ABSTRACT
Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.
KEYWORDS: botulinum neurotoxin, botulism, mass spectrometry
INTRODUCTION
Clostridium botulinum and rare strains of other species of Clostridium bacteria produce botulinum neurotoxins (BoNTs) that cause the disease known as botulism. BoNTs cause descending flaccid paralysis in the body, which can result in death; however, rapid administration of antitoxin prevents progression of the disease (1). BoNTs have seven serologically distinct serotypes labeled A through G. Additional serotypes have been proposed (X, En, and H) but have not been accepted by consensus as new BoNT serotypes within the scientific community. Four serotypes, BoNT/A, /B, /E, and /F, most commonly cause disease in humans (2). BoNTs are structurally a dichain toxin, 150 kDa protein, comprised of an enzymatic light chain and a receptor binding heavy chain. BoNTs inhibit the release of acetylcholine at the neuromuscular junctions by cleaving synaptosomal-associated proteins of 25 kDa (SNAP-25) for BoNT/A and /E and the vesicle-associated membrane proteins (VAMP/synaptobrevin-2) for BoNT/B and /F in the soluble N-ethylmaleimide (SNARE) complex of the synaptic vesicle. The cleavage of these proteins results in paralysis (3–10). Because of the lethal potential of BoNTs and the possible use for bioterrorism purposes, a more sensitive and rapid analytical method is needed for detection to provide a rapid public health response.
The current standard for BoNT detection is the mouse bioassay (MBA) (2), which requires the use of laboratory animals and laboratory personnel trained to recognize signs of botulism in mice. Specimens collected prior to antitoxin administration are injected intraperitoneally into mice, usually two mice per specimen, with and without antitoxin. Mice are then observed for typical signs of botulism for up to 4 days. Although the mouse bioassay has been the gold standard of detection for many years, a more cost-effective and time-efficient method is desired. Additionally, the mouse bioassay has ethical concerns due to animal use.
The Endopep-MS method was designed to detect BoNTs in human clinical specimens (11). This method is an in vitro activity assay for detecting the enzymatic activity of BoNTs. The Endopep-MS method uses serotype-specific antibody-coated magnetic beads that bind and extract the toxin from clinical specimens. The serotype-specific magnetic beads are washed and then incubated with reaction buffer and a serotype-specific peptide substrate. The serotype-specific peptide substrate, optimized to mimic SNAP-25 or VAMP-2 BoNT cleavage sites (12, 13), is then analyzed by mass spectrometry to detect the presence or absence of cleavage products.
The Endopep-MS method has been used successfully for many years as a Clinical Laboratory Improvement Amendments (CLIA)-compliant method (14) with large and expensive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) instrumentation that might not be optimal for state and public health laboratories. However, many of these laboratories are already using a benchtop MALDI-TOF for high-throughput analysis of microorganism identification and could easily transition to use these instruments for traditional mass spectrometric detection that is required for the BoNT Endopep-MS method (15). MALDI-TOF is a rapid analysis method that requires no more than 15 seconds to analyze each sample spot and would be beneficial for a large volume of samples.
The original CLIA-compliant method using more sophisticated mass spectrometers was adapted into a CLIA-compliant method using a benchtop MALDI-TOF. Although the method on the benchtop instrument had higher limits of detection, it was encouraging to see that the method worked on benchtop instruments. This paved the way for a potential method better suited for state public health laboratories.
A similar version of the benchtop BoNT Endopep-MS method, which exhibited good agreement with the mouse bioassay, has been previously published (16). However, public health laboratory networks require a benchtop version of this method with Food and Drug Administration (FDA) clearance or approval. We felt that additional and more extensive studies were required for implementation and FDA approval of a benchtop BoNT Endopep-MS method for public health laboratories nationwide. This paper describes the validation studies in human serum, stool extracts, and culture supernatants to define sensitivity, specificity, selectivity, and robustness needed for proper implementation of the BoNT Endopep-MS method in the Laboratory Response Network for public health response. Few laboratories within the USA currently use BoNT Endopep-MS as a CLIA-compliant method. Proper application of this method will improve national preparedness and response to a BoNT public health crisis.
MATERIALS AND METHODS
Safety considerations
Botulinum neurotoxins are hazardous and require appropriate safety measures. All toxin was handled in select agent-registered space in a level 2 biosafety cabinet with HEPA (high efficiency particulate air) filters to minimize potential exposure from aerosols. All pipette tips used contained filters to minimize aerosolization and contamination.
Chemicals and reagents
BoNT/A, /B, /E, and /F dichain toxins at a concentration of 1 µg/mL were originally obtained from Metabiologics (Madison, WI, USA) and then aliquoted and processed into kits at the Centers for Disease Control and Prevention (CDC) (Atlanta, GA, USA). BoNT/A, B, E, and F complex toxins were purchased from Metabiologics (Madison, WI, USA). Further information on the toxins is included in Table S1. Magnetic beads (obtained from ThermoFisher Scientific, Pittsburgh, PA, USA) were coated with monoclonal antibodies to BoNT/A, /B, /E, and /F as described previously (10). The monoclonal antibody-coated beads were manufactured and processed into kits at CDC (Atlanta, GA, USA). Peptide substrates for BoNT/A, /B, /E, and /F were manufactured and processed into kits at CDC (Atlanta, GA, USA). 10× BoNT reaction buffer was manufactured and processed into kits at CDC (Atlanta, GA, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Human serum, tested and found negative for HIV 1 and 2, HIV-Ag (HIV antigen), HCV, HBsAg (hepatitis B surface antigen), and RPR (rapid plasma reagin) by FDA approved methods, was obtained from Millipore Sigma (Burlington, MA, USA). Peptone yeast glucose broth [tryptone peptone glucose yeast extract (TPGY)] was from Thermo Scientific Remel (Lenexa, KS, USA). Cooked meat medium with glucose, hemin, and vitamin K (CMGS) was from Thermo Scientific Remel (Lenexa, KS, USA). Sulfo-NHS-Biotin was purchased from Thermo Fisher Scientific (Waltham, MA, USA). KingFisher supplies (Microtiter Deepwell 96 plate, 96 tip comb for deep-well magnets, and 96-well microplate—200 µL) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Purchasing information for the interfering substances panel reagents can be viewed in supplementary data (Table S2).
Inclusivity and exclusivity panels
Sensitivity was determined by testing a panel (n = 50) of culture supernatants of known BoNT-producing species of Clostridium, some of which produce more than one serotype of toxin (bivalent): 14 C. botulinum type A strains, 3 C. botulinum type Ab strains, 1 C. botulinum type Ba strain, 12 C. botulinum type B strains, 2 C. botulinum type Bf strains, 6 C. botulinum type E strains, 1 C. butyricum type E strain, 4 C. botulinum type F strains, and 1 C. baratii type F strain.
Specificity was determined by testing a panel (n = 51) of culture supernatants consisting of 24 strains of Clostridium spp. that do not produce botulinum toxin (C. histolyticum, C. tertium, C. absonum, C. sporogenes, C. sordelii, C. septicum, C. tetani, C. perfringens, C. butyricum, C. hastiforme, C. ramosum, C. baratii, C. difficile, C. subterminale, C. bifermentans, C. novyii, and C. haemolyticum), 20 enteric bacteria [Serratia marcescens, Salmonella Newport, Salmonella Typhimurium, Proteus vulgaris, Campylobacter jejuni, Campylobacter fetus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Shigella dysenteriae, Shigella sonnei, Shigella boydii, Shigella flexneri, enterotoxigenic Escherichia coli (ETEC), Yersinia enterocolitica, Aeromonas hydrophila, Citrobacter freundii, Edwardsiella tarda, Providencia stuartii, and Pseudomonas aeruginosa], and 7 strains of BoNT-producing species of Clostridium that produce serotypes C (n = 5), D (n = 1), or G (n = 1).
Preparation of stool extract
Human feces (stool), gender-unspecified pooled and viral negative, were purchased from BioIVT (Waltham, MA, USA) or other equivalent manufacturer. A 1 mL aliquot of 1× phosphate-buffered saline with 0.05% Tween-20 detergent (PBST) or gelatin phosphate-buffered collecting fluid was added for every gram of stool, and the volume of liquid and weight of stool were recorded. Stool was ground and pulverized with either the Stomacher 80 Biomaster (Seward, Port Saint Lucie, FL, USA) at maximum speed for 2 minutes or an equivalent process. The mixture was centrifuged at 27,000 × g in a refrigerated (+2°C–+ 8°C) centrifuge for 20 minutes. The supernatant was transferred to a new centrifuge tube and this process was repeated as many times as needed for clarity of supernatant. The supernatant was filtered through a 0.45 µm filter (Whatman 6876-2504), separated into 1 mL aliquots, and stored at −20°C for up to 3 years for future analysis.
Botulinum neurotoxin extraction
BoNT toxin (/A, /B, /E, and /F) was spiked into 1 mL total volume of matrix, 100 µL of 10× PBST, and 20 µL of serotype-specific antibody-coated beads. Antibodies were produced as described in Kalb et al. (17, 18) and serotype-specific antibody-coated beads were made as described in Kalb et al. (19), in a deep well plate. The deep well plate was capped and placed on a plate shaker at 1,200–1,450 revolutions per minute (rpm) for 1 hour to keep beads in solution for optimal toxin binding. The deep well plate was then uncapped and placed into the KingFisher Flex System (Thermo Fisher Scientific, Waltham, MA, USA) for automated bead washing with two washes containing 1 mL each of 2M NaCl followed by two washes of 1 mL 1× PBST. The beads were eluted from the KingFisher Flex into 80 µL of 18 megOhms H2O in a KingFisher 96 standard PCR plate and removed from the KingFisher Flex.
The following modifications were made to the above method for validation experiments: The limit of detection (LOD) used 500 µL buffer, serum, stool extract, TPGY, or CMGS and 500 µL of serum. BoNT toxin (/A, /B, /E, and /F) was spiked directly into the 96-well plate with toxin levels ranging between 0.2 and 5 mouse lethal dose 50% (mLD50) or 0.4 to 10 mLD50/mL. The cross-reactivity study spiked 1 mLD50, 10 mLD50, and 100 mLD50 of BoNT toxin (/A, /B, /E, and /F), which were added to three separate wells for each serotype in a 96-well plate containing 500 µL of serum and 500 µL stool extract or 1 mL of serum. The exclusivity and toxigenic Clostridium panels had 500 µL of culture supernatant added directly into the 96-well plate containing 500 µL of serum. The interfering substances panel spiked endogenous or exogenous substances at therapeutic levels into 12,000 µL serum or 12,000 µL stool extract. A 500 µL aliquot of spiked matrix was then added directly to 24 wells in the 96-well plate containing 500 µL of serum. BoNT toxin (/A, /B, /E, and /F), at concentrations of 5× the LOD for each serotype, was spiked into 12 of the 24 wells, three for each serotype. The stability study analyzed BoNT/A, /B, /E, and /F in serum spiked with dichain toxin at 10 mLD50/mL, stool extract spiked with complex toxin at 50 mLD50/mL, and culture supernatants (TPGY and CMGS) spiked with complex toxin at 10 mLD50/mL. All samples were stored at +2°C–+8°C between testing time points. The 500 µL spiked matrices were added directly into individual wells in a 96-well plate containing 500 µL of serum. Three aliquots of the spiked samples per time point were tested per serotype, plus one negative/unspiked aliquot. The instrument comparison study evaluated all clinical matrices for toxins BoNT/A, /B, /E, and /F at levels above, below, and at the LOD, as well as negative controls. Each clinical matrix was tested at five replicates, except stool extract, which required 20 replicates because it is the most difficult matrix for experimental purposes. For the reproducibility study and clinical validation study, the serum/stool/culture supernatant was pooled and spiked with BoNT/A, /B, /E, and /F, aliquoted into individual 2.5 mL per serotype per specimen type, and stored at −80°C until shipment to laboratory sites.
Endopep-MS reaction
The serotype-specific antibody-coated beads and 18 megOhms H2O were transferred from the KingFisher 96 standard PCR plate to a PCR tube strip. We then used a PCR separation magnet to remove the 18 megOhms H2O. 1× BoNT reaction buffer was prepared from 100 µL of 10× BoNT reaction buffer, 10 µL of 25 mM dithiothreitol, and 890 µL of 18 megOhms H2O. The serotype-specific antibody-coated beads were then reconstituted in 18 µL of 1× BoNT reaction buffer and 2 µL of serotype-specific peptide substrates, as described in Wang et al. for BoNT/A (12), Barr et al. for BoNT/B (11), Wang et. al. for BoNT/E (13), and Kalb et al. for BoNT/F (20), at a concentration of 0.5 mM for BoNT/A and /E and a concentration of 1 mM for BoNT/B and/F. Samples were then incubated at 37°C for 4 hours with no agitation.
MALDI-TOF detection and data processing
After the incubation, 2 µL of supernatant from the reaction was mixed with 18 µL of MALDI matrix solution. The MALDI matrix consists of α-cyano-4-hydroxy cinnamic acid at 5 mg/mL in 50/50 acetonitrile/deionized water, 0.1% trifluoroacetic acid, and 1 mM ammonium citrate. A 2 µL aliquot of the supernatant and MALDI matrix were spotted onto the Bruker Daltonics MALDI Biotarget 48 well disposable plate (Billerica, MA, USA). The spots were dried in the biological safety cabinet. Mass spectra were collected for each spot by analyzing from 800 to 6,080 m/z in positive ion linear mode on the Bruker microflex (Billerica, MA, USA). An acceleration laser of 10 kV is required for the flexControl method. The AutoXecute method requires an initial laser power of 30% and a maximal laser power of 50%, laser power fuzzy control MS/parent mode “On,” a summation of 500 shots on one sample spot, and random walk movement on the spot.
We used Bruker flexAnalysis software to analyze spectra on the Bruker microflex instrument. The flexAnalysis method encompasses the mass control list and stipulates the following settings: a signal-to-noise ratio (S/N) threshold of 10, centroid for peak detection algorithm, a minimum peak width of 1 m/z, and TopHat setting for baseline subtraction. The mass control list uses the mass ranges in Table 1 for identifying BoNT/A, /B, /E, and /F through a specific Bruker macro known as “SearchBackgroundPeaks.”
TABLE 1.
The m/z (mass-to-charge ratio) ranges of the intact peptide and cleavage products for botulinum neurotoxins A, B, E, F, and F5
| BoNT | Intact peptide | Cleavage product 1 | Cleavage product 2 | |||
|---|---|---|---|---|---|---|
| Minimum (m/z) | Maximum (m/z) | Minimum (m/z)2 | Maximum (m/z)3 | Minimum (m/z)4 | Maximum (m/z)5 | |
| A | 3,280.7 | 3,293.7 | 996.8 | 1,000.8 | 2,302.9 | 2,312.1 |
| B | 4,018.4 | 4,034.6 | 1,756.5 | 1,763.5 | 2,277.7 | 2,286.9 |
| E | 3,607.8 | 3,622.2 | 1,129.2 | 1,133.8 | 2,493.6 | 2,503.6 |
| F | 5,100.8 | 5,121.2 | 1,342.5 | 1,347.9 | 3,777.3 | 3,792.5 |
| F5 | 5,100.8 | 5,121.2 | 1,870.3 | 1,877.7 | 3,248.5 | 3,261.5 |
RESULTS AND DISCUSSION
Sensitivity and robustness
The Endopep-MS method has been used to detect BoNT types A, B, E, and F (the serotypes which commonly affect humans) in clinical specimens of serum, stool extract, and culture supernatants. The Endopep-MS method has serotype-specific substrates that were developed with pre-determined cleavage points for each BoNT (Table 1), so it is possible to determine a positive result with only one cleavage product present for each serotype. As the toxin levels decrease, near the LOD, only one cleavage product might be visible because both cleavage products do not have the same likelihood for ionization by MALDI. LOD was determined by conducting 20 experiments for each serotype and clinical matrix and establishing a 95% CI within those experiments, which also determined the S/N false-positive cut-off for spectra as a value of 10 (Table 2). The LOD of the mouse bioassay, the standard assay, is 5 mLD50/mL–10 mLD50/mL (21). The Endopep-MS method has LODs equivalent and, in some cases, is more sensitive than the mouse bioassay. The LOD for BoNT/A, /B, /E, and /F in all matrices tested was in the range of 0.2 to 5 mLD50 or 0.4 to 10 mLD50/mL (Table 2). Greater sensitivity allows for laboratory confirmation of more true positive results and less false negatives for BoNT clinical samples. The N = 20 LOD experiments determined reproducibility of 98.13%. The confidence interval for all clinical matrices and serotypes was 95%–100%.
TABLE 2.
LOD of botulinum neurotoxin (BoNT)/A, /B, /E, and /F in buffer, serum, TPGY, CMGS, and stool extract determined by N = 20 at a ≥95% CI
| BoNT serotype | Matrix | Toxin | LOD (mLD50) | LOD (mLD50/mL) |
|---|---|---|---|---|
| A | Buffer | Dichain | 0.5 | 0.5 |
| B | Buffer | Dichain | 0.5 | 0.5 |
| E | Buffer | Dichain | 0.5 | 0.5 |
| F | Buffer | Dichain | 0.5 | 0.5 |
| A | Buffer | Complex | 0.5 | 0.5 |
| B | Buffer | Complex | 1 | 1 |
| E | Buffer | Complex | 1 | 1 |
| F | Buffer | Complex | 0.5 | 0.5 |
| A | Serum | Dichain | 1 | 2 |
| B | Serum | Dichain | 0.5 | 1 |
| E | Serum | Dichain | 0.5 | 1 |
| F | Serum | Dichain | 0.2 | 0.4 |
| A | Stool extract | Complex | 2 | 4 |
| B | Stool extract | Complex | 1 | 2 |
| E | Stool extract | Complex | 5 | 10 |
| F | Stool extract | Complex | 0.5 | 1 |
| A | TPGY | Complex | 1 | 2 |
| B | TPGY | Complex | 1 | 2 |
| E | TPGY | Complex | 2 | 4 |
| F | TPGY | Complex | 0.5 | 1 |
| A | CMGS | Complex | 2 | 4 |
| B | CMGS | Complex | 1 | 2 |
| E | CMGS | Complex | 2 | 4 |
| F | CMGS | Complex | 0.5 | 1 |
Specificity
Several studies were designed to determine the specificity of the BoNT Endopep-MS method. These included cross-reactivity studies designed to ensure that a sample positive for one serotype does not give a positive result for other serotypes. They also included an exclusivity study using a panel consisting of culture supernatants of organisms commonly associated with enteric diseases, and non-toxigenic strains of Clostridium.
Cross-reactivity studies determined the ability of the Endopep-MS method to differentiate between BoNT serotypes. The magnetic beads in the Endopep-MS method have serotype-specific antibodies biotinylated to them and should only extract toxin from that specific serotype. Dichain (Table 3) and complex (Table 4) toxin were used to test serotypes BoNT/A, /B, /E, and /F in serum and stool extract. The cross-reactivity panels performed as expected; no cross-reactivity between the serotypes was observed.
TABLE 3.
The cross-reactivity study determined the ability of the serotype-specific antibody-coated beads to differentiate and detect the presence of dichain toxins for botulinum neurotoxin (BoNT)/A, /B, /E, and /F
| Serotype of antibody-coated bead | A 100U | A 10U | A 1U | B 100U | B 10U | B 1U | E 100U | E 10U | E 1U | F 100U | F 10U | F 1U |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | Yes | Yes | Yes | No | No | No | No | No | No | No | No | No |
| B | No | No | No | Yes | Yes | Yes | No | No | No | No | No | No |
| E | No | No | No | No | No | No | Yes | Yes | Yes | No | No | No |
| F | No | No | No | No | No | No | No | No | No | Yes | Yes | Yes |
TABLE 4.
The cross-reactivity study determined the ability of the serotype-specific antibody-coated beads to differentiate and detect the presence of complex toxins for botulinum neurotoxin (BoNT)/A, /B, /E, and /F
| Serotype of antibody-coated bead | A 100U | A 10U | A 1U | B 100U | B 10U | B 1U | E 100U | E 10U | E 1U | F 100U | F 10U | F 1U |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | Yes | Yes | Yes | No | No | No | No | No | No | No | No | No |
| B | No | No | No | Yes | Yes | Yes | No | No | No | No | No | No |
| E | No | No | No | No | No | No | Yes | Yes | Yes | No | No | No |
| F | No | No | No | No | No | No | No | No | No | Yes | Yes | Yes |
Inter-serotype specificity was evaluated by testing known botulinum neurotoxin-producing species of Clostridium (toxigenic Clostridium panel) using non-corresponding toxin assays (e.g., type A culture supernatants were tested on the type B, E, and F assays). Results from all the culture supernatants included in the toxigenic Clostridium panel were 100% concordant with the expected results (Table 5).
TABLE 5.
The inclusivity panel determines the ability of the Endopep-MS method to correctly identify the specific subtypes of BoNT
| Species | Strain | Serotype/subtype | Expected results | Results | |||
|---|---|---|---|---|---|---|---|
| Presence BoNT/A | Presence BoNT/B | Presence BoNT/E | Presence BoNT/F | ||||
| C. botulinum | ATCC 3502 | A1 | A | Yes | No | No | No |
| C. botulinum | CDC 2084 | A1(B5) | A | Yes | No | No | No |
| C. botulinum | CDC 2171 | A2 | A | Yes | No | No | No |
| C. botulinum | CDC 297 | A1 (ha−/orf+) | A | Yes | No | No | No |
| C. botulinum | CDC 39277 | A1(B5) | A | Yes | No | No | No |
| C. botulinum | CDC 40234 | A3 | A | Yes | No | No | No |
| C. botulinum | CDC 42961 | A1(B5) | A | Yes | No | No | No |
| C. botulinum | CDC 4997 | A1 | A | Yes | No | No | No |
| C. botulinum | CDC 53119 | A2 | A | Yes | No | No | No |
| C. botulinum | CDC 59755 | A1(B5) | A | Yes | No | No | No |
| C. botulinum | CDC 62A | A1 | A | Yes | No | No | No |
| C. botulinum | CDC 69A | A1 | A | Yes | No | No | No |
| C. botulinum | FRI-H1A2 | A2 | A | Yes | No | No | No |
| C. botulinum | LOCH MAREE | A3 | A | Yes | No | No | No |
| C. botulinum | CDC 1436 | A2b5 | AB | Yes | Yes | No | No |
| C. botulinum | CDC 41370 | A1b5 | AB | Yes | Yes | No | No |
| C. botulinum | CDC 588 | A1b5 | AB | Yes | Yes | No | No |
| C. botulinum | CDC 657 | B5A4 | AB | No | Yes | No | No |
| C. botulinum | ATCC 51387 | B2 | B | No | Yes | No | No |
| C. botulinum | ATCC 7949 | B2 | B | No | Yes | No | No |
| C. botulinum | CDC 1656 | B1 | B | No | Yes | No | No |
| C. botulinum | CDC 6291 | B2 | B | No | Yes | No | No |
| C. botulinum | CDC 795 | B3 | B | No | Yes | No | No |
| C. botulinum | CDC OKRA | B1 | B | No | Yes | No | No |
| C. botulinum | ECKLUND 17B | B4 | B | No | Yes | No | No |
| C. botulinum | ECKLUND 2B | B4 | B | No | Yes | No | No |
| C. botulinum | HALL 10,007 | B1 | B | No | Yes | No | No |
| C. botulinum | KAP 1 | B4 | B | No | Yes | No | No |
| C. botulinum | PREVOT 1662 | B4 | B | No | Yes | No | No |
| C. botulinum | PREVOT 25 NCASE | B2 | B | No | Yes | No | No |
| C. botulinum | CDC 3281 | B5f2 | BF | No | Yes | No | Yes |
| C. botulinum | CDC 4013 | B5f2 | BF | No | Yes | No | Yes |
| C. botulinum | ALASKA E | E3 | E | No | No | Yes | No |
| C. botulinum | ATCC 9564 | E1 | E | No | No | Yes | No |
| C. botulinum | BELUGA | E1 | E | No | No | Yes | No |
| C. botulinum | CDC 5247 | E2 | E | No | No | Yes | No |
| C. butyricum | CDC 5262 | E4 | E | No | No | Yes | No |
| C. botulinum | CDC 5906 | E2 | E | No | No | Yes | No |
| C. botulinum | VH | E3 | E | No | No | Yes | No |
| C. botulinum | 8G | F1 | F | No | No | No | Yes |
| C. baratii | ATCC 43756 | F7 | F | No | No | No | Yes |
| C. botulinum | VPI 2382 | F6 | F | No | No | No | Yes |
| C. botulinum | CDC 54074 | F5 | F | No | No | No | Yes |
| C. botulinum | LANGELAND | F1 | F | No | No | No | Yes |
An exclusivity panel of culture supernatants consisting of 24 strains of non-toxigenic Clostridium spp., 20 enteric bacteria, and 7 strains of BoNT-producing species of Clostridium that produce serotypes C, D, or G was tested by the Endopep-MS assay to determine specificity. All tested culture supernatants gave negative results in 100% concordance with expected results (Table 6). The ability to correctly identify the absence of BoNT in non-BoNT–producing species of Clostridium is imperative for proper laboratory confirmation.
TABLE 6.
The exclusivity panel determines the ability of the Endopep-MS method to correctly identify the absence of BoNT in non-BoNT–producing species of Clostridium
| Genus, species | Expected results | Results | |||
|---|---|---|---|---|---|
| Presence BoNT/A | Presence BoNT/B | Presence BoNT/E | Presence BoNT/F | ||
| C. botulinum, type C | Negative | No | No | No | No |
| C. botulinum, type C | Negative | No | No | No | No |
| C. botulinum, type C | Negative | No | No | No | No |
| C. botulinum, type C | Negative | No | No | No | No |
| C. botulinum, type C | Negative | No | No | No | No |
| C. botulinum, type D | Negative | No | No | No | No |
| C. botulinum group III, non-toxigenic | Negative | No | No | No | No |
| Clostridium argentinense | Negative | No | No | No | No |
| C. histolyticum | Negative | No | No | No | No |
| C. tertium | Negative | No | No | No | No |
| C. absonum | Negative | No | No | No | No |
| C. sporogenes | Negative | No | No | No | No |
| C. sporogenes | Negative | No | No | No | No |
| C. sordellii | Negative | No | No | No | No |
| C. septicum | Negative | No | No | No | No |
| C. tetani | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. butyricum | Negative | No | No | No | No |
| C. hastiforme | Negative | No | No | No | No |
| C. ramosum | Negative | No | No | No | No |
| C. baratii | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. difficile | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. subterminale | Negative | No | No | No | No |
| C. bifermentans | Negative | No | No | No | No |
| C. novyii | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. perfringens | Negative | No | No | No | No |
| C. haemolyticum | Negative | No | No | No | No |
| Serratia marcescens | Negative | No | No | No | No |
| Salmonella Newport | Negative | No | No | No | No |
| Proteus vulgaris | Negative | No | No | No | No |
| Campylobacter jejuni | Negative | No | No | No | No |
| Vibrio parahaemolyticus | Negative | No | No | No | No |
| Escherichia coli (ETEC) | Negative | No | No | No | No |
| Pseudomonas aeruginosa | Negative | No | No | No | No |
| Salmonella Typhimurium | Negative | No | No | No | No |
| Vibrio vulnificus | Negative | No | No | No | No |
| Shigella dysenteriae | Negative | No | No | No | No |
| Yersinia enterocolitica | Negative | No | No | No | No |
| Camplyobacter fetus | Negative | No | No | No | No |
| Vibrio cholerae | Negative | No | No | No | No |
| Aeromonas hydrophila | Negative | No | No | No | No |
| Shigella sonnei | Negative | No | No | No | No |
| Citrobacter freundii | Negative | No | No | No | No |
| Edwardsiella tarda | Negative | No | No | No | No |
| Providencia stuartii | Negative | No | No | No | No |
| Shigella boydii | Negative | No | No | No | No |
| Shigella flexneri | Negative | No | No | No | No |
Selectivity
The toxigenic Clostridium panel and an interfering substances panel determined the accuracy and reproducibility of the BoNT Endopep-MS method. The toxigenic Clostridium panel is representative of a variety of BoNT subtypes obtained from various botulism outbreaks throughout the years. This panel included culture supernatants of 19 subtypes of BoNT isolated from human clinical samples that were readily available out of 38 total subtypes known. All subtypes of BoNT/A, /B, /E, and /F were identified correctly (Table 5). The ability to identify multiple subtypes with the serotype-specific antibodies and substrates is an advantage and is critical for detection for public health purposes. In a botulism outbreak, the subtype of BoNT is not known and could be a subtype which is not commercially available. These data, in addition to data already published with additional subtypes (22–25), provide evidence that our method could be used successfully to detect BoNT in botulism outbreaks for laboratory confirmation.
The interfering substances panel was designed to test exogeneous and endogenous substances that are commonly present in clinical specimens such as serum and stool extract that could affect the possible outcome of results for the Endopep-MS method. The exogeneous and endogenous substances presented no interference in testing spiked serum and stool extract with the BoNT Endopep-MS assay, with the one exception of sodium phosphate (Table 7). Sodium phosphate, at a concentration of 2.5 g/L, when spiked into stool extract, resulted in negative results for samples tested at 5× the LOD in BoNT/A, /B, /E, and /F.
TABLE 7.
Interfering substances with BoNT/A, /B, /E, and /F in serum and stool extract
| Interfering substance serum | Interference with BoNT/A, /B, /E, and /F | Interfering substance stool extract | Interference with BoNT/A, /B, /E, and /F |
|---|---|---|---|
| Hemoglobin | No | Human whole blood | No |
| Bilirubin conjugated | No | Palmitic acid | No |
| Bilirubin unconjugated | No | Stearic acid | No |
| Globulin (40%) | No | Bovine mucin | No |
| Albumin (60%) | No | Human bile | No |
| Human serum albumin | No | Biles acids kit | No |
| IgG | No | Human urine | No |
| Biotin | No | Bacitracin | No |
| Intralipid | No | Nystatin | No |
| Soybean oil | No | Metronidazole | No |
| Procalcitonin | No | Naproxen sodium | No |
| Rheumatoid factor | No | Bisacodyl | No |
| Human genomic DNA | No | Bismuth subsalicylate | No |
| Triglycerides | No | Calcium carbonate | No |
| Cholesterol | No | Docusate sodium | No |
| Rocuronium | No | Glycerin | No |
| Succinylcholine | No | Hydrocortisone | No |
| Gentamicin | No | Loperamide hydrochloride | No |
| Tetracycline | No | Magnesium hydroxide | No |
| Doxycycline | No | Mineral oil | No |
| Ciprofloxacin | No | Phenylephrine hydrochloride | No |
| Azithromycin | No | Sodium phosphate | Yes |
| Imipenem | No | Nonoxynol-9 | No |
| Levofloxacin | No | Triglycerides | No |
| Vancomycin | No | Cholesterol | No |
| Acetaminophen | No | Doxycycline | No |
| Aspirin | No | ||
| Salicylic acid | No | ||
| Ethanol | No | ||
| Caffeine | No | ||
| Celecoxib | No | ||
| Cetirizine HCl | No | ||
| Dextromethorphan | No | ||
| Dobutamine | No | ||
| Heparin | No | ||
| Ibuprofen | No | ||
| Loratadine | No | ||
| Nicotine | No | ||
| Noradrenaline | No | ||
| Oxymetazoline HCl | No | ||
| Phenylephrine | No | ||
| Prednisolone | No | ||
| Salmeterol | No | ||
| Tiotropium | No | ||
| Dopamine | No | ||
| Furosemide | No | ||
| Epinephrine | No |
The list for interfering substances was determined from the Clinical Laboratory Standards Institute EP07-A2 guideline, with additions made by the US Food and Drug Administration and National Center for Emerging and Zoonotic Infectious Diseases epidemiologists (26). The guidance for the panel is to characterize the susceptibility of assay to all possible interferences. Therefore, sodium phosphate was tested as an exogenous substance, because it is present in most enema solutions at ~1 g to receive clinical stool specimens (27, 28). However, patients with suspected botulism only receive sterile non-bacteriostatic enemas for stool collection, per existing physician instruction (29). Although the Endopep-MS method uses sodium phosphate in gelatin phosphate-buffered collecting fluid and 1× PBST, the limits are below the interference threshold at 6 mg/mL, 0.20 mg/mL, and 1.6 mg/mL (data not shown). Therefore, although the large amounts of sodium phosphate had interference with the detection of BoNT, it would not be a substance present in stool specimens collected for botulism testing. Adoption of the Endopep-MS assay as an alternative to the mouse bioassay for diagnosis of human botulism would not change existing physician guidance for collection of stool specimens in the absence of enema solution or collection of specimens prior to antitoxin administration.
Clinical specimen stability study
A necessary factor for full validation of this assay is a determination of the stability of the toxin in different specimen types as it is transported to the testing laboratory. To determine clinical specimen stability, known quantities of the BoNT/A, /B, /E, and /F were spiked into different specimen types and stored until the testing time points.
These specimen stability studies indicate that this assay can accurately detect toxin in various sample matrices well within the estimated shipping periods to the testing laboratory. Due to the nature of the material, stool was only tested to the 10-day testing point and the assay showed similar agreement with expected results. The assay gave expected results for up to 30 days in serum, stool extract, and culture broth after spiking with known concentrations of toxin (Table 8).
TABLE 8.
Sample stability of BoNT/A, /B, /E, and /F in serum, TPGY, CMGS, and stool extract (stored at 2°–8°C) at multiple timepoints
| BoNT serotype | Matrix | Percent of spiked samples that produced expected results | ||||||
|---|---|---|---|---|---|---|---|---|
| Day 0 | Day 3a | Day 7a | Day 10 | Day 14 | Day 21a | Day 30a | ||
| A | Serum | 67 | 100 | 100 | N/A | 100 | 100 | 100 |
| B | Serum | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| E | Serum | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| F | Serum | 100 | No datab | 100 | N/A | 100 | 100 | 100 |
| Negative | Serum | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| A | Stool extract | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| B | Stool extract | 67 | 100 | 100 | N/A | 100 | 100 | 100 |
| E | Stool extract | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| F | Stool extract | 100 | No datab | 100 | N/A | 100 | 100 | 100 |
| Negative | Stool extract | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| A | Stool | 100 | 100 | 100 | 100 | N/A | N/A | N/A |
| B | Stool | 67 | 100 | 100 | 100 | N/A | N/A | N/A |
| E | Stool | 100 | 100 | 100 | 100 | N/A | N/A | N/A |
| F | Stool | 100 | No datab | 100 | 100 | N/A | N/A | N/A |
| Negative | Stool | 100 | 100 | 100 | 100 | N/A | N/A | N/A |
| A | CMGS | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| B | CMGS | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| E | CMGS | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| F | CMGS | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| Negative | CMGS | 100 | 100 | 100 | N/A | 75 | 100 | 100 |
| A | TPGY | 100 | 100 | 100 | N/A | 100 | 100 | 67 |
| B | TPGY | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
| E | TPGY | 100 | 100 | 100 | N/A | 67 | 0 | 0 |
| F | TPGY | 100 | 100 | 100 | N/A | 100 | 33 | 0 |
| Negative | TPGY | 100 | 100 | 100 | N/A | 100 | 100 | 100 |
Deviation: Because of holidays, the data for “Day 3” were tested at day 2 or 3, data for “Day 10” were tested at day 7 or 8, data for “Day 21” were tested at day 21 or day 24, and data for “Day 30” were tested at day 30 or 32.
Deviation: Negative control for BoNT/F failed, and no data could be reported.
The data in Table 8 indicate some normal variation in stability data in all BoNT serotypes due to method and analyst discrepancy. However, the low stability and recovery of BoNT/E and /F at “Day 21” and “Day 30” in TPGY is possibly due to the trypsin in the broth and the lack of protection of the hemagglutinin proteins to protect BoNT/E and /F toxin (30). These data validate the sample matrices, including transportation timeframes, that can be used for laboratory confirmation of botulism.
Instrument bridging study
Initial validation studies for the BoNT Endopep-MS method were conducted on Bruker microflex instruments. Recently, Bruker Daltonics Inc. released a new version, the Bruker Sirius instrument. To determine equivalence between the Bruker microflex and the Bruker Sirius, a bridging study was designed between the two instruments for the BoNT Endopep-MS method. All samples were analyzed in a side-by-side analysis on the Bruker microflex and the Bruker Sirius. The data from these experiments yielded expected outcomes (Table 9). This bridging study has provided sufficient data to determine equivalency between Bruker microflex and the Bruker Sirius, which gives laboratories multiple instrument options.
TABLE 9.
Comparison of the LOD of BoNT/A, /B, /E, and /F in buffer, serum, TPGY, CMGS, and stool extract on the Bruker microflex mass spectrometer and the Bruker Sirius Smartflex mass spectrometer
| BoNT serotype | Matrix | Toxin | Microflex | Sirius Smartflex | ||
|---|---|---|---|---|---|---|
| LOD (mLD50) | LOD (mLD50/mL) | LOD (mLD50) | LOD (mLD50/mL) | |||
| A | Buffer | Dichain | 0.5 | 0.5 | 0.5 | 0.5 |
| B | Buffer | Dichain | 0.5 | 0.5 | 0.5 | 0.5 |
| E | Buffer | Dichain | 0.5 | 0.5 | 0.5 | 0.5 |
| F | Buffer | Dichain | 0.5 | 0.5 | 0.5 | 0.5 |
| A | Buffer | Complex | 0.5 | 0.5 | 0.5 | 0.5 |
| B | Buffer | Complex | 1 | 1 | 1 | 1 |
| E | Buffer | Complex | 1 | 1 | 1 | 1 |
| F | Buffer | Complex | 0.5 | 0.5 | 0.5 | 0.5 |
| A | Serum | Dichain | 1 | 2 | 1 | 2 |
| B | Serum | Dichain | 0.5 | 1 | 0.5 | 1 |
| E | Serum | Dichain | 0.5 | 1 | 0.5 | 1 |
| F | Serum | Dichain | 0.2 | 0.4 | 0.2 | 0.4 |
| A | Stool extract | Complex | 2 | 4 | 2 | 4 |
| B | Stool extract | Complex | 1 | 2 | 1 | 2 |
| E | Stool extract | Complex | 5 | 10 | 5 | 10 |
| F | Stool extract | Complex | 0.5 | 1 | 0.5 | 1 |
| A | TPGY | Dichain | 1 | 2 | 1 | 2 |
| B | TPGY | Dichain | 1 | 2 | 1 | 2 |
| E | TPGY | Dichain | 2 | 4 | 2 | 4 |
| F | TPGY | Dichain | 0.5 | 1 | 0.5 | 1 |
| A | CMGS | Dichain | 2 | 4 | 2 | 4 |
| B | CMGS | Dichain | 1 | 2 | 1 | 2 |
| E | CMGS | Dichain | 2 | 4 | 2 | 4 |
| F | CMGS | Dichain | 0.5 | 1 | 0.5 | 1 |
Reproducibility
Reproducibility of the method was determined as part of a multicenter evaluation study and was assessed with a panel of spiked serum samples (reproducibility panel). The panel consisted of five serum samples, blind to the operator, containing either no toxin or dichain toxin spiked into serum at five times the limit of detection. Each panel was analyzed for BoNT/A, /B, /E, and /F by two independent operators each in five separate sites (inter-rater reliability), for a total of 10 panels run at each site. One panel was run per day on five separate days per operator (intra-rater reliability). In total, 1,000 tests were performed to assess reproducibility. Table 10 shows the results of the reproducibility study. The correct answer was determined in 99.9% of the runs, demonstrating excellent reproducibility. Some of the runs were deemed invalid due to failure of positive controls, and only one run incorrectly assigned a positive for BoNT/F in a negative sample.
TABLE 10.
Comparison reproducibility data of BoNT/A, /B, /E, and /F at multiple testing sites
| Sample | Number of analytical runs correctly identified | ||||
|---|---|---|---|---|---|
| Site 1 | Site 2 | Site 3 | Site 4 | Site 5 | |
| R1 | 40/40 | 40/40 | 40/40 | 40/40 | 36/36a |
| R2 | 40/40 | 40/40 | 40/40 | 40/40 | 32/32a |
| R3 | 40/40 | 40/40 | 40/40 | 40/40 | 36/36a |
| R4 | 40/40 | 40/40 | 40/40 | 40/40 | 28/28a |
| R5 | 39/40 | 40/40 | 40/40 | 40/40 | 40/40 |
One or more of the runs were invalidated due to positive control failures, thus the number of samples is less than 40.
Clinical validation
A final validation of the method was performed as part of a multicenter evaluation study to assess the ability of multiple operators to correctly identify spiked clinical specimens (clinical panel). The clinical panel consisted of 10 serum, 10 stool extract, 10 CMGS, and 10 TPGY samples, for a total of 40 samples per panel, to which the operator was blind. The samples contained either no toxin, dichain toxin spiked into serum at five times the limit of detection, or complex toxin spiked into stool extract, CMGS, or TPGY at five times the limit of detection. Each panel was analyzed for BoNT/A, /B, /E, and /F at the same five sites used for the reproducibility study described above. In total, 800 tests were performed to assess clinical validation. Table 11 shows the results of the clinical validation study. The correct answer was determined in 99.4% of the runs, demonstrating excellent ability to accurately detect BoNT/A, /B, /E, and /F in spiked clinical specimens. Some of the runs were deemed invalid due to failure of positive controls, and five runs were incorrectly assigned with false positives and negatives.
TABLE 11.
Comparison of the clinical validation data analyzing BoNT/A, /B, /E, and /F at multiple testing sites
| Sample ID | Expected results | Number of samples correctly identified | Total samples correctly identified (%) | ||||
|---|---|---|---|---|---|---|---|
| Site 1 | Site 2 | Site 3 | Site 4 | Site 5 | |||
| 1 | E | E | E | E | E | E | 100 |
| 2 | A | A | A | A | A | A | 100 |
| 3 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 4 | E | E | E | E | E | E | 100 |
| 5 | F | F | F | F | F | F | 100 |
| 6 | B | B | B | B | B | B | 100 |
| 7 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 8 | E | E | E | E | E | E | 100 |
| 9 | A | A | A | A | A | A | 100 |
| 10 | B | B | B | B | B | B | 100 |
| 11 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 12 | F | F | B, F | F | F | F | 95 |
| 13 | E | E | E | E | E | E | 100 |
| 14 | B | B | B | B | B | B | 100 |
| 15 | E | E | E | E | E | E | 100 |
| 16 | A | A | A | A | A | A | 100 |
| 17 | F | F | F | F | F | F | 100 |
| 18 | B | B | B | B | B | B | 100 |
| 19 | A | A | A | A | A | A | 100 |
| 20 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 21 | E | E | E | E | E | E | 100 |
| 22 | A | A | A | A | A | A | 100 |
| 23 | B | B | B, F | B | B | B | 95 |
| 24 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 25 | F | F | A, F | F | F | F | 95 |
| 26 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 27 | A | A | A | A | A | A | 100 |
| 28 | E | E | E | E | E | E | 100 |
| 29 | F | F | F | F | F | Negative | 95 |
| 30 | B | B | B | B | B | B | 100 |
| 31 | A | A | Negative | A | A | A | 95 |
| 32 | F | F | F | F | F | F | 100 |
| 33 | E | E | E | E | E | E | 100 |
| 34 | B | B | B | B | B | B | 100 |
| 35 | E | E | E | E | E | E | 100 |
| 36 | A | A | A | A | A | A | 100 |
| 37 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 38 | B | B | B | B | B | B | 100 |
| 39 | Negative | Negative | Negative | Negative | Negative | Negative | 100 |
| 40 | F | F | F | F | F | F | 100 |
Conclusion
The BoNT Endopep-MS validation, assessing sensitivity, robustness, selectivity, specificity, and reproducibility, demonstrates that this method is a promising technique for public health laboratories for laboratory confirmation of botulism. Public health laboratories will benefit from the Endopep-MS method, which allows for a larger throughput of samples in a shorter period compared with the gold-standard mouse bioassay as the mouse bioassay requires up to 4 days for final results of 10 samples, whereas the Endopep-MS method can be completed in an 8-hour work day. The studies presented in this work demonstrate the ability of the Endopep-MS method to accurately detect BoNTs in human clinical specimens. The use of the BoNT Endopep-MS assay by CDC and other public health laboratories will improve the capacity to respond to botulism outbreaks in the USA, by providing accurate and timely laboratory test results.
ACKNOWLEDGMENTS
We would like to acknowledge several CDC colleagues who have worked with us on this project over the course of the studies. They include Julie Villanueva, Tricia Aden, Shannon York, Stacy Rager, Stephanie Marcum, Tricia Blevins, Laura Walls, and Rachael Cook. In addition, we acknowledge other partners that assisted with this project, including Association of Public Health Laboratories, Virginia Division of Consolidated Laboratories, Texas Department of State Health Services, Maryland Department of Health, Utah Public Health Laboratory, and Washington State Public Health Laboratory.
This project was supported by the Office of Readiness and Response, Centers for Disease Control and Prevention.
The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the US Department of Health and Human Services, or the US Centers for Disease Control and Prevention. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the US Department of Health and Human Services or the US Centers for Disease Control and Prevention.
Contributor Information
Carolina Lúquez, Email: cluquez@cdc.gov.
Suzanne R. Kalb, Email: skalb@cdc.gov.
Erin McElvania, NorthShore University HealthSystem Department of Pathology and Laboratory Medicine, Evanston, Illinois, USA.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/jcm.01629-23.
Tables S1 and S2.
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
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Associated Data
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Supplementary Materials
Tables S1 and S2.