Surveillance for Harmful Algal Bloom Events and Associated Human and Animal Illnesses - One Health Harmful Algal Bloom System, United States, 2016-2018.

Harmful algal bloom events can result from the rapid growth, or bloom, of photosynthesizing organisms in natural bodies of fresh, brackish, and salt water. These events can be exacerbated by nutrient pollution (e.g., phosphorus) and warming waters and other climate change effects (1); have a negative impact on the health of humans, animals, and the environment; and damage local economies (2,3). U.S. harmful algal bloom events of public health concern are centered on a subset of phytoplankton: diatoms, dinoflagellates, and cyanobacteria (also called blue-green algae). CDC launched the One Health Harmful Algal Bloom System (OHHABS) in 2016 to inform efforts to prevent human and animal illnesses associated with harmful algal bloom events. A total of 18 states reported 421 harmful algal bloom events, 389 cases of human illness, and 413 cases of animal illness that occurred during 2016-2018. The majority of harmful algal bloom events occurred during May-October (413; 98%) and in freshwater bodies (377; 90%). Human and animal illnesses primarily occurred during June-September (378; 98%) and May-September (410; 100%). Gastrointestinal or generalized illness signs or symptoms were the most frequently reported (>40% of human cases and >50% of animal cases); however, multiple other signs and symptoms were reported. Surveillance data from harmful algal bloom events, exposures, and health effects provide a systematic description of these occurrences and can be used to inform control and prevention of harmful algal bloom-associated illnesses.

Quantification of Microcystin-LR in Human Urine by Immunocapture Liquid Chromatography Tandem Mass Spectrometry.

Microcystins are toxins produced by many cyanobacteria species, which are often released into waterways during blue-green algal blooms in freshwater and marine habitats. The consumption of microcystin-contaminated water is a public health concern as these toxins are recognized tumor promoters and are hepatotoxic to humans and animals. A method to confirm human exposures to microcystins is needed; therefore, our laboratory has developed an immunocapture liquid chromatography tandem mass spectrometry (LC-MS/MS) method targeting the conserved adda portion of microcystins for the quantitation of a prevalent and highly toxic congener of microcystin, microcystin-LR (MC-LR). An acute exposure method was initially evaluated for accuracy and precision by analyzing calibrators and quality control (QC) samples ranging from 0.500 to 75.0 ng/mL in urine. All calibrators and QC samples characterized were within 15% of theoretical concentrations. An analysis of acutely exposed mouse urine samples using this method identified MC-LR levels from 10.7 to 33.9 ng/mL. Since human exposures are anticipated to result from low-dose or chronic exposures, a high-sensitivity method was validated with 20 calibration curves and QC samples ranging from 0.0100 to 7.50 ng/mL. Relative standard deviations (RSDs) and inaccuracies of these samples were within 15%, meeting United States Food and Drug Administration (FDA) guidelines for analytical methods, and the limit of detection was 0.00455 ng/mL. In conclusion, we have developed a method which can be used to address public health concerns by precisely and accurately measuring MC-LR in urine samples.

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry.

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

Detection of Anatoxins in Human Urine by Liquid Chromatography Triple Quadrupole Mass Spectrometry and ELISA.

Harmful cyanobacterial blooms are becoming more common and persistent around the world. When in bloom, various cyanobacterial strains can produce anatoxins in high concentrations, which, unlike other cyanobacterial toxins, may be present in clear water. Potential human and animal exposures to anatoxins occur mainly through unintentional ingestion of contaminated algal mats and water. To address this public health threat, we developed and validated an LC-MS/MS method to detect anatoxins in human urine to confirm exposures. Pooled urine was fortified with anatoxin-a and dihydroanatoxin at concentrations from 10.0 to 500 ng/mL to create calibrators and quality control samples. Samples were diluted with isotopically labeled anatoxin and solvent prior to LC-MS/MS analysis. This method can accurately quantitate anatoxin-a with inter- and intraday accuracies ranging from 98.5 to 103% and relative standard deviations < 15%, which is within analytical guidelines for mass spectrometry methods. Additionally, this method qualitatively detects a common degradation product of anatoxin, dihydroanatoxin, above 10 ng/mL. We also evaluated a commercial anatoxin-a ELISA kit for potential diagnostic use; however, numerous false positives were detected from unexposed individual human urine samples. In conclusion, we have developed a method to detect anatoxins precisely and accurately in urine samples, addressing a public health area of concern, which can be applied to future exposure events.

Comparison of high-resolution and tandem mass spectrometry for the analysis of nerve agent metabolites in urine.

RATIONALE: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.

Detection of Brevetoxin in Human Plasma by ELISA.

Florida red tides have become more common and persistent in and around the Gulf of Mexico. When in bloom, red tides can produce brevetoxins in high concentrations, leading to human exposures primarily through contaminated food and ocean spray. The research described here includes adapting and validating a commercial brevetoxin water test kit for human plasma testing. Pooled plasma was fortified with a model brevetoxin, brevetoxin 3, at concentrations from 0.00500 to 3.00 ng/mL to generate calibration curves and quality control samples. The quantitative detection range was determined to be 0.0400-2.00 ng/mL brevetoxin 3 equivalents with inter- and intraday accuracies ranging from 94.0% to 109% and relative standard deviations <20%, which is within the US Food and Drug Administration guidelines for receptor-binding assays. Additionally, cross-reactivity was tested using 4 of the 10 known brevetoxins and 12 paralytic shellfish toxins. The cross-reactivity varied from 0.173% to 144% for the commercially available brevetoxin standards and 0% for the commercially available paralytic shellfish toxin standards. Fifty individual unexposed human plasma samples were measured to determine the limit of detection and endogenous interferences to the test. The validated method was used to test 31 plasma samples collected from humans potentially exposed to brevetoxins, detecting 11 positives. This method has been proven useful to measure human exposure to brevetoxins and can be applied to future exposure events.

A cluster of tetrodotoxin poisoning in Oman.

INTRODUCTION: Tetrodotoxin (TTX) is a potent sodium channel blocker, with significant neurotoxicity, found in marine animals like pufferfish and blue-ringed octopus. The severity of toxicity depends on the amount of toxin ingested and the outcome depends on the time-lapse to appropriate medical care. CASES REPORT: We report five patients who presented with tetrodotoxin poisoning after consuming fried internal organs of local pufferfish from the coast of Oman. The patients' clinical manifestations were consistent with the expected TTX toxidrome of perioral and generalized paresthesia, weakness of upper and lower extremities, gastrointestinal manifestations, dyspnea, dysarthria, ascending paralysis, hypotension, bradycardia and coma. The severity varied among the patients who recovered completely except one patient who developed a subarachnoid hemorrhage without underlying aneurysms on computed tomography-angiogram. This complication was potentially related to TTX poisoning and has not been previously reported. In addition to standard supportive management, patients with severe illness should potentially receive the intravenous acetylcholinesterase inhibitor neostigmine, and intermittent dialysis. Urine specimens were sent to CDC in Atlanta, where they were analyzed using online solid phase extraction (SPE) with LC-MS/MS and confirmed the diagnosis in all five cases. DISCUSSION: In general, the patients' clinical manifestations were consistent with the expected TTX toxidrome except patient 3 who developed a subarachnoid hemorrhage early during his clinical course. Two patients received neostigmine and underwent dialysis with complete recovery.

Measurement of Microcystin Activity in Human Plasma Using Immunocapture and Protein Phosphatase Inhibition Assay.

Microcystins are toxic chemicals generated by certain freshwater cyanobacteria. These chemicals can accumulate to dangerous levels during harmful algal blooms. When exposed to microcystins, humans are at risk of hepatic injury, including liver failure. Here, we describe a method to detect microcystins in human plasma by using immunocapture followed by a protein phosphatase inhibition assay. At least 279 microcystins have been identified, and most of these compounds share a common amino acid, the Adda side chain. We targeted this Adda side chain using a commercial antibody and extracted microcystins from human samples for screening and analysis. To quantitate the extracted microcystins, we fortified plasma with microcystin-LR, one of the most well-studied, commonly detected, and toxic microcystin congeners. The quantitation range for the detection of microcystin in human plasma using this method is 0.030-0.50 ng/mL microcystin-LR equivalents. This method detects unconjugated and conjugated forms (cysteine and glutathione) of microcystins. Quality control sample accuracies varied between 98.9% and 114%, with a precision of 7.18-15.8%. Finally, we evaluated plasma samples from a community health surveillance project of Florida residents living or working near harmful algae blooms.

Use of Diagnostic Ions for the Detection of Fentanyl Analogs in Human Matrices by LC-QTOF.

To combat the ongoing opioid epidemic, our laboratory has developed and evaluated an approach to detect fentanyl analogs in urine and plasma by screening LC-QTOF MS/MS spectra for ions that are diagnostic of the core fentanyl structure. MS/MS data from a training set of 142 fentanyl analogs were used to select the four product ions and six neutral losses that together provided the most complete coverage (97.2%) of the training set compounds. Furthermore, using the diagnostic ion screen against a set of 49 fentanyl analogs not in the training set resulted in 95.9% coverage of those compounds. With this approach, lower reportable limits for fentanyl and a subset of fentanyl-related compounds range from 0.25 to 2.5 ng/mL in urine and 0.5 to 5.0 ng/mL in plasma. This innovative processing method was applied to evaluate simulated exposure samples of remifentanil and carfentanil in water and their metabolites remifentanil acid and norcarfentanil in urine. This flexible approach enables a way to detect emerging fentanyl analogs in clinical samples.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of ß-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography-mass spectrometry (LC-MS) methods.

Application of the fentanyl analog screening kit toward the identification of emerging synthetic opioids in human plasma and urine by LC-QTOF.

Human exposures to fentanyl analogs, which significantly contribute to the ongoing U.S. opioid overdose epidemic, can be confirmed through the analysis of clinical samples. Our laboratory has developed and evaluated a qualitative approach coupling liquid chromatography and quadrupole time-of-flight mass spectrometry (LC-QTOF) to address novel fentanyl analogs and related compounds using untargeted, data-dependent acquisition. Compound identification was accomplished by searching against a locally-established mass spectral library of 174 fentanyl analogs and metabolites. Currently, our library can identify 150 fentanyl-related compounds from the Fentanyl Analog Screening (FAS) Kit), plus an additional 25 fentanyl-related compounds from individual purchases. Plasma and urine samples fortified with fentanyl-related compounds were assessed to confirm the capabilities and intended use of this LC-QTOF method. For fentanyl, 8 fentanyl-related compounds and naloxone, lower reportable limits (LRL100), defined as the lowest concentration with 100 % true positive rate (n = 12) within clinical samples, were evaluated and range from 0.5 ng/mL to 5.0 ng/mL for urine and 0.25 ng/mL to 2.5 ng/mL in plasma. The application of this high resolution mass spectrometry (HRMS) method enables the real-time detection of known and emerging synthetic opioids present in clinical samples.

Measurement of Microcystin and Nodularin Activity in Human Urine by Immunocapture-Protein Phosphatase 2A Assay.

Microcystins (MC) and nodularin (NOD) are toxins released by cyanobacteria during harmful algal blooms. They are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A) and cause a variety of adverse symptoms in humans and animals if ingested. More than 250 chemically diverse congeners of MCs have been identified, but certified reference materials are only available for a few. A diagnostic test that does not require each reference material for detection is necessary to identify human exposures. To address this need, our lab has developed a method that uses an antibody to specifically isolate MCs and NOD from urine prior to detection via a commercially available PP2A kit. This assay quantitates the summed inhibitory activity of nearly all MCs and NOD on PP2A relative to a common MC congener, microcystin-LR (MC-LR). The quantitation range for MC-LR using this method is from 0.050-0.500 ng/mL. No background responses were detected in a convenience set of 50 individual urines. Interday and intraday % accuracies ranged from 94%-118% and relative standard deviations were 15% or less, meeting FDA guidelines for receptor binding assays. The assay detected low levels of MCs in urines from three individuals living in close proximity to harmful algal blooms (HABs) in Florida.

Determination of Fentanyl Analog Exposure Using Dried Blood Spots with LC-MS-MS.

Fentanyl, and the numerous drugs derived from it, are contributing to the opioid overdose epidemic currently underway in the USA. To identify human exposure to these growing public health threats, an LC-MS-MS method for 5 µL dried blood spots (DBS) was developed. This method was developed to detect exposure to 3-methylfentanyl, alfentanil, α-methylfentanyl, carfentanil, fentanyl, lofentanil, sufentanil, norcarfentanil, norfentanyl, norlofentanil, norsufentanil, and using a separate LC-MS-MS injection, cyclopropylfentanyl, acrylfentanyl, 2-furanylfentanyl, isobutyrylfentanyl, ocfentanil and methoxyacetylfentanyl. Preparation of materials into groups of compounds was used to accommodate an ever increasing need to incorporate newly identified fentanyls. This protocol was validated within a linear range of 1.00-100 ng/mL, with precision ≤12% CV and accuracy ≥93%, as reported for the pooled blood QC samples, and limits of detection as low as 0.10 ng/mL. The use of DBS to assess fentanyl analog exposures can facilitate rapid sample collection, transport, and preparation for analysis that could enhance surveillance and response efforts in the ongoing opioid overdose epidemic.

Designing traceable opioid material§ kits to improve laboratory testing during the U.S. opioid overdose crisis.

In 2017, the U.S. Department of Health and Human Services and the White House declared a public health emergency to address the opioid crisis (Hargan, 2017). On average, 192 Americans died from drug overdoses each day in 2017; 130 (67%) of those died specifically because of opioids (Scholl et al., 2019). Since 2013, there have been significant increases in overdose deaths involving synthetic opioids - particularly those involving illicitly-manufactured fentanyl. The U.S. Drug Enforcement Administration (DEA) estimates that 75% of all opioid identifications are illicit fentanyls (DEA, 2018b). Laboratories are routinely asked to confirm which fentanyl or other opioids are involved in an overdose or encountered by first responders. It is critical to identify and classify the types of drugs involved in an overdose, how often they are involved, and how that involvement may change over time. Health care providers, public health professionals, and law enforcement officers need to know which opioids are in use to treat, monitor, and investigate fatal and non-fatal overdoses. By knowing which drugs are present, appropriate prevention and response activities can be implemented. Laboratory testing is available for clinically used and widely recognized opioids. However, there has been a rapid expansion in new illicit opioids, particularly fentanyl analogs that may not be addressed by current laboratory capabilities. In order to test for these new opioids, laboratories require reference standards for the large number of possible fentanyls. To address this need, the Centers for Disease Control and Prevention (CDC) developed the Traceable Opioid Material§ Kits product line, which provides over 150 opioid reference standards, including over 100 fentanyl analogs. These kits were designed to dramatically increase laboratory capability to confirm which opioids are on the streets and causing deaths. The kits are free to U.S based laboratories in the public, private, clinical, law enforcement, research, and public health domains.

Detection of α-, ß-, and γ-amanitin in urine by LC-MS/MS using 15N10-α-amanitin as the internal standard.

The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect α-, ß-, and γ-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N10-α-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N10-α-amanitin internal standard, precision and accuracy of α-amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1-200 ng/mL. ß- and γ-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0-200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled α-amanitin with the ability to detect and confirm human exposures to α-, ß-, and γ-amanitin.

Quantitation of saxitoxin in human urine using immunocapture extraction and LC-MS.

AIM: An immunomagnetic capture protocol for use with LC-MS was developed for the quantitation of saxitoxin (STX) in human urine. MATERIALS & METHODS: This method uses monoclonal antibodies coupled to magnetic beads. STX was certified reference material grade from National Research Council, Canada. Analysis was carried out using LC-MS. RESULTS: With an extraction efficiency of 80%, accuracy and precision of 93.0-100.2% and 5.3-12.6%, respectively, and a dynamic range of 1.00-100 ng/ml, the method is well suited to quantify STX exposures based on previously reported cases. CONCLUSION: Compared with our previously published protocols, this method has improved selectivity, a fivefold increase in sensitivity and uses only a third of the sample volume. This method can diagnose future toxin exposures and may complement the shellfish monitoring programs worldwide.

Investigation of dried blood sampling with liquid chromatography tandem mass spectrometry to confirm human exposure to nerve agents.

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL-1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events.

Enhancing the response of alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry by post-column addition of organic solvents.

A method to enhance the signal intensity and signal-to-noise of several alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry (ESI LC-MS/MS) is presented. This class of compound represents the initial metabolites and environmental degradants of the nerve agents: VX, rVX (Russian VX), GB (Sarin), GF (Cyclosarin), and GD (Soman). Compared with the post-column addition of the mobile phase, the post-column addition of aprotic solvents and longer chain alcohols enhance the signal intensity and signal-to-noise ratio (S/N) of the chromatographic peaks by factors of up to 60 and 19, respectively. The post-column addition of water, methanol, and ethanol resulted in little or no relative signal enhancement. It is proposed that the post-column addition of these solvents do not result in the same enhancements due to stabilization of analyte solvation through hydrogen bonding.

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry.

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.

Quantitation of Fentanyl Analogs in Dried Blood Spots by Flow-Through Desorption Coupled to Online Solid Phase Extraction Tandem Mass Spectrometry.

An automated dried blood spot (DBS) elution coupled with solid phase extraction and tandem mass spectrometric analysis for multiple fentanyl analogs was developed and assessed. This method confirms human exposures to fentanyl, sufentanil, carfentanil, alfentanil, lofentanil, α-methyl fentanyl, and 3-methyl fentanyl in blood with minimal sample volume and reduced shipping and storage costs. Seven fentanyl analogs were detected and quantitated from DBS made from venous blood. The calibration curve in matrix was linear in the concentration range of 1.0 ng/mL to 100 ng/mL with a correlation coefficient greater than 0.98 for all compounds. The limit of detection varied from 0.15 ng/mL to 0.66 ng/mL depending on target analyte. Analysis of the entire DBS minimized the effects of hematocrit on quantitation. All quality control materials evaluated resulted in <15% error; analytes with isotopically labeled internal standards had <15% RSD, while analytes without matching standards had 15-24% RSD. This method provides an automated means to detect seven fentanyl analogs, and quantitate four fentanyl analogs with the benefits of DBS at levels anticipated from an overdose of these potent opioids.