely and reliable answers to support critical decision making and countermeasures related to biological warfare agents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBioFire Defense developed the BioThreat Panel v2.5 for use on the FILMARRAY 2.0 instrument for the qualitative detection of a range of biothreat analytes from environmental samples, including: \u003cem\u003eBacillus anthracis, Brucella melitensis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, Rickettsia prowazekii, Yersinia pestis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eEastern equine encephalitis virus, \u003cem\u003eOrthomarburgvirus marburgense, Orthopoxvirus\u003c/em\u003e spp., variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, \u003cem\u003eOrthoebolavirus zairense\u003c/em\u003e, and toxin-encoding genes from \u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003e(botulinum toxin) and \u003cem\u003eRicinus communis\u0026nbsp;\u003c/em\u003e(Ricin toxin). The BioThreat Panel v2.5 for the BIOFIRE FILMARRAY 2.0 has been adopted for environmental surveillance because of its broad capabilities, ease-of-use, speed, and low logistics burden (e.g.,\u003cem\u003e\u0026nbsp;\u003c/em\u003eno cold chain storage required).\u003c/p\u003e\n\u003cp\u003eThe BIOFIRE SPOTFIRE instrument dimensions and high-level specifications.\u003c/p\u003e\n\u003cp\u003eThe BIOFIRE SPOTFIRE instrument represents the next generation of biothreat detection capabilities. Compared with the FILMARRAY 2.0\u003csup\u003e21\u003c/sup\u003e, the SPOTFIRE\u003csup\u003e22\u003c/sup\u003e instrument requires less benchtop space and can be scaled vertically, up to four modules, enabling customizable throughput. In contrast to requiring an external computer to control FILMARRAY 2.0 instruments, the SPOTFIRE instrument contains an integrated, intuitive system interface, making it easy-to-use for a variety of applications (Figure 1). Like the FILMARRAY 2.0, the SPOTFIRE instrument automatically executes all steps of a PCR test, including nucleic acid purification, reverse transcription, PCR amplification, amplicon melt analysis, and report generation.\u003c/p\u003e\n\u003cp\u003eWithin a BIOFIRE pouch consumable, FILMARRAY 2.0 or SPOTFIRE instruments execute automated sample extraction (A), nucleic acid purification (A-D), reverse-transcription and multiplex first-stage PCR (PCR1) (E) and nested individual second-stage PCR (PCR2) reactions (G) in a disposable pouch to evaluate multiple RNA and DNA targets in a single run.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe BioThreat Panel v2.5 consumable with its current chemistry configuration (Figure 2) is compatible with both the FILMARRAY 2.0 and SPOTFIRE instruments. With the corresponding BioThreat Panel v2.5 software package specific to each instrument, BioThreat Panel v2.5 consumables can be tested with both instrument platforms. Each consumable pouch is manufactured with all reagents necessary for nucleic acid purification, reverse transcription, and PCR. Each BIOFIRE pouch consumable contains built-in controls used to demonstrate that the instrument and pouch are performing correctly: 1) internal process controls made up of lyophilized cells from the yeast \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e that are subject to all sample preparation, reverse transcription, and PCR amplification steps, and 2) a PCR control comprised of a synthetic PCR template and corresponding primers manufactured directly into the final PCR stage of each consumable.\u003c/p\u003e\n\u003cp\u003eWith minimal hands-on time, and a run time of approximately one hour on both instrument platforms, the combination of instrument and consumable pouch are used to execute the following steps: nucleic acid purification from the sample (Figure 2A-D), reverse transcription, and amplification in a first-stage PCR reaction, referred to as PCR1 (Figure 2E). Following PCR1, the reaction is diluted (Figure 2F) and flooded across a 102-well array where the primers for the second stage PCR reaction, referred to as PCR2, are pre-spotted (Figure 2G). Each assay is spotted in triplicate on the PCR2 array.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter completion of the nested PCR reaction, the BIOFIRE FILMARRAY 2.0 and SPOTFIRE instruments perform a DNA amplicon melting analysis on the PCR products and measure the fluorescence signal generated in each well. The software then performs several analyses, including analysis of the melt curves and analysis of amplification curves, and assigns a final, qualitative assay result for every well. The BioThreat Panel v2.5 software automatically interprets and returns a result of \u0026ldquo;Detected\u0026rdquo;, \u0026ldquo;Not Detected\u0026rdquo;, or \u0026ldquo;Possible Detection\u0026rdquo; for each pathogen. The interpretation is based on the results of one or more assays for each target pathogen as shown in Table 1 and Supplemental Tables S1-S4.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnalysis of Melt Curves\u003c/em\u003e: The BioThreat Panel v2.5 software evaluates the DNA amplicon melt curves from each well of the PCR2 array. In wells where PCR amplification occurs, the analysis software calculates the melting temperature (Tm) of the DNA melt curve and compares it against a temperature range where Tm values would be expected if the PCR amplicon came from target pathogen nucleic acid. If the Tm falls inside this assay-specific temperature range, the melt curve is called positive. If the software determines that the melt curve is not in the appropriate temperature range, the melt curve is called negative. For an assay to be called positive, at least two out of three possible array wells must contain positive melt curves with similar Tm values. Assays that do not meet these criteria are called negative.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Assay Interpretation Scheme for the BIOFIRE BioThreat Panel v2.5\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"681\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Assays\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssay Detection Requirements for Analyte Detection\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 681px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBACTERIA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis/Bacillus\u0026nbsp;\u003c/em\u003especies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eSee Table S1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrucella melitensis/Brucella\u0026nbsp;\u003c/em\u003especies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eSee Table S2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eBurkholderia mallei/pseudomallei\u003csup\u003e1\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eCoxiella burnetii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eFrancisella tularensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia prowazekii/Rickettsia\u0026nbsp;\u003c/em\u003especies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eSee Table S3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eYersinia pestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eSee Table S4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 681px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVIRUSES\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthoebolavirus zairense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003ePositive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthopoxvirus\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003eVariola virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAll Positive Assays = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003eEastern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003ePositive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003eVenezuelan equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003eAny Positive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003eWestern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003ePositive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 681px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTOXIN-ENCODING GENES\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003e(Botulinum toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003ePositive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 272px;\"\u003e\n \u003cp\u003e\u003cem\u003eRicinus communis\u0026nbsp;\u003c/em\u003e(Ricin toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 256px;\"\u003e\n \u003cp\u003ePositive Assay = \u0026quot;Detected\u0026quot;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCross-reactivity with other \u003cem\u003eBurkholderia\u003c/em\u003e spp. may be observed\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eThe BioThreat Panel v2.5 does not detect toxins directly. Assays on the Panel are designed to detect genomic regions that encode these toxins. It is assumed that crude toxin extracts may contain target pathogen nucleic acids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe BioThreat Panel v2.5 automatically interprets and returns results of \u0026ldquo;Detected\u0026rdquo; or \u0026ldquo;Not Detected\u0026rdquo; for each pathogen. The interpretation is based on the results of one or more assays for each pathogen. This table illustrates how many target assays need to be called positive to produce a \u0026ldquo;Detected\u0026rdquo; result for the target analyte.\u0026nbsp;\u003c/p\u003e"},{"header":"Study Design","content":"\u003cp\u003eThe investigation described herein is composed of two sequential phases. First, the limit of detection (LoD) was estimated for each BioThreat Panel v2.5 target on both the FILMARRAY 2.0 and SPOTFIRE instruments. Second, the LoD was confirmed for each Panel target on both instrument types. All testing was performed with contrived samples, wherein analytes were spiked in a phosphate-buffered saline (PBS) matrix. All work was performed in a certified BSL-2 laboratory following institutional biosafety guidelines. Each sample was tested in parallel on FILMARRAY 2.0 and SPOTFIRE instruments. LoD determination for each analyte was based on the analyses performed by the BioThreat Panel v2.5 panel software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalyte detection refers to the qualitative software interpretation from a FILMARRAY 2.0 or SPOTFIRE pouch run. For some analyte targets, the BioThreat Panel v2.5 software integrates the results of multiple assays to determine whether an analyte is \u0026ldquo;Detected\u0026rdquo;, \u0026ldquo;Not Detected\u0026rdquo;, or \u0026ldquo;Possible Detection\u0026rdquo;. For example, to determine whether \u003cem\u003eFrancisella tularensis\u0026nbsp;\u003c/em\u003eis \u0026ldquo;Detected\u0026rdquo; or \u0026ldquo;Not Detected,\u0026rdquo; the results of two assays are considered: FTT2 and FTT3 (see Table 1). Individual assays may have different specificity, inclusivity, and sensitivity towards a given analyte. The algorithms contained in the BioThreat Panel v2.5 software convert the results of individual assays into a \u0026ldquo;Detected\u0026rdquo;, \u0026ldquo;Not Detected\u0026rdquo;, or \u0026ldquo;Possible Detection\u0026rdquo; call for each analyte. The relationships between BioThreat Panel v2.5 assays and overall interpretations are described in Tables 1 and S1-S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEstimated LoD (eLoD)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LoD estimation phase of this study utilized historical data from initial assay design and development to establish a starting concentration for each analyte. From this starting concentration, five-fold dilutions were tested in four BioThreat Panel v2.5 pouches at each dilution on each instrument type. The eLoD was determined based on the rate of detection at each concentration; the lowest concentration with analyte detection in all four pouches was designated the 1\u0026times; eLoD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLoD Confirmation \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LoD was confirmed for each analyte by testing 20 pouches with samples contrived at the 1\u0026times; eLoD and 20 pouches contrived at a 10-fold dilution lower. Each sample at 1\u0026times; eLoD and each sample at 0.1\u0026times; eLoD was prepared independently and tested on both FILMARRAY 2.0 and SPOTFIRE instruments, in parallel. The LoD for each analyte was confirmed if, at the 1\u0026times; concentration, a \u0026ldquo;Detected\u0026rdquo; result was produced in at least 19/20 pouches and a \u0026ldquo;Detected\u0026rdquo; result was produced in fewer than 19/20 pouches at the 10-fold lower concentration. If either concentration failed to meet these criteria, additional concentrations were tested until the criteria were met. For this study, a \u0026ldquo;Possible Detection\u0026rdquo; interpretation was considered the same as a \u0026ldquo;Not Detected\u0026rdquo; interpretation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalyte selection\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesting was performed for all 16 targets detected by the BioThreat v2.5 Panel with a total of 18 representative analytes used in this study (Table 2). All analytes were quantified in-house using digital PCR (dPCR) prior to use in this study.\u003c/p\u003e\n\u003cp\u003eFor this study, live or inactivated organisms suitable for use in a BioSafety Level 2 facility were the preferred analyte type. Where live or inactivated organisms were unavailable, genomic nucleic acid was the next preferred analyte type. For four analyte targets, \u003cem\u003eEastern equine encephalitis\u003c/em\u003e (EEE) virus, variola virus, \u003cem\u003eClostridium botulinum,\u003c/em\u003e and \u003cem\u003eCoxiella burnetii\u003c/em\u003e, neither intact organism nor genomic nucleic suitable for use in a BioSafety Level 2 facility were available; synthetic templates were used in place of organism or genomic nucleic acid for these target analytes.\u003c/p\u003e\n\u003cp\u003eA single analyte was tested for most BioThreat Panel v2.5 targets as part of this study. Exceptions to this include \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e. For these exceptions where multiple isolates of an organism were tested, the LoD was determined for each strain or virus individually. \u003cem\u003eBacillus anthracis\u003c/em\u003e was tested with both the Ames and Sterne 34F2 strains. The Ames strain is inactivated and expected to produce a positive assay detection for all three assays associated with \u003cem\u003eBacillus anthracis\u003c/em\u003e (Chromosome Element, pXO1, pXO2), thereby returning a \u0026ldquo;\u003cem\u003eBacillus anthracis\u003c/em\u003e Detected\u0026rdquo; interpretation. The Sterne 34F2 strain is missing the pXO2 plasmid and is expected to produce a positive assay detection call for only two of the assays associated with \u003cem\u003eBacillus anthracis\u003c/em\u003e. This will result in a \u0026ldquo;Bacillus species Detected\u0026rdquo; interpretation for this analyte. Two virus types of \u003cem\u003eOrthomarburgvirus marburgense\u0026nbsp;\u003c/em\u003ewere tested: Ravn and Voege. Both viruses trigger both Marburg assays (Marb2 and Marb3) and produce a \u0026ldquo;Marburg virus Detected\u0026rdquo; interpretation.\u003c/p\u003e\n\u003cp\u003eAll analytes were tested individually for LoD estimation and LoD confirmation, except those templates used as a surrogate for variola virus. Variola virus requires four target assays to be positive to return a \u0026ldquo;Variola virus Detected\u0026rdquo; interpretation. Three separate nucleic acid templates were loaded at equivalent concentrations into the pouch together to simulate variola virus; one template is complementary to two assays, and each of the other two templates is complementary to one of the other two target assays. The presence of these nucleic acid templates complementary to the four target assays, enables all assays to be detected and for the panel software to produce a \u0026ldquo;Variola virus Detected\u0026rdquo; interpretation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Analytes Tested for Each Target on the BioThreat v2.5 Panel\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"953\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 185px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTarget Analyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalyte Strain\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVendor ID\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eType of Analyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTargeted Assays\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExpected Interpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eAmes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 1331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eChromosome Element, pXO1, pXO2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eSterne 34F2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-1400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eLive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eChromosome Element, pXO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e species Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrucella melitensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e16M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 0074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Formalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eBRT2, BRT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrucella melitensis\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eBurkholderia pseudomallei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eMSHR 146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 1595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Formalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eBurk2, Burk8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eBurkholderia mallei/pseudomallei\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eCoxiella burnetii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eCHEM-GBK-0485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eCBT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eCoxiella burnetii\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eFrancisella tularensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eSCHU S4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-15753\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Formalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eFTT2, FTT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eFrancisella tularensis\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia prowazekii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eBrienl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 0079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Formalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eRIK2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia prowazekii\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eYersinia pestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCO92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-2717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eGenomic DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eYPT1, YPT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"10\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eViruses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthoebolavirus zairense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eMayinga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-31807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Gamma-irradiated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eEB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eEbola Zaire Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003eEastern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eCHEM-RNA-0039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eEEE01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eEEE Virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eGerman Voege\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-31816\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Gamma-irradiated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eMarb2, Marb3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 204px;\"\u003e\n \u003cp\u003eMarburg virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eRavn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-31819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Gamma-irradiated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eMarb2, Marb3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthopoxvirus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eModified vaccinia ankara (MVA) virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eAttenuated Live\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eOPX2, Var3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eOrthopox genus virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 155px;\"\u003e\n \u003cp\u003eVariola virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eCHEM-GBK-0752\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eOPX2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 204px;\"\u003e\n \u003cp\u003eVariola virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eVar1_BTNIVD_g1QC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eVar1a, Var1b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eVar3_BTNIVD_g1QC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eVar3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003eVenezuelan equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eTrinidad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 0108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Formalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eVEE-MP2, VEE-RC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eVEE virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003eWestern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCBA87 Alpha 025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eAGD 0110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eInactivated (Gamma-irradiated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eWEE01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eWEE virus Detected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eToxin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003e(Botulinum toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eCHEM-RNA-0031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSynthetic Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eBoNTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cem\u003eRicinus communis\u0026nbsp;\u003cbr\u003e\u003c/em\u003e(Ricin toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCastor Bean gDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003eNR-44091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eGenomic DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eRCN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cem\u003eRicinus communis\u0026nbsp;\u003c/em\u003eDetected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eAnalyte strain listed for organism isolates; \u003cem\u003eN/A\u003c/em\u003e listed for synthetic nucleic acid analytes.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eVendor ID or BioFire Defense part number, for synthetic nucleic acid templates.\u003c/p\u003e\n\u003cp\u003eFor each target analyte tested as part of the investigation described herein, additional characteristics of the analyte are listed, including the strain and vendor identification number. The column \u0026ldquo;Type of Analyte\u0026rdquo; refers to whether the analyte is a live or inactivated analyte along with the method of inactivation, genomic nucleic acid, or synthetic nucleic acid. The final two columns indicate the assays on the BioThreat Panel v2.5 that are expected to be positive and the expected interpretation of the panel software when testing the indicated analyte.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSummary of LoD Estimation Testing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach analyte was tested on FILMARRAY 2.0 and SPOTFIRE instruments over a range of concentrations, with four pouches tested on each instrument platform at each concentration. The lowest concentration where the analyte was detected in 4/4 pouches tested was designated as the estimated LoD, unless it was determined through review of amplification curves, that it was unlikely that LoD would be confirmed at that concentration (Table 3). The bolded values in Table 3 represent the initial concentrations used in LoD confirmation testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSummary of LoD Confirmation Testing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 4 lists the confirmed LoD determined for the analytes tested in this study on both FILMARRAY 2.0 and SPOTFIRE instruments. Figures S1-S18 present the Cp and Tm values from individual pouch runs used to confirm the LoD values presented in Table 4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeven analytes demonstrated the same LoD on FILMARRAY 2.0 and SPOTFIRE : \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eAmes, \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eSterne 34F2, \u003cem\u003eBrucella melitensis, Burkholderia pseudomallei, Coxiella burnetii, Franciscella tularensis,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOrthomarburgvirus marburgense\u0026nbsp;\u003c/em\u003eGerman Voege. An additional eight analytes demonstrated similar sensitivity (within 5-fold) on FILMARRAY 2.0 and SPOTFIRE : \u003cem\u003eRickettsia prowazekii, Orthoebolavirus zairense, Orthomarburgvirus marburgense\u0026nbsp;\u003c/em\u003eRavn, modified Vaccinia Ankara virus, variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and \u003cem\u003eRicinus communis\u003c/em\u003e. Two analytes demonstrated a greater than 5-fold improvement in sensitivity on the SPOTFIRE relative to the FILMARRAY 2.0: Eastern equine encephalitis virus, and \u003cem\u003eClostridium botulinum.\u003c/em\u003e A single target analyte demonstrated reduced sensitivity on the SPOTFIRE compared with the FILMARRAY 2.0: \u003cem\u003eYersinia pestis.\u0026nbsp;\u003c/em\u003eA reduction in sensitivity towards \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003ewas expected due to changes in the calling scheme designed to increase the stringency and specificity of the BioThreat Panel v2.5 towards \u003cem\u003eYersinia pestis\u003c/em\u003e (see Discussion).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Confirmed LoD Values for Analytes on the BioThreat v2.5 Panel on FILMARRAY 2.0 and SPOTFIRE\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"924\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 303px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFILMARRAY 2.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 303px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSPOTFIRE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eAmes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eSterne 34F2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.0E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.0E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrucella melitensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e16M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eBurkholderia pseudomallei\u003csup\u003e1\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eMSHR 146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.8E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.8E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.9E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.8E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eCoxiella burnetii\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e7.4E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.9E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.2E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e7.4E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.9E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.2E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eFrancisella tularensis\u003csup\u003e3\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eSCHU S4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.4E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.4E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.4E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.4E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia prowazekii\u003csup\u003e4\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eBrienl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.7E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.0E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.7E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.0E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e7/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eYersinia pestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eCO92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.1E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.2E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.4E+01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.8E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.8E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.1E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.4E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.8E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.8E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthoebolavirus zairense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eMayinga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.7E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.4E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.7E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.4E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003eEastern equine encephalitis virus\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.4E+05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.5E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.9E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e7.4E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.9E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eGerman Voege\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.4E+04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.9E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.4E+04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.9E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e5.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eRavn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.5E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.3E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e6.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.3E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthopoxvirus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eMVA virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003eVariola virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.0E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.0E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.0E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003eVenezuelan equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eTrinidad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.2E+04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.4E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.7E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003eWestern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eCBA87 Alpha 025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.8E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.8E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e9.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003e(Botulinum toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1E+04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e6.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.5E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3.1E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.2E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.2E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.5E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eRicinus communis\u0026nbsp;\u003cbr\u003e\u003c/em\u003e(Ricin toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 78px;\"\u003e\n \u003cp\u003eCastor Bean gDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eConcentration (copies/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0E+01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e2.0E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.0E+02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e8.0E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1.6E+01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003eDetections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e0/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDelayed amplification was observed when testing at 9.6E+01 copies/mL on SPOTFIRE, so it was decided to confirm at the next highest dilution.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eConfirmation testing was performed at 3.0E+04 copies/mL due to a clerical error.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDelayed amplification was observed when testing at 2.4E+01 copies/mL on SPOTFIRE, so it was decided to confirm at the next highest dilution.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eEight pouches were tested on SPOTFIRE at 1.7E+03 copies/mL. Since 7/8 pouches demonstrated analyte detection, a concentration of 1.7E+03 copies/mL was used for confirmation testing.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003eConfirmation testing was initiated at 5.9E+03 copies/mL on FILMARRAY 2.0 and at 7.4E+04 copies/mL on SPOTFIRE due to a clerical error.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u003c/sup\u003eDelayed amplification was observed when testing at 2.9E+03 copies/mL on FILMARRAY 2.0, so it was decided to confirm at the next highest dilution.\u003c/p\u003e\n\u003cp\u003eFor each analyte, the concentrations (in units of copies/mL) tested are indicated, as well as the number of pouches producing the expected interpretation. Cells representing analyte concentrations where all pouches produced the expected interpretation are highlighted in green. The bolded concentrations indicate the concentrations that were used for initial testing during the LoD confirmation phase; assay detection results and expert review of Cp values led to these determinations. Cells shaded in gray with \u003cem\u003eN/A\u0026nbsp;\u003c/em\u003eindicate that additional concentrations were not tested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Confirmed LoD Values for Analytes on the BioThreat v2.5 Panel on FILMARRAY 2.0 and SPOTFIRE\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 275px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTarget Analyte\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalyte Strain\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 210px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConfirmed LoD (copies/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFILMARRAY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSPOTFIRE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eAmes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e8.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e8.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eSterne 34F2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eBrucella melitensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e16M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eBurkholderia pseudomallei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eMSHR 146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.8E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.8E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eCoxiella burnetii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e3.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e3.0E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eFrancisella tularensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eSCHU S4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e2.4E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e2.4E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia prowazekii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eBrienl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.7E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e8.7E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eYersinia pestis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCO92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.4E+01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eViruses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthoebolavirus zairense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eMayinga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.8E+05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e3.7E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eEastern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.9E+05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.4E+03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthomarburgvirus marburgense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eGerman Voege\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.4E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.4E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eRavn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e6.5E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e3.2E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eOrthopoxvirus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eMVA poxvirus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e6.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e3.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eVariola virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e8.0E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eVenezuelan equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eTrinidad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e1.2E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eWestern equine encephalitis virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCBA87 Alpha 025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e2.4E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.8E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eToxin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium botulinum\u0026nbsp;\u003c/em\u003e(Botulinum toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cem\u003eN/A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1E+06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1E+05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cem\u003eRicinus communis\u0026nbsp;\u003c/em\u003e(Ricin toxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eCastor Bean gDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e8.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e4.0E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eAnalyte strain listed for organism isolates; \u003cem\u003eN/A\u003c/em\u003e listed for synthetic nucleic acid analytes\u003c/p\u003e\n\u003cp\u003eThe confirmed LoD in units of copies/mL are listed for each analyte on each instrument platform, FILMARRAY 2.0 and SPOTFIRE. LoD values shaded in blue indicate reduced sensitivity on the SPOTFIRE instrument relative to the FILMARRAY 2.0. LoD values shaded in orange indicate increased sensitivity on the SPOTFIRE instrument relative to the FILMARRAY 2.0. Bold text indicates that differences in confirmed LoD values between the two instrument platforms are greater than 5-fold.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe sensitivity, or limit of detection (LoD), of the FILMARRAY 2.0 and SPOTFIRE instruments for 18 analytes targeted by the BioThreat v2.5 Panel was determined. Seven analytes demonstrated the same LoD on FILMARRAY 2.0 and SPOTFIRE: \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eAmes, \u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003eSterne 34F2, \u003cem\u003eBrucella melitensis, Burkholderia pseudomallei, Coxiella burnetii, Franciscella tularensis,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOrthomarburgvirus marburgense\u0026nbsp;\u003c/em\u003eGerman Voege. An additional eight analytes demonstrated similar sensitivity (within 5-fold) on FILMARRAY 2.0 and SPOTFIRE: \u003cem\u003eRickettsia prowazekii, Orthoebolavirus zairense, Orthomarburgvirus marburgense\u0026nbsp;\u003c/em\u003eRavn, modified Vaccinia Ankara virus, variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and \u003cem\u003eRicinus communis\u003c/em\u003e. Two analytes, Eastern equine encephalitis virus and \u003cem\u003eClostridium botulinum,\u003c/em\u003e demonstrated a greater than 5-fold improvement in sensitivity on the SPOTFIRE relative to the FILMARRAY 2.0. A single target analyte, \u003cem\u003eYersinia pestis,\u003c/em\u003e demonstrated an expected reduction in sensitivity on the SPOTFIRE compared with the FILMARRAY 2.0\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOverall, the SPOTFIRE demonstrated similar (within 5-fold) or improved sensitivity towards all analytes tested, except for \u003cem\u003eYersinia pestis.\u0026nbsp;\u003c/em\u003eA decrease in sensitivity towards \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003ewhen testing on the SPOTFIRE was expected due to changes to the calling scheme being used on the SPOTFIRE compared with the calling scheme on FILMARRAY\u0026nbsp;2.0 (Table S4). The BioThreat Panel v2.5 contains two assays, YPT1 and YPT3, for the detection of \u003cem\u003eYersinia pestis.\u0026nbsp;\u003c/em\u003eWhen testing on the FILMARRAY 2.0, if either YPT1 or YPT3 assays are positive, the software returns a \u0026ldquo;\u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eDetected\u0026rdquo; interpretation; however, on SPOTFIRE, the YPT3 assay must be positive to produce a \u0026ldquo;\u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eDetected\u0026rdquo; interpretation. The YPT1 and YPT3 assays target distinct extrachromosomal plasmids; the YPT1 assay targets the pPCP1 plasmid which is generally more abundant than the pMT1 plasmid targeted by the YPT3 assay\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. The genetic loci targeted by the YPT1 assay, the \u003cem\u003epla\u0026nbsp;\u003c/em\u003egene, previously thought to be specific to \u003cem\u003eYersinia pestis,\u0026nbsp;\u003c/em\u003ehas been detected in other bacterial species\u003csup\u003e26\u003c/sup\u003e. Due to the potential presence of the \u003cem\u003epla\u0026nbsp;\u003c/em\u003egene in bacterial species other than \u003cem\u003eYersinia pestis\u003c/em\u003e, the calling scheme was updated for the SPOTFIRE instrument to reflect the inherent uncertainty whether nucleic acid specific to \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003eis present when only the YPT1 assay is positive. As the YPT1 assay targets the more abundant pPCP1 plasmid compared to the target of the YPT3 assay, the pMT1 plasmid, reduced sensitivity of the BioThreat v2.5 Panel on SPOTFIRE towards \u003cem\u003eYersinia pestis\u0026nbsp;\u003c/em\u003ewith this modified calling scheme is expected.\u003c/p\u003e\n\u003cp\u003eBIOFIRE systems have been adopted for environmental surveillance and infectious disease testing by the US Department of Defense and others because of their broad capability, ease-of-use, speed, and low logistics burden (e.g.,\u003cem\u003e\u0026nbsp;\u003c/em\u003eno cold chain storage required). Earlier generations of BIOFIRE platforms (BIOFIRE FILMARRAY 2.0 and BIOFIRE Torch) have FDA-cleared IVD syndromic panels with more than 170 pathogen target assays and non-IVD panels with more than 100 pathogen target assays. These earlier generation instruments fill a testing need for the most common infections, including respiratory, gastrointestinal, and blood infections, as well as biothreat testing capabilities.\u003c/p\u003e\n\u003cp\u003eThe BIOFIRE SPOTFIRE is the next-in-line system of BIOFIRE instrument\u003csup\u003e22\u003c/sup\u003e. SPOTFIRE panels for the detection of respiratory pathogens have been cleared by the FDA; other SPOTFIRE panels are in development to provide the same testing capabilities available with earlier generations of BIOFIRE platforms. Based on the data presented herein, it is anticipated that users of the BioThreat Panel v2.5 will observe similar levels of sensitivity and functionality upon both the legacy FILMARRAY 2.0 and next-in-line SPOTFIRE instruments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding Statement:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;The work presented in this manuscript was completed without external funding.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests Statement:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;All authors are employees of BioFire Defense LLC.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability Statement:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics, Consent to Participate, and Consent to Publish declarations:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eNot applicable\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical Trial Number:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Not applicable\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions Statement:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Study design and conceptualization: J.N., K.W., and M.K. Study execution: K.P., N.D., D.S., S.P., R.P., C.G., E.K., A.B., W.B., and K.L. Data analysis: J.N., K.W., K.P., and N.D. Manuscript drafting: J.N. and M.K. All authors have read and agreed to the submitted version of the manuscript.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoffey, R., Tegnell, A. \u0026amp; Elgh, F. Biological warfare in a historical perspective. \u003cem\u003eClin. Microbiol. Infect.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 450–454 (2002).\u003c/li\u003e\n\u003cli\u003evan Aken, J. \u0026amp; Hammond, E. Genetic engineering and biological weapons. 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Notes\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 535 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Biotechnology](https://link.springer.com/journal/44340)","snPcode":"44340","submissionUrl":"https://submission.springernature.com/new-submission/44340/3","title":"Discover Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7716200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7716200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Rapid and sensitive detection of biothreat agents represents a critical capability for first responders, federal agencies, and militaries around the world. For nearly a decade, the BIOFIRE® BioThreat Panel v2.5 has been an easy-to use, automated, “lab in a pouch” detection tool utilizing qualitative, multiplexed polymerase chain reaction (PCR) with the BIOFIRE FILMARRAY® 2.0 instrument to detect biothreat agents directly from environmental samples in approximately one hour. The BIOFIRE BioThreat Panel v2.5 detects Bacillus anthracis, Brucella melitensis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, Rickettsia prowazekii, Yersinia pestis, Eastern equine encephalitis virus, Orthomarburgvirus marburgense, Orthopoxvirus spp., variola virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Orthoebolavirus zairense, and toxin-encoding genes from Clostridium botulinum (botulinum toxin) and Ricinus communis (Ricin toxin). Herein we establish performance of the BioThreat Panel v2.5 on the next-in-line system, the BIOFIRE SPOTFIRE®.\nSensitivity of the BioThreat Panel v2.5 was compared between the FILMARRAY 2.0 and SPOTFIRE instruments in parallel by comparing the limit of detection (LoD) for each of the 16 biothreat targets on the panel, including seven bacterial pathogens, seven viral pathogens, and two toxin-encoding genes. The BioThreat Panel v2.5 exhibited equivalent or better sensitivity on SPOTFIRE compared to FILMARRAY 2.0 for all targets, except for Yersinia pestis, which displayed reduced sensitivity due to a modified calling scheme on SPOTFIRE. This study demonstrates that the BioThreat Panel v2.5 can provide sensitive biothreat detection across both BIOFIRE FILMARRAY 2.0 and SPOTFIRE automated PCR instruments.","manuscriptTitle":"Sensitivity Comparison of the BIOFIRE® BioThreat Panel v2.5 on FILMARRAY® 2.0 and SPOTFIRE® Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 14:31:42","doi":"10.21203/rs.3.rs-7716200/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-20T05:55:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T05:27:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T09:13:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12005690174205446792888736256730223708","date":"2026-03-31T04:37:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-28T19:37:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69688640190483201886631773748312673862","date":"2026-03-18T12:23:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2026-03-17T06:45:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T16:54:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34343233988102062600539353307202403724","date":"2025-12-16T22:59:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T16:12:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-05T11:07:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T18:03:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T06:49:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Biotechnology","date":"2025-11-30T05:02:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Biotechnology](https://link.springer.com/journal/44340)","snPcode":"44340","submissionUrl":"https://submission.springernature.com/new-submission/44340/3","title":"Discover Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3526ab0f-e210-4487-b0cc-fd92e63caae2","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T08:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 14:31:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7716200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7716200","identity":"rs-7716200","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}