Asian Neglected Tropical Disease Conference
Asian Neglected Tropical Disease Conference NTDASIA2019 International Research Network ABSTRACTS & PROCEEDINGS NTD ASIA 2019 Pullman Raja Orchid Hotel, Khon Kaen, Thailand NTDASIA2019
Pullman Raja Orchid Hotel Khon Kaen, Thailand 7-9 August 2019 7-9 August 2019 7-9 August
Organized by Tropical Disease Research Center Faculty of Medicine, Khon Kaen University, Thailand Tropical Disease Research Center Fax: +66-43-204359 Tel: +66-43-363113 Faculty of Medicine, Khon Kaen University, Thailand
2 NTDASIA 2019 i
Welcome message Welcome to NTDASIA2019
Dear colleagues and guests,
On behalf of the organizing committee and KKU, the hosting institution, it is my great pleasure to welcome you to Khon Kaen, Thailand, for the 5th Asian Neglected Tropical Diseases Conference (NTDASIA2019).
The Meeting theme this year, “International Research Network” will highlight international cooperation to tackle NTDs based on evidence-based research. The NTDASIA2019 scientific program will foster discussion and hopes to inspire participants from a wide array of themes to initiate collaborations within and across disciplines for the advancement of our field.
The various thematic sessions will showcase important scientific advances and highlight impacts of NTDs in a world of fast changes and complex interactions. The conference program will cover a large range of tropical diseases, especially those endemic to Asia including dengue, helminthic infections, bacterial, viral, fungal and other parasitic diseases. The fields of the diseases include, but not limited to, epidemiology, biology, molecular biology, pathogenesis, clinical, vaccine & drug development and control.
We would like to strongly encourage NTDs academia and Tropical Medicine professionals from Asia and outside to join the conference. Your participation will add valued diversity and greater insight from endemic areas. To help facilitate your participation, the NTDASIA2019 will have no registration and other fees.
The conference will be held in NTDs endemic Khon Kaen, Thailand for two days conference plus a one-day workshop on integrated NTD control. Our website, www.ntdasia.org, has all the details you need to book your place, submit your abstract and find out more about the event.
We greatly look forward to seeing you all at NTDASIA2019 in Khon Kaen, 7-9 August 2019!
Yours sincerely,
Banchob Sripa
Chairman, NTDASIA2019 ii NTDASIA 2019
Asian Neglected Tropical Disease Conference (NTDASIA)
Neglected Tropical Diseases (NTDs) affect over 1 billion people, and cause 170,000 deaths a year in poor and vulnerable populations mainly in tropical countries. WHO has updated a list of NTDs to 20 diseases (2017), seven of which are caused by helminths namely soil-transmitted helminthiases (STH), schistosomiasis, foodborne trematodiases, lymphatic filariasis, taeniasis/cysticercosis, guinea-worm diseases and onchocerciasis. The first five helminths in the list plus other parasites are still endemic in eastern Asian countries, especially the ten member states of the Association of Southeast Asian Nations (ASEAN). ASEAN constitutes an economic powerhouse, yet these countries also harbor a mostly hidden burden of poverty and NTDs. Almost 200 million people live in extreme poverty, mostly in the low or lower middle-income countries of Indonesia, the Philippines, Myanmar, Viet Nam, and Cambodia, and many are affected by at least one NTD.
However, NTDs are also prevalent among upper middle-income ASEAN countries such as Malaysia and Thailand, especially among the indigenous populations. The three major intestinal helminth infections are the most common NTDs; each helminthiasis is associated with approximately 100 million infections in the region. In addition, more than 10 million people suffer from either liver or intestinal fluke infections, as well as schistosomiasis and lymphatic filariasis. However, these Asian helminthic diseases are neglected by WHO, donors, researchers or even local governments which rank the diseases as low priorities in terms of control activities/budgets compared to other emerging or non-communicable diseases. Our Asian Neglected Tropical Diseases Network (NTDASIANET), a group of researchers and practitioners in Asia and experts outside, has been committed to study the biology, disease pathogenesis, diagnosis, treatment, transmission, and control of the Asian NTDs for decades. Our milestone is to improve our understanding of these critically important public health NTDs and provide effective research-based NTD control and elimination.
The main platform for communication and collaboration among members, apart from research, is an annual meeting to update scientific advances in NTD research named Asian Neglected Tropical Disease Conference (NTDASIA). There were 4 NTDASIA conferences over the past years, the latest, NTDASIA2018, was held back to back with ICOPA2018 in Daegu, Korea. This time, NTDASIA2019 will be held at the home city of Khon Kaen, Thailand during 7-9 August 2019. NTDASIA 2019 iii
Conference Committee
Advisory Committee President, Khon Kaen University Dean, Faculty of Medicine, Khon Kaen University Director, the Thailand Research Fund Director, Academic Research Division, the Thailand Research Fund
Organizing Committee
Banchob Sripa Petrus Tang Chairman, NTDASIA2019 Committee
Yukifumi Nawa Thewarach Laha John F. Smith Editor Committee Committee
Sutas Suttiprapa Prasert Saichua Nantana Suwandittakul Committee Committee Committee
Kanin Salao Sirikachorn Tangwattana Sangduan Wannachart Committee Secretariat Secretariat Assistant iv NTDASIA 2019
NTDASIA2019 Proceedings Editorial Board
Prof. Yukifumi Nawa Editor-in-Chief, Parasitology International (Elsevier)
Editor-in-Chief Yukifumi Nawa TDRC, MD-KKU ([email protected])
Editorial Board Overseas Ted (Chia-Kwung) Fan Taipei Medical University, Taiwan ([email protected]) Sung-Jung Hong Chung-Ang University, Korea ([email protected]) Ayako Yoshida VM, University of Miyazaki, Japan ([email protected])
KKU John F. Smith PH-KKU ([email protected]) Prasarn Tangkawattana VM-KKU ([email protected]) Thewarach Laha MD-KKU ([email protected]) Kanin Salao MD-KKU ([email protected]) Sutas Suttiprapa TDRC MD-KKU ([email protected]) Prasert Saichua TDRC MD-KKU ([email protected]) Nantana Suwandittakul TDRC MD-KKU ([email protected]) NTDASIA 2019 v
Keynote speaker Professor Jong-Yil Chai
Professor Jong-Yil Chai President, the World Federation of Parasitologists
Prof. Dr. Jong-Yil Chai graduated from Seoul National University College of Medicine, Seoul, the Republic of Korea and obtained his M.D. in 1976. He finished his Ph.D. at the same university in 1984 majoring clinical parasitology. He has worked at Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine for 40 years as Instructor, Assistant Professor, Associate Professor, and full Professor. Now he is Professor Emeritus at Department of Tropical Medicine and Parasitology, Seoul, Korea. He has been investigating diseases caused by intestinal parasites, including flukes, roundworms, and protozoans for over 40 years in Korea. He also worked in other Asian countries, including China, Thailand, Lao PDR, Cambodia, Vietnam, Myanmar, Indonesia, and in several countries in Africa (especially Tanzania). He has published more than 600 original research articles on parasitology and contributed over 30 books or book chapters. He has been serving as the Editor of Korean Journal of Parasitology (since 2006). He is currently the President of World Federation of Parasitologists (since 2018) and the Treasurer of International Federation for Tropical Medicine (since 2012). He is also serving as the President of Korea Association of Health Promotion (since 2016). vi NTDASIA 2019
Scientific Program Asian Neglected Tropical Disease Conference (NTDASIA2019) “International Research Network” Ballroom 1, Pullman Khon Kaen Raja Orchid, Khon Kaen, Thailand August 7-9, 2019
Wednesday 7 August 2019
8.30-9.00 Registration 9.00-9.15 Opening Ceremony Report: Chairman of the NTDASIA2019 Conference Dr. Banchob Sripa Welcome Address: - Vice President for Research Affairs Dr. Monchai Duangjinda - President, Taiwan Society of Parasitology Dr. Petrus Tang Opening Address: President of Khon Kaen University - Dr. Charnchai Panthongviriyakul
9.15-9.30 Group photo
Session 1 Keynote speaker Chairperson: Dr.Petrus Tang
9.30-10.15 Major Neglected Tropical Diseases in Asia Jong - Yil Chai (President of the World Federation of Parasitologists)
10.15-10.30 Coffee Break
Session 2 Asian neglected tropical diseases I – Zoonotic & foodborne trematodes Chairperson: Dr. Sung-Tae Hong
10.30-10.50 Neglected zoonotic helminths in Vietnam: country emergence and insights into genomics, diagnostics and molecular epidemiology (Thanh Hoa Le, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam) 10.50-11.10 Opisthorchis felineus infection in Western Siberia: update on prevalence, risk factors and impact on human health (Olga Fedorova, Siberian State Medical University, Tomsk, Russia) 11.10-11.30 Is Opisthorchis viverrini infection endemic in Cambodia? (Kazuko Miyamoto, Faculty of Medicine, University of Yamanashi, Japan) NTDASIA 2019 vii
11.30-11.50 Update on Opisthorchis felineus research data (Viatcheslav Mordvinov, Institute of Cytology & Genetics, RAS, Novosibirsk, Russia) 11.50-12.10 The genome of the liver flukeFasciola gigantica present novel protein kinases as drug targets (Timir Tripathi, North-Eastern Hill University, Shillong, India)
12.10-13.20 Lunch
Session 3 Liver fluke International Research Network Chairperson: Dr. Banchob Sripa
13.20-13.40 Oncogenesis of Clonorchis sinensis (Sung-Tae Hong, Seoul National University, Seoul, Korea) 13.40-14.00 Clonorchis sinensis: prepare for and chemotactically migrate to bile duct (Sung-Jong Hong, Chung-Ang University, Seoul, Korea) 14.00-14.20 Experimental model of Opisthorchis felineus liver fluke infection: mechanistic insights into biliary neoplasia formation (Maria Pakharukova, Institute of Cytology & Genetics, RAS, Novosibirsk, Russia) 14.20-14.40 Diagnosis of Clonorchis sinensis infection (Men-Bao Qian, Institute of Parasitic Diseases, Shanghai, PR China) 14.40-15.00 Update on Opisthorchis viverrini glycoconjugates (Sutas Suttiprapa, Tropical Medicine Graduate Program, Faculty of Medicine, Khon Kaen University, Thailand)
15.00-15.15 Coffee break
Session 4 Poster round and relax Chairperson: Dr. Thewarach Laha
15.15-17.00 Poster presentation with snack
18.00-21.00 Welcome Dinner
Thursday 8 August 2019
Session 5 Asian neglected tropical diseases II – Helminth control and consequences Chairperson: Dr. Siti Nursheena Mohd Zain
8.30-8.50 Post-Era MDA: an update on Intestinal Parasitic Infections (IPI) among selected urban poor communities in West Coast of Peninsular Malaysia (Siti Nursheena Mohd Zain, Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia) 8.50-9.10 Diagnosis and drug resistance of human soil-transmitted helminth infections: A public health perspective (Veeranoot Nissapatorn, Walailuk University, Nakhon Si Thammarat, Thailand) viii NTDASIA 2019
9.10-9.30 Assessment of the burden of soil-transmitted helminthiasis after five years of mass drug administration for lymphatic filariasis in Banggai District, Central Sulawesi (Helena Pangaribuan, Ministry of Health Republic Indonesia, Indonesia) 9.30-9.50 Progress toward schistosomiasis elimination in Lao People’s Democratic Republic: a long-term evaluation of impact on integrated control approach (Somphou Sayasone, Laos TPHI, Lao PDR) 9.50-10.10 Deworming among pregnant women in Myanmar: a simple, effective but forgotten public health intervention (Ye-min Htet, Neglected Tropical Diseases Support Center, Task Force for Global Health, Atlanta, USA) 10.10-10.25 Socioeconomic factors associated with prevention measure against filariasis in Banyuasin, South Sumatra (Rini Mutahar, Sriwijaya University, Indonesia) 10.25-10.40 Adverse effects following mass drug administration for lymphatic filariasis elimination in Banyuasin, South Sumatra (Desheila Andarini, Sriwijaya University, Indonesia)
10.40-10.50 Coffee Break
Session 6 Asian neglected tropical diseases III – Vector-borne diseases Chairperson: Dr. Xiaoguang Chen
10.50-11.10 The epidemic and vector control of dengue in China (Xiaoguang Chen, Southern Medical University, Guangzhou, China) 11.30-11.30 Lethal but safe: efficacy of the artificial breeding trap againstAedes albopictus under the laboratory and the field Study (Yugu Xie, Southern Medical University, Guangzhou, China) 11.30-11.50 Current insecticide-resistant status of dengue haemorragic fever vector populations in Indonesia: a systematic review of literature (Shifa Fauziyah, Laboratory of Entomology, Institute of Tropical Disease, Universitas Airlangga, Indonesia) 11.50-12.10 Benefit using transparent wall compare to using not transparent wall of tropic trap anti mosquito in Manado City, North Sulawesi, Indonesia (Joy Albert Sumakul, Health Office Manado, North Sulawesi, Indonesia)
12.10-13.00 Lunch
Session 7 Asian neglected tropical diseases IV – Zoonoses and other NTDs Chairperson: Dr.Keeseon Eom
13.00-13.20 On the origin of Taenia asiatica (Keeseon Eom, Chungbuk National University School of Medicine, Korea) 13.20-13.40 Neurocysticercosis, the parasitic disease in people and pigs: the present situation in Asia and towards eradication from Bali, Indonesia (Akira Ito, Asahikawa Medical University, Japan) NTDASIA 2019 ix
13.40-14.00 Genome evolution of trichomonadidae (Petrus Tang, College of Medicine, Chang Gung University, Taiwan) 14.00-14.15 Development of omics-based new diagnostic tools for schistosomiasis in China (Wei Hu, School of Life Sciences, Fudan University, Shanghai, China) 14.15-14.30 Possible transmission of human schistosomiasis in previously nonendemic and under-reported settings: a rapid evidence product for knowledge synthesis (Khin Thet Wai, Department of Medical Research and Department of Public Health, Myanmar) 14.30-14.45 Rickettsia infection in Sri Lanka. Epidemiology and clinical impacts (Rajapakse RPVJ, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka) 14.45-15.00 Laboratory diagnosis of leptospirosis- a way forward (Noraziah Mohamad Zin, Faculty of Health Sciences, Universiti Kebangsaan)
15.00-15.15 Coffee Break
Session 8 Symposium on “Risk determinants in NTD control: Liver fluke case study”
15.15-15.35 PRECEDE-PROCEED model framework for controlling Opisthorchis viverrini infection: community-based intervention with propensity score matching analysis (Picha Suwannahitatorn, Phramongkutklao College of Medicine, Bangkok, Thailand) 15.35-15.55 M&Ms - Masculinity and Multi-levels of Information Dissemination - are contributing to liver fluke infection risk in Thailand (Yi-Chen Wang, National University of Singapore, Singapore) 15.55-16.15 Sub-district health management evaluation of liver flukes and cholangiocarcinoma surveillance, prevention and control in 7th health Area (Kesorn Thaewnongiew, Office of Disease Prevention and Control Region 7 Khon Kaen, Thailand)
16.15-16.30 Way forwards: Future collaboration in Asian NTDs
· Poster award event · Closing Remark
Dr. Banchob Sripa x NTDASIA 2019
Friday 9 August 2019
8.00 Ready for Departure to Lawa village (40 km from KK) 9.00 Arrive at Lawa Lake
- Group photo at the Lawa Lake front
9.30 Visit Lawa Health Promoting Hospital (HPH) - Welcome address by Director of the HPH - Overview of liver fluke, cancer and the Lawa model (Dr.Banchob Sripa) - Demonstration of “Lawa model” an integrated liver fluke control programme by village health volunteers & enjoy! - Visit Lawa School. Demonstration of “Liver fluke-free school” - Visit fermented fish producing households – the risk food of infection - Group photo
12.00 Lunch at Lawa HPH
13.00 Liver fluke control program at Lawa Lake
- GPS tracking in animal reservoirs (Dr.Sirikachorn Tangkawattana) - Tour around Lawa Lake, rural northeastern environment and lifestyle - Catching fish from the lake (fisherman) - Cooking raw fish spicy salad “koi pla”
15.30 Back to Khon Kaen 1
Oral Presentation 2 NTDASIA 2019 3
O:01 NTDs and Their Public Health Significance in Asia: An Overview
Jong-Yil Chai1,2
Abstract: Neglected Tropical Diseases (NTDs) include a variety 1Institute of Parasitic Diseases, of tropical diseases (12 parasitic diseases and 8 microbial Korea Association of Health diseases) that have been neglected by global health authorities. Promotion, Seoul 07649, Korea Asia is one of the most important continents in which NTDs have 2Department of Tropical Medicine been prevailing. Parasitic diseases occurring in Asia include and Parasitology, Seoul National University College of Medicine, largely 9 kinds; schistosomiasis, leishmaniasis, lymphatic filariasis, Seoul 03080, Korea dracunculiasis, soil-transmitted helminthiases, foodborne e-mail: [email protected] trematodiases, echinococcosis, taeniases/cysticercosis, and scabies, and the remaining 3 occur outside Asia; African trypanosomiasis, Chagas’ disease, and onchocerciasis. All of the 8 microbial diseases occur in Asia; yaws, Buruli ulcer, cholera, dengue/chikungunya fever, leprosy, rabies, trachoma, and deep mycoses. The species of parasites involved are numerous which include Schistosoma japonicum, Leishmania major, L. tropica, L. infantum, L. donovani, Wuchereria bancrofti, Brugia malayi, Dracunculus medinensis, Ascaris lumbricoides, hookworms (Ancylostoma duodenale, Necator americanus), Trichuris trichiura, Strongyloides stercoralis, Opisthorchis viverrini, Clonorchis sinensis, Paragonimus westermani, P. heterotremus, P. skrjabini, P. miyazakii, Metagonimus yokogawai, Heterophyes nocens, Haplorchis taichui, echinostomes, fasciolids, gymnophallids, Echinococcus granulosus, E. multilocularis, Taenia solium, T saginata, T. asiatica, and Sarcoptes scabiei. Microbial diseases include bacterial, viral, and fungal infections. With regard to the prevalence of each NTD in Asia, soil-transmitted helminthiases and foodborne trematodiases are the two major groups of public health concerns.
Keywords: neglected tropical diseases (NTDs), parasitic diseases, microbial diseases, Asia, soil-transmitted helminthiases, foodborne trematodiases 4 NTDASIA 2019
O:02 Neglected Zoonotic Helminths: Country Emergence and Insights into Genomics, Diagnostics and Molecular Epidemiology in Vietnam
Thanh Hoa Le,1,2,* Khue Thi Nguyen,1 Huong Thi Thanh Doan,1,2 1Institute of Biotechnology (IBT), Roan Thi Do,1 Ha Viet Thi Le,1 Nga Thi Bich Nguyen,1 Vietnam Academy of Science and Xuyen Kim Thi Le1 Technology (VAST), 18. Hoang Quoc Viet Rd., Cau Giay, Hanoi, Abstract: Neglected zoonotic helminths (NZHs) affect mainly Vietnam; poor and marginalized populations, ie. low developing countries 2 Graduate University of Science and (LDCs). The NZHs comprise of broad categories of parasites, Technology (GUST), Vietnam involving in the infection of up to 80% of the population of the Academy of Science and Technolog LCDs but not excluding the developed countries. Common (VAST), 18. Hoang Quoc Viet Rd., Cau Giay, Hanoi, Vietnam; nematodes and platyhelminths, including those of the phylum *Presenter e-mail: [email protected] Platyhelminthes (Fasciolidae/ Paragonimidae/ Opisthorchiidae/ Heterophyidae/ Echinostomatidae/ Echinochasmidae; Taeniidae; Diphyllobothriidae, etc); and of Nematoda (Toxocaridae; Ancylostomatidae; Onchocercidae; Gnathostomatidae; Thelaziidae, etc.), have been increasingly reported. All life-cycle forms of the NZHs (eggs, metacercariae, cercariae, adults) found in soils/stools/sputum/fishes/crabs/animals/snails, are associated with our daily lives and difficult to be discriminated by morphology. Genomic analysis of, primarily preferable to, mitochondrial (mtDNA) and ribosomal DNA (rDNA), termed mito- and ribo-genomics, provides reliable tools used for species diagnosis/detection, taxonomic identification, and epidemiology. For common species of the NZHs, selected genetic markers are widely applied for identification of their developmental, adult and hybrid forms from human hosts (human cases) and animals; metacercariae from intermediate hosts (animals and fishes/crabs) and cercariae from snails or soils, if any. Single tube/one-step multiplexing systems (multiplex-PCR; LAMP) and other epidemiologic/ serologic tests are also developed to fix their geographic distribution, regional spread and multi-species infections in the overlapping areas. Molecular/genomic data are also used to simplify the detection of human and animal cases/outbreaks from the common soil-transmitted and food-borne zoonotic species. Over 30 years, nationally in Vietnam, particularly under the support of the NAFOSTED grants, and through our international research network, we have obtained valuable data from the NZHs for: i) mapping geographical and epidemiological distribution; ii) understanding dynamical, spatial, temporal spreading over countries; iii) clarifying taxonomic position/ identification/ evolution; iv) confirming the natural hybridization (introgression), if any; v) providing directions for epidemiological studies and control strategies in Vietnam and worldwide; vi) collaboratively publishing updated data enhancing the understandings on the NZHs (NAFOSTED: 108.02-2017.09/Le TH).
Keywords: genomics, genetic markers, helminths; mitochondrial, ribosomal, zoonotic. NTDASIA 2019 5
O:03 Opisthorchis felineus Infection in Western Siberia: Update on Prevalence, Risk Factors and Impact on Human Health
Olga S. Fedorova,1 Anna V. Kovshirina,1 Sofia V. Mazeina,2 Olga I. Zvonareva,3 Marina M. Fedotova,1 Yulia V. Kovshirina,1 1Department of Faculty Pediatrics, Tatiana S. Sokolova,1 Vyatcheslav A. Petrov,4 Jan Hattendorf,5,6 Siberian State Medical University, Ludmila M. Ogorodova,1 Peter Odermatt5,6 Tomsk, Russian Federation 2Shegarskiy Regional Hospital, Abstract: O. felineus is endemic in Southern and Eastern Europe Tomsk Oblast, Russian Federation 3University of Maastricht, and in large parts of the Russian Federation, particularly in Maastricht, the Netherlands Western Siberia. However, knowledge on the local infection and 4Central Research and Development disease status is scarce. To assess the current O. felineus infection Laboratory, Siberian State Medical prevalence, the risk factors and associated morbidity in rural University, Tomsk, Russian Federation Western Siberia. A cross-sectional study was conducted in 5Swiss Tropical and Public Health randomly selected villages of Shegarskiy district, Tomsk Oblast. Institute, Department of All household members (≥ 7 years) present on the survey day Epidemiology and Public Health, were enrolled (n=600). Two stool samples were examined on Basel, Switzerland 6 helminth eggs using PARASEP® (DiaSys Ltd, UK). Number of eggs University of Basel, Basel, Switzerland per gram (EPG) of feces was recorded. Each study participant was interviewed on risk factors using a pre-tested questionnaires. An abdominal ultrasonography examination of liver and bile ducts was performed by use of mobile, high resolution ultrasound machine (Shenzhen Mindray Bio-Medical Electronics, Co, Ltd). A total of 488 participants completed all examinatoins The O. felineus infection prevalence was 60.3%. The infection was significantly associated with age, gender and well-being. Among significant risk factors are fishing activities, consumption of undercooked fish and alcohol, and presence of dogs in the household. Significant hepatobiliary morbidity was associated with O. felineus infection: periductal fibrosis of intrahepatic bile ducts associated with the infection. Our study showed that O. felineus infection is highly prevalent in rural Western Siberia, and associated with severe hepatobiliar pathology. Results of the study will be used to develop a comprehensive O. felineus infection control program. 6 NTDASIA 2019
O:04 Is Opisthorchis viverrini Infection Endemic in Cambodia?
Miyamoto K,1,* Khieu V,2 Kirinoki M,3 Hirayama N,1 Muraoka M,1 Nakakuki N,1 Chigusa Y3
1 Faculty of Medicine, University of Yamanashi, Japan Abstract: In Thailand and Laos many studies documented high 2 National Helminth Control infection rates ofO. viverrini. In Cambodia, which is the neighbor Program, National Center for country of these countries, the first endemic village (EPR: egg Parasitology, Entomology and positive rate was 26.9% by Kato-Katz) in Takeo province was Malaria Control (CNM), Ministry of found in 2006. Then endemic villages (EPR > 20% by Kato-Katz) Health, Cambodia 3 School of Medicine, Dokkyo have been found in 9 provinces by collaboration survey between Medical University, Japan CNM and Japanese team until 2018. The results of stool exam * Presenter **e-mail: mkazuko@ (SE) by Kato-Katz showed more than 50% positive in some yamanashi.ac.jp villages of Kampong Cham, Tbong Khmum, Prey Veng and Kratie provinces. Most of those villages are located nearby ponds or lakes where the residents obtain fresh water fish. While results of SE of the villages which are located nearby the Mekong River, except the area of Lao border, and the big lake of Tonle Sap showed less 10% positive. Most of the residents in endemic villages love to eat raw fresh-water fish dishes which are called “Prea Trai Chau” or “Bokk Trai Chhau”. We found some kinds of cyprinide fish which were infected byO. viverrini metacercariae and snails which were infected by cercariae in some endemic areas. Liver function survey has been provided in 2 endemic provinces, Kampong Cham and Prey Veng. A few persons who have high EPR were found having some problems in their liver by ultrasound examination.Yes, Opisthorchis viverrini infection is endemic in Cambodia and it is one of the large health issues!
Keywords: Opisthorchis viverrini, endemic, Cambodia NTDASIA 2019 7
O:05 Update on Opisthorchis felineus Research Data
Viatcheslav Mordvinov,* Mariya Pakharukova, Damira Avgustinovich
The Federal Research Center Abstract: Opisthorchiasis caused by the liver flukeOpisthorchis Institute of Cytology and Genetics, felineus is one of the most common helminthic infections in the Siberian Branch of Russian territory of former Soviet Union. O. felineus infection symptoms Academy of Sciences, Novosibirsk include precancerous lesions with the potential for malignancy. 630090, Russia * The highest prevalence of opisthorchiasis is registered in rural Presenter e-mail: mordvin@bionet. nsc.ru areas of Western Siberia, where hard work and difficult living conditions provoke excessive alcohol consumption and contribute to the development of social stress. Our presentation demonstrates that these factors may also increase susceptibility of experimental animals to O. felineus infection. To date, the only drug of choice for the treatment of opisthorchiasis felinea is praziquantel (PZQ). However, PZQ is often not effective enough to eliminate helminths and treatment with PZQ causes side effects indicating toxicity of this drug. Thus, the discovery and development of novel efficacious drugs to control and treat opisthorchiasis felinea is one of the top research priorities. This presentation briefs our strategy for development of anthelmintic recipes. In particular, assessment of the parasitic proteins as potential molecular targets for drug discovery, efficiency of drug combinations and modified solid samples of PZQ for therapy of opisthorchiasis felinea will be discussed. This work was supported by the Russian Science Foundation [18-15-00098].
Keywords: Food-borne trematodes, Opisthorchis felineus, experimental opisthorchiasis, Praziquantel, Tribendimidine, Miconazole, Clotrimazole, combination chemotherapy, Glycyrrhizic acid, mechanochemistry, host physiological state 8 NTDASIA 2019
O:06 The genome of the Liver Fluke Fasciola gigantica Present Novel Protein Kinases as Drug Targets
Timir Tripathi Assistant Professor, Molecular and Structural Biophysics Laboratory, Abstract: Fascioliasis is a neglected food-borne disease caused Department of Biochemistry, by liver flukes (genusFasciola ) and affects more than 200 million North-Eastern Hill University, Shillong -793022 people worldwide. Despite technological advances, little is e-mail: [email protected] known about the molecular biology and biochemistry of the fluke. We present the draft genome ofFasciola gigantica for the first time. The assembled draft genome has a size of ~1.04 Gb with an N50 of 129 kb. A total of 20,858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. In pathway analysis, all the genes of glycolysis, Kreb’s cycle and fatty acid metabolism were found to be present, but the key genes for fatty acid production in fatty acid biosynthesis were missing. This indicates that the fatty acid required for the survival of the fluke may be acquired from the host bile. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control. Eukaryotic protein kinases (ePKs) are regulators of several cellular processes, the most important being phosphorylation. We used various computational and bioinformatics tools to extensively analyze the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/ Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.
Keywords: Genome sequence, liver fluke, trematode, de novo repeats, protein kinases, domain organization, drug targets NTDASIA 2019 9
O:07 Oncogenesis of Clonorchis sinensis
Sung-Tae Hong
Abstract: Clonorchis sinensis is a digenetic trematode that lives in the intrahepatic bile duct of human and other mammals. The International Agency for Research on Cancer reclassified Department of Tropical Medicine C. sinensis in the Group 1 biological carcinogen of human and Parasitology, Seoul National University College of Medicine, cholangiocarcinoma (CCA). The evidence of the carcinogenicity Seoul, Korea of C. sinensis includes increased risk of CCA in clonorchiasis e-mail: [email protected] patients by epidemiological and hospital data, development of CCA in animal experiments, and oncogenic changes of genes and cellular molecules by cell experiments. Animal experiment demonstrated CCA development in Syrian golden hamsters by C. sinensis infection and N-dimethylnitrosamine. Kupffer cells proliferate significantly and play a critical role in the host response at the initial phase ofC. sinensis infection. In vitro cell model studies investigated carcinogenetic changes of the molecules and genes using HuCCT, HEK 293T cells, and H69 cholangiocytes. The stimulated cells by bothC. sinensis soluble products and NDMA proliferate significantly with G2/M shift, and produced E2F1, p-pRb, CDK2, and cyclin B, and increased expression of COX-2. Also, the gap-junction molecules of connexin (CX) 43 and CX 26 increased but CX 32 decreased. NO and other oxidative stresses are produced in the bile duct wall by C. sinensis infection and they may damage DNA of bile duct cells. Excretory-secretory product of C. sinensis suppresses apoptosis and stimulates proliferation of damaged cholangiocytes to be neoplastic. Changed CX characters may promote the neoplastic cells to invade nearby or remote tissues. C. sinensis is a carcinogenic trematode of humans in Asia. 10 NTDASIA 2019
O:08 Clonorchis sinensis: Prepare for and Chemotactically Migrate to Bile Duct
Won Gi Yoo, Shunyu Li, Sung-Jong Hong*,**
Abstract: When ingested by the mammalian hosts, Clonorchis Department of Medical Environmental Biology, Chung-Ang sinensis metacercariae are stagnated in the stomach for a while. University College of Medicine, Then the metacercariae pass down to the duodenum and briefly Seoul, Korea excyst upon contacting to trypsin. The newly excysted juveniles *Presenter **e-mail: hongsj@cau. (CsNEJ) promptly migrate along bile-chemotaxis into the ac.kr intrahepatic bile duct fitting to the body, and grow to ovigerous adults therein. In vitro, the CsNEJs migrated toward bile solution and cholic acid, while in opposite direction from lithocholic acid. The higher concentration of lithocholic acid in the gall bladder may push the CsNEJs to pass by and to migrate up to bile capillaries. Bile-induced neuronal signals were transduced through dopaminergic neurons. The C. sinensis adults (CsAd) were also chemotactic to bile, CA and DCA, but repellent to LCA. This bile-chemotaxis of CsAd was inhibited by dopaminergic antagonists. To thrive in the bile, C. sinensis should keep tight control over bile acids, transportation, importing and exporting. The sodium-bile acid co-transporter (CsSBAT) was identified in the C. sinensis adults. When CsSBAT was inhibited or silenced, bile acids were accumulated in the body and deteriorated survival of C. sinensis adults in bile. The CsSBAT was localized exclusively in the adult flukes; mesenchymal tissues and excretory bladder, but not in outer membrane of tegument and intestine. Organic solute transporter (CsOST) had ordered N- and disordered C-terminus domains corresponding to α- and β-subunits of the mammalian animals. Multidrug resistance protein 4 (CsMRP4) showed two membrane-spanning domains and two nucleotide-binding domains. In the CsNEJs, most of the induced genes, by 37�C incubation, were up-regulateds of energy and amino acid metabolism, signal transduction, transcription and translation, and heat shock proteins. By bile and cholic acid, smaller number of genes were induced and two-thirds were down-regulated. Collectively, it is elucidated that in the stomach the C. sinesis metacercariae are prepared to get in and live in the bile, cholic acid is a major component driving chemotactic migration of the CsNEJs the bile duct. Orchestration of these three bile transporters may pass down bile acids to the excretory bladder. With functioning of the bile transporters, C. sinensis could manage bile acids in the body and live in the bile duct. NTDASIA 2019 11
O:09 Experimental Model of Opisthorchis felineus Liver Fluke Infection: Mechanistic Insights into Biliary Neoplasia Formation
Maria Pakharukova,* Oxana Zaparina, Nina V. Baginskaya, Viatcheslav A. Mordvinov Institute of Cytology and Genetics, Siberian Branch of Russian Academy Abstract: Opisthorchiasis caused by food-borne trematode of Sciences, Laboratory of Molecular Opisthorchis felineus is a substantial public health problem, with Mechanisms of Pathological Processes, Novosibirsk, Russia 1.7 million persons infected worldwide. This disease is associated *Presenter e-mail: [email protected] with hepatobiliary inflammation, cholangiofibrosis, intraepithelial neoplasia, and even cholangiocarcinoma among chronically infected individuals. The report will present data on global changes in host gene expression related to the parasite infection. In particular, (1) differential expression gene analysis of liver transcriptomes (Illumina HiSeq2500) of golden hamsters infected with O. felineus at 1 and 3 months postinfection was carried out. Gene Ontology annotation and functional enrichment analysis indicated that all of the differentially expressed genes were annotated into biological process, cellular component and molecular function. To provide in-depth research and to gain first insights into the mechanism by which O. felineus infection causes precancerous liver lesions (2) the profiles of chronic inflammation and fibrogenesis markers in the dynamics of opisthorchiasis (from 1 to 18 months postinfection) as well as (3) oxidative stress (lipid peroxidation byproducts and 8-hydroxy-2¢-deoxyguanosine) were studied. Our data provide knowledge about global changes in gene expression of O. felineus-infected host liver, from which a complex molecular pathways and pathogenesis-related biological processes are involved in biliary neoplasia formation. This work was supported by the Russian Science Foundation [18-15-00098].
Keywords: Food-borne trematodes, Opisthorchis felineus, experimental model, transcriptome, oxidative stress, chronic inflammation 12 NTDASIA 2019
O:10 Diagnosis of Clonorchis sinensis Infection
Men-Bao Qian,1,2,3,4* Xiao-Nong Zhou1,2,3,4**
Abstract: Clonorchis sinensis infection caused by the ingestion 1 National Institute of Parasitic Diseases, Chinese Center for of raw freshwater fish exerts great burden in East Asia, especially Disease Control and Prevention, in China, South Korea and Vietnam. Diagnosis is important for Shanghai 200025, China. subsequent treatment and intervention. We have systematically 2 Chinese Center for Tropical evaluated the performance of various diagnostic techniques for Diseases Research, Shanghai 200025, China. C. sinensis infection. The Kato-Katz method is widely applied for 3 Key Laboratory of Parasite and both qualitative and quantitative evaluation. Multiple smears Vector Biology, Ministry of Health, from repeated stool samples are needed to increase the Shanghai 200025, China. sensitivity, especially when the infection intensity is low. 4 WHO Collaborating Center for Immunological tests are useful in clinical settings. However, Tropical Diseases, Shanghai 200025, China. current diagnostic kits need further improvement in terms of * Presenter **e-mail: zhouxn1@ obtaining both higher sensitivity and specificity. The screening chinacdc.cn of C. sinensis infection could be achieved through the interview with queries of eating practice, living environment etc., which is simple and useful in the field. Different diagnostic techniques should be applied according to the control stages and targets. Additionally, more researches on diagnostic tools are expected for C. sinensis infection.
Keywords: Clonorchis sinensis, Diagnosis, Kato-Katz method, Immunological test, Screening NTDASIA 2019 13
O:11 Update on Opisthorchis viverrini Glycoconjugates
Sutas Suttiprapa,1,2,*,** Binh T. T Ta, 1 D. Linh Nguyen,3 Prasert Saichua,1,2 Angela van Diepen,3 Cornelis H. Hokke,3 Banchob Sripa2 1 Tropical Medicine Graduate Program, Academic Affairs, Faculty Abstract: The carcinogenic liver fluke Opisthorchis viverrini is of Medicine, Khon Kaen University, endemic in Thailand and neighboring countries including Laos Khon Kaen, Thailand. PDR, Vietnam and Cambodia. Infections with O. viverrini 2 WHO Collaborating Centre for lead to hepatobiliary abnormalities including bile duct Research and Control of Opisthor- chiasis (Southeast Asian Liver cancer-cholangiocarcinoma (CCA). Despite decades of extensive Fluke Disease), Tropical Disease studies, the underlying mechanisms of how this parasite Research Center, Department of survives in the bile duct and causes disease are still unclear. Pathology, Faculty of Medicine, Glycoconjugates play an important role in host-parasite Khon Kaen University, Khon Kaen, interaction. Previous studies showed N- and O-glycan structures Thailand 3 Department of Parasitology, of the total extracts of juvenile and adult worms. In this study, Leiden University Medical Center, we further characterize glycans on the tegument and Albinusdreef 2, 2333 ZA Leiden, excretory-secretory products of the parasite. The results showed The Netherlands that there are different structure and abundancy of glycans in * Presenter **e-mail: sutasu@kku. ac.th different part of the worm. Some uncharacterized glycan structures were also detected. Further studies are needed to fully characterize these glycans and elucidate their roles in host-parasite interaction and pathogenesis of liver fluke infection.
Keywords: Liver fluke,Opisthorchis viverrini, glycoconjugates 14 NTDASIA 2019
O:12 Post- MDA Era: An Update on Intestinal Parasitic Infections (IPI) Among Selected Urban Poor Communities in the West Coast of Peninsular Malaysia
Institute of Biological Sciences, Norhidayu Sahimin,* Nur Sabrina Abd Khalil, Faculty of Science, University of ** Malaya, 50603, Kuala Lumpur, Siti Nursheena Mohd Zain Malaysia * Presenter **e-mail: nsheena@ Abstract: STHs have been recognized as a major public health um.edu.my problem in low socioeconomic communities worldwide. The National Helminth Control Program through mass drug administration (MDA) was initiated in Malaysia in 1974 to eradicate STHs until it was discontinued in 1983. The control strategy of the Ministry of Health, Malaysia, was an integrated national environment program that included education, sanitation and anthelminthic treatment to school-children only. Therefore, this study was conducted to re-assess STHs infection 30 years after discontinuation of MDA program among the urban poor communities of the PPR flats and urban slums in the states of federal territories Kuala Lumpur, Selangor and Malacca with the objectives to determine the current incidence rate of intestinal parasitic infection and associated risk factors. A total of 206 stool samples were successfully collected and screened for both helminth and protozoan infections using formalin ethyl-acetate concentration technique and Ziehl-Neelsen’s staining method. Overall, 18.9% (n=39) infections were recorded among the residents with at least one helminth and/or protozoan (n=35; 17%). The roundworm Ascaris lumbricoides was the most dominant helminth infection (n=32, 15.5%), whereas Hymenolepis nana infection was seen in only one case (n=1; 0.5%). The most of protozoan infections (n=21; 10.2%) were Cryptosporidium spp. (n=20; 9.7%) infection, and Giardia sp. Infection was seen in only one case (n=1; 0.5%). None of the risk factors (intrinsic and extrinsic) were significantly associated with infections. The low prevalence in the study cohort is indicative of the government’s successful effort to provide clean water sources and good sanitation facilities to the general population at large in addition to good personal hygiene practices.
Keywords: Post- MDA Era, intestinal parasitic infections, Malaysia NTDASIA 2019 15
O:13 Diagnosis and Drug Resistance of Human Soil-Transmitted Helminth Infections: A Public Health Perspective
Polydor Ngoy Mutombo,1,2 Nicola W.Y. Man,1 Peter Nejsum,3 Robert Ricketson,4 Catherine A. Gordon,5 Gemma Robertson,6 1 School of Public Health and Archie C.A. Clements,7 Nathalie Chacón-Fonseca,8 Community Medicine, UNSW Veeranoot Nissapatorn,9,10*,** Joanne P. Webster, 11 Medicine, UNSW, Sydney, NSW, Mary-Louise McLaws1 Australia 2 Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Abstract: Soil-transmitted helminth (STH) infections represent Australia a major public health problem globally, particularly among 3 Department of Clinical Medicine, socio-economically disadvantaged populations. Detection of Faculty of Health, Aarhus University, STH infections is often challenging, requiring a combination of Aarhus, Denmark 4 diagnostic techniques to achieve acceptable sensitivity and Hale O’mana’o Biomedical Research, Division of Emerging specificity, particularly in low infection-intensity situations. The Pathogens, Edmond, OK, United microscopy-based Kato-Katz remains the most widely used States method but has low sensitivity in the detection of, for instance, 5 Molecular Parasitology Laboratory, Strongyloides spp. infections, among others. Antigen/antibody Infectious Diseases Division, QIMR Berghofer Medical Research Institute, assays can be more sensitive but are parasite species-specific. Brisbane, QLD, Australia Highly sensitive PCR methods have been developed to be 6 Public and Environmental Health, multiplexed to allow multi-species detection. Novel diagnostic Forensic and Scientific Services, tests for all STH species are needed for effective monitoring, Department of Health, Brisbane, evaluation of chemotherapy programmes, and to assess the QLD, Australia 7 Faculty of Health Sciences, Curtin potential emergence of parasite resistance. This review discusses University, Perth, WA, Australia available diagnostic methods for the different stages of STH 8 Soil-Transmitted Helminths control programmes, which vary in sensitivity and spectrum of Section, Tropical Medicine Institute, detection requirements, and tools to evaluate drug efficacy and Tropical Medicine Department, Faculty of Medicine, Central resistance. University of Venezuela, Caracas, Venezuela Keywords: soil-transmitted helminth, human infection, diagnosis 9 School of Allied Health Sciences, and drug resistance Walailak University, Nakhon Si Thammarat, Thailand 10 Research Excellence Center for Innovation and Health Products (RECIHP), Walailak University, Nakhon Si Thammarat, Thailand 11 Centre for Emerging, Endemic and Exotic Diseases (CEEED), Department of Pathobiology and Population Sciences, Royal Veterinary College, University of London, London, United Kingdom * Presenter **e-mail: veeranoot. [email protected]; [email protected] 16 NTDASIA 2019
O:14 Assessment of the Burden of Soil-Transmitted Helminthiasis After Five Years of Mass Drug Administration for Lymphatic Filariasisin Banggai District, Central Sulawesi 1 Ministry of Health Republic Indonesia 2 Banggai District Health Office, Helena Ullyartha,1,*,** Yudit Labatjo,2 Nurmasita Datu Adam,2 Central Sulawesi Anang S Otoluwa2 * Presenter **e-mail: helenartha@ yahoo.co.uk Abstract: Mass drug administration (MDA) for lymphatic filariasis (LF) has often been replaced with specific interventions targeting soil-transmitted helminthiases (STH) in co-endemic areas. Banggai District, Central Sulawesi, is co-endemic for LF and STH. Therefore, we have evaluated the epidemiological situation of STH in this area, with special reference to determine whether administration of albendazole for the STH control should be continued in the absence of further MDA for lymphatic filariasis. In this area, MDA for LF has been done in 2014-2018. The district was classified as one evaluation units (EUs). Microsoft Excel computer tool entitled the Survey Sample Builder (SSB) tool was used for sampling. Clusters sample survey school-based) were implemented for 54 primary schools in Evaluation Unit (EU). Kato Katz method was used to detect the presence of the eggs of STH in fecal samples. The results showed the detection rate of any STH (Ascaris, Trichuris or hookworm) eggs in children was 10.8%. Most of STH detected were mild infections, particularly with Ascaris (5.4%) and also with Trichuris (7%), whereas hookworm infection was only 0.9%. Overall, the intensity of STH infection remains mild degree, and no heavy infections were detected. These preliminary results suggest that annual community MDA with albendazole is a promising strategy for controlling STH and LF.
Keywords: Assessment, soil-transmitted helminthiasis, lymphatic filariasis, Banggai Distract, Central Sulawesi NTDASIA 2019 17
O:15 Progress Toward Schistosomiasis Elimination in Lao People’s Democratic Republic: A Long-Term Evaluation of Impact on Integrated Control Approach
Lao Tropical and Public Health Somphou Sayasone Institute, Ministry of Health, Vientiane Capital, Lao People’s Abstract: Schistosoma mekongi is currently endemic southern Democratic Republic Laos and northern Cambodia. In Laos, as early as the 1980ties, e-mail: somphou.sayasone@yahoo. its public health importance was recognized and preventive com chemotherapy-regular mass treatment of the affected communities-using praziquantel has been the key strategy. In Lao PDR, the interventions started in the late 1980s, but suffered several interruptions which permitted transmission to resume. Today, a number of small foci continue to show substantial prevalence rates. The re-emergence calls for further sustained control. We evaluated a long-term impact of an integrated control strategy using echohealth principles as strategy of choice to control schistosomiasis in four villages of Khong district, Champasack province, southern Lao PDR. First, a baseline study was carried out in 2012 to assess prevalence and risk factors for schistosomiasis. After, the villages were randomly assigned into two groups, e.g., control villages (receiving only Mass Drug Administration) and intervention villages (receiving MDA plus improving access to latrine, treatment of animal reservoirs and health education to habitants. Finally, we evaluated the impact on infection from 2014 until 2018. We observed that the prevalence of S. mekongi dropped significantly in the intervention villages (23.6% to 10.9%) when compared to control one (22.2% to 17.9%) 6 months after intervention (mid 2014. The prevalence continued to drop dramatically for intervention villages in the following years, e.g., 2015 (7.6%), 2016 (2.4%) when the decrease in prevalence was slower in the control villages, e.g., 12.1% in 2015, 9.6% in 2016. By the end of 2016, the control villages received the intervention package and the prevalence of the disease were at 2.1% in 2017 and 4.5% in 2018 for intervention group and 2.9% in 2017 and 4.3% for control villages, respectively. We conclude that integrated intervention impacted on the prevalence of schistosomiasis at the faster rate when compared to the traditional intervention using only MDA. Hence, it might be an ideal option to accelerate the elimination of schistosomiasis and achieve the WHO target of eradication of the disease by 2030. 18 NTDASIA 2019
O:16 Deworming Among Pregnant Women in Myanmar: A Simple, Effective but Forgotten Public Health Intervention
Ye Min Htet,1,2,*,** Kathrine Gass2 1 Rollins School of Public Health, Emory University, Atlanta, The Abstract: Hookworm and Trichuris trichiura cause anaemia, United States of America which is particularly harmful for pregnant women. The World 2 Neglected Tropical Diseases Health Organization recommends deworming using single dose Support Center, Task Force for albendazole (400 mg) or mebendazole (500 mg) after the first Global Health, Atlanta, The United trimester as part of the antenatal care (ANC) package in endemic States of America areas. The aim of this descriptive study is to make policy makers * Presenter **e-mail: yeminhtet. aware of important gaps in deworming coverage of pregnant [email protected] women and to identify areas needing further investigation. This study reports deworming among pregnant women in Myanmar in comparison to ANC uptake, disaggregated by residence, States/Regions, education, and wealth quantile. Data was obtained from the Myanmar Demographic and Health Survey 2015-2016. A nationally representative sample of 3583 women ages 15-49, with a live birth in the five years preceding the survey, were asked whether they received ANC from a skilled provider and and if they took deworming medication during their most recent pregnancy. Results showed 80.7% of women received ANC and 55.3% of women took deworming medication. The distribution of women receiving deworming treatment was consistently lower than that of ANC across all variables. By States/Regions, percent receiving ANC ranged from 68.1% in Shan to 94.6% in Yangon, while that of deworming ranged from 39% in Shan to 66.8% in Yangon. Whereas more educated women seek ANC (89.8% and 99.5% among secondary and beyond, vs. 56.1% among non-educated), the women receiving deworming remained low (59.7%, and 54% among secondary and beyond, vs. 39.5% among no education). ANC seeking women increased linearly from the poorest (67.1%) to the richest (98.1%) but deworming status did not improve by wealth quantile, and remained virtually the same (52.6% in the lowest to 53.9% in the highest group). By residence, 94.4% of women from urban area received ANC, but only 59.8% percent residing in urban area reported taking deworming medication. This study has identified that a significant proportion of pregnant women are not receiving a low-cost deworming intervention and are consequently at risk for adverse health outcomes. The reasons for this low uptake of deworming treatment among pregnant women, including who are seeking ANC, should be explored. Access to deworming during ANC should be examined at the service delivery points. Improving the awareness, training, recording and reporting linked to deworming, as part of the standard ANC package, is recommended.
Keywords: Deworming, soil-transmitted helminths, ante-natal care, pregnant women, Myanmar NTDASIA 2019 19
O:17 Socioeconomic Factors Associated with Prevention Measure Against Filariasis in Banyuasin, South Sumatra
Rini Mutahar,1* Desheila Andarini,2 Amrina Rosyada,3 Novrikasari2 1 Epidemiology Department, Faculty of Public Health, Abstract: Filariasis is an infectious disease by filarial worms Universitas Sriwijaya 2 transmitted through mosquito bites. Symptoms usually occur Occupational Health and Safety Department, Faculty of Public in the form of swelling in certain areas in the flow of lymph Health, Universitas Sriwijaya vessels, enlargement of the legs/feet (elephantiasis) or arms 3 Biostatistics Department, Faculty and enlargement of the scrotum/vagina that is permanent if not of Public Health, Universitas prevented. Prevention efforts have been carried out in Sriwijaya * Presenter email: rini_mutahar@ combating filariasis such as Mass Drug Administration (MDA) fkm.unsri.ac.id and preventative blood tests conducted by individuals. The aim of this study was to analyze factors associated with prevention measure against Filariasis in Banyuasin, South Sumatra. This study used a cross-sectional design, with a total sample of 200 family heads. The results of the study showed that as many as 30% of respondents had a defiant act. Furthermore, majority of the respondents use drug, mosquito nets and mosquito repellent to avoid mosquito bites. They also have a habit of going out at night, and never use finger surveys. Respondents who have poor knowledge, higher education levels, and high economic status are at higher risk of not having good debates. Information and education about health workers can contribute to reducing stigma in the community and the side effects of drugs so that the community can improve community services to get a better environment.
Keywords: lymphatic filariasis, diseases prevention, mass drug administration (MDA), socioeconomic factor 20 NTDASIA 2019
O:18 Adverse Effects Following Mass Drug Administration for Lymphatic Filariasis Elimination in Banyuasin
Rini Mutahar,1 Desheila Andarini,2,*,** Amrina Rosyada,3 1 Epidemiology Department, Novrikasari4 Faculty of Public Health, Universitas Sriwijaya Abstract: Filariasis or commonly referred to as elephantiasis is 2 Occupational Health and Safety, Faculty of Public Health, Universi- an infectious disease by filarial worms likeWuchereria bancrofti, tas Sriwijaya Brugia malayi and Brugia timori. These parasites are transmitted 3 Biostatistic , Faculty of Public to humans through mosquito bites and then develop into adult Health, Universitas Sriwijaya worms in the lymph glands. Despite several rounds of Mass Drug 4 Occupational Health and Safety, Administration (MDA) as an elimination strategy of Lymphatic Faculty of Public Health, Universitas Sriwijaya Filariasis, adverse drug reactions following MDA are still a major *Presenter ** e-mail: desheila_ factor of poor treatment adherence in Indonesia. This study [email protected] assessed the coverage and occurrence of adverse drug reactions following MDA. Population-based cross-sectional surveys were conducted in a cluster random sample of the population target of MDA in Banyuasin district of South Sumatera, Indonesia. Data were obtained through interviews and presented in descriptive statistics. A total 200 individuals were interviewed, 49 persons 24,5% did not consumed the drug. Out of 49 persons, 44,9% did not consumed drug because community stigma, 40,8% lack of information and 12,2% have a history of disease. From 151 persons who consumed the drug, 53 persons 26,5% has adverse drug reactions. The adverse drug reactions were 23,8% headache, 7,3% drowsiness, 3,3% nausea and 0,7% vomiting. Information and education from health workers may contribute to reduce stigma in community and fear of drug side effects.
Keywords: Lymphatic Filariasis, Mass Drug Administration (MDA), Adverse effects, Coverage NTDASIA 2019 21
O:19 The Epidemic and Vector Control of Dengue in China
Xiao-Guang Chen
Abstract: The presentation reviews the epidemic of dengue in Department of Pathogen Biology, the past 40 years in China and analyzed the factors correlated School of Public Health, Southern with the epidemic. The advances in vector mosquitoes control Medical University, Guangzhou, of dengue in China is also introduced. China e-mail: [email protected] Keywords: Aedes albopictus, dengue, China 22 NTDASIA 2019
O:20 Lethal but Safe: Efficacy of the Artificial Breeding Trap Against Aedes albopictus under the Laboratory and the Field Study
Tong Liu, Yu-gu Xie,* Feng Lin, Wen-qiang Yang, Li-hua Xie, Department of Pathogen Biology, Xing-hua Su, Xiao-Guang Chen** Guangdong Provincial Key Laboratory of Tropical Disease Abstract: Mosquito-borne diseases cause an increasing health Research, School of Public Health, burden around the world, which highlights the importance of Southern Medical University, Guangzhou, Guangdong, China effective vector control. In addition, viable approach based on * Presenter **e-mail: xgchen2001@ permanent egg-collection using an exceptional ovitrap and hotmail.com long-lasting larvicide is a good option. In this study, we performed laboratory and field tests for a noval lethal ovitrap named artificial breeding trap (ABT). It is a BLOCK sustainably release larvicides which is consisted of the larvicide Bacillus thuringiensis var. israelensis (Bti) and pyriproxyfen (PPF) in biodegradable carrier (polyethylene glycol-co-hexadecyl) and it is aimed at Aedes mosquitoes. In the laboratory, it showed high mortality against larvae stages (>90%) indicating that ABT mainly aimed at larvae and complete inhibitory effects on pupation and eclosion of pupae (100%) could last over six months. In the 4-month field study, eggs of Aedes albopictus were always collected by the ABT in the study site, and occasionally Culex ones. In total, 88,090 eggs were caught during the intervention: the number of mosquito eggs in the first to the fourth month is 28,987, 25,974, 22,826 and 10,303, and 1,077, 933, 792 and 344 for each ABT. We confirmed 89% reduction in Ae. albopictus female adult abundance. After the intervention, the mosquito population in the field had produced 85 times higher resistance to deltamethrin, while it had just developed resistance to PPF but still remains sensitive to Bti. Our results demonstrated that this combination can be an inexpensive, and low-maintenance Aedes solution, which can also be a recommendation in container-inhabiting mosquitoes control toolbox.
Keywords: lethal ovitrap, Bacillus thuringiensis var. israelensis, pyriproxyfen, Aedes albopictus NTDASIA 2019 23
O:21 Current Insecticide-Resistant Status of Dengue Haemorragic Fever Vector Populations in Indonesia: A Systematic Review of Literature
Shifa Fauziyah1,2 1 Laboratory of Entomology, Abstract: Aedes aegyptiand Aedes albopictus have been known Institute of Tropical Disease, as the vectors of DHF in Indonesia. Both of this species are Universitas Airlangga, East Java, distributed depending on geographical condition and Indonesia 2 Tropical Medicine Department, environmental factors. Since having found in 1968, DHF still is School of Medicine, Universitas a major public health problem in Indonesia. Frequently, DHF Airlangga, Indonesia cases give impact on many aspects such as economy, culture, e-mail: shifafauziyah1996@gmail. social, and others. Some control programs have already been com conducted for larval reduction, which is often called as 3M. However, there’s no evaluation on vector status against commonly used insecticides. This study was conducted through a systematic review of the literature. Available data based on the published article from PubMed showeds current insecticide resistant status of DHF in Indonesia. We found some report that shows high frequency of insecticide resistance both of the aforementioned Aedes species, and also their genetic polymorphism. Mutations were detected inAedes from Central Java, Yogyakarta, Semarang, and Denpasar. Evaluation of current insecticide resistant status are important to avoid ineffective use of insecticide that can lead to maintenance dengue virus in nature. Research about insecticide-resistant status in Indonesia is still limited, while distribution of DHF cases is very wide. This problem should be considered more seriously by national health workers.
Keywords: dengue, Indonesia, insecticide, resistance 24 NTDASIA 2019
O:22 Benefit Using Transparent Wall Compare to Using not Transparent Wall of Tropic Trap Anti Mosquito in Manado City, North Sulawesi, Indonesia
Health Office, Manado City, North Joy Albert Sumakul Sulawesi Province, 95119, Indonesia. e-mail: [email protected] Abstract: Mosquitoes are small insects that are dangerous because some of them are the vectors of parasites and viruses that cause contagious diseases such as malaria, dengue, Japanese encephalitis, filariasis, chikungunya, yellow fever, zika. In mosquito eradication programs in several countries, ovitrap and gravitrap with dark, invisible walls have been used as containers. From September to October 2015 a comparative survey was conducted using mosquito traps with dark-colored and transparent containers to compare the efficacy of mosquito trapping. Experiments were carried out for a 30-day period using two types of mosquito traps; one prepared with transparent container and the other with non-transparent container. The results showed that the number of mosquito larvae in the transparent container was higher than that in the non-transparent container. Promotion and development of mosquito eradication program to prepare mosquito trap using transparent wall water container anti mosquito larva is needed to reduce contagious diseases transmitted by mosquitoe vectors.
Keywords: mosquito population, mosquito larvae, contagious diseases, mosquito trap, transparency, container NTDASIA 2019 25
O:23 On the Origin of Taenia asiatica
Dongmin Lee & Keeseon Eom*
Abstract: Baer (1940), noting the host specificity, proposed that human Taenia tapeworms are highly associated with human’s livestock domestication. Hoberg (2001) suggested that human Department of Parasitology, Parasite Taenia tapeworms were inherited from the ancestors of humans Research Center and Parasite Resource Bank, Chungbuk National from Africa based on the morphological phylogenetic University School of Medicine, relationships. According to the morphological taxonomy,Taenia Cheongju 28644, Korea asiatica is closely related to lion’s Taenia simbae. On the other *e-mail: [email protected] hand, molecular biological phylogeny does not support this. This study examines the early hypothesis on the origin of human Taenia tapeworms by focusing on T. asiatica through the comparison of the phylogeny and the coevolutionary interpretation. The origin ofT. asiatica was traced and discussed through evidences of the geographical distribution, intermediate host animals, phylogenetic data, movement of humans and livestocks with comparative analysis of other major human Taenia tapeworms: T. solium and T. saginata
Keywords: Taenia asiatica, origin, hypothesis, out of africa, out of asia 26 NTDASIA 2019
O:24 Neurocysticercosis, the Parasitic Disease in People and Pigs: The Present Situation in Asia and Towards Eradication from Bali, Indonesia
Department of Parasitology and Akira Ito Laboratory of NTDs, Asahikawa Medical University, Asahikawa 078-8510, Japan Abstract: Neurocysticercosis (NCC) is transmitted from humans to humans with eggs of the pork tapeworm, Taenia solium. T. solium causes two different diseases in humans. One is intestinal taeniasis caused by ingestion of cysticercus/cysticerci, the larval stage of this parasite in pork. The other is tissue cysticercosis (CC) including NCC caused by ingestion of eggs released from people suffering from taeniasis. Taeniasis is a food- or meat-borne, but basically asymptomatic, parasitic zoonosis and focally endemic in remote poor villages in Asia where local people live with free roaming pigs. Pigs’ main dish is human feces! Thus, people suffering from taeniasis are called tapeworm carriers. In contrast, CC is potentially the most lethal parasitic communicable disease from humans (tapeworm carriers) to humans (carriers and many others) with eggs. Pigs are the main intermediate host of T. solium, whereas humans are the exclusive definitive host ofT. solium, but simultaneously aberrant, intermediate hosts. Historically, this parasite was conceived to be originally the human parasite and the life cycle was completed by “cannibalism” before the intermediate host switching happened from humans to pigs. NCC is now spreading globally, since tapeworm carriers are travelling to big cities, especially capital cities in their home countries and even in foreign countries to get better salary through globalization. NCC cases are accidentally confirmed in urban areas where better medical cares are available. The present situation of NCC in Asia with traditional human lifestyles is overviewed as an introduction and followed by the main topic on the ongoing project “towards eradication of NCC from the tropical resort island, Bali, Indonesia”. NTDASIA 2019 27
O:25 Genome Evoluation of Trichomonadidae
Petrus Tang
Abstract: Since the first sequenced Trichomonas vaginalis genome become available in 2007, more and more studies showed conflicting evidences between T. vaginalis genetic Department of Parasitology, College identity and phenotypic diversity. For the interest in of Medicine, Chang Gung University, Taoyuan 333, Taiwan understanding the genetic basis of genome evolution in e-mail: [email protected] Trichomadidae, we compared the whole genome sequencing data of 10 different T. vaginalis isolates with the reference G3 isolate. Based on the mapping rate to G3 genome, the isolates can be separated into three clusters of 98%-99% (6 isolates), 58%-78% (3 isolates) and 3% (1 isolate). Bioinformatics analysis revealed that the genome structure and gene content of most isolates is very similar to G3 except the isolate that have the lowest mapping rate. Blastx of 8,517 de novo assembled contigs longer than 1 kb from RNAseq dataset of this isolate against the G3 reference proteome showed that 6,990 contigs have significant amino acid sequence homology. In order to clarify why the DNA sequence homology of this specific isolate with G3 at such a low level can encode proteome similar to G3, we elucidated the 87.2 Mbp genome of this isolate at ~200-fold coverage. Genome annotation from 528 scaffolds identified 20,585 genes. While approximately 93.76% of the annotated genes are single exon genes, more than a thousand genes contain intron. Analysis of synonymous codon usage pattern showed that this isolate prefer to use codons that contain G/C in the third codon position. However, there is no significant difference on the abundance of tRNA species between G3 and this isolate. Gene-order collinearity analysis against G3 only identified 13 collinear blocks and 77 gene pairs respectively. This result excluded the possibility that the G3 genome is duplicated from this isolate. In summary, we identified T.a vaginalis isolate that have completely different genomic organization compared with G3 but can encode similar proteome. Whole genome sequencing of this dwarf T. vaginalis genome illustrates challenges on the taxonomy and genome evolution of Trichomonadidae.
Keywords: Trichomonas vaginalis, genome evolution 28 NTDASIA 2019
O:26 Development of Omics-Based New Diagnostic Tools for Schistosomiasis in China
Wei Hu,1,2,*,** Bin Xu,2 Yan Lu,2 Junhu Chen,2 Jian Li,1 Qinghua Hong,1 Zhiqiang Qin,2 Jing Xu,2 Jiaxu Chen,2 Zheng Feng,2 Xiao-Nong Zhou2 1State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Abstract: Schistosomiasis japonica remains one of the public Contemporary Anthropology, health problems in Southeastern Asia. Sensitive diagnosis is Collaborative Innovation Center of crucial for the control and prevention of the disease. Therefore, Genetics and Development, School to identify the antigenic antigens and the released nucleic acids of Life Sciences, Fudan University, Shanghai, 200438, China is the key to develop rapid, sensitive and specific diagnostic 2National Institute of Parasitic techniques. The omics technology provides an advanced way to Diseases, Chinese Center for find the diagnostic antigen and nucleic acid markers. Firstly, the Disease Control and Prevention, genome and transcriptome information provide a large number Key Laboratory of Parasite and of expressed genes and species-specific, high copy genomic Vector Biology of the Chinese Ministry of Health, WHO sequences. Through the bioinformatics analysis on the genome Collaborating Center for Malaria, and transcription data, secreted proteins, highly expressed Schistosomiasis and Filariasis, proteins and membrane proteins with the potential for diagnostic Shanghai, 200025, China purpose could be searched, as well as the species specific and *Presenter, **email: huw@fudan. edu.cn high copy number of DNA fragments. Secondly, the genome and transcriptome provides a large number of protein-coding genes, which made it possible to identify new diagnostic antigens by the proteomic methods. On the other hand, the omics techniques create new opportunities for identification of circulating antigens. Through the omics studies, we have found some promising target antigens, among them one recombinant protein was use to develop a colloidal-gold immuno-chromatography assay (GICA) strip to detect the specific antibody induced by schistosome infection. The GICA showed similar sensitivity and higher specificity when compared with the other diagnostic systems reported using the crude antigens of worm source. The use of recombinant antigen for the diagnostic approach has the advantages in quality control and product standardization, and in reducing the cost, of which the worm source antigens has been soaring in recent years. Therefore, the recombinant reagent may be popularized in the field use instead of worm source reagents. Meantime, the new nucleic acid markers found from genome sequences showed potential value in early diagnosis and evaluation of chemotherapy. It is believed that the omics studies may provide new and efficient ways to develop new diagnostic tools.
Keywords: Schistosoma japonicum, omics, target screening, diagnostic tool NTDASIA 2019 29
O:27 Possible Transmission of Human Schistosomiasis in Previously Non-Endemic and Under-Reported Settings: A Rapid Evidence Product for Knowledge Synthesis
1 Department of Medical Research, Khin Thet Wai,1,*,** Kay Thwe Han,2 Toe Thiri Aung3 Yangon, Myanmar 2 Parasitology Research Division, Abstract: The emergence of schistosomiasis infection in previously Department of Medical Research, non-endemic settings is worthwhile to study to support the Yangon, Myanmar elimination goal of World Health Organization (WHO). Thus, this 3 Central Epidemiology Unit, fast tracking review underscored the evidence-based strategies Department of Public Health, Nay in the establishment of schistosomiasis control programme in Pyi Taw, Myanmar Myanmar as a health sector response. The WHO rapid review * Presenter **e-mail: khinthetwaid- methodology to strengthen health policy and systems (2017) [email protected] was used. PubMed/MEDLINE and EMBASE search engines identified published studies that informed schistosomiasis in Myanmar (n=5) between 2011 and mid-2019. In addition, the available reports of meeting, field assessment and field survey (n=4) and conference presentations (n=2) served as grey literature within the same period. Two researchers selected the studies related to schistosomiasis in Myanmar verified by the programme manager as a knowledge user. Data inclusive of descriptions as well as quantitative findings were extracted by one researcher and validated by the second researcher to mitigate the risk of bias. The anecdotal evidence of schistosomiasis was distinctive for rural areas of different ecological zones in Myanmar starting from the year 2012 that covered poverty-stricken sites. The prevalence estimates by stool examination in three study sites varied from <3% in 2012 to 11% in 2019. Between 2016 and 2019, in the inland sites of the coastal region in the western part of Myanmar, there was an increase in reported number of cases among hospitalized children and adults up to 890 diagnosed by ELISA test kit and treated. Potential snail vectors were identified from fresh water collections but still unconfirmed. However, possible transmission was noted in that region and in other sites that required interruption. Diagnostic methods used in field assessment included urinary antigen testing (Point-of-Care Circulating Cathodic Antigen) apart from Kato-Katz thick smears for stool examination. As a knowledge generation strategy, current rapid review synthesized findings and assessed the validity of research evidence of schistosomiasis in Myanmar. Further recommendations for policy/program included the necessity for an integrated plan to verify schistosome species and snail vector mapping, access to more sensitive diagnostics to confirm prevalence estimates, treatment of cases and introduction of effective control measures to suppress morbidity and to prevent transmission potential by health literacy promotion.
Keywords: transmission, schistosomiasis 30 NTDASIA 2019
O:28 Rickettsia Infection in Sri Lanka, Epidemiology and Clinical Impacts
Rajapakse RPVJ & Karunathilake KJK
Abstract: Rickettsiae, a group of alpha-proteobacteria are Department of Veterinary Pathobiology, Faculty of Veterinary obligate intracellular parasites of eukaryotic cells. Three Medicine and Animal Science, recognized rickettsial disease entities, Spotted Fever Group University of Peradeniya. Sri Lanka (SFG), Typhus Group (TG) and Scrub Typhus Group (STG) are e-mail: [email protected] prevalent in different regions of the world and Sri Lanka. This presentation describes the serological, clinical manifestations, epidemiology with identification of causative species of the human rickettsial infection in the central region of the Sri Lanka. Total of 2,940 patients from 2014 to 2018 with clinical features suggestive of rickettsial infection admitted to the medical unit of the Teaching Hospital, Peradeniya were included in this study. IgG of antibody level was examined by IFT using Rickettsia conori antigen. Clinical data was taken from the hospital recordings and clinical history records. There were 1,640 male patients (54%) and 1300 of females. Mean age of the study group was 44 years (SD 19.4). Confirmation of the diagnosis was made on IFA, IgG titer and clinical data. Serologically, 1882 (64%) patients belong to the high titre seropositive group, 764 (26%) to the low titre seropositive group and 294 (10%) belong to the negative seroreactivity group. All the patents in the high titre seropositive group had fever and skin rash. Other main symtoms include headache (89%) Myalgia (77%) and arthralgia (66%) mainly of large joints. Common physical signs were conjunctival injection (62%). Pallor (10%), cervical lymphadenopathy (8.6%), hepatomegaly (16%) and lung creptiations (13%). Majority of the group had throbocytopenoa (58%) and few had low heamoglobin (10%). Leukcocytosis (26%) was more common compared to leukopenia (13%) and some had elevated Aspartate Transaminase (52%) and Alamine Transaminase (55%) levels. Skin rash characterized by discrete maculopapular pattern and dusky erythematous hue was distributed mainly the limbs, back of the chest, anterior abdomen and soles. Common variations were fern leaf shapes, blackish discoloration and gangrene of the skin. None of the patients had classical eschars and 6% had only possible tick bite marks. A series of patients predominately of the older age group had obscure neurological manifestations such as altered level of consciousness, extrapyramidal manifestations, tremor, rigidity, and dyskinesia. This study reaffirmed the presence of rickettsioses in the study region with predominate of SFG. It is likely that the prevailing environmental condition and the host or vector factors are more conductive for infections by SFG in the region. NTDASIA 2019 31
O:29 Laboratory Diagnosis of Leptospirosis - a Way Forward
Noraziah Mohamad Zin,1,*,** Mohd Asif Mohd Sukri,1 Siti Norlia Othman,2 Fatin Ridzuana Abd Rahman1
1 Abstract: Clinical symptoms of leptospirosis include febrile Programme of Biomedical illness that can be mistaken for other infectious diseases. Sciences, Faculty of Health Sciences, Universiti Kebangsaan Therefore, leptospirosis is usually diagnosed late and further Malaysia, Jalan Raja Muda Abdul complicated by the nature of Leptospira sp. that is fastidious Aziz, 50300, Kuala Lumpur, and slow growth in culture. Current laboratory diagnostic tests Malaysia for detection of leptospirosis include microscopic agglutination 2 Virology Serology Unit, test (MAT), enzyme-linked immunosorbent assay (ELISA), Department of Medical molecular detection, isolation of Leptospira sp. by culture and Microbiology and Immunology, immunohistochemistry method. MAT is a current reference Pusat Perubatan Universiti standard for leptospirosis. In this test, diluted serum from Kebangsaan Malaysia, Jalan Yaacob patient suspected with leptospirosis is mixed with liveLeptospira Latif, Bandar Tun Razak, 56000, Cheras, Kuala Lumpur Malaysia sp. serovers and agglutination reaction is read under dark field * Presenter **e-mail: noraziah.zin@ microscopy. Endpoint titre is read at dilution in which 50% of ukm.edu.my agglutination occurs. Samples from acute and convalescent periods are tested in MAT and a fourfold increase of titre is required for confirmatory result. Detection of IgM in suspected patient can also be conducted using ELISA. However, ELISA is usually only used for screening purpose and confirmatory test using other methods must be performed. Molecular detection of leptospirosis using polymerase chain reaction (PCR) towards specific genes such as housekeeping genes of Leptospira sp. is rapid, sensitive and specific. PCR can be expensive as it requires reagents and thermocycler to operate but it is available in most laboratory nowadays. Development of loop-mediated isothermal amplification (LAMP) procedure that employs constant temperature reaction is economically needed. Direct microscopic identification of Leptospira sp. also can be employed for diagnosis purpose by culture from suspected leptospirosis patient. Nevertheless, culture of Leptospira sp. is not feasible for rapid identification as it is time consuming and laborious. Another method is examination of tissue samples from deceased patients using immunohistochemistry method. In this method, tissue is stained and Leptospira sp. can be identified under microscope. As leptospirosis symptoms are usually not conclusive and can be mistaken for other diseases, there is a need for development of novel reliable, rapid, sensitive and high specificity method for laboratory diagnosis of leptospirosis. Molecular tests using PCR particularly using LAMP procedure may be considered as alternative supporting methods if immuno-rapid test failed to produce positive results.
Keywords: Leptospirosis, Leptospira sp., microscopic agglutination test, ELISA, Leptospira culture, immunohistochemistry, polymerase chain reaction, loop-mediated isothermal amplification 32 NTDASIA 2019
O:30 PRECEDE-PROCEED Model Framework for Controlling Opisthorchis viverrini Infection: Community-Based Intervention with Propensity Score Matching Analysis.
1 Department of Parasitology, Picha Suwannahitatorn, 1,*,** Phramongkutklao College of Medicine, Thailand * Presenter **e-mail: atnox25@ Abstract: Fieldwork conducted in Ta-Kadarn Subdistrict in live.com Chachoengsao Province during 2009-11 indicated that prevalence and incidence of Opisthorchis viverrini (OV) infection were surprisingly high considering the studied areas were outside the endemic Northeastern region of Thailand. Consumption of uncooked fish menu,Koi pla, increased risk for acquiring infection. PRECEDE-PROCEED model framework was applied in the community in 2012 to comprehensively engage the communities with the aim of reducing OV infection incidence through behavioral modification by decreasing Koi pla consumption. Intervention and control groups were matched by propensity score matching. The intervention group participated in community-derived mutual agreement plan to discontinue Koi pla consumption. After the 17-month study period, both groups were evaluated for OV infection by stool examination and Koi pla consumption was assessed by questionnaire. The results showed that community intervention significantly reduced incidence of OV infection by 63% (95% CI: 7-85), p = 0.03 and reduced Koi pla consumption by 46% (95% CI: 1 - 71), p = 0.04. Following the PRECEDE-PROCEED framework, community participatory action including community-derived intervention was considered adaptive and practically suited to locals’ lifestyles where consumption behaviors were complex involving traditional beliefs, attitudes and diverse cultural backgrounds. Local health volunteers from those communities could also play an important role as a reinforcing factor. The PRECEDE-PROCEED model framework also provided a comprehensive approach for intervention/evaluation planning.
Keywords: PRECEDE-PROCEED, propensity score matching, intervention, community,Opisthorchis viverrini NTDASIA 2019 33
O:31 M&Ms - Masculinity and Multi-Levels of Information Dissemination -are Contributing to Liver Fluke Infection Risk in Thailand
Yi-Chen Wang,1,* Carl Grundy-Warr,1 Paiboon Sithithaworn,2 Miles Kenney-Lazar,1 Jutamas Namsanor2 1Department of Geography, National University of Singapore, Abstract: Infections with the liver fluke Opisthorchis viverrini Singapore 2 (Ov) is a major public health problem in Southeast Asia, Department of Parasitology, Khon Kaen University, Thailand particularly in the Mekong region. This foodborne parasite has *Presenter e-mail: [email protected] infected an estimate of 10 million people in the region through the consumption of raw or undercooked freshwater fish. There has been considerable amount of efforts dedicating to Ov diagnosis and control programs in Thailand. While more and more people are aware of the associated health risks, consumption of raw fish dishes has not ceased and numerous pockets of the northeastern provinces still report high Ov prevalence. This study examines the roles of social-cultural and structural factors contributing toOv infection risk. Surveys and interviews were conducted in four villages in southern Kalasin Province. Despite awareness campaign was recently conducted in the villages, approximately 32% of the participants reported that they would continue to consume raw fish, mainly because of the delicious taste and family tradition. Participants have further mentioned that social gathering, convenient to prepare, and affordability as the reasons for consumption. Some male participants have thought that eating raw foods would enable them to gain strength, much like tigers and lions. Such believe underscores that raw fish consumption is culturally linked to masculinity. Although about 70% of the participants have linked to Ov infection to cancer, misconceptions of liver fluke life cycle, Ov infection health consequence, and food safety remain. This is likely attributed to the structural factor of multiple levels of information dissemination from government and university medical professionals to local health center officers, health volunteers and villagers. For disease prevention to be most effective, health campaigns must take the influences of masculinity and multi-levels of information dissemination M&M into consideration.
Keywords: Liver fluke infection, social-cultural factors, raw attitude, information dissemination 34 NTDASIA 2019
O:32 Sub-District Health Management Evaluation of Liver Flukes and Cholangiocarcinoma Surveillance, Prevention and Control in Thailand’s 7th Health Region.
1 Office of Disease Prevention and Kesorn Thaewnongiew,1,*,** Cherdpong Mongkonsin,1 Control, Region 7, Ministry of 1 1 Public Health, Khon Kaen Sumalee Chantaluk, Sujinun Treedetch, 2 3 2 Bureau of General Communicable Oranard Watthanawong, Supaporn Lamoonsil Diseases, Department of Disease Control, Ministry of Public Health, Abstract: This study aimed to evaluate sub-district health Khon Kaen. management of Liver flukes and cholangiocarcinoma surveillance 3 Regional Health Promotion Centre systems in Thailand’s 7th health region across four dimensions 7, Ministry of Public Health, namely input, process, output and feedback. The study was conducted Khon Kaen. in sub-districts where prevalence of Liver fluke was more than 10 * Presenter **e-mail: Kesthaew@ percent in 2015. Twenty-nine sub-districts were randomly sampled hotmail.com from 42 of those. Data was collected from 5 groups; 1) health officers in health promoting hospitals (29), 2) presidents of sub-district administration organizations (22), 3) teachers in primary school (87), 4) students in primary school (year 4-6) who were able to have stool examination and 5) community members (750). Data were gathered through questionnaires, interviews, and document review and analyzed using descriptive statistics, namely frequency, percentage, mean and standard deviation. Input evaluation showed that all sub-districts had clear policies and used local resources in implementing health plans and providing knowledge to all related networks. All sub-districts surveyed had databases for those above 40 years old a and 62.1% had learning center in their community. Process evaluation found that 72.4% had sub-district health plans, all had campaigns against eating cooked fish, 86.2% had environment risk reduction plans and 89.7% had teaching and learning related to Liver Flukes in schools and 93.1% had home visit and caring for patients. Regarding, outcomes evaluation revealed that there were four dimensions which were 1) people dimension; infection rate among people reduced 5 % when compared with year 2014. 68.9% had Liver Flukes screening in people age more than 15 years old. Liver Fluke and cholangiocarcinoma screening and referral for treatment was fast. In addition, all sub-districts provided palliative care for all patients. 2) Local waste reduction domain; there were no waste reduction in the community. 3) Students domain; 43.7 % had knowledge at high level. 20.2% had positive health belief and 16.2 % cared for themselves to prevent Liver Flukes while 0.8 % were infected Liver Flukes. 4) Innovation domain; 96.6% of sub-district health management had cholangiocarcinoma risk surveillance every six months. Feedback evaluation revealed that 82.8% had p action plans based on evaluation feedback and 69% improved plans for action. Thus, the study indicated that staff who are responsible for developing sub-district health management in Liver Flukes and cholangiocarcinoma surveillance, prevention, and problem solving have to improve waste management and review teaching and learning in school. They also should encourage people to eat cooked fish, Improve excretion management and school curriculums and changing local citizens’ behavior. 35
Poster Presentation 36 37
List of Poster Presentation P:01 Identification and Characterization of Glyceraldehyde 3-Phosphate Dehydrogenase 39 from Fasciola gigantica
P:02 SjCB2, a Cysteine Protease that Plays a Role in the Cercariae Invasion of Schistosoma 40 japonicum P:03 An Improved Genome Assembly of the Fluke Schistosoma japonicum 41 P:04 Co-Dispersal of the Blood Fluke Schistosoma japonicum and Homo sapiens in the 42 Neolithic Age P:05 Metagenomic Sequencing and Untargeted Metabolomic Profiling Reveal Mouse 43 Gut Microbiome and Metabolite Changes Caused by Schistosoma japonicum Infection P:06 Dynamic Transcriptomes Identify Biogenic Amines and Insect-Like Hormonal 44 Regulation for Mediating Reproduction in Schistosoma japonicum P:07 Opisthorchis viverrini Extracellular Vesicles Interact with Cholangiocytes and 45 Reduce Parasite Burden in a Hamster Model of Infection P:08 Diagnostic Value of a Urine Antigen Assay to Assess Treatment Outcomes and 46 Reinfection in Human Opisthorchiasis P:09 Areca catechu L. reduce Opisthorchis viverrini motility 47 P:10 Apoptosis and Necroptosis of Rat Brain Induced by Angiostrongylus cantonensis 48 P:11 Expression of Hepatic Fibrosis in Hamster and Mice withOpisthorchis viverrini Infection 49 P:12 Distribution of Mast Cell in the Biliary Vicinity of Hamster and Mice with 50 Opisthorchis viverrini Infection P:13 Goblet Cell Hyperplasia in Biliary Epithelium of Hamster and Mouse with 51 Opisthorchis viverrini Infection P:14 Experimental Opisthorchiasis: Mitochondrial Antioxidant SKQ1 Reduces the Biliary 52 Neoplasia and Inflammation Markers P:15 A Novel Cell Line Derived from Experimental Opisthorchiasis felinea Associated 53 Cholangiocarcinoma P:16 Expression of VCP and Its Application in Cholangiocarcinoma 54 P:17 ABCC1 and CASP4 as Predictive Markers for Survival in Cholangiocarcinoma Patients 55 P:18 CCDC25 in the Sera as a Potential Screening/Diagnostic Biomarker for 56 Cholangiocarcinoma. P:19 Antiproliferative Effect of Tiliacora triandra Leaf Powder Ethanolic Extract on 57 Cholangiocarcinoma Cell Lines P:20 Risk of Opisthorchis viverrini Infection Among Villages Surrounding Nong Han 58 Lake, Sakon Nakhon Province P:21 Intensity of Opisthorchis viverrini Metacercariae in Freshwater Cyprinid Fish in 59 Nakhon Phanom Province, Northeast Thailand P:22 Seasonal Variation of the Infection Rate of Gnathostoma spinigerum Larvae in 60 Asian Swamp Eels (Monopterus albus) from Yangon, Myanmar 38
P:23 The Status of Human Hookworm Infections Among Residents Along the Mekong 61 River in Preah Vihear, Cambodia P:24 Molecular Identification of Anisakis Larvae Extracted by Gastrointestinal 62 Endoscopy from Health Check-up Patients in Korea P:25 Infection Status of Anisakid Larvae in Anchovies (Engraulis japonica) Purchased 63 from the Local Fishery Market Near the Southern Sea in Korea P:26 The Infection of Echinochasmus caninus n. comb. (Trematoda: Echinostomatidae) 64 in the Riparian People Along the Mekong River in Khammouane Province, Lao PDR P:27 MaxEnt Modeling of Soil-Transmitted Helminth Infection Distributions in Thailand 65 P:28 Ecological Niche Model Based on Maximum Entropy for Mapping Distribution of 66 Bithynia siamensis goniomphalos, First Intermediate Host Snail of Opisthorchis viverrini in Thailand P:29 Syndemic of Opisthorchiasis and Leptospirosis in Thailand: A Nationalwide Analysis 67 P:30 Enhancing Attraction of the Vector Mosquito,Aedes albopictus, by Using a Novel 68 Synthetic Odorant Blend P:31 Morphological Identification of Mosquito Vector Associated with Mangrove Forest 69 in Chachoengsao Province, Thailand P:32 Epidemiology of Dengue Infection and Circulating Dengue Serotypes in 70 Hospitalized Dengue Patients; A Decade Trend from 2009 to 2018 P:33 Fast Emerging Insecticide Resistance in Aedes albopictus in Guangzhou, China: 71 Alarm to the Dengue Epidemic P:34 Nix is a Male-Determining Factor in the Asian Tiger Mosquito Aedes albopictus 72 P:35 Development of an Efficient DNA-Launched Dengue Virus Serotype 3 Replicon 73 System Based on a Clinical Isolate for Antiviral Compound Discovery P:36 The Study of Association Between Polymorphism of Drug Resistant Genes of 74 Leishmania spp. P:37 Bacterial Profiles and Antibacterial Susceptibility Patterns from a Canine Model of 75 Kidney Stone Disease P:38 Phytochemical Screening and Antioxidant Activity of Ethanolic Leaf Extract of Sida acuta 76 P:39 Antimicrobial Susceptibility Patterns and Prevalence of Bacteria Isolated from 77 Participants with Chronic Kidney Disease in a Rural Thai Population P:40 Crystallization byEscherichia coli and Proteus mirabilis in Artificial Urine 78 P:41 Construction and Expression of Dengue Nonstructural 1 (NS1) Protein in 79 Lactobacillus casei P:42 Diclofenac Suppresses Proliferation of Cholangiocarcinoma Cell Lines 80 P:43 Leishmania martiniquensis Induces Activation and Increased Survival of Human 81 Neutrophil P:44 Expression of p53, Bcl-22, Granulin, and PCNA in the Liver of Opisthorchis viverrini 82 Infected Hamsters: A Comparison Between Single Infection and Repeated Infection Plus Praziquantel Administration NTDASIA 2019 39
P:01 Identification and Characterization of Glyceraldehyde 3-Phosphate Dehydrogenase from Fasciola gigantica
Purna B. Chetri,1,* Rohit Shukla,1 Timir Tripathi1 1 Molecular and Structural Abstract: Fasciola gigantica is an important food-borne Biophysics Laboratory, Department trematode responsible for the hepatobiliary disease, commonly of Biochemistry, North Eastern Hill University, Shillong -793022, India known as fascioliasis. In F. gigantica, the glyceraldehyde *Presenter e-mail: purnachetri2010 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the @gmail.com glycolytic pathway and catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the closely relate fluke F. hepaticaGAPDH. The gapdh gene was cloned and expressed in E. coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~37 kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in case of FgGAPDH, similar to the human GAPDH; thus the conformational topology of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.
Keywords: Fasciola gigantica, liver fluke, glyceraldehyde 3-phosphate dehydrogenase, activity, modeling, docking, quenching. 40 NTDASIA 2019
P:02 SjCB2, a Cysteine Protease that Plays a Role in the Cercariae Invasion of Schistosoma japonicum
Bingkuan Zhu,1* Fang Luo,1 Qunfeng Wu,1 Wenbing Yang,1 1 State Key Laboratory of Genetic Chengsong Sun,1 Bin Xu,2 Xiaojin Mo,2 Yongdong Li,3 Wei Hu1,2,** Engineering, Ministry of Education Key Laboratory for Biodiversity Abstract: Cercariae invasion is the first step for the schistosome Science and Ecological to infect the definitive host. Compare to S. mansoni and Engineering, Ministry of Education S. haematobium, the invasion speed of S. japonicum is much Key Laboratory of Contemporary Anthropology, School of Life faster, which implies differential invasion strategies were used Science, Fudan University, by S. japonicum. It is known that S. mansoni mainly depends on Shanghai 200433, China elastases to complete cercariae invasion. But for S. japonicum, 2 National Institute of Parasitic there is no evidence that any specific enzyme is involved in this Diseases, Chinese Center for process. In this work, we proved that a cysteine protease-SjCB2 Disease Control and Prevention, participates in the invasion process of S. japonicum cercariae Key Laboratory of Parasite and Vector Biology of China Ministry of invasion. The recombinant SjCB2 expressed in Escherichia coli Health, WHO Collaborating Centre was used to produce the polyclonal antibody against rSjCB2, for Tropical Diseases, Joint which analyze the native expression and tissue location of SjCB2 Research Laboratory of Genetics in cercariae. Active enzyme of SjCB2 was obtained by thePichia and Ecology on Parasite-host pastoris expression system. Enzyme activity and inhibitory test Interaction, Chinese Center for with inhibitor E-64 were investigated by the fluorogenic Disease Control and Prevention & Fudan University, Shanghai 200025, substrate Z-FR-MCA. Enzyme activity analysis reveals that SjCB2 China is a typical cysteine protease, the optimum pH value was 4.0, 3 Key Laboratory of the optimum temperature was 37°C. The enzyme activity can Organo-Pharmaceutical Chemistry, be totally inhibited by the cysteine protease inhibitor E-64 at 10 Gannan Normal University, μM concentration. The zymogen and active enzyme forms of Ganzhou 341000, China SjCB2 in the soluble cercariae extract were identified by *Presenter **e-mail huw@fudan. western-blot with antibody against rSjCB2. Furthermore, the edu.cn. immunofluorescence assay showed that SjCB2 was prominently presented in cercariae post-acetabular penetration glands and their ducts, indicating that it could be released during the invasion process. In addition, we found that SjCB2 was able to degrade main components of the skin, such as collagen, keratin, elastin and fibronectin; the immune molecules, like IgG, IgA, IgE and Complement C3. Furthermore, we found the worm burden had a 22.94% reduction after cercariae incubation with antibody against rSjCB2 before invasion the skin, which indicated that SjCB2 participates in the invasion process ofS. japonicum cercariae. These results suggest that SjCB2 was stored in the acetabular gland of cercariae and will be released during invasion process, then it will help the penetrating larva degrading the skin barrier and immune system components, which provide a new clue to understand the mechanism behind S. japonicum cercariae invasion.
Keywords: Schistosoma japonicum, cysteine protease, SjCB2, enzyme activity, cercariae invasion NTDASIA 2019 41
P:03 An Improved Genome Assembly of the Fluke Schistosoma japonicum
Fang Luo,1, #,* Mingbo Yin,1,2 # Xiaojin Mo,2, # Chengsong Sun,1 Qunfeng Wu,1 Bingkuan Zhu,1 Manyu Xiang,1 Jipeng Wang,1 Yi Wang,1 Ting Zhang,1 Bin Xu,2 Huajun Zheng,3 Zheng Feng,2 1 Wei Hu1, 2** State Key Laboratory of Genetic Engineering, Ministry of Education Key Laboratory for Biodiversity Abstract: Schistosoma japonicum is a parasitic flatworm that Science and Ecological causes human schistosomiasis, which is a significant cause of Engineering, Ministry of Education morbidity in China and the Philippines. A single draft genome Key Laboratory of Contemporary was available for S. japonicum, yet this assembly is very Anthropology, School of Life fragmented and only covers 90% of the genome, which make it Science, Fudan University, Songhu Road 2005, Shanghai, China difficult to be applied as a reference in functional genome 2 National Institute of Parasitic analysis and genes discovery. In this study, we present a Diseases, Chinese Center for high-quality assembly of the fluke S. japonicum genome by Disease Control and Prevention, combining 20 G (~53X) long single molecule real time sequencing Key Laboratory of Parasite and Vector Biology of China Ministry of reads with 80 G (~ 213X) Illumina paired-end reads. This Health, WHO Collaborating Centre improved genome assembly is approximately 370.5 Mb, with for Tropical Diseases, Joint contig and scaffold N50 length of 871.9 kb and 1.09 Mb, Research Laboratory of Genetics representing 142.4-fold and 6.2-fold improvement over the and Ecology on Parasite-host released WGS-based assembly, respectively. Additionally, our Interaction, Chinese Center for Disease Control and Prevention & assembly captured 85.2% complete and 4.6% partial eukaryotic Fudan University, 207 Rui Jin Er Benchmarking Universal Single-Copy Orthologs. Repetitive Road, Shanghai 200025, China elements account for 46.80% of the genome, and 10,159 of the 3 Chinese National Human Genome protein-coding genes were predicted from the improved Center at Shanghai, Shanghai 201203, China genome, of which 96.5% have been functionally annotated. *Presenter ** e-mail: huw@fudan. Lastly, using the improved assembly, we identified 20 significantly edu.cn #authors contributed equally expanded gene families in S. japonicum, and those genes were primarily enriched in functions of proteolysis and protein glycosylation. Using the combination of PacBio and Illumina Sequencing technologies, we provided an improved high-quality genome of S. japonicum. This improved genome assembly, as well as the annotation, will be useful for the comparative genomics of the flukes and more importantly facilitate the molecular studies of this important parasite in the future.
Keywords: Schistosoma japonicum, PacBio sequencing, genome assembly, genome annotation 42 NTDASIA 2019
P:04 Co-Dispersal of the Blood Fluke Schistosoma japonicum and Homo sapiens in the Neolithic Age
Mingbo Yin,1 Hong-Xiang Zheng,1,* Jing Su,1 Zheng Feng,2 1State Key Laboratory of Genetic Donald P. McManus,3 Xiao-Nong Zhou, 2 Li Jin,1,4 Wei Hu1,2,*,** Engineering and Ministry of Education Key Laboratory of Abstract: The global spread of human infectious diseases is of Contemporary Anthropology, considerable public health and biomedical interest. Little is Collaborative Innovation Center of Genetics and Development, School known about the relationship between the distribution of of Life Sciences, Fudan University, ancient parasites and that of their human hosts. Schistosoma Shanghai, 200438, China japonicum is one of the three major species of schistosome 2 National Institute of Parasitic blood flukes causing the disease of schistosomiasis in humans. Diseases, Chinese Center for Disease Control and Prevention, The parasite is prevalent in East and Southeast Asia, including Key Laboratory of Parasite and the People’s Republic of China, the Philippines and Indonesia. Vector Biology of the Chinese We studied the co-expansion of S. japonicum and its human Ministry of Health, WHO definitive host. Phylogenetic reconstruction based on complete Collaborating Center for Malaria, mitochondrial genome sequences showed that S. japonicum Schistosomiasis and Filariasis, Shanghai, 200025, China radiated from the middle and lower reaches of the Yangtze 3QIMR Berghofer Medical Research River to the mountainous areas of China, Japan and Southeast Institute, Brisbane, Queensland Asia. In addition, the parasite experienced two population 4006, Australia expansions during the Neolithic agriculture era, coinciding with 4Chinese Academy of Sciences Key Laboratory of Computational human migration and population growth. The data indicate that Biology, CAS-MPG Partner the advent of rice planting likely played a key role in the spread Institute for Computational Biology, of schistosomiasis in Asia. Moreover, the presence of different SIBS, CAS, Shanghai, 200021, subspecies of Oncomelania hupensis intermediate host snails China in different localities in Asia allowed S. japonicum to survive in *Presenter **email: [email protected] new rice-planting areas, and concurrently drove the intraspecies divergence of the parasite.
Keywords: Schistosoma japonicum, complete mitochondrial genome, co-expansion, Homo sapiens, Neolithic Age, rice agriculture NTDASIA 2019 43
P:05 Metagenomic Sequencing and Untargeted Metabolomic Profiling Reveal Mouse Gut Microbiome and Metabolite Changes Caused by Schistosoma japonicum Infection
1 Zhongshan School of Medicine, Yue Hu,1, 2, 3,* Jiansong Chen,4, 5 Hongli Zhou,1, 2, 3 Yiyue Xu,1, 2 1, 2, 3 1, 2, 3 6 Sun Yat-sen University, Guangzhou Ping Huang, Yubin Ma, Minzhao Gao, 510080, China 1, 2 4, 5 1, 2, 3,** Shaoyun Cheng, Haiyun Zhou, Zhiyue Lv 2 Key Laboratory of Tropical Disease Control (Sun Yat-sen Abstract: Schistosoma japonicum is one of the etiological agents University), Ministry of Education, of schistosomiasis, a widespread zoonotic parasitic disease, Guangzhou 510080, China however, the mechanism of the balanced co-existence between 3 Provincial Engineering the host immune system and S. japonicum as well as their Technology Research Center for Biological Vector Control, complex interaction remains unclear. In this study, the 16S rRNA Guangzhou 510080, China gene sequencing combined with metagenomic sequencing 4 School of Life Sciences, Sun approach, as well as UPLC-MS metabolomics profiling were Yat-sen University, Guangzhou applied to demonstrate the gut microbiome community 510275, China structure changes during schistosomiasis progression, the 5 Instrumental Analysis and functional interactions between the gut bacteria and Research Center, Sun Yat-sen University, Guangzhou 510275, S. japonicum infection in BALB/c mice, and the dynamic China metabolite profiling of the host. The results showed that both 6 Department of Gastroenterology, gut microbiome and the metabolites were significantly altered The Fifth Affiliated Hospital of Sun in different time after the infection. The decreases of richness Yat-sen University, Zhuhai 519000, and diversity and the obviously differed composition of gut China * ** microbiota were observed when compared to the uninfected Presenter e-mail: lvzhiyue@mail. status. The numbers of the bacteria which could produce sysu.edu.cn short-chain fatty acids decreased while the populations of opportunistic pathogens which could raise the risk of infections increased. Besides, metagenomic analysis illuminated AMPK signaling pathway and chemokine signaling pathway were significantly perturbed after the infection. Phosphatidylcholine and colfosceril palmitate in serum as well as xanthurenic acid, naphthalenesulfonic acid and pimelylcarnitine in urine might be the metabolic biomarkers due to their promising diagnostic potential at the early stage of the infection. Also, alternations of sphingolipid and glycerophospholipid metabolism were discovered in the infection. The present study might provide further understanding of the mechanisms during schistosome infection in aspects of gut microbiome and metabolites and facilitate the discovery of new targets for early diagnosis and prognostic. Further validations of potential biomarkers in human populations are necessary, and the exploration of interactions among S. japonicum, gut microbiome and metabolites need more future information.
Keywords: Schistosoma japonicum, gut microbiome, metabolomics, 16S rRNA, UPLC-MS 44 NTDASIA 2019
P:06 Dynamic Transcriptomes Identify Biogenic Amines and Insect-Like Hormonal Regulation for Mediating Reproduction in Schistosoma japonicum
1State Key Laboratory of Genetic Jipeng Wang,1,2 Ying Yu,1 Haimo Shen,2 Tao Qing,1 Engineering, Ministry of Education 1 1 2 1 Key Laboratory of Contemporary Yuanting Zheng, Qing Li, Xiaojin Mo, Shuqi Wang, 1 1 2 1 1 Anthropology, Collaborative Nana Li, Riyi Chai, Bin Xu, Mu Liu, Jian Li, 1 3 4 Innovation Center for Genetics and Ruixiang Zhang, Paul J. Brindley, Donald P. McManus, Development, School of Life Zheng Feng,2 Leming Shi,1 Wei Hu1,2,*,** Sciences, Fudan University, Shanghai 200438, China Abstract: Eggs produced by the mature female parasite are 2 Key Laboratory of Parasite and responsible for the pathogenesis and transmission of Vector Biology of the Chinese Ministry of Health, WHO schistosomiasis. Female schistosomes rely on a unique Collaborating Center for Malaria, male-induced strategy to accomplish reproductive development, Schistosomiasis and Filariasis, a process that is incompletely understood. Here we map detailed National Institute of Parasitic transcriptomic profiles of male and female Schistosoma Diseases, Chinese Center for Disease Control and Prevention, japonicum across eight time points throughout the sexual Shanghai 200025, China developmental process from pairing to maturation. The dynamic 3Department of Microbiology, gene expression pattern data reveal clear sex-related Immunology & Tropical Medicine, characteristics, indicative of an unambiguous functional division Research Center for the Neglected between males and females during their interplay. Cluster Diseases of Poverty, School of Medicine & Health Sciences, analysis, in situ hybridization and RNAi assays indicate that males George Washington University, likely use biogenic amine neurotransmitters through the nervous Washington DC 20037, USA system to control and maintain pairing with females. In addition, 4 Molecular Parasitology Laboratory, the analyses indicate that reproductive development of females Immunology Department, QIMR Berghofer Medical Research involves an insect-like hormonal regulation. These data sets and Institute, Brisbane, Queensland analyses serve as a foundation for deeper study of sexual 4006, Australia development in this pathogen and identification of novel *Presenter **email: huw@fudan. anti-schistosomal interventions. edu.cn Keywords: Schistosoma japonicum, dynamic transcriptome, reproduction development, pairing, hormonal regulation NTDASIA 2019 45
P:07 Opisthorchis viverrini Extracellular Vesicles Interact with Cholangiocytes and Reduce Parasite Burden in a Hamster Model of Infection
1 Department of Parasitology, Sujittra Chaiyadet,1,* Javier Sotillo,3, 5 Watchara Krueajampa,1 1 2 4 Faculty of Medicine, Khon Kaen Sophita Thongsen, Paul J. Brindley, Banchob Sripa, University, Khon Kaen, Thailand 3 1,** Alex Loukas, Thewarach Laha 2 Department of Microbiology, Immunology and Tropical Abstract: Infection with the liver fluke Opisthorchis viverrini Medicine, and Research Center for remains a public health problem in Southeast Asia and is the Neglected Diseases of Poverty, major risk factor of cholangiocarcinoma. Despite the interaction School of Medicine & Health of some O. viverrini secreted soluble molecules with host cells Sciences, George Washington University, Washington, DC, United have been well characterized, the specific interactions of States of America O. viverrini secreted extracellular vesicles (Ov-EVs) are still 3 Centre of Biodiscovery and unclear. Here we investigated the content of Ov-EVs, the Molecular Development of mechanism of the internalization into the host cell and also the Therapeutics, Australian Institute of Tropical Health and Medicine, potential of these EVs as well as the tetraspanin 2 (Ov-TSP-2) in James Cook University, Australia a hamster vaccine model. Ov-EVs can be internalized by 4 Department of Pathology, Faculty cholangiocytes and modulate their activation, promoting the of Medicine, Khon Kaen University, production of IL-6 and inducing cell proliferation. By performing Khon Kaen, Thailand 5 an analysis of the different compartments of Ov-EVs, we National Microbiology Centre, Instituto De Salud Carlos III, identified several proteins including the tetraspanins Ov-TSP-2, Madrid, Spain Ov-TSP-3. The co-incubation of Ov-EVs with antibodies against *Presenter **email: thewa_la@kku. these tetraspanins blocked their internalization resulting in ac.th the suppression of the internalization and cell proliferation. Furthermore, hamsters vaccinated with EVs and two recombinant proteins encoding the large extracellular loop (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) formulated with alum protected against O. viverrini, as shown by a reduction in worm recovery associated with an increase of the total IgG response to parasite infection, highlighting an important role in vivo. Hence, vaccination with Ovr -TSP-2, rOv-TSP-3, and Ov-EVs can reduce the intensity of O. viverrini infection by blocking a key host-parasite interaction.
Keywords: Opisthorchis viverrini, Opisthorchiasis, extracellular vesicles, tetrasapnins, vaccination 46 NTDASIA 2019
P:08 Diagnostic Value of a Urine Antigen Assay to Assess Treatment Outcomes and Reinfection in Human Opisthorchiasis
Chanika Worasith,1,2* Chompunoot Wangboon,3 1 Department of Parasitology, Jiraporn Sithithaworn,4 Anchalee Techasen,2,5 Faculty of Medicine, Khon Kaen Raynoo Thanan,2,6 Narong Khuntikeo, 2,7 Paiboon Sithithaworn 1,2** University, Khon Kaen 40002, Thailand Abstract: Urine antigen assay is a sensitive method for diagnosis 2 Cholangiocarcinoma Research Institute, Khon Kaen University, of opisthorchiasis and the performance is comparable to Khon Kaen 40002, Thailand copro-antigen detection. However, confirmation of genuine 3 School of Preclinical Science, infection in case of urine antigen positive but fecal egg-negative Institute of Science, Suranaree is required. In this study, we applied the urine assay method in University of Technology, Nakhon monitoring the outcomes and reinfection of O. viverrini after Rachasima 30000, Thailand 4 Faculty of Medicine, curative chemotherapy with praziquantel (PZQ). With reference Mahasarakham University, to the baseline study, assessment for new or incidence of Mahasarakham 44000, Thailand infection was also performed. In the subjects who had both egg 5 Faculty of Associated Medical and urine antigen positive, similar cure rates of PZQ (100%) at Sciences, Khon Kaen University, Khon Kaen 40002, Thailand 4 weeks post treatment were observed by fecal examination or 6 Department of Biochemistry, urine assay. In a long term followed up study at one-year post Faculty of Medicine, Khon Kaen PZQ treatment, the average reinfections with O. viverrini were University, Khon Kaen 40002, 16.8% by faecal examination and 27.7% by urine assay. The Thailand observed rates of reinfection were associated with local 7 Department of Surgery, Faculty of Medicine, Khon Kaen University, transmission levels in terms of the baseline prevalence and Khon Kaen 40002, Thailand intensity of O. viverrini infection. The data observed in this study * Presenter **e-mail: paib_sit@kku. indicate that urine antigen assay can be used not only for ac.th diagnosis and screening of infection but also for evaluation and follow-up after chemotherapy in opisthorchiasis. Because of the ease-of-use and non-invasive sample collection, the urine antigen assay is an effective tool for control and elimination of opisthorchiasis.
Keywords: Opisthorchis viverrini, monoclonal antibody-based enzyme-linked immunosorbent assay, praziquantel, follow-up, reinfection NTDASIA 2019 47
P:09 Areca catechu L. reduce Opisthorchis viverrini motility
Jarawee wannachart,1* Kamonsot Kumpangnil,1 Nantaya Krasuaythong,2 Nongnuch Kanharat,2 Ratana Leksomboon 2** 1 Biomedical Science Program, College of Medicine and Public Abstract: Cholangiocarcinoma caused by infection with liver Health Ubon Ratchathani fluke Opisthorchis viverrini, is a major health problem of University, Warin Chamrap, North-eastern, Thailand. Humans are infected by ingestion of Ubon Ratchathani 34190, Thailand 2 raw or undercooked cyprinid fish containing metacercariae. The College of Medicine and Public Health Ubon Ratchathani larvae of liver fluke will migrate through common bile duct after University, Warin Chamrap, metacercariae were excysted in small intestine. In this study, we Ubon Ratchathani 34190, Thailand have evaluated effects of the crude extract of Areca catechu L. *Presenter **e-mail: ratana_tlek@ against the motility of newly excysted juveniles (NEJs) of yahoo.ac.th O. viverrini on relative motility (RM) assay. The NEJs were incubated in RPMI-1640 medium containing 200 µg/ml, 1 mg/ ml, 5 mg/ml and 10 mg/ml of A. catechu L. crude extract. The RM values were evaluated after 0, 30, 60, 90, 120, 150 and 180 min incubation using scoring under the stereomicroscope. The control group were untreated. The results showed at 0 minutes (before treatment), O. viverrini has a good active. The crude extract of A. catechu L. treatment groups showed rapid decrease of motility at 30 min incubation. The initial reduction of motility occurred at 30 minutes and the increase at 60 minutes in crude extract at the concentration of 200 µg/ml, 1 mg/ml and 5 mg/ml. Additionally, the parasites incubated in 10 mg/ml concentration of crude extract rapidly reduced the RM values from the 60 to 90 min and died during 150 and 180 min. This study suggests that crude extract A. catechu L. has inhibition effects of the NEJs movement. Hence, the crude extract of A. catechu L., may exert effect against O. viverrini. It should be further studied for the effects of the crude extract of A. catechu L. in animal models, which is currently in the experimental stage.
Keywords: Opisthorchis viverrini, Areca catechu L., reduce motility 48 NTDASIA 2019
P:10 Apoptosis and Necroptosis of Rat Brain Induced by Angiostrongylus cantonensis
Zhou Hongli,1, 2, 3,* Chen Zhe,1, 2 Ma Yubin,1, 2, 3 Hu Yue,1, 2, 3 Huang Ping, 1, 2, 3 Zhang Mengying, 1,2,3 Wu Zhongdao,1, 2, 3 Lv Zhiyue1, 2, 3,** 1 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Abstract: Angiostrongylus cantonensis infection can lead to 2 Key Laboratory of Tropical eosinophils meningoencephalitis. The hosts of A. cantonensis Disease Control (Sun Yat-sen can be divided into permissive and non-permissive hosts, which University), Ministry of Education, presented different pathological outcomes. Our previous study Guangzhou, 510080, China 3 Provincial Engineering Technology indicated that A. cantonensis infection could cause the apoptosis Research Center for Biological and necroptosis of neuron, microglia and astrocytes in the Vector Control, Guangzhou, parenchymal and hippocampal regions of mice brain tissues. 510080, China However, it remains unknown that whether there is cell death * ** Presenter e-mail: lvzhiyue@mail. in the brain of permissive host infected with A. cantonensis. We sysu.edu.cn use RT - qPCR, western blot and immunofluorescence to determine the types of cell death in rats brain, results showed that the levels of apoptosis marker caspase-2 and necroptosis marker RIP3 went up along with disease progression, climbed rapidly at 14 dpi, peaked at 21 dpi and 28 dpi, but strikingly declined at 35 dpi when the worms migrated from brain to lung. Moreover, no changes in the mRNA or protein levels of LC3B, caspase-1 and caspase-11, indicating that apoptosis and necroptosis, but not autophagy and pyroptosis, occurred in the brain tissues of rat infected with A. cantonensis. Flow cytometry and transmission electron microscopy (TEM) further confirm the apoptosis and necroptosis of neuron, astrocyte and microglia in rat brain. Altogether, we showed for the first time that A. cantonensis infection caused the apoptosis and necroptosis of microglia and astrocytes in the parenchymal and hippocampal regions in permissive host, further demonstrating the pathogenesis of A. cantonensis infection and providing potential therapeutic targets for the management of angiostrongyliasis.
Keywords: Angiostrongylus cantonensis, rats, apoptosis, necroptosis NTDASIA 2019 49
P:11 Expression of Hepatic Fibrosis in Hamster and Mice with Opisthorchis viverrini Infection
Khao Keonam,1,3,* Woro Danur Wendo,1,3 Theerayuth Thongrin,1,3 Suwit Balthaisong,2,3 1 Faculty of Veterinary Medicine, Prasarn Tangkawattana,1 Sutas Sutthiprapa,2,3 Khon Kaen University, Khon Kaen Sirikachorn Tangkawattana1,3,** 40002, Thailand 2 Faculty of Medicine, Khon Kaen Abstract: Periductal fibrosis in hepatobiliary system is University, Khon Kaen 40002, Thailand defined as an antecedent finding prior to the development of 3 Tropical Disease Research Center, cholangiocarcinoma (CCA) in chronic opisthorchiasis. It is a Faculty of Medicine, Khon Kaen matter of fact that CCA occurs only in the susceptible host, but University, Khon Kaen 40002, not in the non-susceptible host. Hepatic fibrosis is believed to Thailand *Presenter **e-mail: sirikach@kku. be a subsequent finding following the invasion of periductal ac.th fibrosis into the surrounding hepatic stroma of each portal triad. Hence, distribution of hepatic fibrosis should be more extensive in the susceptible animal than in the non-susceptible animals. Regards, level of hepatic fibrosis in the Opisthorchis viverrini infected susceptible animal using male Syrian golden hamsters and the non-susceptible animal using Balb-C mice were compared. Five animals of each group were sacrificed at day 1, 2, and week 1, 2, 4 and 8 after the O. viverrini infection. Sirius red histochemistry was employed for the evaluation of hepatic fibrosis in their liver and biliary tract samples. Although hepatic fibrosis occurred in both animal groups, grade of the lesion was seemingly higher in the infected hamster than in the infected mice. This finding suggested that hepatic fibrosis should be considered as a precedent lesion in line with the CCA development in the susceptible infected host.
Keywords: fibrosis, Opisthorchis viverrini, hamster, mice 50 NTDASIA 2019
P:12 Distribution of Mast Cell in the Biliary Vicinity of Hamster and Mice with Opisthorchis viverrini Infection
Theerayut Thongrin,1,3,* Khao Keonam,1,3 Woro Danur Wendo,1,3 1 Faculty of Veterinary Medicine, Prasarn Tangkawattana,1 Sutas Suttiprapa,2,3 Khon Kaen University, Khon Kaen Sirikachorn Tangkawattana2,3,** 40002, Thailand 2 Faculty of Medicine, Khon Kaen Abstract: Accumulation of mast cells at the infection site is University, Khon Kaen 40002, Thailand commonly observed in any parasitic infection. During the initial 3 Tropical Disease Research Center, phase of Opisthorchis viverrini infection, numerous mast cells Faculty of Medicine, Khon Kaen are chemotactically recruited to infiltrate into the periductal University, Khon Kaen 40002, area and portal triad following the worm exposure to the biliary Thailand system. Afterward, eosinophils are subsequently drawn into the *Presenter **e-mail: sirikach@kku. ac.th perimeter of each worm containing bile duct. This cellular infiltration is presumed to associate with periductal fibrosis which is known to be the precedent lesion prior to the development of cholangiocarcinoma (CCA). Besides, chemicals released from these inflammatory cells could also injure the biliary cell either at the genetic or epigenetic level. Since CCA develops only in the susceptible host, the fibrotic lesion in the susceptible host should be more intense than the non-susceptible host. Regards, this lesion in the O. viverrini infected hamsters as the susceptible animal model and mice as the non-susceptible animal model was observed and compared. Five animals of each group were sacrificed at day 1, 2, and week 1, 2, 4 and 8 post-infection. Histochemistry using Alcian blue-Safranin O staining was employed to observe mast cells being vicinal to the periductal fibrotic lesion in the liver and biliary system of both animal groups. Grading and distribution of this inflammatory cell were further evaluated. It was apparently that mastocytic infiltration into the biliary system of both groups was observable since day 1 post-infection. Cellular distribution was generally intense in the lamina propria but sparsely in the biliary epithelium. The inflammatory reaction was gradually increased until week 8 in hamster, but maintained at low grade through the whole infection period in mice. This study suggested that mast cell may play important role as an underlying factor leading to the injury and proliferation of biliary cells.
Keywords: mast cell, Opisthorchis viverrini, hamster, mice NTDASIA 2019 51
P:13 Goblet Cell Hyperplasia in Biliary Epithelium of Hamster and Mouse with Opisthorchis viverrini Infection
Woro Danur Wendo,1,3,* Theerayuth Thongrin,1,3 Khao Keonam,1,3 Suwit Balthaisong,2,3 Sutas Suttiprapa,2,3 1 Faculty of Veterinary Medicine, Prasarn Tangkawattana,1 Sirikachorn Tangkawattana2,3,** Khon Kaen University, Khon Kaen 40002, Thailand 2 Abstract: Maturation process ofOpisthorchis viverrini is known Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, to complete concurrently with inflammation of the biliary system Thailand of the susceptible host. But in the non-susceptible host, such 3 Tropical Disease Research trematode cannot extend to a full development of which its Laboratory, Department of premature worm is probably expulsed from the biliary system Pathology, Faculty of Medicine, prior to provoking the inflammation. In the aspect of Khon Kaen University, Khon Kaen 40002, Thailand host-trematode interaction, increase of mucin production is *Presenter ** e-mail: sirikach@kku. believed to be a major candidate of innate mucosal immunity ac.th to intervene such process. Since goblet cell is responsible for mucin secretion, hyperplasia of goblet cell in the biliary epithelium should be more prominent in the non-susceptible host. To prove this assumption, hyperplastic degree of goblet cell in biliary epithelium of golden Syrian hamster as a susceptible animal and mouse as a non-susceptible animal with O. viverrini infection was compared. Animals of both groups were sacrificed on days 1, 2, 7, 14, 28, and 56 to collect liver and biliary tract. Alcian blue-safranin O staining was applied to identify goblet cell. The hyperplastic figure in both group commenced on day 28 post-infection. As a result, degree of hyperplasia was higher in the mouse group, but the degree of lesion was more apparent in the hamster group. These findings suggested that goblet cell hypertrophy as a part of mucosal immunity could play a certain protective role against the O. viverrini infection.
Keywords: goblet cell hyperplasia, Opisthorchis viverrini, hamster, mice 52 NTDASIA 2019
P:14 Experimental Opisthorchiasis: Mitochondrial Antioxidant SKQ1 Reduces the Biliary Neoplasia and Inflammation Markers
Oxana G. Zaparina,1,*,** Anna V. Kovner,1 1 Institute of Cytology and Genetics, Viatcheslav A. Mordvinov,1 Maria Y. Pakharukova1,2 Siberian Branch of Russian Academy of Sciences, Laboratory Abstract: Opisthorchiasis caused by the liver flukeOpisthorchis of Molecular Mechanisms of felineus is widespread in Asia and Europe. This disease is Pathological Processes, Novosibirsk 630090, Russia accompanied by liver disorders including periductal fibrosis, 2 Novosibirsk State University, cholangiofibrosis, biliary neoplasia with the potential of Novosibirsk 630090, Russia cholangiocarcinoma. To study the possible role of oxidative * ** Presenter e-mail: zp.oksana.93@ stress in the pathogenesis of opisthorchiasis we assessed lipid gmail.com peroxidation markers accumulation, DNA damage in infected hamsters from 1 to 18 months postinfection and the effect of mitochondrial antioxidant SKQ1 (10- (6’-Plastoquinonyl) decyltriphenylphosphonium)). We showed time-dependent accumulation of inflammatory, lipid peroxidation (MDA, HNE) and oxidative DNA damage (8-OHdG) markers. Moreover, liver histopathology, including inflammation, dysplasia, periductal fibrosis were assessed using morphometry analysis. The histopathology of liver i.e. biliary neoplasia and lipid peroxidation markers, and serum biochemistry were significantly attenuated by SKQ1 treatment. Thus, reactive oxygen species play a pivotal role in the pathogenesis of opisthorchiasis, probably mediating the processes of inflammation and dysplasia of the bile ducts. SKQ1 has a hepatoprotective effect on the liver by reducing oxidative stress.
Keywords: oxidative stress, reactive oxygen species, inflammation, neoplasia, Opisthorchis felineus. NTDASIA 2019 53
P:15 A Novel Cell Line Derived from Experimental Opisthorchiasis felinea Associated Cholangiocarcinoma
Galina A. Minkova,1,* Aleksandr G. Shilov,1 Dmitriy V. Ponomarev,1 Maria N. Lvova,1 Svetlana A. Romanenko,2 1 Institute of Cytology and Genetics Maria Y. Pakharukova,1 Viatcheslav A. Mordvinov1 SB RAS, Novosibirsk 630090, Russia 2 Abstract: Cholangiocarcinoma (CCA) is one of the most serious Institute of Molecular and Cellular Biology SB RAS, Novosibirsk complications of opisthorchiasis. It is an aggressive tumor that 630090, Russia is difficult to treat and diagnose at an early stage due to lack of *Presenter e-mail: maksimova@ specific symptoms and specific tumor markers. Furthermore, bionet.nsc.ru because of poor response to drug therapy research for new effective treatment and approach is required. Mechanisms of cholangiocarcinogenesis associated with O. felineus infection are not studied. The aim of the work was establishment of cholangiocarcinoma cell line and determination of CCA biomarkers. A cell line HamCCA-1 was derived from the CCA tissue of O. felineus-infected and dimethylnitrosamine-treated Syrian hamsters Mesocricetus auratus. To validate HamCCA-1 cell line, standard and potential tumor markers of human CCA, such as keratin 7 (CK7), vimentin (VIM), annexin A1 (ANXA1) and exostosin 1 (EXT1) were identified. Immunocytochemistry analysis of the cell culture revealed positive staining of all specific markers. Tumorigenicity of HamCCA-1 cells was demonstrated by allograft transplantation in hamster. Histological examination of transplanted tumors showed glandular structure similar to the primary CCA in hamster liver. In conclusion, the models can be used for studying cholangiocarcinogenesis, as well as for searching CCA treatments and methods for early diagnosis of the disease. This work was supported by Russian Science Foundation [18-15-00098].
Keywords: Opisthorchis felineus, cholangiocarcinoma, biomarkers, model, cell line, translational medicine 54 NTDASIA 2019
P:16 Expression of VCP and Its Application in Cholangiocarcinoma
Son D.A. Truong,1,*,** Tanakorn Proungvitaya,1 Sittiruk Roytrakul,2 Temduang Limpaiboon,1,3 Daraporn Chua-on,1 and Siriporn Proungvitaya1,3 1 Centre of Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Abstract: Cholangiocarcinoma (CCA) is the second most common Medical Sciences, Khon Kaen primary liver cancer and, CEA and CA19-9 often use for CCA University, Khon Kaen 40002, routine diagnosis, but they are low sensitivity and low specificity. Thailand. Overexpression of Valosin-containing protein (VCP) occurs in 2 National Center for Genetic Engineering and Biotechnology, many carcinomas and often correlates with the progression, National Science and Technology prognosis, and metastatic potential of carcinoma. However, the Development Agency, Pathumthani specific function the of VCP in human CCA remains unclear. In 12120, Thailand. our previous study, VCP is a candidate mitochondrial protein 3 Cholangiocarcinoma Research which expressed only in papillary CCA tumor. This study aims to Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, evaluate VCP as a diagnostic marker in CCA. VCP expression in Thailand. papillary and nonpapillary CCA tissues was analyzed using mass *Presenter **e-mail: trdason@gmail. spectrometry (MS) and western blot (WB), respectively. Then, com VCP expression between CCA and adjacent tissues was compared by immunohistochemistry (IHC) from Human Protein Atlas (HPA). To predict secretory protein nature of VCP, SecretomeP by using NN-score >0.5, SignalP by using likelihood ratio, European Molecular Biology Open Software Suite (EMBOSS) by using the value of the minimum weight at 3.5, and the Plasma Proteome Database (PPD), which is one of the most abundant resources on plasma proteins, were used. Based on HPA, the results of IHC revealed that VCP expression in CCA tissues was higher than in adjacent tissues. VCP was the highest expression in all of four mitochondrial proteins in papillary CCA tissue by MS. However, western blot results indicated that the VCP expressed in both papillary and nonpapillary CCA tissues. EMBOSS predicted that there were two signal peptide cleavage sites in the VCP sequence. Also, VCP can be present in the plasma or serum by using PPD. The significance expression of VCP in CCA tissue was demonstrated, and we can be found VCP in plasma or serum by using bioinformatics tools, which might be a candidate diagnostic biomarker of CCA patients. Therefore, VCP should be further investigated in the serum of CCA patients.
Keywords: Cholangiocarcinoma, mitochondrial protein, diagnosis biomarker, and valosin-containing protein. NTDASIA 2019 55
P:17 ABCC1 and CASP4 as Predictive Markers for Survival in Cholangiocarcinoma Patients
Mang Ngaih Ciin,1,* Tanakorn Proungvitaya,1, 2 Sittiruk Roytrakul,3 Temduang Limpaiboon,1, 2 Siriporn Proungvitaya,1,2,**
1 Centre of Research and Abstract: Cholangiocarcinoma (CCA) is the second most common Development of Medical Diagnostic primary hepatobiliary cancer and these patients have a meager Laboratories (CMDL), Faculty of prognosis and short-term survival. The majority of patients Associated Medical Sciences, presents with unresectable disease and have a survival of less Khon Kaen University, Khon Kaen than 12 months following diagnosis. This study aimed to 40002, Thailand 2 Cholangiocarcinoma Research investigate the candidate proteins that associated with the Institute, Faculty of Medicine, Khon survival in CCA patients. Serum samples from 6 CCA patients Kaen University, Khon Kaen 40002, with lymph node metastasis were analyzed using mass Thailand 3 spectrometry. The patients were divided into two groups: National Center for Genetic Engineering and Biotechnology 3 patients in short and 3 patients in long survival group. The (BIOTEC), National Science and median value (12.3 months) was used as a cut off for survival Technology Development Agency time of patients. Using Jvenn proteomic software, 40 unique (NSTDA), Pathumthani 12120, proteins were expressed in short survival group and 88 proteins Thailand * ** were uniquely observed in long survival group. STITCH version Presenter e-mail: sirpat@kku. ac.th 5.0 was used to predict the interaction of unique proteins with cancer survival related factors. ABCC1 and RPS10 showed an association to cisplatin and doxorubicin in short survival group but the association was stronger with ABCC1. In long survival group, CASP4, CCNB3, GFT2I, MCM9 and LIG1 were interacted with cisplatin and doxorubicin, and direct interaction was found only with CASP4. ABCC1 is a multidrug resistance associated protein and CASP4 involved in inducing apoptotic process. In conclusion, ABCC1 and CASP4 would be the valuable markers for predicting the survival of CCA patients.
Keywords: survival, cholangiocarcinoma, mass spectrometry 56 NTDASIA 2019
P:18 CCDC25 in the Sera as a Potential Screening/Diagnostic Biomarker for Cholangiocarcinoma.
Ravinnipa Chanakankun,1,*,** Tanakorn Proungvitaya,1 1 Centre of Research and Daraporn Chua-on,1 Temduang Limpaiboon,1,2 Development of Medical Diagnostic Sittiruk Roytrakul,3 Apinya Jusakul,1,2 Attapol Titapun,4 Laboratories, Faculty of Associated Apiwat Jarearnrat,4 Siriporn Proungvitaya1,2 Medical Sciences, Khon Kaen University, Khon Kaen 40002, Abstract: Recently, we identified that coiled-coil domain Thailand. 2 Cholangiocarcinoma Research containing 25 (CCDC25) is overexpressed in cholangiocarcinoma Institute, Faculty of Medicine, Khon (CCA) tissues but not in the adjacent normal tissues. This present Kaen University, Khon Kaen 40002, study investigated whether serum CCDC25 level can be a Thailand. potential marker for CCA diagnosis. For this purpose, using 3 National Center for Genetic Engineering and Biotechnology, bioinformatic tools, the results showed that CCDC25 can be National Science and Technology secreted into plasma/serum via a non-conventional pathway. Development Agency, Pathumthani Subsequently, the CCDC25 levels in the sera of patients with CCA 12120, Thailand (n=141) and healthy controls (HC; n=72) were measured using 4 Department of Surgery, Faculty of a quantitative dot blot assay based on the standard curve Medicine, Khon Kaen University, Khon Kaen 40002, Thailand created using recombinant CCDC25 protein. The results *Presenter **e-mail: c.ravinnipa@ showed serum CCDC25 level in the CCA group (0.28±0.06 ng/ gmail.com µl) was significantly higher compared with that in HC group (0.0017±0.0008 ng/µl). Receiver operating characteristic curve analysis revealed high sensitivity and specificity of serum CCDC25 level to differentiate CCA and healthy control at 93.0 and 100%, respectively. When correlation analyses were performed between serum CCDC25 level and CEA or CA19-9, no correlation was not observed, this suggests that CCDC25 is an independent biomarker different from CEA or CA19-9. Moreover, 23 CCA tissues were retrieved among 141 CCA cases to validate CCDC25 expression, CCDC25 expression in cancer tissues was moderately correlated with the serum CCDC25 level. To conclude, the serum CCDC25 level may be a promising screening and diagnostic marker for differential diagnosis- be tween CCA and healthy control.
Keywords: CCA, CCDC25, serum, screening, diagnosis. NTDASIA 2019 57
P:19 Antiproliferative Effect of Tiliacora triandra Leaf Powder Ethanolic Extract on Cholangiocarcinoma Cell Lines
Arunta Samankul,1,* Gusiri Senawong,1 Banchob Sripa,2 Thanaset Senawong1,** 1 Department of Biochemistry, Faculty of Science, Khon Kaen Abstract: The northeastern of Thailand has the highest University, Khon Kaen 40002, cholangiocarcinoma (CCA) incidence in the world. The infection Thailand 2 Department of Pathology, Faculty of Opisthorchis has been classified as a risk factor of the disease. of Medicine, Khon Kaen University, Furthermore, over half of the patients are diagnosed at the Khon Kaen 40002, Thailand terminal stage and very low survival rate. Therefore, the *Presenter **e-mail: sthanaset@kku. development of new drug treatment methods is also necessary ac.th for the development of treatment methods for cholangiocarcinoma. In this study, antiproliferative effect of T. triandra leaf powder ethanolic extract on cholangiocarcinoma cell lines (KKU-M214 and KKU-100 cells) was investigated by MTT assay. Apoptosis induction and cell cycle arrest were analyzed by flow cytometry. The gene expression of apoptosis and cell cycle arrest were analyzed by western blotting. Phenolic acid contents of T. triandra leaf powder ethanolic extract were analyzed by high performance liquid chromatography (HPLC) and total phenolic contents were analyzed by Folin reagent. T. triandra leaf powder ethanolic extract inhibited proliferation of both KKU-M214 and KKU-100 cells in a dose- and time-dependent manner, with IC50 values of 90.50 ± 9.62, 9.09 ± 0.15 and 7.86 ± 0.05 µg/ml for KKU-M214 cells and 115.32 ± 9.53, 13.67 ± 1.44 and 8.59 ± 0.36 µg/ml for KKU-100 cells at exposure times of 24, 48 and 72 h, respectively. Moreover, T. triandra leaf powder ethanolic extract induced apoptosis of KKU-M214 cells and increased population of KKU- 100 cells at G0/G1 phase. T. triandra leaf powder ethanolic extract up-regulated Ac-H3 expression but down-regulated p-ERK, p53, Bax, CDK4 and Bcl2 expression in KKU-M214 cells. T. triandra leaf powder ethanolic extract up-regulated Ac-H3, p21 and Bax expression but down-regulated p-ERK, p53, CDK4 and Bcl2 expression in KKU-100 cells. HPLC analysis six phenolic acids including p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids of the extract were identified. Total phenolic content as 33.09±3.17 mg gallic acid equivalent/g of dry extract. The results of this study suggest the possibility to develop T. triandra leaf extract as chemotherapeutic or chemoprevention drug for cholangiocarcinoma treatment in the future.
Keywords: Ya-nang, Tiliacora triandra, cholangiocarcinoma, antiproliferative effect 58 NTDASIA 2019
P:20 Risk of Opisthorchis viverrini Infection Among Villages Surrounding Nong Han Lake, Sakon Nakhon Province
Kulwadee Suwannatrai,1,*,** Natenapa Phanomkhet,1 1 Department of Biology, Faculty of Apiporn Thinkhamrop Suwannatrai2 Science and Technology, Sakon Nakhon Rajabhat University, Sakon Abstract: Opisthorchis viverrini is a major cause of cholangio- Nakhon 47000, Thailand carcinoma in Northeast Thailand. Sakon Nakhon Province is an 2 Department of Parasitology, Faculty of Medicine, Khon Kaen endemic area of O. viverrini infection which had the highest University, Khon Kaen 40002, prevalence of O. viverrini infection in Thailand of the surveyed Thailand population in 2005, despite the attempted control of * ** Presenter e-mail: kulwadeesnru@ opisthorchiasis for more than 20 years. This study aimed to gmail.com investigate risk of O. viverrini infection among villages surrounding Nong Han lake, Sakon Nakhon Province during June 2018 to July 2019. Bithynia siamensis goniomphalos snails were randomly collected from 20 villages in wet- and dry-season and examined for trematode infection by cercarial shedding and crushing methods. One thousand two hundred sixty of 28,482 snails were infected with trematodes as amphistome, cystocercous, furcocercous, virgulate, parapleurolophocercous, mutabile and O. viverrini. Sixty-six snails with prevalence of O. viverrini infection 0.21 - 2.21% were found from 5 villages ekal naH gnoN gnola detacol. Moreover, the highest prevalence of O. viverrini infection was found in the cool dry and wet season. These results indicated that Nong Han communities are risk areas of O. viverrini infection.
Keywords: Opisthorchis viverrini, Bithynia siamensis goniomphalos, Nong Han Lake, Sakon Nakhon Province NTDASIA 2019 59
P:21 Intensity of Opisthorchis viverrini Metacercariae in Freshwater Cyprinid Fish in Nakhon Phanom Province, Northeast Thailand
Sriwipa Chuangchaiya,1,* Sirinapa Prathummang,1 Nonglak Laoprom,2 Zulkarnain Md Idris3,** 1 Faculty of Public Health, Kasetsart University, Chalermphrakiat Sakon Abstract: Opisthorchis viverrini, a fish-borne zoonotic trematode, Nakhon Province Campus, 47000 requires cyprinid freshwater fish as one its intermediate host. Sakon Nakhon, Thailand 2 Faculty of Science and Engineer- Opisthorchiasis is endemic in parts of Southeast Asia, including ing, Kasetsart University, Chaler- North and Northeastern Thailand. In this region, the transmission mphrakiat Sakon Nakhon Province by fish intermediate hosts has received little attention. Campus, 47000 Sakon Nakhon, Therefore, a survey was performed to investigate the intensity Thailand 3 Department of Parasitology and of O. viverrini metacercariae (OV-MC) on wild cyprinid fish from Medical Entomology, Faculty of five districts in Nakhon Phanom Province, Northeast Thailand. Medicine, Universiti Kebangsaan A cross-sectional survey was conducted on 2,149 fishes Malaysia, 56000 Kuala Lumpur, consisting of 12 species randomly procured from rivers and local Malaysia * ** markets. Fish were collected in each of the summer, rainy and Presenter e-mail: zulkarnain. [email protected] winter seasons from January - December 2018. Fish were identified, counted, weighed and digested using pepsin-HCL. Samples were examined for OV-MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. In overall, the mean intensity of OV-MC were 0.3 and 9.1 cysts per fish and per kg of fish, respectively. OV-MC were found in six species of fish i.e.Anematichthys repasson, Hampala dispar, Henicorhynchus lineatus, Mystacoleucus marginatus, Osteochilus vittatus and Thynnichthys thynnoides. Among the five districts, different mean intensity of OV MC per fish/per kg of fish were observed: highest in Muang (0.5/13.7), similar in Na Kae (0.1/4.5), Na Tom (0.1/4.1) and Renu Nakhon (0.1/3.8), and no OV-MC in Si Songkhram. When stratified by season, the mean intensity of OV-MC were 2- and 3-folds higher in summer than both rainy and winter seasons for number fish and per kg of fish, respectively. Among the fish species, the mean intensity of OV-MC ranged from 0.005 to 0.9 per fish (or 2.8 to 21.9 per kg of fish) where H. lineatus harboured the highest OV-MC. In conclusion, OV-MC in cyprinid fish are abundant in Nakhon Phanom, with the intensity rate varying according to district, season and fish species.
Keywords: Opisthorchis viverrini, cyprinid fish, metacercariae, intensity, Thailand 60 NTDASIA 2019
P:22 Seasonal Variation of the Infection Rate of Gnathostoma spinigerum Larvae in Asian Swamp Eels (Monopterus albus) from Yangon, Myanmar
1 Department of Parasitology, Aung Phyo Wai,1,*,** Thidarut Boonmars,1 Faculty of medicine, Khon Kaen 2 2 University, Khon Kaen 40002, Ratchadawan Aukkanimart, Pranee Sriraj Thailand 2 Department of Traditional Abstract: Asian swamp eel (Monopterus albus) is consumed in Medicine, Faculty of Natural Southeast Asian counties to prepare special dishes, and is Resources, Rajamangala University famous as the intermediate host of a zoonotic nematode of Technology ISAN Sakon Gnathostoma species. In addition,M. albus can be a host for an Compus, Sakon Nakhon 47160, array of parasites such as acanthocephalans, nematodes, Thailand * Presenter **e-mail: phyothethein@ trematodes, cestodes, or even protozoan parasite such as gmail.com (agphyowai@kkumail. trypanosome species. In Myanmar where human gnathostomiasis com) is endemic, Gnathostoma spinigerum larvae were reported to be found in snakehead fish, catfish and Asian swamp eels. In this study, the infection rate and the intensity of infection of G. spinigerum larvae in Asian swamp eels from local markets of Yangon, Myanmar were monitored to see the seasonal variation of Gnathostoma infection rates in eels. In addition to the routine screening and counting ofGnathostoma larvae under a dissection microscope, validation of the species identification was confirmed using scanning electron microscopy and molecular method with ITS2 as a marker. Monitoring was carried out from 2016 to 2019 except for 2018. A total of 883 G. spinigerum third-stage larvae were collected from 596 eels in the study period. During the raining season, 121 Asian swamp eels were examined and 81(66.9%) were infected with 372 larvae (3.04/ eel). In the cold season, 88 (40.9%) out of 215 eels were infected with 350 larvae (1.63/eel). In the hot season, 74 (28.5%) out of 260 eels were infected with 161 G. spinigerum larvae (0.62/eel). The highest number of larvae found in one eel was 69. As an overall, both the infection rate and the infection intensity of G. spinigerum larvae in Asian swamp eels was higher in the raining season than in the cold and hot seasons. The average infection rate and the intensity of G. spinigerum larvae in the fish intermediate host, M. albus, in Myanmar were lower comparing with other ASEAN countries. Further etiological and epidemiological studies should be done in rural areas where Asian swamp eels are commonly consumed. Nationwide education and advertisement for the awareness of the risk of human infection is necessary.
Keywords: Asian swamp eels, Gnathostoma spinigerum, Seasonal variation, Yangon, Myanmar NTDASIA 2019 61
P:23 The Status of Human Hookworm Infections Among Residents Along the Mekong River in Preah Vihear, Cambodia
Bong-Kwang Jung,1,*,** Taehee Chang,1 Sooji Hong,1 Hyejoo Shin,1 Hyemi Song,1 Jeong-Gyu Lee,1 1 Institute of Parasitic Diseases, Keon-Hoon Lee,1 Jong-Yil Chai1,2 Korea Association of Health Promotion (KAHP), Seoul, Korea Abstract: Hookworm infection is one of the most common 2 Department of Tropical Medicine parasitic infections, which recognized as soil-transmitted and Parasitology, Seoul National University College of Medicine, helminthiasis or neglected tropical diseases. The largest Seoul, Korea proportion of hookworm infection occurs in developing *Presenter **e-mail: mulddang@ countries in Southeast Asia, especially. We investigated gmail.com prevalence of intestinal parasites among 1,156 people in Preah Vihear and Stung Treng province in May 2018 to identify infection status of intestinal parasites. Total hookworm infection rate was 12.1% (134/1,156), 12% (43/359) in Preah Vihear province and 11.4% (91/797) in Stung Treng province. Through this examination with fecal specimen, we could specify a village where people are highly infected with intestinal parasites. The prevalence of hookworm species infection is 22.5% (20/89) among residents living in the border village of Cambodia and Lao PDR (Kampong Sangkae, Preah Vihear province, Cambodia). To confirm the diversity and substantial geographical distributions of hookworm in the area, worm purgation was conducted in December 2018. Individuals who were confirmed to be infected with intestinal parasites by fecal specimen were treated with 40 mg/kg Praziquantel and 5 mg/kg Pyrantel pamoate, followed by purgation with MgSO4. From 9 people who appeared positive with hookworms, 65 adult hookworms were discharged by diarrhea. Intensity of infection was 7.2 per person among infected inhabitants. Sequencing of the hookworms by PCR product targeting CO1 gene identified hookworm species. Among 65 adult worms, 90% (59/65) was Necator americanus, and 10% (6/65) was Ancylostoma ceylanicum. This study has drawn different conclusions from the previous studies conducted in Cambodia since the region is located on the border, which has a unique regional characteristic. Though hookworm infection is generally considered as asymptomatic but, chronic and heavy infections can cause anemia and malnutrition which adversely affect socioeconomic development in developing countries. Considering the fact that worms are prevalent in developing countries with poor environmental conditions, environmental factors need to be investigated further to prevent hookworm infections among inhabitants.
Keywords: Necator americanus, Ancylostoma ceylanicum, Preah Vihear, Cambodia 62 NTDASIA 2019
P:24 Molecular Identification of Anisakis Larvae Extracted by Gastrointestinal Endoscopy from Health Check-up Patients in Korea
Sooji Hong,1,*,** Hyemi Song,1, Bong-Kwang Jung,1 1 Institute of Parasitic Diseases, Jaeeun Cho,1 Taehee Chang,1 Sun Huh,2 Jong-Yil Chai,1,3 Korea Association of Health Promotion, Seoul 07649, Korea Abstract: Anisakiasis is a zoonotic disease induced by anisakid 2 Department of Parasitology and nematodes, and endoscopic inspection is used for a Institute of Medical Education, Hallym University College of diagnosis or remedy for it. Anisakis simplex, A. physeteris, and Medicine, Chuncheon 24252, Korea Pseudoterranova decipiens had been reported to be the major 3Department of Tropical Medicine species causing human infections, particularly, in Japan. and Parasitology, Seoul National However, in Korea, recent studies strongly suggested that University College of Medicine, Seoul 03080, Korea A. pegreffii is the major species of human infections. To support *Presenter **email: nnaya@naver. this suggestion, we collected anisakid larvae (n=20) from 20 com human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.
Keywords: Anisakis pegreffii, Hysterothylacium aduncum, Gastrointestinal Endoscopy, Internal Transcribed Spacer (ITS), Cox2 NTDASIA 2019 63
P:25 Infection Status of Anisakid Larvae in Anchovies (Engraulis japonica) Purchased from the Local Fishery Market Near the Southern Sea in Korea
1Institute of Parasitic Diseases, Taehee Chang,1,*,** Sooji Hong,1 Hyejoo Shin,1 Bong-Kwang 1 1 1,2 Korea Association of Health Jung, Jeong-Gyu Lee, Jong-Yil Chai Promotion (KAHP), Seoul, Korea 2Department of Tropical Medicine Abstract: Anisakiasis (anisakidosis) refers to a foodborne and Parasitology, Seoul National zoonosis caused by ingesting the raw marine fish or squids University College of Medicine, infected with anisakid nematodes belong to Anisakidae and Seoul, Korea *Presenter **email: redwood10@ Raphidascarididae families including the genera Anisakis, naver.com Pseudoterranova, and Contracecum. Furthermore, it has been reported that Hysterothylacium sp. is zoonotic nematode that can cause clinical symptoms in humans. In this study, we developed the test methods for the diagnosis of anisakid larvae from marine fish including digestion method, microscopic examination, and genetic sequencing. Also, we evaluated the various PCR conditions using primer sets designed for distinguishing the species among the various anisakid larvae. Furthermore, we investigated the prevalence of anisakid nematodes in anchovies (Engraulis japonica) purchased from a market in Tongyeoung, Southern coastal area of Korea which accounts for more than 50% of total anchovy production in Korea. Anchovies were delivered to the Institute of Parasitic Diseases to be investigated by microscopic examinations. A total of 32 anisakid larvae were collected from 100 anchovies. Sequencing of the anisakid larvae by PCR product targeting COX2 gene identified anisakid larvae species. Among 32 larvae, 66% (21/32) were Anisakis pegreffii and several species of the genus Hysterothylacium, namely, and Hysterothylacium aduncum (9/32, 28%), Hysterothylacium sinense (1/32, 3%) and Hysterothylacium sp. (1/32, 3%) were also identified in the present study. Thus, A. pegreffii and Hysterothylacium sp. are the major species of anisakid nematodes in anchovies of the southern coast of Korea. Since a portion of anchovies purchased from the local fishery market was infected with anisakid nematodes, it is better to cook (higher than 60°C) or freeze (under -20°C) before intake.
Keywords: Anisakis pegreffii, Hysterothylacium sp., Engraulis japonica, Korea 64 NTDASIA 2019
P:26 The Infection of Echinochasmus caninus n. comb. (Trematoda: Echinostomatidae) in the Riparian People Along the Mekong River in Khammouane Province, Lao PDR
1 Institute of Parasitic Diseases, Hyejoo Shin,1* Bong-Kwang Jung,1 Taehee Chang,1 Korea Association of Health 2 3 4 Promotion, Seoul 07649, Korea Woon-Mok Sohn, Keeseon S. Eom, Tai-Soon Yong, 5 6 2 Department of Parasitology and Duk-Young Min, Bounlay Phammasack, 6 7 1,8,* Tropical Medicine, and Institute of Bounnaloth Insisiengmay, Han-Jong Rim, Jong-Yil Chai * Health Sciences, Gyeongsang National University College of Abstract: Adult worms of Echinochasmus caninus n. comb. Medicine, Jinju 52727, Korea (Verma, 1935) (Trematoda: Echinostomatidae) (syn.Episthmium 3 Department of Parasitology and Medical Research Institute, Parasite caninum Yamaguti, 1958) were recovered from 11 riparian Resource Bank, Chungbuk National people who resided along the Mekong River in Khammouane University School of Medicine, Province, Lao PDR. Fecal examinations done by the Kato-Katz Cheongju 28644, Korea technique, the cases revealed eggs of Opisthorchis viverrini/ 4 Department of Environmental minute intestinal flukes, hookworms, and in 2 cases echinostome Medical Biology, Institute of Tropical Medicine and Arthropods eggs. To recover the adult helminths, praziquantel 30-40 mg/kg of Medical Importance Resource and pyrantel pamoate 10-15 mg/kg in a single dose were given Bank, Yonsei University College of and purged with magnesium salts. Various species of trematodes Medicine, Seoul 03722, Korea (including O. viverrini and Haplorchis spp.), cestodes, and 5 Department of Microbiology and Immunology, Eulji University nematodes were recovered from their diarrheic stools. Among School of Medicine, Daejeon the trematodes, small echinostome flukes (n = 42; av. 3.8 34824, Korea specimens per case) of 0.7-1.2 mm in length are subjected in 6 Department of Hygiene and this study. They were morphologically characterized by having Prevention, Ministry of Health, 24 collar spines interrupted dorsally and anterior extension of Vientiane, Lao PDR 7 Department of Parasitology, Korea vitellaria from the cirrus sac or genital pore level to the posterior University College of Medicine, end of the body. Particularly based on this extensive distribution Seoul 02841, Korea of vitellaria, the specific diagnosis was made asEchinochasmus 8 Department of Tropical Medicine caninus. The cases were co-infected with various other helminth and Parasitology, Seoul National University College of Medicine, parasites; thus, clinical manifestations specific for this Seoul 03080, Korea echinostome infection were difficult to determine. The present *Presenter ** email: [email protected] paper describes for the first time human E. caninus infections in Lao PDR. Our reported cases marked the 4th-14th human infections with this echinostome around the world following the 3 previous cases reported from Thailand.
Keywords: Echinchasmus caninus, Echinostome, Khammouane, Lao PDR NTDASIA 2019 65
P:27 MaxEnt Modeling of Soil-Transmitted Helminth Infection Distributions in Thailand
Jukkrid Chaiyos,1 Kulwadee Suwannatrai,2 Kavin Thinkhamrop,3 Khanittha Pratumchart,1 Chanisala Sereewong,1 Smarn Tesana,1 Sasithorn Kaewkes,1 Banchob Sripa,4,5, Thitima Wongsaroj,6 1 Department of Parasitology, Apiporn T. Suwannatrai1,*,** Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Abstract: Soil-transmitted helminths (STHs), i.e. Ascaris Thailand lumbricoides, Trichuris trichiura, hookworms, and Strongyloides 2 Department of Biology, Faculty of stercoralis, are widely distributed in tropical and subtropical Science and Technology, Sakon areas. The aim of the study was to identify the effects of Nakhon Rajabhat University, Sakon Nakhon 47000, Thailand environmental and climatic factors on distribution patterns of 3 Data Management and Statistical STHs and to develop a risk map for STH infections under current Analysis Center, Faculty of Public environmental and climate regimes in Thailand. Geographical Health, Khon Kaen University, information systems (GIS), remote sensing, and Maximum Khon Kaen 40002, Thailand 4 WHO Collaborating Centre for Entropy (MaxEnt) algorithm software were used to determine Research and Control of the significant factors and to create predictive risk maps for STH Opisthorchiasis, Tropical Disease infections in Thailand. The disease data from Thailand covered Research Laboratory, Khon Kaen the years from 1969 to 2014, while environmental and climatic 40002, Thailand 5 data were compiled from the Worldclim database, MODIS Department of Pathology, Faculty of Medicine, Khon Kaen University, satellite imagery, Soilgrids, and ISCGM. The models predicted Khon Kaen 40002, Thailand that STHs occur mainly in southern Thailand. Mean annual 6 Department of Disease Control, precipitation was the factor most affecting the current Bureau of General Communicable distribution of A. lumbricoides, T. trichiura, and S. stercoralis. Diseases, Ministry of Public Health, Nonthaburi 11000, Thailand Land cover class was the main predictor for the distribution of * Presenter **e-mail: apiporn@kku. S. stercoralis and important for hookworms. Altitude was the ac.th dominant factor affecting the distribution of hookworms, and mean temperature of the wettest quarter was significantly associated with A. lumbricoides distribution. A predicted distribution map of STHs to identify environmental risk factors in Thailand is presented. This work provides a model for use in STH monitoring and health planning not only in Thailand but also in other countries with similar disease conditions.
Keywords: soil-transmitted helminths, geographic information system, remote sensing, ecological niche modeling 66 NTDASIA 2019
P:28 Ecological Niche Model Based on Maximum Entropy for Mapping Distribution of Bithynia siamensis goniomphalos, First Intermediate Host Snail of Opisthorchis viverrini in Thailand 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Khanittha Pratumchart,1,*,** Kulwadee Suwannatrai,2 Thailand Chanisala Sereewong,1 Kavin Thinkhamrop,3 Jukkrid Chaiyos,1 2 Department of Biology, Faculty of Thidarat Boonmars,1 Apiporn T. Suwannatrai1 Science and Technology, Sakon Nakhon Rajabhat University, Sakon Nakhon 47000, Thailand Abstract: Opisthorchiasis is fish-borne disease endemic in 3 Data Management and Statistical Cambodia, Laos, Vietnam, Myanmar and Thailand. The Analysis Center, Faculty of Public distribution of Opisthorchis viverrini coincides with that of the Health, Khon Kaen University, intermediate hosts as determined by environmental and climatic Khon Kaen 40002, Thailand *Presenter **e-mail: Jiewkhanittha factors. In this study, we used a geographic information system @gmail.com (GIS), remote sensing (RS) and maximum entropy (MaxEnt) algorithm software to predict the distribution of Bithynia siamensis goniomphalos in Thailand on the basis of environmental and climatic factors. The MaxEnt model forB. s. goniomphalos was excellent, with average test AUC values of 0.89. The predicted distribution of B. s. goniomphalos was affected by altitude, land cover, normalized difference vegetation index (NDVI), precipitation in the driest month (BIO 14), land surface temperature (LST) and soil pH. The areas suitable for B. s. goniomphalos were mostly in Northeast Thailand and some northern parts of the country. The presence of B. s. goniomphalos was significantly decrease associated with increasing altitude and increasing NDVI value. Opisthorchiasis prevalence data were overlaid on the map of predicted B. s. goniomphalos distribution. Villages where there was a high probability of the occurrence of B. s. goniomphalos were those with the highest prevalent of O. viverrini. Opisthorchiasis prevalence was directly proportional to the likelihood of snail occurrence as predicted by the model. This ecological niche model provides a model for use to predict Bithynia snail distribution and hence to provide a basis for future work of opisthorchiasis prevention in opisthorchiasis-endemic countries.
Keywords: Bithynia siamensis goniomphalos, Opisthorchis viverrini, MaxEnt Ecological niche model, Thailand NTDASIA 2019 67
P:29 Syndemic of Opisthorchiasis and Leptospirosis in Thailand: A Nationalwide Analysis
Muhammad Almanfaluthi,1,2* Stefani Widodo,1,2 Sutas Suttiprapa,1 Thitima Wongsaroj,3 Banchob Sripa1** 1 WHO Collaborating Centre for Research and Control of Abstract: Opisthorchiasis caused by Opisthorchis viverrini is an Opisthorchiasis (Southeast Asian important foodborne trematodiasis in Thailand, Laos and Liver Fluke Disease), Faculty of Cambodia. Interestingly, the opisthorchiasis endemic region Medicine, Khon Kaen, Thailand 2 Faculty of Medicine, University of overlaps with an area of leptospirosis emergence. Syndemic Muhammadiyah Purwokerto, (interaction) of opisthorchiasis and leptospirosis and its Indonesia association with socio-economic determinants, occupation risk 3 Department of Disease Control, factors, reservoir hosts and flooding in Thailand needs to be Ministry of Public Health, Thailand *Presenter **e-mail:banchob@kku. explored. A confirmed incidence case (provincial aggregate) of ac.th opisthorchiasis and leptospirosis for years 2009 and 2014 were obtained from Ministry of Public Health of Thailand. A Bayesian multivariate Poisson with intrinsic conditional autoregressive (iCAR) and Besag-York-Mollié (BYM) modeling was used to explore the impact of opisthorchiasis toward leptospirosis. Principle component analysis (PCA) were deployed to reduce multidimensionality of associate risk factors and nested correlation analysis for subset group. Ophisthorchiasis increase the risk of leptospirosis 3·8% (95% credible interval [CrI] 3·6-4·0%) per-unit percentage, Nationally. The maps of posterior relative risk demonstrated evidence of opisthorchiasis massively alter the risk of leptospirosis in provincial level (synergistic syndemic). Socio-economic and occupation factors significantly influence disease syndemic with moderate effect sizes as -·4, and .5 respectively (p-value <0·001). Northeast region significantly not only has the highest poverty level but also the highest proportion for occupational risk. Cat and dog play crucial role maintaining these syndemic (r-value ·78 and ·6 for both diseases with p-value <0·001). This study provides the syndemic evidence of opisthorchiasis and leptospirosis, particularly in Northeast Thailand. Socio-economic and occupation parameter are highly associated with syndemic tailing to the characteristics of neglected tropical diseases whereas the role of reservoir host highlights the importance of interdisciplinary approach such as one-health for sustainable disease control.
Keywords: Syndemic, opisthorchiasis, leptospirosis, Thailand 68 NTDASIA 2019
P:30 Enhancing Attraction of the Vector Mosquito, Aedes albopictus, by Using a Novel Synthetic Odorant Blend
Lihua Xie,1,* Wenqiang Yang,1 Hongmei Liu,1 Tong Liu,1 1 Department of Pathogen Biology, Yugu Xie,1 Feng Lin,1 Guofa Zhou,2 Xiaohong Zhou,1 Guangdong Provincial Key Kun Wu,1 Jinbao Gu,1 Guiyun Yan,2 Xiao-Guang Chen1,** Laboratory of Tropical Disease Research, School of Public Health, Abstract: The Asian tiger mosquito, Aedes albopictus, an Southern Medical University, Guangzhou, China. increasingly relevant arboviral vector, has spread worldwide. 2 Program in Public Health, However, currently available tools are limited in terms of University of California Irvine, effective monitoring of vector populations and accurate Irvine, California, United States of determination of the extent of viral transmission, especially America before and during outbreaks. Therefore, it is essential to develop *Presenter **e-mail: xgchen2001@ hotmail.com novel monitoring and surveillance tools, particularly those that target adult mosquitoes and enhance the trapping efficiency for Ae. albopictus. In this study, a variety of human body odorants associated with different types of mosquito olfactory receptors were selected, and their attractiveness to Ae. albopictus was tested by a four-arm olfactometer. In the olfactometer experiments, the attractiveness of the selected candidate compounds at varying dilutions was poor when the individual compounds were used alone.The optimal compatibility and proportion of the odorants, Mix-5, was observed via orthogonal design analyses. In the laboratory, the average numbers of female Ae. albopictus mosquitoes attracted by the synthetic odorant blend Mix-5 were 27.00 and 27.50, compared with 12.00 and 14.83 for the control, when using Mosq-ovitraps and Electric Mosquito Killers, respectively. In the field, the effectiveness of generic BG-Lure, Mix-5 and a control treatment was compared with a baited Biogents Sentinel trap (BGS-trap) using a Latin square design. The average number ofAe. albopictus female mosquitoes trapped by Mix-5 was 9.67 females/trap, whereas the average numbers for BG-Lure and the control were 7.78 and 4.47, respectively. The lure also played an important role in attracting Culex quinquefasciatus mosquitoes, and the average numbers of Cx. quinquefasciatus female mosquitoes attracted by Mix-5, BG-Lure and the control were 18.78, 25.11, and 12.22, respectively. In conclusion, a human odor-based bait blend was developed and exhibited enhanced effectiveness at attractingAe. albopictus This blend can be used to monitor and trap Dengue vector mosquitoes in Chinese cities.
Keywords: olfaction, host-seeking behavior, attractive odor blend, orthogonal design, mosquito traps, Aedes albopictus NTDASIA 2019 69
P:31 Morphological Identification of Mosquito Vector Associated with Mangrove Forest in Chachoengsao Province, Thailand
Somsanith Chonephetsarath, 1,* Chamnarn Apiwathnasorn,1 Sungsit sungvornyothin,1 Arnon payakkapol,1 Thipruethai 1 Department of Medical Entomology, phanitchat,1 Yudthana Samung,1 Chadchalerm Raksakoon,1 Faculty of Tropical Medicine, Rutcharin Potiwat1,** Mahidol University, Bangkok 10400, Thailand. 2 Abstract: The medical important mosquito vector in Thailand Department of Chemistry, Faculty of Science, Kasetsart University, are including Culex sp., Aedes sp., Anopheles sp. and Mansonia Bangkok 10900, Thailand sp. etc. The Culex sp. are important vector of Japanese * Presenter **e-mail: rutcharin. encephalitis virus (JEV) and Aedes sp. are primary vector of [email protected] dengue, yellow fever, chikungunya and Zika virus. Normally, the mosquito vector has a different breeding site that related to natural ecology and water container. To clarify the mosquito vector that circulated in Mangrove forest in Chachoengsao Province, Thailand. We collected the mosquito larvae and maintenance it in insectarium until the adult stage. All mosquito was separated raring and identified using the pictorial keys. Even though the previously research showed the important mosquito vector of dengue virus such as Aedes spp. (Aedes aegypti, Aedes albopictus and Aedes scutellaris) in this area but we did not find the Aedes scutellaris inside the mangrove forest during these periods. Only Aedes dux that presents as a predominant species in this area and we also found the other mosquito vector of Japanese encephalitis virus,Culex spp.; Culex sitiensaround the forest. The Aedes dux was found inside the mangrove forest approximately 81.85% and Culex brevipalpis found 1.53% around outside the mangrove forest. Both of Aedes dux and Culex brevipalpis have never reported in this area. Finding of Aedes dux and Culex brevipalpis are supported the vector control strategy and virus transmission via this species are rarely reported.
Keywords: Aedes sp., Culex sp., Mangrove forest, dengue virus, mosquito vector 70 NTDASIA 2019
P:32 Epidemiology of Dengue Infection and Circulating Dengue Serotypes in Hospitalized Dengue Patients; A Decade Trend from 2009 to 2018
1 Virology Research Division, Theingi Win Myat,1,*,** Hlaing Myat Thu,1 Khin Saw Than,2 Department of Medical Research, 2 2 3 4 Myanmar Hlaing Mya Win, Zaw Than Htun, Moe Moe San, Ye Myint Kyaw, 3 5 1 2 Department of Medicine, Yangon Aye Mya Theingi Win, Khine Wuttyi Thin, Moh Moh Htun General Hospital, Myanmar 3 Tropical Medicine, Yangon Abstract: Dengue is the most important arthropod-borne viral General Hospital, Myanmar disease of public health significance. Myanmar is one of the 4 Department of Pediatrics, Yangon dengue endemic countries with epidemic peaks occur every 2-3 Children Hospital, Myanmar years and all four dengue serotypes are circulating. The 5 University of Medicine (1), increased epidemic activity and the co-circulation of multiple Yangon, Myanmar serotypes increase the possibility of genetic changes and diversity *Presenter **email: drtheingiwin- in virus populations. Emergence of new viral strains and a change [email protected] in serotype is particularly important to bring about a surge of dengue cases as well as the severity of the disease. Continuous and comprehensive surveillance upon epidemiology and prevailing dengue serotypes is crucial. A hospital-based cross-sectional survey was carried out continuously from 2009 to 2018 at Yangon Children Hospital (YCH) and Yangon General Hospital (YGH). Blood samples were collected from admitted dengue patients, screened by ICT (SD BIOLINE) for serological confirmation, serotyped by virus isolation/RT-PCR. Patient’s demographic and clinical information were collected in case report forms for epidemiological information. Results showed that, every year, an average of 900 dengue cases (ranging ~800- 1200) was enrolled in the study. Among them, 60-90% was serologically confirmed dengue infections. Dengue infection was found to be the most prevalent in 5-9 years old children followed by 1-4 year group, 10-12 year group and least affected in >25 year old group, accounting <1%. Throughout 2009 to 2013, and 2016, secondary dengue infection accounted for nearly two third of confirmed dengue infection (range of 62% to 83%). In 2014, primary and secondary dengue infections were equally distributed. In 2015, 2017 and 2018, early dengue infection (NS1Ag alone positive) was increasingly observed, accounting 38-53%. Dengue virus serotype 1 (DENV 1) was predominant continuously from 2009 to 2016 except for the year 2010 when DENV 3 was predominant. In 2017, DENV 4 was predominant; however, it was changed in 2018 to DENV 3. DENV 1 was found to be associated with milder form of dengue (DHF GI) whereas DENV 2 and 3 with severe form (DHF II and DSS). Continuous and comprehensive surveillance is essential tool for prediction of outbreaks and severity of the disease and a better way to plan for control measures.
Keywords: dengue, epidemiology of dengue, dengue serotypes, YCH and YGH, Myanmar NTDASIA 2019 71
P:33 Fast Emerging Insecticide Resistance in Aedes albopictus in Guangzhou, China: Alarm to the Dengue Epidemic
Xinghua Su,1 Yijia Guo,1,* Jielin Deng,1 Jiabao Xu,1 Guofa Zhou,2 Tengfei Zhou,1 Yiji Li,1 Daibin Zhong,2 1 Department of Pathogen Biology, Ling Kong,1 Xiaoming Wang,1 Min Liu,1 Kun Wu,1 School of Public Health, Southern Guiyun Yan,1,2,** Xiao-Guang Chen1,** Medical University, Guangzhou, China 2 Abstract: Dengue is one of the most serious mosquito-borne Program in Public Health, University of California, Irvine, infectious diseases in the world. Aedes albopictus is the most Irvine, CA, United States of invasive mosquito and one of the primary vectors of dengue. America Vector control using insecticides is the only viable strategy to *Presenter **e-mail: guiyuny@uci. prevent dengue virus transmission. In Guangzhou, after the 2014 edu (GY); xgchen2001@hotmail. com (XGC) pandemic, massive insecticides have been implemented. Massive use of insecticides may lead to the development of resistance but there are few reports on the status of insecticide resistance in Guangzhou after 2014. In this study, Ae. albopictus were collected from four districts with varied dengue transmission intensity in Guangzhou from 2015 to 2017. Adult Ae. albopictus insecticide susceptibility status against deltamethrin (0.03%), permethrin (0.25%), DDT (4%), malathion (0.8%) and bendiocarb (0.1%) was determined by the standard WHO tube test and larval resistance bioassays were conducted using temephos, Bacillus thuringiensis israelensis (Bti), pyriproxyfen (PPF) and hexaflumuron. Mutations at the voltage- gated sodium channel (VGSC) gene and acetylcholinesterase (AChE) gene were analyzed. The effect of cytochrome P450s on the resistance of Ae. albopictus to deltamethrin was tested using the synergistic agent piperonyl butoxide (PBO). The results showed that Ae. albopictus populations have rapidly developed very high resistances to multiple commonly used insecticides at all study areas except malathion, Bti and hexaflumuron. We found 1534 codon mutations in VGSC gene were significantly correlated with the resistance to pyrethroids and DDT, and 11 synonymous mutations were also found in the gene. The resistance to deltamethrin can be significantly reduced by PBO but generated cross-resistance to PPF. Fast emerging resistances in Ae. albopictus affect mosquito management and threaten the prevention and control of dengue, similar to the resistance in Anopheles mosquitoes has prevented the elimination of malaria and call for timely and guided insecticide management.
Keywords: insecticide resistance, Aedes albopictus, dengue, China 72 NTDASIA 2019
P:34 Nix is a Male-Determining Factor in the Asian Tiger Mosquito Aedes albopictus
Yijie Zhao,1 Peiwen Liu,1 Binbin Jin,1 Xiaocong Li,1 Jinbao Gu,1 James K. Biedler,2 Zhijian Jake Tu,2 Xiao-Guang Chen1,*,**
1 Department of Pathogen Biology, Guangdong Provincial Key Abstract: Aedes albopictus, also known as the Asian tiger Laboratory of Tropical Disease mosquito, is the most invasive mosquito vector of viral Research, School of Public Health, pathogens. In recent years, Ae. albopictus has rapidly spread Southern Medical University, worldwide including Europe and it poses a serious public health Guangzhou, Guangdong 510515, threat due to its potential to spread a number of infectious China 2 Department of Biochemistry and diseases including dengue, chikungunya, and Zika. The initial the Fralin Life Science Institute, signal that governs sex determination is highly variable among Virginia Tech, Blacksburg, VA insects. A homolog of Nix, the male-determining factor in Aedes 24061, US aegypti, was previously found in the Ae. albopictus. Here we *Presenter **e-mail: xgchen2001@ hotmail.com showed that the Ae. albopictus Nix (AalNix) is more complex in gene structure and splice isoforms than its Ae. aegypti homolog (AaeNix). AalNix shows a similar transcription profile compared to AaeNix. CRISPR/Cas9-mediated knockouts of AalNix in vivo and in the Ae. albopictus C6/36 cells lead to a shift of dsx and
fru splicing towards the female isoforms. G0 knockout males showed feminization and deformities including feminized antennae, absence or partial absence of gonocoxites, gonostyli, testes and accessory glands, and the formation of ovaries. Therefore, we have shown that despite ~70 MY of divergence, Nix functions as a conserved male-determining factor in the two most important arboviral vectors, namely Ae. aegypti and Ae. albopictus. Only females bite and transmit these disease-causing viruses. This study provides the foundation for genetic manipulations that produce male-only progeny to achieve mosquito population suppression, or to significantly reduce the cost and augment the scale of any mosquito control strategies that requires the separation of males from females.
Keywords: M-factor, sex-determination, CRSPR-Cas9, feminized NTDASIA 2019 73
P:35 Development of an Efficient DNA- Launched Dengue Virus Serotype 3 Replicon System Based on a Clinical Isolate for Antiviral Compound Discovery
1 Institute of Human Virology and Yang Yang,1, 2 Yiyi Chen,1, 2 Mingxiao Chen,1, 2 Yi-Ping Li1, 2, *, ** Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Abstract: Dengue virus (DENV) is one of the most important China. mosquito-transmitted viruses classifying into the Flavivirus 2 Key Laboratory of Tropical genus of the Flaviviridae family. To date, five serotypes of DENV Disease Control of Ministry of have been identified, of which serotypes 1-4 overall have been Education, Sun Yat-sen University, Guangzhou, China. reported in more than 120 countries, and its infection in humans * Presenter **e-mail: lyiping@mail. leads to symptoms that vary from a self-limited dengue fever sysu.edu.cn (DF) to life-threatening diseases, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). There is no specific antiviral therapy for DENV infection so far. The DENV replicon reporter system is an important tool for the study of DENV replication and drug development. In 2009-2010, DENV serotype 3 (DENV-3) reemerged in China, and the emerging strains and lineages of DENV-3 are increasingly being reported and are evidentially associated with severe dengue cases. Here, we constructed a DNA-launched subgenomic replicon containing 5’UTR-Capsid and NS1-NS5 sequences from a DENV-3 clinical isolate, using the transcriptional control of the immediate-early promoter of cytomegalovirus (CMV). Following transfection of BHK21 cells with replicon DNA plasmid and neomycin selection, we established BHK21 cells harboring DENV-3 replicon steadily expressing high signals of luciferase reporter. Sequence analysis of DENV-3 RNA extracted from replicon-containing BHK21 cells that have been passaged continuously for 120 days revealed four synonymous mutations in NS2B, NS3, NS4B, and NS5, respectively. Using the replicon system, we confirmed that antiviral activity of mycophenolic acid (EC50=1.7±0.11 μM), a known inhibitor for DENV. In the screening of a library containing 400 drugs that have been approved for clinical use, we identified 3 drugs that exhibited inhibitory effect for DENV-3 replicon. These drugs are using in clinic for its antitumor and/or antibacterial activities, and the molecular mechanisms of its antiviral effect against DENV are ongoing. Together, a DNA- launched DENV-3 replicon system based on a clinical isolate represents an approach feasible for molecular manipulation and a reliable tool for the study of DENV and drug discovery.
Keywords: dengue virus, replicon, drug discovery 74 NTDASIA 2019
P:36 The Study of Association Between Polymorphism of Drug Resistant Genes of Leishmania spp.
Sirinan Kimcharoensuk,1,* Suradej Siripattanapipong,1 1Department of Microbiology, Mathirut Mungthin,2 Teerasak E-kobon3,** Faculty of Science, Mahidol University, Bangkok 10400, Abstract: Leishmaniasis is a group of diseases caused by Thailand flagellated protozoan parasites of the genusLeishmania , which 2Department of Parasitology, Phramongkutklao College of are spread by the bite of infected female phlebotomine Medicine, Bangkok 10400, sandflies, commonly infects animals and human. Visceral Thailand leishmaniasis (VL) is the most severe clinical manifestation 3 Department of Genetics, Faculty of caused by four Leishmania species including L. donovani, Science, Kasetsart University, Bangkok 10900, Thailand L. infantum, L. martiniquensis and L. siamensis. Thailand is the * Presenter **e-mail: fscitse@ku. endemic area of leishmaniasis causing by L. martiniquensisand ac.th L. siamensis. The basic information on several biological of novel Leishmania species has not been studied. To study the basic biology and the association between antileishmanial drug resistance and polymorphisms of drug target, Nucleotide sequences of L. donovani and L. infantum genes which reported to resistant to Antimonials (SSG) and Miltefosine (MIL) were retrieved from GenBank database. The nucleotide sequences alignment between sequences of drug target genes of L. donovani and L. infantum, and L. siamensis and L. martiniquensis genomes were performed using BLAST tool in Genieous program. The matched score of each gene were graded by considering from E-value, Score blast, Percentage of coverage, and Percentage of identical site. The results showed S-adenosylhomocysteine hydrolase, Trypanothione synthetase, Trypanothione reductase, Alpha-adaptin like protein and Folylpolyglutamate synthetase were the top five highest scores of resistant genes. In conclusion, the association between the polymorphism of drug target genes of L. martiniquensis and L. siamensis were predicted and determined. Moreover, the data on genetic characteristics of resistant genes might be useful for further drug efficacy studies.
Keywords: Leishmaniasis, Leishmania spp, drug resistant genes, mutational analysis, Thailand NTDASIA 2019 75
P:37 Bacterial Profiles and Antibacterial Susceptibility Patterns from a Canine Model of Kidney Stone Disease
Nahathai Uttamamul,1,2,* Aroonlug Lulitanond,1,2 Lumyai Wonglakorn,3 Patcharee Boonsiri,4 Supranee Jitpean,5 1Centre for Research and Phitsamai Saisud,1,2 Vitoon Prasongwattana,3 Development of Medical Diagnostic Ratree Tavichakorntrakool1,2,** Laboratories, Faculty of Associated Medical Science, Khon Kaen Abstract: Urinary tract infections (UTIs) are normally known to University, Khon Kaen, Thailand 2 be related with kidney stone disease, especially struvite stone. Department of Clinical The most common type of infection-induced stone in human Microbiology, Faculty of Associated Medical Science, Khon Kaen and canine (a mammal model) is composed of magnesium University, Khon Kaen, Thailand ammonium phosphate. For a long time, the analysis of bacterial 3Clinical Microbiology Unit, profiles in kidney stone disease from animal models especially Srinagarind Hospital, Khon Kaen canine remains changes. Therefore, the aims of this study were University, Khon Kaen, Thailand to evaluate the bacterial profiles and antibacterial susceptibility 4Department of Biochemistry, patterns of uropathogenic bacteria isolated from urine and Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand kidney stone of canines. The urine and kidney stone samples 5Division of Surgery, Faculty of were collected by cystocentesis and nephrotomy in the same Veterinary Medicine, Khon Kaen canine, respectively. A total of 4 canines (2 males and 2 females) University, Khon Kaen, Thailand diagnosed as kidney stone and treated with surgical removal *Presenter **e-mail: ratree.t@gmail. between 2017 and 2018 at the Khon Kaen University Veterinary com Teaching Hospital were recruited. Both samples were immediately cultured on blood agar and MacConkey agar. All the bacterial isolates were identified and evaluated for the antibacterial susceptibility testing by disc diffusion method. Chemical compositions of stones were performed by using by Fourier-transformed infrared spectroscopy (FT-IR). One of all canines was free from bacteria in both samples and which chemical compositions of stone was calcium oxalate. From three canines, which stone types were magnesium ammonium phosphate, had a total of 14 bacterial isolates (4 from urine and 10 from stone) cultured from their samples. The most common bacteria found in urine and stone matrices were Staphylococcus spp. (S. pseudintermedius, S. aureus, S. lentus, S. xylosus and S. auricularis) and Proteus mirabilis, respectively. These bacterial were urea-splitting bacteria that may related with the struvite stone. Multi-drugs resistance was frequently found inP. mirabilis. However, all isolated bacteria in this study were susceptible to gentamicin. This canine model may help to explain the bacterial profiles and antibacterial patterns in patients with kidney stone disease. The mechanism of canine and human with struvite stone should be further study in the future.
Keywords: canine, bacterial profiles, kidney stone, antibacterial susceptibility patterns, urea-splitting bacteria 76 NTDASIA 2019
P:38 Phytochemical Screening and Antioxidant Activity of Ethanolic Leaf Extract of Sida acuta
Nitis Smanthong,1,2,* Chularut Prariyachatigul,1,2 Porntip Pinlaor,1,2 Jureerut Daduang,1 Phitsamai Saisud,1,2 Patcharee Boonsiri,3 Ratree Tavichakorntrakool1,2,** 1 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Abstract: Sida acuta (SA) is an erect perennial shrub, commonly Medical Sciences, Khon Kaen found in the northeast of Thailand. SA has many biological University, Khon Kaen, Thailand activities such as wound healing, diuretic and antimicrobial 2 Department of Clinical properties. This study aimed to investigate the phytochemical Microbiology, Faculty of Associated constituents and antioxidant activity of SA extract. The leaves Medical Sciences, Khon Kaen University, Khon Kaen, Thailand of SA were collected from Khon Kaen province and extracted by 3 Department of Biochemistry, ethanol. The total phenolic and flavonoids contents of SA, Faculty of Medicine, Khon Kaen determined by Folin-Ciocalteau and aluminium chloride University, Khon Kaen, Thailand methods, respectively, were 45.53±1.52 µg GAE/mg dry weight *Presenter **e-mail: ratree.t@gmail. com and 370.06±13.60 µg QE/mg dry weight. Ferric reducing power of SA was 109.43±1.10 µg/mL Fe(II)/mg dry weight. The radical scavenging activity as determined by 2,2-diphenyl-1-picrylhy- drazyl (DPPH) method showed that % DPPH scavenging was 52.10±3.36 %. Gas chromatography-mass spectroscopy analysis revealed that SA was composed of linoleic acid, n-hexadecanoic acid, phytol, 2-methoxy-4-vinylphenol, glycerol 1-monooleate, di-alpha-tocopherol and 5-hydroxymethylfurfural. High performance liquid chromatography analysis also revealed that SA was consisted of catechin, chlorogenic acid, rutin and ferulic acid. The various bioactive compounds of SA should be further studied for possibility of developing of a novel agent such as antibacterial agent.
Keywords: Sida acuta, phytochemical constituents, antioxidant activity NTDASIA 2019 77
P:39 Antimicrobial Susceptibility Patterns and Prevalence of Bacteria Isolated from Participants with Chronic Kidney Disease in a Rural Thai Population
1 Centre for Research and Ratree Tavichakorntrakool,1,2,3,** Wiyada Chumpol,1,2,3,* 1,3 2,3 2,3 Development of Medical Diagnostic Aroonlug Lulitanond, Ubon Cha’on, Sirirat Anutrakulchai, Laboratories, Faculty of Associated 1,2 4 4 Nattaya Sae-ung, Patcharee Boonsiri, Lumyai Wonglakorn, Medical Sciences, Khon Kaen 5,6 Visith Thongboonkerd University, Khon Kaen, Thailand, 2 Chronic Kidney Disease Abstract: Urinary tract infection in patients with chronic kidney Prevention in the Northeast of disease (CKD) caused by multidrug resistant has been increased Thailand, Khon Kaen University, Khon Kaen, Thailand in difference areas. The analysis of the prevalence, etiology, 3 Faculty of Associated Medical antimicrobial susceptibility patterns of bacterial isolates from Sciences, Khon Kaen University, urine of patients with CKD in rural areas is scarce. In this study, Khon Kaen, Thailand, we examined extensively the identification of uropathogenic 4 Faculty of Medicine, Khon Kaen bacteria in urine and studied their antimicrobial susceptibility University, Khon Kaen, Thailand, 5 Medical Proteomics Unit, Office patterns. Urine samples were collected from participants with for Research and Development, CKD in a rural area of Northeastern Thailand. Identification of Faculty of Medicine Siriraj bacterial isolates was done by colony morphology and standard Hospital, Mahidol University, biochemical test. The antibacterial susceptibility test of these Bangkok, Thailand, 6 Center for Research in Complex bacteria was done by broth microdilution method. Based on Systems Science, Mahidol bacterial isolates, we divided the subjects into two main groups: University, Bangkok, Thailand 204 CKD participants without bacterial isolates and 44 CKD *Presenter **e-mail: ratree.t@gmail. participants with bacterial isolates. From the 44 particitpants com had a total of 47 bacterial isolates cultivated from their urine samples. The most two common bacteria isolated from urine were Escherichia coli (51.06 %) and Staphylococcus spp. (23.40 %). Multiple antimicrobial resistances were commonly found in these bacteria isolates. E. coli was highly resistant to ampicillin (95.83 %) and trimethoprim/sulfamethoxazole (54.16 %). Staphylococcus spp. was resistant to penicillin (36.36%) and oxacillin (27.27%). This result indicated that E. coli and Staphylococcus spp. isolates from these subjects were multidrug resistance. The antimicrobial resistance and virulence factors of these bacteria need to be further investigated
Keywords: Chronic kidney disease, urinary tract infection, antimicrobial susceptibility patterns, uropathogenic bacteria 78 NTDASIA 2019
P:40 Crystallization by Escherichia coli and Proteus mirabilis in Artificial Urine
Krittaya Saelee,1,2* Aroonlug Lulitanond,1,2 Nattaya Sae-Ung,1,3 Patcharee Boonsiri,4 Vitoon Prasongwatana,4 Visith Thongboonkerd,5 Ratree Tavichakorntrakool1,2** 1 Centre for Research and Development of Medical Diagnostic Abstract: Due to its high recurrent rate, kidney stone disease Laboratories, Faculty of Associated Medical Sciences, Khon Kaen remains a healthcare problem worldwide. Crystallization is an University, Khon Kaen, Thailand important step in stone genesis. Many formulas of artificial urine 2 Department of Clinical (AU) were used to perform the crystallizationin vitro. However, Microbiology, Faculty of Associated in these AU formulas, lack of amino acids, which normally Medical Sciences, Khon Kaen University, Khon Kaen, Thailand present in human urine, may affect the crystallization. This study 3 Department of Clinical Hematology, aimed to compare the crystallization by Escherichia coli and Faculty of Associated Medical Proteus mirabilis isolated from stones of stone formers in Sciences, Khon Kaen University, AU-Siriraj and modified AU-Siriraj, which added Luria-Bertani Khon Kaen, Thailand broth as amino acid sources. The bacteria were cultured in each 4 Department of Biochemistry, Faculty of Medicine, Khon Kaen AU incubated at 37ºC for 0, 48, and 96 hr. The crystal images University, Khon Kaen, Thailand were taken by inverted light microscope. The pH values of AU 5 Medical Proteomics Unit, Office were measured by pH indicator. Results showed that E. coli was for Research and Development, able to induce crystals in modified AU-Siriraj but not in AU-Siriraj. Faculty of Medicine Siriraj Hospital, Mahidol University, While P. mirabilis was able to induce crystals in both AU. The pH Bangkok, Thailand values of each AU were not much different. However, the pH * Presenter **e-mail: ratree.t@ (mean ±SEM) of modified AU-Siriraj cultured by E. coli at 96 hr gmail.com was higher than AU-Siriraj (8.5 ± 0.0 vs 8.0 ± 0.0) and the pH (mean ±SEM) of AU-Siriraj cultured by P. mirabilis at 48 hr was higher than modified AU-Siriraj (9.5 ± 0.0 vs 9.0 ± 0.0). In conclusion, E. coli was able to induce small crystals only in modified AU-Siriraj, in the presence of amino acids, while P. mirabilis was able to induce crystals in amino acid-free and amino acid presented in AU. Therefore, amino acid sources in modified AU-Siriraj might affect crystallization E.by coli.
Keywords: crystallization, amino acids,Escherichia coli, Proteus mirabilis, artificial urine NTDASIA 2019 79
P:41 Construction and Expression of Dengue Nonstructural 1 (NS1) Protein in Lactobacillus casei
Nutchanon Duangdech,1,* Supranee Phanthanawiboon,1 Chonlatip Pipattanaboon,1 Marutpong Panya,2 1Department of Microbiology and Viraphong Lulitanond 1,** Research and Diagnostic Center for Emerging Infectious Diseases,
Abstract: Dengue nonstructural 1 (NS1) is one of an attractive Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand candidate antigen to be incorporated in dengue vaccine due to 2College of Medicine and Public its ability to elicit protective immunity that can cross-react with Health, Ubon Ratchathani University, all types of dengue virus. In this study, the codon-optimized NS1 Ubon Ratchathani, Thailand gene was cloned in our Escherichia coli-L.casei shuttle vector *Presenter **e-mail: viraphng@ gmail.com using a constitutive promoter of lactate dehydrogenase gene, pRCEID-LC13.9. The two recombinant plasmid containing NS1 gene, pRCEID-LC13.9:NS1 without signal peptide and pRCEID-LC13.9:USP:NS1 carried the signal peptide, Usp45, were constructed and were each transformed in the expression host, L. casei RCEID02. It was found that the recombinant L. casei carrying pRCEID-LC13.9:NS1 and that carrying pRCEID-LC13.9: USP:NS1 can successfully expressed NS1 protein as intracellular and secreted form as determined by immunoblotting using anti-NS1 antibody.
Keywords: lactic acid bacteria, Lactobacillus casei, dengue nonstructural 1 protein, heterologous protein expression. 80 NTDASIA 2019
P:42 Diclofenac Suppresses Proliferation of Cholangiocarcinoma Cell Lines
Kamonrot Kumpangnil,1,* Kanjana Pangjit1, Latiporn Udomsuk1, Jarawee Wannachart1, Ratana Leksomboon1,**
1 College of Medicine and Public Health, Ubon Ratchathani Abstract: Cholangiocarcinoma (CCA) is a major public problem, University, Sathonmak Road, which has the highest incidence of morbidity and mortality in Ubon Ratchathani, Thailand the Greater Mekong Subresion countries, especially the *Presenter **e-mail: ratana_tlek@ Northeast Thailand. Lipid metabolism is a hallmark of yahoo.com tumorigenesis. Hence, alteration of lipid metabolism enhances proliferation of tumor cells. Previously, aspirin was reported to reduce the risk of CCA because of its anti-proliferative and anti-inflammatory effects, leading to anti-tumor activity. Aspirin is able to inhibit the lipid synthesis in CCA, thereby suppresses the production of cell membrane material of cancer cells to decrease CCA cell proliferation. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) in the same group of aspirin but its side effect is less than that of aspirin. In this study, we are interested in the effect of diclofenac on the proliferation of the human CCA cell lines, KKU-M139 and KKU-M214. Cell proliferation was measured using MTT assay, while lipid synthesis was determined using oil red O staining. The results showed that diclofenac suppressed the viability of the CCA KKU-M139 and KKU-M214 cells. However, it did not affect lipid synthesis determined by oil red O staining. These findings suggest that diclofenac has a promising potential as an anticancer proliferation agent for CCA treatment.
Keywords: cholangiocarcinoma, proliferation, diclofenac NTDASIA 2019 81
P:43 Leishmania martiniquensis Induces Activation and Increased Survival of Human Neutrophil
Nanthicha Inrueangsri1,*, Parima Boontanom1, Saovanee Leelayoova2, Suradej Siripattanapipong1, 1Department of Microbiology, Fabien Loison1, ** Faculty of Science, Mahidol University, Bangkok 10400, Abstract: Leishmania spp. are intracellular parasites that target Thailand 2Department of Parasitology, myeloid cells, particularly monocytes and macrophages. Disease Phramongkutklao College of manifestations range from asymptomatic, self-healing, to Medicine, Bangkok 10400, chronic and severe disease, depending on the Leishmania spp. Thailand and host immune factors. No vaccine is currently available. *Presenter **e-mail: Fabien.loi@ Neutrophils are myeloid cells that are rapidly recruited to the mahidol.ac.th site of Leishmania infection and play protective or detrimental roles depending on the Leishmania spp. and the parasite and disease stages. The involvement of neutrophils in the defense and pathology of leishmaniasis is well established but is highly dependent on the Leishmania spp. Endemic cases of leishmaniasis in Thailand involve predominantly spp. of the Leishmania enriettii complex. The response of human neutrophils to exposure to Leishmania martiniquensis, the most common endemic species in Thailand, have not been studied to date. The aim of this work was to characterize the activation, phagocytic activity, reactive oxygen species (ROS) production, and survival of human neutrophils upon interaction with L. martiniquensis metacyclic promastigotes, isolated using peanut lectin agglutination. Exposure of neutrophils to either un-opsonized or serum-opsonized L. martiniquensis induced increased expression of CD11b and CD66b, and decreased CD62L levels, indicating human neutrophil activation and recognition of the parasite. This was associated with production of a high level of ROS. Interestingly, neutrophil survival was increased by the co-culture with promastigotes. This suggested that L. martiniquensis was able to modulate neutrophil survival. This has been described for other species such as L. major, and is believed to be a strategy often use by pathogens to survive and facilitate dissemination in the host.
Keywords: Leishmania martiniquensis, metacyclic promastigotes, human neutrophils, flow cytometry, immunofluorescence 82 NTDASIA 2019
P:44 Expression of p53, Bcl-22, Granulin, and PCNA in the Liver of Opisthorchis viverrini Infected Hamsters: A Comparison Between Single Infection and Repeated Infection Plus Praziquantel Administration 1Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Songkiad Upontain,1,3,* Piya Sereerak,6 Thewarach Laha,2,3 2 Faculty of Medicine, Khon Kaen Banchob Sripa,2,3 Prasarn Tangkawattana,1 Anudep Rangsipipat,4 University, Khon Kaen 40002, Chanokchon Setthawongsin,5 Sirikachorn Tangkawattana1,3,** Thailand 3WHO Collaborating Centre for Research and Control of Abstract: Opisthorchiasis caused by Opisthorchis viverrini Opisthorchiasis (Southeast Asian, (O. viverrini) is an important public problem in the Greater Liver Fluke Disease) Tropical Mekong Subresion countries. Habitual eating of raw cyprinid Disease Research Center, Khon fish dishes and unaware taking of praziquantel (PZQ), an Kaen University, Khon Kaen 40002, Thailand effective anthelminthic against this trematode, are the major 4Faculty of Veterinary Science, causes of repeated infection of the people in these endemic Chulalongkorn University, Bangkok areas. This 15-week experiment was conducted in two groups 10300, Thailand of five Syrian golden hamsters—Group 1 with singleO. viverrini 5 Faculty of Veterinary Technology, infection, and Group 2 with three consecutive rounds of Kasetsart University, Bangkok 10900, Thailand infection and praziquantel (PZQ) administration. To both groups 6Department of National Park, of animals, subcarcinogenic dose of NDMA (12.5 ppm) was Wildlife and Plant Conservation, given in the drinking water. Histopathology and expression of Surat Thani 84000, Thailand p53, Bcl-2, granulin (GRN) and proliferating cell nucleus antigen *Presenter **e email: sirikach@kku. ac.th (PCNA) in liver samples were studied. The results showed that there was no evidence of cholangiocarcinoma (CCA) development in all hamsters of both groups. Although histopathological changes in both groups were similar, proliferative biliary lesions such as bile duct hyperplasia, bile duct dysplasia, and periductal fibrosis were more severe in the hamsters of Group 1. Average PCNA index and abnormal proliferation of biliary epithelial cells in Group 2 were significantly higher than those in Group 1 (p-value = 0.014). Average percentage of Bcl-2 expression in Group 2 was higher than in Group 1. Immunostaining of p53 and GRN was negative in both groups. These results suggest that three repeated rounds of infections followed by PZQ treatments in a period of 15 weeks with subcarcinogenic dose of NDMA did not induce CCA development. Late response of CCA involving genes, such as p53 and GRN, might not be altered so that neotransformation did not occur within the study period.
Keywords: repeated infection,Opisthorchis viverrini, p53, Bcl-2, PCNA, hamster 83
Proceedings 84 85
List of Proceedings
PO:01 Construction and Expression of Dengue Nonstructural 1 (NS1) 87 Protein in Lactobacillus casei
PO:02 Diclofenac Suppresses Proliferation of Cholangiocarcinoma Cell Lines 92
PO:03 Leishmania martiniquensis Induces Activation and Increased Survival of Human 97 Neutrophil
PO:04 Morphological Identification of Mosquito Vectors in and Around Mangrove 105 Forest in Chachoengsao Province, Thailand
PO:05 Expression of p53, Bcl-22, Granulin, and PCNA in the Liver of Opisthorchis 109 viverrini Infected Hamsters: A Comparison Between Single Infection and Repeated Infection Plus Praziquantel Administration 86 NTDASIA 2019 87
PO:01 Construction and Expression of Dengue Nonstructural 1 (NS1) Protein in Lactobacillus casei
Nutchanon Duangdech,1,* Supranee Phanthanawiboon,1 Chonlatip Pipattanaboon,1 Marutpong Panya,2 1,** and Viraphong Lulitanond 1Department of Microbiology and Research and Diagnostic Center for Abstract: Dengue nonstructural 1 (NS1) is one of an attractive Emerging Infectious Diseases, candidate antigen to be incorporated in dengue vaccine due to Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand its ability to elicit protective immunity that can cross-react with 2College of Medicine and Public all types of dengue virus. In this study, the codon-optimized NS1 Health, Ubon Ratchathani University, gene was cloned in our Escherichia coli-L.casei shuttle vector Ubon Ratchathani, Thailand using a constitutive promoter of lactate dehydrogenase gene, *e-mail: [email protected] ** - : . pRCEID-LC13.9.The two recombinant plasmid containing NS ; e mail viraphng@gmail com gene, pRCEID-LC13.9:NS1 without signal peptide and pRCEID-LC13.9:USP:NS1 carried the signal peptide, Usp45, were constructed and were each transformed in the expression host, L. casei RCEID02. It was found that the recombinant L. casei carrying pRCEID-LC13.9:NS1 and that carrying pRCEID-LC13.9: USP:NS1 can successfully expressed NS1 protein as intracellular and secreted form as determined by immunoblotting using anti-NS1 antibody.
Keywords: Lactic acid bacteria, Lactobacillus casei, dengue nonstructural 1 protein, Heterologous protein expression.
Introduction: Dengue virus (DENV) is transmitted to humans by Aedes mosquitoes. The DENV proteins consists of 3 structural proteins (i.e. capsid, envelope, and membrane) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Among these proteins, nonstructural 1 protein (NS1) secreted from DENV-infected cells can stimulate antiviral immune response. Several studies demonstrated that immunization with NS1 protein or passively transferred with anti-NS1 antibodies can provide protection in DENV-infected mice. In addition, anti-NS1 can cross-react with all types of dengue virus1 . Due to the efficacy and safety of the present licensed dengue vaccine require further evaluation by partial protection against all four DENV serotype infection. The vaccine was claimed to induce antibody-dependent enhancement (ADE) a high risk to develop severe dengue disease and might be the cause of increases hospitalizations2, 3. Therefore, the development for a better and safer dengue vaccine is still needed. NS1 is a promising alternative for the development of such dengue vaccine. According to vaccine vehicle, lactic acid bacteria (LAB) is one of 88 NTDASIA 2019
an attractive expression system to be used as an alternative vaccine vehicle as many studies found that LAB can elicit both localized and systemic immune response through oral administration 4. Thus, in this study we aim to clone and express Dengue serotype 2 NS1 gene in Lactobacillus casei as intracellular and secreted form.
Methodology: Codon optimization of NS1 gene in L. casei The native NS1 gene sequence of Dengue virus 2 strain 16681 (Genbank accession no. JN692493.1) was optimized based on the codon usage ofL. casei using a reference database L. casei ATCC334 (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=321967). The codon-optimized NS1 gene was synthesized (GenScript, NJ, USA) with 5’- and 3’- end have AatII (GACGTC) and NdeI (CATATG) sites. Upstream of NS1 gene were added with lactate dehydrogenase promoter (pLDH) and ribosome binding site (RBS) and downstream of the gene was added with the transcription termination (TT) (Fig. 1). The synthetic gene construct was supplied as a clone using pUC57 as the backbone vector and was designated as pUC57:NS1 was shown in Fig. 2.
Figure 1. Schematic overview of the codon-optimized NS1 gene construct contains the lactate dehydrogenase promoter (pLDH), ribosome binding site (RBS), NS1 gene and the transcription terminator (TT).
Construction of the recombinant plasmid containing NS1 gene for intracellular expression The codon-optimized NS1 construct was separated from pUC57:NS1 by AatII/NdeI-digestion and cloned into pRCEID-LC13.9 5, the Escherichia coli-L.casei shuttle vector. The new construct carrying NS1 gene was designed as pRCEID-LC13.9:NS1 (Fig. 2), was propagated in E. coli XL1-Blue and further verified for the correctness byAat II and NdeI digestion followed by DNA sequencing.
Construction of the recombinant L. casei expressing NS1 protein for secretory expression For expression of the secreted NS1 protein, the signal peptide (SP) must be inserted upstream of the NS1 gene in order to translocate the expressed protein across the plasma membrane into the medium. In this study, Usp45 signal peptide were selected. This signal peptide was originally derived from Lactococcus lactis subsp. CremorisMG1363 6 The cloning steps for pRCEID-LC13.9: USP:NS1 was shown in Fig. 3. In brief, Usp45 fragment separated from XmaI-digested pRCEID-LC13.9:USP:M2e:HBc 4 was ligated to XmaI-digested pRCEID-LC13.9:NS1 to generate recombinant plasmid, pRCEID-LC13.9:USP:NS1. The recombinant plasmidwas propagated in E. coli XL1-Blue and verified for the correctness byXma I and PstI digestion followed by DNA sequencing. NTDASIA 2019 89
Expression and detection of NS1 protein in L. casei as intracellular and secreted form pRCEID-LC13.9:NS1 and pRCEID-LC13.9:USP:NS1 derived from the above construction were each transformed in the expression host, L. casei RCEID02. To examine for expression of NS1 protein by the corresponding transformants, each transformant was cultured in MRS broth supplemented with erythromycin (2.5 μg/ml) at 37˚C until the optical density (OD600) reached 3.0. The supernatant and bacterial cells were separately collected by centrifugation. The bacterial cells were lysed by sonicate samples for 3 x 30 sec with a 5 min (Ultrasonic Homogenizer, Sonic VCX750). Proteins from supernatants were precipitated by using trichloroacetic acid (TCA) and pelleted by centrifugation. The expression of NS1 protein both in the crude cell lysates and in the supernatants were determined by western blotting using mouse monoclonal anti-NS1 antibody.
Figure 2. Construction of the recombinant pRCEID-LC13.9:NS1.Amp r, Eryr, ori and repA1 indicate ampicillin resistant gene, erythromycin resistant gene, origin of replication and repliconA1, respectively. 90 NTDASIA 2019
Figure 3. Construction of the recombinant pRCEID-LC13.9:USP:NS1. Ampr, Eryr, ori and repA1 indicate ampicillin resistant gene, erythromycin resistant gene, origin of replication and repliconA1, respectively.
Results: In this study two recombinant plasmids, i.e. pRCEID-LC13.9:NS1 and pRCEID-LC13.9: USP:NS1 were constructed as details above. These two recombinant plasmids were verified for the correctness by restriction enzyme digestion and DNA sequencing and were each electrotransformed into L. casei RCEID02 for NS1 expression. As shown in Fig. 4A lane 1, NS1 protein as intracellular form can be detected in the crude cell lysate derived from the recombinant bacteria carrying pRCEID-LC13.9:NS1. Whereas in Fig. 4B lane 3, NS1 can be detected in the supernatant derived from the recombinant bacteria carrying pRCEID-LC13.9:USP:NS1 but not in those carrying pRCEID-LC13.9:NS1 (Fig. 4b, lane 2).
Figure 4. Western blot analysis of NS1 protein expressed in recombinant L. casei RCEID02 as intracellular and secreted form. (A) NS1 protein detected in crude cell lysate. Lane1 = L. casei RCEID02 containing pRCEID-LC13.9:NS1, Lane2 = DENV2 strain 16681 containing NS1 gene, Lane3 = L. casei RCEID02 containing pRCEID-LC13.9. (B) NS1 protein detected in the supernatant. Lane1 = L. casei RCEID02 containing pRCEID-LC13.9, Lane2 = L. casei RCEID02 containing pRCEID- LC13.9:NS1, Lane3 = L. casei RCEID02 containing pRCEID-LC13.9:USP:NS1. NTDASIA 2019 91
Discussion: Heterologous protein can be expressed in LAB in three different forms, i.e. intracellular, cell wall-anchored (CWA) and secreted form. For expression of protein as CWA and secreted form, the recombinant expression plasmid needed to incorporate the signal peptide motif upstream of the expressed gene to translocate the expressed protein across the bacterial membrane into the medium. In addition, CWA domain is needed to insert downstream of the expressed gene for covalent attachment of expression protein to peptidoglycan of LAB. In this study, we successfully expressed DENV NS1 as intracellular and secreted forms in L. casei for the first time. Our study indicated that Usp45 can be used as signal peptide for NS1 expression as secreted form. Next step of our work is to construct recombinant plasmid for expression NS1 as cell-wall anchored form by selection of appropriate CWA domain to insert downstream of NS1 gene in this construct. Lastly, three forms of NS1 expression in L. casei will be evaluated for their efficacies to elicit anti-NS1 in animal model.
Conclusion: In conclusion, in this study demonstrated that the recombinant L. casei RCEID02 containing pRCEID-LC13.9:NS1 and pRCEID-LC13.9:USP:NS1 that can successfully cloning and expression NS1 protein as intracellular form and secreted form, respectively. That could lead to use as live bacterial vector for their use in delivery of antigen and secretion of therapeutic or alternative dengue vaccine candidates for future human use.
Acknowledgment: This study was supported by the Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University
References: 1. Chen H-R, Lai Y-C, Yeh T-M.Journal of biomedical science.2018; 25: 58-. 2. Liu Y, Liu J, Cheng G.Emerging microbes & infections.2016; 5: e77-e. 3. Aguiar M, Stollenwerk N, Halstead SB.PLoS Negl Trop Dis.2016; 10: e0005179. 4. Themsakul S, Suebwongsa N, Mayo B, Panya M, Lulitanond V.FEMS Microbiol Lett.2016; 363: 5. Panya M, Lulitanond V, Tangphatsornruang S, Namwat W, Wannasutta R, Suebwongsa N, et al.Appl Microbiol Biotechnol.2012; 93: 261-72. 6. Van Asseldonk M, Rutten G, Oteman M, Siezen RJ, de Vos WM, Simons G.Gene.1990; 95: 155-60. 92 NTDASIA 2019
PO:02 Diclofenac Suppresses Proliferation of Cholangiocarcinoma Cell Lines
Kamonrot Kumpangnil,1,* Kanjana Pangjit1, Latiporn Udomsuk1, Jarawee Wannachart1, Ratana Leksomboon1**
1 College of Medicine and Public Health, Ubon Ratchathani Abstract: Cholangiocarcinoma (CCA) is a major public problem, University, Sathonmak Road, which has the highest incidence of morbidity and mortality in Ubon Ratchathani, Thailand the Greater Mekong Subresion countries, especially the **e-mail: [email protected] Northeast Thailand. Lipid metabolism is a hallmark of tumorigenesis. Hence, alteration of lipid metabolism enhances proliferation of tumor cells. Previously, aspirin was reported to reduce the risk of CCA because of its anti-proliferative and anti-inflammatory effects, leading to anti-tumor activity. Aspirin is able to inhibit the lipid synthesis in CCA, thereby suppresses the production of cell membrane material of cancer cells to decrease CCA cell proliferation. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) in the same group of aspirin but its side effect is less than that of aspirin. In this study, we are interested in the effect of diclofenac on the proliferation of the human CCA cell lines, KKU-M139 and KKU-M214. Cell proliferation was measured using MTT assay, while lipid synthesis was determined using oil red O staining. The results showed that diclofenac suppressed the viability of the CCA KKU-M139 and KKU-M214 cells. However, it did not affect lipid synthesis determined by oil red O staining. These findings suggest that diclofenac has a promising potential as an anticancer proliferation agent for CCA treatment.
Keywords: Cholangiocarcinoma, proliferation, diclofenac
Introduction: Cholangiocarcinoma (CCA) is cancer originated in the biliary epithelial cells1. This cancer is the major cause of the death in the Northeast of Thailand. CCA in this region most likely caused by liver fluke infection (Opisthorchis viverrini) 2. Normally, the characteristics of cancer cells can increase the number of cells and CCA as well that demonstrated poor prognosis.
From the previous evidence, Aspirin drug has been shown inhibit process of lipid synthesis resulted not only to fatty acid and phospholipids synthesis reduction but cell proliferate suppression also3. Nevertheless, Aspirin have a serious adverse effects that included internal organ bleeding,etc 4. NTDASIA 2019 93
Consequently, Diclofenac (NSAIDs) has been widely used to replace aspirin with the same outcome but lesser side effect. There are evidence illustrated that diclofenac inhibit an enzyme synthesize prostaglandin caused inflammation namely cyclooxygenase5. However, the information of diclofenac to CCA treatment and mode of action have been limited so far. Accordingly, we would like to know the preliminary effect of diclofenac to cell proliferation and liposynthesis of CCA cell line. In order to prove these effects, we were performed MTT assay for monitors CCA cell proliferation and oil red O staining test for measured liposynthesis. We expect that the results presented here may facilitate improvements in the potential of Diclofenac to suppress CCA cell proliferation.
Methodology: Cell Culture Two human intrahepatic CCA cell lines; namely KKU-M139 and KKU-M214 were used in this study. All cell lines were established from CCA patients residing in opisthorchiasis endemic areas in Northeastern Thailand at department of pathology, faculty of medicine, Khon Kaen University. KKU-M139 was developed from a patient with squamous carcinoma and KKU-M214 were derived from patients with moderately differentiated adenocarcinoma, respectively. All cell lines were cultured in Ham’s F12, containing 100 U/ml penicillin and 100 µg/ml streptomycin with 10% fetal 6 bovine serum at 37 °C with 5% CO2 .
Cell viability assay Viability of CCA cell lines was determined using MTT assay. Briefly, 100 µL of cell suspension (2×104 cells/well) were seeded in a well of 96-well flat-bottomed microtiter plates and incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. Then, the KKU-M139 and KKU-M214 cells were treated with various concentrations (125 µM - 2 mM) of diclofenac incubated at 37°C for 48 hours. The culture medium was subsequently removed and 100 µL medium containing 100 µL MTT solution was added to each well. The plates were further incubated for 4 hours at
37°C in a humidified 5% CO2 atmosphere. Absorbency was measured at 540 nm using a microplate reader (EZ read 2000 microplate reader, biochrom). The concentrations of drug required to inhibit cell proliferation by 50% (IC50) was determined by plotting the percentage of cell growth viability versus the concentrations of the chemotherapeutic drug as described previously7.
Oil red O staining Oil Red O staining was performed according to the protocol of the manufacturer. Briefly, KKU-M139 and KKU-M214 cells were seeded in 24 well plates (at 2 × 104 cells/well) and incubated overnight. Then cells were treated with various concentrations of diclofenac (0, 500 µM and 2 mM). Cells were washed with phosphate buffer saline (PBS) and PBS was removed completely. Then cells were fixed with 10% formalin at room temperature for at least 1 hour. Cells were washed with ddH2O. Then, 60% isopropanol was added to the well and let the cells dry completely at room temperature (If necessary, a hairdryer was used to dry cells). Add the Oil Red O working solution and incubate at room temperature for 10 min. Cells were washed four times with ddH2O, then fat droplets in the stained cells were visualized and photographed using a ZEISS AXIO Imager.A2 axiocam HRc microscope. 94 NTDASIA 2019
Statistical Analysis All data were expressed as mean ± SE of three independent experiments. Significant differences between groups were analyzed by Student’s t-test using SPSS statistical software, version 16.0 (SPSS, Chicago, IL, USA). Statistical significance was indicated by *p<0.05, **p<0.01 or ***p<0.001.
Results: Cytotoxic effect of Diclofenac on KKU-M139 and KKU-M214 The effect of diclofenac on the viability of KKU-M139 and KKU-M214 cells was assessed using MTT assay. As shown in Figure 1, a dose dependent decrease of cell viability was observed after
diclofenac treatment. The inhibitory concentration (IC50) value for KKU-M139 and KKU-M214 cells was 481.56 µM and 455.57 µM, respectively.
Figure 1. Effects of diclofenac on cell viability of KKU-M139 and KKU-M214 cell lines.
The effect of diclofenac on cell and nuclear morphology After incubation with various concentrations of diclofenac for 48 hours, morphological alterations of KKU-M139 and KKU-M214 cell lines were observed under a bright field inverted microscope. Untreated control KKU-M139 cells were polygonal in shape (Figure2A). Exposure of KKU-M139 cells to diclofenac resulted in retraction, rounding and detachment of some sensitive cells from the surface. Membrane blebbing and nuclear morphological changes typical of cell injury or cell damage were observed (Figure2B).
Untreated control KKU-M214 cells were polygonal in shape (Figure2C). Treatment of KKU-M139 cells with diclofenac also induced retraction, rounding and detachment of some sensitive cells from the surface. Membrane blebbing and nuclear morphological changes typical of cell injury or cell damage were also observed (Figure2D). NTDASIA 2019 95
Figure 2. Morphological changes of KKU-M139 and KKU-M214 cell lines treated with diclofenac. (A) Untreated control KKU-M139 cells (B1, B2) KKU-M139 cells treated with 500 µM and 2 mM diclofenac, respectively (C) Untreated control KKU-M214 cells (D1, D2) KKU-M214 cells treated with500 µM and 2 mM diclofenac, respectively (x 20 magnifications)
Oil red O staining The results of oil red O staining is shown in Figure 3. After treatment of KKU-M139 and KKU-M214 cell lines with diclofenac, there was no difference in oil red O staining patters between diclofenac-treated and un-treated KKU-M139 and KKU-M214 cells. Both diclofenac-treated and untreated KKU-M139 and KKU-M214 cell lines have equal lipid droplets in the cytoplasm under a microscope.
Figure 3. Oil red O staining of KKU-M139 and KKU-M214 cell lines treat with diclofenac. (A) Untreated control KKU-M139 cells (B1, B2) KKU-M139 cells treated with 500 µM and 2 mM diclofenac (C) Untreated control KKU-M214 cells (D1, D2) KKU-M214 cells treated with 500 µM and 2 mM diclofenac (x 40 magnifications) 96 NTDASIA 2019
Discussion and Conclusion: Diclofenac is a phenylacetic acid derivative inhibiting cyclooxygenase COX-2 and prostaglandin E2 synthesis. Diclofenac has well-documented chemopreventive and anti-prolferation properties against cancer cells such as colorectal cancer, neuroblastoma, ovarian cancer and other cancers5. In this study, we found that diclofenac suppressed proliferation of KKU-M139 and KKU-M214 cell lines. Observation under a microscope revealed that diclofenac treatment induced typical morphological changes of cell injury and damage. Our findings were similar to the previous report of aspirin-induced inhibition of the viability of human CCA cell line8.
On the other hand, in this study, there are no significant inhibition of lipogenesis determined by Oil red O staining. Diclofenac might not be able to suppress the level of lipid droplets deposition in KKU-M139 and KKU-M214 cell lines. Our data are inconsistent with the previous study showing aspirin-induced reduction of lipogenesis in HCC cells3, suggesting different mechanisms of diclofenac and aspirin on cell viability.
In summary, the present results showed that diclofenac can inhibit proliferation of KKU-M139 and KKU-M214 cells lines. There is no significant inhibition in lipogenesis determined by Oil Red O staining. Since information about the use of diclofenac in the treatment of CCA is limited, further study on the knowledge of the mechanisms of action of diclofenac should be continually evolved.
Acknowlegdements: We would like to thank Tropical Disease Research Laboratory, Faculty of Medicine, Khon Kaen University, for providing laboratory facilities.
References: 1. Huether, A., Höpfner, M., Baradari, V., Schuppan, D., & Scherübl, H. Biochem Pharmacol. 2007;73(9), 1308-1317. 2. Thunyaharn, N., Promthet, S., Wiangnon, S., Suwanrungruang, K., & Kamsa-ard, S. Asian Pacific J Cancer Prevent. 2013;14(11), 7029-7032. 3. Yang, G., Wang, Y., Feng, J., Liu, Y., Wang, T., Zhao, M., . . . Zhang, X. Biochem Biophysl Res Comm. 2017;486(3), 827-832. 4. Warner, T. D., Nylander, S., & Whatling, C. (2011). Br J clin pharmacol. 2011;72(4), 619-633. 5. Pantziarka, P., Sukhatme, V., Bouche, G., Meheus, L., & Sukhatme, V. P. Ecancermedicalscience. 2016;10. 6. Namwat, N., Sripa, B., Loilome, W., Bhudisawadi, V., & Tassaneeyakul, W. Srinagarind Medical J. 2007;22(3), 230-234. 7. Laupattarakasem, P., Sripa, B., & Laupattarakasem, W. Srinagarind Medical J. 2007;22(4), 339-345. 8. Boueroy, P., Aukkanimart, R., Boonmars, T., Sriraj, P., Ratanasuwan, P., Juasook, A., . . . Wongkham, S. Asian Pacific J Cancer Prevent. 2017;18(11), 3091. NTDASIA 2019 97
PO:03 Leishmania martiniquensis Induces Activation and Increased Survival of Human Neutrophil
Nanthicha Inrueangsri1, *, Parima Boontanom1, Saovanee Leelayoova2, Suradej Siripattanapipong1, 1Department of Microbiology, Fabien Loison1, ** Faculty of Science, Mahidol University, Bangkok 10400, Thailand Abstract: Leishmania spp. are intracellular parasites that target 2 myeloid cells, particularly monocytes and macrophages. Disease Department of Parasitology, Phramongkutklao College of manifestations range from asymptomatic, self-healing, to Medicine, Bangkok 10400, chronic and severe disease, depending on the Leishmania spp. Thailand and host immune factors. No vaccine is currently available. *e-mail: Nanthicha.inr@student. Neutrophils are myeloid cells that are rapidly recruited to the mahidol.edu; **e-mail: Fabien.loi@ site of Leishmania infection and play protective or detrimental mahidol.ac.th roles depending on the Leishmania spp. and the parasite and disease stages. The involvement of neutrophils in the defense and pathology of leishmaniasis is well established but is highly dependent on the Leishmania spp. Endemic cases of leishmaniasis in Thailand involve predominantly spp. of the Leishmania enriettii complex. The response of human neutrophils to exposure to Leishmania martiniquensis, the most common endemic species in Thailand, have not been studied to date. The aim of this work was to characterize the activation, phagocytic activity, reactive oxygen species (ROS) production, and survival of human neutrophils upon interaction with L. martiniquensis metacyclic promastigotes, isolated using peanut lectin agglutination. Exposure of neutrophils to either un-opsonized or serum-opsonized L. martiniquensis induced increased expression of CD11b and CD66b, and decreased CD62L levels, indicating human neutrophil activation and recognition of the parasite. This was associated with production of a high level of ROS. Interestingly, neutrophil survival was increased by the co-culture with promastigotes. This suggested thatL. martiniquensis was able to modulate neutrophil survival. This has been described for other species such as L. major, and is believed to be a strategy often use by pathogens to survive and facilitate dissemination in the host.
Keywords: Leishmania martiniquensis, metacyclic promastigotes, human neutrophils, flow cytometry, immunofluorescence
Introduction:Leishmania infections in Thailand have long been seen as imported cases. However, a number of endogenous cases have been reported in the past decade, and two responsible species have been identified: Leishmania 98 NTDASIA 2019
martiniquensis and Leishmania siamensis (orientalis). The majority of infections have been attributed to Leishmania martiniquensis. Leishmania spp. have an extracellular stage (promastigotes) transmitted by the bite of sandflies (Sergentomyia spp.), and an intracellular stage (amastigotes) replicating inside myeloid cells, particularly macrophages. Manifestations of Leishmania infection range from asymptomatic and self-healing, to chronic and severe disease depending on the parasite species and host immune factors. Neutrophils are myeloid cells rapidly recruited to the site of Leishmania infection and involved in the clearance of the parasite. However, PMN role in leishmaniasis has recently received renewed attention as some Leishmania spp. subdue neutrophils to act as intermediate host cells. Some Leishmania spp., such as L. major, have been shown to use neutrophils as ‘Trojan horses’ to infect macrophages and escape the immune response11. Others, such as L. mexicana, have been shown to support the replication of amastigotes9. As neutrophil contribution to the outcome of leishmania infection is species dependent, it is important to characterize the interaction betweenL. martiniquensis and human neutrophils. This study aimed to describe human neutrophil activation, phagocytosis, respiratory burst and survival in response to metacyclic promastigotesL. martiniquensis in vitro.
Methodology: Parasite culture, isolation, and staining Leishmania martiniquensis (MHOM/TH/2011/PG, zymodeme MON-229) was a kind gift from Major General Prof. Saovanee leelayoova. The parasites were grown in Schneider’s insect medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) at 25±2°C. Growth curves, cell viability and identification of the different stages were performed. Increased proportion of metacyclic promastigotes was found during the stationary phase (day 6-7). Stationary phase cultures were used to isolate the metacyclic promastigotes (PNA-) from the other stages (PNA+) after centrifugation. In some experiments, two million PNA (-) promastigotes were labeled using the membrane dye PKH26 (2 μM, Sigma-Aldrich).
Neutrophil isolation Buffy coats from healthy donors were provided by Rajavithi Hospital with approval from MUCER (MU-CIRB 2018/009.1701). Red blood cells were sedimented in 3% dextran-0.9% NaCl solution. The leukocyte-rich fraction was layered over Ficoll-Hypaque solution and pelleted by centrifugation. Neutrophil pellets were washed and resuspended RPMI+ 5% FBS. The purity of the neutrophils (>95%) was controlled after Giemsa staining and cytospin.
Infection of neutrophils with the parasites Isolated human neutrophils (1 million cells, primed or not with 1 ng/ml of TNF-α for 20 minutes) were seeded in 24-well plate and incubated with un-opsonized or 10% serum-opsonized PNA (-) promastigotes at different MOI.
Flow cytometric measurement of surface expression of neutrophils Infected cells were collected and washed twice with cold PBS (400 x g, 4°C, 5 min). PMNs were stained with anti-CD11b-PerCP (Immunotool), anti-CD66b-FITC (BD Bioscience), and antiCD62L-PE (Immunotool) antibodies for 45 minutes at 4 ˚C in the dark, washed and finally, fixed. The samples were run on a Beckman-Coulter CYTOFLEX and data were analyzed using the CytoExpert software (Beckman-Coulter).
Quantifying parasite uptake by human neutrophils Unprimed neutrophils (1 million cells) were seeded in a 24-well plate and opsonized PKH26- labeled L. martiniquensiswere added at MOI 1:1, 1:5, 1:10. PMN and parasites were co-incubated NTDASIA 2019 99 at 4 ˚C or 37˚C for 2 hours. Cells were then washed and fixed with 4% paraformaldehyde. The neutrophil-parasite interaction was quantified using flow cytometry. Alternatively, wells containing coverslips were used to perform immunofluorescence. Coverslips were washed with PBS, permeabilized with 0.5% Triton X-100 for 1 minute, and then washed again with PBS. After blocking (3% BSA + 5% AB human serum in PBS) for 30 minutes at RT, neutrophils were stained with mouse anti-human MPO antibody (1:500 in blocking buffer for overnight). The coverslips were washed with PBS and stained with anti-mouse IgG Alexa 488 (1:2,000 in blocking buffer for 1 hour at RT), washed once more, counterstained with Hoechst 3342 (1 µM in PBS), washed with sterile water, and mounted in anti-fade reagent. Immunofluorescent images were taken using a fluorescent Nikon ECLIPSE 80i microscope and the number of uninfected cells and surface-bound and intracellular parasites were counted using Fiji12.
Detection of reactive oxygen species upon parasite interaction Unprimed neutrophils (106 cells) were stained with 1 μM dihydrorhodamine 123 (DHR 123, Invitrogen) and co-cultured with opsonized PKH26 labelled L. martiniquensis for 30 minutes. ROS production is measured by the conversion of DHR123 to fluorescent rhodamine 123 (Rho 123) in response to oxidation. The reaction was stopped on ice and cells were stained using CD11b marker to gate specifically on neutrophils. CD11b expression and the Rho 123 mean fluorescence intensity (MFI) of PKH26+ (infected) and PKH26- (bystander) neutrophils were measured by flow cytometry.
Measurement of neutrophil apoptosis After neutrophil-leishmania co-culture, cells were collected and washed twice with cold PBS and resuspended in Annexin V binding buffer before staining with Annexin V (Immunotool) and Propidium Iodide (PI) (BioLegend) for 30 minutes at 4 ˚C in the dark. The percentage of live neutrophils (Annexin V – and PI -) and the percentage of neutrophil apoptosis (Annexin V + cells) were quantified by flow cytometry.
Results and discussion: Characterization of growth and differentiation in vitro, isolation of promastigotes In order to study the neutrophil responses to L. martiniquensis, we isolated infectious stage of the parasite. In vitro culture of L. martiniquensis was performed in Schneider’s insect medium supplemented with 10% FBS to measure growth rate, cell viability (data not shown), and stages differentiation (Fig. 1A). We identified all major developmental stages2, starting from procyclic promastigote, nectomonad, leptomonads, and the infectious metacyclic promastigotes (Fig. 1B, i-iv). The proportion of metacyclic promastigotes was highest (16%) during the stationary phase on day 10, with 62% cell viability. Siripattanapipong and colleagues described a similar percentage of promastigotes with higher cell viability on day 6 using 20% FBS in the culture medium16. Therefore, we supplemented the parasites with 20% FBS and collected the parasites at day 6 in our later experiments. To isolate metacyclic promastigotes, peanut lectin agglutination was performed. It has been previously used for the enrichment of metacyclic promastigotes ofL. major5 and L. infantum1. Cells (200*106) were collected on day 6 and incubated with 50 µg/ml of peanut lectin. The non-agglutinated cells (PNA-) were leptomonads and metacyclics, as shown in figure 1C. Moreover, preliminary results showed that PNA- cells could infect and replicate in human macrophages (data not shown). Therefore, peanut lectin agglutination is a valid method for the isolation of L. martiniquensis infectious promastigotes. 100 NTDASIA 2019
Figure 1: Leishmania martiniquensis promastigotes culture and differentiation.(A) Proportion of the different developmental stages ofL. martiniquensis cultured in Schneider’s insect medium + 10% FBS (initial density: 500,000 cells/ml), n=3; Mean ± SEM. (B) The different stages were stained (Giemsa solution) and identified under microscopy. Procyclic promastigotes in rosette (i), nectomonads (ii), leptomonads (iii), and metacyclic promastigotes (iv). (C) Stained PNA- promastigotes following PNA agglutination.
Neutrophils are activated in the presence of L. Martiniquensis To investigate the phenotypic response of human neutrophils in the presence ofL. martiniquensis in vitro, we measured neutrophil activation using the surface expression of CD11b (Mac-1), CD66b, and CD62L (L-selectin) using flow cytometry. These markers are constitutively expressed on the surface of neutrophils. CD11b and CD66b are normally upregulated in activated neutrophils, whereas CD62L expression is decreased. To mimic the proinflammatory environment found at the sandfly bite site7, we primed neutrophils using TNFa17. The complement system is part of the innate immunity and plays an important role in pathogen clearance. Therefore, unprimed neutrophils were incubated with promastigotes opsonized or not with human serum. Unprimed neutrophils co-incubated with L. martiniquensis showed increased expression of CD11b and CD66b, and a marked decrease in CD62L expression. The change of expression of CD62L was statistically significant in a MOI-dependent manner. Similar trends were observed for CD11b and CD66b, but additional replicates are required to confirm these initial observations (Fig. 2). For CD66b and CD62L at MOI 1:10, the activation was comparable to the positive control fMLP. These results suggested that resting neutrophils were activated by coculture with L. martiniquensis. Furthermore, it suggested that PMN directly recognized PAMPs derived from leishmania, as they were activated in the presence of unopsonized parasites.
Figure 2: The expression of surface activation markers on unprimed neutrophils in the presence of unopsonized or opsonized L. martiniquensis. Unprimed human neutrophils were co-incubated with unopsonized or serum-opsonized PNA- promastigotes at different MOI (1:1, 1:5, 1:10) for 2 h. Flow cytometry was performed to measure the surface expression of CD11b (A), CD66b (B), CD62L (C). The increased surface levels of CD11b and CD66b, as well as the decreased level of CD62L, suggest that PMNs were activated in the presence of the promastigotes. N≥2; Mean ± SEM, *,**,*** p < 0.05 (One way ANOVA). NTDASIA 2019 101
L. martiniquensis is phagocytosed by human neutrophils The major function of neutrophils is to phagocytose microbes and kill them. To examine the uptake of L. martiniquensisby human neutrophils, we used flow cytometry and immunofluorescence. Isolated neutrophils were incubated with opsonized PKH26-labeled L. martiniquensis at different MOI for 2 hours, 37°C. The results of this experiment are presented in Figure 3A. The percentage of PMN interacting with the parasites was determined by measuring the proportion of PKH-26+/ CD11b+ cells. Approximately 40% of PMNs were associated with fluorescent leishmania at MOI 1:5 and 70% at MOI 1:10 (Fig. 3B). This result is comparable to previous reports using other Leishmania spp.6. As flow cytometry does not allow the differentiation of internalized and cell surface-bound parasites, PKH26+ PMN have either internalized or are bound to L. martiniquensis. When the interaction was performed at 4 °C to prevent phagocytosis, a smaller number of PKH26+ PMN was detected (approximately 25% at MOI 1:5, data not shown). Thus, it suggested that the parasites could be up taken by the neutrophils. Controls Co-culture
Figure 3: Flow cytometric analysis of L. martiniquensis – human PMNs interaction.PMNs were incubated with opsonized PKH26-L. martiniquensis (MOI 1:1, 1:5, 1:10) at 37°C for 2 h. (A) Neutrophil associated fluorescence measured by flow cytometry. SSC/FSC was used for neutrophils and Leishmania gating (top panel). Control neutrophils did not express red fluorescence, whereas the parasite-associated neutrophils showed positive in PKH26 signal (co-culture, bottom panel). (B) Quantification of (A), the percentage of cell-cell interaction were increased in dose dependent of MOI. N≥2; Mean ± SEM, *,** p < 0.05 (One way ANOVA).
We performed immunofluorescence to determine the number of surface-bound or internalized parasites. Human neutrophils were seeded on a cover slip and then exposed to opsonized PKH26-labeled L. martiniquensis. After 2 hours, the samples were washed and stained for MPO (granule marker) and DNA (Hoechst 3342). Uninfected neutrophils (Fig. 4A, i), adherent parasites (red fluorescence attached to the surface of PMNs, Fig. 4A, ii), and internalized parasites (yellow co-localization inside the PMNs, Fig. 4A, iii) were counted. As shown in figure 4B, both surface-bound and infected PMNs were increased in a dose dependent manner. The discrepancy in the amount of cell-cell interaction between the immunofluorescence and the flow cytometry assays are likely due to the limited number of cells counted in the IF experiment and will be addressed in the future. Nevertheless, the results from flow cytometry and immunofluorescence showed that L. martiniquensis was phagocytosed by human neutrophils. 102 NTDASIA 2019
Figure 4: Quantification of PKH26-labelled Leishmania uptake by human PMNs. After co-incubation between human neutrophils and PKH26-stained L. martiniquensis (red), cells were stained with anti-myeloperoxidase (green), Hoechst 3342 (blue). (A) Representative immunofluorescence staining of uninfected neutrophil (i), adherent parasites (ii, red arrows), and internalized parasites (iii, yellow arrow), scale= 10 mm. (B) Quantification of A. Percentage of uninfected, surface – bound parasites and infected PMNs were quantified at MOI 1:5 and 1:10. N=2, at least 30 cells were counted at 100x.
Interaction with L. martiniquensis induced neutrophil respiratory burst We hypothesized that the phagocytosis of L. martiniquensis by human PMN also induced the production reactive oxygen species (ROS) by neutrophils during their interaction, as shown for other species6,8. We measured PMN’s oxidative burst with the fluorescent indicator DHR123 and flow cytometry. CD11b+ was used for gating neutrophils. The level of Rho123 was determined in PKH26+ (infected) and PKH26- (bystander) neutrophils (Fig. 5A). We exposed PMNs with L. martiniquensis at MOI 1:5 in the presence of serum for 30 minutes, then quantified Rho123 fluorescence intensity as shown in figure 5B. A significant level of ROS was measured in PKH26+ PMNs compared to control and bystander PMNs, suggesting that the respiratory burst was mediated by cell-cell interaction and not the release of soluble mediators either by PMN or
leishmania. To determine if ROS other than H2O2 (detected by DHR123) are also produced, future experiments are planned to use the luminol-isoluminol assay. The effect of phagocytosis and ROS production on L. martiniquensis survival need to be investigated next, as some leishmania spp. have been shown to survive intracellularly in PMN4.
Figure 5: Interaction withL. martiniquensis induced neutrophil respiratory burst. (A) Neutrophils were gated on SSC/FSC (A, i) and CD11b expression (A, ii). CD11b+/PKH26+ and CD11b+/PKH26- (A, iii) cells reactive oxygen species content (A, iv and v) was measured by measuring the green fluorescence of RHO123. (B) ROS levels generated by unprimed neutrophils were determined in Leishmania associated PMNs (PKH26(+)) or bystander cells (PKH26(-)) upon interaction with the parasite at MOI 1:5. N=3; Mean ±SEM, *** p < 0.05. NTDASIA 2019 103
Neutrophil survival was increased by the co-incubation with L. martiniquensis Apoptosis is one of the mechanisms maintaining neutrophil homeostasis and leading to the resolution of inflammation after infection. It was previously reported that Leishmaniaupon spp. infection, neutrophil apoptosis was either accelerated or delayed, depending on the parasite species or the origin of the neutrophils13. The modulation of neutrophil survival is one of the strategies used by different leishmania species to subvert the immune system and facilitate infection4. We performed co-cultures of parasites and PMNs for 9 h and 24 h, at MOI 1:5. Apoptotic neutrophils express phosphatidylserine (PS) that can be detected using Annexin V staining. Therefore, we stained cells after coculture using PI/Annexin V and quantified the number of live cells (Annexin V negative) and apoptotic cells (Annexin V positive) by flow cytometry. As presented in figure 6, little to no apoptosis was detected at 9 hours in all conditions. However, after 24 hours, only ~6 % of the control neutrophils were alive. Interestingly, L. martiniquensis increased neutrophil survival at 24 h to ~28%. This effect was independent of leishmania opsonization (Fig. 6B). Therefore, it suggested that L. martiniquensis produces molecules that modulate neutrophil survival, as shown for L. major14, and it has also been shown that Leishmania spp. express chemotactic factors10, such as ILC and PSG, that act as neutrophil chemoattractants and activators. The identification of the exact factor(s) producedL. by martiniquensis to activate PMN and prolong their survival will be of interest, as well as the characterization of the anti-apoptotic pathways triggered in response. Importantly, it will be necessary to clarify whether L. martiniquensis survives within neutrophils, as it is a strategy used by several species13. However, the consequences and the importance of the increased survival of PMN for the parasites are beyond the scopes of this study.
Figure 6: Increased of neutrophil survival in the presence of the parasites. Unprimed PMNs were incubated with L. martiniquensis at MOI 1:5. The percentage of live (AnnexinV-/PI-) PMNs was measured by flow cytometry at 9 hours (6 hours of coculture) and 24 hours (21 hours of coculture). n=3; Mean ± SEM, * p < 0.05.
Conclusions: L. martiniquensis triggers neutrophil activation as evidenced by the upregulation of CD11b, CD66b, and the loss of surface 62L expression, even in absence of the serum. This suggests that human neutrophils recognize L. martiniquensis and get activated directly. Serum-opsonized L. martiniquensis was phagocytosed by the neutrophils. Significant levels of ROS were produced by the neutrophil associated with serum-opsonized PKH26-labeled L. martiniquensis. Serum-opsonized and un-opsonized L. martiniquensis were able to modulate 104 NTDASIA 2019
neutrophil survival. The delay of neutrophil apoptosis might contribute to a strategy often use by Leishmania spp. to survive and spread in the host, as described for L. major.
Acknowledgements: We would like to thank to Prof. Saowanee Leelayoova who kindly gave us L. martiniquensis for this work. My special gratitude is expressed to Prof. Sukhathida Ubol who provided us with the macrophages. Thanks to the technicians who prepared us the buffy coat. This research project was financially supported by DPST scholarship to Nanthicha Inrueangsri, and the Thailand Research Fund (TRF-RSA5880021) to Fabien Loison.
References: 1. Alcolea PJ, Alonso A, Sánchez-Gorostiaga A, Moreno-Paz M, Gómez MJ, Ramos I, Parro V, Larraga V. Genomics. 2009;93(6):551-64. 2. Bates PA, Rogers ME. Current molecular medicine. 2004;4(6):601-9. 3. Carlsen ED, Hay C, Henard CA, Popov V, Garg NJ, Soong L. Infection and immunity. 2013;81(11):3966-74. 4. Carlsen ED, Jie Z, Liang Y, Henard CA, Hay C, Sun J, de Matos Guedes H, Soong L. Journal of innate immunity. 2015;7(4):354-63. 5. Da Silva R, Sacks DL. Infection and immunity. 1987;55(11):2802-6. 6. Davis RE, Thalhofer CJ, Wilson ME. Journal of immunological techniques in infectious diseases. 2016;5(3). 7. Dey R, Joshi AB, Oliveira F, Pereira L, Guimarães-Costa AB, Serafim TD, de Castro W, Coutinho-Abreu IV, Bhattacharya P, Townsend S, Aslan H. Cell host & microbe. 2018;23(1): 134-43. 8. Falcão SA, Weinkopff T, Hurrell BP, Celes FS, Curvelo RP, Prates DB, Barral A, Borges VM, Tacchini-Cottier F, de Oliveira CI. PLoS neglected tropical diseases. 2015;9(3):e0003601. 9. Hurrell BP, Beaumann M, Heyde S, Regli IB, Müller AJ, Tacchini‐Cottier F. Journal of leukocyte biology. 2017;102(5):1187-98. 10. Hurrell BP, Regli IB, Tacchini-Cottier F. Trends in parasitology. 2016 May 1;32(5):392-401. 11. Laskay T, Van Zandbergen G, Solbach W. Trends in microbiology. 2003;11(5):210-4. 12. Martínez-López M, Soto M, Iborra S, Sancho D. Frontiers in microbiology. 2018;9:883. 13. Regli IB, Passelli K, Hurrell BP, Tacchini-Cottier F. Frontiers in immunology. 2017;8:1558. 14. Sarkar A, Aga E, Bussmeyer U, Bhattacharyya A, Möller S, Hellberg L, Behnen M, Solbach W, Laskay T. Medical microbiology and immunology. 2013;202(1):25-35. 15. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY. Nature methods. 2012 Jul;9(7):676. 16. Siripattanapipong S, Boontanom P, Leelayoova S, Mungthin M, Tan-ariya P. Acta tropica. 2019 Sep 1;197:105039. 17. Volk AP, Barber BM, Goss KL, Ruff JG, Heise CK, Hook JS, Moreland JG. Journal of innate immunity. 2011;3(3):298-314. NTDASIA 2019 105
PO:04 Morphological Identification of Mosquito Vectors in and Around Mangrove Forest in Chachoengsao Province, Thailand
Somsanith Chonephetsarath,1* Chamnarn Apiwathnasorn,1 Sungsit sungvornyothin,1 Arnon Payakkapol,1 1 1 Thipruethai phanitchat, Yudthana Samung, 1Department of Medical Chadchalerm Raksakoon,2 Rutcharin Potiwat1** Entomology, Faculty of Tropical Medicine, Mahidol University, Abstract: The medically important mosquito vectors in Thailand Bangkok 10400, Thailand. includes Culex spp., Aedes spp., Anopheles spp., Mansonia spp. 2Department of Chemistry, Faculty etc. The Culex spp. are important vectors of Japanese encephalitis of Science, Kasetsart University, Bangkok 10900, Thailand virus (JEV) and Aedes spp. are the primary vectors of dengue, *e-mail: nithsomsanith.ch@gmail. yellow fever, chikungunya and Zika virus. Normally, mosquito com; **e-mail: rutcharin.pot@ vectors have different breeding sites that are related to natural mahidol.ac.th ecology and water container. According to the previous study, three dengue vectors were found in the same study area. Therefore, to confirm the mosquito vectors in the mangrove forest in Chachoengsao Province, Thailand, we collected mosquito larvae and maintained in the insectarium until they developed to the adult stage. Adult mosquitoes were separated and identified using the pictorial keys.Aedes dux is a predominant (81.9%) species which was found inside the mangrove forest followed by Culex brevipalpis (1.5%). The results provide valuable information for vector control strategy and virus transmission from mangrove forest.
Keywords: Aedes spp., Culex spp., mangrove forest, dengue virus, mosquito vector
Introduction:The climate of Thailand is appropriate for mosquito rearing especially of Aedes spp. mosquitoes, which can be found in an indoor and outdoor environment, and it can grow in both clean and brackish water. There are three medically important Aedes spp. members in Thailand, including Ae. aegypti, Ae. albopictus and Ae. scutellaris 1. Aedes spp. are the important vector of Yellow fever, Dengue, Chikungunya and Zika viruses 2,3. For Ae. albopictus is a primary vector of chikungunya virus and also is a secondary vector to support dengue virus transmission 4,5.
Mangrove forests is an important place because it provides breeding sites for mosquitoes for diseases distribution 6. It provides abundant and diverse habitats for aquatic insects including pest and disease vector species, and it has traditionally been a neglected area of ecological research, particularly regarding mosquitoes. The presence of mosquitoes in mangrove 106 NTDASIA 2019
is regard as a threat to humankind although the risk itself is insignificant. The previous study showed the biting pattern of Aedes mosquitoes in mangrove forests mimics the biting habits of mosquitoes in urban and pre-urban areas, which peaked at dawn and dusk, but showed greater potential for biting activity throughout the day, especially in the disturbed areas7 .
Another mosquito vector that is associated with saline-brackish ground pools mangrove forests is Cx. sitiens, which has been known as the vector of medically and veterinary important arthropod- borne diseases. It is a potential vector of the Japanese encephalitis virus (JEV) transmission in Australia and Malaysia 8,9. Previous research has recorded the presence of a few vectors of mosquito-borne pathogens in mangrove forests 1. Although these mosquitoes may not be directly associated with these habitats, but they are the important vectors of dengue virus transmission. The present study aimed to assess distribution of mosquito species and population during dry season, which may be used as the supporting data to control mosquito-vector transmission in future.
Methodology: Sample collection The study was conducted in Mangrove forest, Chachoengsao Province, Thailand in triplicated time within the same container such as jars and flower pots around mangrove forest during dry season in 2019 1. The larvae of mosquitoes from the field were collected using WHO standard dipping method (WHO 1992) and transfer to the laboratory 10.
Mosquito rearing Larval stage were fed round about 200 per plastic tray (6 x 36 x 26cm) (height; length width). The adult mosquitoes were maintained about 200-300 per each cage size: 30 x 30 x 30 cm (height, length, width). Fish food solution was given to larval stage (1 time/day) until they have developed to pupa stage, then they were kept in a cage fed with 10% sugar solution (1 time/week) until emergence of adults 1. All mosquito colonies were kept in an insectary of the Entomological Laboratory for Dengue Virus Infection (ELDVI) with 12 hours of day/light cycle with control condition 27°C ± 2°C and a relative humidity (RH) of 65 ± 10%.
Mosquito identification All adult stage mosquitoes were separated and pinned according to the laboratory procedure. The complete samples were taken a photo using Nikon AZ100M camera before comparing the morphological characteristics of mosquito-based on pictorial identification keys from Huang 1972 11 and Rattanarithikul R et al.12-15. The mosquito body part were identified and taken picture to show differentiation of each species.
Ethical consideration This study was approved by the Tropical Medicine Ethics Committee, Faculty of Tropical Medicine, Mahidol University. Permission for animal care and use of Aedes aegypti mosquitoes was approved by the Mahidol University-Institute Animal Care and Use Committee, Mahidol University.
Results and Discussion: A total of 2,215 larvae were collected three times in the same mangrove forest area and then maintained until fully develop to the adult stage in our laboratory. The adult stages were identified and found that these mosquitoes belonged to two genera,Aedes and Culex NTDASIA 2019 107
(Table 1). In this study we found both of them in different container and water quality such as pH ranging 8.04 – 8.94, total dissolved solids (TDS) ranging 221 – 826 ppm, and dissolved oxygen (DO) ranging 115.7 – 192.7 ppm. The water quality also affected the oviposition behavior of female mosquitoes 16,17. Their results showed the important vector of dengue virus: Ae. aegypti, and Japanese encephalitis virus;Cx. sitiens from the mangrove forest. Our data revealed that Ae. dux are the predominant species in this area. This species is difficult to colonize in the laboratory and the information in relation to vector-borne diseases is rarely reported. Therefore, in our experiments, we also tried to feed both Ae dux and Cx. brevipalpis using artificial membrane feeding, but they did not uptake a blood (unpublished data) 18. According to the previous research, Ae. aegypti, Ae. albopictus and Ae. scutellaris mosquitoes were found in the mangrove forest 1. However, we did not find the same species of dengue vector in this area exceptAe. aegypti and Ae. dux are presenting as dominant species.
Table 1: Number of each mosquito species collected in the study area Larvae collected Percentage of mosquito Mosquito Species 1st time 2nd time 3rd time collection (%) Aedes dux 819 536 458 81.9% Aedes aegypti 252 - - 11.4% Culex brevipalpis 34 - - 1.5% Culex sitiens 118 - - 5.3% Total 1,223 536 458 2,215
Figure 1. The mosquitoes collected from the mangrove forest, Chachoengsao Province, Thailand. (A) Ae. aegypti, (B) Ae. dux, (C) Cx. brevipalpis, and (D) Cx. sitiens.
A total of 2,215 adult mosquitoes from 2 genera and 4 species were identified (Figure1), and over 81.9% of the total mosquitoes are Ae. dux (Table 1). We also found Cx. brevipalpis and Cx. sitiens 1.5% and 5.3%, respectively. Normally,Cx. sitienshave been found in many saline-water container or mangrove forest and it was a dominant species in disturbed area around the mangrove forest 108 NTDASIA 2019
in Kedah, Malaysia 7. Interestingly, we have found Cx. brevipalpis and Ae. dux which were never been identified in the previously report in the same study area1 . These species might be silence vectors of many diseases associated with mangrove forests. Therefore, discovery of Ae. dux and Cx. brevipalpis might be considered in the vector control strategy future, because viral transmission via this species are rarely reported.
Acknowledgements: This work was partially supported by the Faculty of Tropical Medicine, Young researcher Fund (Fiscal year 2018). We would like to thank Dr. Sylvia Meek Scholarships, Malaria Consortium that giving us a great opportunity and support our study. We thank Medical Entomology Laboratory staff for technical assistance.
References: 1. Sumruayphol S, Apiwathnasorn C. Acta Trop. 2016;159:1-10. 2. Phumee A, Chompoosri J. Sci Rep. 2019; 9(1):5257. 3. Thavara U, Tawatsin A. Southeast Asian J Trop Med Public Health. 2009;40(5):951-62. 4. Potiwat R, Komalamisra N. Southeast Asian J Trop Med Public Health. 2011;42(6):1388 94. 5. Rohani A, Potiwat R. Southeast Asian J Trop Med Public Health. 2009;40(3):443-8. 6. Dale P, Knight J. Wetlands Ecol Manage. 2008;16:255-276. 7. TNS TI, A Kassim NF. Trop Med Infect Dis. 2018;3(3):1-8. 8. Van-den-Hurk AF, Ritchie SA. Emerging infectious diseases. 2008;14(11):1736-1738. 9. Vythilingam I, Oda K. J Am Mosq Control Assoc. 1995;11(1):94-8. 10. World Health Organization.1992:1-77 11. YM H. Contributions of the American Entomological Institute. 1972;9(1):1-109. 12. Rattanarithikul R, Harbach R. Southeast Asian J Trop Med Public Health. 2010;41 Suppl 1: 1-225. 13. Rattanarithikul R, Harbach R. Southeast Asian J Trop Med Public Health. 2005;36 Suppl 2: 1-97. 14. Rattanarithikul R, Harrison B. Southeast Asian J Trop Med Public Health. 1988;20:21. 15. Rattanarithikul R, Harrison B. Southeast Asian J Trop Med Public Health. 2005;36 Suppl 1: 1-80. 16. Ma M, Huang M. Acta Trop. 2016;163:121. 17. Wetzel RG. Elsevier Science. 2001:1-1006. 18. Gunathilaka N, Ranathunge T. Biomed Res Int. 2017;2017:1-7. NTDASIA 2019 109
PO:05 Expression of p53, Bcl-22, Granulin, and PCNA in the Liver of Opisthorchis viverrini Infected Hamsters: A Comparison Between Single Infection and Repeated Infection Plus Praziquantel Administration 1Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen Songkiad Upontain,1,3,* Piya Sereerak,6 Thewarach Laha,2,3 40002, Thailand 2 Banchob Sripa,2,3 Prasarn Tangkawattana,1 Faculty of Medicine, Khon Kaen Anudep Rangsipipat,4 Chanokchon Setthawongsin,5 University, Khon Kaen 40002, Thailand Sirikachorn Tangkawattana1,3,** 3WHO Collaborating Centre for Research and Control of Abstract: Opisthorchiasis caused by Opisthorchis viverrini Opisthorchiasis (Southeast Asian, (O. viverrini) is an important public problem in the Greater Liver Fluke Disease) Tropical Mekong Subresion countries. Habitual eating of raw cyprinid Disease Research Center, Khon fish dishes and unaware taking of praziquantel (PZQ), an Kaen University, Khon Kaen 40002, Thailand effective anthelminthic against this trematode, are the major 4Faculty of Veterinary Science, causes of repeated infection of the people in these endemic Chulalongkorn University, Bangkok areas. This 15-week experiment was conducted in two groups 10300, Thailand 5 of five Syrian golden hamsters—Group 1 with singleO. viverrini Faculty of Veterinary Technology, Kasetsart University, Bangkok infection, and Group 2 with three consecutive rounds of 10900, Thailand infection and praziquantel (PZQ) administration. To both groups 6Department of National Park, of animals, subcarcinogenic dose of NDMA (12.5 ppm) was Wildlife and Plant Conservation, given in the drinking water. Histopathology and expression of Surat Thani 84000, Thailand * ** p53, Bcl-2, granulin (GRN) and proliferating cell nucleus antigen Presenter; e email: sirikach@kku. ac.th (PCNA) in liver samples were studied. The results showed that there was no evidence of cholangiocarcinoma (CCA) development in all hamsters of both groups. Although histopathological changes in both groups were similar, proliferative biliary lesions such as bile duct hyperplasia, bile duct dysplasia, and periductal fibrosis were more severe in the hamsters of Group 1. Average PCNA index and abnormal proliferation of biliary epithelial cells in Group 2 were significantly higher than those in Group 1 (p-value = 0.014). Average percentage of Bcl-2 expression in Group 2 was higher than in Group 1. Immunostaining of p53 and GRN was negative in both groups. These results suggest that three repeated rounds of infections followed by PZQ treatments in a period of 15 weeks with subcarcinogenic dose of NDMA did not induce CCA development. Late response of CCA involving genes, such as p53 and GRN, might not be altered so that neotransformation did not occur within the study period.
Keywords: repeated infection,Opisthorchis viverrini, p53, Bcl-2, PCNA, hamster 110 NTDASIA 2019
Introduction: Opisthorchiasis, caused by Opisthorchis viverrini (O. viverrini), is an important public health problem in the Greater Mekong Subresion countries, especially in the wetland, river basin, and lake areas of Thailand, Lao PDR, Vietnam, and Cambodia6. People in these regions are known to acquire the infection through their habitual eating of raw or undercooked cyprinid fish and its short fermenting products that carry metacercariae of O. viverrini24,26. Chronic and repeated infection of this trematode has been alarmed to be a risk factor associated with the incurrence of cholangiocarcinoma (CCA)23. The inflammatory responses to both physical and chemical injuries by the liver fluke in patients with chronic and repeated infection could break down their biliary apparatus to incur the accumulative genetic and epigenetic abnormalities. Appearance of biliary proliferation and periductal fibrosis, which are accepted as the semeiotic clues of these abnormalities, have been determined to be the precedent evidences of CCA22.
Thus far, WPRO27 has recommended a single dose of 40 mg/kg body weight of praziquantel (PZQ) to treat opisthorchiasis in all endemic countries. Although the medicine is proven to be highly effective against the infection, the infection rate of opisthorchiasis seemingly never subsides. Our yearly surveys in the endemic area of Khon Kaen, Thailand, revealed that re-infection rate reached nearly 90% in patients receiving such medicinal treatment13,20. Reluctance to refrain from eating raw fish dishes and availability to easily access to this medicine are probably the underlying causes of the repeated infection4. Each individual patient usually decides to take this medicine without prescription. The repeated use of this medicine can be seriously problematic since it is claimed to be a risk factor of CCA. This claim is usually based on the experiments of repeated O. viverrini infection together with multiple PZQ treatment in animal model18,19. Such recurrent infection could induce or enhance inflammatory cells to synthesize nitric oxide which is molecularly harmful to bile duct epithelium leading to the initiation of CCA19.
Mutation ofp53 gene has been considered as a major infliction during the development of various cancers in humans and animals10. Some cell-cycle regulatory proteins, such as cyclin D1 and Bcl2, were also recruited into the p53-mediated pathway10. Regards, the whole genome sequencing study of CCA could indicate a significant role of p53 in the incurrence of O. viverrini-associated CCA11. Co-aberration ofp53 and Bcl-2 overexpression was likewise regarded as a causative factor in the onset of CCA10. Granulin (GRN) was another protein of interest in cancer development because its overexpression could be detected in many types of cancer6. Thus, this study aims to detect immunohistochemically the aberrant expression of p53, Bcl-2, and GRN, and PCNA in hamsters being induced by single O. viverrini infection vs. three consecutive rounds of infection and PZQ administration. Histopathological changes of the intrahepatic biliary apparatus were also shown.
Materials and Methods: Hamsters The study was approved by the Animal Ethics Committee of Khon Kaen University according to the Ethics of Animal Experimentation of the National Research Council of Thailand (ACUC KKU 55/2554). Two groups of 5 Syrian golden hamsters were used--Group 1 was fed orally with 50 O. viverrini metacercariae without PZQ treatment, and Group 2 was scheduled for 3 consecutive infections by oral feeding of 50 O. viverrini metacercariae on weeks 1, 5, 10, and PZQ treatments on weeks 4, 9, and 14 (Figure 1). The animals could access to drinking water containing a subcarcinogenic dose of NDMA (12.5 ppm) ad libitum. All animals were sacrificed to collect the liver and biliary apparatus on week 15 (Figure 1). NTDASIA 2019 111
Figure 1. Experimentation schedule
Histopathogical study Based on Lvova et al.27, histopathological classification of biliary changes was classified into bile duct proliferation (increase in number of biliary cells in small bile ducts), bile duct hyperplasia (increase in number of biliary cells in large bile ducts), periductal fibrosis, biliary metaplasia (types of transformed biliary cells), and CCA (cancer of biliary cells).
Immunohistochemical studies for p53, Bcl-2, granulin, and PCNA The tissue sections were deparaffinized in xylene in an incubator at 65oC for 30 min, and rehydrated in a decending series of ethanol as routine histological study. Antigen was retrieved in 0.1M sodium citrate in a high-pressure cooker for 5 min. Non-specific bindings were blocked with 5% normal horse serum for 30 min, after which the sections were probed with the primary antibody
(Table 1) in PBS, 0.05% NaN3 at room temperature overnight. Subsequently, tissue sections were incubated with the secondary antibodies (Table 1) in PBS for 1 hr. After several washes in PBS, positive signals were detected using 0.03% diaminobenzidine (DAB) in 0.003% hydrogen peroxide in Tris-HCl pH 7.2, as the chromogenic substrate. Counterstaining with Mayer’s hematoxylin was performed before mounting the slides. Reference samples for positive and negative controls were listed in Table 1.
Table 1 List of antibody (Ab), dilutions, and reference samples Primary Ab Dilution Secondary Ab Dilution Positive control Negative control Monoclonal mouse 1: 100 Rabbit anti-mouse 1: 300 Human colon cancer Normal hamster anti-human p53 liver Polyclonal rabbit 1: 200 Goat anti-rabbit 1: 300 Hamster CCA Normal hamster anti-Bcl-2 liver Polyclonal rabbit 1: 100 Goat anti-rabbit 1: 300 Human hepatoma Normal hamster anti-human GRN liver Polyclonal rabbit 1: 200 Goat anti-rabbit 1: 300 Hamster CCA Normal hamster anti-PCNA liver Note: Antibodies were purchased from Abcam plc., Cambridge, UK, except monoclonal mouse anti-human p53 (Dako, DO-7, USA) and polyclonal rabbit anti-human GRN (HPA008763, Sigma-Aldrich, St. Louis, MO). 112 NTDASIA 2019
Data analysis Evaluation of the histopathological study was described qualitatively. The results of PCNA, p53, granulin and Bcl-2 were formulated in percent (%= positive biliary cells x 100/total biliary cells). Statistical differences among each group and treatment period were compared by Mann-Whitney U test because at the end of experimental samples in each group was uneven. P values of <0.05 were considered to be statistically significant.
Results: Histopathological and immunohistochemical study The hamsters in both groups expressed similar histopathological changes, especially proliferative biliary lesions, such as bile duct hyperplasia and dysplasia. Few periductal fibrosis existed at the location where parasites lived. In comparison with Group 1, the liver of the hamsters in Group 2 showed less severe proliferative changes of biliary epithelium, such as bile duct hyperplasia and dysplasia, and very few periductal fibrosis (Figure 2). Cystic formation and CCA were not found in both groups.
Average PCNA index and expression of Bcl-2 in the hamsters of Group 1 were significantly higher than that in Group 2 (p-value = 0.014). Immunostainings of p53 and GRN in both groups were negative (Figure 3).
Figure 2. Histopathological features of the biliary tract in hamsters. Degree of proliferative changes in Group 1 (A) was higher than that in Group 2 (B). Proliferative biliary cells with strongly positive PCNA staining (C) and slightly positive staining of Bcl-2 (E) in Group 1 were demonstrated. Staining for p53 (D) and GRN (F) in the biliary epithelial lining cells of both groups was negative. Adult fluke was occasionally seen in bile duct (F). Original magnification = 10x (C, E), 20x (F), 40x (A, B, D). NTDASIA 2019 113
Figure 3. Expression of PCNA, p53, Bcl-2 and granulin proteins in the liver of Group 1 (G1) and Group 2 (G2) hamsters. PCNA and Bcl-2 expression was much higher in G1 than in G2. Expression of P53 and granulin of both groups was not detectable.
Discussion and Conclusion: Immunohistopathology of biliary epithelial cells of hamsters having sole O. viverrini infection (Group 1) and three consecutive O. viverrini infections with PZQ treatments (Group 2) was compared. Proliferative lesions in this study were similar to those in Lvova et al.27 indicating an inflammatory reaction along with the presence of the parasitic infection. However, development of CCA was not detected within this 15-week study period. A much longer period is surely required. The study of Hanpanich et al.5 in hamster having multiple O. viverrini infection and PZQ medication also could not demonstrate CCA by the week 40 of the experiment. However, Lvova et al.27 showed that tumor formation in O. viverrini-NDMA induction model commenced on the month 3 post-infection. Sub-carcinogenic dose of NDMA could expedite the tumorigenesis. Since this study lasted only 15 weeks, accumulation of genetic abnormalities probably was not high enough to reach the neotransformation point. Indicators of this abnormalities are necessary to be revealed.
PCNA is a nuclear antigen that is expressed at the initiation of cell proliferation during the DNA synthesis phase14. PCNA immunohistochemistry has been effectively used in the evaluation of cell proliferation. In this experiment, positive staining of PCNA indicated the response of biliary cells to the parasitic induction. Thus, the significantly higher PCNA expression of Group 1 than that of Group 2 indicated that Group 1 has a higher biliary cell proliferation than Group 2. This result was contradicted to our prior expectation that PCNA should be expressed higher in the hamsters with repeating infections and treatments than in the hamsters with single infection. It is possible that the biliary cells might require a longer parasitic induction period to provoke aggravating cellular responses. A 15-week induction as in this experiment might not be sufficient to promote rational proliferation. p53 gene is commonly found in various kind of cancers including CCA. Normally, p53 functions in contrary to wild-type Bcl-2. While p53 protein induces apoptosis, Bcl-2 protein blocks this process through diverting p53 activity from the apoptotic induction2. The connection of p53 and Bcl-2 appears in the apoptotic pathway in which p53 regulates both direct and indirect activity 114 NTDASIA 2019
of the proteins in Bcl-2 family8. p53 is also known as a transcription factor that controls the cell cycle and a tumor suppressor. Mutation of this tumor suppressor gene is the most frequent genetic alteration found in many cancers, including CCA13. Alterations of this gene could signify its induction role in cholangiocarcinogenesis15. Additionally, overexpression of p53 protein is related to poor prognosis of CCA1,9. Bcl-2 is considered as a major regulator of cell apoptosis3. It is known as anti-apoptotic gene that inhibits apoptosis either directly through controlling via MAC or indirectly through other proteins25. Overexpression of Bcl-2 has been found in many cancers including CCA6. The gene product could inhibit apoptosis and promote cancer cell to survive, grow, invade, and metastasize. Recently, Bcl-2 protein is believed to regulate cell differentiation, autophagy, mitochondrial fusion/fission, and senescence3. In this study, p53 protein was not detectable in all liver tissues of both groups. Expression of Bcl-2 in biliary cells of hamsters in Group 1 was higher than those in Group 2. These findings suggested that apoptotic activity of the hepatic biliary cells in Group 1 might be concordance with the amount of proliferation marker, PCNA. However, the hamsters in both groups did not develop any cancer. This could explain the negative results ofp53 which is a late response gene in cancer development12. GRN is also another late response gene evaluated in this experiment. GRN proteins usually function in relation with the transmission of intracellular signals. Expression of this gene in cancer is to promote cell proliferation, anti-apoptosis, and carcinogenesis and stimulate angiogenesis as well7. Overexpression of GRN has been reported in metastatic stage and related to poor prognosis21. The absence of GRN product in both experimental groups was consistent with the nonappearance of CCA.
In conclusion, progressive histopathological lesions of the Group1 hamster is higher than that of the Group 2 hamster. Immunohistochemical results were concordance with the lesions. Multiple PZQ treatment could relieve the parasitic infection so that the lesion progression is lessened. Therefore, the repeated infections and treatments may not be the potent risk factor in CCA induction. A long-term study is required to approve this preliminary assumption.
Acknowledgements: The authors would like to thank Faculty of Veterinary Medicine, KKU and Staffs from WHO Collaborating Centre for Research and Control of Opisthorchiasis (Southeast Asian Liver Fluke Diseases)/Tropical Disease Research Center (KKU) for supporting the equipment and laboratory and technical assistance. This study was financially supported by the Graduate School KKU Year 2017.
References: 1. Ahrendt SA, Rashid A, Chow JT, Eisenberger CF, Pitt HA, Sidransky D. J Hepatobiliary Pancreat Surg. 2000;7:426-431. 2. Chiou SK, Rao L, White E. Mol. Cell Biol. 1994;14:2556–2563. 3. Ding X, Zhang Y, Huang T, Xu G, Peng C, Chen G, Kong B, Friess H, Shen S, Lv Y, Roberts LR, Wang L, Zou X. Am J Cancer Res. 2019;9(3):546–561. 4. Grundy-Warr C, Andrews RH, Sithithaworn P, Petney TN, Sripa B, Laithavewat L. Parasitol Int. 2012;61:65–70. 5. Hanpanich P, Laha T, Sripa B, Mairiang E, Sereerak P, Upontain S, Tangkawattana P, Brindley PJ, Tangkawattana S. Parasitol Int. 2017;66(4):464-470. 6. Harnois D, Que F, Larusso N, Gores A. Hepatology. 1997;26:4. 7. He Z, Bateman A. J Mol Med. 2003;81:600–612. 8. Hemann MT, Lowe SW. Cell Death & Differentiation. 2006;13:1256–1259. NTDASIA 2019 115
9. Horie S, Endo K, Kawasaki H, Terada T. Virchows Arch 2000;437:25-30. 10. Jarnagin WR, Klimstra DS, Hezel M, Gonen M, Fong Y, Roggin K, Cymes K, DeMatteo RP, D’Angelica M, Blumgart LH, Singh B. J Clin Oncol. 2006;1;24(7):1152-60. 11. Jusakul et al. American Association for Cancer Research. 2017;doi:10.1158/2159-8290. CD-17-0368. 12. Kamsa-ard S,Luvira V,Pugkhem, Luvira V,Thinkhamrop B, Suwanrungruang k, Bhudhisawasdicorresponding V. BMC Cancer. 2015;15: 776. 13. Khan SA, Thomas HC, Toledano MB, Cox IJ, Taylor-Robinson SD. Liver Int 2005;25:704–716. 14. Leonardi E, Girlando S, Serio G, Mauri FA, Perrone G, Scampini S, Dalla Palma P, Barbareschi M. J. Clin. Pathol. 1992;45 (5):416–419. 15. Liu XF, Zhang H, Zhu SG, Zhou XT, Su HL, Xu Z, Li SJ. World J Gastroenterol. 2006;12(29): 4706-4709. 16. Lvova MN, Tangkawattana S, Balthaisong S. Parasitol Int. 2012;61:167-72. 17. Okaro AC, Deery AR, Hutchins RR, Davidson BR. J Clin Pathol 2001;54:927–932. 18. Pinlaor S, Hiraku Y, Yongvanit P, Tada-Oikawa S, Ma N, Pinlaor P, Sithithaworn P, Sripa B, Murata M, Oikawa S, Kawanishi S. Int J Cancer. 2006;1:119(5):1067-72. 19. Pinlaor S, Ma N, Hiraku Y, Yongvanit P, Semba R, Oikawa S, Murata M, Sripa B, Sithithaworn P, Kawanishi S. Carcinogenesis. 2004;25:1535–1542. 20. Sithithaworn P, Ziegler AD, Grundy-Warr C, Andrews RH, Petney TN. Lancet Infect Dis. 2012;12:588. 21. Smout MJ, Laha T, Mulvenna J, Sripa B, Suttiprapa S, Jones A, Brindley PJ, Loukas A. PLoS Pathog. 2009;5:e1000611. 22. Sripa B, Brindley PJ, Mulvenna J, Laha T, Smout MJ, Mairiang E, Bethony JM, Loukas A. Trends Parasitol. 2012;10:359-407. 23. Sripa B, Kaewkes S, Sithithaworn P. PLoS Med. 2008;4:e201. 24. Sripa B, Pairojkul C. Curr Opin Gastroenterol. 2008;24(3):349–56. 25. Tsujimoto Y. Genes Cells. 1998;3(11):697-707. 26. Watanapa P, Watanapa WB. Br J Surg. 2002;89(8):962–70. 27. WPRO. World Health Organization Regional Office for Western Pacific, Manila Philippines. 2017;16. 116 NTDASIA 2019 117
List of Participants Achara Phumyen Achariya Sailasuta Chulabhorn International College of Department of Pathology, Faculty of Veterinary Medicine, Thammasat University Science, Chulalongkorn University, Thailand (Rangsit Center), Thailand E-mail: [email protected] E-mail: [email protected]
Agatha ranindita kirana Candra Akarapon Chantongsri Indonesia International Institute for Life Khon Kaen University, Thailand sciences (i3L), Jakarta, Indonesia E-mail: [email protected] E-mail: [email protected]
Akira Ito Alessia Leonetti Department of Parasitology and Laboratory of Faculty of Medicine, Khon Kaen University, NTDs, Asahikawa Medical University, Japan Thailand E-mail: [email protected] E-mail: [email protected]
Amita Kulawong Amonrat Jumnainsong Udon Thani Rajabhat University, Thailand Faculty of Associated Medical Sciences, E-mail: [email protected] Khon Kaen University, Thailand E-mail: [email protected]
Anang samudera Otoluwa Anat Buarat Banggai District Health Office, Indonesia Faculty of Associated Medical Sciences, E-mail: [email protected] Khon Kaen University, Thailand E-mail: [email protected]
Anchalee Techasen Anggita Ratri Pusporini Faculty of Associated Medical Sciences, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Apiporn T. Suwannatrai Ardhina Nugrahaeni Department of Parasitology, Faculty of Khon Kaen University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Aroonlug Lulitanond Aroonwadee Chanawong Faculty of Associated Medical Sciences, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] 118 NTDASIA 2019
List of Participants (Cont.) Arunnee Sangka Arunta Samankul Faculty of Association Medical Sciences, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Aung Tun Aung Phyo Wai Ministry of Health and Sports, Myanmar Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Banchob Sripa Basripuzi Nurul Hayyan Hassan Basri Tropical Disease Research Laboratory, Faculty Universiti Malaysia Kelantan, Malaysia of Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Bingkuan Zhu Bong-kwang Jung Fudan University, China Institute of Parasitic Diseases, E-mail: [email protected] Korea Association of Health Promotion, Korea South E-mail: [email protected]
Chadchalerm Raksakoon Chalida Chuenchom Kasetsart University, Thailand Tropical Disease Research Laboratory, Faculty E-mail: [email protected] of Medicine, Khon Kaen University, Thailand E-mail: [email protected]
Chanakan Jantawong Chanakarn Loiklung Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Chanika Worasith Chatanun Eamudomkarn Department of Parasitology, Faculty of Khon Kaen University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Chatchai Sarachai Chee Dhang Chen Faculty of Veterinary Medicine, Chiang Mai University of Malaya, Malaysia University, Chiangmai, Thailand E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 119
List of Participants (Cont.) Chuchat Kamollerd Chularut Prariyachatigul Faculty of Veterinary Medicine, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Darika Sirisudtha Daroon Kotimanusvanij Thakhantho hospital, Thailand Faculty of Medicine, Khon Kaen E-mail: [email protected] University, Thailand E-mail: [email protected]
Desheila Andarini Doolyawat Kladkempetch Universitas Sriwijaya, Indonesia Faculty of Veterinary Medicine, E-mail: [email protected] Chiang Mai University, Thailand E-mail: [email protected]
Dung Bui Thi Dyna Doum Institute of Ecology and Biological Resources, Khon Kaen University, Thailand Vietnam Academy of Science and Technology, E-mail: [email protected] Vietnam E-mail: [email protected]
Fang Luo Galina Minkova Fudan University, China Institute of Cytology and Genetics SB RAS, Russia E-mail: [email protected] E-mail: [email protected]
Helena Ullyartha Pangaribuan Hendra Kurniawan Infectious Diseases Unit Research Center & Faculty of Medicine, Community Health Development National Khon Kaen University, Thailand Institute Health Research & Development E-mail: [email protected] (NIHRD) Ministry of Health Republic Indonesia, Indonesia E-mail: [email protected]
Hongli Zhou Hye Joo Shin Zhongshan School of Medicine, Korea Association of Health Promotion, Sun Yat-sen University, China Korea South E-mail: [email protected] E-mail: [email protected]
Isobel Thompson Jarawee Wannachart Khon Kean University, Thailand College of Medicine and Public Health, E-mail: [email protected] Ubon Ratchathani University, Thailand E-mail: [email protected] 120 NTDASIA 2019
List of Participants (Cont.) Jeerapapon Khonprom Jeonggyu Lee Udon Thani Rajaphat University, Thailand Institute of Parasitic Diseases, E-mail: [email protected] Korea South E-mail: [email protected]
Jielin Deng Jitrawee Luengtrairat Department of Pathogen Biology, School of Ching Mai University, Thailand Public Health, Southern Medical University, E-mail: [email protected] China E-mail: [email protected]
Jittiyawadee Sripa John F Smith College of Medicine and Public Health, Faculty of Public Health, Ubon Ratchathani University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Jong-Yil Chai Joy Albert Sumakul Institute of Parasitic Diseases, Health Office, Indonesia Korea Association of Health Promotion, E-mail: [email protected] Korea South E-mail: [email protected]
Jumaporn Sonongbua Jutamas Namsanor Khon Kaen University, Nong Khai Campus, Khon Kaen University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Juthawan Nawakakam Kamonrot Kumpangnil Udon Thani Rajabhat University, Thailand College of Medicine and Public Health E-mail: [email protected] Ubon Ratchathani University, Thailand E-mail: [email protected]
Kanaporn Maisao Kanchana Nakhapakorn Udon Thani Rajabhat University, Thailand Mahidol University, Thailand E-mail: [email protected] E-mail: [email protected]
Kanin Salao Kannawee Swangneat Department of Microbiology, Faculty of Khon Kaen University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 121
List of Participants (Cont.) Kanokwan Bootyothee Kantima Sirisantimethakom Khon Kaen University, Thailand Kalasin University, Thailand E-mail: [email protected] E-mail: [email protected]
Kavin Thinkhamrop Kazuko Miyamoto Faculty of Public Health, University of Yamanashi, Japan Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Keeseon Eom Kesinee Saranrittichai Chungbuk National University School of Khon Kaen University, Thailand Medicine, Korea South E-mail: [email protected] E-mail: [email protected]
Kesorn Thaewnongiew Khanittha Pratumchart Office of Disease Prevention and Control, Khon Kaen University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Khao Keonam Khin Thet Wai Faculty of Veterinary Medicine, Department of Medical Research, Myanmar Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Khwancheewa Wattanatripop Kitti Intuyod Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Kittiya Wittayapipath Krittaya Saelee Khon Kaen University, Thailand Faculty of Associated Medical Sciences, E-mail: [email protected] Khon Kaen University, Thailand E-mail: [email protected]
Krongkarn Watakulsin Kulwadee Suwannatrai Department of Microbiology, Sakon Nakhon Rajabhat University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] 122 NTDASIA 2019
List of Participants (Cont.) Kunnika Kuaha Lakhanawan Charoensuk Faculty of Associated Medical Sciences, Department of Clinical Pathology, Faculty of Khon Kaen University, Thailand medicine Vajira hospital, Navamindradhiraj E-mail: [email protected] University, Thailand E-mail: [email protected]
La-or Juntrima Li hua Xie College of Medicine and Public Health, Department of Pathogen Biology,School of Ubon Ratchathani University, Thailand Public Health, Southern Medical University E-mail: [email protected] Guang Zhou, Guang Dong ,P.R.China E-mail: [email protected]
Li peng Tan Lianawati Lianawati Faculty of Veterinary Medicine, Universiti Khon Kaen University, Thailand Malaysia Kelantan, Malaysia E-mail: [email protected] E-mail: [email protected]
Lorina Ulag Mang Ngaih Ciin Indonesia International Institute for Life Khon Kaen University, Thailand Sciences, Indonesia E-mail: [email protected] E-mail: [email protected]
Maria Pakharukova Md. Siddikur Rahman Institute of Cytology and Genetics SB RAS, Department of Medical Microbiology, Russia Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Men-Bao Qian Mohamed Shahid National Institute of Parasitic Diseases, Chinese Khon Kaen University, Thailand Center for Disease Control and Prevention, China E-mail: [email protected] E-mail: [email protected]
Mohd Faizal Ghazali Muhammad Almanfaluthi UNISZA, Malaysia University of Muhammadiyah Purwokerto, E-mail: [email protected] Indonesia E-mail: [email protected]
Muntinee Lertpanprom Nahathai Uttamamul Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 123
List of Participants (Cont.) Nantana Suwandittakul Nanthicha Inrueangsri Tropical Medicine Program, Faculty of Mahidol University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Narong Pleerux Naruemon Sitthichot Faculty of Geoinformatics, Phramongkutklao College Medicine, Burapha University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Natchita Chungkanchana Nathkapach Rattanapitoon Faculty of Associated Medical Sciences, Parasitic Disease Research Center, Institute of Khon Kaen University, Thailand Medicine, Suranaree University of Technology, E-mail: [email protected] Thailand E-mail: [email protected]
Nattaya Watwiengkam Ngoy Mutombo Veterinary sciences of Mahasarakam Independent Consultant, Australia University, Thailand E-mail: [email protected] E-mail: [email protected]
Nicha Charoensri Nikom Srikacha Faculty of Associated Medical Sciences, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Nitis Smanthong Noraziah Mohamad Zin Faculty of Associated Medical Sciences, Programme of Biomedical Science, Malaysia Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Nurmasita Datu Adam Nutchanon Duangdech Banggai District Health Office, Indonesia Department of Microbiology, Faculty of E-mail: [email protected] Medicine, Khon Kaen University, Thailand E-mail: [email protected]
Oei Stefani Yuanita Widodo Olga Fedorova University of Muhammadiyah Purwokerto, Siberian State Medical University, Russia Indonesia E-mail: [email protected] E-mail: [email protected] 124 NTDASIA 2019
List of Participants (Cont.) Ornuma Haonon Oxana Zaparina Khon Kaen University, Thailand Institute of Cytology and Genetics SB RАS, E-mail: [email protected] Russia E-mail: [email protected]
Parichat Sattayarak Patcharin Phuwilert Chaiyaphum Rajabhat University, Thailand Mahasarakham University, Thailand E-mail: [email protected], E-mail: [email protected] [email protected]
Patpicha Arunsan Paweesuda Choosung Department of Parasitology, Faculty of Khon Kaen University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Petrus Tang Phuangphet Waree Molee Department of Parasitology, Chang Gung Naresuan University,Thailand University, Taiwan E-mail: [email protected] E-mail: [email protected]
Picha Suwannahitatorn Pornphutthachat Sota Phramongkutklao College of Medince, Thailand Tropical Disease Research Laboratory, Faculty E-mail: [email protected] of Medicine, Khon Kaen University, Thailand E-mail: [email protected]
Porntip Pinlaor Prasarn Tangkawattana Faculty Associated Medical Science, Faculty of Veterinary Medicine, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Prasert Saichua Prissadee Thanaphongdecha Tropical Medicine Program, Faculty of Faculty of Medicine, Khon Kaen University, Medicine, Khon Kaen University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Purna Chetri Putri Ramadhani Putri North Eastern Hill University (NEHU), Faculty of Public Health, Khon Kaen University, Shillong, India Thailand E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 125
List of Participants (Cont.) Qin Yang Qinyan Chen Faculty of Public Health, Guangxi Key Laboratory for the Prevention Khon Kaen University, Thailand and Control of Viral Hepatitis Guangxi Zhuang E-mail: [email protected] Autonomous Region Centre for Disease Prevention and Control, China E-mail: [email protected]
Radhakrishnan Muthukumar Rajapakse Peramune Vedikkarage Jayanthe Khon Kaen University, Thailand Faculty of Veterinary Medicine and Animal E-mail: [email protected] Science, University of Peradeniya, Sri Lanka E-mail: [email protected]
Raksawan Deenonpoe Ratana Leksomboon Department of Pathology, Faculty of Medicine, Ubon Ratchathani University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Ratree Tavichakorntrakool Ravinnipa Chanakankun Khon Kaen University, Thailand Faculty Associated Medical Sciences, E-mail: [email protected] Khon Kaen University, Thailand E-mail: [email protected]
Rini Mutahar Rungtiwa Dangtakot Sriwijaya University, Indonesia Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Rutcharin Potiwat Saeranee Siriphak Department of Medical Entomology, Faculty of Khon Kaen University, Thailand Tropical Medicine, Mahidol University, Thailand E-mail: [email protected] E-mail: [email protected]
Sakonsupa Pichetworakool Samnang Chin Chiang Mai University, Thailand Royal Government of Cambodia, E-mail: [email protected] Cambodia E-mail: [email protected]
Sangduan Wannachart Santisith Khiewkhern Tropical Disease Research Laboratory, Faculty Mahasarakham University, Thailand of Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] 126 NTDASIA 2019
List of Participants (Cont.) Sararat Hattakam Sasisopa Singhanetr Suranaree University of Technology, Thailand Chiang Mai University, Thailand E-mail: [email protected] E-mail: [email protected]
Shafariatul Akmar Ishak Shifa Fauziyah Universiti Kebangsaan Malaysia, Malaysia Universitas Airlangga, Indonesia E-mail: [email protected] E-mail: [email protected]
Sirikachorn Tangkawattana Sirinan Kimcharoensuk Faculty of Veterinary Medicine, Mahidol University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Sirinapa Prathummang Siriporn Proungvitaya Kasetsart University, Thailand Department of Clinical Chemistry, Faculty of E-mail: [email protected] Associated Medical Science, Khon Kaen University, Thailand E-mail: [email protected]
Siti Nursheena Mohd Zain Somchai Pinlaor University of Malaya, Malaysia Department of Parasitology, Faculty of E-mail: [email protected] Medicine, Khon Kaen University, Thailand E-mail: [email protected]
Somphou Sayasone Somsanith Chonephetsarath Lao Tropical and Public Health Institute, Laos Department of Medical Entomology, Faculty of E-mail: [email protected] Tropical Medicine, Mahidol University, Thailand E-mail: [email protected]
Son d.a. Truong Songkiad Upontain Faculty of Associated Medical Sciences, Faculty of Veterinary Medicine, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Sooji Hong Sothear San Institute of Parasitic Diseases, Faculty of Public Health, Korea South Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 127
List of Participants (Cont.) Sriwipa Chuangchaiya Sudarat Onsurathum Kasetsart University, Thailand Department of Parasitology, Faculty of E-mail: [email protected] Medicine, Khon Kaen University, Thailand E-mail: [email protected]
Sujittra Chaiyadet Suksanti Prakobwong Faculty of Medicine, Khon Kaen University, Udon Thani Rajabhat University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Sung-Jong Hong Sung-Tae Hong Department of Medical Environmental Biology, Department of Tropical Medicine and Chung-Ang University College of Medicine Parasitology, Seoul National University E-mail: [email protected] College of Medicine, Korea South E-mail: [email protected]
Suriyani Tan Sutas Suttiprapa Tropical Medicine Program, Faculty of Tropical Medicine Graduate Program, Medicine, Khon Kaen University, Academic Affairs, Faculty of Medicine, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Sutharut Pituk Taehee Chang Udontani Rajabhat University, Thailand Institute of Parasitic disease, Korea Association E-mail: [email protected] of Health Promotion, Korea South E-mail: [email protected]
Tanakorn Proungvitaya Tanapat Ratanapakorn Faculty of Associated Medical Science, Faculty of Medicine, Khon Kaen University, Khon Kaen University, Thailand Thailand E-mail: [email protected] E-mail: [email protected]
Thamayanthi Nada Raja Thanh Hoa Le Malaria Research Centre, UNIMAS, Malaysia Institute of Biotechnology (IBT) and Graduate E-mail: [email protected] University of Science and Technology (GUST) belonging to Vietnam Academy of Science and Technology (VAST) in Hanoi, Vietnam E-mail: [email protected]
Thatsanapong Pongking Theerayut Thongrin Khon Kaen University, Thailand Faculty of Veterinary Medicine, E-mail: [email protected] Khon Kaen University, Thailand E-mail: [email protected] 128 NTDASIA 2019
List of Participants (Cont.) Theingi Win Myat Therachon Kamollerd Department of Medical Research, Faculty of Veterinary Medicine, Myanmar Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Thewarach Laha Thunyamas Guntawang Department of Parasitology, Faculty of Chiang Mai University, Thailand Medicine, Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Tidaratt Sittisak Tilly Fox Chiang Mai University, Thailand University of Liverpool, United Kingdom E-mail: [email protected] E-mail: [email protected]
Timir Tripathi Tri Maharani Department of Biochemistry, Daha husada hospital, Indonesia North-Eastern Hill University, India E-mail: [email protected] E-mail: [email protected]
Vannarith Nhem Veeranoot Nissapatorn Faculty of Nursing, Khon Kaen University, School of Allied Health Sciences, Thailand Walailak University, Thailand E-mail: [email protected] E-mail: [email protected]
Veha Khun Viatcheslav Mordvinov Prash Kosamak Hospital, Cambodia The Federal Research Center Institute of E-mail: [email protected] Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Russia E-mail: [email protected]
Vickneshwaran Muthu Vipat Kuruchittham Zoonosis Sector, Disease Control Division, Southeast Asia One Health University Network Malaysia (SEAOHUN), Thailand E-mail: [email protected] E-mail: [email protected]
Virak Khieu Wachiraporn Donsa National Center for Parasitology, Entomology Tropical Disease Research Laboratory, Faculty and Malaria Control, Ministry of Health, of Medicine, Khon Kaen University, Thailand Cambodia E-mail: [email protected] E-mail: [email protected] NTDASIA 2019 129
List of Participants (Cont.) Wansiri Weeraphongthongchai Wei Hu Faculty of Medicine, School of Life Sciences, Fudan University, China Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Wilasinee Srisanyong Wilawan Pumidonming Kalasin University, Thailand Department of Microbiology and Parasitology, E-mail: [email protected] Faculty of Medical Science, Naresuan University, Thailand E-mail: [email protected]
Wiyada Chumpol Worapol Aengwanich Faculty of Associated Medical Sciences, Faculty of Veterinary Medicine, Khon Kaen University, Thailand Mahasarakham University, Thailand E-mail: [email protected] E-mail: [email protected]
Worawan Chumpia Woro Danur Wendo Faculty of Associated Medical Sciences, Faculty of Veterinary Medicine, Khon Kaen University, Thailand Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Xiaoguang Chen Xueyan Wang Department of Pathogen Biology, School of Guangxi Key Laboratory for the Prevention Public Health, Southern Medical University, and Control of Viral Hepatitis Guangxi Zhuang China Autonomous Region Centre for Disease E-mail: [email protected] Prevention and Control, China E-mail: [email protected]
Ye Min Htet Yeni Indriyani Neglected Tropical Disease Support Center, Khon Kaen University, Thailand Task Force for Global Health, Atlanta, USA E-mail: [email protected] E-mail: [email protected]
Yichao Yang Yi-Chen Wang Department of Parasitic Disease Prevention Department of Geography, Guangxi Zhuang Autonomous Region Centre National University of Singapore, for Disease Prevention and Control, China Singapore E-mail: [email protected] E-mail: [email protected]
Yi-jia Guo Yijie Zhao Department of Pathogen Biology, School of Department of Pathogen Biology, School of Public Health, Southern Medical University, Public Health, Southern Medical University, China China E-mail: [email protected] E-mail: [email protected] 130 NTDASIA 2019
List of Participants (Cont.)
Yingsi Lai Yiping Li School of Public Health, Zhongshan School of Medicine, Sun Yat-sen University, China Sun Yat-sen University, China E-mail: [email protected] E-mail: [email protected]
Yudha Puratmaja Yue Hu Faculty of Public Health, Sun Yat-sen University, China Khon Kaen University, Thailand E-mail: [email protected] E-mail: [email protected]
Yugu Xie Yukifumi Nawa Department of Pathogen Biology, Key Tropical Disease Research Laboratory, Faculty Laboratory of Tropical Disease Research of of Medicine, Khon Kaen University, Thailand Guangdong Province, School of Public Health, E-mail: [email protected] Southern Medical University, China E-mail: [email protected]
Yupawadee Tasako Zetty norhana Balia Yusof Khon Kaen University, Thailand Universiti Putra Malaysia, Malaysia E-mail: [email protected] E-mail: [email protected]
Zhihua Jiang Zulkarnain Md Idris Department of Parasitic Disease Prevention Universiti Kebangsaan Malaysia, Malaysia Guangxi Zhuang Autonomous Region Centre E-mail: [email protected] for Disease Prevention and Control, China E-mail: [email protected] NTDASIA 2019 131 TROPICAL MEDICINE GRADUATE PROGRAM-KKU
Institute: Khon Kaen University Contact Info: Tel: +66-43-363113 Fax: +66-43-348375 email: [email protected]
WELCOME MESSAGE Why study in Khon Kaen? • Situated in the heart of Northeast On behalf of the Faculty of Medicine, Khon Kaen University, it is my pleasure to Thailand, an endemic area for welcome you to the Interna=onal Graduate Program in Tropical Medicine. The neglected tropical diseases program was established in 2012 and offers an interdisciplinary curriculum, • An urban area within easy reach of comprising two different tracks each for a Master of Science or a Doctor of affected communities (10-30 min drive) Philosophy in Tropical Medicine. The program was established to give students an • Fully equipped modern research opportunity to pursue their specific interests in biomedical sciences, community facilities at Khon Kaen University, a health and clinical tropical infec=ous diseases. Therefore, Tropical Medicine students National Research University of Thailand will become specialized in mul=ple disciplines as they will contribute to liver fluke • Strong research teams with world and other neglected tropical disease research relevant to Southeast Asia. renowned experts in various tropical With the advantage of longstanding collabora=ons with several tropical ins=tutes diseases across the world, we provide students with challenging research, world-renowned • A day’s drive to Laos, Vietnam and Cambodia, other affected Lower professors, and top-ranked scien=sts in tropical medicine as supervisors/co- Mekong countries served by the supervisors. Students also have opportuni=es to do parts of their research work in medical facilities in Khon Kaen Khon Kaen, Thailand, or in other Mekong countries at regional partner ins=tutes. • World class conference center with a Students can also experience modern laboratories in advanced countries such as complete range of accommodations Australia, UK, German, Japan, Singapore, Switzerland and the USA. (3 to 5 star hotels) • Only 40 min flying time from Bangkok - On behalf of both faculty and staff, we welcome you to study in our Tropical the heart of Southeast Asia Medicine Graduate Program at the Faculty of Medicine, Khon Kaen University.
Sincerely, Program coordinator: Banchob Sripa Dr.Sutas Suttiprapa Director, Tropical Medicine Graduate Program E-mail: [email protected]
ADDRESS: Tropical Disease Research Laboratory, Department of Pathology, Medicine, Khon Kaen University, THAILAND Facebook: TDRLab/ Tropical132 MedicineNTDASIA Graduate 2019 Program, Faculty of Medicine, Khon Kaen University, Thailand - Destination for NTD research & study
MSc in Tropical Medicine Student scholarship Thailand are field research sites in cooperation with local health offices. Study program ( 2 years) There will be several studentships available for foreign students upon Type A1 36 credit hours (thesis only) competition. Current research activities Type A2 36 credit hours Mekong Opisthorchiasis Control All research expenses are covered by • Coursework a minimum of 24 credits Initiative project grants. Thesis 12 credits • Innovative Strategies for Sustainable Current research grants Control of Asian Schistosomiasis and PhD in Tropical Medicine Tropical Medicine Research Center Other Helminth Zoonoses through Study program (3-4 years) Grant (NIAID, NIH, USA) Socio-Ecosystem-Based Type A1.1 48 credit hours (thesis only) National Cancer Institute (NIH, USA) Interventions Pathogenesis, carcinogenesis and Type A2.1 48 credit hours (from MSc) Eco-EID, IDRC (Canada) • ecology of opisthorchiasis in Thailand Coursework a minimum of 12 credits National Research University - KKU • Helicobacter - -indcued Thesis 36 credits Grant Opisthorchis cholangiocarcinoma 72 credit hours (from BSc) National Health Security Office Grant Type A2.2 • Helminth control using EcoHealth/ Coursework a minimum of 24 credits International collaborations One Health approach Thesis 48 credits George Washington University, USA • Metabolic syndrome and helminths Examples of courses: James Cook University, Australia • Epidemioloy of leptospirosis in endemic areas 359701 Tropical Medicine University of Liverpool, UK Melioidosis and more 359711 Tropical Disease Control Institute of Medical Research, Australia • 359712 Experiences in Tropical Health Swiss Tropical and Public Health Regional partners 364701 Medical Parasitology Institute, Switzerland 364711 Medical Entomology • National Institute of Parasitic Global Health Asia Diseases, Shanghai, PR China 362713 Infection and Immunity School of Public Health, University of Hanoi Medical University, Vietnam 374754 Community Epidemiology • California Berkeley, USA 379794 Clinical Tropical Infectious • National Institute of Hygiene and National University of Singapore Epidemiology, Vietnam Diseases Center of Malariology, Parasitology 381703 Health Systems Management Research facilities • and Entomology, Lao PDR Tropical Disease Research Laboratory How to apply (TDRC) is the main laboratory of the • Center for Parasitology, Entomology and Malaria Control, Cambodia For details, please visit the link:http:// program. Endemic areas of Northeast inter.gs.kku.ac.th • Ministry of Public Health, Thailand
CHRISTINA KIM The first PhD graduate of Tropical Medicine International Graduate Program. “Being from a small town in Cleveland, Ohio, I never imagined myself moving to Thailand, a tropical country, to study Tropical Medicine. The Tropical Medicine Graduate Program at Khon Kaen University has exceeded my expectations as a new program. I am constantly challenged by my multidisciplinary coursework, my co-workers in the Tropical Disease Research (TDR) Laboratory and teachers; world renown leading experts in Medical Parasitology, Pathology, Public Health, Biomedical Sciences, and Medicine. The learning and experience I am gaining by choosing Khon Kaen, Thailand and the TDR laboratory could not be replaced by any other form of study. I feel challenged, humbled, and blessed every day!, not to mention the food is great!”
ADDRESS: Tropical Disease Research Laboratory, Department of Pathology, Medicine, Khon Kaen University, THAILAND http://tdr.kku.ac.th/
Printed by : KLUNG NANA VITHYA PRESS LIMITED PARTNERSHIP Tel. +66-43-466444, +66-43-466860-61 Asian Neglected Tropical Disease Conference
Asian Neglected Tropical Disease Conference NTDASIA2019 International Research Network ABSTRACTS & PROCEEDINGS NTD ASIA 2019 Pullman Raja Orchid Hotel, Khon Kaen, Thailand NTDASIA2019
Pullman Raja Orchid Hotel Khon Kaen, Thailand 7-9 August 2019 7-9 August 2019 7-9 August
Organized by Tropical Disease Research Center Faculty of Medicine, Khon Kaen University, Thailand Tropical Disease Research Center Fax: +66-43-204359 Tel: +66-43-363113 Faculty of Medicine, Khon Kaen University, Thailand