Early transmission patterns of coronavirus disease 2019 (COVID-19) in travellers from Wuhan to Thailand, January 2020.

We report two cases of coronavirus disease 2019 (COVID-19) in travellers from Wuhan, China to Thailand. Both were independent introductions on separate flights, discovered with thermoscanners and confirmed with RT-PCR and genome sequencing. Both cases do not seem directly linked to the Huanan Seafood Market in Hubei but the viral genomes are identical to four other sequences from Wuhan, suggesting early spread within the city already in the first week of January.

Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex.

Phylogenetic analysis of 11 genetic loci and results from many genotyping studies revealed significant genetic diversity with the pathogenic Cryptococcus gattii/Cryptococcus neoformans species complex. Genealogical concordance, coalescence-based, and species tree approaches supported the presence of distinct and concordant lineages within the complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and five species within C. gattii. The type strain of C. neoformans CBS132 represents a serotype AD hybrid and is replaced. The newly delimited species differ in aspects of pathogenicity, prevalence for patient groups, as well as biochemical and physiological aspects, such as susceptibility to antifungals. MALDI-TOF mass spectrometry readily distinguishes the newly recognized species.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

A new species of Megalaria (Ramalinaceae, Ascomycota) from Thailand, and recognition of subgenus Catillochroma.

Tropical regions harbor a substantial diversity of lichenized fungi, but face numerous threats to their persistence, often even before previously unknown species have been described and their evolutionary relationships have been elucidated. Megalaria (Ramalinaceae) is a lichen-forming genus of fungi that produces crustose thalli, and includes a number of lineages occupying tropical rain forests; however, taxonomic and phylogenetic work on this clade is limited. Here we leverage both morphological and sequence data to describe a new species from the tropics, M.pachaylenophila. This taxon forms a crustose thallus, lacks secondary metabolites, and occurs in mangrove forests of Thailand. We supplemented molecular data from this species with data from other species, including two genera related to and occasionally included in Megalaria, namely Catillochroma and Lopezaria. Our analyses revealed Catillochroma species form a monophyletic group embedded within Megalaria, and we therefore recognize this clade at the subgeneric level. Since we only included the type species of Lopezaria in this study, we refrain from proposing a taxonomic conclusion for that clade at the moment. Several taxonomic combinations are made to reflect phylogenetic evidence supporting the inclusion of these species in Megalaria.

Draft Genomes of Six Wild Poisonous Mushrooms.

Foodborne illnesses caused by wild mushroom poisoning occur globally and have led to food safety concerns. Here, we reported de novo genome assemblies of the six most commonly encountered toxic mushrooms in Thailand. These comprised Amanita brunneitoxicaria, Cantharocybe virosa, Chlorophyllum molybdites, Entoloma mastoideum, Pseudosperma sp. and Russula subnigricans. The nuclear genome sizes of these species ranged from 40 to 77 Mb, with the number of predicted genes ranging from 5,375 to 14,099. The mitogenome sizes varied from 41,555 to 78,907 bp. The resulting draft genomes of these poisonous mushrooms provide insights into toxin-related genes that may be used to establish genetic markers for monitoring mushroom poisoning outbreaks.

Prevalence and antimicrobial susceptibility of Campylobacter isolated from retail chickens in Thailand.

Campylobacter is an important foodborne pathogen causing bacterial gastroenteritis worldwide; however, there has been a lack of information over the past decade on its occurrence, antibiotic susceptibility and genetic diversity in Thailand. Poultry meat is considered as a reservoir for transmission of Campylobacter to humans. This study determines the prevalence and antimicrobial resistance patterns of Campylobacter spp. on chicken samples purchased from 50 local wet markets and supermarkets in central Thailand. Of the 296 samples, 99 (33.5%) were contaminated with C. jejuni, 54 (18.2%) were C. coli and 15 (5.1%) were contaminated with both species. Antibiotic resistance rate is higher among C. coli isolates; 100%, 76.8%, 37.7%, 36.2% and 13.0% were resistant to quinolones, cyclines, macrolides, clindamycin and gentamicin, respectively. Most of the C. jejuni isolates were resistant to quinolones (79.8%) and cyclines (38.6%) whereas resistance to macrolides, clindamycin and gentamicin was found to be 1.8%. Multi-drug resistance (i.e. to three or more unrelated antimicrobials) was detected in 37.7% of C. coli and 1.8% of C. jejuni isolates. This study has revealed high contamination rates and alarming levels of antimicrobial resistance in Campylobacter spp. isolated from retail chicken samples in Thailand, suggesting the necessity of implementing interventions to reduce its prevalence from farm to table in the country.

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

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.

Molecular identification of poisonous mushrooms using nuclear ITS region and peptide toxins: a retrospective study on fatal cases in Thailand.

Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

A cross-sectional study of exposure to mercury in schoolchildren living near the eastern seaboard industrial estate of Thailand.

BACKGROUND: Industrial activity in Thailand's coastal areas has significantly increased mercury concentrations in seawater, causing accumulation through the food chain. Continuous exposure to mercury has been linked to bioaccumulation in living organisms and potential adverse health effects in children. METHODS: Blood samples were collected from 873 schoolchildren aged 6-13 years living in four sites near the eastern seaboard industrial estates of the Gulf of Thailand in 2011. Total mercury level in whole blood (Hg-B) was compared with standard reference values. RESULTS: Mean (± standard deviation) concentrations of Hg-B from schoolgirls (2.19 ± 0.5 µg/L; n = 405) and schoolboys (2.29 ± 0.3 µg/L; n = 468) did not exceed the regulatory limits of the United States Environmental Protection Agency (US EPA), the German Commission on Human Biological Monitoring (HBM I, II) or Clarke's analysis of drugs and poisons reference values. Nevertheless, 67 children (34 girls and 33 boys) had individual values that exceeded the lowest of these standards (4 µg/L). CONCLUSION: The relatively low concentrations of Hg-B detected in this study suggested a relatively low risk for schoolchildren. However, 67 children had elevated mean total Hg-B concentrations, especially in the two sites located nearest the industrial area. This information may serve as an early warning of the potential for pollution to affect children living around industrial areas. Further regular monitoring, including studies assessing the health impact of mercury pollution in this region of Thailand, is to be encouraged.

Serum concentrations of organochlorine pesticides p,p'-DDE in adult Thai residents with background levels of exposure.

In Thailand, DDT was banned for agriculture use in 1983 and for malaria vector control in 1999. However, contamination by DDT and its primary metabolite, p,p'-DDE remains serious environmental and human health concerns. The main focus of this study were i) to investigate serum concentrations of p,p'-DDE and p,p'-DDT as exposure biomarkers for potential adverse health effect in adult Thai residents and ii) to compare the associations of BMI, thyroid hormones, cholesterol, triglycerides and fasting blood sugar levels in human serum with the concentrations of these pesticides. In a total of 1,137 participants were measured blood serum for analyses of p,p'-DDE and p,p'-DDT. The geometric mean concentration (95% confidence interval) for serum total p,p'-DDE concentration was 1,539 (1,242-1,837) ng/g lipid and 1,547 (1,293-1,806) ng/g lipid in adult males and females, respectively. Furthermore, the total amount of serum p,p'-DDE concentration significantly correlated with plasma glucose levels. Neither p,p'-DDE nor p,p'-DDT was significantly associated with serum thyroid hormones levels. Additionally, the high p,p'-DDE/DDT ratio indicates that the exposure is due past rather than recent use of DDT.

The sister-group relationships of the largest family of lichenized fungi, Parmeliaceae (Lecanorales, Ascomycota).

Parmeliaceae is the largest family of lichen-forming fungi. In spite of its importance for fungal diversity, its relationships with other families in Lecanorales remain poorly known. To better understand the evolutionary history of the diversification of lineages and species richness in Parmeliaceae it is important to know the phylogenetic relationships of the closest relatives of the family. A recent study based on two molecular loci suggested that either Protoparmelia s. str. or a group consisting of Gypsoplaca and Protoparmelia s. str. were the possible sister-group candidates of Parmeliaceae, but that study could not distinguish between these two alternatives. Here, we used a four-locus phylogeny (nuLSU, ITS, RPB1, MCM7) to reveal relationships of Parmeliaceae with other potential relatives in Lecanorales. Maximum likelihood and Bayesian analyses showed that Protoparmelia is polyphyletic, with Protoparmelia s. str. (including Protoparmelia badia and Protoparmelia picea) being most closely related to Parmeliaceae s. str., while the Protoparmelia atriseda-group formed the sister-group to Miriquidica. Gypsoplaca formed the sister-group to the Parmeliaceae s. str. + Protoparmelia s. str. clade. Monophyly of Protoparmelia as currently circumscribed, and Gypsoplaca as sister-group to Parmeliaceae s. str. were both significantly rejected by alternative hypothesis testing.

Phylogenetic classification at generic level in the absence of distinct phylogenetic patterns of phenotypical variation: a case study in graphidaceae (ascomycota).

Molecular phylogenies often reveal that taxa circumscribed by phenotypical characters are not monophyletic. While re-examination of phenotypical characters often identifies the presence of characters characterizing clades, there is a growing number of studies that fail to identify diagnostic characters, especially in organismal groups lacking complex morphologies. Taxonomists then can either merge the groups or split taxa into smaller entities. Due to the nature of binomial nomenclature, this decision is of special importance at the generic level. Here we propose a new approach to choose among classification alternatives using a combination of morphology-based phylogenetic binning and a multiresponse permutation procedure to test for morphological differences among clades. We illustrate the use of this method in the tribe Thelotremateae focusing on the genus Chapsa, a group of lichenized fungi in which our phylogenetic estimate is in conflict with traditional classification and the morphological and chemical characters do not show a clear phylogenetic pattern. We generated 75 new DNA sequences of mitochondrial SSU rDNA, nuclear LSU rDNA and the protein-coding RPB2. This data set was used to infer phylogenetic estimates using maximum likelihood and Bayesian approaches. The genus Chapsa was found to be polyphyletic, forming four well-supported clades, three of which clustering into one unsupported clade, and the other, supported clade forming two supported subclades. While these clades cannot be readily separated morphologically, the combined binning/multiresponse permutation procedure showed that accepting the four clades as different genera each reflects the phenotypical pattern significantly better than accepting two genera (or five genera if splitting the first clade). Another species within the Thelotremateae, Thelotrema petractoides, a unique taxon with carbonized excipulum resembling Schizotrema, was shown to fall outside Thelotrema. Consequently, the new genera Astrochapsa, Crutarndina, Pseudochapsa, and Pseudotopeliopsis are described here and 39 new combinations are proposed.

Using phylogenetic and coalescent methods to understand the species diversity in the Cladia aggregata complex (Ascomycota, Lecanorales).

The Cladia aggregata complex is one of the phenotypically most variable groups in lichenized fungi, making species determination difficult and resulting in different classifications accepting between one to eight species. Multi-locus DNA sequence data provide an avenue to test species delimitation scenarios using genealogical and coalescent methods, employing gene and species trees. Here we tested species delimitation in the complex using molecular data of four loci (nuITS and IGS rDNA, protein-coding GAPDH and Mcm-7), including 474 newly generated sequences. Using a combination of ML and Bayesian gene tree topologies, species tree inferences, coalescent-based species delimitation, and examination of phenotypic variation we assessed the circumscription of lineages. We propose that results from our analyses support a 12 species delimitation scenario, suggesting that there is a high level of species diversity in the complex. Morphological and chemical characters often do not characterize lineages but show some degree of plasticity within at least some of the clades. However, clades can often be characterized by a combination of several phenotypical characters. In contrast to the amount of homoplasy in the morphological characters, the data set exhibits some geographical patterns with putative species having distribution patterns, such as austral, Australasian or being endemic to Australia, New Zealand or Tasmania.