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. 2019 Nov 26;36(3):239–248. doi: 10.1007/s43188-019-00024-2

Phylogenetic evidence revealed Cantharocybe virosa (Agaricales, Hygrophoraceae) as a new clinical record for gastrointestinal mushroom poisoning in Thailand

Sittiporn Parnmen 1,✉, Nattakarn Nooron 1, Siriwan Leudang 1, Sujitra Sikaphan 1, Dutsadee Polputpisatkul 1, Achariya Rangsiruji 2
PMCID: PMC7351933  PMID: 32685428

Abstract

Epidemiological data showed increasing incidence rates of gastrointestinal (GI) mushroom syndrome in Thailand. This study therefore, aimed to identify suspected GI toxin-containing mushrooms using DNA sequence analyses of the internal transcribed spacer (ITS) region and the large subunit (LSU) of nuclear ribosomal DNA. GI toxins were also identified using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). 39 patients presented with poisoning symptoms, including nausea, vomiting, fatigue, abdominal pain, circulatory disturbances and diarrhea after ingesting wild mushrooms. The latent periods varied from 30 min to 4 h, but mostly between 1 and 2 h. Results of the ITS sequence-based identification revealed high similarities for the obtained clinical mushroom samples with the genus Cantharocybe H.E. Bigelow & A.H. SM. Maximum likelihood and Bayesian summary trees of combined ITS and LSU data confirmed that these toxic mushroom samples ingested by the patients belonged to Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar. Detection of GI toxins using LC-QTOF-MS method revealed the presence of coprine in C. virosa. This study described the first outbreak of C. virosa poisoning in Thailand which resulted in severe cases of gastrointestinal irritation. To prevent such poisoning cases it is essential to educate the public not to gather any unidentified or unfamiliar wild mushrooms.

Keywords: Cantharocybe virosa, Coprine, GI toxin-containing mushroom, ITS, LSU, LC-QTOF-MS

Introduction

Wild mushroom food poisoning cases of more than 1300 exposures were reported to the Department of Disease Control, Ministry of Public Health, Thailand, during January to December 2018 (http://www.boe.moph.go.th/boedb/surdata/index.php). The peak time of mushroom poisoning was in the rainy season, extending from May to August. Based on epidemiological data incidence rates of mushroom poisoning were higher in the north and northeast regions of the country.

Approximately 1978 species have been recorded in a checklist of Thai mushrooms (Basidiomycetes). Of these however, the number of poisonous mushrooms was uncertain [1]. A few mushroom samples obtained from clinical poisoning cases were identified as Amanita brunneitoxicaria Thongbai, Raspé & K.D. Hyde, A. digitosa Boonprat. & Parnmen, A. exitialis Zhu L. Yang & T.H. Li, A. fuliginea Hongo, A. pyriformis Boonprat. & Parnmen, A. virosa (Fr.) Bertillon, Chlorophyllum molybdites (G. Mey.) Massee and C. globosum (Mossebo) Vellinga [2–6]. Other cases of poisoning were suspected to be caused by mushrooms in the genus Cantharocybe H.E. Bigelow & A.H. SM. which has not been recorded in the checklist. Within this genus, three known species, namely C. brunneovelutina Lodge, Ovrebo & Aime, C. gruberi (A.H. Sm.) H.E. Bigelow & A.H. Sm. and C. virosa (Manim. & K.B. Vrinda) T.K.A. Kumar have been reported worldwide [7–9].

Mushroom poisoning has been classified based on types of syndrome and primary organ systems affected. It consists of six types of clinical manifestations, including cytotoxic mushroom poisoning, neurotoxic mushroom poisoning, myotoxic mushroom poisoning, metabolic/endocrine toxicity mushroom poisoning, gastrointestinal irritant mushroom poisoning and miscellaneous adverse reactions to mushrooms [10]. Our recent studies revealed that gastrointestinal (GI) toxin-containing mushrooms were the main cause of poisoning cases in Thailand [4]. Ingestion of these toxic mushrooms results in GI illness including nausea, vomiting, abdominal cramping and diarrhea. These GI symptoms however, appear as the first phase in all types of mushroom poisoning [11, 12]. This leads to difficulties in accurate identification of mushroom poisoning types.

Hence, the present study focused on detection of GI toxin-containing mushroom samples using two molecular markers, namely the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) ribosomal DNA. Several studies revealed that these species-specific markers were useful for mushroom identification [4, 13, 14]. Furthermore, GI toxins were determined using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

Materials and methods

Mushroom samples and DNA extraction

Specimens of unconsumed mushroom remains were obtained from clinically reported cases during 2014–2018. Total genomic DNA was isolated using PureLink™ Genomic DNA Mini Kit (Invitrogen, USA) according to manufacturer’s guide. DNA samples were PCR amplified and sequenced using ITS1F and ITS4 primers [15, 16] and LROR, LR5 and LR6 primers [17] for ITS and LSU, respectively. By using Mastercycler® gradient (Eppendorf, Germany) PCR reactions were carried out for 34 cycles with PCR profiles of 45 s at 94 °C (denaturation), 45 s at 52–55 °C (annealing) and 1.30 min at 72 °C (extension) with final extension of 72 °C for 10 min. Each PCR reaction of 25 µl contained 9.5 µl of OnePCR™ (GeneDirex®, Korea) reaction mixture with fluorescence dye, 2.5 µl of 10 µM of each primer, 1 µl of genomic DNA template and 9.5 µl of nuclease-free water. Amplification products were cleaned using PureLink™ Quick Gel Extraction Kit (Invitrogen, USA) and eluted with 35 µl of nuclease-free water. DNA sequencing analyses were performed by Macrogen Inc. in South Korea.

Fungal identification and microscopy

The nuclear ITS sequences generated from the clinical mushroom samples were compared for nucleotide similarity against the GenBank database using BLAST search of NCBI [18]. Morphological characteristics of the specimens were examined by using a low magnification stereomicroscope (HumaScope Stereo), while anatomical characteristics were observed on free-hand sections using a compound microscope (HumaScope LightLED) with 40–1000 magnifications.

Sequence alignments and phylogenetic analyses

Details of specimens and GenBank accession numbers are listed in Table 1. Outgroup and representative mushroom samples belonging to the Cuphophylloid clade were selected based on the previous studies [7, 9]. Sequence alignments were carried out using Geneious R8 (http://www.geneious.com/). Ambiguously aligned portions of the ITS and LSU sequences were then removed using Gblocks with less stringent setting [19, 20]. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian approaches as implemented on the CIPRES portal [21]. ML analysis was performed in RAxML 8.2.10 [22] using the GTRGAMMA model. Branch support was estimated based on 1000 bootstrap replicates. Bayesian analysis on the other hand, was performed using a variant of Markov Chain Monte Carlo (MCMC) method in MrBayes 3.2.2 [23] with GTR + G model. Number of generations was ten million. The first 25% were discarded as burn in and a majority rule consensus tree was constructed. Only clades that received bootstrap support ≥ 75% under ML and posterior probabilities ≥ 0.95 were considered as strongly supported. Phylogenetic trees were depicted using the program FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Table 1.

Details of samples, localities and GenBank accession numbers

Taxon Country ITS LSU
Ampulloclitocybe clavipes USA AY789080 AY639881
Cantharocybe brunneovelutina Belize KX452404 HM588721
Cantharocybe gruberi USA DQ200927 DQ234540
Cantharocybe gruberi USA – AF261530
Cantharocybe gruberi USA – AF261529
Cantharocybe gruberi Spain JN006422 JN006420
Cantharocybe virosa D217, Thailand MH827009 MH820127
Cantharocybe virosa D214, Thailand MH827010 MH820128
Cantharocybe virosa DMSC10891, Thailand MH827011 MH820129
Cantharocybe virosa D231, Thailand MH827012 MH820130
Cantharocybe virosa D232, Thailand MH827013 MH820131
Cantharocybe virosa D221, Thailand MH827014 MH820132
Cantharocybe virosa D295, Thailand MH827015 MH820133
Cantharocybe virosa D251, Thailand MH827016 MH820134
Cantharocybe virosa D287, Thailand MH827017 MH820135
Cantharocybe virosa D289, Thailand MH827018 MH820136
Cantharocybe virosa D290, Thailand MH827019 MH820137
Cantharocybe virosa D297, Thailand MH827020 MH820138
Cantharocybe virosa D298, Thailand MH827021 MH820139
Cantharocybe virosa D323, Thailand MH827022 MH820140
Cantharocybe virosa D354, Thailand MH827023 MH820141
Cantharocybe virosa Bangladesh – KX452406
Cantharocybe virosa Bangladesh KX452403 KF303143
Cantharocybe virosa India KX452405 JX101471
Cuphophyllus acutoides var. pallidus USA KF291096 KF291097
Cuphophyllus adonis Chile KF291035 KF291036
Cuphophyllus aurantius Puerto Rico KF291099 KF291100
Cuphophyllus basidiosus USA DQ486684 DQ457651
Cuphophyllus borealis USA HM020684 HM026552
Cuphophyllus canescens USA DQ486685 DQ457652
Cuphophyllus flavipes Japan KF291044 KF291045
Cuphophyllus griseorufescens New Zealand GU233328 GU233423
Phyllotopsis nidulans China GQ142019 GQ142039
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New accession numbers obtained in this study are in bold

Sample extraction and screening of GI toxins using liquid chromatography quadrupole time-of-flight mass spectrometry

Five grams of mushroom samples were extracted using methanol and incubated at 60 °C for 10 min, followed by centrifugation at 14,000 rpm for 5 min. Purification of the supernatant was carried out as described previously [5]. Mushroom extracts were analyzed on the Dionex UPLC systems (Bruker) equipped with an Ultimate 3000 RS pump, autosampler and column compartment coupled to the Impact II QTOF-MS system (Bruker). Chromatographic separation was conducted on an Acclaim RSLC 120 C18 column (2.1 mm × 100 mm diameter, 2.2 μm particle size) at flow rate of 0.2 ml/min. The injection volume was 5 µl. The column was eluted using the following binary gradient solutions: mobile phases A (10% methanol: 5 mM ammonium formate: 0.01% formic acid) and B (methanol: 5 mM ammonium formate: 0.01% formic acid) with flow rate of 0.20 ml/min: 99:1 at 1 min, 61:39 at 3 min, flow rate of 0.40 ml/min: 0.1:99.9 at 14 min, flow rate of 0.48 ml/min: 99:1 at 16 min and 100% A at 19 min. The mass detection was set in positive electrospray mode and scan ion mode within the mass range of 50–1500 Da. Parameters employed were as follows: capillary voltage at 4.5 kV, nebulizer at 2.0 bar, dry gas at 8.0 l/min, dry temperature at 180 °C, ion energy at 5.0 eV and collision energy at 4.0 eV. The column temperature was set to 30 °C. The LC-QTOF-MS data was manipulated using the HyStar software (version 4.1, Bruker). The targets of interest were putatively identified using in-house libraries based on molecular ions reported in the literatures [11, 12].

Results

Clinical symptoms

Of 39 patients reviewed, 23 were male (age range 18–88 years) and 16 were female (age range 11–73 years). The most prevalent symptoms in all patient respondents were nausea, vomiting, abdominal pains and circulatory disturbances as well as occasional diarrhea (Fig. 1). The latent periods ranged from 30 min to 4 h after ingesting the mushrooms. All patients recovered fully after timely treatment.

Fig. 1.

Fig. 1

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Clinical manifestations of patients after ingestion of Cantharocybe virosa

Fungal identification

Fifteen clinical mushroom samples (D) were delivered to the Toxicology Center, photographed and kept at − 70 °C (Fig. 2). Results of ITS BLAST search showed the highest pairwise identities for all samples tested with scores ranging from 93 to 99%. They indicated that these samples were closely related to Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar from Bangladesh (KX452403) and India (KX452405).

Fig. 2.

Fig. 2

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Cantharocybe virosa obtained from poisoning cases. a Basidiomata with pileus surface and longitudinal section showing unchanged solid context (D289: poisoning case in 2017), b Remnant mushroom samples harvested by patient (D232: poisoning case in 2014), c Trichoderm pileipellis, d Cheilocystidia and e Basidiospores

Key for identification of Cantharocybe virosa

  1. Basidiomata medium-sized........2

  2. Basidiomata medium-sized to large (45–100 mm), pale grayish brown; basidiospores 6.5–11(− 12) × 5–7 μm, subglobose to broadly ellipsoid, hyaline, thin-walled, smooth, inamyloid..............Indian C. virosa [7]

  3. Basidiomata medium-sized (26–75 mm), grayish brown; basidiospores 9.0–10(− 11) × 6–8 μm, ellipsoid, hyaline, thin-walled, smooth, inamyloid........Thai C. virosa

  4. Basidiomata medium-sized (50–80 mm), dark brown to grayish brown; basidiospores 8.0–10(− 11) × 5–7 μm, subglobose to broadly ellipsoid, hyaline, thin-walled, smooth, inamyloid........Bangladesh C. virosa [9]

Phylogenetic analyses

Thirty new sequences (15 each from ITS & LSU) were generated for this study and aligned with sequences obtained from GenBank (Table 1). Single-locus ML analyses were performed (Fig. 3). Since no conflicts were found, a combined dataset of the ITS and LSU sequences were analyzed using both the ML and Bayesian approaches. A matrix of 1857 unambiguously aligned nucleotide position characters (ITS: 778 characters & LSU: 1079 characters) was constructed. The ML analysis of the combined dataset yielded a tree with the final optimization likelihood of lnL = − 10,909.206, while for the Bayesian inference the likelihood parameters in the samples possessed the mean value (± standard deviation) of lnL = − 10,379.505 (± 0.09). The topology of the trees from the ML and Bayesian analyses did not show any conflict and hence, only the ML tree is shown here with the ML bootstrap support (BS) ≥ 75% and the posterior probabilities ≥ 0.95. All clinical mushroom samples are clustered in a strongly supported clade with C. virosa from Bangladesh as well as a holotype specimen from India (Fig. 4). Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys is a well-supported sister taxon of all three species of Cantharocybe.

Fig. 3.

Fig. 3

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Maximum likelihood phylogenies based on individual datasets of ITS and LSU sequences. Bootstrap values ≥ 75% are given above branches

Fig. 4.

Fig. 4

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Maximum likelihood tree depicting relationships within the genus Cantharocybe based on a combined dataset of ITS and LSU sequences. Maximum likelihood bootstrap values ≥ 75% are shown above branches and Bayesian posterior probabilities ≥ 0.95 are indicated as bold branches. Newly generated sequences are highlighted in bold. Asterisk indicates a DNA sequence of holotype specimen of C. virosa from India

Screening of GI toxins using LC-QTOF-MS

Suspected GI toxins were putatively identified based on the molecular ions and isotope pattern matching. Our results confirmed the presence of toxic coprine in the unconsumed samples of C. virosa. The molecular ion peak obtained at 11.4 min possessing accurate mass of m/z 203.1067 was corresponded to a chemical substance with the empirical formula C8H14N2O4 (molecular weight: 202.21 g/mol) whose isotope pattern best matches to theoretical abundances (Fig. 5).

Fig. 5.

Fig. 5

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Tentative identification of coprine using LC-QTOF-MS. a Retention time of [M + H] + of coprine and b Isotope abundances of three isotopes

Discussion

In Thailand, high incidence rates of mushroom poisoning usually occur due to misconceptions of ethnomycological knowledge and misidentification between edible and poisonous species. Moreover, mushroom hunters sometimes collect wild mushrooms in unfamiliar areas.

Retrospective analysis of mushroom intoxication cases from the Toxicology Center revealed that GI toxins were most commonly encountered (more than 50%), followed by hepatotoxins and neurotoxins. The GI toxin-containing mushrooms normally are the false parasols in the genera Chlorophyllum Massee, Entoloma P. Kumm. and some wild boletes, whereas hepatotoxic and neurotoxic mushrooms include noxious Amanita Dill. Ex Boehm. and Inocybe (Fr.) Fr., respectively [3–5, 24]. In this study, we focused on new poisoning cases of the GI toxin-containing mushrooms in the genus Cantharocybe during 2014–2018. Epidemiological investigations of Cantharocybe poisoning showed the regional occurrence in the following orders of northern (80%), western (13%) and rarely in the northeastern (7%) areas. Such incidence took place predominantly during May to October. The remnants of unconsumed mushroom samples from the patients and the surveillance and rapid response team: SRRT (Department of Disease Control) were delivered to our laboratory. Results based on ITS BLAST search revealed that all mushroom samples were close to Cantharocybe virosa. By comparison with the ITS sequence of the holotype specimen of C. virosa [9], nucleotide positions varied between 0 and 15 sites (93–99%).

Phylogenetic analyses based on a combined dataset of two loci confirmed that all clinical mushroom samples belong to a robust clade of C. virosa. Our resulting phylogenetic trees were in agreement with that of Hosen et al. [9]. Cantharocybe brunneovelutina is closely related to C. virosa, while C. gruberi forms a basal taxon. Morphologically, Thai C. virosa is a medium to large agaric mushroom that is characterized by a convex to applanata-shaped, dry with cracked margin, grayish brown cap; adnate to decurrent, crowded and white lamellae; stipe concolorous with cap, cylindrical cover with pruina, cottony mycelium at the base, interior solid and unchanging when cut or bruised. On the other hand, C. gruberi and C. brunneovelutina can be distinguished from C. virosa by certain characteristics such as pileus colors and surfaces, patterns of cheilocystidia as well as sizes of basidiospores [9, 25]. In addition, these three species are separated geographically [9, 25]. Cantharocybe gruberi is disjunctly distributed in North America and Europe, whereas C. brunneovelutina occurs in Central America, and C. virosa is only known from South Asia including Bangladesh, India and Thailand.

Although several groups of the GI toxin-containing mushrooms have been reported the toxins leading to the GI symptoms remain largely unknown. In Thailand, a number of suspected GI toxin-containing mushrooms include Amanita digitosa, A. pyriformis, Chlorophyllum molybdites and C. globosum [4, 6].

Reports of C. virosa poisoning are quite rare. However, there was an outbreak in the Kerala state of India in 2006 [26]. Villagers were mistaken that the mushrooms collected were some edible species in the genera Tricholoma (Fr.) Staude and Termitomyces R. Heim. Most patients were encountered with the severe gastrointestinal irritation. For the poisoning cases in Thailand, some patients had ingested what they believed to be an edible species of Termitomyces (Lyophyllaceae) and Macrocybe crassa (Sacc.) Pegler & Lodge (Tricholomataceae). Other patients had misconceptions that the toxic mushrooms tarnished rice when boiled.

According to Bresinsky and Besl [12] the gastrointestinal syndrome usually presents a short latent period (15 min to 2 or 4 h) after consuming the mushrooms. The symptoms include nausea, vomiting, abdominal pain and diarrhea. In the cases of C. virosa poisoning, all patients showed short latent periods of 30 min to 4 h. The dominant symptoms were similar to those caused by the other GI toxin-containing mushrooms, except induced circulatory disturbances which were observed in some patients. For the patients with circulatory disturbances, the poisoning symptoms were shown after ingesting C. virosa within an hour. They experienced nausea, vomiting, abdominal pains and feeling of tightness with shortness of breath. These symptoms appeared to be partially resemble coprine-like syndrome after consumption of the common ink cap, Coprinopsis atramentarius (Bull.) Fr. [11, 12]. Other mushrooms that produce disulfiram-like effects have also been reported. These include Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys, Echinoderma asperum (Pers.) Bon, Pholiota squarrosa (Vahl) P. Kumm., Rubroboletus pulcherrimus (Thiers & Halling) D. Arora, N. Siegel & J.L. Frank and Suillellus luridus (Schaeff.) Murrill [10, 11, 27].

Interestingly, our results based on LC-QTOF-MS analysis revealed the presence of coprine in all samples of C. virosa. The type of poisoning syndrome caused by this toxin belongs to the major group 4 (metabolic/endocrine toxicity mushroom poisoning) which can be further classified into group 4B (disulfiram-like mushroom poisoning) [10]. Coprine poisoning can be induced by alcohol, and the onset of symptoms includes rash, sweating, gastrointestinal system effects and arrhythmias which can occur within a few minutes to 2 h. Coprine is a precursor of 1-aminocyclopropanol, a toxic substance which blocks the normal breakdown of alcohol to acetic acid by inhibiting the enzyme aldehyde dehydrogenase. Excess concentration of acetaldehyde in blood causes facial flushing, throbbing headache and a sign of circulatory disturbances including tachycardia, shortness of breath, fall in blood pressure and collapse [11, 12, 28]. In this study however, the patients reported that they consumed the mushrooms without alcohol before or after meals. In desperate for immediate pain relief we presumed that these patients may have taken a common stomachic mixture which unfortunately contains 6.65% (v/v) alcohol concentration. This may have triggered the severe alcohol-related toxicity of coprine.

As a part of the National Institute of Health, the Department of Medical Sciences has contributed to raise public awareness on issues of wild mushroom foraging to prevent mushroom food poisoning in Thailand. Although ingestion of poisonous C. virosa is not life-threatening it is necessary to educate local communities to take precautions for harvesting the wild mushrooms by rapid morphological diagnoses and to seek timely medical attention in case of poisoning. Further studies are required for the identification of specific GI toxins and other unknown compounds present in C. virosa.

Acknowledgements

This work was financially supported by the Department of Medical Sciences, Ministry of Public Health for the project “Molecular genetic databases of poisonous mushrooms for clinical toxicology” (Project code: 6662). The authors wish to thank Mr. Sathaporn Ramchiun and Bruker application specialist (Thailand) for their useful suggestions on toxin identification as well as the Bureau of Drug and Narcotic for allowing us to use the instrument and facilities in the laboratory.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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