Porntip Laummaunwai

A Cross-Sectional Study on Intestinal Parasitic Infections in Rural Communities, Northeast Thailand

Despite the existence of effective anthelmintics, parasitic infections remain a major public health problem in Southeast Asia, including Thailand. In rural communities, continuing infection is often reinforced by dietary habits that have a strong cultural basis and by poor personal hygiene and sanitation. This study presents a survey of the prevalence of intestinal parasitic infections among the people in rural Thailand. The community-based cross-sectional study was conducted in villages in Khon Kaen Province, northeastern Thailand, from March to August 2013. A total of 253 stool samples from 102 males and 140 females, aged 2-80 years, were prepared using formalin-ethyl acetate concentration methods and examined using light microscopy. Ninety-four individuals (37.2%) were infected with 1 or more parasite species. Presence of parasitic infection was significantly correlated with gender (P=0.001); nearly half of males in this survey (49.0%) were infected. Older people had a higher prevalence than younger members of the population. The most common parasite found was Opisthorchis viverrini (26.9%), followed by Strongyloides stercoralis (9.5%), Taenia spp. (1.6%), echinostomes (0.4%), and hookworms (0.4%). The prevalence of intestinal protozoa was Blastocystis hominis 1.6%, Entamoeba histolytica 0.8%, Entamoeba coli 0.8%, Balantidium coli 0.4%, Iodamoeba bütschlii 0.4%, and Sarcocystis hominis 0.4%. Co-infections of various helminths and protozoa were present in 15.9% of the people. The present results show that the prevalence of parasitic infections in this region is still high. Proactive education about dietary habits, personal hygiene, and sanitation should be provided to the people in this community to reduce the prevalence of intestinal parasite infections. Moreover, development of policies and programs to control parasites is needed.

Anti-inflammatory, antioxidant and hepatoprotective effects of Thunbergia laurifolia Linn. on experimental opisthorchiasis

Thunbergia laurifolia Linn (Rang Chuet) possesses antioxidant and anti-inflammatory properties as well as anticancer activities. The aim of the present study was to evaluate the efficacy of T. laurifolia in reducing inflammation from pathological changes in Syrian hamsters infected with the human liver fluke Opisthorchis viverrini. Hamster groups were also administered N-nitrosodimethylamine (NDMA) and treated with T. laurifolia. Light microscopic observation of histopathological changes, liver function tests for alanine transaminase (ALT) and alkaline phosphatase (ALP) and kidney function tests for blood urea nitrogen (BUN) and creatinine were performed. Antioxidant effects of both fresh and dried Rang Chuet solutions were observed. Analysis of the histopathological changes showed anti-inflammatory properties, both in the case of O. viverrini infection or with NDMA administration, by reducing the aggregation of inflammatory cells surrounding the hepatic bile ducts as indicated by normal serum ALT, ALP, BUN and creatinine levels in treated Syrian hamsters. The present study found that fresh and dried Rang Chuet solutions clearly reduced the inflammatory cells in both O. viverrini-infected and NDMA-administered groups and was correlated with the total antioxidant capacity. These findings suggest that T. laurifolia possesses antioxidant and anti-inflammatory properties and that its application may be useful for prevention of the inflammatory process, one of the risk factors of O. viverrini-associated cholangiocarcinoma (CCA).

Down-Regulated Expression of HSP70 in Correlation with Clinicopathology of Cholangiocarcinoma

Cholangiocarcinoma is a crucial health problem in northeast Thailand. Although rare, it is a highly fatal disease and the prognosis of CCA patients is very poor. To determine if expression of specific genes is useful for diagnosis and prognosis for CCA. We examined the expression of HSP70, HSP90, RB1, cyclin D1, and HDAC6 in 50 resections of human CCA tissues by quantitative real-time PCR. The expression of HSP70, RB1, and HDAC6 was “dominant down-regulation,” while the expression of cyclin D1 and HSP90 was “dominant up-regulation.” There were no correlations between RB1, cyclin D1, HSP90, and clinicopathological parameters such as status, histology type, histological grading, stage of CCA, and metastasis. A significant association was found between HDAC6 and CCA staging (p = 0.000), CCA gross type and HSP70 (p = 0.046) as well as RB1 expression (p = 0.046). Patients with down-regulation of HSP70 had significantly poorer prognosis than those in the up-regulation group (p = 0.002). Expression of HSP70 may be useful as a new prognostic marker for CCA.

Molecular Detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.

Tumor-Related Gene Changes in Immunosuppressive Syrian Hamster Cholangiocarcinoma

The results of a previous study demonstrated that prednisolone enhanced cholangiocarcinogenesis. Therefore, to clarify molecular changes during immunosuppressive cholangiocarcinogenesis, Syrian hamsters were divided into 8 groups: uninfected controls; immunosuppressed Syrian hamsters using prednisolone (P); normal Syrian hamsters administered N-nitrosodimethylamine (ND); immunosuppressed Syrian hamsters administered N-nitrosodimethylamine (NDis); normal Syrian hamsters infected with Opisthorchis viverrini (OV); immunosuppressed Syrian hamsters infected with O. viverrini (OVis); normal Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCA); and immunosuppressed Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCAis). Syrian hamster livers were used for analysis of tumor-related gene expression and immunohistochemistry through cytokeratin 19 (CK19) and proliferating cell nuclear antigen (PCNA) staining. The tumor-related gene expression results show that CCAis groups at all time points exhibited upregulation of COX-2, IL-6, SOD1, CAT and iNOS and downregulation of p53, which correlated with the predominant expression of CK19 and PCNA in liver tissue. These results suggest that prednisolone enhances cholangiocarcinoma development, which was confirmed by molecular changes.

A Hospital-Based Study of Epidemiological and Clinical Data on Blastocystis hominis Infection

Blastocystis hominis is a foodborne protozoan found in the human feces worldwide. One hundred and ninety-nine individuals with stool samples positive for B. hominis were identified from a pool of 14,325 patient stools collected between 2003 and 2010 from Srinagarind hospital in Thailand. The medical records of patients were reviewed for demographic and clinical data. Of the 85 patients (42.7%) who had B. hominis infection with no co-infections, 42.5% experienced gastrointestinal symptoms. Abdominal pain is the most frequently observed symptom followed by diarrhea. Strongyloides stercolaris and Opisthorchis viverrini were the predominant parasitic co-infections in blastocystosis patients. The infection rates of B. hominis were high during the rainy season. Most B. hominis-infected patients (94%) had underlying diseases; malignancy and chronic diseases were equally top ranked (35.3%) which indicated that B. hominis is an opportunistic protozoan.

Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails.

Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies.

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Effects of fermentation time and low temperature during the production process of Thai pickled fish (pla-som) on the viability and infectivity of Opisthorchis viverrini metacercariae

Contamination of a popular fermented fish dish, pla-som, by Opisthorchis viverrini metacercariae (OVMC) is a possible cause of carcinogenic liver fluke infection in Thailand. Affected individuals are at risk of bile duct cancer, which is a major health problem for people in the Greater Mekong Subregion. In order to investigate concerns about food safety, we studied the effects of fermentation time and low temperature on the viability and infectivity of OVMC during the pla-som production process. Pla-som was prepared at room temperature for up to 1 week in duplicate experiments using cyprinid freshwater fish obtained from an O. viverrini-endemic area. OVMC were then isolated and identified under a stereomicroscope. Complete and viable OVMC were found on days 1-4 of fermentation, while their morphology was degenerated thereafter. After OVMC were fed to hamsters, the percentage of the worm recovery after 1 to 2 months of infection was 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively. In order to measure the effect of low temperature on OVMC, fish were kept in a refrigerator (4 °C) for up to five days and then subsequently fermented for three days. In fish stored in a refrigerator for 1 and 2 days, viable OVMC were clearly observed and were able to infect hamsters, a worm-recovery percentage of 3.3% and 12.7%, respectively. By contrast, in pla-som prepared from fish stored for 3 to 5 days, OVMC were degenerated and could not infect the host. In conclusion, pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing can prevent O. viverrini infection. This study may increase awareness of fermented-fish dish preparation to prevent liver fluke infection.

Evaluation of IgG4 Subclass Antibody Detection by Peptide-Based ELISA for the Diagnosis of Human Paragonimiasis Heterotrema

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.