toxins Article Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine Candace S. Bever 1 , Kenneth D. Swanson 2, Elizabeth I. Hamelin 2 , Michael Filigenzi 3 , Robert H. Poppenga 3, Jennifer Kaae 4, Luisa W. Cheng 1,* and Larry H. Stanker 1 1 Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA; [email protected] (C.S.B.); [email protected] (L.H.S.) 2 Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA; [email protected] (K.D.S.); [email protected] (E.I.H.) 3 California Animal Health and Food Safety Laboratory System, University of California, 620 West Health Sciences Drive, Davis, CA 95616, USA; msfi[email protected] (M.F.); [email protected] (R.H.P.) 4 Pet Emergency and Specialty Center of Marin, 901 E. Francisco Blvd, San Rafael, CA 94901, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-510-559-6337 Received: 30 January 2020; Accepted: 12 February 2020; Published: 15 February 2020 Abstract: 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 β-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. Keywords: lateral flow immunoassay; amatoxins; amanitins; point-of-care; mushroom poisoning Key Contribution: The study demonstrates the utility of the lateral flow immunoassay for the rapid detection of amatoxins in urine. 1. Introduction Distinguishing toxic mushrooms from non-toxic ones is highly challenging, even for expert mycologists. Techniques to properly identify a mushroom include detailed morphological examination of the mushroom body, substrate identification, and knowledge of the location and the season. The toxins often associated with lethal cases are cyclopeptides, and more specifically amanitins (most commonly α-amanitin (α-AMA), β-AMA, and γ-AMA, collectively referred to as amatoxins) [1] (Figure1). Amatoxins are found in a few species of mushrooms from di fferent genera, including Amanita, Galerina, and Lepiota [2]. Amatoxins are highly resistant to degradation, and on the cellular Toxins 2020, 12, 123; doi:10.3390/toxins12020123 www.mdpi.com/journal/toxins Toxins 2020, 12, 123 2 of 10 Toxins 2020, 12, x FOR PEER REVIEW 2 of 9 level they inhibit transcription by binding to RNA polymerase II. As little as 0.1 mg/kg body weight of body weight of amatoxins may cause death [3,4], and this amount can be found in a single Amanita amatoxins may cause death [3,4], and this amount can be found in a single Amanita phalloides. phalloides. Compound R1 R2 R3 α-amanitin NH2 OH OH β-amanitin OH OH OH γ-amanitin NH2 OH H (a) (b) Figure 1. Chemical structures of the amatoxin variants examined in this paper, (a) molecular structure Figureof the 1. amanitin, Chemical (b structures) R-group of designations the amatoxin for variants each variant. examined in this paper, (a) molecular structure of the amanitin, (b) R-group designations for each variant. Consumption of toxin-containing mushrooms can result in a range of symptoms, from mild to life-threateningConsumption of [5 toxin-containing,6]. The presumptive mushrooms diagnosis can forresult amatoxin in a range poisoning of symptoms, is based from on amild history to life-threateningof consuming wild[5,6]. mushroomsThe presumptive (if known), diagnosis presentation for amatoxin of delayed poisoning gastroenteritis, is based on elevateda history liver of consumingenzyme levels, wild and mushrooms ruling out other(if known), gastrointestinal presentati diseaseson of ordelayed conditions gastroenteritis, [6]. To distinguish elevated amatoxin liver enzymepoisonings, levels, the and presence ruling of anout amatoxin other gastrointestin in an intoxicatedal diseases patient’s or urineconditions would [6]. provide To distinguish a definitive amatoxindiagnosis. poisonings, For dogs, obtainingthe presence a history of an amatoxin of mushroom in an ingestion intoxicated is rare, patient’s making urine diagnosis would provide even more a definitivechallenging. diagnosis. There areFor only dogs, a fewobtaining laboratories a history capable of mushroom of testing ingestion biological is specimensrare, making for diagnosis amatoxins evento confirm more challenging. human or animal There exposures,are only a few and labora even whentories available,capable of test testing results biological might not specimens be available for amatoxinssoon enough to confirm to help guidehuman treatment. or animal Although exposures, there and are even no when FDA-approved available, antidotes,test results early might diagnosis, not be availableaggressive soon immediate enough to supportive help guide care,treatment. and a Al rangethough of potentialthere are no therapies FDA-approved can potentially antidotes, improve early diagnosis,patient outcomes aggressive [6– 11immediate]. supportive care, and a range of potential therapies can potentially improveFor patient both humans outcomes and [6–11]. dogs, the first symptoms of amatoxin poisonings usually appear 6–24 h afterFor ingestion both humans of an and amatoxin-containing dogs, the first symptoms mushroom of amatoxin [6]. By thispoisonings time, amatoxins usually appear have already6-24 h afterbegun ingestion damaging of an the amatoxin-containing liver and kidneys. Basedmushroom on toxicological [6]. By this time, studies, amatoxins amatoxins have disappear already begun rapidly damagingfrom the serum,the liver but and are kidneys. detectable Based in urineon toxicolo up togical 4 days studies, after amatoxins ingestion [12disappear–14]. In rapidly human from urine, thetoxin serum, concentrations but are detectable decreased in over urine time, up andto 4 thedays highest after ingestion concentrations [12–14]. observed In human were urine, 4820 ngtoxin/mL concentrationsfor α-AMA and decreased 7103 ng/ mLover for time,β-AMA and the [12 highest]. Because concentrations of the relative observed ease of obtainingwere 4820 a ng/mL urine sample, for α- AMAand theand longer 7103 ng/mL duration for ofβ-AMA detectability [12]. Because of amatoxins of the relative in urine ease compared of obtaining to serum, a urine urine sample, seems and an theobvious longer sample duration matrix of detectability for performing of amatoxins rapid amatoxin in urine analysis. compared Sensitive, to serum, rapid, urine and seems easy-to-perform an obvious samplemethods matrix are needed for performing to detect amatoxins rapid amatoxin for the early analysis. diagnosis Sensitive, of toxin rapid, poisoning and [easy-to-perform9,15,16]. methodsCurrent are needed methods to ofdetect chemical amatoxins detection for ofthe amatoxins early diagnosis in urine of include toxin poisoning liquid chromatography–mass [9,15,16]. spectrometryCurrent methods (LC-MS) of methods chemical [ 17detection–23] and of antibody-based amatoxins in urine enzyme-linked include liquid immunosorbent chromatography– assays mass(ELISAs) spectrometry [24,25]. LC-MS (LC-MS) methods methods require [17–23] sample and extractionantibody-based and expensive enzyme-linked equipment, immunosorbent while ELISA assaysmethods (ELISAs) require [24,25]. specialized LC-MS equipment. methods Methodsrequire sa ofmple both extraction types typically and expensive take a few equipment, hours to complete. while ELISALateral methods flow immunoassay require specialized (LFIA) formatsequipment. utilize Methods some of of the both reagents types typically used for ELISA,take a few but hours the entire to complete.test can be Lateral completed flow immunoassay in minutes and (LFIA) requires formats no specialized utilize some equipment. of the reagents used for ELISA, but the entireWe havetest can recently be completed developed in minu an LFIAtes and for requires the detection no specialized of mushroom equipment. amatoxins [26]. As the sensitivityWe have of therecently LFIA developed allows it to an detect LFIA as for little the as de 10tection ng/mL, of we mushroom hypothesized amatoxins that this [26]. test As would the sensitivitybe useful of for the urine LFIA analysis allows init instancesto detect as of little mushroom as 10 ng/mL, poisonings. we hypothesized To test this that hypothesis, this test wewould first beconducted useful for analysis urine analysis of urine in samples instances that of weremushro