First report and molecular identification of Opisthorchis viverrini infection in human communities from Lower Myanmar.

Opisthorchis viverrini is endemic in the South East Asian region, especially in Cambodia, Lao People's Democratic Republic, Vietnam and Thailand, but there have been no previous records from Myanmar. During stool surveys of rural populations in three regions of Lower Myanmar, Opisthorchis-like eggs were found in 34 out of 364 (9.3%) participants by stool microscopy after using the modified formalin-ether concentration technique. DNA was extracted from these positive stool samples and a portion of the mitochondrial cytochrome c oxidase subunit I (cox1) gene was amplified using the polymerase chain reaction and then sequenced. DNA sequences, successfully obtained from 18 of 34 positive samples (Bago Region, n = 13; Mon State, n = 3; Yangon Region, n = 2), confirmed that the eggs were of O. viverrini. Sequences showed 99.7% identity with O. viverrini mitochondrial cox1 (GenBank accession no. JF739555) but 95%, 88.7%, 82.6% and 81.4% identities with those of Opisthorchis lobatus from Lao People's Democratic Republic (GenBank accession nos. HQ328539-HQ328541), Metorchis orientalis from China (KT239342), Clonorchis sinensis from China (JF729303) and Opisthorchis felineus from Russia (EU921260), respectively. When alignement with other Opisthorchiidae trematodes, 81% similarity with Metorchis bilis from Czech Republic (GenBank accession nos. KT740966, KT740969, KT740970) and Slovakia (GenBank accession nos. KT740971-KT740973), 84.6% similarity with Metorchis xanthosomus from Czech Republic (GenBank accession no. KT740974), 78.6% similarity with M. xanthosomus from Poland (GenBank accession no. KT740968) and 82.2% similarity with Euamphimerus pancreaticus from Czech Republic (GenBank accession no. KT740975) were revealed. This study demonstrated, for the first time, O. viverrini from rural people in Myanmar using molecular methods and is an urgent call for surveillance and control activities against opisthorchiasis in Myanmar.

First Molecular Identifications of Necator americanus and Ancylostoma ceylanicum Infecting Rural Communities in Lower Myanmar.

Hookworms are enteric parasitic roundworms infecting an estimated 400 million persons worldwide. Herein, we provide the first molecular identifications of human hookworms from certain parts of rural Lower Myanmar. DNA was extracted from hookworm-positive stool samples, as determined by microscopy. DNA sequences of the partial internal transcribed spacer 1, full length 5.8S gene, and partial internal transcribed spacer 2 were determined and compared with available hookworm sequences from public databases. Of the 11 polymerase chain reaction-positive samples, eight (Bago Region, N = 4; Mon State, N = 4) yielded sequences with high similarity to those of Necator americanus A further three sequences (Mon State, N = 2; Bago Region, N = 1) showed high similarity with those of Ancylostoma ceylanicum The latter is primarily a parasite of dogs and represents a zoonosis. Given that different species of hookworms exhibit different epidemiological and biological characteristics, accurate identification is essential for the planning and execution of effective control programs for hookworm infections.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

First molecular identification and report of genetic diversity of Strongyloides stercoralis, a current major soil-transmitted helminth in humans from Lao People's Democratic Republic.

Strongyloidiasis is a major soil-transmitted helminth (STH) disease that affects people worldwide. We present updated data on prevalence in the Lao People's Democratic Republic (Lao PDR) in 2015, arising from a community cross-sectional helminthiasis survey. Fecal samples were collected from 327 individuals across three provinces in Lao PDR (Luang Prabang in the north, Khammouane in the center, and Champasack in the south). Agar plate culture and Kato-Katz methods were used to examine duplicate stool samples from each participant to detect Strongyloides stercoralis and co-infecting helminths. Overall prevalences of S. strercoralis human hookworm, Taenia spp., Trichuris trichiura, Ascaris lumbricoides, and Enterobius vermicularis were 41.0, 28.1, 4.9, 4.0, 1.5, and 0.9 %, respectively. The prevalence of miscellaneous trematodiases (including opisthorchiasis) was 37.9 % and of Schistosoma mekongi infection was 0.3 %. Strongyloidiasis is a current major STH disease in Lao PDR. We also report the molecular-phylogenetic identification of S. stercoralis adult males collected from 40 representative human strongyliodiasis fecal samples. DNA was extracted, amplified, and sequenced from a portion of the mitochondrial cox1 gene and the nuclear 18S ribosomal DNA. Phylogenetic analyses indicated that all specimens sequenced belonged to S. stercoralis (Bavay, 1876) Stiles and Hassall, 1902. The cox1 sequences exhibited great diversity (24 haplotypes) in Lao PDR. This is the first molecular identification and report of genetic diversity of S. stercoralis in humans from Lao PDR. An effective parasite control program is needed to reduce the serious health impacts.

Detection and quantification of Wuchereria bancrofti and Brugia malayi DNA in blood samples and mosquitoes using duplex droplet digital polymerase chain reaction.

Lymphatic filariasis, a mosquito-borne disease, is still a major public health problem in tropical and sub-tropical countries. Effective diagnostic tools are required for identification of infected individuals, for epidemiological assessment, and for monitoring of control programs. A duplex droplet digital polymerase chain reaction (ddPCR) was conducted to differentiate and quantify Wuchereria bancrofti DNA by targeting the long DNA repeat (LDR) element and Brugia malayi DNA by targeting the HhaI element in blood samples and mosquito vectors. The analytical sensitivity and specificity were evaluated. Our results indicated that the duplex ddPCR assay could differentiate and quantify W. bancrofti and B. malayi DNA from blood samples and mosquitoes. DNA from a single larva in 50 µl of a blood sample, or in one mosquito vector, could be detected. The analytical sensitivity and specificity for W. bancrofti are both 100 %. Corresponding values for B. malayi are 100 and 98.3 %, respectively. Therefore, duplex ddPCR is a potential tool for simultaneous diagnosis and monitoring of bancroftian and brugian filariasis in endemic areas.

Three Human Gnathostomiasis Cases in Thailand with Molecular Identification of Causative Parasite Species.

Human gnathostomiasis is one of the important food-borne parasitic zoonoses. The disease is caused by a spirurid roundworm of the genus Gnathostoma. Here, we describe three parasitological confirmed cases of human gnathostomiasis, caused by Gnathostoma spinigerum, in a hospital in Thailand during 2004-2012. Clinical characteristics, treatment, and outcome of cases were revealed. Parasites were accidentally recovered from patients and morphologically identified as Gnathostoma species. Confirmed diagnosis and identification of causative parasite species was made by DNA extraction of the recovered worms, followed by a polymerase chain reaction (PCR) of the second internal transcribed spacer region (ITS2) of DNA and the partial cytochrome c oxidase subunit 1 (cox-1) gene. Sequences corresponding to ITS2 and cox-1 were similar to G. spinigerum. To our knowledge, this study represents the first molecular confirmation that recovered G. spinigerum is a causative agent of human infection in Thailand.

PGE2 signaling and its biosynthesis-related enzymes in cholangiocarcinoma progression.

Prostaglandin E2 (PGE2) involves in progression of various chronic inflammation-related cancers including cholangiocarcinoma (CCA). This study aimed to determine the role of PGE2 signaling, its biosynthesis-related enzymes in a clinical prognosis, and their targeted inhibition in CCA progression. The immunohistochemical staining of cyclooxygenase (COX)-1, COX-2, mPGES-1, EP1, and EP4 was examined in CCA tissues, and their expressions were compared with clinicopathological parameters. The effect of PGE2 on levels of its signaling molecules was examined in CCA cell lines using proteome profiler array. The suppression of mPGES-1 using a small-molecule inhibitor (CAY10526) and small interfering RNA (siRNA) was determined for growth and migration ability in CCA cells. The results indicated that strong expressions of COX-1, COX-2, mPGES-1, EP1, and EP4 were found in CCA tissues as 87.5, 47.5, 52.5, 55, and 80 % of frequencies, respectively. High mPGES-1 expression was significantly correlated with tumor stages III-IV (p = 0.001), lymph node metastasis (p = 0.004), shorter survival (p = 0.009), and prognostic indicator of CCA patients (HR = 2.512, p = 0.041). Expressions of COX-1, COX-2, and EP receptors did not correlate with data tested from patients. PGE2 markedly enhanced protein levels of integrinα6, VE-cadherin, Jagged1, and Notch3, and CAY10526 suppressed those protein levels as well as PGE2 production in CCA cells. CAY10526 and siRNA mPGES-1 markedly suppressed mPGES-1 protein levels, growth, and migration abilities of CCA cell lines. In conclusion, PGE2 signaling strongly promotes CCA progression. Therefore, inhibition of PGE2 synthesis by suppression of its biosynthesis-related enzymes could be useful for prevention and treatment of CCA.

MOLECULAR EVIDENCE OF SPIROMETRA ERINACEIEUROPAEI INFECTION IN SNAKES PTYAS KORROS FROM LAO PDR AND THAILAND AND FROGS HOPLOBATRACHUS RUGULOSUS FROM MYANMAR.

Sparganosis is a parasitic disease in humans and animals caused by plerocercoid larvae (spargana) of the genus Spirometra. Spirometra erinaceieuropaei is the major causative agent of the disease in Asian countries. However, molecular evidence of the causative parasite species in animals remains lacking. A total of 19 spargana specimens were obtained from frogs, Hoplobatrachus rugulosus, collected from Myanmar and snakes, Ptyas korros, from Lao PDR and Thailand. A partial sequence of mitochondrial cytochrome c oxidase subunit1 gene (cox1) was amplified, sequenced, and the phylogenetic relationship was constructed using maximum likelihood method. Results revealed that the level of nucleotide variations in the partial cox1 sequence (429 bp) among the spargana ranged 0-3.5%, with 15 variable sites. Phylogenetic analysis indicated that all spargana specimens were S. erinaceieuropaei. This is the first report of S. erinaceieuropaei in P. korros from Lao PDR and Thailand and H. rugulosus from Myanmar. The results emphasize the need for prevention and control of sparganosis in these regions.

Anti-inflammatory agents suppress the prostaglandin E2 production and migration ability of cholangiocarcinoma cell lines.

Prostaglandin E2 (PGE2), one of the products catalyzed by cyclooxygenases (COXs), could actuate several pathways implicated in chronic inflammation-related cancer, including apoptosis evasion, cell proliferation, migration and angiogenesis. We hypothesized that blocking of PGE2 production might be an effective strategy to attenuate the progression of cholangiocarcinoma (CCA). Thus, the aim of this study was to examine the effects of two anti-inflammatory agents, meloxicam, a selective COX-2 inhibitor, and xanthohumol, a natural plant extract, on PGE2 production and migration ability of human CCA cell lines. The results showed that 100 µM of meloxicam and 10 µM of xanthohumol suppressed the PGE2 level in the cultured media and wound-induced migration of human CCA cell lines, M139 and M214. The present results revealed that meloxicam and xanthohumol have potential to suppress PGE2 production and cell migration. These findings may offer alternative approaches for chemoprevention and therapy of CCA.

Cytokines released from activated human macrophages induce epithelial mesenchymal transition markers of cholangiocarcinoma cells.

Stromal-epithelial interactions are important for carcinogenesis. Once cancerous lesions develop, a chronically inflamed tumor microenvironment promotes migration and invasion of tumor cells. Multiple immune cell populations are involved in inflammatory processes, including tumor-associated macrophages (TAMs) which have been proposed as major contributors to tumor progression. The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells trans-differentiate and acquire an invasive mesenchymal phenotype. As EMT represents a crucial step in disease progression, it is important to investigate the mechanisms regulating this step. We aimed to identify the profiles of cytokines produced by activated human macrophages and to demonstrate effects on the expression of EMT-related genes in human cholangiocarcinoma (CCA) cell lines. Our results showed that LPS-activated macrophages produced and secreted IL4, IL6, IL10, TNF-α and TGF-ß1. After addition of macrophage conditioning media to CCA cells, expression of epithelial markers E-cadherin and CK-19 was significantly reduced, whereas the expression of mesenchymal markers, S100A4 and MMP9 was strongly induced. Taken together, various cytokines secreted by activated macrophages could induce EMT by altering the expression of EMT-related genes in CCA.