Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease

BACKGROUND: Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response. METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients. RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed. CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-015-0316-2) contains supplementary material, which is available to authorized users

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Phylogeny and taxonomy of Staurothele (Verrucariaceae, lichenized ascomycetes) from the karst of northern Vietnam

The crustose genus Staurothele (Verrucariaceae, Ascomycota) is a common component of the lichen flora from subneutral to alkaline silicate rocks in temperate to cold-temperate climates. Our field study in the karst system of northern Vietnam showed that it is also common on dry to humid limestone in the wet tropics. Molecular data revealed that species of Staurothele from Vietnam belong to an unnamed clade sister to the genus Endocarpon, together with the tropical Australian species Staurothele pallidopora and Staurothele diffractella, a North American species recently transferred to Endocarpon based on molecular data. The genus Willeya is here resurrected for this clade of crustose epilithic Staurothele with pale ascospores. Eight new combinations are proposed and three new species of Willeya are described from Vietnam. Sampling tropical members of a lichen family previously mostly known from temperate areas contributed significantly to improving its generic classification

lectotypification of Polystichum atroviridissimum and P. fimbriatum

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Asplenium serratifolium (Aspleniaceae), a New Fern Species from Central Vietnam Based on Morphological and Molecular Evidence

Asplenium serratifolium (Aspleniaceae), a new fern species from central Vietnam, is described and illustrated. The new species is characterized by plants 10-18 cm tall, laminae pinnatipartite, lobe margins entire or with shallow teeth, and veins simple or forked. Molecular phylogenetic analysis based on five plastid markers (atpB, rbeL, rps4, rps4-trnS, and trnL-F) indicate that the new species is closely related to A. ensiforme

Chlorohiptage (Tetrapteroids, Malpighiaceae), a distinct new genus endemic to Vietnam based on morphological and molecular data

Background and aims – Vietnam is one of the leading diversity centres for Asian Malpighiaceae, comprising 24 accepted species and three native genera (i.e. Aspidopterys, Hiptage, and Tristellateia). During recent fieldwork towards completing the taxonomic revision of Malpighiaceae for the Flora of Vietnam, we have collected specimens from two populations that could not be placed in any of the three native genera of this family. We performed morphological and molecular phylogenetic studies to test the generic placement of those specimens. Material and methods – We sampled 27 genera (including the Asian Acridocarpus, Aspidopterys, Brachylophon, Hiptage, Stigmaphyllon, and Tristellateia, out of a total of 75) of Malpighiaceae representing all phylogenetic clades comprising paleotropical lineages (i.e. acridocarpoids, bunchosioids, tetrapteroids, malpighioids, and stigmaphylloids), the unusual specimen recently collected in Vietnam, and the two genera of Elatinaceae as outgroups. Maximum Likelihood analysis was carried out based on a molecular matrix alignment of the internal transcribed spacer marker (ITS). Comprehensive morphological analyses were also carried out based on the collected specimens and additional herbarium specimens. Key results – Our molecular phylogeny strongly supported the unusual specimen from Vietnam placed as sister to Hiptage in the tetrapteroid clade. Key morphological traits differing these Vietnamese specimens from Hiptage were identified related to the floral bud imbrication, shape, colour, posture of sepals and petals, length of stamen filaments, number of styles, and shape of mericarps, allowing the proposition of a new monospecific genus. Conclusions – Chlorohiptage vietnamensis is proposed as a new monospecific genus closely related to Hiptage but differing in several key morphological traits. A complete morphological description is provided alongside photographic illustrations and notes on its conservation, distribution, ecology, etymology, and taxonomy. Additionally, an updated identification key to the genera of the tetrapteroid clade is provided

Three new species of the fern genus Arachniodes (Dryopteridaceae) from Vietnam

Three new species of the fern genus Arachniodes (Dryopteridaceae) are described from Vietnam: A. daklakensis, A. longicaudata, and A. quangnamensis. The first two occur in Central Highlands and Central Vietnam, respectively, while the last is found in the north (Bavi, Langson and Tamdao) as well as in the central Vietnam (Nghean, Quangbinh, Quangtri)

Pteridryaceae: A new fern family of Polypodiineae (Polypodiales) including taxonomic treatments

We undertook phylogenetic analyses to resolve the relationships of Pteridrys and related taxa based on six plastid markers (atpA, atpB, matK & rps16-matK, rbcL, rps4 & rps4-trnS, and trnL & trnL-F) and nuclear pgiC. We included 195 accessions representing approximately 147 species in 38 genera, and seven of the nine families in Polypodiineae (eupolypods I). Tectariaceae s.l. (i.e., Arthropteris, Draconopteris, Hypoderris, Malaifilix, Pteridrys, Tectaria, and Triplophyllum in addition to Polydictyum) is recovered as monophyletic (97% maximum likelihood bootstrap value), but with low (<50%) maximum parsimony jackknife value. The family Tectariaceae s.l. is therefore the only family in ferns without a corresponding non-parametric-based strong support in spite of our data totaling 9616 aligned base pairs. Tectariaceae s.l. can not be unambiguously recognized by any of the 13 morphological characters analyzed. However, if the clade composed of Draconopteris, Malaifilix, Polydictyum, and Pteridrys (DMPP) is recognized as a distinct family, at least four morphological characters enable the distinction of the DMPP clade from its sister clade. Considering the uncertainty in the monophyly, the diagnosability, and the deep divergence, we propose to establish a new family, Pteridryaceae, to accommodate the DMPP clade. Species of Pteridryaceae share mostly the following characteristics: erect to suberect rhizomes, reduced basal pinnae, anastomosing or free venation, absence of catenate hairs at the leaf surface, and perine ornamentation lacking spines or spinules. Identification keys are provided for the four genera and 31 species of the DMPP clade (or Pteridryaceae). Reflecting the presented results, the recognition of Arthropteridaceae is the preferred taxonomic status of the Arthropteris clade