Spirometra species from Asia: Genetic diversity and taxonomic challenges.

Despite considerable controversy concerning the taxonomy of species within the genus Spirometra, human sparganosis and spirometrosis mainly in Asia and Europe has long been confidently ascribed to Spirometra erinaceieuropaei. Recently, the mitochondrial genomes of purported "S. erinaceieuropaei", "Spirometra decipiens" and "Spirometra ranarum" from Asia have been determined. However, it has been pointed out that the morphological criteria used for identifying these species are unsuitable and thus these identifications are questionable. In the present study, therefore, Spirometra samples from Asia were re-examined based on mitochondrial cytochrome c oxidase subunit 1 gene sequences and the identification of these species was discussed. Haplotype network and phylogenetic analyses revealed that: i) two distinct Spirometra species, Type I and Type II, are present in Asia and neither of which is close to likely European "S. erinaceieuropaei"; ii) Type I is genetically diverse and widely distributed, however Type II is known so far from Japan and Korea; iii) "S. decipiens" and "S. ranarum" reported from Asia are conspecific with Type I; iv) Type I is probably conspecific with Spirometra mansoni, and Type II may represent an undescribed species.

Molecular identification and genetic diversity of Gnathostoma spinigerum larvae in freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Gnathostomiasis, an emerging food-borne parasitic zoonosis in Asia, is mainly caused by Gnathostoma spinigerum (Nematoda: Gnathostomatidae). Consumption of raw meat or freshwater fishes in endemic areas is the major risk factor. Throughout Southeast Asia, including Thailand, Lao PDR, Cambodia, and Myanmar, freshwater fish are often consumed raw or undercooked. The risk of this practice for gnathostomiasis infection in Lao PDR, Cambodia, and Myanmar has never been evaluated. Here, we identified larvae of Gnathostoma species contaminating freshwater fishes sold at local markets in these three countries. Public health authorities should advise people living in, or travelling to, these areas to avoid eating raw or undercooked freshwater fishes. Identification of larvae was done using molecular methods: DNA was sequenced from Gnathostoma advanced third-stage larvae recovered from snakehead fishes (Channa striata) and freshwater swamp eels (Monopterus albus). Phylogenetic analysis of a portion of the mitochondrial cytochrome c oxidase subunit I gene showed that the G. spinigerum sequences recovered from southern Lao PDR, Cambodia, and Myanmar samples had high similarity to those of G. spinigerum from China. Sequences of the nuclear ribosomal DNA internal transcribed spacer 2 region closely resembled sequences of G. spinigerum from Thailand, Indonesia, the USA, and central Lao PDR. This is the first molecular evidence of G. spinigerum from freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Reduced ADAM8 levels upon non-surgical periodontal therapy in patients with chronic periodontitis.

OBJECTIVE: To determine effect of non-surgical periodontal treatment on a disintegrin and metalloproteinase 8 (ADAM8) levels in gingival crevicular fluid (GCF) of patients with chronic periodontitis (CP) in comparison with those of patients with gingivitis and to find correlations between ADAM8 levels and clinical parameters. DESIGN: Twenty-two and eleven patients with CP and gingivitis, respectively, were examined for four clinical parameters, probing depth, clinical attachment level, gingival and plaque indices. GCF from the selected gingivitis or periodontitis sites with distinct severities was sampled by Periopaper strips. The non-surgical treatments, including scaling and/or root planing and oral hygiene instruction, were provided for all patients. Clinical measurements and GCF sampling were repeated at three months after the treatments. ADAM8 concentrations were analyzed by ELISA and normalized by GCF volumes or total protein amounts. RESULTS: All patients exhibited significant improvement of almost every clinical parameter after treatment, whereas the median ADAM8 concentrations were significantly decreased at the moderate and severe periodontitis sites of patients with CP (p < 0.05). Moreover, the significantly positive correlations between ADAM8 concentrations and four clinical parameters were found in both moderate and severe groups (p < 0.05). CONCLUSION: ADAM8 concentrations were decreased by non-surgical periodontal therapy in patients with chronic periodontitis at the moderate and severe sites and were correlated with four clinical parameters, implying that GCF ADAM8 levels reflect inflammatory and bone-resorbing activities in the periodontal pocket.

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand, Lao PDR, and Myanmar.

Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides, has a worldwide distribution. Here, we have provided the first molecular identification of Ascaris eggs and adults recovered from humans and pigs in Thailand, Lao PDR, and Myanmar. We amplified and sequenced nuclear ribosomal DNA (ITS1 and ITS2 regions) and mitochondrial DNA (cox1 gene). Sequence chromatograms of PCR-amplified ITS1 region revealed a probable hybrid genotype from two human ascariasis cases from Chiang Mai Province, northern Thailand. All complete ITS2 sequences were identical and did not differ between the species. Phylogenetic trees and haplotype analysis of cox1 sequences showed three clusters with 99 haplotypes. Forty-seven samples from the present study represented 14 haplotypes, including 7 new haplotypes. To our knowledge, this is the first molecular confirmation of Ascaris species in Thailand, Lao PDR, and Myanmar. Zoonotic cross-transmission of Ascaris roundworm between pigs and humans probably occurs in these countries.

Human liver fluke Opisthorchis viverrini (Trematoda, Opisthorchiidae) in Central Myanmar: New records of adults and metacercariae identified by morphology and molecular analysis.

Opisthorchis-like metacercariae were found in cyprinoid fish, Puntius brevis, bought from markets in the Bago region, Central Myanmar. Adult worms recovered from experimentally-infected hamsters resembled Opisthorchis viverrini. DNA was extracted from adults and metacercariae. A portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the internal transcribed spacer 2 (ITS2) regions were amplified using the polymerase chain reaction and then sequenced. The sequences confirmed that the flukes were O. viverrini. In phylogenetic analyses, sequences of O. viverrini, including our new sequences, clustered in a group with high bootstrap support for ITS2 (80%) and the cox1 gene (99%). Interestingly, ITS2 and cox1 sequences of O. viverrini and O. lobatus were very similar, raising a question about the identity of the latter. This is the first report of O. viverrini in cyprinoid fish in Central Myanmar, and only the second report of the species in Myanmar. It is an urgent warning against consuming raw or semi-cooked freshwater fish dishes. Development of an effective food-safety strategy should be provided for the prevention and control of opisthorchiasis and other foodborne diseases.

O-GlcNAcylation in oral squamous cell carcinoma.

BACKGROUND: Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked ß-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. METHODS: Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFRtyr1173 , and anti-phosphorylated-Aktser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. RESULTS: The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 were significantly higher in OSCC than normal tissues (P < .001 and P < .01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 (P < .01 and P < .001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. CONCLUSION: Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC.

Overexpression of ADAM9 in oral squamous cell carcinoma.

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) has been shown in various types of cancer. Some studies have reported inconclusive findings regarding chromosomal aberrations in the ADAM9-containing region and ADAM9 expression in oral cancer. Therefore, in this study, ADAM9 protein expression was determined and compared between oral squamous cell carcinoma (OSCC) and normal oral tissues, and between oral cancer cell lines and human oral keratinocytes (HOKs). In total, 34 OSCC and 10 healthy paraffin-embedded tissue sections were probed with an anti-ADAM9 antibody, and the immunohistochemical score was determined by multiplying the percentage of positively stained cells with the intensity score. Four different oral cancer and eight independent HOK cell lines were cultured, and the expression of membrane ADAM9 and active ADAM9 at 84 kDa in these cell lines was assayed by flow cytometry and western blot hybridization, respectively. The results showed that the median immunohistochemical score of ADAM9 expression in OSCC tissues was significantly greater than that in normal tissues (P<0.001). Furthermore, among OSCC cases, intense staining of ADAM9 expression was detected in well-differentiated and in moderately-differentiated OSCC; ADAM9 expression was also correlated with an increased degree of cell differentiation (r=0.557; P=0.001). Expression of membrane ADAM9 was present in 3/4 cancer cell lines. Expression of active ADAM9 varied among all the tested cell lines, but significantly higher ADAM9 expression was present in certain cancer cell lines than those in HOKs (P<0.05). In summary, ADAM9 expression is enhanced in OSCC and oral cancer cell lines, suggesting its role in the pathogenesis of oral cancer. Similar to the overexpression of ADAM9 in well-differentiated prostate cancer, high degrees of ADAM9 expression have also been observed in well-differentiated OSCC.

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.