Genomic characterization of Aeromonas spp. isolates from striped catfish with motile Aeromonas septicemia and human bloodstream infections in Vietnam.

Aeromonas spp. are commonly found in the aquatic environment and have been responsible for motile Aeromonas septicemia (MAS) in striped catfish, resulting in significant economic loss. These organisms also cause a range of opportunistic infections in humans with compromised immune systems. Here, we conducted a genomic investigation of 87 Aeromonas isolates derived from diseased catfish, healthy catfish and environmental water in catfish farms affected by MAS outbreaks in eight provinces in Mekong Delta (years: 2012-2022), together with 25 isolates from humans with bloodstream infections (years: 2010-2020). Genomics-based typing method precisely delineated Aeromonas species while traditional methods such as aerA PCR and MALDI-TOF were unable identify A. dhakensis. A. dhakensis was found to be more prevalent than A. hydrophila in both diseased catfish and human infections. A. dhakensis sequence type (ST) 656 followed by A. hydrophila ST251 were the predominant virulent species-lineages in diseased catfish (43.7 and 20.7 %, respectively), while diverse STs were found in humans with bloodstream infections. There was evidence of widespread transmission of ST656 and ST251 on striped catfish in the Mekong Delta region. ST656 and ST251 isolates carried a significantly higher number of acquired antimicrobial resistance (AMR) genes and virulence factors in comparison to other STs. They, however, exhibited several distinctions in key virulence factors (i.e. lack of type IV pili and enterotoxin ast in A. dhakensis), AMR genes (i.e. presence of imiH carbapenemase in A. dhakensis), and accessory gene content. To uncover potential conserved proteins of Aeromonas spp. for vaccine development, pangenome analysis has unveiled 2202 core genes between ST656 and ST251, of which 78 proteins were in either outer membrane or extracellular proteins. Our study represents one of the first genomic investigations of the species distribution, genetic landscape, and epidemiology of Aeromonas in diseased catfish and human infections in Vietnam. The emergence of antimicrobial resistant and virulent A. dhakensis strains underscores the needs of enhanced genomic surveillance and strengthening vaccine research and development in preventing Aeromonas diseases in catfish and humans, and the search for potential vaccine candidates could focus on Aeromonas core genes encoded for membrane and secreted proteins.

A new snake of the genus Dendrelaphis Boulenger, 1890 (Squamata: Colubridae) from the coastal area of southern Vietnam.

We describe a new species of Dendrelaphis from the coastal area in southern Vietnam based on morphological data and nucleotide sequences from one partial mitochondrial gene (Cytb). Dendrelaphis binhi sp. nov. is diagnosed by the following morphological characters: body scale rows 13 at neck and midbody, 9-11 before vent; vertebral scale row feebly enlarged; ventrals 154-158 in males and 161-170 in females; subcaudals 95-106 in males and 95-102 in females; one supralabial touching the eye; 18 or 19 maxillary teeth; hemipenis spinose, reaching 10th or 11th SC, with a slender papilla; cloacal plate divided; dorsum dark brown; and a white stripe along the lower flank. The new species differs from its congeners by an uncorrected p-distance in Cytb sequences of at least 8.1%.

A new species of the genus Rhabdophis Fitzinger, 1843 (Squamata: Colubridae) from the Hoang Lien range, northwest Vietnam.

We describe a new species of the Natricinae genus Rhabdophis Fitzinger, 1843 from the Hoang Lien range, northwest Vietnam. The new species is distinct from all congeneric species on the grounds of morphometric and molecular data. The new species is most similar to Rhabdophis leonardi in terms of morphology but can be distinguished from it based on differences in maxillary tooth count, scalation, and genetic data. A sequence on GenBank from a Rhabdophis specimen collected in Honghe, Yunnan, China was identical to the species we describe, and it is likely that the new species is not restricted to Vietnam. As a priority, future work should focus on determining the distribution of this species, as well as understanding population and life history traits such as reproductive rate.

A new species of Leptobrachella (Anura, Megophryidae) from Mount Pu Ta Leng, northwest Vietnam.

The Hoang Lien Range in northwest Vietnam is known to harbour five species of Leptobrachella, a specious genus of terrestrial frogs. We collected specimens of Leptobrachella from Mount Pu Ta Leng on the second highest peak in the Hoang Lien Range and use morphological and molecular data to show that this population represents a previously undescribed species which we name Leptobrachella graminicola sp. nov. after the abundance of calling males on sedge-like plants. This new species is closely related to L. bourreti but the new species is distinguished from L. bourreti and other congeneric species by a combination of the following characters: (1) a body size range of 23.124.6 mm in six adult males and 28.632.9 mm in five adult females; (2) skin on dorsum smooth, with many tubercles and lacking dermal ridges; (3) toes with rudimentary webbing and broad lateral fringes; (4) belly white with brown spots; (5) throat dark brown with light grey-brown flecks and spots; (6) a row of large white spots on the outer edge of the tarsus extending from the heel to the inner metatarsal tubercle, sometimes forming a long white stripe; and (7) a bicoloured iris. The new, high-elevation species is likely to be range-restricted and threatened by habitat loss and the activities of tourists.

Amolops caelumnoctis Rao amp; Wilkinson, 2007, a Junior Synonym of A. splendissimus Orlov amp; Ho, 2007 (Amphibia: Anura: Ranidae).

Amolops splendissimus Orlov and Ho, 2007 and A. caelumnoctis Rao and Wilkinson, 2007 were described almost simultaneously from either side of the China-Vietnam border. The two species share a strong morphological resemblance, and their taxonomic distinctiveness has been questioned, yet no one has confirmed the taxonomic relationship and status between the two taxa. To resolve this taxonomic issue, we collected additional topotypic and near-topotypic specimens of A. splendissimus and A. caelumnoctis from both China (A. caelumnoctis: Wenshan County, Yunnan Province; type locality Luchun County, Yunnan Province), and Vietnam (A. splendissimus: Tam Duong District, Lai Chau Province; type locality Mount Ky Quan San, Bat Xat, Lao Cai Province). Molecular analysis based on a 16S rRNA fragment revealed minimal genetic divergences between the two taxa (0.0%0.4% uncorrected p-distance), and both species are closely related to A. viridimaculatus (2.1%2.3%) and A. medogensis (3.5%3.7%). Morphological comparisons between the newly collected specimens and the original descriptions of both species further support the lack of distinctiveness of the two species, hence, we conclude that A. caelumnoctis is a junior synonym of A. splendissimus.

Liquid biopsy uncovers distinct patterns of DNA methylation and copy number changes in NSCLC patients with different EGFR-TKI resistant mutations.

Targeted therapy with tyrosine kinase inhibitors (TKI) provides survival benefits to a majority of patients with non-small cell lung cancer (NSCLC). However, resistance to TKI almost always develops after treatment. Although genetic and epigenetic alterations have each been shown to drive resistance to TKI in cell line models, clinical evidence for their contribution in the acquisition of resistance remains limited. Here, we employed liquid biopsy for simultaneous analysis of genetic and epigenetic changes in 122 Vietnamese NSCLC patients undergoing TKI therapy and displaying acquired resistance. We detected multiple profiles of resistance mutations in 51 patients (41.8%). Of those, genetic alterations in EGFR, particularly EGFR amplification (n = 6), showed pronounced genome instability and genome-wide hypomethylation. Interestingly, the level of hypomethylation was associated with the duration of response to TKI treatment. We also detected hypermethylation in regulatory regions of Homeobox genes which are known to be involved in tumor differentiation. In contrast, such changes were not observed in cases with MET (n = 4) and HER2 (n = 4) amplification. Thus, our study showed that liquid biopsy could provide important insights into the heterogeneity of TKI resistance mechanisms in NSCLC patients, providing essential information for prediction of resistance and selection of subsequent treatment.

A new species of Ahaetulla Link, 1807 (Squamata: Colubridae) from the Mekong Delta, Vietnam.

A new vine snake, genus Ahaetulla, from Soc Trang Province, southern Vietnam is described based on morphological data and nucleotide sequences from COI and Cytb. Ahaetulla rufusoculara sp. nov. is diagnosed by the following morphological characters: bright red eyes; snout without dermal appendage; internasal separated from supralabial by loreal; body scale rows 15-15-13; ventrals 186190 in males and 182185 in females; subcaudals 143153 in males and 128 or 129 in females; 8 (rarely 9) supralabials, fourth and fifth or fourth to sixth entering orbit; 1214 maxillary teeth; hemipenis short, reaching 6th or 7th SC; cloacal plate divided; dorsum bright green; and yellow or white stripe along the lower flank. The new species differs from its congeners by an uncorrected p-distance in COI and Cytb sequences of at least 7.7% and 7.5%, respectively.

Gekko phuyenensis sp. nov. (Squamata: Gekkonidae), a new species from southern Vietnam.

A new species of the genus Gekko Laurenti, 1768 is described from Phu Yen Province, southern Vietnam based on morphological and molecular evidence. Gekko phuyenensis sp. nov. differs from other congeners by its moderate size, with a snout-vent length of 90.292.8 mm; nares in contact with rostral; internasal 1; interorbitals 35 or 36; ventrals 3032; dorsal tubercle rows 9 or 10; precloacal pores 12 or 13 in males and precloacal pits 13 in female; subdigital lamellae under first finger 1821, fourth finger 19 or 20, first toe 1820, and fourth toe 2123; subcaudals median enlarged; no webbing between toes; and the absence of tubercles on hind limbs. Molecular phylogenetic analyses of mitochondrial gene COI (681 bp) using the geckos from Phu Yen and its congeners in southern Vietnam resolve the new species as the sister lineage to G. cf. grossmanni from Khanh Hoa Province. Genetic distances between G. phuyenensis sp. nov. and its congeners in southern Vietnam range from 16.924.2%. The discovery of this new species raises the number of known species of Gekko in Vietnam to 14.

A new species of Limnonectes (Amphibia: Anura: Dicroglossidae) from Vietnam.

A new species of Limnonectes from Phu Yen Province, South-central Vietnam is described based on morphological and molecular differences. Morphologically, the new species is distinguishable from its congeners on the basis of a combination of the following diagnostic characters: SVL 49.8-77.9 mm in males, 40.8-61.8 mm in females; males with moderately enlarged head (HL/SVL 0.47, HW/SVL 0.48); head slightly broader than long (HL/HW 0.97 in males, 0.95 in females); vomerine teeth present; external vocal sacs absent; rostral length short (RL/SVL 0.15); tympanum invisible; dorsal surface of head, body and flanks with ridges and tubercles; dorsal surface of tibia and foot distinctly tuberculate, covered with moderately dense, small, low tubercles; supratympanic fold present; dorsolateral fold absent; Finger I with nuptial pad, composed of minute spines on dorsal surface and medial edge in males; toes webbed to distal of terminal phalanx, webbing formula I0-0II0-0III0-0IV0--0V0; in life, dorsum yellowish brown with dark brown markings; ventral surface white with brown markings. In phylogenetic analyses, the new species from Vietnam and Limnonectes fastigatus as well as L. kiziriani form a clade, which is sister to L. fragilis from China, although the nodal support values for the set of relationships are insignificant from all analyses.

A new skink of the genus Scincella Mittleman, 1950 (Squamata: Scincidae) from southern Vietnam.

A new skink of the genus Scincella Mittleman, 1950, from Binh Phuoc Province, southern Vietnam is described based on morphological data, including hemipenial characters, and nucleotide sequences from COI. The following morphological characters diagnose Scincella baraensis sp. nov.: relatively small size in adults (snout-vent length up to 49.2 mm); when limbs adpressed, toes do not or just reach fingers; 30 smooth midbody scale rows and dorsal scales not enlarged; 66-70 paravertebral scales; 64-66 ventral scale rows; 7 supralabials, the fifth and sixth below the center of the eye; enlarged temporal scales 1+2; nuchals present; ear with small auricular lobules; 18-20 smooth lamellae beneath toe IV; hemipenis smooth, short, and unforked; dorsum with faint black dots. The new species differs from its congeners by an uncorrected p-distance in COI sequences of at least 16.2%.

A description of the tadpole of the Critically Endangered Botsford's leaf-litter frog (Leptobrachella botsfordi) with comments on the distribution and conservation status of the species.

We describe the tadpole of the Botsford's leaf-litter frog Leptobrachella botsfordi, a species endemic to Mount Fansipan in northwest Vietnam, for the first time. Tadpoles of this species were found in steep, fast-flowing streams at elevations between 2500-2600 m asl. We also report a previously unknown locality for this species on Mount Fansipan, which increases the species' known Extent of Occurrence from 8 km2 to 36 km2. Our findings will inform subsequent conservation initiatives for this poorly known and highly threatened species.

A description of the tadpole of Megophrys "Brachytarsophrys" intermedia (Smith, 1921).

Megophrys Kuhl and Van Hasselt are an Asian radiation of terrestrial frogs which are usually associated with montane forest (Mahony et al. 2017). The Annam horned frog (Megophrys intermedia) is reported from montane evergreen forest in Vietnam and Lao People's Democratic Republic (Stuart 2005; Hendrix et al. 2008; Nguyen et al. 2009; Tran 2013; Orlov et al. 2015; IUCN 2017; Mahony et al. 2017). The IUCN Red List assessment for the species reports an elevational range of 782-1500 m asl (IUCN 2017) although the type locality was reported to be above 1500 m asl (Smith 1921), and Tran (2013) collected a specimen at 1515 m asl. Megophrys intermedia is assessed as Least Concern (IUCN 2017) and little is known about the reproductive behaviour and life history of the species. In Lam Dong Province (Vietnam), male M. intermedia call in March (Tran 2013); on the Langbian Plateau (Vietnam), male specimens are reported to call by day and by night from March to May and specimens called from within deep crevices between boulders in streams (Smith 1921); in Xe Kong Province (Laos), males call or guard egg clutches that are laid in water in July (Stuart 2005).

The tadpoles of five Megophrys Horned frogs (Amphibia: Megophryidae) from the Hoang Lien Range, Vietnam.

Frogs in the genus Megophrys are an Asian radiation of stream-breeding frogs. The tadpoles of many Megophrys species are undescribed; those that are described are often dubiously allocated to species by association with post metamorphic specimens at collection sites and without supportive molecular data. We provide detailed descriptions of the larvae of five species of Megophrys from the Hoang Lien Range in northwest Vietnam: Megophrys fansipanensis, M. gigantica, M. hoanglienensis, M. jingdongensis and M. maosonensis. Tadpoles from different subgenera differ from each other via a combination of patternation in life, oral disc shape and tail morphology but given the small sample size, and limited number of species it is unlikely that these differences can be applied more widely to delineate subgenera. Morphological differences between tadpoles from species within the subgenus Panophrys were insufficient to clearly delineate all species. The ability to identify tadpoles is likely to advance our understanding of the frog fauna in mainland southeast Asia.

Ultra-Deep Massive Parallel Sequencing of Plasma Cell-Free DNA Enables Large-Scale Profiling of Driver Mutations in Vietnamese Patients With Advanced Non-Small Cell Lung Cancer.

Population-specific profiling of mutations in cancer genes is of critical importance for the understanding of cancer biology in general as well as the establishment of optimal diagnostics and treatment guidelines for that particular population. Although genetic analysis of tumor tissue is often used to detect mutations in cancer genes, the invasiveness and limited accessibility hinders its application in large-scale population studies. Here, we used ultra-deep massive parallel sequencing of plasma cell free DNA (cfDNA) to identify the mutation profiles of 265 Vietnamese patients with advanced non-small cell lung cancer (NSCLC). Compared to a cohort of advanced NSCLC patients characterized by sequencing of tissue samples, cfDNA genomic testing, despite lower mutation detection rates, was able to detect major mutations in tested driver genes that reflected similar mutation composition and distribution pattern, as well as major associations between mutation prevalence and clinical features. In conclusion, ultra-deep sequencing of plasma cfDNA represents an alternative approach for population-wide genetic profiling of cancer genes where recruitment of patients is limited to the accessibility of tumor tissue site.

A little frog leaps a long way: compounded colonizations of the Indian Subcontinent discovered in the tiny Oriental frog genus Microhyla (Amphibia: Microhylidae).

Frogs of the genus Microhyla include some of the world's smallest amphibians and represent the largest radiation of Asian microhylids, currently encompassing 50 species, distributed across the Oriental biogeographic region. The genus Microhyla remains one of the taxonomically most challenging groups of Asian frogs and was found to be paraphyletic with respect to large-sized fossorial Glyphoglossus. In this study we present a time-calibrated phylogeny for frogs in the genus Microhyla, and discuss taxonomy, historical biogeography, and morphological evolution of these frogs. Our updated phylogeny of the genus with nearly complete taxon sampling includes 48 nominal Microhyla species and several undescribed candidate species. Phylogenetic analyses of 3,207 bp of combined mtDNA and nuDNA data recovered three well-supported groups: the Glyphoglossus clade, Southeast Asian Microhyla II clade (includes M. annectens species group), and a diverse Microhyla I clade including all other species. Within the largest major clade of Microhyla are seven well-supported subclades that we identify as the M. achatina, M. fissipes, M. berdmorei, M. superciliaris, M. ornata, M. butleri, and M. palmipes species groups. The phylogenetic position of 12 poorly known Microhyla species is clarified for the first time. These phylogenetic results, along with molecular clock and ancestral area analyses, show the Microhyla-Glyphoglossus assemblage to have originated in Southeast Asia in the middle Eocene just after the first hypothesized land connections between the Indian Plate and the Asian mainland. While Glyphoglossus and Microhyla II remained within their ancestral ranges, Microhyla I expanded its distribution generally east to west, colonizing and diversifying through the Cenozoic. The Indian Subcontinent was colonized by members of five Microhyla species groups independently, starting with the end Oligocene-early Miocene that coincides with an onset of seasonally dry climates in South Asia. Body size evolution modeling suggests that four groups of Microhyla have independently achieved extreme miniaturization with adult body size below 15 mm. Three of the five smallest Microhyla species are obligate phytotelm-breeders and we argue that their peculiar reproductive biology may be a factor involved in miniaturization. Body size increases in Microhyla-Glyphoglossus seem to be associated with a burrowing adaptation to seasonally dry habitats. Species delimitation analyses suggest a vast underestimation of species richness and diversity in Microhyla and reveal 15-33 undescribed species. We revalidate M. nepenthicola, synonymize M. pulverata with M. marmorata, and provide insights on taxonomic statuses of a number of poorly known species. Further integrative studies, combining evidence from phylogeny, morphology, advertisement calls, and behavior will result in a better systematic understanding of this morphologically cryptic radiation of Asian frogs.

Actionable Mutation Profiles of Non-Small Cell Lung Cancer patients from Vietnamese population.

Comprehensive profiling of actionable mutations in non-small cell lung cancer (NSCLC) is vital to guide targeted therapy, thereby improving the survival rate of patients. Despite the high incidence and mortality rate of NSCLC in Vietnam, the actionable mutation profiles of Vietnamese patients have not been thoroughly examined. Here, we employed massively parallel sequencing to identify alterations in major driver genes (EGFR, KRAS, NRAS, BRAF, ALK and ROS1) in 350 Vietnamese NSCLC patients. We showed that the Vietnamese NSCLC patients exhibited mutations most frequently in EGFR (35.4%) and KRAS (22.6%), followed by ALK (6.6%), ROS1 (3.1%), BRAF (2.3%) and NRAS (0.6%). Interestingly, the cohort of Vietnamese patients with advanced adenocarcinoma had higher prevalence of EGFR mutations than the Caucasian MSK-IMPACT cohort. Compared to the East Asian cohort, it had lower EGFR but higher KRAS mutation prevalence. We found that KRAS mutations were more commonly detected in male patients while EGFR mutations was more frequently found in female. Moreover, younger patients (<61 years) had higher genetic rearrangements in ALK or ROS1. In conclusions, our study revealed mutation profiles of 6 driver genes in the largest cohort of NSCLC patients in Vietnam to date, highlighting significant differences in mutation prevalence to other cohorts.

Evaluation of a Liquid Biopsy Protocol using Ultra-Deep Massive Parallel Sequencing for Detecting and Quantifying Circulation Tumor DNA in Colorectal Cancer Patients.

The identification and quantification of actionable mutations are critical for guiding targeted therapy and monitoring drug response in colorectal cancer. Liquid biopsy (LB) based on plasma cell-free DNA analysis has emerged as a noninvasive approach with many clinical advantages over conventional tissue sampling. Here, we developed a LB protocol using ultra-deep massive parallel sequencing and validated its clinical performance for detection and quantification of actionable mutations in three major driver genes (KRAS, NRAS and BRAF). The assay showed a 92% concordance for mutation detection between plasma and paired tissues and great reliability in quantification of variant allele frequency.

A new skink of the genus Scincella Mittleman, 1950 (Squamata: Scincidae) from Ba Den Mountain, Tay Ninh Province, southern Vietnam.

Ba Den is an isolated mountain in southern Vietnam and home to two endemic species of lizards. Herein, we describe another endemic species, a new skink of the genus Scincella Mittleman, 1950, from the area based on morphological data, including hemipenial characters and nucleotide sequences of COI. The following morphological characters diagnose Scincella badenensis sp. nov.: medium size in adults (snout-vent length up to 64.4 mm); toes reach to fingers when limbs adpressed; midbody scale rows 32-36, smooth; paravertebral scales 67-71; dorsal scales not enlarged; ventral scale rows 68-74; supraoculars four; prefrontals in broad contact with one another; loreal scales two; tympanum deeply sunk, without auricular lobules; two enlarged anterior temporal scales; smooth lamellae beneath toe IV 18-20; pair of enlarged precloacal scales; hemipenes short, smooth and forked near the tip with two short lobes and two small terminal papillae; no dorsal pattern in males; and females with black interruptive vertebral line. The new species differs from its congeners by at least 10.4% uncorrected p-distance in COI sequences.

A new species of Acanthosaura Gray 1831 (Reptilia: Agamidae) from central Vietnam.

Herein, we describe the new agamid species Acanthosaura phongdienensis sp. nov. from central Vietnam based on morphological and genetic data. Males of the new medium-sized species have a snout-vent length of up to 77.4 mm and females up to 64.7 mm. In both genders, the tail is longer than the snout-vent length. A spine occurs on each side of the neck, the rostral scute is entire, and the nuchal and dorsal crests are continuous. A postorbital spine is about half as long as the diameter of the eye-ball, and small lateral scales intermix with large, keeled scales whose tips point backwards and upwards. The new species differs genetically from the closely related species A. brachypoda, A. coronata, and A. lepidogaster by uncorrected p-distances of 13.6%, 21.7%, and 12.8%, respectively. This is the 13th species of Acanthosaura and the eighth species in the genus from Vietnam.

A new species of the genus Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from Tay Nguyen Plateau, Central Vietnam.

We describe a new species of Microhyla from Tram Lap forest, Gia Lai Province, Central Vietnam based on morphological, molecular, and acoustic data. The new species resembles M. butleri morphologically, but differs from all congeners by a combination of the following morphological attributes: (1) medium-sized adult snout-vent length 25.2-27.0 mm in 15 males and 30.5 mm in a single female; (2) body habitus moderately stocky; (3) head flat, snout rounded, slightly prominent in ventral profile; (4) dorsum and flanks slightly shagreened with evenly scattered tiny tubercles, ventral skin smooth; (5) first finger well developed, more than one-half the length of the second finger; (6) tips of three outer fingers slightly enlarged, forming weak disks and tips of all toes distinctly dilated into wide disks with narrow peripheral grooves; (7) finger and toe disks with dorsal median longitudinal grooves; (8) three palmar tubercles and two metatarsal tubercles; (9) tibiotarsal articulation of adpressed limb reaching slightly beyond the orbit; (10) webbing formula: I 1¾-2 II 1½-2¾ III 2-31/3 IV 3»-1½ V; (11) in life, chin and throat yellowish to bright-orange with tiny dark brown speckling laterally; and (12) a call consisting of 15-26 pulses with a dominant frequency of 1.8-2.2 kHz (recorded at 18.5ºC). We also provide a preliminary genealogy of Microhyla based on analysis of a 2644 bp fragment of 12S-16S rRNA mitochondrial DNA. Based on the examed data, the new species and M. butleri are sister-species (genetic p-distance: 9.0%) and it can be distinguished from M. butleri by its morphology (size, webbing on toes, color) and advertisement call. Interspecific genetic p-distances between the new species and its congeners vary from 9.0% to 14.8%. Microhyla aurantiventris sp. nov. occurs in evergreen montane tropical forests at elevations around 1200 m a.s.l. and is known only from the type locality. The new species appears to be threatened due to intensive logging and agriculture plantation.