THE EPIDEMIOLOGY OF PORK-BORNE PARASITIC ZOONOSES IN RURAL COMMUNITIES IN THE CENTRAL HIGHLANDS OF VIETNAM Dinh Ngoc Nguyen http://orcid.org/0000-0002-2028-2186 Submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy May 2018 Faculty of Veterinary and Agricultural Sciences The University of Melbourne ABSTRACT Background Food-borne parasitic zoonoses impact negatively on humans in terms of health and loss of productivity as well as economically, through loss of market access and trade. Taenia and Trichinella are two of the most important meat-borne zoonoses globally. In the Central Highlands of Vietnam, living standards are poor; open defaecation via use of outdoor latrines is commonly practiced and livestock access to these latrine areas is, for the most part, unrestricted. Moreover, the consumption of undercooked or raw pork dishes is common traditional practice. These factors combined are highly conducive for the transmission of several pork-borne parasitic zoonoses, including taeniasis, Taenia solium cysticercosis and trichinellosis. In this context, developing and validating diagnostic assays to study the epidemiology of these pork-borne parasites within these communities allows risk factors for exposure and infection to be ascertained and this, in turn, allows community-based control programs to be better targeted. Methodology/Principle findings This project was conducted as a cross-sectional study in Buon Don, Krong Nang and M’Drak district in Dak Lak province in the Central Highlands of Vietnam. A combination of parasitological, serological and molecular diagnostic methods were utilised to determine the levels of infection of Taenia spp. in humans and Trichinella spp. in pigs, and seroexposure to T. solium in humans and pigs. The data were analysed in relation to host, management and environmental risk factors using frequentist and Bayesian multivariable analyses. In Chapter 2, a systematic review on previous research on taeniasis, T. solium cysticercosis and trichinellosis was performed and showed that little to no data are available on the prevalence of these food-borne zoonoses in the central and southern areas of the country. Moreover, utilisation of tests with either low sensitivity or poor specificity for the diagnosis of taeniasis in humans and T. solium cysticercosis in humans and pigs produced either an over- or underestimation of their TP. To address this, a Bayesian model was used to estimate the TP of taeniasis and Taenia solium i cysticercosis and demonstrated a true prevalences of up to 25% and 24%, respectively for specific rural ‘hotspots’. In Chapter 3, a multiplex Taq-Man probe-based real-time PCR for the species-specific detection of three Taenia species in human stool was developed and its diagnostic parameters compared with the Kato-Katz thick smear and coproantigen ELISA assay using field samples collected as part of a cross-sectional survey in Dak Lak province. The overall AP of human taeniasis by the multiplex qPCR was 6.7% (95% CI 4.4 to 10). The sympatric existence of Taenia solium, Taenia asiatica and Taenia saginata in Dak Lak province was confirmed for the first time, as was the extension of the known distribution of T. asiatica to southern Vietnam. Risk factors associated with Taenia spp. infection included the routine consumption of undercooked beef and pork, routine consumption of pork tongue, and personal observation of Taenia proglottids in stool. Chapter 4 describes a cross-sectional study on the seroprevalence of human T. solium cysticercosis in Dak Lak determined by two diagnostic assays, the apDia-ELISA, a commercial antigen detection assay and the LLGP-EITB antibody detection assay. The seroprevalence of exposure to T. solium in humans was 5% (95% CI 3 to 8), while the apDia-ELISA, detected circulating T. solium antigens in 7.3% (95% CI 4.9 to 10.8) of the sampled population. Consuming raw vegetables, drinking water sourced from lakes, streams or ponds, and outdoor defaecation were associated with increased T. solium exposure risk in humans. In Chapter 5, the seroexposure of pigs to T. solium cysticercosis in Dak Lak was determined using rT24H and native LLGP antigens in series, in an EITB format. The seroprevalence of exposure to T. solium was low at 0.94 (95% CI 0.51 to 1.68). Coprophagy of human faeces and scavenging for food were factors significantly associated with T. solium exposure in pigs. In Chapter 6, the contribution of all modifiable risk factors for taeniasis, T. solium exposure in humans and pigs is explored and this was estimated to range from 24% to 77%. Spatial analyses demonstrated that human- or pig-T. solium exposure-positive households were more likely to be surrounded by other exposure-positive households in some study locations. This spatial aggregation of human exposure-positive households ii was hypothesised to be due to a combination of demographic, anthropological and micro environmental factors, whilst scavenging for food and coprophagy of human faeces are likely to drive the aggregation of pig exposure-positive households. Finally, no Trichinella larvae were detected in the confined and free-roaming pig populations using the artificial digestion technique on tongue, diaphragm and masseter muscle. The estimated TP of Trichinella was low, ranging from 0.09 to 0.80% in domestic pigs. Further diagnostic screening using serology was beyond the scope of this current project but advocated for future research. Conclusions This thesis has contributed significantly to a better understanding of the epidemiological characteristics of Taenia spp. infection, and seroexposure to T. solium and Trichinella spp. infection among humans and pigs in the Central Highlands of Vietnam. This information will assist local government and residents to develop appropriate methods and strategies to reduce or mitigate the burden of these diseases in Dak Lak. iii DECLARATION This is to certify that I. the thesis comprises only my original work towards the PhD except where indicated the text II. due acknowledgement has been made in the text to all other materials used III. the thesis is fewer than 100,000 words in length, exclusive of tables, figures, bibliographies and appendices ……………… Dinh Ngoc Nguyen Faculty of Veterinary and Agricultural Sciences The University of Melbourne iv PREFACE This study outcomes presented here were achieved by self-effort with the dedicated support from colleagues and students at Tay Nguyen University, local medical staff and veterinarians in Dak Lak, Vietnam that assisted with fieldwork, and from experts in Australia, Belgium and the USA that assisted laboratory and statistical analysis. The results herein are original work conducted during the duration of my PhD and has not been not published previously except for the references cited in the text. This thesis is composed of a combination of peer reviewed published papers, manuscripts that are currently under review and those in preparation. This thesis content does not contain in any part work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. Peer-reviewed articles published in international scientific journals Ng-Nguyen D, Stevenson MA, Traub RJ. A systematic review of taeniasis, cysticercosis and trichinellosis in Vietnam. Parasit Vectors 2017;10:150. Ng-Nguyen D, Stevenson MA, Dorny P, Gabriël S, Vo T Van, Nguyen VAT, Phan TV, Hii SF, Traub RJ. Comparison of a new multiplex real-time PCR with the Kato Katz thick smear and copro-antigen ELISA for the detection and differentiation of Taenia spp. in human stools. PLoS Negl Trop Dis 2017;11:e0005743. Ng-Nguyen D, Stevenson MA, Breen K, Phan T Van, Nguyen V-AT, Vo T Van, et al. The epidemiology of Taenia spp. infection and Taenia solium cysticerci exposure in humans in the Central Highlands of Vietnam. BMC Infect Dis 2018;18:527. Ng-Nguyen D, Noh J, Breen K, Stevenson MA, Handali S, Traub RJ. The epidemiology of porcine Taenia solium cysticercosis in communities of the Central Highlands in Vietnam. Parasit Vectors. 2018;11:360. v Ng-Nguyen D, Traub RJ, Nguyen V-AT, Breen K, Stevenson MA. Spatial distribution of Taenia solium exposure in humans and pigs in the Central Highlands of Vietnam. PLoS Negl Trop Dis 2018;12:e0006810. Conference proceedings and seminars given Ng-Nguyen D, 2017. The epidemiology of pork-borne parasitic zoonoses in rural communities in the central highlands of vietnam. PhD Oration, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Australia, 30 November 2017. Ng-Nguyen D, Stevenson MA, Handali S, Noh J, Traub RJ, 2017. Individual- and environmental-level risk factors of Taenia solium cysticercosis in the Central Highlands of Vietnam. Annual Conference of the Australian Society for Parasitology, Leura, Australia, 26-29 June 2017. Ng-Nguyen D, Stevenson MA, Traub RJ. Individual- and environmental-level risk factors for taeniasis and taenia solium cysticercosis in the central highlands of Vietnam. International Congress for Tropical Medicine and Malaria, Brisbane, Australia, 18-22 September 2016. Ng-Nguyen D, 2015. The epidemiology of pork-borne parasitic zoonoses in rural communities in the central highlands of Vietnam. Confirmation of Candidature, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, Australia, 24 February 2015. vi ACKNOWLEDGEMENTS This thesis is dedicated my parents, Nguyen Manh Dong and Pham Thi Nhung, and Aunt, Nguyen Thi Boc, who always provided their endless love and blessings throughout my life. This thesis is especially dedicated to my beloved wife, Nguyen Thi Van-Anh, for her constant encouragement, limitless giving, great sacrifice and help and support during my PhD study; to my kids, Nguyen Ngoc Dang Khoa and Nguyen Ngoc Thao Vy, for their endurance owing to their lack of a Father’s care during my period away from home. It is with deepest gratitude that I acknowledge my supervisors, Prof. Rebecca Justine Traub and Prof.